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Published in final edited form as: Aging Cell. 2012 Jul 3;11(5):783–793. doi: 10.1111/j.1474-9726.2012.00842.x

Nutrient-Dependent Requirement for SOD1 in Lifespan Extension by Protein Restriction in Drosophila melanogaster

Xiaoping Sun 1,#, Toshimitsu Komatsu 1,2,#, Jinhwan Lim 1, Mara Laslo 1, Jason Yolitz 1, Cecilia Wang 1, Luc Poirier 1, Thomas Alberico 1, Sige Zou 1,*
PMCID: PMC3444681  NIHMSID: NIHMS383135  PMID: 22672579

Summary

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.

Introduction

Dietary nutrients are among the most influential environmental factors in aging (Fontana et al. 2010). Dietary restriction (DR) without malnutrition increases lifespan in many species. Several conserved pathways mediate the effect of DR, including target of rapamycin (TOR), insulin-like signaling and sirtuin pathways, in yeast, worms, flies and mice (Fontana et al. 2010). Dietary yeast or certain essential amino acids appear to play a more prominent role than other nutrients in modulating lifespan in Drosophila and rodents. Protein restriction promotes longevity through reducing TOR signaling (Zoncu et al. 2011). The effect of essential amino acids on Drosophila lifespan is partly mediated by insulin-like signaling (Grandison et al. 2009). Besides calorie and protein contents, dietary nutrient composition is critical for lifespan. Nutritional parametric studies using diets with different amounts of sugar and protein indicate that lifespan is primarily modulated by relative amounts of sugar and protein in several fly models, including Drosophila and the Mexican fruit fly Anastrepha ludens (Carey et al. 2008; Lee et al. 2008). However, not much is known about the mechanisms by which dietary composition modulates lifespan.

The oxidative stress theory of aging states that reactive oxygen species (ROS), mainly generated as by-products in mitochondria during aerobic metabolism, induce an accumulation of oxidative damage to macromolecules in the cell, which in turn causes aging and eventually death (Salmon et al. 2010). Consistent with this hypothesis, numerous strategies designed to reduce oxidative damage often result in lifespan extension. For example, overexpression of superoxide dismutase 1 (SOD1), a major cytosolic enzyme responsible for scavenging highly toxic superoxide radicals, in certain tissues increases lifespan in Drosophila, while sod1 knockdown reduces lifespan in flies and mice (Phillips et al. 1989; Elchuri et al. 2005; Landis & Tower 2005). Lifespan extension induced by DR or genetic modification is often associated with reduced protein, lipid and DNA oxidation (Fontana et al. 2010). Numerous genomic studies have indicated that many genes regulated by age involve oxidative stress response (Zahn & Kim 2007; Zhan et al. 2007). Moreover, ROS play an import role in onset and progression of age-related diseases, such as type 2 diabetes and neurodegenerative diseases. Reduction of antioxidant genes, such as sod1 and sod2, accelerates pathological progression of age-related diseases in animal disease models (Salmon et al. 2010). Overexpression of antioxidant genes can delay the manifestation of age-related diseases and increase lifespan in animal disease models (Salmon et al. 2010).

The relative importance of ROS in aging and the underlying mechanisms, however, remain elusive. There is compelling evidence against oxidative stress theory of aging in its simplest form. In C. elegans, mutations of each or combinations of five sod genes do not lead to lifespan shortening, even though mutant worms are sensitive to acute oxidative challenges (Doonan et al. 2008). Reduction of antioxidant genes, such as sod2, does not necessarily shorten lifespan in mice, either (Salmon et al. 2010). Cells of these mutant mice do accumulate higher levels of oxidative damage to macromolecules, and are sensitive to acute oxidative challenges. Increasing expression of antioxidant genes often is not sufficient to promote longevity in wild type animals and the role of oxidative stress in modulating lifespan and healthspan depends on environment and genetic background (Salmon et al. 2010).

How ROS mediates or modulates the effect of diet composition on lifespan remains unclear. Given the complex relationship among nutrient metabolism, oxidant generation and detoxification, we postulate that the effect of diets on lifespan is modulated by ROS in a context dependent manner. Here we investigated the role of SOD1 in modulating lifespan in response to diets containing different amounts of sugar and yeast extract (SY) in Drosophila. Yeast extract is the only protein source in SY-based diets. We also tested the effects of rapamycin and an antioxidant N-acetylcysteine (NAC) on lifespan. Rapamycin has been shown to increase lifespan in mice and flies (Harrison et al. 2009; Bjedov et al. 2010), and NAC can promote longevity in Drosophila (Brack et al. 1997). Our findings reveal the interaction between SOD1 and dietary nutrients in modulating lifespan, demonstrate diet-dependent effects of rapamycin and NAC on lifespan, and support the context-dependent role of ROS in aging.

Results

Dietary calorie and composition affect lifespan, acute stress resistance and accumulated oxidative damage

To investigate the mechanisms underlying lifespan modulation by diet, we first measured lifespan of wild type Canton S flies fed five SY diets varying in calorie content and SY ratio. We called these base, low-calorie (L-C), high-calorie (H-C), high sugar-low protein (HS-LP) and low sugar-high protein (LS-HP) diets, respectively (Tables 1 and S2). The base diet is routinely used as a standard SY diet, and the other diets have been used in dietary studies by many Drosophila labs (Bass et al. 2007; Skorupa et al. 2008). The L-C diet contained a comparable level of yeast extract to the HS-LP diet and, therefore, both were considered as protein restriction diets relative to the base diet. The L-C diet had a comparable level of sugar to the LS-HP diet, and both were sugar restriction diets relative to the base diet. The base, HS-LP and LS-HP diets differed only in SY ratio, but had the same amount of total combined sugar and yeast extract and, hence, similar amount of calorie content, since sugar and yeast extract have similar calorie content by weight (Bass et al. 2007). Therefore, these diets allowed assessing the effects of dietary calorie and composition on lifespan.

