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 organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; 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). 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 evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. 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.

Extended Data Figure 1

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). Expt. #1, m_veh = 18.9 (n = 87), m_α-KG = 25.8 (n = 96); Expt. #2, m_veh = 17.5 (n = 119), m_α-KG = 25.4 (n = 97); Expt. #3, m_veh = 16.3 (n = 100), m_α-KG = 26.1 (n = 104). m, mean lifespan (days of adulthood); n, number of animals tested. 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). m, mean lifespan (days of adulthood); n, number of animals tested. 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 rpm. 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. 10 L4 wildtype 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.

Extended Data Figure 2

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 (2 independent experiments); similar to α-KG treated worms shown in figure 2g. d, α-KG does not affect the electron flow through the electron transport chain. OCR from isolated mouse liver mitochondria at basal (pyruvate and malate as Complex I substrate and Complex II inhibitor, respectively, in 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 of octyl α-KG, whereas Complex V was inhibited (see Fig. 2h) by the same treatment (2 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.

Extended Data Figure 3

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 quantitated 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 (n.s.) between control worms treated with oligomycin and CeTOR(RNAi) worms treated with vehicle, nor between vehicle and α-KG treated CeTOR(RNAi) worms, consistent with independent experiments in Fig. 4b-c; also, oligomycin does not augment autophagy in CeTOR(RNAi) worms (if anything, there may be a small decrease*); by t-test, two-tailed, twosample unequal variance. Bars indicate the mean.

Extended Data Figure 4

a, The atp-2(RNAi) worms have higher levels of 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). ROS levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA). Since 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. Fresh stock was prepared every time prior to use. For measuring ROS in worm lysates, a working concentration of H₂DCF-DA at 30 ng/mL was hydrolyzed 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 2', 7'-dichlorofluorescein (DCF). DCF fluorescence was read at excitation / emission of 485 / 530 nm using SpectraMax MS (Molecular Devices). H₂O₂ was used as positive control (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 “Assay for ATP levels in C. elegans” (see 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 the 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 4 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 rpm 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).

Extended Data Figure 5

Lifespans of α-KG supplemented a, N2 worms, m_veh = 17.5 (n = 119), m_α-KG = 25.4 (n = 97), P < 0.0001; or aak-2(ok524) mutants, m_veh = 13.7 (n = 85), m_α-KG = 17.1 (n = 83), P < 0.0001. b, N2 worms fed gfp RNAi control, m_veh = 18.5 (n = 101), m_α-KG = 23.1 (n = 98), P < 0.0001; or daf-16 RNAi, m_veh = 14.3 (n = 99), m_α-KG = 17.6 (n = 99), P < 0.0001. c, N2 worms, m_veh = 21.5 (n = 101), m_α-KG = 24.6 (n = 102), P < 0.0001; hif-1(ia7) mutants, m_veh = 19.6 (n = 102), m_α-KG = 23.6 (n = 101), P < 0.0001; vhl-1(ok161) mutants, m_veh = 20.0 (n = 98), m_α-KG = 24.9 (n = 100), P < 0.0001; or egl-9(sa307) mutants, m_veh = 16.2 (n = 97), m_α-KG = 25.6 (n = 96), P < 0.0001. m, mean lifespan (days of adulthood); n, number of animals tested. 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 alleles have been used: ia4 (shown in Fig. 3g) is a deletion over several introns and exons; ia7 (shown above) is an early stop codon, causing a truncated protein. Both alleles have the same effect on lifespan . We tested both alleles for α-KG longevity and obtained the same results.

Extended Data Figure 6

a, Phosphorylation of S6K (T389) was decreased in U87 cells treated with octyl α-KG, but not in cells treated with octanol control. 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 mTOR, as either oligomycin or a higher concentration of octyl α-KG was required for increasing 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. Same results were obtained using U87 cells. 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). Number of GFP::LGG-1 containing puncta was quantitated 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 (for d) or 3 h (for 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 (for d) or 70 °C for 10 min (for 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), mTOR (Cell Signaling, 2972), or GAPDH (Ambion, AM4300). ImageJ was used to quantify the mTOR/GAPDH and ATP5B/GAPDH ratios. Susceptibility of the mTOR 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).

Extended Data Figure 7

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 quantitated 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).

Figure 1. α-KG extends the adult lifespan of C. elegans

a, α-KG extends the lifespan of adult worms in the metabolite longevity screen. 8 mM was used for all metabolites. b, Structure of α-KG. c, Dose response of α-KG in longevity. d–e, α-KG extends the lifespan of worms fed bacteria that have been d, ampicillin-arrested, m_veh = 19.4 (n = 80), m_α-KG = 25.1 (n = 91), P < 0.0001 (log-rank test); or e, γ-irradiation-killed, m_veh = 19.0 (n = 88), m_α-KG = 23.0 (n = 46), P < 0.0001 (log-rank test). f, α-KG does not further extend the lifespan of ogdh-1(RNAi) worms, m_veh = 21.2 (n = 98), m_α-KG = 21.1 (n = 100), P = 0.65 (log-rank test). m, mean lifespan (days of adulthood); n, number of animals tested.

