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).

All procedures involving animals were performed according to guidelines approved by the Northwestern University Institutional Animal Care and Use Committee (IACUC).
1. Mouse Models
<ol>
	<li>Use 8-12 week-old mice in the exercise training. To detect exercised-induced autophagy in vivo, use the GFP-LC3 transgenic mice (C57BL/6 background) for imaging studies and C57BL/6 mice for biochemical analyses.</li>
</ol>
2. Exercise-induced Autophagy
<ol>
	<li>Treadmill setup - fo...

<ol>
	<li>Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., Ohsumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+autophagy+in+response+to+nutrient+starvation+using+transgenic+mice+expressing+a+fluorescent+autophagosome+marker.">In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.</a> Mol Biol Cell. 15, 1101-1111 (2004).</li><li>Tracy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BNIP3+is+an+RB/E2F+target+gene+required+for+hypoxia-induced+autophagy.">BNIP3 is an RB/E2F target gene required for hypoxia-induced autophagy.</a> Molecular and cellular biology. 27, 6229-6242 (2007).</li><li>Mizushima, N., Komatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+renovation+of+cells+and+tissues.">Autophagy: renovation of cells and tissues.</a> Cell. 147, 728-741 (2011).</li><li>Nikoletopoulou, V., Papandreou, M. 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J., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+induces+autophagy+in+peripheral+tissues+and+in+the+brain.">Exercise induces autophagy in peripheral tissues and in the brain.</a> Autophagy. 8, 4 (2012).</li><li>Kuramoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+activation+by+novel+inducers+prevents+BECN2-mediated+drug+tolerance+to+cannabinoids.">Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids.</a> Autophagy. 12, 1460-1471 (2016).</li><li>Dougherty, J. P., Springer, D. A., Gershengorn, M. 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Autophagy is a catabolic process that provides energy and reduces cytotoxicity by lysosomal degradation of cytoplasm components or damaged organelles. Studying autophagy is important to understand the regulation of cellular homeostasis and the mechanisms of stress response. New models and methodologies are emerging in the research field15, to study how impaired autophagy contributes to numerous pathological processes16,17.
<p class="jo...

Autophagy is an evolutionarily conserved degradation pathway, which is induced in response to various stress conditions such as starvation and hypoxia1,2. During autophagy, double-membrane vesicles, called autophagosomes, incorporate unnecessary or damaged subcellular components and transport them into lysosomes for degradation3. Basal autophagy is essential for cellular function and organism development, and impaired basal autophagy is implicated in many disorders, including neurodegeneration, tumorigenesis and type 2 diabetes4,5,6.
The best-known physiological autophagy inducer is starvation. However, it has two major limitations. First, starvation takes a long period to effectively induce autophagy in animals, e.g., 48 hr of food restriction in mice in most organs. Second, starvation barely induces brain autophagy, due to a relatively stable nutrient supply in the brain. In fact, it is also difficult to detect autophagy induction by small-molecule inducers, as many drugs cannot pass the blood brain barrier. Thus, to better analyze the function of autophagy activation in disease pathogenesis, we recently discovered that exercise is a more potent physiological method to induce autophagy in a short period of time7,8,9. Compared with starvation, autophagy is effectively induced by treadmill running as fast as 30 min. Thus, exercise is a convenient and potent physiological approach to study the mechanism of autophagy in mediating health benefits and preventing diseases.
There are several protein markers for the detection of autophagy activity, including LC3 and p62. LC3 (microtubule-associated protein 1A/1B-light chain 3) is a cytosolic protein (LC3-I form) that is conjugated to PE (phosphatidylethanolamine) upon autophagy induction. PE-lipidated LC3 (LC3-II form) is recruited onto autophagosomal membranes and can be used to visualize autophagosomes when labeled with GFP. Its translocation from the cytosol to punctate structures of autophagosomes under microscopy is an indication of autophagy induction. p62 is a cargo receptor for autophagy substrates (such as ubiquitination proteins), and is incorporated into autophagosomes as well. Since the protein is degraded in lysosomes along with autophagosomes, its levels can be used to measure the autophagy flux. Here we show how to use these markers to quantify autophagy in different mouse tissues induced by aerobic exercise, including forced exercise (treadmill) and voluntary exercise (running wheels). The same procedures can also be applied to in vivo measurement of autophagy after treatment of other inducers.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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.

This protocol describes two different methods to induce autophagy in mouse tissues by aerobic exercise: a total of 90 min of forced exercise on a multi-lane treadmill proceeded by two days of acclimation; or two weeks of voluntary exercise on a running wheel used by single-housed mice. In each exercise protocol, we can measure the autophagy flux by fluorescence microscopy and western blot analysis in various organs.
We used a tr...

