Feeding state regulates pheromone‐mediated avoidance behavior via the insulin signaling pathway in Caenorhabditis elegans
2018; Springer Nature; Volume: 37; Issue: 15 Linguagem: Inglês
10.15252/embj.201798402
ISSN1460-2075
AutoresLeesun Ryu, YongJin Cheon, Yang Hoon Huh, Seondong Pyo, Satya P. Chinta, Hongsoo Choi, Rebecca A. Butcher, Kyuhyung Kim,
Tópico(s)Circadian rhythm and melatonin
ResumoArticle19 June 2018free access Transparent process Feeding state regulates pheromone-mediated avoidance behavior via the insulin signaling pathway in Caenorhabditis elegans Leesun Ryu Department of Brain and Cognitive Sciences, DGIST, Daegu, Korea Search for more papers by this author YongJin Cheon Department of Brain and Cognitive Sciences, DGIST, Daegu, Korea Search for more papers by this author Yang Hoon Huh Electron Microscopy Research Center, Korea Basic Science Institute, Cheongju-si, Chungcheongbuk-do, Korea Search for more papers by this author Seondong Pyo Department of Brain and Cognitive Sciences, DGIST, Daegu, Korea Search for more papers by this author Satya Chinta Department of Chemistry, University of Florida, Gainesville, FL, USA Search for more papers by this author Hongsoo Choi Robotics Engineering Department, DGIST, Daegu, Korea Search for more papers by this author Rebecca A Butcher Department of Chemistry, University of Florida, Gainesville, FL, USA Search for more papers by this author Kyuhyung Kim Corresponding Author [email protected] orcid.org/0000-0002-9943-5092 Department of Brain and Cognitive Sciences, DGIST, Daegu, Korea Search for more papers by this author Leesun Ryu Department of Brain and Cognitive Sciences, DGIST, Daegu, Korea Search for more papers by this author YongJin Cheon Department of Brain and Cognitive Sciences, DGIST, Daegu, Korea Search for more papers by this author Yang Hoon Huh Electron Microscopy Research Center, Korea Basic Science Institute, Cheongju-si, Chungcheongbuk-do, Korea Search for more papers by this author Seondong Pyo Department of Brain and Cognitive Sciences, DGIST, Daegu, Korea Search for more papers by this author Satya Chinta Department of Chemistry, University of Florida, Gainesville, FL, USA Search for more papers by this author Hongsoo Choi Robotics Engineering Department, DGIST, Daegu, Korea Search for more papers by this author Rebecca A Butcher Department of Chemistry, University of Florida, Gainesville, FL, USA Search for more papers by this author Kyuhyung Kim Corresponding Author [email protected] orcid.org/0000-0002-9943-5092 Department of Brain and Cognitive Sciences, DGIST, Daegu, Korea Search for more papers by this author Author Information Leesun Ryu1, YongJin Cheon1, Yang Hoon Huh2, Seondong Pyo1, Satya Chinta3, Hongsoo Choi4, Rebecca A Butcher3 and Kyuhyung Kim *,1 1Department of Brain and Cognitive Sciences, DGIST, Daegu, Korea 2Electron Microscopy Research Center, Korea Basic Science Institute, Cheongju-si, Chungcheongbuk-do, Korea 3Department of Chemistry, University of Florida, Gainesville, FL, USA 4Robotics Engineering Department, DGIST, Daegu, Korea *Corresponding author. Tel: +82 53 785 6124; E-mail: [email protected] EMBO J (2018)37:e98402https://doi.org/10.15252/embj.201798402 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Animals change sensory responses and their eventual behaviors, depending on their internal metabolic status and external food availability. However, the mechanisms underlying feeding state-dependent behavioral changes remain undefined. Previous studies have shown that Caenorhabditis elegans hermaphrodite exhibits avoidance behaviors to acute exposure of a pheromone, ascr#3 (asc-ΔC9, C9). Here, we show that the ascr#3 avoidance behavior is modulated by feeding state via the insulin signaling pathway. Starvation increases ascr#3 avoidance behavior, and loss-of-function mutations in daf-2 insulin-like receptor gene dampen this starvation-induced ascr#3 avoidance