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 - forced exercise
	<ol>
		<li>Acclimate and train the mice on a 10&#176; uphill open treadmill for 2 days. On day 1, exercise the mice for 5 min at 8 m/min, and on day 2, exercise the mice for 5 min at 8 m/min followed by another 5 min at 10 m/min10. Encourage the mice to run by gentle hand nudge.</li>
		<li>On the 3rd day, let the mice undergo a single bout of running for 90 min on the 10&#176; uphill treadmill, starting at the speed of 10 m/min. After 40 min, increase the treadmill speed at a rate of 1 m/min every 10 min for a total of 30 min, and then increase the speed at the rate of 1 m/min every 5 min until it reaches 17 m/min, for a total of 90 min of exercise time and 1,070 meters of running distance.</li>
		<li>Set the electric stimulus at a low intensity range (1.2 Hz), and encourage the mice to run to avoid repeated foot shock.</li>
		<li>For tissue collection, euthanize the mice by isoflurane inhalation, followed by cervical dislocation immediately after exercise.&#160;Other approved euthanasia methods can also be used, such as CO2 or I.P. injection of other anesthetics. The level of autophagy does not seem to be affected by methods of euthanasia.</li>
	</ol>
	</li>
	<li>Running wheel setup - voluntary exercise
	<ol>
		<li>Place a mouse-running wheel (11.4 cm diameter) inside a cage with a single-housed mouse.</li>
		<li>Verify the running capacity using a bike odometer. Set up the wheel parameter [distance (in mm) per full rotation of the wheel] at 358 (11.4*&#960;). Measure the running distance after 24 hr.</li>
		<li>Let the mouse run voluntarily for 2 weeks with the running wheel. For tissue collection, sacrifice the mouse in the morning after the running period.</li>
	</ol>
	</li>
</ol>
3. Autophagy Flux Evaluation
<ol>
	<li>To measure the autophagic flux under resting and exercise conditions, treat mice with the autophagy inhibitor chloroquine for three days and compare them to mice treated with PBS.
	<ol>
		<li>Dissolve chloroquine in PBS (5 mg/ml), and inject chloroquine into mice intraperitoneally at the dose of 50 mg/kg/day for three consecutive days. Sacrifice mice and collect tissues 3 hr after the last injection.</li>
		<li>For exercised mice, pretreat them with chloroquine for two consecutive days at the dose of 50 mg/kg/day. On the third day, inject the mice with the same concentration of chloroquine, and after 90 min let them run on the treadmill for another 90 min.</li>
		<li>Euthanize the mice by isoflurane inhalation, followed by cervical dislocation. Collect tissues immediately after running, so that the total incubation time of chloroquine is the same as control (resting) mice (3 hr). 
		NOTE: Alternatively, use a single injection of chloroquine to determine the autophagy flux in mouse tissues.</li>
	</ol>
	</li>
</ol>
4. Tissue Harvest and Fixation
<ol>
	<li>Prior to tissue harvest prepare phosphate buffered saline (PBS) and fresh 4% paraformaldehyde (PFA, caution: personal protection equipment required) in PBS at a final pH of 7.4. Store both solutions at 4 &#176;C.</li>
	<li>Fill 30 ml syringe with 15 - 20 ml of 4% PFA (for GFP-LC3 mice). Connect the syringe with a 20 G catheter needle.</li>
	<li>After the exercise, euthanize the mice by isoflurane inhalation, followed by cervical dislocation.</li>
	<li>Place the animal on a clean work surface on its back. Cut and open the thoracic cavity by cutting the diaphragm to expose the heart.</li>
	<li>Make a small incision on the liver to let the blood come out, and inject a total of 15 ml 4% PFA slowly into the right ventricle of the heart via the catheter, until the lung and liver turn completely pale. Perfuse the mouse immediately after the exercise session, using a syringe pump with a constant flow rate of 90 ml/hr.</li>
	<li>After perfusion, remove the needle and collect tissues of interest (e.g. brain11 and skeletal muscle). For skeletal muscle, dissect the vastus lateralis. Briefly, pull the skin of the leg backwards to expose the muscles, localize the quadriceps femoris12, and dissect out vastus lateralis, which is the external muscle attached to the upper portion of the femoral bone.</li>
	<li>For further fixation and dehydration, place the tissues of GFP-LC3 mice in 4% PFA for 24 hr at 4 &#176;C, and then transfer them to 15% sucrose-PBS at 4 &#176;C overnight or until the tissues have settled, and then to 30% sucrose-PBS at 4 &#176;C overnight or for longer storage. Keep the samples in the dark as much as possible as they may be sensitive to strong light.</li>
	<li>For western blot analysis snap freeze the tissues from C57BL/6 mice directly in liquid nitrogen, and store them at -80 &#176;C.</li>
