rBTI extends Caenorhabditis elegans lifespan by mimicking calorie restriction

Experimental Gerontology 67 (2015) 62–71

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rBTI extends Caenorhabditis elegans lifespan by mimicking calorie restriction Jiao Li, Xiaodong Cui, Zhuanhua Wang ⁎, Yuying Li Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, PR China

a r t i c l e

i n f o

Article history: Received 3 February 2015 Received in revised form 1 May 2015 Accepted 5 May 2015 Available online 7 May 2015 Section Editor: Werner Zwerschke Keywords: rBTI CR Lifespan ROS Insulin/IGF-1 signaling

a b s t r a c t Buckwheat trypsin inhibitor (BTI) is a low molecular weight polypeptide extracted from buckwheat. This study examined the effects of BTI on the lifespan of Caenorhabditis elegans (C. elegans) and investigated the mechanism involved. Our results showed that recombinant BTI (rBTI) extended life expectancy by mimicking calorie restriction (CR) in C. elegans. rBTI promoted formation of reactive oxygen species (ROS) via increasing respiration, induced activities of ROS defense enzymes by activating DAF-16, and increased oxidative stress resistance and survival rates. The inhibition of ROS signal by antioxidants reduced rBTI-mediated longevity by up to 65%. Moreover, it was shown that the disruption of daf-2 abolished the extension of the lifespan and the increased ROS. Taken together, these data indicate that rBTI-mediated longevity mimics CR by down-regulating insulin/IGF-1 signaling (IIS) pathway, implying that BTI has the potential to be a novel anti-aging drug. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Buckwheat, one of medicinal and edible cereal crops, contains a rich supply of amino acids, abundant vitamins B1 and B2, dietary fiber, proteins, minerals and vitamin P (Skrabanja et al., 2001). In recent years, the physiological functions and medicinal properties of buckwheat have been widely studied. However, the studies on buckwheat proteins, especially on buckwheat protease inhibitors, have mainly focused on their antitumor activities (Cui et al., 2013; Ferreira et al., 2013; Liu et al., 2001). It has been well known that buckwheat can reduce levels of blood sugar, lower cholesterol, and protect against heart disease (Hosaka et al., 2014; Lee et al., 2012; Stringer et al., 2013), partly due to its high level of protease inhibitors. All these properties of buckwheat protease inhibitors are associated with aging. And it has been demonstrated that some antiglycemic or cholesterol-lowering drugs can significantly extend the lifespan in Caenorhabditis elegans (De Haes et al., 2014a), whereas very little work has been conducted on the effect of protease inhibitors on aging. Buckwheat trypsin inhibitor (BTI) derived from plant seeds belongs to the potato I-type inhibitor family. It is a polypeptide composed of 69

Abbreviations: BTI, buckwheat trypsin inhibitor; CR, calorie restriction; IIS, insulin/IGF-1 signaling; rBTI, recombinant buckwheat trypsin inhibitor; DR, dietary restrictions; STI, soybean trypsininhibitor;Cur,curcumin; PFK,phosphofructokinase; GK, glucokinase;PK,pyruvate kinase; CRM, calorie restriction mimetic. ⁎ Corresponding author. E-mail address: [email protected] (Z. Wang).

http://dx.doi.org/10.1016/j.exger.2015.05.001 0531-5565/© 2015 Elsevier Inc. All rights reserved.

amino acids and has a molecular weight of 7.9 kDa. The recombinant buckwheat trypsin inhibitor (rBTI) was prepared previously via cloning, expression and one-step affinity purification in our laboratory (Zhang et al., 2007). rBTI has the same amino acid sequence and properties as BTI, and our previous work showed that rBTI can, in addition to its anticancer effects, also extend the lifespan of fruit flies by increasing their ability to anti-oxidative stress. However, the mechanisms by which rBTI exerts its effects are unclear. Aging is one of the most risky factors for diseases affecting the world population, and slowing age-related degeneration would greatly improve the quality of human life. Calorie restriction (CR), being defined as a 10–50% reduction of ad libitum calorie uptake in the absence of malnutrition, is so far the most convincing intervention to delay both aging and the occurrence of age related diseases in a variety of organisms (Fontana et al., 2010). Currently, the most typical CR is dietary restrictions (DR) (Speakman and Mitchell, 2011). It is highly unlikely that DR will become a realistic or popular life choice for most human subjects given the level of restraint required. Consequently, significant research is focused on identifying compounds that could bestow the benefits of CR without the obligation to adhere to stringent reductions in daily food intake. Several such compounds, including rapamycin, metformin and resveratrol, have been identified as potential CR mimetics. Although these compounds show similar effects, they act through different genes to elicit corresponding effects on the lifespan (Blagosklonny, 2007; Mouchiroud et al., 2010; Selman, 2014). Here, we hypothesized that rBTI could elicit its beneficial effects by mimicking CR. Because there are some similar physiology effects between CR and

J. Li et al. / Experimental Gerontology 67 (2015) 62–71

buckwheat, such as low blood glucose, low fat content, anticancer, resistance to oxidative stress (Hosaka et al., 2014; Lee et al., 2012; Speakman and Mitchell, 2011; Stringer et al., 2013), and buckwheat contain rich BTI. We used C. elegans (which is widely used in the study of aging) as a model to investigate effects of rBTI and mechanism involved in the present study. Firstly, we explored whether rBTI could induce some similar effects with CR in C. elegans. We then conducted a preliminary study on which pathway is implicated in rBTI-mediated CR effect. Our data not only provide direct evidences that rBTI mimics CR but also lay a theoretical foundation for future studies on rBTI as a potential anti-aging drug.