behavior. DAF-2 and its downstream signaling molecules, including the DAF-16 FOXO transcription factor, act in the ascr#3-sensing ADL neurons to regulate synaptic transmission to downstream target neurons, including the AVA command interneurons. Moreover, we found that starvation decreases the secretion of INS-18 insulin-like peptides from the intestine, which antagonizes DAF-2 function in the ADL neurons. Altogether, this study provides insights about the molecular communication between intestine and sensory neurons delivering hunger message to sensory neurons, which regulates avoidance behavior from pheromones to facilitate survival chance. Synopsis Feeding state modulates ascr#3 pheromone avoidance behavior of Caenorhabditis elegans via an interplay between DAF-2 insulin-like receptor/DAF-16 FOXO signaling axis in ascr#3 sensing ADL neurons, and regulation of intestinal secretion of the DAF-2-inhibitory insulin-like peptide INS-18. Starvation increases ascr#3 avoidance behavior. DAF-2 insulin-like receptor acts in the ascr#3-sensing ADL neurons to regulate synaptic transmission to downstream target neurons. Starvation decreases the secretion of INS-18 insulin-like peptides from the intestine, which antagonizes DAF-2 function in the ADL neurons. Introduction Acute and chronic adaptations to the ever-changing external and internal environments are crucial for animal survival. Internal metabolic status induced by satiety and hunger has been shown to modulate animal physiology and behavior (see review Mayer, 2011). Particularly, depending on feeding status, animals change sensory responses, leading to altered behavioral outcomes (Magni et al, 2009; Nassel & Winther, 2010; Palouzier-Paulignan et al, 2012). These effects of feeding state on the sensory system have been widely reported in invertebrate and vertebrate systems. For example, increased food intake reduces monkey visual responses, causing decreased behavioral response to food sources (Critchley & Rolls, 1996). In another example, starvation promotes food-seeking behavior via direct modulation of Drosophila olfactory neurons (Root et al, 2011). However, molecular or circuit mechanisms underlying feeding state-regulated sensory responses still remain largely uncharacterized. The nematode Caenorhabditis elegans has a well-defined nervous system with only 302 neurons that mediate a broad spectrum of sensory behaviors, such as chemosensation, nociception, thermosensation, foraging, and feeding. These behaviors are plastic and can be modulated by the animal's feeding state (see review Sengupta, 2013). For example, acute starvation alters patterns of locomotion and decreases the rates of pharyngeal pumping and egg laying (Trent et al, 1983; Avery & Thomas, 1997; Sawin et al, 2000; Hills et al, 2004). In addition, the presence or absence of food acutely affects chemosensory behaviors; presence of food enhances avoidance behavior to repulsive chemicals (Ezcurra et al, 2011), whereas absence of food increases adaptation to repeated exposure of attractive odorants and noxious soluble chemicals, including copper and glycerol (Colbert & Bargmann, 1997; Ezcurra et al, 2016). Chronic starvation has shown to also affect chemosensory behaviors and gene expression of chemosensory signaling genes (Saeki et al, 2001; Gruner et al, 2014, 2016). However, little is known about the mechanisms underlying starvation-mediated changes of chemosensory behaviors. Insulin level reflects feeding state of an animal and directly regulates sensory responses (Fadool et al, 2000; Lacroix et al, 2008). In Drosophila, starvation increases attractive behaviors to favorable odors and decreases avoidance behaviors to unfavorable odors via the change of neuronal activities in the olfactory neurons by regulating insulin signaling (Root et al, 2011; Ko et al, 2015). In C. elegans, INS-1, an insulin-like peptide, regulates olfactory response to odorants and thus