Table 1.

Lifespan of Canton S, sod1 RNAi and its widl type control flies fed different diets.

Genotype Sex SY Diet 1 # of flies
per trial		 Mean±SE
(d)2 		% Changes of
mean lifespan 3 		p value
(logrank) 4 		Maxi±SE
(d)2,5 		% Changes of maxi
lifespan3
Canton S ♂ Base 198 54.3±1.0 - - 71.0±0.4 -
(Wild type) High calorie 160 41.2±1.0 −24.1% <0.001 59.4±0.7 −16.3%
Low calorie 198 68.6±0.8 26.3% <0.001 87.2±0.8 22.8%
LS-HP 200 32.1±0.5 −40.9% <0.001 42.8±0.3 −39.7%
HS-LP 200 68.9±0.6 26.9% <0.001 82.3±0.4 15.9%
♀ Base 200 34.0±0.7 - 51.7±1.1 -
High calorie 196 24.4±0.5 −28.2% <0.001 38.2±0.9 −26.1%
Low calorie 200 44.3±0.9 30.3% <0.001 67.2±1.1 30.0%
LS-HP 199 22.8±0.5 −32.9% <0.001 34.2±0.8 −33.8%
HS-LP 198 59.5±1.2 75.0% <0.001 82.6±1.1 59.8%
UAS-sod1 IR/+ ♂ Base 103 43.7±0.9 - - 58.7±0.7 -
(Wild type control) High calorie 98 33.7±0.8 −22.9% <0.001 43.5±1.1 −25.9%
Low calorie 99 58.9±1.1 34.8% <0.001 77.4±0.9 31.9%
LS-HP 98 27.6±0.7 −36.8% <0.001 38.4±0.6 −34.6%
HS-LP 93 63.7±1.2 45.8% <0.001 80.4±1.6 37.0%
♀ Base 99 34.6±0.8 - 48.9±0.7 -
High calorie 100 21.7±0.5 −37.3% <0.001 30.5±0.6 −37.6%
Low calorie 104 42.1±0.9 21.7% <0.001 59.4±1.1 21.5%
LS-HP 100 17.9±0.3 −48.3% <0.001 24.0±0.6 −50.9%
HS-LP 93 65.4±1.8 89.0% <0.001 84.0±0.6 71.8%
da-Gal4/+ ♂ Base 196 48.3±0.9 - 62.6±0.5 -
(Wild type control) High calorie 197 37.2±0.5 −23.0% <0.001 46.2±0.7 −26.20%
Low calorie 195 62.5±0.9 29.4% <0.001 78.3±0.4 25.10%
LS-HP 199 24.2±0.5 −49.9% <0.001 38±0.2 −39.30%
HS-LP 176 63.9±0.8 32.3% <0.001 80.6±0.6 28.80%
♀ Base 179 34.1±0.5 - 46.5±0.9 -
High calorie 152 22.8±0.4 −33.1% <0.001 32.8±1.0 −29.50%
Low calorie 172 49.3±0.9 44.6% <0.001 68.2±0.6 46.70%
LS-HP 170 17.6±0.3 −48.4% <0.001 25.3±0.9 −45.60%
HS-LP 189 64.5±1 89.1% <0.001 80.8±0.5 73.80%
UAS-sod1 IR/+; da-Gal4/+ ♂ Base 99 19.4±0.5 - - 29.2±0.8 -
(sod1 RNAi) High calorie 102 17.7±0.4 −8.8% <0.001 23.9±0.7 −18.2%
Low calorie 97 43.8±0.9 125.8% <0.001 57.0±0.9 95.2%
LS-HP 100 11.0±0.2 −43.3% <0.001 15.7±0.3 −46.2%
HS-LP 97 22.6±0.7 16.5% <0.001 38.1±2.1 30.5%
♀ Base 100 24.3±0.4 - - 30.7±1.0 -
High calorie 105 19.4±0.2 −20.2% <0.001 22.8±0.2 −25.7%
Low calorie 97 32.6±0.6 34.2% <0.001 43.5±0.7 41.7%
LS-HP 101 17.4±0.4 −28.4% <0.001 23.2±0.5 −24.4%
HS-LP 97 50.3±0.8 107.0% <0.001 64.7±1.0 110.7%
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1

SY represents sugar:yeast extract; LS-HP: low sugar-high protein diet; HS-LP: high sugar-low protein diet.

2

Lifespan is expressed as mean±Standard error (SE) in days (d).

3

Percentage changes were referenced to flies fed the base diet.

4

P values were based on comparing flies fed the base diet to genotype- and gender-matched flies on any other diet by logrank test.

5

Maxi refers to maximum lifespan, which was calculated as mean lifespan of the top 10% longest-live flies.