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washed three times, immediately grinded with a homogenizer on ice in a final volume of 500 μL of M9 buffer plus phenylmethylsulfonyl fluoride (PMSF). Reactive oxygen species were quantified utilizing 200 μL of extract incubated in 500 μL of a 2 mM H2-DCF-DA (2′-7′-dichlorofluoresceindiacetate) solution in M9 buffer plus PMSF, for 30 min at 20 °C. Each sample was read in triplicate (200 μL each) in flat bottom 96-well plate by a fluorescence spectrophotometer reader (Tecan AG, Männedorf, Switzerland) at automatic microplate reader excitation/emission wavelengths of 485 and 528 nm. Different readings were carried out over a time frame of 2 h to ensure that the reaction was proceeding properly; the initial fluorescence was always subtracted from the final reading.

2. Methods 2.5. Activities of antioxidant enzymes 2.1. C. elegans strains, maintenance, and lifespan assays C. elegans strains used in this study were provided by the Caenorhabditis Genetics Center (CGC, University of Minnesota, USA) including CF1038 [daf-16(mu86)], TJ356 [zls356 (daf-16p::GFP + rol-6)], DA2123 (adls2122[lgg-1p::GFP::lgg-1 + rol-6(su1006)]). Wild-type N2, CB1368 [daf-2(e1368)], and Escherichia coli OP50 as a food source were obtained from the Research Institute of Applied Biology, Shanxi University (Taiyuan, China). Maintenance and synchronization (Lewis and Fleming, 1995) were the same as described previously and were performed in the absence of 5-fluoro-2′-deoxyuridine (FUdR). Lifespan assays (McColl et al., 2008) were performed in the presence of FUdR (Sigma-Aldrich, St. Louis, MO, USA). All experiments were performed at 20 °C and all worms were maintained on OP50 bacteria unless stated otherwise. According to the final concentrations indicated in the text, each of the drugs used in this study were added to autoclaved agar at 50 °C. Nacetylcysteine (NAC) was used at a final concentration of 5 mM from a 0.5 M aqueous stock. Animals were exposed to the drugs from late-L4 larvae in all experiments unless stated otherwise. Nematodes were transferred to fresh plates every 3–6 days. The maximum lifespan is the day when the last animal died in this study. The first day of adulthood is shown as day 3 in survival curves unless stated otherwise. The survival curves represent the percentage of worms that were considered to be alive at the given time points. Plots are representative of three independent experiments. 2.2. Pharynx-pumping rate assay Pharynx-pumping rate of animals growing on a plate with E. coli OP50 and FUdR was scored by observing pharyngeal contractions per minute in 15 animals under an Olympus stereomicroscope at room temperature as previously described (Huang et al., 2004). Worms were treated with rBTI as described in the lifespan assays.

Three days after hatching age-synchronized, wild type C. elegans worms (N2) were incubated with 0, 10 μM rBTI on NGM with 75 μM FUdR. Extracts from worms were assessed for antioxidant enzyme activities (SOD, CAT) and the GSH content using standard photometric assays (Schulz et al., 2007) with minor modifications. 2.6. AMP:ATP ratio quantification Determinations of ATP and AMP for C. elegans-derived samples were performed by Water 1525 Binary HPLC Pump (Waters Corporation, Milford, United Kingdom) as previously described with minor modifications (Schulz et al., 2007). About 1000 synchronized young adult wild type worms were transferred to 6 NGM plates with 10 μM or without rBTI, and cultured at 20 °C for 24 h. Then, worms were pooled and washed twice with M9 buffer. After that, worm samples were suspended with 2 mM of boiling MgSO4 and incubated in boiling water for 10 min. The samples were then sonicated for 10 min, and briefly centrifuged. The supernatants were collected and filtered through a 0.22 μm filter and analyzed by reverse-phase HPLC. Samples were separated in an Accucore XL C-18 250 × 4.6 mm 5 μm column (Thermo Fisher Scientific Inc. of Waltham, Massachusetts, USA) by a mobile phase containing 50 mM potassium phosphate buffered saline (pH 6.5). Nucleotide was detected at 254 nm with a Varian Pro Star detector. Peak areas were caculated with Breeze software. Nucleotide identities were confirmed by co-migration with AMP and ATP standards (Sigma-Aldrich, St. Louis, MO, USA). 2.7. Respiration assays

Resistance to lethal oxidative stress derived from juglone was determined as previously described (Van Raamsdonk et al., 2010) with minor modifications. Six day (after L4)-old N2 nematodes were transferred manually to fresh NGM plates containing 240 μM juglone (Sigma-Aldrich, St. Louis, MO, USA) spotted with a lawn of OP50 followed by daily determination of the survival rate until all nematodes were dead. As described for the lifespan analysis, worms were counted as censored in case of internal hatching, crawling off, and bursting. The survival curves were plotted as described above.

