Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis

Abstract

Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.

Figure 1. Exercise induces autophagy in skeletal and cardiac muscle

a, b, Representative images of GFP–LC3 puncta (autophagosomes) in skeletal (vastus lateralis) (a) and cardiac (b) muscle from GFP–LC3 transgenic mice at serial time points after exercise. Scale bar, 20 μm. c, Quantification of data (mean ± s.e.m. of 10 tissue sections) in a and b. **P < 0.01, ***P < 0.001 (one-way ANOVA). d, Western blot analysis of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in indicated tissue from mice at rest (−) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively. e, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice at indicated time points after exercise. IP, immunoprecipitate; WB, western blot; WCL, whole cell lysates.

Figure 2. Non-phosphorylatable BCL2 AAA knock-in mutations block BCL2 phosphorylation, BCL2–beclin 1 dissociation, and starvation- and exercise-induced autophagy

a, Analysis of BCL2 phosphorylation (detected by anti-BCL2 immunoprecipitation and autoradiography of ³²P-labelled cells) and beclin 1 co-immunoprecipitation with BCL2 in wild-type (WT) or BCL2 AAA MEFs grown in normal media or subjected to 4 h Earle’s balanced salt solution (EBSS) starvation. p-BCL2, phospho-BCL2. b, Quantification of GFP–LC3 puncta (autophagosomes) in MEFs of indicated genotype in normal growth conditions or starvation conditions. Data represent mean ± s.e.m. for 100 cells per well of triplicate samples per condition. Similar results were observed in three independent experiments. c, Representative images of GFP–LC3 puncta (autophagosomes) in skeletal and cardiac muscle of GFP–LC3 wild-type and GFP–LC3 BCL2 AAA mice before exercise, after 80 min exercise, or after 75% of maximal exercise capacity. Scale bar, 20 μm. d, Quantification of data (mean ± s.d. of 4 mice per experimental group) in c. e, Western blot analysis of LC3-I/II and p62 levels in indicated tissue from mice of indicated genotype at rest (−) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively. f, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice of indicated genotype at rest (−) or after 30 min of exercise. WCL, whole cell lysates. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA for comparison between groups; †P < 0.001, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes.

Figure 3. BCL2 AAA mice show deficient exercise endurance and alterations in muscle glucose metabolism

a, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 5 mice per group. b, Representative haematoxylin and eosin (H & E) and periodic acid-Schiff (PAS) staining in tibialis anterior muscle sections from mice of indicated genotype. Scale bar, 20 μm. c, d, Plasma glucose (c) and insulin (d) levels in mice of indicated genotype at rest, after 80 min exercise (~900 m), or maximal exercise. Data represent combined mean ± s.e.m. for 9–11 mice per group from three independent cohorts; similar results were observed in each cohort. e, Representative images of GLUT4 immunofluorescence staining in vastus lateralis muscle of mice of indicated genotype before and after maximal exercise. Scale bar, 20 μm. For b and e, similar results were observed in 3 mice per group. f, Western blot analysis of AMPK phosphorylation (p-AMPK (Thr 172)) in vastus lateralis muscle lysates from mice of indicated genotype at indicated time after exercise. g, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 4–6 male mice per group. h, Soleus muscle ¹⁴C-deoxyglucose uptake during treadmill exercise in mice of indicated genotype. Data represent mean ± s.e.m. of 3 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA for comparison between groups; †P < 0.05, ††P < 0.01, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes. NS, not significant.

Figure 4. Long-term exercise training protects wild-type but not BCL2 AAA mice from HFD-induced glucose intolerance

a, b, Oral glucose tolerance test (OGTT) before (a, week 0) and after (b, week 4) 4 weeks of HFD. c, d, OGTT (c) and serum leptin and adiponectin levels (d) after 8 weeks of daily exercise. For a–d, results represent the mean ± s.e.m. for 4–5 mice per group. E, exercise; NE, no exercise; RD, regular diet. *P < 0.05, **P < 0.01, (c, one-way ANOVA; d, Wilcoxon rank test). NS, not significant.