Relative to the base diet, L-C and HS-LP diets extended mean lifespan by 26.3% and 26.9%, respectively in Canton S males, and 31.3% and 75.0% respectively in females, while the LS-HP diet shortened lifespan by 40.9% in males and 32.9% in females (Table 1 and Fig. 1A–B). Relative to the L-C diet, the HS-LP diet did not significantly affect lifespan in males, but did increase mean lifespan by 34.3% in females, while the LS-HP diet shortened mean lifespan by 53.2% in males and 48.6% in females (Tables 1 and S1–S2). Similar patterns were observed for maximum lifespan. Similar lifespan patterns were also observed in another two control strains (Fig. 2C–F), indicating that these dietary responses are not strain-specific. Our findings suggest that calorie or protein restriction extends lifespan in Drosophila and protein plays a more critical role in modulating lifespan than sugar, which are consistent with the findings from other labs using various fly strains (Lee et al. 2008; Skorupa et al. 2008).

Fig. 1.

Fig. 1

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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.

Fig. 2

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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.

We assessed the effects of dietary calorie content and composition on food intake using the capillary feeder (CAFE) method (Ja et al. 2007) ( Fig. S1A–B). In 5-d old flies, the HS-LP diet slightly decreased food intake, while the LS-HP diet did not significantly change food intake, relative to the base diet. The L-C diet increased food intake by 2–3 fold relative to the base diet, suggesting an attempt by flies to compensate for the L-C diet by increasing consumption in volume. Therefore, calorie intake of flies on the L-C diet was about 50–75% of those on the base diet instead of 25% as indicated by the calorie difference between these two diets. The overall patterns of food intake were similar between 5- and 15- day old males or females.

We next determined the effect of diet on flies’ resistance to acute oxidative stress by challenging them with paraquat. 10-d old Canton S flies fed the LS-HP diet displayed the highest resistance to paraquat relative to any other diet, while flies fed the HS-LP diet were most sensitive to paraquat (Fig. 1C–D). The L-C diet decreased resistance to paraquat in males but not females relative to the base diet. These results suggest that lower SY ratios increase resistance to oxidative stress in flies, and DR may lead to increased sensitivity to oxidative stress in males. Aconitase activity is commonly used as a biomarker of oxidative damage (Lind et al. 2006). We therefore quantified cytosolic and mitochondrial aconitase (c-Acon and m-Acon) activities to assess accumulated oxidative damage in flies. The c-Acon/m-Acon ratio was ~25% lower in males fed the HS-LP diet relative to the base and L-C diets (Fig. 1E–F). The c-Acon/m-Acon ratio was not different between males fed the base and L-C diets. These findings suggest that cytosolic oxidative damage is increased by the HS-LP diet relative to other diets. Taken together, these data indicate that oxidative stress resistance is not necessarily tightly correlated with diet-modulated longevity and the role of ROS on lifespan is context dependent.

SOD1 is required for lifespan extension by the HS-LP diet but not the L-C diet

Given the difference in resistance to acute oxidative stress and oxidative damage displayed by flies on different diets, we postulated that SOD1 might modulate lifespan of flies in response to diet. We first investigated whether ubiquitous overexpression of SOD1 affects lifespan of flies responding to diet. The ubiquitously expressed da-Gal4 driver significantly increased SOD1 protein, activity and SOD1/SOD2 activity ratio from the UAS-SOD1 transgene, when compared to two controls, UAS-SOD1/+ and da-Gal4/+ (Fig. S2A–F). Lifespan patterns of SOD1 overexpression flies were not significantly different from diet-matched controls (Fig. S2G–J and Table S3), suggesting that SOD1 overexpression is not sufficient to alter lifespan response of flies to diet.

To investigate the requirement of SOD1, we measured lifespan in a sod1 knockdown mutant induced by RNA interference (RNAi), in which double stranded sod1 RNA from a transgene construct, UAS-sod1 inverted repeat (UAS-sod1IR), was induced by da-Gal4. Both control flies, UAS-sod1IR/+ and da-Gal4/+, showed similar lifespan patterns as Canton S flies in response to diet (Fig. 2A–D). The sod1RNAi flies (UAS-sod1IR/+; da-Gal4/+) had much lower SOD1 mRNA, protein and activity levels relative to control flies, UAS-sod1IR/+ and da-Gal4/+ (Fig. 3 and S3). The sod1RNAi flies were also short-lived and highly sensitive to paraquat (Fig. 2, Table 1 and Fig. S4). However, lifespan pattern of sod1RNAi males but not females differed than control flies in response to diet (Fig. 2, Tables 1 and S4–S10). The HS-LP diet increased mean lifespan only slightly by 16.5% in sod1RNAi males relative to the base diet, and decreased lifespan by 49.6% relative to the L-C diet (Table 1). sod1 knockdown using a gut-specific Gal4 driver (cad-Gal4) was not sufficient to alter lifespan pattern of flies responding to diet (Fig. S5 and Table S3), suggesting that lifespan pattern changes in sod1RNAi males require broad sod1 knockdown or at least knockdown outside the gut. The gut result also indicates that lifespan pattern changes in sod1RNAi males were not due to genetic background. Otherwise, flies with sod1 knockdown in gut alone should have behaved similarly as sod1RNAi males. Food intake patterns in sod1RNAi flies were similar to their respective control and Canton S flies (Fig. S1), indicating that lifespan pattern changes were not due to any difference in food intake.

Fig. 3.

Fig. 3

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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.

To confirm the observation made from sod1RNAi, lifespan of sod1 null mutant (sod1n108) flies were measured. The overall lifespan patterns of sod1−/− flies in response to diet were similar to sod1RNAi flies, except for some difference in females (Fig. 2G–H, and Tables S3, S11–S12). In contrast to sod1RNAi females, the HS-LP diet did not increase lifespan in sod1−/− females relative to the base diet, but did shorten lifespan in sod1−/− females relative to the L-C diet. Since the HS-LP diet had more sugar than the L-C diet, our findings suggest that SOD1 interacts with macronutrients to modulate lifespan in flies, and sugar plays a more critical role than protein in modulating lifespan of sod1 mutant flies.

