Nutrient-dependent requirement for SOD1 in lifespan extension by protein restriction in Drosophila melanogaster

Abstract

Reactive oxygen species (ROS) modulate aging and aging-related diseases. Dietary composition is critical in modulating lifespan. However, how ROS modulate dietary effects on lifespan remains poorly understood. Superoxide dismutase 1 (SOD1) is a major cytosolic enzyme responsible for scavenging superoxides. Here we investigated the role of SOD1 in lifespan modulation by diet in Drosophila. We found that a high sugar-low protein (HS-LP) diet or low-calorie diet with low-sugar content, representing protein restriction, increased lifespan but not resistance to acute oxidative stress in wild-type flies, relative to a standard base diet. A low sugar-high protein diet had an opposite effect. Our genetic analysis indicated that SOD1 overexpression or dfoxo deletion did not alter lifespan patterns of flies responding to diets. However, sod1 reduction blunted lifespan extension by the HS-LP diet but not the low-calorie diet. HS-LP and low-calorie diets both reduced target of rapamycin (TOR) signaling and only the HS-LP diet increased oxidative damage. sod1 knockdown did not affect phosphorylation of S6 kinase, suggesting that SOD1 acts in parallel with or downstream of TOR signaling. Surprisingly, rapamycin decreased lifespan in sod1 mutant but not wild-type males fed the standard, HS-LP, and low-calorie diets, whereas antioxidant N-acetylcysteine only increased lifespan in sod1 mutant males fed the HS-LP diet, when compared to diet-matched controls. Our findings suggest that SOD1 is required for lifespan extension by protein restriction only when dietary sugar is high and support the context-dependent role of ROS in aging and caution the use of rapamycin and antioxidants in aging interventions.

Fig. 1

The effect of diet on lifespan and stress resistance in Canton S flies. (A and B) Lifespan of flies fed five sugar-yeast extract (SY) diets. (C and D) Survival of 10-d old flies fed four SY diets when challenged with 20 mM paraquat. (E) Activities of cytosolic aconitase (c-Acon) and mitochondrial aconitase (m-Acon) in males fed three SY diets. (F) Ratios of c-Acon/m-Acon activity indicate the levels of cytosolic oxidative damage. LS-HP, low sugar-high protein diet; H-C, high-calorie diet; Base, standard base diet; L-C, low-calorie diet; and HS-LP, high sugar-low protein diet. The error bars indicate standard errors. p values were based on comparisons relative to the base diet. ^*p<0.05; ^#p<0.01; ^&p<0.001 by Student’s t-test.

Fig. 2

The effect of diet on lifespan in sod1 mutant flies. (A and B) Lifespan of control flies (UAS-sod1IR/+) fed five SY diets. (C and D) Lifespan of another control flies (da-Gal4/+) fed five SY diets. (E and F) Lifespan of sod1RNAi flies with ubiquitous knockdown of sod1 (UAS-sod1IR/+; da-Gal4/+) fed five SY diets. (G and H) Lifespan of sod1^−/− (sod1ⁿ¹⁰⁸) flies fed four SY diets. All abbreviations are the same as in Fig. 1.

Fig. 3

The effects of sod1RNAi and diet on expression of representative stress-related genes. Relative transcript levels of four genes were shown for two controls (UAS-sod1IR/+ and da-Gal4/+) and sod1RNAi (UAS-sod1IR/+; da-Gal4/+) males fed four SY diets. All transcripts were normalized to rp49. a. u, arbitrary unit. All other abbreviations are the same as in Fig. 1. The error bars indicate standard errors. For sod1, ^&p<0.001 indicates the difference between sod1RNAi flies and diet-matched controls; for catalase, Irp-1B and prx2540, ^*p<0.05 indicates the difference between genotype-matched flies fed the HS-LP and base diets; for prx2540, ^#p<0.01 indicates the difference between sod1RNAi and control flies fed the HS-LP diet. n=4–6. All p values were from Student’s t-test.

Fig. 4

The role of dFoxo on lifespan patterns of flies responding to diet. (A to D) Lifespan of control flies (dfoxo²¹/+ and dfoxo²⁵/+).(E and F) Lifespan of dfoxo^−/− flies (dfoxo²¹/dfoxo²⁵). All abbreviations are the same as in Fig. 1.

Fig. 5

The effects of diet and sod1RNAi on oxidative damage in males. (A and B) Relative levels of 4-HNE-protein adducts were normalized to β-actin level in control (UAS-sod1IR/+) and sod1RNAi (UAS-sod1IR/+; da-Gal4/+) males fed three SY diets. (C and D) Activities of cytosolic aconitase (c-Acon) and mitochondrial aconitase (m-Acon) and c-Acon/m-Acon ratios were measured for sod1RNAi and control males fed three SY diets. (E, F and G) Lifespan of sod1RNAi and control males fed three SY diets supplemented with 0, 100 (low) and 1000 (high) µg/ml of N-acetylcysteine (NAC), respectively. WT, wild type. All other abbreviations are the same as in Fig. 3. The error bars indicate standard errors. ^*p<0.05; ^#p<0.01 are for comparisons relative to the base diet by Student’s t-test.

Fig. 6

The effects of diet and sod1RNAi on TOR signaling and a proposed model. (A) The levels of S6 Kinase (S6K) and S6K phosphorylation (pS6K) in control (UAS-sod1IR/+) and sod1RNAi (UAS-sod1IR/+; da-Gal4/+) males fed three SY diets. (B) pS6K/S6K ratios in sod1RNAi and control males on three SY diets. (C, D and E) Lifespan of sod1RNAi and control males fed the base, L-C and HS-LP diets supplemented or not with 200 µM rapamycin (rapa), respectively. (F) A speculative model shows SOD1 modulates the effect of reactive oxygen species (ROS) generated by high sugar in the HS-LP diet or low TOR signaling in the HS-LP and L-C diets through mitochondrial electron transport chain (mito. ETC). Lifespan is modulated by interactions among dietary nutrients, ROS and SOD1. All abbreviations are the same as in Fig. 3. The error bars indicate standard errors. ^*p<0.05; ^#p<0.01; ^&p<0.001 are for comparisons relative to the base diet for genotype matched flies by Student’s t-test.