affects their olfactory behavior (Chalasani et al, 2010). However, a systemic analysis to investigate how insulin signaling regulates the sensory behavior by food availability in the whole organism context has not been performed yet. Caenorhabditis elegans produces a complex pheromone mixture composed of small chemicals called ascarosides. Subsets of ascarosides have been shown to regulate many aspects of nematode development and behavior including dauer formation and sensory perception (Jeong et al, 2005; Butcher et al, 2007; Edison, 2009; Macosko et al, 2009). Previously, we showed that adult hermaphrodites exhibit acute avoidance to the ascaroside ascr#3, which is mediated by the nociceptive ADL neurons (Jang et al, 2012). Moreover, early experience of ascr#3 modulates ascr#3 avoidance as adults, indicating that ascr#3 avoidance behaviors are plastic (Hong et al, 2017). Here, we show that ascr#3 avoidance is further modulated by feeding state. We found that prolonged starvation enhances ascr#3 avoidance behaviors of adult hermaphrodites. DAF-2 insulin-like signaling acts in the ADL ascr#3-sensing neurons to mediate starvation-induced ascr#3 avoidance by upregulation of the expression level of synaptic molecules via DAF-16 FOXO. Moreover, we also found that prolonged starvation inhibits secretion of an insulin-like peptide INS-18 from the intestine, which antagonizes the function of DAF-2 in ADL. Taken together, these results indicate that prolonged starvation affects the secretion of intestinal insulin-like peptides, which may function via a DAF-2 receptor to regulate the synaptic output of pheromone-sensing neurons and thus the pheromone avoidance behavior. Results Starvation alters avoidance behaviors to ascr#3 via the DAF-2 insulin-like receptor To investigate whether ascr#3 avoidance behaviors are affected by feeding state, we fed or starved young adult animals for 3, 6, or 24 h and examined acute avoidance to 100 nM ascr#3 (Fig 1A). We found that animals starved for longer than 6 h showed increased ascr#3 avoidance, compared to animals well-fed and animals starved for only 3 h (Fig 1B). Increased ascr#3 avoidance appeared to be mediated by increased long reversals but not short reversals or omega turns in starved animals (Fig EV1A; Hong et al, 2017). The increased ascr#3 avoidance in the animals starved for 6 h was reversed to normal level, after 24 h of re-feeding (Fig 1C). To further examine the effects of starvation on ascr#3 avoidance, we tested eat-2 loss-of-function mutant animals. eat-2 encodes nicotinic acetylcholine receptor subunit of which mutations cause decreased pharyngeal pumping, resulting in a dietary restriction state (Lakowski & Hekimi, 1998; Lopez et al, 2013). We found that ascr#3 avoidance was increased in eat-2 mutants, similar to that of wild-type animals starved for 6 h (Fig EV1B). These results indicate that feeding state influences avoidance behavior to ascr#3. Figure 1. An insulin/IGF-1-like receptor, daf-2, is required for increase in ascr#3 avoidance under starvation conditions Experimental scheme of ascr#3 avoidance assay depending upon starvation. 50–100 young adult animals are washed and placed on seeded (fed: colored in black) or non-seeded (starved: colored in gray) plates for each duration: 3, 6, and 24 h; then, various concentrations of ascr#3 are delivered to the front of a freely moving forward animal to measure avoidance frequencies responding to ascr#3. Fraction reversing of fed and starved animals in response to ascr#3 exposure. n = 50–70. ***P < 0.001 and ****P < 0.0001 (one-way ANOVA Bonferroni's test). Fraction reversing of re-fed animals from 6-h starvation in response to ascr#3 exposure. A 24-h re-feeding period reverses ascr#3 avoidances to well-fed status. n = 40–80. *P < 0.05 (one-way ANOVA Dunnett's Test). Fraction reversing of daf-2 mutant animals in fed and starved status in response to ascr#3 exposure. n = 