Oxygen consumption rates were measured using a dissolved oxygen electrode (Boqu Corporation, Shanghai, China) as previously described (Van Raamsdonk et al., 2010). Age-synchronized animals were obtained as described above. First-day adult worms were plated on NGM plates containing the corresponding treatment and covered with E. coli OP50. At the time of measurement, animals were washed three times with M9 buffer and resuspended in 20 mL of M9 buffer in a clamber. The dissolved oxygen electrode was inserted into this chamber, and oxygen partial pressure was monitored at 20 °C for 30 min. The change in oxygen levels during this period was calculated as oxygen consumption. Samples were carefully recovered from the chamber, centrifuged, and homogenized in a homogenizer for 30 s at 3000 rpm. After centrifugation at 8000 rpm for 5 min, the supernatant was removed and used for protein quantification using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China).

2.4. Quantification of ROS content

2.8. RNA isolation and qRT-PCR

ROS measurement followed a protocol described elsewhere (Schiavi et al., 2013) with appropriate modifications as noted here. Populations of ~1000 synchronized nematodes that had grown to the indicated time points in the presence or absence of the drugs were collected in M9 buffer,

Total RNA was isolated from synchronized adult worms which were treated as described in the text using Trizol reagent (TaKaRa Biotechnology Co., Ltd., Dalian, China). RNA purity was checked using UV absorbance (260/280 ratio). cDNA was synthesized with primer Oligo(dT)20

2.3. Juglone stress resistance assay in C. elegans

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using a SuperScript III First-Strand Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. RT-PCR reactions were performed with 20 μL of Power SYBR PCR Master Mix (QIAGEN, Germany), in triplicate for each sample. PCR reactions were carried out in a Real Time PCR Machine 7500 Fast (Applied Biosystems, USA) under the conditions of 95 °C for 5 min for PCR initial heat activation, followed by 40 cycles of 95 °C for 10 s for denaturation, and 60 °C for 30 s for combined annealing/extension. The reaction products were analyzed using the onboard software of the Real Time PCR machine. The target genes' transcriptional expression levels were normalized to the internal control act-1. The primers used for qRT-PCR information are listed in Tables S1 through S5. All experiments were repeated three times, and consistent results were obtained from those independent experiments. 2.9. Key enzyme activity assay in glycolysis and lipase activity assay Populations of ~1000 synchronized animals that had grown to the given time points (in the presence or absence of rBTI) were collected in M9 buffer, washed three times, immediately grinded with a homogenizer on ice in a final volume of 500 μL of M9 buffer. CHAPS was added at 1% final concentration to the resulting homogenate; the mixture was kept on ice for 15 min and centrifuged at 14000 rpm for 10 min. The supernatant as the enzyme was next cleared by a second centrifugation step. Phosphofructokinase (PFK) activity was measured by quantifying ADP using HPLC. Pyruvate kinase (PK) and glucokinase (GK) activities were measured with enzyme-linked colorimetry assay using a UV spectrophotometer (751-GW). See Supplemental Experimental Procedures for details. Lipolytic activity was measured with a colorimetric assay kit (BioAssay Systems, USA) and samples (biological triplicates) were prepared as previously described (Lapierre et al., 2011). All enzyme activities were scaled to the protein concentration of the worm extract. 2.10. Autophagy and lipid quantification The level of autophagy (autophagosomal accumulation) was assessed by observing GFP positive foci using the translational reporter strain GFP::LGG-1. Fat content was quantified by fixing animals and staining them with Oil Red O. See Supplemental Experimental Procedures for details. 2.11. Statistical analyses Data are expressed as means ± SD unless otherwise indicated. Statistical analyses for all data except lifespan and stress resistance

