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The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR

Abstract

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms1,2. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits3,4. Recently, several metabolites have been identified that modulate ageing5,6; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS)7. The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution8,9. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan10,11,12,13. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

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Figure 1: α-KG extends the adult lifespan of C. elegans.
Figure 2: α-KG binds and inhibits ATP synthase.
Figure 3: α-KG longevity is mediated through ATP synthase and the dietary restriction/TOR axis.
Figure 4: Inhibition of ATP synthase by α-KG causes a conserved decrease in TOR pathway activity.

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Acknowledgements

We thank S. Lee, M. Hansen, B. Lemire, A. van der Bliek, S. Clarke, T. K. Blackwell, R. Johnson, J. E. Walker, A. G. W. Leslie, K. N. Houk, B. Martin, J. Lusis, J. Gober, Y. Wang and H. Sun for advice and discussions. J. Avruch for the let-363 RNAi vector; J. Powell-Coffman for strains and advice; and K. Yan for technical assistance. Worm strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). We thank the NIH for traineeship support of R.M.C. (T32 GM007104), M.Y.P. (T32 GM007185), B.L. (T32 GM008496) and M.N. (T32 CA009120). X.F. is a recipient of the China Scholarship Council Scholarship. G.C.M. was supported by Ford Foundation and National Science Foundation Graduate Research Fellowships.

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Authors and Affiliations

Authors

Contributions

Lifespan assays were performed by R.M.C., M.P. and E.H.; DARTS-mass spectrometry by S.D. and B.L.; DARTS-western blots by M.Y.P., H.H. and R.M.C.; mammalian cell experiments by X.F. and H.H.; mitochondrial respiration study design and analyses by L.V. and K.R.; enzyme kinetics and analyses by R.M.C. and J.H.; confocal microscopy by V.S.M., G.C.M. and A.R.F.; ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI/MS/MS) by J.X.W. and S.A.T.; compound syntheses by G.D. and M.E.J.; other analyses by H.H., X.F., M.Y.P., D.B., R.M.C., E.H., G.J., G.M.S., C.K. and A.Q. S.A.W., F.F., M.N., A.S.K., H.A.G., H.R. Chang, K.F.F., F.G., M.J., S.A.T., A.S., D.B., H.R. Christofk, C.F.C., M.A.T., M.E.J., L.V., K.R., A.R.F. and M.P. provided guidance, specialized reagents and expertise. J.H. conceived the study. R.M.C. and J.H. wrote the paper. R.M.C., X.F. and J.H. analysed data. All authors discussed the results, commented on the studies and contributed to aspects of preparing the manuscript.

Corresponding author

Correspondence to Jing Huang.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Supplementation with α-KG extends C. elegans adult lifespan but does not change the growth rate of bacteria, or food intake, pharyngeal pumping rate or brood size of the worms.

a, Robust lifespan extension in adult C. elegans by α-KG. 8 mM α-KG increased the mean lifespan of N2 by an average of 47.3% in three independent experiments (P < 0.0001 for every experiment, by log-rank test). Experiment 1, mean lifespan (days of adulthood) with vehicle treatment (mveh) = 18.9 (n = 87 animals tested), mα-KG = 25.8 (n = 96); experiment 2, mveh = 17.5 (n = 119), mα-KG = 25.4 (n = 97); experiment 3, mveh = 16.3 (n = 100), mα-KG = 26.1 (n = 104). b, Worms supplemented with 8 mM α-KG and worms with RNAi knockdown of α-KGDH (encoded by ogdh-1) have increased α-KG levels. Young adult worms were placed on treatment plates seeded with control HT115 E. coli or HT115-expressing ogdh-1 dsRNA, and α-KG content was assayed after 24 h (see Methods). c, α-KG treatment beginning at the egg stage and that beginning in adulthood produced identical lifespan increases. Light red, treatment with vehicle control throughout larval and adult stages (m = 15.6, n = 95); dark red, treatment with vehicle during larval stages and with 8 mM α-KG at adulthood (m = 26.3, n = 102), P < 0.0001 (log-rank test); orange, treatment with 8 mM α-KG throughout larval and adult stages (m = 26.3, n = 102), P < 0.0001 (log-rank test). d, α-KG does not alter the growth rate of the OP50 E. coli, which is the standard laboratory food source for nematodes. α-KG (8 mM) or vehicle (H2O) was added to standard LB media and the pH was adjusted to 6.6 by the addition of NaOH. Bacterial cells from the same overnight OP50 culture were added to the LB ± α-KG mixture at a 1:40 dilution, and then placed in the 37 °C incubator shaker at 300 r.p.m. The absorbance at 595 nm was read at 1 h time intervals to generate the growth curve. e, Schematic representation of food preference assay. f, N2 worms show no preference between OP50 E. coli food treated with vehicle or α-KG (P = 0.85, by t-test, two-tailed, two-sample unequal variance), nor preference between identically treated OP50 E. coli. g, Pharyngeal pumping rate of C. elegans on 8 mM α-KG is not significantly altered (by t-test, two-tailed, two-sample unequal variance). h, Brood size of C. elegans treated with 8 mM α-KG. Brood size analysis was conducted at 20 °C. Ten L4 wild-type worms were each singly placed onto an NGM plate containing vehicle or 8 mM α-KG. Worms were transferred one per plate onto a new plate every day, and the eggs laid were allowed to hatch and develop on the previous plate. Hatchlings were counted as a vacuum was used to remove them from the plate. Animals on 8 mM α-KG showed no significant difference in brood size compared with animals on vehicle plates (P = 0.223, by t-test, two-tailed, two-sample unequal variance). Mean ± s.d. is plotted in all cases.