40–60. Fraction reversing of well-fed wild-type animals and daf-2 mutants in response to 100, 200, and 400 nM ascr#3. n = 60. ***P < 0.001 (Bonferroni's test). Fraction reversing of wild-type animals, daf-2 mutants, and daf-2 mutants expressing unc-14p::daf-2 cDNA (neurons) or sre-1p::daf-2 cDNA (ADL) in response to 500 nM ascr#3. daf-2 cDNA expression in neuron and ADL restores the defect of ascr#3 avoidance in daf-2 mutants. n = 60. *P < 0.05 (Dunnett's test). Fraction reversing of wild-type animals, daf-2 mutants, and daf-2 mutants expressing sre-1p::daf-2 cDNA (ADL) in fed and starved conditions in response to ascr#3. n = 40–70. **P < 0.01 and ****P < 0.0001 (Bonferroni's test). Data information: All error bars represent ± SEM. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Effect of DAF-2/insulin-like receptor and feeding status on ascr#3 avoidance Fraction reversing of wild-type animals exhibiting short reversal (left) and omega turn under different feeding conditions (right). n = 40–80. Fraction reversing of wild-type animals and eat-2 mutants, daf-2 mutants, and eat-2;daf-2 double mutants under fed conditions. n = 80–110. **P < 0.01 and ****P < 0.0001 (Dunnett's Test). Fraction reversing of daf-2 alleles, e1368, and e1370 in response to 500 nM ascr#3 in fed conditions. n = 50–70. **P < 0.01 and ***P < 0.001 (Dunnett's test). Fraction reversing of wild-type and daf-2 mutant animals on on-food or off-food conditions. n = 30–50. *P < 0.05 (Bonferroni's test). The number of transgenic animals expressing sre-1p::gfp in ADL and ASJ. n = 31. Data information: All error bars represent ± SEM. Download figure Download PowerPoint Since DAF-2/insulin-like signaling has been reported to modify effects of dietary restriction (Lemieux & Ashrafi, 2015), we investigated whether DAF-2 regulates starvation-mediated increase in ascr#3 avoidance. We tested ascr#3 avoidance of daf-2(e1370) reduction-of-function mutants under well-fed and starved conditions (Kimura et al, 1997) and found that this mutation not only suppressed increased ascr#3 avoidance in animals starved for 6 h or 24 h, but also decreased ascr#3 avoidance in well-fed animals (Fig 1D and E). ascr#3 avoidance was also decreased in well-fed daf-2(e1368) mutant animals carrying another reduction-of-function allele (Fig EV1C). In addition, daf-2 mutation suppressed the enhancement of ascr#3 avoidance in eat-2 mutants (Fig EV1B). Furthermore, increased ascr#3 avoidance in the presence of food was also abolished in daf-2 mutants (Fig EV1D; Jang et al, 2012). These results suggest that DAF-2/insulin-like signaling modulates feeding state-dependent alteration of ascr#3 avoidance. We next examined where DAF-2 acts to control ascr#3 avoidance. We expressed daf-2 cDNA under the control of upstream regulatory sequences of unc-14 (pan-neuronal; Ogura et al, 1997) or sre-1 (ADL marker) gene (Fig EV1E; Jang et al, 2012; Gruner et al, 2014; Hong et al, 2017). Pan-neuronal expression or ADL-specific expression of daf-2 cDNA fully rescued the defects of ascr#3 avoidance in daf-2 mutants (Fig 1F). Moreover, the expression of daf-2 cDNA in ADL also restored increased avoidance behaviors to ascr#3 in animals starved for 6 h or 24 h (Fig 1G). Taken together, these observations indicate that DAF-2/insulin-like signaling acts in the ascr#3-sensing ADL neurons to regulate ascr#3 avoidance behaviors in a feeding state-dependent manner. DAF-2 signaling mediates Ca2+ responses, not in the ADL sensory neurons, but in the AVA interneurons upon ascr#3 exposure Previously, works showed that ADL neurons exhibit transient Ca2+ responses to acute ascr#3 exposure in a dose-dependent manner (Jang et al, 2012). Therefore, altered ascr#3 avoidance in daf-2 mutants may be due to decreased ADL Ca2+ responses to ascr#3 exposure. In order to test the hypothesis, we measured the intracellular Ca2+ dynamics of ADL upon ascr#3 exposure using the genetically