assays in C. elegans were performed by Student's t test (unpaired, two-tailed) after testing for equal distribution of the data and equal variances within the data set. For comparing significant distributions between different groups in the lifespan assays and stress resistance assays, statistical calculations were carried out using the log rank test. All calculations were performed using SPSS version 13.0 (demo version, Armonk, NY). A p value below 0.05 was considered as statistically significant. 3. Results 3.1. rBTI extends the lifespan of C. elegans under normal culture conditions To investigate whether rBTI could extend the lifespan in C. elegans, we analyzed the survival rate of C. elegans on NGM plates containing rBTI. Results showed that, compared with the control group, the nematode lifespan was extended by 3.1, 11.5, and 21.2% in worms treated with 2.5, 5 and 10 μM rBTI from the L4 stage onwards, respectively (Fig. 1A; Table 1). Compared with a result achieved with 100 μM curcumin (Cur), the lifespan was extended 1.2% further in worms treated with 10 μM rBTI (Fig. 1A; Table 1). Several reports showed Cur can extend the lifespan significantly in C. elegans (Alavez et al., 2011). Simultaneously, to compare the effect of rBTI and other similar substances, we also assessed the effect of soybean trypsin inhibitor (STI) on the worms' lifespan. The longevity effect induced by rBTI was stronger than STI (Fig. 1B; Table 1). In summary, we found that rBTI can extend the lifespan of C. elegans, and this effect is dose-dependent in certain range of concentration. Based on results, we identified 10 μM rBTI as the optimal dosage. 3.2. rBTI does not affect the food intake in C. elegans In this study, we hypothesized rBTI mimic CR. To determine rBTI is merely an interesting phenocopy of CR rather than an actual CR, we investigated the effect of rBTI on food intake by examining pumping rate in C. elegans. We observed that, compared with the control group, the pumping rate didn't show a significant difference after treatment with rBTI (Fig. 2). This result indicated rBTI did not affect the food intake in C. elegans. 3.3. rBTI induces endogenous ROS defense enzymes and promotes stress resistance in C. elegans In many studies on the prevention of aging including CR, the extended lifespan has been associated with increased stress resistance (Chen et al., 2013; Xiong et al., 2014). Here, to test whether worms treated with rBTI had increased resistance to oxidative stress, we exposed

Fig. 1. rBTI extends the lifespan of C. elegans under normal culture conditions. Three days after hatching age-synchronized, wild type C. elegans worms (N2) were plated on fresh NGM with 75 μM FUdR. (A) The NGM contain 0, 2.5, 5, 10 μM rBTI and 100 μM Cur except FUdR. (B) The NGM contain 0, 2.5, 5, 10 μM rBTI, 10 μM STI except FUdR. Plots are representative of three independent experiments.

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Table 1 Lifespan assay results and statistical analyses.a Strain (solvent)

Maximum lifespan (d) (±SEM)

Mean lifespan (d) (±SEM)

n

N2 (control) N2 (100 μM Cur) N2 (2.5 μM rBTI) N2 (5 μM rBTI) N2 (10 μM rBTI) N2 (2.5 Μm STI) N2 (5 μM STI) N2 (10 μM STI) N2 (control + juglone) N2 (10 μM rBTI + juglone) daf-2(e1368) (control) daf-2(e1368) (10 μM rBTI) daf-16(mu86) (control) daf-16(mu86) (10 μM rBTI) N2 (control + NAC) N2 (10 μM rBTI + NAC)

27.2 ± 0.6 31.3 ± 0.5 28.5 ± 0.9 30.3 ± 0.9 31.7 ± 0.9 28 ± 0.5 28.7 ± 0.8 29.5 ± 0.4 12.5 ± 0.8 15.4 ± 0.6 37.2 ± 0.6 37.6 ± 0.3 18.3 ± 0.5 19.5 ± 0.4 29.3 ± 0.4 29.6 ± 0.2

17.3 ± 0.3 21.2 ± 0.2 18.6 ± 1.1 20.0 ± 1.0 21.4 ± 0.7 18.8 ± 0.2 18.8 ± 1.0 19.1 ± 0.7 6.8 ± 0.8 7.9 ± 0.6 26.2 ± 0.7 26.5 ± 0.4 14.8 ± 0.6 16.1 ± 0.4 16.6 ± 0.5 17.9 ± 0.3

5 3 3 3 5 3 3 3 3 3 3 3 3 3 3 3

p value versus control

b0.01 b0.01 b0.001 n.s. (0.351) b0.05 b0.01 b0.01 n.s. (0.421) n.s. (0.124) n.s. (0.221)

a

Summary of maximum and mean lifespans and statistical analysis (p values) for lifespan experiments, including all strains and different treatments displayed in Figs. 1A–B, 3A, 4D, 6A–B. Maximum and mean lifespans summarize means of n independent experiments. n.s. = not significant.