Source data

Extended Data Figure 2 α-KG binds to the β subunit of ATP synthase and inhibits the activity of complex V but not the other ETC complexes.

a, Western blot showing protection of the ATP-2 protein from Pronase digestion upon α-KG binding in the DARTS assay. The antibody for human ATP5B (Sigma, AV48185) recognizes the epitope 144IMNVIGEPIDERGPIKTKQFAPIHAEAPEFMEMSVEQEILVTGIKVVDLL193 that has 90% identity to the C. elegans ATP-2. The lower molecular weight band near 20 kDa is a proteolytic fragment of the full-length protein corresponding to the domain directly bound by α-KG. b, α-KG does not affect complex IV activity. Complex IV activity was assayed using the MitoTox OXPHOS Complex IV Activity Kit (Abcam, ab109906). Relative complex IV activity was compared to vehicle (H2O) controls. Potassium cyanide (Sigma, 60178) was used as a positive control for the assay. Complex V activity was assayed using the MitoTox Complex V OXPHOS Activity Microplate Assay (Abcam, ab109907). c, atp-2 RNAi worms have lower oxygen consumption compared to control (gfp in RNAi vector), P < 0.0001 (t-test, two-tailed, two-sample unequal variance) for the entire time series (two independent experiments); similar to α-KG-treated worms shown in Fig. 2g. d, α-KG does not affect the electron flow through the ETC. Oxygen consumption rate (OCR) from isolated mouse liver mitochondria at basal (pyruvate and malate as complex I substrate and complex II inhibitor, respectively, in the presence of FCCP) and in response to sequential injection of rotenone (Rote; complex I inhibitor), succinate (Succ; complex II substrate), antimycin A (AA; complex III inhibitor), ascorbate/tetramethylphenylenediamine (Asc/TMPD; cytochrome c (complex IV) substrate). No difference in complex I (C I), complex II (C II) or complex IV (C IV) respiration was observed after 30 min treatment with 800 µM octyl α-KG, whereas complex V was inhibited (see Fig. 2h) by the same treatment (two independent experiments). e, f, No significant difference in coupling (e) or electron flow (f) was observed with either octanol or DMSO vehicle control. g, h, Treatment with 1-octyl α-KG or 5-octyl α-KG gave identical results in coupling (g) or electron flow (h) assays. Mean ± s.d. is plotted in all cases.

Source data

Extended Data Figure 3 Treatment with oligomycin extends C. elegans lifespan and enhances autophagy in a manner dependent on let-363.

a, Oligomycin extends the lifespan of adult C. elegans in a concentration-dependent manner. Treatment with oligomycin began at the young adult stage. 40 µM oligomycin increased the mean lifespan of N2 worms by 32.3% (P < 0.0001, by log-rank test); see Extended Data Table 2 for details. b, Confocal images of GFP::LGG-1 puncta in L3 epidermis of C. elegans with vehicle, oligomycin (40 µM) or α-KG (8 mM), and number of GFP::LGG-1-containing puncta quantified using ImageJ. Bars indicate the mean. Autophagy in C. elegans treated with oligomycin or α-KG is significantly higher than in vehicle-treated control animals (t-test, two-tailed, two-sample unequal variance). c, There is no significant difference (NS) between control worms treated with oligomycin and let-363 RNAi worms treated with vehicle, nor between vehicle- and α-KG-treated let-363 RNAi worms, consistent with independent experiments in Fig. 4b, c; also, oligomycin does not augment autophagy in let-363 RNAi worms (if anything, there may be a small decrease, as indicated by an asterisk); by t-test, two-tailed, two-sample unequal variance. Bars indicate the mean. Photographs were taken at ×100 magnification.