encoded calcium sensor GCaMP3 (Chronis et al, 2007; Jang et al, 2012). We found that the Ca2+ activities of ADL were unaltered in daf-2 mutants, compared to wild-type animals, when animals were exposed to either 100 nM or 500 nM ascr#3 (Fig 2A). Previously, it was shown that Ca2+ transients of ADL upon ascr#3 exposure were not affected by starvation for 6 h (Gruner et al, 2014). These results indicate that the loss of DAF-2 and starvation for 6 h do not affect sensory response of ADL to ascr#3. Figure 2. DAF-2 affects Ca2+ transients not in the ADL ascr#3-sensing neurons but their downstream target neurons Ca2+ transients of ADL upon ascr#3 exposure in wild-type animals and daf-2 mutants. Ca2+ transients to 100 nM ascr#3 of ADL (left), the average peak percentage changes in fluorescence upon 100 nM ascr#3 exposure (middle), and the dose–response curve of the average peak percentage changes in fluorescence Ca2+ peaks upon 100 and 500 nM ascr#3 exposure (right). n = 7–12. Ca2+ transients of AVA upon 500 nM ascr#3 exposure in wild-type animals, daf-2 mutants, and daf-2 mutants expressing sre-1p::daf-2 cDNA (ADL). Ca2+ transients in response to ascr#3 in AVA (left), the average peak percentage changes in fluorescence upon 100 nM ascr#3 exposure (right). n = 10. ***P < 0.001 (Dunnett's test). Heat-map images of Ca2+ transients in AVA upon 500 nM ascr#3 exposure in wild-type animals (left), daf-2 mutants (middle), and daf-2 mutants expressing sre-1p::daf-2 cDNA (right). Each row represents Ca2+ responses of individual animals to ascr#3 exposure. n = 10. Data information: All error bars represent ± SEM. Download figure Download PowerPoint The ADL sensory neurons form chemical synapses onto the AVA, AVD, and AIB interneurons, which trigger backward movements by activating DA or VA motor neurons (Chalfie et al, 1985; White et al, 1986). The signals from ADL neurons mainly transit to AVA but not AVD and AIB upon ascr#3 exposure (Hong et al, 2017). Thus, we next monitored Ca2+ transients of AVA in daf-2 mutants and found that the Ca2+ transients of AVA upon ascr#3 exposure were abolished in daf-2 mutants. Similar to the ascr#3 avoidance defects, ascr#3 Ca2+ transient defects in daf-2 mutants were fully rescued by daf-2 cDNA expression in ADL (Fig 2B and C), indicating that DAF-2 acts in ADL to regulate ascr#3 responses of the ADL downstream target neurons. DAF-2 signaling regulates chemical synaptic transmission from ADL The finding that loss of DAF-2 function decreases ascr#3 responses in ADL downstream neurons prompted us to examine the effects of DAF-2 signaling on chemical synaptic transmission from ADL. We first examined synaptic densities in the ADL neurons by expressing functional YFP-tagged SNB-1/synaptobrevin, which is a component of SNARE (soluble NSF attachment protein receptor) proteins and has been used as a pre-synaptic marker in C. elegans (Nonet et al, 1998; Shen & Bargmann, 2003; Sieburth et al, 2005; Noma & Jin, 2015). We measured fluorescence intensity of SNB-1::YFP in areas near the nerve ring where ADL and AVA connect each other (White et al, 1986). We found that, in wild-type animals, the expression of SNB-1::YFP was detected in the processes of the ADL neurons close to the nerve ring regions (Figs 3A and B, and EV2A and B). However, the fluorescence level of SNB-1::YFP was significantly decreased in daf-2 mutants (Figs 3A and B, and EV2A and B). We also monitored the expression of RAB-3 Ras GTPase protein, which plays key roles in synaptic vesicle release (Nonet et al, 1997). In wild-type animals, RAB-3 proteins were consistently detected in the soma and processes of the ADL neurons (Figs 3A and C, and EV2C). Similar to SNB-1, the mCherry-tagged RAB-3 level in ADL was also decreased in both the soma and the processes of daf-2 mutants (Figs 3A and C, and EV2C). The promoter activity of the sre-1 gene was not affected by daf-2 mutations and starvation (Fig EV2D and