Figure 2. α-KG binds and inhibits ATP synthase

a, DARTS identifies ATP5B as an α-KG-binding protein. Red arrowhead, protected band. b, DARTS confirms α-KG binding specifically to ATP5B. c, Inhibition of ATP synthase by α-KG (released from octyl α-KG; Supplementary Notes). This inhibition was reversible (not shown). d–g, Reduced ATP levels in (d) octyl α-KG treated normal human fibroblasts (**P = 0.0016, ****P < 0.0001; by t-test, two-tailed, two-sample unequal variance) and (e) α-KG treated worms (day 2, P = 0.969; day 8, *P = 0.012; by t-test, two-tailed, two-sample unequal variance). Decreased oxygen consumption rates in (f) octyl α-KG treated cells (***P = 0.0004, ****P < 0.0001; by t-test, two-tailed, two-sample unequal variance) and (g) α-KG treated worms (P < 0.0001; by t-test, two-tailed, two-sample unequal variance). RLU, relative luminescence unit. h, α-KG, released from octyl α-KG (800 µM), decreases state 3, but not state 4o or 3u (P = 0.997), respiration in mitochondria isolated from mouse liver. The respiratory control ratio is decreased in the octyl α-KG (3.1 ± 0.6) vs. vehicle (5.2 ± 1.0) (*P = 0.015; by t-test, two-tailed, two-sample unequal variance). i, Eadie-Hofstee plot of steady-state inhibition kinetics of ATP synthase by α-KG (produced by in situ hydrolysis of octyl α-KG). [S] is the substrate (ADP) concentration, and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol (vehicle control) or octyl α-KG. α-KG (produced from octyl α-KG) decreases the apparent V_max (53.9 to 26.7) and K_m (25.9 to 15.4), by nonlinear regression least squares fit. Number of independent experiments, c–i: 2. Mean ± s.d. is plotted in all cases.

Figure 3. α-KG longevity is mediated through ATP synthase and the DR/TOR axis

Effect of α-KG on the lifespan of mutant or RNAi worms: a, atp-2(RNAi), m_veh = 22.8 (n = 97), m_α-KG = 22.5 (n = 94), P = 0.35; or RNAi control, m_veh = 18.6 (n = 94), m_α-KG = 23.4 (n = 91), P < 0.0001. b, daf-2(e1370), m_veh = 38.0 (n = 72), m_α-KG = 47.6 (n = 69), P < 0.0001. c, eat-2(ad1116), m_veh = 22.8 (n = 59), m_α-KG = 22.9 (n = 40), P = 0.79. d, CeTOR(RNAi), m_veh = 25.1 (n = 96), m_α-KG = 25.7 (n = 74), P = 0.95; or gfp RNAi control, m_veh = 20.2 (n = 99), m_α-KG = 27.7 (n = 81), P < 0.0001. e, daf-16(mu86), m_veh = 13.4 (n = 71), m_α-KG = 17.4 (n = 72), P < 0.0001; or N2, m_veh = 13.2 (n = 100), m_α-KG = 22.3 (n = 104), P < 0.0001. f, pha-4(zu225), m_veh = 14.2 (n = 94), m_α-KG = 13.5 (n = 109), P = 0.55. g, hif-1(ia4), m_veh = 20.5 (n = 85), m_α-KG = 26.0 (n = 71), P < 0.0001; or N2, m_veh = 21.5 (n = 101), m_α-KG = 24.6 (n = 102), P < 0.0001. m, mean lifespan (days of adulthood); n, number of animals tested. P-values were determined by the log-rank test. Number of independent experiments: RNAi control (6), atp-2 (2), CeTOR (3), N2 (5), daf-2 (2), eat-2 (2), pha-4 (2), daf-16 (2), hif-1 (5).

Figure 4. Inhibition of ATP synthase by α-KG causes conserved decrease in TOR pathway activity

a, Decreased phosphorylation of mTOR substrates in U87 cells treated with octyl α-KG or oligomycin. Similar results were obtained in HEK-293, normal human fibroblasts, and MEFs (not shown). b, Increased autophagy in animals treated with α-KG or RNAi for atp-2 or CeTOR. c, GFP::LGG-1 puncta quantitated using ImageJ (Methods). 2–3 independent experiments. Bars indicate the mean. ****P < 0.0001; n.s., not significant (t-test, two-tailed, two-sample unequal variance). d, α-KG levels are increased in starved worms. **P < 0.01 (t-test, two-tailed, two-sample unequal variance). Mean ± s.d. is plotted. e, Model of α-KG-mediated longevity.