</ol>
5. Imaging Analysis of GFP-LC3 Puncta
<ol>
	<li>Tissue process
	<ol>
		<li>Pre-treat the tissues (previously fixed in PFA and stored in 30% sucrose) in embedding medium such as &#34;Optimal Cutting Temperature compound&#34; (OCT) for a few min in a labeled small petri dish.</li>
		<li>Transfer the tissue in a labeled cryomold containing enough fresh OCT to cover the tissue. Orient the sectioning surface toward the bottom of the cryomold (cross section for the vastus lateralis muscle and sagittal section for the hemi-brain). Avoid formation of bubbles.</li>
		<li>Freeze the samples by placing the cryomold for a few min in a covered foam cooler filled with dry ice, until the OCT turns white. Wrap individual samples in labeled foils, seal them in a plastic bag, and store them at -80 &#176;C overnight or longer.</li>
		<li>Use a cryostat to cut sample sections at a thickness of 10 &#181;m, and mount the sections on the slides. Store the slides at -20 &#176;C, and always keep the slides away from light whenever possible.</li>
	</ol>
	</li>
	<li>Slide preparation
	<ol>
		<li>Remove slides from the freezer and thaw them at room temperature. Cover the tissue section with a drop of mounting media with DAPI. Place a coverslip of appropriate size on top and avoid bubble formation.</li>
		<li>Use nail polish to seal the edges of the coverslip, allow nail polish to dry in the dark at room temperature, and then store the slides in a light-tight container at 4 &#176;C, or proceed with imaging capture of GFP-LC3 puncta.</li>
	</ol>
	</li>
	<li>Quantification of GFP - LC3 puncta by fluorescence microscopy
	<ol>
		<li>Perform epifluorescence microscopy, capturing pictures in the same condition (exposure and settings) for all the samples. For GFP, use an emission wavelength of 525 nm; for DAPI, use 490 nm. Acquire at least ten images per sample using a 60X objective for vastus lateralis and the frontal cortex region of the brain.</li>
		<li>Quantify the number of GFP-LC3 puncta in a tissue area of 2,500 &#181;m2 in each image for statistical analysis, blinded for experimental groups. Calculate the average number from 10 images of each sample, using the automatic measurement function of &#34;Object Count&#34;. Settings for the vastus lateralis muscle are: Threshold L = 10, H = 200; 2X smooth; 1X clean, and settings for the brain frontal cortex are: Threshold L = 11, H = 200; 2X smooth; 1X clean.</li>
	</ol>
	</li>
</ol>
6. Western Blot Analysis on Autophagy Markers
<ol>
	<li>Tissue process
	<ol>
		<li>Prepare the lysis buffer: 50 mM Tris-HCl; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; protease inhibitor and phosphatase inhibitor (freshly added).</li>
		<li>Remove the tissue of C57BL/6 mice from -80 &#176;C storage. Cut the muscle tissue into small pieces with a razor blade prior to the following process. Add 800 &#181;l (for hemi-brain) or 500 &#181;l (for vastus lateralis) of cold lysis buffer to the sample. Homogenize the sample at 4 &#176;C with a homogenizer at a medium speed.</li>
		<li>Fully lyse the homogenized mixture for another 30 min to 1 hr at 4 &#176;C with rotation.</li>
		<li>Spin down the homogenate at 12,000 x g for 10 min at 4 &#176;C, discard the pellet, and collect the supernatant in a new tube.</li>
	</ol>
	</li>
	<li>Autophagy analysis
	<ol>
		<li>Determine the protein concentration of tissue lysates using the BCA Protein assay kit according to the manufacturer&#39;s specifications. Normalize each sample to the same concentration.</li>
		<li>Combine the sample with the 2x Laemmli sample buffer at a 1:1 ratio, boil it at 95 &#176;C for 5 - 10 min, and proceed with western blot analysis on autophagy markers13,14, such as p62 (anti-p62 antibody, 1:500 dilution) and LC3 (anti-LC3 antibody, 1:500 dilution). Boil the samples immediately after addition of the sample buffer, as longer incubation on ice may generate multiple degraded bands of LC3.</li>
		<li>Quantify p62 and LC3 bands by densitometry analysis using ImageJ, and normalize the value to the corresponding actin band.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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.
Nutrient deprivation (starvation) and pharmacological induction are commonly used approaches to induce autophagy in vitro and in vivo. These methods, especially for animal models, may show adverse effects that can influence the overall results. For example, since it requires at least 48 hr of starvation to induce a detectable level of autophagy, the animals may not have energy needed for regular motor activity, which affects the outcome of many subsequent behavioral studies. Yet pharmacological inducers may also lead to side effects due to their lack of specificity to the autophagy pathway. The major autophagy inducer rapamycin and its derivatives suppress mTOR activity and cause metabolic dysfunction and immunosuppression18,19,20, which should be taken into consideration in the experimental design for long-term treatment. Thus, we have been working on more physiological and robust ways for autophagy activation in animal models.
