Activating Autophagy by Aerobic Exercise in Mice

Summary

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.

Abstract

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.

Introduction

Autophagy is an evolutionarily conserved degradation pathway, which is induced in response to various stress conditions such as starvation and hypoxia^1,2. 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 diabetes^4,5

Protocol

All procedures involving animals were performed according to guidelines approved by the Northwestern University Institutional Animal Care and Use Committee (IACUC).

1. Mouse Models

1. 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.

2. Exercise-induced Autophagy

1. Treadmill setup - forced exercise
   1. Acclimate and train the mice on a 10° uphill open treadmill for 2 days. On day 1, exercise the mice for 5 min ....

Results

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.......

Discussion

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 processes^16,17.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Treadmill	Columbus Instruments	150-RM Exer 3/6
Mouse running wheel	Super Pet	100079365	diameter 11.4 cm
Odometer	Bell	DASHBOARD 100
Syringe pump	KD Scientific	KDS100
Fluorescence microscope	Nikon	Model: inverted microscope ECLIPSE
Cryostat	Leica	CM 1850UV
Homogenizer	IKA	003737001 / Model: T10 Basic S1
Chloroquine	CAYMAN CHEMICAL COMPANY	14194
Parafolmaldehye	SIGMA-ALDRICH	P6148	Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer.
Mounting media	Vector Laboratories	H-1200
p62 antibody	BD Biosciences	610833
LC3 antibody	Novus Biologicals	NB100-2220
2x Laemmli Sample Buffer	Bio-Rad Laboratories	161-0737
ImageJ	NIH