Source data

Extended Data Figure 4 Analyses of oxidative stress in worms treated with α-KG or atp-2 RNAi.

a, The atp-2 RNAi worms have higher levels of 2′,7′-dichlorofluorescein (DCF) fluorescence than gfp control worms (P < 0.0001, by t-test, two-tailed, two-sample unequal variance). Supplementation with α-KG also leads to higher DCF fluorescence, in both HT115- (for RNAi) and OP50-fed worms (P = 0.0007 and P = 0.0012, respectively). Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA). As whole worm lysates were used, total cellular oxidative stress was measured here. H2DCF-DA (Molecular Probes, D399) was dissolved in ethanol to a stock concentration of 1.5 mg ml−1. Fresh stock was prepared every time before use. For measuring ROS in worm lysates, a working concentration of H2DCF-DA at 30 ng ml−1 was hydrolysed by 0.1 M NaOH at room temperature for 30 min to generate 2′,7′-dichlorodihydrofluorescein (DCFH) before mixing with whole worm lysates in a black 96-well plate (Greiner Bio-One). Oxidation of DCFH by ROS yields the highly fluorescent DCF. DCF fluorescence was read at excitation/emission of 485/530 nm using SpectraMax MS (Molecular Devices). H2O2 was used as positive control (data not shown). To prepare the worm lysates, synchronized young adult animals were cultivated on plates containing vehicle or 8 mM α-KG and OP50 or HT115 E. coli for 1 day, and then collected and lysed as described in Methods. Mean ± s.d. is plotted. b, There was no significant change in protein oxidation upon α-KG treatment or atp-2 RNAi. Oxidized protein levels were determined by OxyBlot. Synchronized young adult N2 animals were placed onto plates containing vehicle or 8 mM α-KG, and seeded with OP50 or HT115 bacteria that expressed control or atp-2 dsRNA. Adult day 2 and day 3 worms were collected and washed four times with M9 buffer, and then stored at −80 °C for at least 24 h. Laemmli buffer (Biorad, 161-0737) was added to every sample and animals were lysed by alternate boil/freeze cycles. Lysed animals were centrifuged at 14,000 r.p.m. for 10 min at 4 °C to pellet worm debris, and supernatant was collected for OxyBlot analysis. Protein concentration of samples was determined by the 660 nm Protein Assay (Thermo Scientific, 1861426) and normalized for all samples. Carbonylation of proteins in each sample was detected using the OxyBlot Protein Oxidation Detection Kit (Millipore, S7150).

Source data

Extended Data Figure 5 Lifespan extension by α-KG in the absence of aak-2, daf-16, hif-1, vhl-1 or egl-9.

a, Lifespans of α-KG-supplemented N2 worms, mveh = 17.5 (n = 119), mα-KG = 25.4 (n = 97), P < 0.0001; or aak-2(ok524) mutants, mveh = 13.7 (n = 85), mα-KG = 17.1 (n = 83), P < 0.0001. b, N2 worms fed gfp RNAi control, mveh = 18.5 (n = 101), mα-KG = 23.1 (n = 98), P < 0.0001; or daf-16 RNAi, mveh = 14.3 (n = 99), mα-KG = 17.6 (n = 99), P < 0.0001. c, N2 worms, mveh = 21.5 (n = 101), mα-KG = 24.6 (n = 102), P < 0.0001; hif-1(ia7) mutants, mveh = 19.6 (n = 102), mα-KG = 23.6 (n = 101), P < 0.0001; vhl-1(ok161) mutants, mveh = 20.0 (n = 98), mα-KG = 24.9 (n = 100), P < 0.0001; or egl-9(sa307) mutants, mveh = 16.2 (n = 97), mα-KG = 25.6 (n = 96), P < 0.0001. P values were determined by the log-rank test. Number of independent experiments: N2 (8), hif-1 (5), vhl-1 (1) and egl-9 (2); see Extended Data Table 2 for details. Two different hif-1 mutant alleles27 have been used: ia4 (shown in Fig. 3g) is a deletion over several introns and exons; ia7 (shown here) is an early stop codon, causing a truncated protein. Both alleles have the same effect on lifespan27. We tested both alleles for α-KG longevity and obtained the same results.

