Octopamine enhances oxidative stress resistance through the fasting‐responsive transcription factor DAF‐16/FOXO in C. elegans
Abstract
Dietary restriction regimens lead to enhanced stress resistance and extended life span in many species through the regulation of fasting and/or diet‐responsive mechanisms. The fasting stimulus is perceived by sensory neurons and causes behavioral and metabolic adaptations. Octopamine (OA), one of the Caenorhabditis elegans neurotransmitters, is involved in behavioral adaptations, and its levels are increased under fasting conditions. However, it remains largely unknown how OA contributes to the fasting responses. In this study, we found that OA administration enhanced organismal resistance to oxidative stress. This enhanced resistance was suppressed by a mutation of the OA receptors, SER‐3 and SER‐6. Moreover, we found that OA administration promoted the nuclear translocation of DAF‐16, the key transcription factor in fasting responses, and that the OA‐induced enhancement of stress resistance required DAF‐16. Altogether, our results suggest that OA signaling, which is triggered by the absence of food, shifts the organismal state to a more protective one to prepare for environmental stresses.
Introduction
Organisms in the wild live in fluctuating environments. Thus, organisms sense and process environmental signals to help them prepare to adapt their behavior. Appropriate responses to environmental changes are essential for organismal survival. The regulation of the life span by the nervous system was originally identified in Caenorhabditis elegans by a study using mutants defective in sensory perception (Apfeld & Kenyon 1999). Subsequently, many sensory neurons, including gustatory, olfactory, and thermosensory neurons, were shown to affect the life span (Alcedo & Kenyon 2004; Lee & Kenyon 2009). Recent studies have shown that genetic manipulations in neuronal cells are sufficient to increase the life span (Durieux et al. 2011; Taylor & Dillin 2013; Douglas et al. 2015; Leiser et al. 2015). Thus, the nervous system may play an important role in the life span regulation in response to environmental changes.
Temperature and food are the two essential environmental factors that modulate the organismal life span (Fontana et al. 2010; Kenyon 2010). In C. elegans, low temperatures at the adult stage promote longevity (Klass 1977), and a cold‐sensitive transient receptor potential (TRP) channel in neuronal cells contributes to cold‐induced longevity (Xiao et al. 2013). Many forms of dietary restriction regimens, including calorie restriction (CR) and intermittent fasting (IF), extend the life span in C. elegans (Kenyon 2010). SKN‐1, the ortholog of NRF2, in neurons plays an important role in CR‐induced longevity (Bishop & Guarente 2007). Recently, the fasting stimulus has been shown to be an important regulator of life span (Longo & Mattson 2014). Indeed, the IF regimen, repetitions of periods of two days of feeding and two days of fasting, is one of the most effective dietary restriction regimens to extend the life span and enhance the resistance to heat and oxidative stress in C. elegans (Honjoh et al. 2009).
In C. elegans, four neurotransmitter amines (serotonin, dopamine, tyramine, and octopamine) modulate behaviors and metabolism in response to food availability (Sulston et al. 1975; Horvitz et al. 1982; Alkema et al. 2005; Chase & Koelle 2007). Octopamine (OA) is considered the counterpart of noradrenalin in invertebrates. OA levels are increased under fasting conditions (Suo et al. 2009), and OA signaling regulates gene expression through cAMP response element‐binding protein (CREB) under fasting conditions (Suo et al. 2006). However, the role of OA in regulation of life span and oxidative stress response remained unclear.
In this study, we found that OA administration enhances oxidative stress resistance, which requires OA receptors (SER‐3 and SER‐6) and DAF‐16. Our analyses showed that OA administration promotes DAF‐16 nuclear translocation and induces genomewide transcriptome alterations in a DAF‐16‐dependent manner. These results suggest that the fasting stimulus elicits octopamine release from the nervous system, which increases oxidative stress resistance via DAF‐16 nuclear accumulation.