E; Gruner et al, 2014). We further examined the fluorescence level of OCR-2::mCherry in daf-2 mutants. ocr-2 encodes a TRPV channel that acts in the ADL neurons to mediate ascr#3 avoidance (Jang et al, 2012). We found that OCR-2 level was not altered in daf-2 mutants (Fig EV2F). These results suggest that DAF-2 specifically regulates the protein levels of pre-synaptic proteins in the ADL neurons, and then, the reduction in these protein levels in daf-2 mutants leads to the lack of AVA neuronal responses which eventually result in the suppression of the pheromone avoidance behaviors upon ascr#3 exposure. Figure 3. DAF-2 regulates synaptic transmissions in ADL A. Representative images of wild-type animals (left) and daf-2 mutants (right) expressing sre-1p::snb-1 cDNA::yfp (top) and sre-1p::mCherry::rab-3 cDNA (bottom). The scale bar is 10 μm. B, C. Relative fluorescence intensity of wild-type animals and daf-2 mutant animals expressing sre-1p::snb-1 cDNA::yfp (B) and sre-1p::mCherry::rab-3 cDNA (C). n = 59–60. ****P < 0.0001 (unpaired Student's t-test). D. Relative fluorescence intensity of sre-1p::snb-1 cDNA::yfp of wild-type animals in fed and starved conditions for 3, 6, and 24 h. n = 37–80. **P < 0.01 and ****P < 0.0001 (Bonferroni's test). E. Relative fluorescence intensity of sre-1p::snb-1 cDNA::yfp of daf-2 mutants in fed and starved conditions. n = 20–30. F. Fraction reversing of wild-type animals and daf-2 mutant animals expressing sre-1p::TeTx and sre-1p::pkc-1(gf). n = 50–90. *P < 0.05, **P < 0.01, and ***P < 0.001 (Dunnett's test). ++P < 0.01 (unpaired Student's t-test). Data information: All error bars represent ± SEM. Tops and bottoms of boxes indicate the 25th and 75th percentiles, respectively; whiskers represent 10th–90th percentile. Median is indicated by a horizontal line and the average is marked by "+" in the box. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Quantification of SNB-1::YFP and mCherry::RAB-3 and gene expression of sre-1 promoter upon daf-2 mutation or starvation A. Relative fluorescence intensity of integrated animals expressing sre-1p::snb-1 cDNA::yfp. n = 30. ***P < 0.0001 (unpaired Student's t-test). B, C. Quantification of fluorescence intensity of wild-type and daf-2 mutant animals expressing sre-1p::snb-1 cDNA::yfp (B) and sre-1p::mCherry::rab-3 cDNA (C) analyzed using ImageJ software. n = 13–41. ***P < 0.001 (unpaired Student's t-test). D. Relative fluorescence intensity of daf-2 mutants expressing sre-1p::gfp. n = 50. E. Relative fluorescence intensity of transgenic animals expressing sre-1p::mCherry in fed and starved conditions. n = 29–35. F. Relative fluorescence intensity of wild-type and daf-2 mutant animals expressing sre-1p::ocr-2 genome::mcherry. n = 41. Data information: All error bars represent ± SEM. Tops and bottoms of boxes indicate the 25th and 75th percentiles, respectively; whiskers represent 10th–90th percentile. Median is indicated by a horizontal line, and the average is marked by "+" in the box. Download figure Download PowerPoint We then examined the SNB-1::YFP level in starved wild-type animals and found that the SNB-1 level was increased in ADL after 6- and 24-h starvation (Fig 3D). Moreover, daf-2 mutation also suppressed the increase in SNB-1 level in starved wild-type animals (Fig 3E). Together, these results indicate that starvation status may regulate ADL synaptic transmission via the expression levels of synaptic vesicle molecules. Chemical synaptic outputs of ADL mediate avoidance behaviors to ascr#3 (Jang et al, 2012; Hong et al, 2017). Consistent with the previous report, blocking of chemical synaptic transmission from ADL by expressing tetanus toxin light chain (TeTx) decreased ascr#3 avoidance in wild-type animals which appeared not to be further decreased by daf-2 mutation (Fig 3F; Jang et al, 2012). We next increased the chemical synaptic output of ADL by expressing gain-of-function