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 procedures waarbij dieren werden uitgevoerd volgens de richtlijnen van de Northwestern University Institutional Animal Care en gebruik Comite (IACUC) goedgekeurd. 1. muismodellen <ol><li> Gebruik 8-12 weken oude muizen in de lichamelijke oefening. Om uitgeoefend geïnduceerde autofagie in vivo te detecteren, gebruik maken van de GFP-LC3 transgene muizen (C57BL / 6 achtergrond) voor de beeldvorming studies en C57BL / 6 muizen voor biochemische analyses. </li></ol> 2. Exercise...

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Autofagie is een katabool proces dat energie levert en vermindert cytotoxiciteit door lysosomale afbraak van cytoplasma onderdelen of beschadigde organellen. Bestuderen autofagie belangrijk voor de regulatie van cellulaire homeostase en de mechanismen van stress respons begrijpen. Nieuwe modellen en methodieken zijn in opkomst op het gebied van onderzoek 15, om te bestuderen hoe verstoorde autofagie bijdraagt aan tal van pathologische processen 16, <sup class="x...

Autofagie is een evolutionair geconserveerd afbraakroute, die wordt geïnduceerd in reactie op verschillende stress-aandoeningen zoals honger en hypoxie 1, 2. Tijdens autofagie, dubbel membraan blaasjes, genaamd autofagosomen, nemen onnodige of beschadigde subcellulaire componenten en vervoeren ze in lysosomen voor degradatie 3. Basale autofagie is essentieel voor cellulaire functie en organisme ontwikkeling en verminderde basale autofagie is betrokken bij vele stoornissen, waaronder neurodegeneratie, tumorigenese en type 2 diabetes 4, 5, 6. De bekendste fysiologische autofagie inducer is de honger. Het heeft echter twee belangrijke beperkingen. Ten eerste, verhongering duurt een lange periode om autofagie effectief te veroorzaken bij dieren, bijvoorbeeld, 48 uur van voedsel beperking in muizenIn de meeste organen. Ten tweede, verhongering nauwelijks induceert hersenen autofagie, als gevolg van een relatief stabiele toevoer van voedingsstoffen in de hersenen. In feite is het ook moeilijk om autofagie detecteren van kleine moleculen inductoren, zoals veel geneesmiddelen de bloed-hersenbarrière niet kan passeren. Dus, om beter de functie van autofagie activering in pathogenese analyseren we onlangs ontdekt dat de oefening is een meer potente fysiologische methode autofagie induceren in een korte tijd 7, 8, 9. Vergeleken met hongersnood, wordt autofagie effectief geïnduceerd door loopband lopen zo snel als 30 min. Aldus oefening is een gemakkelijke en krachtige fysiologische benadering om het mechanisme van autofagie bestuderen mediëren gezondheidsvoordelen en voorkomen van ziekten. Er zijn verschillende eiwit markers voor de detectie van autofagie activiteit, waaronder LC3 en p62. LC3 (microtubule-geassocieerde eiwit 1A / 1B-light chain 3) is een cytosolisch eiwit (LC3-I te vormen) dat is geconjugeerd aan PE (fosfatidylethanolamine) bij autofagie. PE-gelipideerde LC3 (LC3-II vorm) wordt gerekruteerd op autophagosomal membranen en kan worden gebruikt om autofagosomen zichtbaar wordt bij gelabeld met GFP. De translocatie van het cytoplasma naar structuren van autofagosomen punctata onder microscopie is een indicatie van autofagie. p62 is een lading receptor voor autofagie substraten (zoals ubiquitinatie eiwitten), en is opgenomen in autofagosomen ook. Omdat het eiwit wordt afgebroken in lysosomen met autofagosomen, kan het niveau worden gebruikt om de flux te meten autofagie. Hier laten we zien hoe deze markers te gebruiken om autofagie te kwantificeren in verschillende muis weefsels veroorzaakt door aërobe oefening, met inbegrip van gedwongen oefening (loopband) en vrijwillige inspanning (loopwielen). Dezelfde werkwijzen kunnen ook worden toegepast op in vivo meten van autofagie na behandeling van andere induceerders.

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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.

Dit protocol beschrijft twee verschillende methodes om autofagie te induceren in de muis weefsels door aërobe oefening: een totaal van 90 min van gedwongen oefening op een multi-lane loopband voorafgegaan door twee dagen van acclimatisatie; of twee weken van de vrijwillige oefening op een loopwiel gebruikt door single-gehuisvest muizen. Iedere testprotocol, kunnen we de autophagy flux door fluorescentie microscopie en western blot analyse in verschillende organen te meten. <p class=...

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

Activating Autophagy by Aerobic Exercise in Mice

Autofagie activatie is gunstig bij de preventie van een aantal ziekten. Een van de fysiologische technieken induceren autofagie in vivo lichaamsbeweging. Hier laten we zien hoe autofagie activeren door aërobe oefening en meet autofagie niveaus in muizen.

Het activeren van Autofagie door Aerobic Oefening in Muizen

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

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

Meting van insuline- en contractie-gestimuleerde glucoseopname in geïsoleerde en geïncubeerde volwassen skeletspieren van muizen

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 ...