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

Activating Autophagy by Aerobic Exercise in Mice

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

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

The overall goal of this experiment is to physiologically activate autophagy in mice by forced or voluntary running, and then measure the levels of autophagy in specific tissues. This method can help answer key questions about autophagy, in particular, how autophagy and action contributes to the beneficial effects mediated by aerobic exercise. The main advantage of this technique is that physical exercise is a fast aerobic sway between autophagy in vivo using mice.For this experiment, start with eight to twelve week old C57 black six mice, harboring a trans gene to detect exercise induced autophagy in vivo, known as GFP-LC3. For details on the use of mice treadmills, see the following JoVE publication. Set the electrical stimulus of the treadmill to low intensity.On their first two sessions, acclimate the mice to a ten degree uphill open treadmill. On the first day, exercise the mice for five minutes with the treadmill running at eight meters per minute. On the second day, run the mice for ten minutes, initially at eight meters per minute, and after five minutes, ramp up the speed to ten meters per minute.On the third day, run the mice for a full 90 minutes. Start the treadmill at ten meters per minute and encourage the mice to run using gentle nudges to avoid repeated foot shocks. It is critical to observe and manipulate the mice so they don't receive too many electric shocks during the 90 minute run.Throughout the run, use your fingers to encourage the mice to stay on the treadmill. After 40 minutes, 6increase the speed by one meter per minute. After 50 minutes, increase the speed by one meter per minute again.After 60 minutes, increase the speed by another meter per minute. After 70 minutes, start increasing the speed every five minutes so that the last five minutes of the 90 minute run are 17 meters per minute. After the end of the trial, immediately euthanize the mice for tissue collection if desired.For this test, house the mice individually. Use the same strain as previously described. Prepare an 11.4 centimeter diameter mouse running wheel with an attached bike odometer.Each rotation should measure 358 millimeters of travel. Move the mouse into the cage containing the wheel and let the mouse use it freely for two weeks. Every 24 hours, record the distance run by the mouse from the odometer.After the two weeks, harvest tissues for analysis as needed. To measure the autophagic flux, treat the mice with the autophagy inhibitor, chloroquine, for three days. Dissolve the chloroquine in PBS at five milligrams per milliliter and inject it into mice intraperitoneally at the dose of 50 micrograms per gram.Give the mice one injection daily for three consecutive days and harvest tissues three hours after the last injection. For the treadmill exercised mice, start the 90 minute run 90 minutes after the injection on the third day so the run ends three hours after the injection. Then euthanize the mice and immediately harvest their tissues.Prepare to profuse an animal within 90 minutes of being exercised by preparing the fixative. Load a 30 milliliter syringe with 15 to 20 milliliters of freshly made four percent paraformaldehyde and connect the syringe with a 20 gauge catheter needle. Attach the loaded syringe pump and set it to 90 milliliters per hour of continuous flow.With the animal on a clean work surface, in a supine position, cut open the thoracic cavity through the diaphragm to expose the heart. Now, insert the catheter needle into the right ventricle. Next, make a small incision on the liver to drain the blood and start the pump.Inject the fixative until the lung and liver turn completely pale. Usually, 15 milliliters of fixative will suffice. After the profusion, remove the needle and collect tissues of interest.To collect skeletal muscle, dissect the vastus lateralis. Briefly pull the skin of the leg backwards to expose the muscles and localize the quadriceps femoris. Then, dissect out the vastus lateralis, which is the external muscle attached to the upper portion of the femoral bone.For further fixation and dehydration, place the harvested tissues in four percent paraformaldehyde for 24 hours at four degrees celsius. The next day, transfer the tissues to 15 percent sucrose PBS and let them soak at four degrees celsius overnight or until they have settled in the solution. Then, transfer the tissues to 30 percent sucrose PBS at four degrees celsius for overnight or longer.Keep the tissues in the dark as much as possible during these baths. Next, place the tissues in an embedding medium such as OCT and cut them into smaller pieces if necessary. Then, allow them to acclimate for a few minutes in a small petri dish.Afterwards, transfer them to cryomolds with enough OCT to cover. In the molds, orient the sectioning surfaces toward the bottom and avoid forming bubbles. Then, freeze the samples in a covered foam cooler filled with dry ice until the OCT turns white.Now, wrap individual samples in labeled foils, seal them in a plastic bag and store them at negative 80 degrees celsius until they can be processed. Using GFP tagged LC3 as a reporter system, autophagy induction was observed in mice exercised as described. After autophagy stimulation, LC3 translocated from the cytosol to the autophagosome in punctuate structures.Exercised mice had many more GFP-LC3 puncta in both skeletal muscle and the cerebral cortex compared to resting condition mice. Western blot analysis using an LC3 antibody showed that exercise significantly induced the generation of lipid conjugative LC3-II, suggesting that there was increased conversion of cytosolic LC3-I into LC3-II. Next, the degradation of the autophagic cargo receptor, p62, was measured.90 minutes of treadmill activity caused a higher degradation of p62 in skeletal muscle than the resting condition. This effect was rescued by an injection with chloroquine prior to exercise. As chloroquine is an inhibitor of lysosomal degradation, these data indicate that the observed phenomena is due to an elevated autophogagic flux rather than a block in autophagosome degradation.After its development, this technique had precursors in the field of autophagy to explore the effects on the mechanism of exercise in the degradation of metabolism and animal behaviors. While attempting this procedure, it's important to monitor the mice while they are running. Following this procedure, are the methods like electric microscopy.Animal microscopy can be performed in order to answer additional questions like exercise in select autophagy pathways, such as mitophagy and lipophagy.

activating-autophagy-by-aerobic-exercise-in-mice

Research

JoVE Journal

Biology

11.7K visualizações.  Northwestern University. The overall goal of this experiment is to physiologically activate autophagy in mice by forced or voluntary running, and then measure the levels of autophagy in specific tissues. This method can help answer key questions about autophagy, in particular, how autophagy and action contributes to the beneficial effects mediated by aerobic exercise. The main advantage of this technique is that physical exercise is a fast aerobic sway between autophagy in vivo using mice.For this experiment, ...