doi: 10.1038/ncomms3300.
Overexpression of Atg5 in mice activates autophagy and extends lifespan
Affiliations
- PMID: 23939249
- PMCID: PMC3753544
- DOI: 10.1038/ncomms3300
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Overexpression of Atg5 in mice activates autophagy and extends lifespan
Nat Commun.
2013.
Free PMC article
Abstract
Autophagy has been implicated in the ageing process, but whether autophagy activation extends lifespan in mammals is unknown. Here we show that ubiquitous overexpression of Atg5, a protein essential for autophagosome formation, extends median lifespan of mice by 17.2%. We demonstrate that moderate overexpression of Atg5 in mice enhances autophagy, and that Atg5 transgenic mice showed anti-ageing phenotypes, including leanness, increased insulin sensitivity and improved motor function. Furthermore, mouse embryonic fibroblasts cultured from Atg5 transgenic mice are more tolerant to oxidative damage and cell death induced by oxidative stress, and this tolerance was reversible by treatment with an autophagy inhibitor. Our observations suggest that the leanness and lifespan extension in Atg5 transgenic mice may be the result of increased autophagic activity.
Figures
(a) Kaplan–Meier survival curves of combined male and female WT and Atg5 Tg mice no. 25 (P<0.001; n=65 for WT mice and n=70 for Atg5 Tg mice). (b,c) Lifespan extension in male Atg5 Tg mice no. 25 (P<0.001; n=32 for WT mice and n=36 for Atg5 Tg mice no. 25) (b) and female Atg5 Tg mice no. 25 (P<0.001; n=33 for WT mice and n=34 for Atg5 Tg mice no. 25) (c). (d–f) Kaplan–Meier survival curves of combined male and female WT and Atg5 Tg mice no. 43 (d), no. 47 (e) and no. 471 (f) (P<0.001; n=25 for WT mice and n=30 for Atg5 Tg mice). P-values were calculated using the log-rank test. (g) Expression levels of Atg5 and Atg12-Atg5 conjugate, the conversion of LC3 I into LC3 II, and p62 level in the hearts of Atg5 Tg mice. Whole-tissue extracts were prepared from 3-month-old Atg5 Tg mice and WT littermates, and analysed by western blotting using the indicated antibodies. β-Actin served as a control. (h) Densitometric analysis of the signals on the blots shown in (g). The bars represent the mean±s.d. (n=3).
(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a). (c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P<0.0001 versus control; Student’s t-test. (e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and Atg5/GFP-LC3 Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0). (f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.
(a) Representative physical pictures of 18-month-old WT and Atg5 Tg mice fed with a regular diet. (b) Body weight chart of ageing-induced obesity in WT and Atg5 Tg mice. The mice were fed with a regular diet for 24 months (n=180-–250) and their body weights were monitored monthly. The bars represent the mean±s.d. ***P<0.0001 versus control; Student’s t-test. (c) Average daily oral food intake normalized to body weight, measured on regular diet. The food intake of mice (n=18–24) was measured for 2 weeks at 3-, 6-, 12- and 18-month-old age. (d) Quantification of gonadal fat pads from 18-month-old mice (n=11). ***P<0.0001 versus control; Student’s t-test. (e,f) Quantification of visceral (e) and subcutaneous (f) fat pads of 18-month-old mice (n=6). ***P<0.0001 versus control; Student’s t-test. (g) Enhanced glucose tolerance of Atg5 Tg mice. WT and Atg5 Tg mice were starved overnight and then given with an i.p injection of glucose (1 mg g−1 body weight). The results are the mean±s.d. of 12-week-old WT and Atg5 Tg mice (n=12). *P<0.01, **P<0.001, ***P<0.0001 versus control; Student’s t-test. (h) Enhanced insulin sensitivity of Atg5 Tg mice. WT and Atg5 Tg mice were starved for 6 h and then i.p injected with 0.75 IU soluble insulin. *P<0.01, **P<0.001 versus control; Student’s t-test. (i) Plasma levels of triglycerol (TG) in WT and Atg5 Tg mice. The mice were fasted for 3 h before measurement. Young mice (4-week old, n=8); old mice (18-month old, n=8). ***P<0.0001 versus control; Student’s t-test. (j) Plasma leptin levels in WT and Atg5 Tg mice (n=8–12). ***P<0.0001 versus control; Student’s t-test. (k) GSH/GSSG ratio in the liver (n=4 males, three times trial). **P<0.001, ***P<0.0001 versus control; Student’s t-test. (l) Respiration rates of mitochondria in WT and Atg5 Tg MEFs (n=2). (m) Wire-hang endurance test of Atg5 Tg and WT mice (n=10). All the bars represent the mean±s.d. ***P<0.0001 versus control; Student’s t-test.
(a) Comparison of Atg12-Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis. MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13, after which cell extracts were examined by western blot analysis using anti-Atg5 and anti-β-actin antibodies. (b) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no. 6 by rapamycin and/or Baf.A1. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf.A1, and cell extracts were then analysed by western blotting. The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β-actin. (c,d) Increased resistance of Atg5 Tg MEFs to oxidative stress. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated for 24 h with 50, 100, 200 and 300 μM H2O2 (c) or 300 μM H2O2 in the absence or presence of 5 mM 3-MA or 20 nM Baf.A1 (Baf.A1) (d). Cell viability was assessed by propidium iodide staining. The values are the mean±s.e.m. (n=3). **P<0.001, ***P<0.0001 versus control; Student’s t-test. (e) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress. Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope. Scale bars, 50 μm. (f) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf.A1, and cell extracts were analysed by western blotting. About 10 and 30 μg of proteins were used for LC3 (upper panel) and p62 (bottom panel) detection, respectively. β-Actin served as a control.
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