Recently we and others have identified exercise as an effective, faster and safer autophagy inducer in vivo7,8,9. Both forced exercise by treadmill and voluntary exercise by running wheel have been used to analyze the effects of exercise on autophagy activation, with variations in exercise duration and intensity21,22. For example, an hour of treadmill running (starting at speed of 10 m/min to a maximum of 40 m/min) induces abundant LC3 lipidation in skeletal muscle21, and longer-term voluntary wheel running for 3 months or 4 - 5 weeks also increases basal autophagy and enhances the expression of autophagy proteins in skeletal muscle21,22.
Here we described and compared the two methods (treadmill and running wheel) to exercise mice, and presented optimized shorter protocols with high efficiency in autophagy induction. Each approach has pros and cons. A single bout of 90 min of forced exercise on treadmill is sufficient to induce autophagy in skeletal muscle and brain. We have done a time course of treadmill exercise7, and found that the exercise conditions described here induce the maximal level of autophagy in mouse skeletal muscle, which is also validated by others23. Under these conditions, mice undergo aerobic exercise; as previously reported, aerobic exercise is induced by prolonged running with gradual increases in speeds on a treadmill 24,25,26, whereas the anaerobic condition is obtained by a short duration of high intensity exercise, which increases muscle mass but does not necessarily enhance exercise endurance27,28. Furthermore, the running speed reported here in this protocol positively correlates with oxygen consumption capacity, by indirect methods to assess the aerobic capacity29. Therefore, aerobic exercise on a treadmill is a fast and effective way to induce autophagy.
However, forced running in an enclosed space may exert stress to mice. Thus, to avoid giving additional stress to animals, we use finger nudges or wire tassels as a method to keep mice running, instead of the built-in electric shock. Compared to the treadmill, the use of running wheel has many advantages. It is less stressful, does not require researchers&#39; observation time, and is convenient to study long-term effects of autophagy activation. Yet exercise on a running wheel is voluntary and thus generates variability in terms of running distance and speed among different mice or the same mouse on different days. Therefore, it is necessary to run mice on a wheel for an extended period of time (e.g., several weeks) to minimize the individual variability. Importantly, the approach also requires using an odometer at night to verify that the mouse is actually running. We measured the average running distance of wild-type C57BL/6 mice over a period of 2 weeks, and found that they run approximately 1 km/night at the beginning of training (day 1) and can run up to 8 - 10 km/night at day 14. Overall, the running wheel is an effective method to stimulate autophagy in most organs, although, it is harder to capture autophagosome accumulation in the skeletal muscle compared to using the treadmill. The reason is that skeletal muscle has a high autophagic flux and a fast autophagosome degradation rate, which requires immediate tissue harvesting after running to detect the autophagy induction by GFP-LC3 puncta measurement. In either protocol, rapid PFA perfusion and fixation of the tissues after animal euthanasia is a critical step of the procedure to preserve the autophagosome structures that are otherwise easily degraded during dissection.
This protocol demonstrates how to use aerobic exercise to stimulate autophagy in mouse tissues, including brain and skeletal muscle. These methods can be used to study the mechanisms of the autophagy pathway in the maintenance of tissue function, such as mitochondrial maintenance23, and to study the long-term effects of autophagy induction on the regulation of behavior and health of animal disease models, such as enhancing the analgesic effects of cannabinoids9. Both the treadmill and running wheel methods induce a good level of autophagy; yet it is important to consider their differences when choosing the best approach to meet different research goals.