Results
Octopamine administration enhances oxidative stress resistance in an SER‐3‐ and SER‐6‐dependent manner
To test whether OA administration extends C. elegans life span by mimicking fasting conditions, we treated animals with OA constantly or intermittently and measured the life span. Both OA treatments failed to reproducibly increase the life span (see Table 1), suggesting that OA is not sufficient for life span extension. Then, we investigated whether OA administration is sufficient to enhance oxidative stress resistance and found that OA administration enhanced oxidative stress resistance in a dose‐dependent manner (Fig. 1A, Table 2). As the body size and the pharyngeal pumping rate of the animals decreased under fasting conditions (Horvitz et al. 1982; Alkema et al. 2005), we examined whether OA administration also induced these phenotypic changes. The animals treated with OA were not smaller than the control animals (Fig. 1B). Moreover, although the pumping rate was suppressed after 2 h of OA administration as previously reported (Horvitz et al. 1982), there were no significant differences in the pumping rate between control animals and the OA‐treated animals after 48 h of OA administration (Fig. 1C). These results suggest that OA administration enhances oxidative stress resistance without inducing other phenotypes caused by fasting.
| OA treatment | Trial | Mean life span (days) | % of extension by OA | P value (log‐rank test) | Number of animals | ||
|---|---|---|---|---|---|---|---|
| Control | OA treated | Control | OA treated | ||||
| Aaa
Treatment A: constant OA administration at 5 mg/mL.
|
#1 | 18.3 | 20.6 | 12.6 | 0.050 | 59 | 58 |
| #2 | 20.3 | 19.6 | −3.5 | 0.058 | 60 | 60 | |
| #3 | 19.9 | 23.9 | 20.0 | <0.0001 | 60 | 59 | |
| #4 | 21.7 | 24.7 | 13.6 | <0.0001 | 60 | 60 | |
| #5 | 25.6 | 30.0 | 17.1 | <0.0001 | 57 | 60 | |
| #6 | 23.6 | 24.8 | 4.8 | 0.13 | 58 | 56 | |
| #7 | 21.4 | 22.8 | 6.6 | 0.26 | 57 | 59 | |
| Bbb
Treatment B: intermittent OA administration at 5 mg/mL.
|
#1 | 19.9 | 22.8 | 14.4 | 0.0006 | 60 | 30 |
| #2 | 21.7 | 24.6 | 13.2 | 0.0002 | 60 | 60 | |
| #3 | 25.6 | 30.4 | 18.9 | <0.0001 | 57 | 60 | |
| #4 | 23.6 | 25.2 | 6.6 | 0.031 | 58 | 55 | |
| #5 | 21.4 | 23.3 | 8.8 | 0.097 | 57 | 57 | |
| #6 | 22.7 | 21.1 | −7.3 | 0.021 | 47 | 57 | |
| #7 | 20.4 | 20.7 | 1.3 | 0.10 | 54 | 63 | |
| #8 | 22.4 | 21.3 | −5.1 | 0.022 | 60 | 60 | |
| #9 | 23.5 | 23.1 | −1.7 | 0.061 | 76 | 73 | |
- a Treatment A: constant OA administration at 5 mg/mL.
- b Treatment B: intermittent OA administration at 5 mg/mL.
| Strain | Trial | OA concentration (mg/mL) | % survivors (17 h) | P valueaa
Compared with the 0 mg/mL OA treatment in each trial.