To determine temporal requirement of SOD1 for lifespan extension by the HS-LP diet and further control genetic background, we used a RU486 inducible gene-switch Gal4, da-GS-Gal4 (da-GSG), to ubiquitously knockdown sod1 in adult flies (McGuire et al. 2004). Feeding RU486 significantly reduced SOD1 protein level in da-GSG/UAS-sod1IR flies fed four SY diets, but not necessarily decreased lifespan in females (Fig. S6A– B and Table S14). Lifespan patterns in control males and females without RU486 induction of sod1 knockdown in response to diet were similar as wild type Canton S, UAS-sod1IR/+ and da-Gal4/+ flies (Fig. S6C and E, and Tables S14–15). Males with sod1 knockdown in adult alone had a similar lifespan pattern in response to diet as sod1RNAi males (Fig. 6D and Tables S14). The HS-LP diet shortened lifespan in males with adult sod1 knockdown relative to the L-C diet and slightly to the base diet. Females with adult sod1 knockdown had a similar lifespan pattern as sod1RNAi females except for flies fed the HS-LP diet (Fig. S6F, and Tables S15). The HS-LP diet decreased lifespan in females with adult sod1 knockdown relative to the L-C diet. These results suggest that SOD1 is required in the adult stage to modulate lifespan in response to diet.

Fig. 6.

Fig. 6

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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.

Dietary nutrients and SOD1 affect transcript levels of aging-related genes

To elucidate gene networks involved in lifespan modulation by the interaction between dietary nutrients and SOD1, transcript levels of representative genes involved in aging, metabolism and stress response were measured for sod1RNAi and its control males on four SY diets. SOD1 transcript, protein and activity levels were not significantly different among wild type flies fed these diets, but were significantly reduced by >80% in sod1RNAi flies when compared to diet matched controls (Fig. 3 and Fig. S3), suggesting that SOD1 is not regulated by dietary nutrients.

Most genes we tested did not show significant changes at transcript level by diet or sod1RNAi, and none of them showed any difference between males fed the base and L-C diets (Fig. 3 and S6). However, transcript levels of catalase and iron regulatory protein 1B (Irp-1B), a cytosolic aconitase, were reduced by the HS-LP diet relative to other diets in genotype matched flies (Fig. 4). adenosine 3(ade3) involved in purine metabolism and oxidative stress response and phosphoenolpyruvate carboxykinase (Pepck) involved in gluconeogenesis were down-regulated by the HS-LP diet but up-regulated by the LS-HP diet relative to the base or L-C diet in genotype-matched flies (Fig. S7). Pepck was further down-regulated in sod1RNAi flies relative to diet-matched wild type controls. The HS-LP diet down-regulated, while the LS-HP diet up-regulated a peroxiredoxin gene, Prx2540, relative to the base or L-C diet in genotype matched males (Fig. 3). The HS-LP diet further down-regulated Prx2540 in sod1RNAi males relative to diet-matched wild type flies. Taken together, our findings indicate that the HS-LP but not the L-C diet reduces expression of genes involving stress response. This is consistent with requirement for SOD1 in lifespan extension by the HS-LP but not the L-C diet, suggesting that oxidative stress differentially modulates lifespan of flies in response to diet.

Fig. 4.

Fig. 4

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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.

dFOXO is not required for lifespan extension by either HS-LP or L-C diet

Since the HS-LP diet and sod1RNAi decrease expression of Pepck, a target of dFOXO in insulin-like signaling, we measured lifespan of a trans-allelic null mutant of dfoxo (dfoxo21/dfoxo25) fed four SY diets (Junger et al. 2003). dfoxo−/− flies were short-lived relative to wild type controls, but their lifespan patterns responding to diet were similar to their wild type controls (Fig. 4A–F). These findings suggest that dFOXO is not required for the lifespan pattern of flies in response to diet.

ROS is differentially involved in lifespan extension by the HS-LP and L-C diets

To further investigate the involvement of ROS as modulators of lifespan in response to diet, we assessed lipid oxidation in flies fed the base, HS-LP and L-C diets by measuring the levels of 4-Hydroxynonenal (4-HNE)-protein adducts, a lipid oxidation marker (Tsai et al. 1998), in males. 4-HNE-protein level was significantly higher in both wild type and sod1RNAi males fed the HS-LP diet relative to genotype matched flies on the base or L-C diet (Fig. 5A and B). No significant difference was observed between males fed the base and L-C diets. 4-HNE-protein level was not statistically significantly different between wild type and sod1RNAi males, either. In females, 4-HNE-protein level was not significantly different among the control UAS-sod1IR/+ flies fed the base, HS-LP and L-C diets (Fig. S8). These findings suggest that the HS-LP diet increases oxidative damage in males but not females relative to other diets.

Fig. 5.

Fig. 5

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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.