them to juglone, which is known to act as a stressor and to be capable of inducing mitochondrial ROS formation. We measured the lifespan of individuals using previously described methods, and observed a 16.3% significant longer lifespan in worms treated with 10 μM rBTI under conditions of oxidative stress, compared with the control group (Fig. 3A; Table 1). This result indicated that the worms treated with 10 μM rBTI had increased resistance to oxidative stress. It was repeatedly reported that CR is capable of inducing stress defense mechanisms, particularly those which are involved in the detoxification of ROS, such as radical-scavenging enzymes. This is why CR could increase resistance to oxidative stress. So we measured changes in SOD and CAT activities and also the GSH content. These substances are responsible for the removal of ROS in vivo. The results showed that, compared with the control, SOD activity, CAT activity and GSH content increased significantly by 154%, 49% and 29%, respectively, after rBTI treatment for 1 day (Fig. 3B–D). However, compared with the control, SOD activity decreased by 25.3%, CAT activity increased weakly, by 12.9%, and GSH level increased 38.4% on day 2 post treatment. On day 3, SOD and CAT activity decreased by 60.3% and 32.8%, while GSH increased by 95.2%. On day 4, SOD, CAT and GSH had decreased by 29.4%, 18.7% and 39.5%, respectively. These findings were beyond our expectations. Thus, we continued the test for longer period of time. To our surprise, enzymatic activities and GSH content remained at decreased levels (12.5%, 16.7% and 41.8%) on day 7 (Fig. 3B–D).

3.4. rBTI increases AMP:ATP ratio and respiration, induces a transient ROS signal We hypothesized that rBTI elicits its beneficial effects by mimicking CR, and CR has been linked to increased AMP:ATP ratio, respiration and transient ROS signal (Masoro, 2005; Selman, 2014). As reported by others (Boveris and Chance, 1973; Kemp et al., 2003), increased AMP could induce respiration, and ROS generation is increased by respiration, as inevitable by-product of respiration. Our results indicated that, upon 10 μM rBTI treatment, a significant increase in AMP:ATP ratio (57%) and respiration (25%) was observed (Fig. 4A, B). Next, to intuitively observe changes in ROS after treatment with rBTI, we directly measured ROS levels in vivo using fluorescent probes. We repeated the exposure of worms to 10 μM rBTI for 1, 2, 3, 4 and 7 days to explain the changes in ROS defense enzymes. We found that, compared with the control group, ROS levels increased up to 60% in worms treated with 10 μM rBTI on day 1 (Fig. 4C); the spike in ROS levels of worms treated with 10 μM rBTI on day 2 was increased by 4.7%, and was reduced by 38% on day 3 and 20% on day 4 (Fig. 4C). The accumulation of ROS is decreased by ~ 20% in worms treated for 7 days (Fig. 4C). 3.5. rBTI-mediated longevity is dependent on ROS signal To verify whether ROS signal plays an important role in rBTImediated longevity as in CR-mediated longevity, we exposed nematodes to 10 μM rBTI and 1 mM NAC, a membrane-permeable glutathione precursor known to ameliorate the effects of ROS by mimicking the radical-scavenging potential of reduced glutathione. It was noted that the pretreatment of NAC reduced rBTI-mediated longevity by up to 65% (Fig. 4D; Table 1), suggesting that rBTI-mediated longevity is dependent on ROS, at least partly. 3.6. rBTI down-regulates insulin/IGF-1 signaling pathway and activates DAF-16

Fig. 2. rBTI does not affect the food intake in C. elegans. There was no significant change in pharyngeal pumping rate after treatment with 10 μM rBTI for 1 day in C. elegans. Relative values are depicted and values are given as mean ± SD.

It is well known that the insulin/IGF-1 signaling (IIS) is a canonical pathway regulating lifespan in C. elegans. It could exert its effect on the lifespan via regulating a wide variety of cellular stress-responses. With regard to oxidative stress, transcription factors DAF-16 and SKN-1 play an important role. In view of the fact that rBTI increased stress resistance in C. elegans, we tested the effect of rBTI on IIS pathway and the transcriptional activities of DAF-16 and SKN-1.

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Fig. 3. rBTI induces endogenous ROS defense enzymes and promotes stress resistance in C. elegans. (A) The lifespan analysis of C. elegans under oxidative stress. Three days after hatching age-synchronized, wild type C. elegans worms (N2) were plated on fresh NGM with 75 μM FUdR and 240 μM juglone. The NGM contain 0 μM, 10 μM rBTI except FUdR and juglone. Plots are representative of three independent experiments. (B) Change in SOD activity. (C) Change in CAT activity. (D) Change in GSH content. In all panels, relative values are depicted. All values are given as mean ± SD. p-Value was calculated using Student's t-test. *p b 0.05, **p b 0.01, ***p b 0.001 versus respective controls.

We used qRT-PCR analysis to examine transcriptional expression of genes involved in the IIS pathway, namely daf-2, age-1, and akt-1. It was noted that daf-2 mRNA levels decreased ~ 78% in worms treated with

10 μM rBTI on day 1, compared with the control (Fig. 5A). At the same time, age-1 and akt-1 mRNA levels decreased by 27%, while daf-16 mRNA increased ~ 3-fold in worms treated with 10 μM rBTI on day 1,

Fig. 4. rBTI increases AMP:ATP ratio and respiration, induces a transient ROS signal, and rBTI-mediated longevity is dependent on ROS signal. (A) Change in AMP:ATP ratio. (B) Change in respiration rate. (C) Change in ROS level. In all panels, relative values are depicted. All values are given as mean ± SD. p-Value was calculated using Student's t-test. *p b 0.05, **p b 0.01, ***p b 0.001 versus respective controls. (D) The lifespan analysis of C. elegans in pretreatment of nematodes with NAC treated with 0, 10 μM rBTI. Plots are representative of three independent experiments.