(log‐rank test) |
Number of animals |
|---|---|---|---|---|---|
| N2 | #1 | 0 | 5 | – | 40 |
| 5 | 20 | 0.0022 | 40 | ||
| 20 | 42.5 | <0.0001 | 40 | ||
| #2 | 0 | 10 | – | 40 | |
| 5 | 32.5 | 0.0083 | 40 | ||
| 20 | 55 | <0.0001 | 40 | ||
| #3 | 0 | 25 | – | 40 | |
| 5 | 32.5 | 0.052 | 40 | ||
| 20 | 22.5 | 0.21 | 40 | ||
| #4 | 0 | 38.5 | – | 39 | |
| 5 | 67.5 | <0.0001 | 40 | ||
| 20 | 80 | <0.0001 | 40 | ||
| #5 | 0 | 32.5 | – | 40 | |
| 5 | 65 | <0.0001 | 40 | ||
| 20 | 69.2 | <0.0001 | 39 | ||
| #6 | 0 | 27.5 | – | 40 | |
| 5 | 55 | 0.0054 | 40 | ||
| 20 | 62.5 | 0.0004 | 40 | ||
| #7 | 0 | 40 | – | 40 | |
| 5 | 64.1 | 0.0012 | 39 | ||
| 20 | 75 | 0.0002 | 28 | ||
| #8 | 0 | 55 | – | 40 | |
| 5 | 70 | 0.0023 | 40 | ||
| 20 | 90 | <0.0001 | 40 | ||
| #9 | 0 | 25 | – | 40 | |
| 5 | 40 | 0.042 | 40 | ||
| 20 | 85 | <0.0001 | 40 | ||
| octr‐1 (ok371) VC224 | #1 | 0 | 27.5 | – | 40 |
| 5 | 52.5 | 0.0032 | 40 | ||
| 20 | 87.5 | <0.0001 | 40 | ||
| #2 | 0 | 20 | – | 40 | |
| 5 | 50 | <0.0001 | 40 | ||
| 20 | 62.5 | <0.0001 | 40 | ||
| #3 | 0 | 47.5 | – | 40 | |
| 5 | 70 | 0.0026 | 40 | ||
| 20 | 62.5 | 0.0031 | 40 | ||
| ser‐3 (ok2007) RB1631 | #1 | 0 | – | – | 40 |
| 5 | – | 0.23 | 40 | ||
| 20 | – | 0.0001 | 39 | ||
| #2 | 0 | 0 | – | 40 | |
| 5 | 2.5 | 0.31 | 40 | ||
| 20 | 5 | 0.077 | 40 | ||
| #3 | 0 | 2.5 | – | 40 | |
| 5 | 10.8 | 0.20 | 37 | ||
| 20 | 43.6 | 0.0008 | 39 | ||
| ser‐6 (tm2146) FX02146 | #1 | 0 | 5 | – | 40 |
| 5 | 5 | 0.27 | 40 | ||
| 20 | 30 | 0.0031 | 40 | ||
| #2 | 0 | 2.5 | – | 40 | |
| 5 | 2.5 | 0.023 | 40 | ||
| 20 | 27.5 | 0.022 | 40 | ||
| #3 | 0 | 45 | – | 40 | |
| 5 | 25 | 0.095 | 40 | ||
| 20 | 50 | 0.062 | 40 | ||
| ser‐3;ser‐6 (ok2007;tm2146) | #1 | 0 | 10.3 | – | 39 |
| 5 | 15 | 0.43 | 40 | ||
| 20 | 27.5 | 0.0061 | 40 | ||
| #2 | 0 | 32.5 | – | 40 | |
| 5 | 12.5 | 0.045 | 40 | ||
| 20 | 25 | 0.60 | 40 | ||
| #3 | 0 | 17.5 | – | 40 | |
| 5 | 5 | 0.18 | 40 | ||
| 20 | 30 | 0.24 | 40 |
- a Compared with the 0 mg/mL OA treatment in each trial.
OCTR‐1, SER‐3, and SER‐6 are OA receptors (Mills et al. 2012). We examined the requirements of these three receptors for OA‐enhanced resistance to oxidative stress. Among the mutations of three OA receptors, the deletion of ser‐3 and ser‐6 suppressed OA‐induced increase in the resistance to oxidative stress (Fig. 1D,E). Additionally, the double mutation of ser‐3 and ser‐6 substantially suppressed the OA‐enhanced resistance to oxidative stress (Fig. 1D,E). Together, OA administration enhanced oxidative stress resistance in an OA receptor (SER‐3 and SER‐6)‐dependent manner.