We also quantified c-Acon and m-Acon activities in sod1RNAi males and their controls on the standard base, HS-LP and L-C diets (Lind et al. 2006). c-Acon/m-Acon ratio was lower in sod1RNAi males relative to diet matched controls, indicating elevated oxidative damage by sod1RNAi (Fig. 5C and D, and Fig. S9). Consistent with the results in Canton S, no significant difference in m-Acon activity was observed between sod1RNAi males and their wild type controls on any diet (Fig. 5C and D), whereas the HS-LP diet further reduced c-Acon/m-Acon ratio by ~25% relative to the base or L-C diet in genotype matched males. No significant change was observed between sod1RNAi males fed the base and L-C diets. The m-Acon protein levels in Canton S, sod1RNAi, da/Gal4/+ and UAS-sod1IR/+ males remained unchanged among genotype matched males fed four SY diets (Supplementary Fig. S9C–G), suggesting that the Aconitase protein level is not affected by diet. These findings suggest that cytosolic oxidative damage is increased by the HS-LP diet relative to other diets.

Considering different effects of the HS-LP and L-C diets on oxidative damage, we postulated that antioxidant feeding might differentially affect lifespan in flies on different diets. To this end, we fed antioxidant NAC to adult flies. Relative to non-supplemented controls, NAC at 100 µg/ml slightly increased lifespan in wild type males fed the base diet by ~10% (p<0.001), but did not affect lifespan in wild type flies fed the HS-LP and L-C diets, while NAC at 1000 µg/ml slightly shortened lifespan in wild type flies fed the HS-LP diet by ~8% (p<0.001), but did not alter lifespan in wild type flies fed the base and L-C diets (Fig. 5E–G and Table 2). In contrast, NAC at both 100 and 1000 µg/ml did not change lifespan in sod1RNAi males fed the standard and L-C diets. However, NAC at 1000 µg/ml but not 100 µg/ml increased lifespan in sod1RNAi males fed the HS-LP diet by ~15% (p<0.01). NAC at both 100 and 1000 µg/ml did not extend lifespan either sod1RNAi or its wild type control UAS-sod1IR/+ females fed any of the base, HS-LP and L-C diets (Fig. S10A–C). In addition, NAC at 1000 µg/ml did not affect average daily food intake in both males and females (Fig. S11A–F). Moreover, NAC at 1000 µg/ml did not affect the SOD1 protein and activity levels and SOD1/SOD2 ratio when comparing diet-matched males (Fig. S12). This suggests that the effect of NAC on lifespan is unlikely due to food intake or changes in SOD1 protein and activity levels. Taken together, our results suggest that sod1 reduction blunts lifespan extension by the HS-LP diet through increased oxidative damage, and this suppression can be partially alleviated by antioxidant treatment in a gender-dependent manner.

Table 2.

Lifespan of sod1 RNAi flies and their controls fed rapamycin and N-acetyl cysteine.

Genotype Compound
conc.		 Sex Base diet Low calorie diet High sugar-low protein diet
# of
flies		 Mean±SE
(d)1, 2 		% Changes of
lifespan3 		# of
flies		 Mean±SE
(d)1, 2 		% Changes of
lifespan3 		# of
flies		 Mean±SE
(d)1, 2 		% Changes of
lifespan3
Rapamycin
UAS-sod1 IR/+ 0 µM ♂ 157 51.9±1.0 155 66.5±0.8 169 64.5±0.8
   (Wild type) 200 µM 148 55.0±1.5& 6.0% 145 58.7±1.4* −11.7% 180 63.0±1.1 −2.3%
0 µM ♀ 112 30.8±0.5 119 38.4±0.6 206 66.8±0.6
200 µM 115 38.8±1.2& 25.6% 137 60.1±1& 56.5% 206 61.3±1.1 −8.3%
sod1RNAi 0 µM ♂ 260 18.6±0.6 212 48.9±0.8 244 26.6±0.9
200 µM 238 14.6±0.3& −21.5% 196 23.8±0.6& −51.3% 282 19.6±0.9& −26.3%
0 µM ♀ 160 23.8±0.2 128 32.7±0.5 113 32.5±0.4
200 µM

NAC		 161 22.3±0.4 −6.4% 116 36.5±0.6& 11.9% 112 35.5±0.4& 9.4%
UAS-sod1 IR/+ 0 µg/ml ♂ 125 53.6±1.3 139 72.5±1.0 145 74.6±1.2
   (Wild type) 100 µg/ml 152 59.0±1.1& 10.1% 155 71.1±0.9 −1.9% 149 74.4±1.0 −0.3%
1 mg/ml 138 56.4±1.1 5.2% 132 70.9±1.0 −2.2% 128 68.7±1.0& −7.9%
0 µg/ml ♀ 166 33.2±0.7 162 43.7±0.9 149 68.6±0.8
100 µg/ml 159 31.0±0.5# −6.60% 157 42.5±0.9 −2.7% 152 67.4±0.9 −1.7%
1 mg/ml 152 33.0±0.7 −0.60% 159 38.6±0.9& −11.7% 152 68.2±0.8 −0.6%
sod1RNAi 0 µg/ml ♂ 147 21.8±0.5 155 53.2±1.2 150 27.2±0.7
100 µg/ml 146 22.0±0.6 0.9% 146 53.1±0.8 −0.2% 111 28.5±1.0 4.8%
1 mg/ml 140 20.8±0.5 −4.6% 147 44.2±1.0 −16.9% 129 31.2±1.1# 14.7%
0 µg/ml ♀ 156 24.6±0.3 163 36.5±0.6 150 46.0±0.8
100 µg/ml 161 26.0±0.3 5.70% 159 35.5±0.6 −2.7% 156 40.0±0.7& −13.0%
1 mg/ml 164 24.4±0.3 −0.80% 153 29.9±0.6& −18.1% 156 36.4±0.6& −20.9%
Open in a new tab
1

Lifespan is expressed as mean±Standard error (SE) in days (d).

