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).

Alle Verfahren mit Tieren wurden von der Northwestern University Institutional Animal Care und Use Committee (IACUC) zugelassen nach Richtlinien durchgeführt. 1. Mausmodelle <ol><li> Verwenden Sie 8-12 Wochen alten Mäusen in der Ausübung Ausbildung. Zur Erkennung ausgeübt induzierten Autophagie in vivo, verwenden Sie das GFP-LC3 - transgenen Mäusen (C57BL / 6 Hintergrund) für die Bildgebung Studien und C57BL / 6 - Mäuse für biochemische Analysen. </li></ol> 2. Übung indu...

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Autophagie ist ein katabolischen Prozess, der Energie liefert, und reduziert die Zytotoxizität von lysosomalen Abbau von Cytoplasma-Bestandteile oder beschädigte Organellen. Autophagie Studium ist wichtig, dass die Regulation der zellulären Homöostase und die Mechanismen der Stressreaktion zu verstehen. Neue Modelle und Methoden entstehen 15, im Bereich der Forschung zu untersuchen , wie beeinträchtigte Autophagie zu zahlreichen pathologischen Prozessen beiträgt 16,</...

Autophagy ist ein evolutionär konservierten Abbauweg, die in Reaktion auf verschiedene Stressbedingungen, wie Hunger und Hypoxie 1, 2 induziert wird. Während der Autophagie, Doppelmembranbläschen, die so genannte Autophagosomen, integrieren unnötige oder subzellulären Komponenten beschädigt und transportieren sie in die Lysosomen zum Abbau 3. Basal autophagy ist wesentlich für die zelluläre Funktion und Entwicklung des Organismus und die Beeinträchtigung der basalen autophagy wird in vielen Störungen in Verbindung gebracht, einschließlich Neurodegeneration, Tumorgenese und Typ 2 Diabetes 4, 5, 6. Die bekannteste physiologischen Autophagie Induktor Hunger. Es hat jedoch zwei wesentliche Beschränkungen. Zuerst nimmt Hunger eine lange Zeit , um effektiv Autophagie bei Tieren, zum Beispiel 48 Stunden von Lebensmitteln Einschränkung bei Mäusen induzierenin den meisten Organen. Zweitens induziert Verhungern kaum Gehirn autophagy, aufgrund einer relativ stabilen Nährstoffversorgung im Gehirn. In der Tat ist es auch autophagy Induktion von kleinen Molekül-Induktoren zu detektieren schwierig, da viele Medikamente die Blut-Hirn-Schranke passieren können. So um besser auf die Funktion der Autophagie Aktivierung in der Pathogenese der Erkrankung zu analysieren, haben wir entdeckt , vor kurzem , dass die Übung ein potenter physiologische Methode ist die Autophagie in einem kurzen Zeitraum 7, 8, 9 zu induzieren. Verglichen mit Hunger wird Autophagie effektiv induziert durch Laufband so schnell wie 30 Minuten läuft. Somit Übung ist eine bequeme und potente physiologische Ansatz, den Mechanismus der Autophagie zu studieren Nutzen für die Gesundheit bei der Vermittlung und Prävention von Krankheiten. Es gibt mehrere Proteinmarker zum Nachweis von autophagy Aktivität, einschließlich LC3 und p62. LC3 (Mikrotubuli-assoziierten Protein 1A / 1B-Licht chain 3) eine cytosolische Protein (LC3-I bilden), die an PE (Phosphatidylethanolamin), auf autophagy Induktions konjugiert ist. PE-lipidierter LC3 (LC3-II-Form) auf autophagosomalen Membranen rekrutiert und kann verwendet werden, Autophagosomen sichtbar zu machen, wenn sie mit GFP-markierten. Seine Translokation aus dem Cytosol Strukturen Autophagosomen unter Mikroskopie punktiert ist ein Hinweis auf autophagy Induktion. p62 ist ein Fracht Rezeptor für autophagy Substrate (wie Ubiquitinierung Proteine), und wird in Autophagosomen als auch eingearbeitet. Da das Protein in Lysosomen mit Autophagosomen abgebaut wird, kann dessen Ebenen die autophagy Fluss zu messen, verwendet werden. Hier zeigen wir, wie diese Marker zu verwenden, die Autophagie in verschiedenen Geweben der Maus durch Aerobic-Übungen induziert zu quantifizieren, einschließlich Zwangs Bewegung (Laufband) und Kür (Laufräder). Die gleichen Verfahren können auch zur in - vivo - Messung von autophagy nach der Behandlung von anderen Induktoren angewendet werden.

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		<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>
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		<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>
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			<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>
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			<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>
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		<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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		<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>
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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.

Dieses Protokoll beschreibt zwei verschiedene Methoden Autophagie in Geweben der Maus durch Aerobic-Übungen zu induzieren: insgesamt 90 min von Zwangs Übung auf einer mehrspurigen Tretmühle von zwei Tagen der Eingewöhnung verlief; oder zwei Wochen nach der Kür auf einem Laufrad durch einzeln untergebracht Mäuse verwendet. In jeder Trainingsprotokoll, können wir die autophagy Fluss durch Fluoreszenzmikroskopie und Western Blot-Analyse in verschiedenen Organen zu messen. <p clas...

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

Activating Autophagy by Aerobic Exercise in Mice

Autophagy Aktivierung ist bei der Prävention von einer Reihe von Krankheiten nützlich. Eine der physiologischen Ansätze Autophagie in vivo zu induzieren körperliche Bewegung ist. Hier zeigen wir, wie die Autophagie von Aerobic-Übungen zu aktivieren und die Autophagie Ebenen bei Mäusen messen.

Die Aktivierung Autophagy von Aerobic-Übung bei Mäusen

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

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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, ...

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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

Laktat-Dehydrogenase Sequestrierung Assay – Eine einfache und zuverlässige Methode zur Bestimmung Bulk Selbstverdauende Sequestration Aktivität in Säugerzellen

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

Messung der insulin- und kontraktionsstimulierten Glukoseaufnahme in isolierten und inkubierten reifen Skelettmuskeln von Mäusen

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 ...