Octopamine‐enhanced oxidative stress resistance is dependent on DAF‐16
To investigate whether well‐known stress responsive pathways are required for the OA‐enhanced resistance to oxidative stress, we carried out RNAi of daf‐16, the insulin/IGF‐1 signaling effector, and skn‐1, a transcription factor that functions in oxidative stress responses. Knockdown of daf‐16, but not knockdown of skn‐1, suppressed OA‐enhanced oxidative stress resistance (Fig. 2A, Table 3). Additionally, the ablation of daf‐16 also completely suppressed the OA‐enhanced resistance to oxidative stress (Fig. 2B,C, Table 4). These results indicate that DAF‐16 is required for the OA‐enhanced resistance to oxidative stress.
| Trial | RNAi | OA concentration (mg/mL) | P valueaa
Compared with the 0 mg/mL treatment in each RNAi condition.
(log‐rank test) |
Number of animals |
|---|---|---|---|---|
| #1 | Control RNAi (empty vector) | 0 | – | 30 |
| 5 | 0.0006 | 30 | ||
| 10 | 0.0003 | 30 | ||
| daf‐16 RNAi | 0 | – | 30 | |
| 5 | 0.69 | 30 | ||
| 10 | 0.43 | 30 | ||
| skn‐1 RNAi | 0 | – | 30 | |
| 5 | 0.025 | 18 | ||
| 10 | <0.0001 | 30 | ||
| #2 | Control RNAi | 0 | – | 30 |
| 5 | 0.0005 | 30 | ||
| daf‐16 RNAi | 0 | – | 30 | |
| 5 | 0.84 | 30 | ||
| skn‐1 RNAi | 0 | – | 30 | |
| 5 | 0.019 | 30 | ||
| #3 | Control RNAi | 0 | – | 30 |
| 5 | 0.052 | 30 | ||
| daf‐16 RNAi | 0 | – | 28 | |
| 5 | 0.11 | 30 | ||
| skn‐1 RNAi | 0 | – | 30 | |
| 5 | 0.015 | 30 |
- a Compared with the 0 mg/mL treatment in each RNAi condition.
| Strain | Trial | OA concentration (mg/mL) | % survivors (13 h) | P valueaa
Compared with 0 mg/mL OA treatment in each trial.
(log‐rank test) |
Number of animals |
|---|---|---|---|---|---|
| N2 | #1 | 0 | 27.5 | – | 40 |
| 5 | 32.5 | 0.099 | 40 | ||
| 20 | 70 | <0.0001 | 40 | ||
| #2 | 0 | 15 | – | 40 | |
| 5 | 40 | 0.0003 | 40 | ||
| 20 | 66.7 | <0.0001 | 39 | ||
| #3 | 0 | 45 | – | 40 | |
| 5 | 62.5 | 0.037 | 40 | ||
| 20 | 82.5 | <0.0001 | 40 | ||
| daf‐16 (mu86) CF1038 | #1 | 0 | 0 | – | 40 |
| 5 | 0 | 0.31 | 40 | ||
| 20 | 0 | 0.44 | 40 | ||
| #2 | 0 | 7.5 | – | 40 | |
| 5 | 7.5 | 0.74 | 40 | ||
| 20 | 32.5 | 0.0014 | 40 | ||
| #3 | 0 | 2.5 | – | 40 | |
| 5 | 7.5 | 0.25 | 40 | ||
| 20 | 15 | 0.0049 | 40 |
- a Compared with 0 mg/mL OA treatment in each trial.
