Sirt3 (sirtuin 3) is an NAD-dependent deacetylase localized to mitochondria. Sirt3 expression is increased in mouse muscle and liver by starvation, which could protect against the starvation-dependent increase in oxidative stress and protein damage. Damaged proteins and organelles depend on autophagy for removal and this is critical for cell survival, but the role of Sirt3 is unclear. To examine this, we used Sirt3-KO (knockout) mouse embryonic fibroblast cells, and found that, under basal conditions, Sirt3-KO cells exhibited increased autophagy flux compared with WT (wild-type) cells. In response to nutrient deprivation, both WT and KO cells exhibited increased basal and ATP-linked mitochondrial respiration, indicating an increased energy demand. Both cells exhibited lower levels of phosphorylated mTOR (mammalian target of rapamycin) and higher autophagy flux, with KO cells exhibiting lower maximal mitochondrial respiration and reserve capacity, and higher levels of autophagy than WT cells. KO cells exhibit higher phospho-JNK (c-Jun N-terminal kinase) and phospho-c-Jun than WT cells under starvation conditions. However, inhibition of JNK activity in Sirt3-KO cells did not affect LC3-I (light chain 3-I) and LC3-II levels, indicating that Sirt3-regulated autophagy is independent of the JNK pathway. Caspase 3 activation and cell death are significantly higher in Sirt3-KO cells compared with WT cells in response to nutrient deprivation. Inhibition of autophagy by chloroquine exacerbated cell death in both WT and Sirt3-KO cells, and by 3-methyadenine exacerbated cell death in Sirt3-KO cells. These data suggest that nutrient deprivation-induced autophagy plays a protective role in cell survival, and Sirt3 decreases the requirement for enhanced autophagy and improves cellular bioenergetics.
- c-Jun N-terminal kinase (JNK)
- sirtuin 3
Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GABARAPL1, γ-aminobutyric acid (A) receptor-associated protein-like 1; HBSS, Hanks buffered saline solution; JNK, c-Jun N-terminal kinase; KO, knockout; LAMP1, lysosome-associated membrane protein 1; LC3, light chain 3; 3-MA, 3-methyladenine; MEF, mouse embyronic fibroblast; Mfn, mitofusin; mTOR, mammalian target of rapamycin; OCR, oxygen consumption rate; p70S6K, p70 S6 kinase; Sir, silent information regulator; Sirt, sirtuin; WT, wild-type
- © The Authors Journal compilation © 2013 Biochemical Society