2

Significant changes are marked with superscripted symbols. P values were based on comparing flies fed the rapamycin or N-acetylcysteine (NAC) supplemented diet to genotype- SY diet- and gender-matched non-supplemented controls by logrank test.

*

p <0.05;

#

p <0.01;

&

p <0.001.

3

Percentage changes were relative to diet-, gender- and genotype matched non-supplemented controls.

SOD1 and TOR signaling in modulating lifespan extension by the HS-LP diet

Reduction of TOR signaling by protein restriction promotes longevity, and the ratio of phosphorylated S6 kinase (pS6K) to total S6K reflects TOR complex 1 (TORC1) activity (Zid et al. 2009; Zoncu et al. 2011). We found that the HS-LP and L-C diets significantly reduced the pS6K/S6K ratio in both wild type and sod1RNAi males relative to the base diet (Fig. 6A and B). The base diet has a higher protein level than the HS-LP and L-C diet. Therefore, our findings are consistent with the literature showing that high protein diets reduce the pS6K/S6K ratio (Zoncu et al. 2011). No significant difference in the pS6K/S6K ratio was observed between diet-matched wild type and sod1RNAi males (Fig. 6 and S13). Together with lifespan results, our findings suggest that lifespan extension by the HS-LP or L-C diet is partly mediated by TOR signaling but differentially modulated by SOD1.

To further investigate the interaction between TOR signaling and SOD1, we measured lifespan of sod1RNAi males and their controls fed the base, L-C and HS-LP diets supplemented or not with 200 µM rapamycin, a TOR activity inhibitor. Rapamycin did not affect food intake in both males and females fed any SY diet (Fig. S11). In wild type males, 200 µM rapamycin slightly increased lifespan in males fed the base diet by ~6% (p<0.05) and decreased lifespan in males fed the L-C diet by ~12% (p<0.001), but did not alter lifespan in males fed the HS-LP diet, relative to diet-matched non-supplemented controls (Fig. 6C–D and Table 2). Rapamycin increased lifespan in control females fed the base and L-C diets but not the HS-LP diet (Fig. S10D–F). In contrast, 200 µM rapamycin decreased mean lifespan in sod1RNAi males and females on all three diets by >20% (p<0.001) relative to genotype-matched non-supplemented controls (Fig. 6C–D, Table 2 and Fig. S10D–F). Rapamycin fed sod1RNAi males on the L-C diet were still longest lived relative to those on the base and HS-LP diets.

To further determine the relationship between TOR signaling and SOD1, we measured SOD1 protein and activity levels in control UAS-sod1IR/+ males treated with rapamycin. Rapamycin did not alter SOD1 protein level in males fed the base, HS-LP and L-C diets (Fig. S12A–C). Rapamycin did not affect SOD2 activity in flies fed any of these SY diets, or SOD1 activity and SOD1/SOD2 ratio in flies fed the base diet, when compared to diet-matched non-supplemented controls (Fig. S12D–F). However, rapamycin significantly reduced SOD1 activity and SOD1/SOD2 ratio in flies fed the HS-LP and L-C diets (Fig. S12D–F). Taken together, these findings suggest that SOD1 acts downstream of or in parallel with TOR signaling to modulate lifespan of male flies but not necessarily females in response to diets.

Discussion

Dietary nutrients are potent environmental factors in aging (Fontana et al. 2010). ROS are critical molecules modulating lifespan and aging related diseases (Salmon et al. 2010). However, the relationship between dietary nutrients and ROS in modulating lifespan remains unclear. Our study reveals several lines of evidence that provide insight into the nutrient-dependent role of ROS in lifespan modulation. First, protein restriction diets, the HS-LP and L-C diets, increase lifespan but reduce resistance to oxidative stress, while the LS-HP diet has the opposite effect, relative to the base diet in wild type flies. This is consistent with the literature showing that dietary composition is a key determinant of lifespan and protein plays a more prominent role than sugar in Drosophila (Lee et al. 2008; Skorupa et al. 2008). This is also consistent with many studies indicating that longevity is not always positively associated with increased stress resistance (Burger et al. 2007; Salmon et al. 2010). Second, sod1 reduction suppresses lifespan extension by the HS-LP but not L-C diet. The two diets differ in sugar content, indicating that SOD1 is required for lifespan extension induced by protein restriction when sugar level is high. This reveals a novel role of sugar in modulating lifespan. Third, Lifespan extension by protein restriction either through the HS-LP or L-C diet is associated with reduced TOR signaling. Feeding rapamycin decreased lifespan in sod1RNAi but not much in wild type flies, suggesting an interaction between SOD1 and TOR signaling in modulating lifespan. Lastly, the HS-LP but not L-C diet induces more oxidative damage and reduces expression of some antioxidant genes relative to the base diet. Antioxidant NAC can increase lifespan in sod1RNAi flies fed the HS-LP diet but shorten lifespan in sod1RNAi flies on the L-C diet, indicating a nutrient-dependent role of ROS in modulating lifespan. Moreover, we have observed that male and female wild type and sod1 mutant flies respond differently to diet, which is consistent with the literature showing dimorphic lifespan patterns in flies (Tower 2006). In summary, our findings suggest that dietary nutrients have profound effects on oxidative damage in the cell and the role of ROS on lifespan is nutrient and gender dependent. This supports a revised free radical hypothesis of aging that categorizes ROS as both detrimental and beneficial factors in aging (Salmon et al. 2010).