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Fig. 5. rBTI down-regulates insulin/IGF-1 signaling pathway and activates DAF-16. The target genes' transcriptional expression levels were quantified using qRT-PCR and normalized to the internal control act-1. (A) The mRNA levels of genes involved in IIS pathway. (B) The target genes of DAF-16 and SKN-1 transcriptional expression. The data for at least three independent experiments were pooled. The mean normalized RNA level for each gene in control group worms was set as 1. All values are given as mean ± SD. p-Value was calculated using Student's t-test. *p b 0.05, **p b 0.01, ***p b 0.001 versus respective controls. (C) DAF-16::GFP relocalization. Compared with control group, worms treated with 10 μM rBTI produces a clear relocalization of DAF16::GFP to the nuclei of intestinal cells.

and skn-1 mRNA decreased slightly, compared to the control (Fig. 5A). These results showed that rBTI indeed down-regulated the IIS pathway. Considering the fact that the change in mRNA level doesn't really reflect the activity of transcription factor, we measured the transcriptional activities of DAF-16 and SKN-1 by examining transcriptional expression of their most reliable downstream target genes. The sod-3 and gsh-px are classical readout for DAF-16 activation and gst-4 and gcs-1 for SKN-1 (Tullet et al., 2008; Xiong et al., 2014). As expected, we observed that sod-3 and gsh-px mRNA levels increased about 2.3 fold and 1.5 fold in worms treated with 10 μM rBTI on day 1, compared with the control

group, but there was no significant change in gst-4 and gcs-1 (Fig. 5B), which suggested that rBTI activates DAF-16 but not SKN-1. When DAF-16 is activated, it would translocate to nuclei from cytoplasm (Henderson and Johnson, 2001). For further proof, rBTI induced constitutive nuclear localization of a DAF-16 fusion protein (Fig. 5C). 3.7. daf-2 and daf-16 are required for rBTI-mediated longevity Considering that rBTI has an effect on daf-2 and daf-16, the most significant components that are involved in the IIS pathway regulated

Fig. 6. daf-2 and daf-16 are required for rBTI-mediated longevity. (A) The lifespan analysis of daf-2(e1368) nematodes treated with 0, 10 μM rBTI. (B) The lifespan analysis of daf-16(mu86) nematodes treated with 0, 10 μM rBTI. Plots are representative of three independent experiments.

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lifespan, we wonder whether rBTI-mediated longevity is dependent on daf-2 and daf-16 in worms. We used two worm strains carrying a deletion in genes daf-2 and daf-16, respectively. As shown in Fig. 6, rBTI treated animals didn't significantly increase the mean lifespan of daf-2 and daf-16 mutants, suggesting that the rBTI-induced lifespan extension requires daf-2 and daf-16. 3.8. daf-2 is required for the induction of ROS As our testing showed that ROS signal, daf-2 and daf-16 are all required for the rBTI-induced lifespan. And it has been reported that both ROS and daf-2 can activated DAF-16 directly (Landis and Murphy, 2010; Putker et al., 2013). Moreover, ROS may function as signaling molecules that promote the lifespan by regulating daf-2 (Bloch-Damti and Bashan, 2005; Finkel and Holbrook, 2000), and impaired or downregulated daf-2 may induce the ROS signal and extend the lifespan (Zarse et al., 2012). We questioned what kind of the relationship between IIS down regulation, ROS induction and the longevity in our study. We found rBTI almost equally down-regulated IIS pathway in pretreatment of nematodes with NAC, compared with untreated worms, daf-2 mRNA levels decreased ~65% in worms treated with 10 μM rBTI for 1 day, compared with the control (Fig. 7A), while rBTI did not induce ROS in daf-2 mutant worms. The ROS content just increased 5% in daf-2 mutant worms treated with 10 μM rBTI for 1 day, compared with control group, and no significant difference was observed (Fig. 7B). Taken together, these data indicate that inhibitor of IIS signaling pathway induces ROS rather than ROS signal inhibits the IIS pathway. 3.9. rBTI decreases glycolysis, increased lipolysis, and induced autophagy in C. elegans Exactly how they generate the health benefits remains open for debate, however CR results in promoted oxidative stress resistance, enhanced autophagy, and a generalized shift from carbohydrate to fat metabolism, all of which could be essential components of the beneficial effects (Selman, 2014; Speakman and Mitchell, 2011). As described previously, our experiments showed that worms treated with rBTI showed increased oxidative stress resistance. Here, we made a preliminary exploration on the other two signatures. Results of qRT-PCR showed that transcriptional expression of key enzymes involved in glycolytic metabolism generally declined (Fig. 8A). And direct measurement of those enzymes in worm extracts also showed that phosphofructokinase (PFK) and hexokinase (HK) activities decreased 47.6% and 7.9%, respectively, while pyruvate kinase (PK) activity increased roughly one-third (Fig. 8B). Clearly, test results

