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. 2014 Oct;13(5):869-78.
doi: 10.1111/acel.12248. Epub 2014 Jul 9.

mTORC2-SGK-1 acts in two environmentally responsive pathways with opposing effects on longevity

Masaki Mizunuma  1 , Elke Neumann-Haefelin, Natalie Moroz, Yujie Li, T Keith Blackwell
Affiliations

mTORC2-SGK-1 acts in two environmentally responsive pathways with opposing effects on longevity

Masaki Mizunuma et al. Aging Cell. 2014 Oct.

Abstract

The nematode worm Caenorhabditis elegans provides a powerful system for elucidating how genetic, metabolic, nutritional, and environmental factors influence aging. The mechanistic target of rapamycin (mTOR) kinase is important in growth, disease, and aging and is present in the mTORC1 and mTORC2 complexes. In diverse eukaryotes, lifespan can be increased by inhibition of mTORC1, which transduces anabolic signals to stimulate protein synthesis and inhibit autophagy. Less is understood about mTORC2, which affects C. elegans lifespan in a complex manner that is influenced by the bacterial food source. mTORC2 regulates C. elegans growth, reproduction, and lipid metabolism by activating the SGK-1 kinase, but current data on SGK-1 and lifespan seem to be conflicting. Here, by analyzing the mTORC2 component Rictor (RICT-1), we show that mTORC2 modulates longevity by activating SGK-1 in two pathways that affect lifespan oppositely. RICT-1/mTORC2 limits longevity by directing SGK-1 to inhibit the stress-response transcription factor SKN-1/Nrf in the intestine. Signals produced by the bacterial food source determine how this pathway affects SKN-1 and lifespan. In addition, RICT-1/mTORC2 functions in neurons in an SGK-1-mediated pathway that increases lifespan at lower temperatures. RICT-1/mTORC2 and SGK-1 therefore oppose or accelerate aging depending upon the context in which they are active. Our findings reconcile data on SGK-1 and aging, show that the bacterial microenvironment influences SKN-1/Nrf, mTORC2 functions, and aging, and identify two longevity-related mTORC2 functions that involve SGK-regulated responses to environmental cues.

Keywords: SKN-1/Nrf; aging; mTORC2; microbiome; rictor; serum- and glucocorticoid-regulated kinase.