We propose the following model for the nutrient-dependent role of SOD1 and ROS on lifespan (Fig. 6F). In wild type flies, low TOR signaling in flies on the HS-LP and L-C diets increases activity of mitochondrial electron transport chain and produces more ROS, which induce defense response at the organismal level through mitohormesis and promote longevity. Although a higher sugar level in the HS-LP diet induces more ROS, ROS are partially detoxified by fully functional SOD1, which consequently results in no obvious detrimental effect of sugar on lifespan in wild type flies. In contrast, high ROS and oxidative damage induced by the HS-LP not the L-C diet are insufficiently alleviated when SOD1 activity is low, although low protein levels in the HS-LP and L-C diets still reduce TOR signaling and promote longevity. This difference leads to differential lifespan response of sod1 mutant flies to the HS-LP and L-C diets. Consistent with this model, further reduction of TOR signaling by rapamycin shortens lifespan in flies with low SOD1 activity, and NAC has prolongevity effect on sod1RNAi flies fed the HS-LP but not other diets.

Reduction of TOR signaling promotes longevity through increasing mitochondrial biogenesis, autophagy and paradoxically ROS production (Pan et al. 2011; Zoncu et al. 2011). ROS modulates the effect of TOR on chronological lifespan in yeast (Pan et al. 2011). We show here that both HS-LP and L-C diets decrease relative phosphorylation of S6K, and hence reduce TOR signaling relative to the base diet, which is consistent with previous studies showing protein restriction reduces TOR signaling (Bjedov et al. 2010; Zoncu et al. 2011). An interesting observation from our study is that sod1RNAi blunts lifespan extension by the HS-LP diet, but the pS6K/S6K ratio is not affected by sod1 reduction, suggesting a genetic interaction between SOD1 and TOR signaling. This interaction is further supported by our observation that rapamycin decreases lifespan in sod1RNAi flies, but not wild type flies. Taken together, our findings suggest that SOD1 modulates lifespan of flies in response to diets through a parallel pathway of TOR signaling or downstream of S6K or both.

Glucose plays an important role in modulating lifespan, and aberrant glucose metabolism is a major cause of many metabolic syndromes in humans, such as type 2 diabetes. In C. elegans, impairment of glucose metabolism either by 2-deoxyglucose (2-DG), a glycolysis inhibitor, increases lifespan probably by inducing mitochondrial respiration (Schulz et al. 2007). Increasing availability of glucose shortens lifespan in worms, partly by modulating insulin-like signaling (Lee et al. 2009). Unexpectedly, 2-DG treatment increases the ROS level in worms and pretreatment of worms with antioxidants blunts 2-DG induced lifespan extension. Based on mitohormesis hypothesis of aging (Ristow & Zarse 2010), it has been proposed that 2-DG extends lifespan by inducing ROS formation in mitochondria and subsequently a positive hormetic response to increase defense capacity at the organism level. However, glucose-treated worms also have an elevated ROS level, but shorter lifespan. The role of ROS in mediating the effect of 2-DG and glucose on lifespan in worms, therefore, remains unclear.

Nutrient geometric studies in Drosophila indicate that sugar by itself plays a much less prominent role than protein in modulating lifespan (Lee et al. 2008; Skorupa et al. 2008). Consistent with this view, our study indicates that increased SY ratio does not necessarily decrease, and can even increase lifespan in wild type flies. Further, reduction of insulin-like signaling, involved in glucose metabolism, by dfoxo deletion has minimal impact on lifespan of flies responding to diet. However, sod1 reduction blunts lifespan extension by the HS-LP diet, suggesting that the detrimental effect of sugar on lifespan is alleviated by SOD1. Several stress-related genes, such as catalase, ade3 and Prx2540, are downregulated in males fed the HS-LP diet relative to other diets, and the HS-LP diet induces higher levels of oxidative damage especially in sod1RNAi flies. Catalase is a major enzyme to detoxify H2O2. Ade3 is induced upon oxidative stress and during aging (Landis et al. 2004). Peroxiredoxin belongs to a family of detoxification genes encoding enzymes to remove H2O2 (Radyuk et al. 2001). How these genes and antioxidant system interact with dietary nutrients to modulate lifespan remains to be determines. Our findings suggest that the defense system against oxidative stress is somewhat suppressed by the HS-LP diet. Consistent with this view, NAC feeding increases lifespan in sod1RNAi flies on the HS-LP diet but not other diets. On the other hand, NAC shortens lifespan in sod1RNAi flies on the L-C diet, suggesting that the L-C diet promotes survival through ROS-induced hormesis as in the case of 2-DG treated worms. Together with previously published results in multiple species, our findings reveal an important role of sugar in aging. Our study further uncovers the unique role of SOD1 in mitigating detrimental effects of sugar, suggesting that the effect of sugar on lifespan depends on the antioxidant system.