for PK activity were inconsistent with the qRT-PCR results. We speculated that an accumulation of the PK-activators fructose-1, 6-diphosphate and phosphoenolpyruvate may lead to the increased PK activity. Because we found an increase in pyruvate carboxylase transcriptional expression and a decrease in fructose-1, 6-bisphosphatase transcriptional expression caused the accumulation of fructose-1, 6-diphosphate and phosphoenolpyruvate in gluconeogenesis (Figure S1) in following experiments. PFK is a key rate-limiting enzyme, and PK catalyzes the ultimate and irreversible reaction in glycolysis, so the decrease of their activities or transcriptional expression would be predicted to the declined glycolysis activity. Moreover, we found evidence for increased lipolysis, because compared with control group, lipl-4 encoding a triglyceride lipase was up-regulated 2.6 fold (Fig. 8C), lipase activity was increased 28% (Fig. 8D), and the fat storage was decreased 15.4% (Fig. 8E) in rBTI-treated worms. Additionally, we observed obvious autophagosome (GFP::LGG-1 punta), although the transcriptional expression of the autophagy related gene almost had no significant change (Fig. 8F, G). Though we observed that rBTI induced some beneficial effects related delay aging, it is not clear that whether they mediated the ability of rBTI to extend the lifespan in worms. We will do further research in a follow-up experiment in the future (Fig. 9). 4. Discussion CRMs are defined as pharmaceutical or naturally occurring compounds that may mimic the metabolic state of CR (Ristow and Schmeisser, 2014). One of the best studied compounds in this area is 2-deoxy-glucose (DOG). However, significant toxic effect was observed in rats following chronic ingestion of DOG (Minor et al., 2010). It is therefore necessary to look for safe and effective compounds from natural substances. Along this line, BTI is a good choice. Our data clearly showed that rBTI extends the lifespan in C. elegans by mimicking CR. It has been repeatedly reported that CR is capable of induce mitochondrial metabolism to transiently increased formation of ROS and other stressors, leading to a secondary increase in stress defense, cumulating in reduced net stress levels and possibly extended lifespan. The process of a lifespan extension based on mitochondrial oxidative stress is known as mitohormesis (Ristow and Schmeisser, 2014). Our study showed rBTI performed similar metabolic state of CR, such as increased AMP:ATP ratio and respiration induced transient ROS signal and so on. Moreover, we also proved that the ROS signal was required for rBTImediated longevity. Although the changes in SOD, CAT and GSH were unexpected, the similar overall trend for ROS explained their changes well. As the mitohormetic effect, the change of ROS induced the changes of SOD,

Fig. 7. daf-2 is required for the induction of ROS. (A) daf-2 transcriptional expression level in nematodes after treatment with 0, 10 μM rBTI. (B) The ROS content analysis of daf-2(e1368) nematodes after treatment with 0, 10 μM rBTI. In all panels, relative values are depicted. All values are given as mean ± SD. p-Value was calculated using Student's t-test. *p b 0.05, **p b 0.01, ***p b 0.001 versus respective controls.

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Fig. 9. The mechanism of rBTI-mediated longevity. Addition of rBTI induces ROS formation via inhibiting insulin/IGF-1 signaling, leading to increased antioxidase activity and stress resistance, cumulating in extension of life expectancy, which mimics CR effect. At the same time, rBTI can decrease the activity of glycolysis, reduce fat storage, and induce autophagy in C. elegans. But it is not clear whether they mediate the ability of rBTI to extend the lifespan in worms. The dashed line indicates the mechanisms are not completely known, while the solid arrows indicate the mechanisms are certain in our study.