Environmental inputs, including fasting, promote DAF‐16 nuclear accumulation (Henderson & Johnson 2001; Honjoh et al. 2009). Therefore, we examined whether OA administration could mimic the fasting‐induced nuclear accumulation of DAF‐16. To this end, we evaluated the nuclear accumulation of DAF‐16 using DAF‐16::GFP expressing transgenic animals (TJ356). The OA administration induced DAF‐16 nuclear accumulation in a dose‐dependent manner (Fig. 3A,B). Our experiments indicated that 5 mg/mL OA induced DAF‐16 nuclear accumulation to an extent comparable to the fasting‐induced DAF‐16 nuclear accumulation (Fig. 3A,B). Our results suggest that OA administration enhances oxidative stress resistance by inducing DAF‐16 nuclear accumulation.
Octopamine‐induced transcriptional changes are mediated by DAF‐16 and octopamine receptors
DAF‐16 activation or DAF‐16 nuclear accumulation induces dramatic changes in the expression of genes that are involved in cellular stress response, metabolism, and autophagy, which are important for the regulation of the life span and organismal stress response. To identify genes involved in the OA‐enhanced resistance to oxidative stress, we carried out microarray analysis. We identified 398 OA‐regulated genes whose expression levels were increased more than twofold or decreased less than 0.5‐fold after OA administration in N2 (Fig. 4A,B). Among the OA‐regulated genes, we identified 269 DAF‐16‐dependent genes and 248 OA receptor‐dependent genes, in which the extent of the OA‐induced change in their expression level was reduced by more than one quarter in daf‐16 and ser‐3; ser‐6 mutants, respectively, compared to that in N2 (Fig. 4A). The DAF‐16‐dependent genes greatly overlapped the OA receptor‐dependent genes (Fig. 4A), suggesting that OA receptors and DAF‐16 act in the same pathway under OA‐treated conditions. These results suggest that OA administration enhances oxidative stress resistance through the regulation of DAF‐16‐mediated changes in gene expression in the stress response.
Discussion
An increasing number of studies indicate that the nervous system and fasting stimuli are important for the regulation of organismal stress resistance and life span (Longo & Mattson 2014; Uno & Nishida 2016). However, how the fasting‐responsive neurotransmitter octopamine contributes to the fasting responses remains unclear. In this study, we show that OA administration markedly enhances oxidative stress resistance in an OA receptor‐dependent manner. Our analyses also uncovered that OA administration promotes DAF‐16 nuclear accumulation and induces genomewide transcriptional changes in a DAF‐16‐dependent manner. Together, the fasting stimulus elicits OA release from the nervous system, which enhances oxidative stress resistance via DAF‐16 nuclear translocation.
Although OA administration does not significantly extend the life span, it markedly enhances oxidative stress resistance. These results suggest that OA may have both beneficial and adverse effects. Indeed, a recent study has shown that neuronal AMPK activation‐induced longevity is mediated by the inhibition of OA release, suggesting that OA has a negative effect on longevity (Burkewitz et al. 2015). More recently, it has been shown that OA administration enhances thermotolerance (Furuhashi & Sakamoto 2016) and promotes lipid hydrolysis (Tao et al. 2016) in C. elegans. Based on these studies, including ours, OA signaling could account for some of the responses to fasting.
Our results show that the enhancement of resistance to oxidative stress after 2 days of OA administration requires OA receptors (SER‐3 and SER‐6), suggesting that the OA‐enhanced resistance to oxidative stress does not result from side effects of OA administration. Moreover, it has been reported that OA administration enhances thermotolerance in a DAF‐16‐dependent manner (Furuhashi & Sakamoto 2016), which is consistent with our conclusion that OA administration enhances oxidative stress resistance through the activation of DAF‐16 via OA receptors. Taken together, OA‐DAF‐16 axis has an important role in organismal stress resistance. A recent report (Furuhashi & Sakamoto 2016) showed that OA administration does not enhance resistance to the oxidant hydrogen peroxide (H2O2), in apparent contrast to our result that OA administration enhances resistance to the oxidant paraquat. This difference may result from the difference in the oxidant used (H2O2 vs. paraquat) or the difference in the duration of OA treatment (1 day vs. 2 days). Our analysis shows that OA administration promotes DAF‐16 nuclear accumulation and induces transcriptional changes of the stress response genes. As the fasting stimulus also promotes DAF‐16 nuclear accumulation and induces transcriptional changes in the stress response genes (Honjoh et al. 2009; Uno et al. 2013), the OA‐DAF‐16 axis also functions under fasting conditions. The downstream targets of the OA receptors, which regulate DAF‐16 localization, remain to be determined. Additional studies are needed to understand the detailed mechanisms of OA actions.