Source data

Extended Data Figure 6 α-KG decreases TOR pathway activity but does not directly interact with TOR.

a, Phosphorylation of S6K (T389) was decreased in U87 cells treated with octyl α-KG, but not in cells treated with octanol control. The same results were obtained using HEK-293 and MEF cells. b, Phosphorylation of AMPK(T172) is upregulated in WI-38 cells upon complex V inhibition by α-KG, consistent with decreased ATP content in α-KG-treated cells and animals. However, this activation of AMPK appears to require more severe complex V inhibition than the inactivation of mammalian TOR, as either oligomycin or a higher concentration of octyl α-KG was required for increasing phospho (P)-AMPK whereas concentrations of octyl α-KG comparable to those that decreased cellular ATP content (Fig. 2d) or oxygen consumption (Fig. 2f) were also sufficient for decreasing P-S6K. The same results were obtained using U87 cells. Samples were subjected to SDS–PAGE on 4–12% Bis-Tris gradient gel (Invitrogen, NP0322BOX) and western blotted with specific antibodies against P-AMPK T172 (Cell Signaling, 2535S) and AMPK (Cell Signaling, 2603S). c, α-KG still induces autophagy in aak-2 RNAi worms; **P < 0.01 (t-test, two-tailed, two-sample unequal variance). The number of GFP::LGG-1 containing puncta was quantified using ImageJ. Bars indicate the mean. d, e, α-KG does not bind to TOR directly as determined by DARTS. HEK-293 (d) or HeLa (e) cells were lysed in M-PER buffer (Thermo Scientific, 78501) with the addition of protease inhibitors (Roche, 11836153001) and phosphatase inhibitors (50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 2 mM Na3VO4). Protein concentration of the lysate was measured by BCA Protein Assay kit (Pierce, 23227). Chilled TNC buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM CaCl2) was added to the protein lysate, and the protein lysate was then incubated with vehicle control (DMSO) or varying concentrations of α-KG for 1 h (d) or 3 h (e) at room temperature. Pronase (Roche, 10165921001) digestions were performed for 20 min at room temperature, and stopped by adding SDS loading buffer and immediately heating at 95 °C for 5 min (d) or 70 °C for 10 min (e). Samples were subjected to SDS–PAGE on 4–12% Bis-Tris gradient gel (Invitrogen, NP0322BOX) and western blotted with specific antibodies against ATP5B (Santa Cruz, sc58618), mammalian TOR (Cell Signaling, 2972) or GAPDH (Ambion, AM4300). ImageJ was used to quantify the mammalian TOR/GAPDH and ATP5B/GAPDH ratios. Susceptibility of the mammalian TOR protein to Pronase digestion is unchanged in the presence of α-KG, whereas, as expected, Pronase resistance in the presence of α-KG is increased for ATP5B, which we identified as a new binding target of α-KG. f, Increased autophagy in HEK-293 cells treated with octyl α-KG was confirmed by western blot analysis of MAP1 LC3 (Novus, NB100-2220), consistent with decreased phosphorylation of the autophagy-initiating kinase ULK1 (Fig. 4a).

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Extended Data Figure 7 Autophagy is enhanced in C. elegans treated with ogdh-1 RNAi.

a, Confocal images of GFP::LGG-1 puncta in the epidermis of mid-L3 stage, control or ogdh-1 knockdown C. elegans treated with vehicle or α-KG (8 mM). b, Number of GFP::LGG-1 puncta quantified using ImageJ. Bars indicate the mean. ogdh-1 RNAi worms have significantly higher autophagy levels, and α-KG does not significantly augment autophagy in ogdh-1 RNAi worms (t-test, two-tailed, two-sample unequal variance). Photographs were taken at ×100 magnification.

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Extended Data Table 1 Enriched proteins in the α-KG DARTS sample
Extended Data Table 2 Summary of lifespan data

Supplementary information

Supplementary Information

This file contains Supplementary Notes and additional references. (PDF 242 kb)

Example of a vehicle-treated day 16 adult animal

The animal had lost all motility in the body and could only move its head slowly. (WMV 2122 kb)

Example of a α-KG-treated day 16 adult animal

The animal remained youthful and exhibited full body movements. (WMV 2200 kb)

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Chin, R., Fu, X., Pai, M. et al. The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR. Nature 510, 397–401 (2014). https://doi.org/10.1038/nature13264

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