Figure 1.
The mitochondrial TCA cycle with the glyoxylate shunt and malate dismutation.
Numbered reactions are catalyzed by 1. fumarase, 2a. isocitrate lyase, 2b. malate synthase, 3. mitochondrial succinate dehydrogenase/fumarate reductase (complex II), 4. soluble fumarate reductase, 5. cytoplasmic malate dehydrogenase, 6. mitochondrial malate dehydrogenase, and 7. malic enzyme. It is unknown if the glyoxylate shunt is present in mitochondria, peroxisomes, or glyoxysomes in C. elegans.
Figure 2.
Lifespan analysis of C. elegans strains in the presence of TCA cycle intermediates.
10 mM concentrations of malate, succinate, or fumarate were present where indicated. (A) Malate (log-rank p<0.001) or fumarate (log-rank p<0.001), but not succinate (log-rank p = 0.72) extended lifespan of N2 worms. (B) Neither malate (log-rank p = 0.97) nor succinate (log-rank p = 0.88) extended lifespan of sir-2.1(ok434) worms. (C) No effect of malate treatment (log-rank p = 0.22) on lifespan and decreased lifespan with succinate treatment (log-rank p<0.001) in eat-2(ad1116) worms. (D) Neither malate (log-rank p = 0.83) nor succinate (log-rank p = 0.22) extended lifespan of daf-16(mgDf50) worms. (E) 10 mM malate (log-rank p = 0.08) did not extend the lifespan of HSF-1 mutant hsf-1(sy441) worms. (F) Neither 10 mM malate (log-rank p = 0.18) nor 10 mM succinate (log-rank p = 0.10) extended lifespan of AMPK mutant aak-2(ok524) worms.
Table 1.
Summary of lifespan experiments.
Figure 3.
Adding malate to the media increased lifespan of C. elegans RNAi knockdown strains.
(A) 10 mM malate increased lifespan of skn-1 RNAi knockdown N2 worms (log-rank p = 0.03). (B) 10 mM malate increased the lifespan of malic enzyme (men-1) RNAi knockdown N2 worms (log-rank p<0.001). (C) 10 mM malate increased the lifespan of mitochondrial malate dehydrogenase (mdh-1) RNAi knockdown N2 worms (log-rank p<0.001). (D) 10 mM malate increased the lifespan of cytoplasmic malate dehydrogenase F46E10.10 RNAi knockdown N2 worms (log-rank p = 0.006).
Figure 4.
Malate-induced lifespan extension requires fumarase and the glyoxylate cycle.
(A) 10 mM malate treatment did not alter the lifespan of glyoxylate cycle gei-7 RNAi knockdown N2 worms (log-rank p = 0.24). (B) 10 mM fumarate treatment decreased the lifespan of glyoxylate cycle gei-7 RNAi knockdown N2 worms (log-rank p = 0.02). (C) 10 mM malate treatment did not alter the lifespan of fumarase (fum-1) RNAi knockdown N2 worms (log-rank p = 0.49). (D) 10 mM fumarate treatment increased the lifespan of fumarase (fum-1) RNAi knockdown N2 worms (log-rank p<0.001).
Figure 5.
Malate treatment did not increase the lifespan of sdha-2 and F48E8.3 RNAi knockdown worms.
(A) 10 mM malate increased the lifespan of complex II flavoprotein (sdha-1) RNAi knockdown N2 worms (log-rank p<0.001). (B) 10 mM malate did not increase the lifespan of complex II flavoprotein (sdha-2) RNAi knockdown N2 worms (log-rank p = 0.95). (C) 10 mM malate decreased the lifespan of soluble fumarate reductase F48E8.3 RNAi knockdown N2 worms (log-rank p = 0.002). (D) 10 mM malate increased the lifespan of FAD synthase (flad-1) RNAi knockdown N2 worms (log-rank p<0.001).
Figure 6.
Effects of malate, fumarate, and succinate on thermotolerance, oxidative stress, and DAF-16::GFP nuclear translocation.
(A) 10 mM malate increased thermotolerance (log-rank p<0.001), while 10 mM succinate (log-rank p = 0.03) and 10 mM fumarate (log-rank p = 0.12) had smaller protective effects. C. elegans were grown at 20°C and then upshifted to 38°C. (B) 10 mM malate, fumarate, or succinate treatment decreased GST-4::GFP fluorescence in the absence (*p<0.05 compared to untreated N2) and in the presence of 10 mM paraquat (# p<0.05 compared to paraquat treated N2). (C) 10 mM malate, fumarate, or succinate treatment increased the nuclear translocation of DAF-16::GFP.
Figure 7.
Malate treatment increased C. elegans NAD and NADPH levels more than succinate.
A. Relative NAD and NADH levels in day 4 worms cultured with 10 mM malate, 10 mM succinate, or no addition (*p<0.05 compared to control). B. Relative NADP and NADPH levels in day 4 worms cultured with 10 mM malate, 10 mM succinate, or no addition (*p<0.05 compared to control).
Figure 8.
The effect of malate, fumarate, and succinate on respiration, ATP, and ΔΨ in C. elegans.
(A) The effect of 10 mM malate, fumarate, or succinate treatment on oxygen consumption in day 4 N2 worms (p<0.001). (B) The effect of 10 mM malate, fumarate, or succinate treatment on ATP levels in day 4 N2 worms (p<0.001). (C) The effect of 10 mM malate, fumarate, or succinate or 10 µM FCCP treatment on ΔΨ in day 2 N2 worms.
Table 2.
Effect of 10 mM malate on C. elegans ATP levels.