SOD1 is implicated in human degenerative diseases, such as amyotrophic lateral sclerosis (ALS) (Rosen et al. 1993; Polymenidou & Cleveland 2008; Salmon et al. 2010). High levels of ROS and oxidative damage are associated with pathological progression of metabolic diseases, such as type 2 diabetes. Increasing glucose intake induces ROS formation and shortens lifespan in worms (Lee et al. 2009). sod1−/− mice are short-lived with impaired pancreatic function and glucose homeostasis (Elchuri et al. 2005; Wang et al. 2011). Although rapamycin promotes survival and delays onset of degeneration in several mouse models of degenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s diseases, rapamycin exacerbates motor neuron degeneration and shortens lifespan in a human ALS SOD1G93A mouse model (Mendelsohn & Larrick 2011; Zhang et al. 2011). Consistent with those studies, our findings indicate that both high sugar and rapamycin are detrimental to lifespan in sod1RNAi flies. It has been proposed that deleterious effect of rapamycin on lifespan in SOD1G93A mouse model is due to hyper-activation of autophagy pathway. This might explain our observation of lifespan shortening effect of rapamycin on sod1RNAi flies. Considering that rapamycin promotes glucose uptake (Zhu et al. 2011), detrimental effect of rapamycin on sod1RNAi flies may be partly due to increased glucose uptake by rapamycin and consequently increased oxidative damage. These findings support the notion that increasing cellular glucose uptake in conventional treatment of type 2 diabetes may have less beneficial effects than expected (Lee et al. 2009). Furthermore, high sugar diets may have deleterious effects in ALS patients, especially those with mutations in SOD1, and beneficial effect of rapamycin is nutrient-dependent.

Experimental Procedures

Fly media and stocks

Wild-type Canton S strain, da-Gal4 (w1118; P{w[+mW.hs]=GAL4-da.G32},3) and CAD-Gal4 (y1w1118; P{GawB}cadmd509/CyO; MKRS/TM2) were obtained from the Bloomington Drosophila Stock Center. UAS-sod1IR (w1; P{UAS-Sod1.IR}F103/SM5), UAS-SOD1 (w1; P{UAS-SOD1.A}B36) and sod1 null mutant (w1; sod1n108), da-GSG, dfoxo21 and dfoxo25 stocks were provided by J. Hu, E. Hafen and V. Monnier. Flies were maintained on standard cornmeal agar medium at 25±1 °C, 60±5% humidity and a 12:12 h light/dark cycle. Compositions of the SY diets are shown in Table S3. Rapamycin was dissolved in ethanol before being added into the SY diets to the final concentration of 200 µM. NAC was dissolved in water and then added into the SY diets to the final concentrations of 100 and 1000 µg/ml of food.

Lifespan, food intake and stress assays

Lifespan measurements were performed for wild type Canton S, sod1RNAi, sod1RNAi controls and sod1n108 flies. Both da-Gal4 and UAS-sod1IR stocks were backcrossed with w1118 more than five times and maintained as heterozygous lines, w1118; da-Gal4/+ and w1118; UAS-sod1IR /+, respectively, before being used in lifespan assays. Lifespan assays for sod1RNAi and its control UAS-sod1IR/+ were repeated in three separate trials shown in Table 1 and 2. For experiments to assess the effects of sod1 knockdown in adult alone, da-GSG/UAS-sod1IR adult flies were treated with or without 200 µM RU486. Lifespan assays for da-GSG/UAS-sod1IR adult flies were repeated twice (Supplementary Table S13). Each lifespan trial was conducted with 100–200 flies in 6–10 vials for each treatment.

Food intake was measured once every 24 hrs for three constitutive days using the CAFE method (Ja et al. 2007). Paraquat assay was conducted with adult flies of 7–14 days old on 0, 2, 5 or 20 mM paraquat in 5% sucrose solution. 6–10 vials each with 20 flies were used in each paraquat assay.

Biochemical assays

The SOD and Aconitase activity assays were based on the methods described previously (Beauchamp & Fridovich 1971; Phillips et al. 1989; Lind et al. 2006). At least three replicates were done for each treatment. The images were quantified with the ImageQuant™ TL software in the Typhoon TRIO+ Variable Mode Imager.

Quantitative real-time PCR (qPCR)

Total RNA was isolated from 10-day old flies. qPCR was performed with the Step-One plus system from Applied Biosystems. The sequence information of primers is listed in Table S16. Relative mRNA levels were normalized to rp49.

Western blot analysis

Proteins were extracted from flies exposed to the SY diets for 14 days. Primary antibodies and working dilutions were anti-SOD1 (Abcam, 1:2000 dilution), anti-phosphorylated S6K (Cell Signaling, 1:2000), anti-S6K (provided by T. P. Neufeld, 1:2000), anti-4-HNE-protein adduct (CalBiochem, 1:4000) and mouse anti-β actin (Abcam, 1:2000). Secondary antibodies were goat polyclonal antibody to rabbit IgG (HRP) and goat polyclonal antibody to mouse IgG (HRP) at 1:3000-5000 dilution (Abcam).

Statistical analysis

Statistical analyses were performed using the PASW Statistics 18 (IBM). The data were presented as mean ± standard error. The difference in lifespan was assessed by logrank test. All other data were analyzed by Student’s t-test, or one- or two-way analysis of variance (ANOVA). A p value of < 0.05 was considered statistically significant.

Supplementary Material

Supp Fig S1-S13
Supp Table S1-S16

Acknowledgements

We thank J. Hu for technical help, E. Spangler, M. Driscoll and P. Rapp for critical reading of the manuscript. This study was supported by the IRP of the National Institute on Aging, NIH to S.Z..

Footnotes

Author Contributions

X.S.,T.K. and S.Z. conceived, designed and performed experiments, and wrote the manuscript. J.L., M.L., J.Y., C. W., L.P. and T.A. performed experiments.

Conflict of Interests

All authors declare no conflict of interest.

Supporting Information

Supplemental materials include detailed experimental procedures, Table S1–16 and Figures S1–13.

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Associated Data

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Supplementary Materials

Supp Fig S1-S13
NIHMS383135-supplement-Supp_Fig_S1-S13.pdf (1.3MB, pdf)
Supp Table S1-S16
NIHMS383135-supplement-Supp_Table_S1-S16.pdf (164.5KB, pdf)

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