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Figures

Figure 1
Figure 1
Loss of rict-1 function increases oxidative stress resistance dependent upon food source and SKN-1. (A) rict-1 RNAi increased resistance to oxidative stress from Arsenite (As). (B, C) Effect of rict-1 mutation on As stress resistance, assayed in animals maintained on OP50 (B) or HT115 (C). (D, E) Effects of rict-1 mutation on resistance to oxidative stress from tert-butyl hydrogen peroxide (TBHP), assayed as in (B, C). In these and other experiments, the rict-1(mg451) and skn-1(zu135) alleles were analyzed unless otherwise indicated. Both rict-1(mg451) and skn-1(zu135) are probable null alleles (WormBase) (Soukas et al., 2009). Mean survival is shown in parentheses. All stress analyses were performed at 20 °C. Representative experiments are shown, with replicates, statistics, and percent changes in survival time provided in Tables S1 (A), S2 (B, C), and S3 (D, E).
Figure 2
Figure 2
Influence of temperature and food source on rict-1 lifespan effects. (A, B) Effects of rict-1(mg451) and skn-1(zu135) mutations on lifespan at 25 °C, in animals propagated on OP50 (A) or HT115 (B). (C–F) Lifespan analysis of rict-1(mg451) and sgk-1(ok538) mutants at 20 °C (C and D) or 15 °C (E and F), in animals propagated on OP50 (C and E) or HT115 (D and F). sgk-1(ok538) is a presumed null allele (Hertweck et al., 2004). Note that the effects of rict-1(mg451) and sgk-1(ok538) mutation were similar. Analyses of rict-1(mg451) and sgk-1(ok538) mutants performed in parallel at 25 °C are presented in Fig. 4D,E. Panels A–D each show a composite of three replicates, with mean adult lifespans indicated in parentheses. Statistics for individual and pooled experimental data are provided in Tables S4 (A, B), S5 (C, D), and S6 (E, F) (Supporting information). These lifespan experiments are compared with others performed at the same temperatures in Tables S21 and S22 (Supporting information).
Figure 3
Figure 3
Food source dependence of sgk-1 oxidative stress resistance. (A, B) Arsenite (As) resistance deriving from sgk-1(ok538) and rict-1(mg451) mutants in response to HT115, assayed in animals maintained on OP50 (A) or HT115 (B). Mean survival is shown in parentheses. All stress analyses were performed at 20 °C. Representative experiments are shown, with replicates, statistics, and percent changes in survival time provided in Table S7 (Supporting information).
Figure 4
Figure 4
RICT-1/mTORC2 affects lifespan by acting through SGK-1. (A–C) skn-1 but not daf-16 is required for rict-1 or sgk-1 knockdown to increase lifespan. RNAi treatments were performed only during adulthood at 25 °C. Composites of three replicates are shown, with mean lifespans indicated in parentheses. (D, E) Similar effects of food source on rict-1(mg451) and sgk-1(ok538) lifespan. Statistics and individual experimental data are provided in Tables S8 (A–C) and S9 (D and E) (Supporting information). These lifespan experiments are compared with others performed at the same temperature in Table S21 (Supporting information). (F) SKN-1C is phosphorylated by SGK-1, dependent upon rict-1. Wild-type (WT) animals and rict-1(mg451) mutants were fed with OP50, HT115, or OP50 supplemented with l-Tryptophan, as indicated. SGK-1::GFP was immunoprecipitated from Caenorhabditis elegans lysates with anti-GFP antibodies, quantified by Western blotting and used to phosphorylate purified GST-fused SKN-1C with [γ-32P]ATP. (G) Quantification of the SKN-1 phosphorylation of two independent experiments (ImageJ) normalized to the levels in WT that were fed OP50. Error bars represent ±SD.
Figure 5
Figure 5
Effects of liquid culture and dietary restriction (DR) on rict-1 mutants. (A, B) rict-1(mg451) mutants respond robustly to DR in a liquid bacterial dilution protocol (Experimental Procedures). A600 = 3 is designated as ad libitum feeding. (C) rict-1(mg451) lifespan is not increased by HT115 vs. OP50 feeding in liquid culture (Student’s t-test P < 0.0001). Ad libitum feeding at 20 °C is shown. Note that under these conditions, rict-1(mg451) lifespan is only modestly reduced compared with wild-type (WT). Statistical analysis is presented in Tables S10 (A, B), S11 (A, B), and S12 (C) (Supporting information).
Figure 6
Figure 6
RICT-1/mTORC2 and SGK-1 act in two pathways that affect lifespan oppositely. (A) A working model for how RICT-1/mTORC2 and SGK-1 influence Caenorhabditis elegans longevity. RICT-1/mTORC2 directs SGK-1 to activate DAF-16 in the TRPA-1 temperature-sensing pathway and inhibit SKN-1 in an interaction that is modulated by bacterially produced signals. These signals regulate SKN-1 downstream or in parallel to its phosphorylation by SGK-1. A single interconnected pathway is arbitrarily shown, but while mTORC2 and SGK-1 inhibit SKN-1 in the intestine, neuronal mTORC2/RICT-1 is required for longevity mediated by the TRPA-1 pathway. See text for details. (B, C) The sgk-1(gf) mutation blocked lifespan extension from adulthood rict-1 RNAi. (D, E) sgk-1(gf) dramatically reduced the lifespan extension that rict-1 mutants experienced when fed HT115 instead of OP50. (F, G) sgk-1(gf) failed to increase lifespan in rict-1(mg451) mutants, which are short-lived at 20 °C. (H, I) Effects of rict-1 and sgk-1 RNAi on lifespan in sgk-1(gf) mutants. Compare to (G) to observe the dramatic difference between effects of rict-1 mutation and RNAi, and note that sgk-1 RNAi did not interfere with the lifespan increase in sgk-1(gf) relative to wild-type (WT) (H, I). (J) Lifespan is reduced by neuronal rict-1 RNAi. RNAi was performed in a strain in which the dsRNAi transporter mutation sid-1 is rescued specifically in neurons (Calixto et al., 2010). A composite of three replicates is shown in B–E, H, and I, with mean lifespans indicated in parentheses. Statistics and all individual experiments are described in Tables S13 (B, C), S14 (D, E), S15 (F, G), S16 (H, I), and S17 (J) (Supporting information). These lifespan experiments are compared with others performed at the same temperatures in Tables S21 and S22 (Supporting information).
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