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><tbody><tr><td>Treadmill</td><td>Columbus Instruments</td><td>150-RM Exer 3/6</td><td></td></tr><tr><td>Mouse running wheel</td><td>Super Pet</td><td>100079365</td><td>diameter 11.4 cm</td></tr><tr><td>Odometer</td><td>Bell</td><td>DASHBOARD 100</td><td></td></tr><tr><td>Syringe pump</td><td>KD Scientific</td><td>KDS100</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>Model: inverted microscope ECLIPSE</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 1850UV</td><td></td></tr><tr><td>Homogenizer</td><td>IKA</td><td>003737001 / Model: T10 Basic S1</td><td></td></tr><tr><td>Chloroquine</td><td>CAYMAN CHEMICAL COMPANY</td><td>14194</td><td></td></tr><tr><td>Parafolmaldehye</td><td>SIGMA-ALDRICH</td><td>P6148</td><td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer.</td></tr><tr><td>Mounting media</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>p62 antibody</td><td>BD Biosciences</td><td>610833</td><td></td></tr><tr><td>LC3 antibody</td><td>Novus Biologicals</td><td>NB100-2220</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>Bio-Rad Laboratories</td><td>161-0737</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></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 transgenic mouse line expressing GFP-tagged LC3 as a reporter system to monitor autophagy by exercise1. Upon autophagy induction, LC3 translocates from the cytosol to the autophagosome in punctate structures. After sectioning, formation of GFP-LC3 puncta can be directly visualized by fluorescence microscopy (Figure 1A). Alternatively, autophagosome structures can also be immunostained by an LC3 antibody for imaging. Either 90 min of treadmill exercise or 2 weeks of voluntary running increased the number of GFP-LC3 puncta in both skeletal muscle (vastus lateralis) and cerebral cortex, compared to the resting condition (Figure 1B). It should be noted that the frontal cortex region has been the major region in the brain where autophagy is clearly induced by either method so far. Exercise also induced the conversion of LC3 from the cytosolic form (LC3-I) to the lipid-conjugated form (LC3-II), which can be detected by western blot analysis (Figure 1C).
Exercise-induced increment of autophagosomes (represented by LC3-II and GFP-LC3 puncta) is due to an elevated autophagic flux, rather than a block in autophagosome degradation, assessed by the use of inhibitors of lysosomal degradation, such as bafilomycin A1 or chloroquine. Here we measured the degradation of the autophagic cargo receptor p62 as an example. Exercise (90 min of treadmill) caused a higher degradation of p62 in skeletal muscle than the resting condition, which was rescued by injection with chloroquine prior to exercise (Figure 2). The similar results are also observed with voluntary exercise by running wheels. Thus, aerobic exercise by treadmill or running wheels induces autophagy in vivo, measured by the steady-state level of LC3 and the degradation of p62.
<img alt="Figure 1" src="/files/ftp_upload/55099/55099fig1.jpg" /> 
Figure 1. Aerobic Exercise Induces Autophagy in Mouse Tissues. Representative images (A) and quantification (B) of GFP-LC3 puncta in skeletal muscle (vastus lateralis) and brain (frontal cortex) of GFP-LC3 transgenic mice under the control condition (resting), after 90 min of forced exercise (treadmill) or after 2 weeks of voluntary exercise (wheel). Results represent mean &#177; s.e.m of 10 pictures per mouse. N = 5 mice. The following emission wavelengths were used: GFP - 525 nm; DAPI - 490 nm. (C) Western blot detection of LC3 in skeletal muscle (vastus lateralis) from rested and exercised (by treadmill) mice. Quantification data represent the level of LC3-II normalized to actin (left) and the ratio of LC3-II to LC3-I (right). N = 3 mice. Statistic is comparing each value to the control sample. Results represent mean &#177; s.e.m. *, P &#60; 0.05; **, P &#60; 0.01; ***, P &#60; 0.001, t-test. Scale bar, 25 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55099/55099fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55099/55099fig2.jpg" /> 
Figure 2. Exercise Increases Autophagy Flux in Mouse Skeletal Muscle. (A) Western blot detection of p62 in vastus lateralis from rested and exercised mice in the presence or absence of the lysosomal inhibitors chloroquine. (B) (Left) Quantification analysis of p62 normalized to the corresponding actin band. (Right) The p62 flux is determined by subtracting the normalized densitometric value of PBS-treated p62 from that of chloroquine-treated p62. Results represent mean &#177; s.e.m. N = 3 mice. *, P &#60; 0.05, t-test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55099/55099fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