CAT and GSH (Zarse et al., 2012). The transiently increased ROS levels act to induce a vaccination-like response within the individual cell to lead to reduced ROS levels in a time-resolved manner and better stress defense in the steady state. As for the slight difference in SOD, CAT, and GSH, it may be because they remove different radicals. SOD mainly removes O2− and H2O2, while CAT and GSH mainly target OHˉ. CRMs have similar beneficial biological effects. Caution is due, however, in referring to CR, because different methods to induce CRM in C. elegans act through different genes to elicit corresponding effects on the lifespan (Bishop and Guarente, 2007; Greer and Brunet, 2009; Panowski et al., 2007). For example aspirin extends the lifespan of C. elegans via DAF-16 in CR pathway (Wan et al., 2013), and metformin promotes the lifespan through mimicking CR via the peroxiredoxin PRDX-2 (De Haes et al., 2014b). Four pathways have been implicated in mediating the CR effect, and these are the insulin like growth factor (IGF-1)/insulin signaling (IIS) pathway, the sirtuin pathway, the adenosine monophosphate (AMP) activated protein kinase (AMPK) pathway and the target of rapamycin (TOR) pathway. For example, DOG mediates the CR effect by activating AMPK pathway (Schulz et al., 2007), while rapamycin mimic CR via TOR pathway (Tsang et al., 2007), the phytochemical resveratrol is found to be CR mimetic as it potentially slows aging and certainly delays age-related diseases by activating sirtuins (Agarwal and Baur, 2011). Here, our results showed rBTI mediated the CR effect by DAF-16 and IIS pathway, the down-regulation of IIS pathway induced ROS, and ROS signal activated DAF-16 which upregulated the related target genes and ultimately induced stress defense. As previously reported, D-glucosamine mimics CR, as an inhibitor of both hexokinase and glucokinase, blocking glucose to form pyruvate and ATP (Weimer et al., 2014). In addition, resveratrol and metformin have been found to inhibit mitochondrial complex I of the ETC, acting as potential CRMs (Agarwal and Baur, 2011; De Haes et al., 2014b). Although the current findings indicating that rBTI induces similar effect to CR and it is dependent on daf-2, we have not found the fundamental reason that rBTI induces ROS or mimic CR. Here, we suspect that rBTI, as a micro-molecule polypeptide, might play a similar role with the insulin-like peptides. Because, we found rBTI have very similar

structures with insulin, especially the functional domain (Wang et al., 2011). daf-2 gene, orthologs of the mammalian insulin/IGF-1 receptor (InR), can be activated or inhibited by some insulin-like peptides. Some insulin-like peptides expressed in C. elegans were reported. For example, DAF-28 and INS-7 are two agonists (Li et al., 2003; Malone et al., 1996; Murphy et al., 2007), and INS-1 is an antagonist (Pierce et al., 2001). But now, some natural substances that have antagonism or activation on daf-2 are scarcely reported. Another possibility is that rBTI, as a trypsin inhibitor, causes a specific shortage of amino acids and a down regulation of protein synthesis rates with concomitant extension of the lifespan. In mice, it has been shown that a reduced nutritional protein content extent lifespan as it was shown for casein restriction in Drosophila (Min and Tatar, 2006). Pep-2 deletion which reduces the uptake of peptides, induces similar effects with rBTI, determines an increase in the lifespan and stress tolerance and synergizes with reduced insulin signaling (Meissner et al., 2004). Among these two possible explanations, we tend to agree with the former case, because our experiment showed STI cannot significantly extend the lifespan in C. elegans as a trypsin inhibitor. We need to do further study and analysis on this issue in future. Changes in several catabolic pathways in C. elegans treated with rBTI such as a decrease in glycolysis, and an increase in lipolysis and autophagy also occurred in animals with CR (Speakman and Mitchell, 2011). Further experiments will be needed to fully explore their roles of in the rBTI-induced lifespan extension. Our results suggest that rBTImediated decrease in glycolysis may be the direct reason that caused the lower AMP:ATP ratio, while increase in lipolysis and autophagy are likely a compensatory mechanism that is not directly related to rBTI induced longevity. In conclusion, our results showed that rBTI induced a transient ROS signaling for the adaptive induction of endogenous stress defense to extend the lifespan as CR does, and this process was dependent on daf-2. Besides, we found that the glycolysis was decreased, while lipolysis and autophagy were increased in worms treated with rBTI. This study suggested that rBTI had the potential to be a natural CRM to delay aging, and rBTI longevity pathway could be conserved in other organisms, including humans. However, the fundamental cause of the rBTIinduced lifespan remains unclear and further studies are needed.

Acknowledgments The authors acknowledge the National Natural Sciences Foundation of China (grant nos. 31171659 and 31300653) supported this project. The authors acknowledge Prof. Enbo Ma (Research Institute of Applied Biology, Shanxi University) for the kind donation of C. elegans strain N2 and E. coli OP50. Some C. elegans strains were provided by CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40OD010440).

Appendix A. Supplementary data Supporting Information detailed description of materials and methods; tables listing the primers used for the quantitative real-time reverse transcription polymerase chain reaction; and figure showing the transcriptional expression of genes encoding key enzymes in gluconeogenesis in C. elegans. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.exger.2015.05.001.

Fig. 8. rBTI decreases glycolysis, increases lipolysis, and induces autophagy in C. elegans. (A) The mRNA levels of genes encoding key enzyme in glycolysis. (B) The activity of key enzyme in glycolysis. (C) The mRNA levels of lipase genes. (D) The activity of lipase. (E) rBTI-treated worms showed reduced fat storage. (F) The mRNA levels of autophagy genes. (G) rBTI induced autophagy in C. elegans. adls2122 strains treated with rBTI produce obvious GFP::LGG-1-positive foci. (A, C, D, F) The target genes' transcriptional expression levels were quantified using qRT-PCR and normalized to the internal control act-1. All values are given as mean ± SD. Relative values are depicted. p-Value was calculated using Student's t-test. *p b 0.05, **p b 0.01, ***p b 0.001 versus respective controls.

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