Experimental procedures
C. elegans strains
All strains were maintained at 20 °C on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 as previously described (Brenner 1974). The following strains were used in this study: N2, wild type; VC224, octr‐1(ok371); RB1631, ser‐3(ok2007); FX02146, ser‐6(tm2146); TJ356, zIs356[daf‐16::gfp; rol6].
Life span assay
We carried out an OA administration life span assay as follows. Worms from synchronized eggs were raised in normal conditions, and young adult animals were transferred to NGM plates that contained 200 μg/mL 5‐fluoro‐2′‐deoxyuridine (FUdR; Sigma‐Aldrich, St. Louis, MO, USA) with or without 5 mg/mL octopamine hydrochloride (Sigma‐Aldrich). Approximately 60 animals were transferred to FUdR‐containing NGM plates seeded with UV‐treated OP50. The day on which the animals were transferred to FUdR‐containing NGM plates was defined as t = 0 days. We scored death events every other day. Animals were scored as dead if they failed to respond to touch by a pick. The log‐rank test was used to evaluate differences in survival between the two groups and was carried out using graphpad prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA).
Oxidative stress assay after octopamine administration
Animals from synchronized eggs were raised in normal conditions, and young adult animals were transferred to NGM plates that contain octopamine hydrochloride (Sigma‐Aldrich) (5, 10, and 20 mg/mL). After the 2 days of OA administration, two 5‐day‐old animals were transferred to each well (60‐well plate, Greiner Bio‐one, Frickenhausen, Germany) containing 20 μL of 300 mm paraquat [methyl viologen (Nacalai Tesque Inq., Kyoto, Japan)] in M9 buffer. Twenty replicates per condition were assayed. After 9 h of treatment, the plates were monitored almost every 2 h to document the number of animals that were alive, dead, or censored.
Fluorescence microscopy
In DAF‐16 localization assays, worms expressing DAF‐16::GFP were synchronized and raised under normal conditions for 48 h, followed by additional 24 h on NGM plates containing 200 μg/mL FUdR. Then, animals were transferred to plates with or without octopamine. After 18 h of octopamine treatment, worms were fixed with 4% paraformaldehyde (Nacalai Tesque Inq., Kyoto, Japan) in PBS for 3 min at room temperature. After washing samples twice with PBS, animals were observed with Axioplan2.
Microarray analysis
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from frozen 5‐day‐old N2, daf‐16, and ser‐3;ser‐6 mutants treated with or without 20 mg/mL OA. cDNA synthesis from the total RNA was carried out using a GeneChip 3′ IVT PLUS Reagent Kit according to the manufacturer's protocol. RNA degradation and cRNA elongation and fragmentation were verified with an Agilent 2100 Bioanalyzer. The fragmented cRNA was hybridized using a GeneChip C. elegans Genome Array (Affymetrix, Santa Clara, CA, USA) at 45 °C for 16 h. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned chip images were analyzed with Affymetrix GeneChip Command Console version 2.0 (AGCC) and processed using default settings. The Affymetrix output (CEL files) was imported into genespring GX 11.0.2 (Agilent Technologies, Palo Alto, CA, USA) microarray analysis software for the presentation of expression profiles. Expression signals of probe sets were calculated using RMA (robust multiarray average, as implemented in GeneSpring GX). The log of ratio mode was used for all analyses (GeneSpring GX).
Acknowledgements
We thank members of the Nishida laboratory for their technical comments and helpful discussion. C. elegans strain ser‐6 was provided by National BioResource Project, and the others used in this study were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health NCRR.