12.1K Views.  Northwestern University.  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. 

Video: Activating Autophagy by Aerobic Exercise in Mice

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

Behavior

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

Cancer Research

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Medicine

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

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

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

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

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.

This paper details monitoring autophagy flux to identify new molecules by cell-based imaging screening.

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

A Chronic High-Intensity Interval Training and Diet-Induced Obesity Model to Maximize Exercise Effort and Induce Physiologic Changes in Rats

Compared to continuous-moderate or low-intensity training, high-intensity interval training (HIIT) is a more time-efficient alternative method that results in similar physiologic benefits. This paper presents a HIIT protocol that can be used to assess various health markers in a Sprague-Dawley rat model of diet-induced obesity. Female Sprague Dawley rats aged 21 days old were randomly assigned to the following groups: control (CON, n = 10), exercise-trained (TRN, n = 10), high-fat diet (HFD, n = 10), and high-fat diet/exercise training (HFD/TRN, n = 10). The control diets consisted of commercial laboratory chow with 10% kilocalories (kcal) from fat (3.82 kcal/g), and the high-fat diets (HFD) consisted of 45% kcal from fat (4.7 kcal/g). The animals had ad libitum access to their assigned diet throughout the study. After an 8 week diet induction period, the exercise cohorts completed four HIIT sessions per week for 8 weeks. Each HIIT session consisted of 10 intervals of 1 min sprints/2 min rest using a rodent treadmill with a motor-driven belt. After the 8 weeks of training, the animals were sacrificed for tissue collection. The results revealed no differences in the distance run between the TRN and HFD/TRN groups, and the training speed steadily increased over the duration of the study, with a final running speed of 115 cm/s and 111 cm/s for the TRN and HFD/TRN groups, respectively. The weekly caloric intake was decreased (p &#60; 0.05) in the TRN group relative to the CON group but increased (p &#60; 0.05) in the HFD/TRN group relative to the HFD group. Lastly, the animals on the HFD had greater (p &#60; 0.05) adiposity, and the trained animals had reduced (p &#60; 0.05) adiposity relative to controls. This protocol demonstrates an efficient method to evaluate the effects of HIIT on various physiologic outcomes in a diet-induced obesity model.

This paper presents the morphometric responses and training performance outcomes of a high-intensity interval training (HIIT) protocol in a Sprague-Dawley rat model of diet-induced obesity. The purpose of this protocol was to maximize exercise intensity and determine the physiologic responses to HIIT in lean and obese rats.

Compared to continuous-moderate or low-intensity training, high-intensity interval training (HIIT) is a more time-efficient alternative method that ...

Activating Autophagy by Aerobic Exercise in Mice

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