mutation of pkc-1 protein kinase C gene which promotes synaptic vesicle fusion (Sieburth et al, 2007; Tsunozaki et al, 2008). We found that the expression of pkc-1(gf) enhanced avoidance behavior to ascr#3 in wild-type animals (Fig 3F). The daf-2 mutant animals expressing sre-1p::pkc-1(gf) also suppressed the enhancement of ascr#3 avoidance (Fig 3F). These results support that DAF-2 signaling regulates ascr#3 avoidance by modulating the synaptic activity in ADL. Taken together, these results indicate that feeding status and DAF-2 insulin signaling may regulate ADL synaptic transmission via the change in synaptic vesicle protein levels. PI3K/AKT/FOXO act downstream of DAF-2 in ADL to regulate ascr#3 avoidance behavior In canonical insulin signaling, DAF-2 signals through AGE-1 (phosphoinositide 3-kinase), PDK-1 (phosphoinositide-dependent kinase), AKT-1/AKT-2 (Akt/protein kinase B family), DAF-18 (PTEN), and DAF-16/FOXO (Kimura et al, 1997; Ogg & Ruvkun, 1998; Engelman et al, 2006; Murphy & Hu, 2013). We investigated whether these genes affect DAF-2-mediate ascr#3 avoidance behaviors as well as protein levels of SNB-1 in ADL. Similar to daf-2 mutants, age-1 or akt-1 mutants exhibited decreased avoidance to ascr#3 and SNB-1 level in ADL (Fig 4A and B). However, the mutations in akt-2, pdk-1, or daf-18 did not affect ascr#3 avoidance (Fig 4A). Figure 4. PI3K/AKT/FOXO pathway acts downstream of daf-2 signaling in ADL to mediate ascr#3 avoidance Fraction reversing of wild-type animals, age-1 mutants, akt-1 mutants, akt-2 mutants, and pdk-1 mutants in response to 500 nM ascr#3. n = 40–90. *P < 0.05 and **P < 0.01 (Dunnett's test). Relative fluorescence intensity of transgenic animals expressing sre-1p::snb-1 cDNA::yfp, including daf-2 mutants, age-1 mutants, akt-1 mutants, daf-16 mutants, daf-16;daf-2 double mutants, and daf-16 mutants expressing sre-1p::daf-16 cDNA (ADL). n = 37–218. *, **, and **** present different from wild type at P < 0.05, P = 0.01, and P < 0.0001 (Dunnett's test). ++P < 0.01 and ++++P < 0.0001 (unpaired Student's t-test). Tops and bottoms of boxes indicate the 25th and 75th percentiles, respectively; whiskers represent 10th–90th percentile. Median is indicated by a horizontal line, and the average is marked by "+" in the box. Fraction reversing of daf-16 mutant animals in fed and starved conditions in response to ascr#3 exposure. n = 30–40. Fraction reversing of wild-type animals, daf-2 mutants, daf-16 mutants, and daf-16;daf-2 double mutants in response to ascr#3. n = 50–80. *P < 0.05 and **P < 0.01 (Dunnett's test). Fraction reversing of wild-type animals, daf-16 mutants, and daf-16 mutants expressing sre-1p::daf-16 cDNA (ADL). n = 70. *P < 0.05 (Dunnett's test). Data information: All error bars represent ± SEM. Download figure Download PowerPoint We next tested daf-16, a major downstream target of daf-2, and found that the mutation of daf-16 increased ascr#3 avoidance behavior under well-fed conditions (Figs 4C and D, and EV3A). Furthermore, we found that this increased ascr#3 avoidance in daf-16 mutants was maintained under starved conditions (Fig EV3A). SNB-1 level in ADL was increased in daf-16 mutants (Fig 4B), and daf-16;daf-2 double mutants showed increased ascr#3 avoidance and SNB-1 level similar to that seen in daf-16 mutants (Fig 4B and D). The expression of daf-16 cDNA isoform(a) in ADL rescued the ascr#3 avoidance and SNB-1 level phenotypes of daf-16 mutants (Fig 4B and E). These results indicate that daf-16 antagonizes daf-2 function cell autonomously in ADL to regulate ascr#3 avoidance and the expression of ADL synaptic proteins. However, ascr#3 avoidance was unaltered in the mutants of a NRF transcription factor/skn-1, which is another downstream factor to daf-2 (Fig EV3B; Ewald et al, 2015), suggesting a specific role of daf-16 in daf-2-mediated ascr#3 avoidance. Taken together, these results further suggest that the DAF-2/AGE-1/AKT-1/DAF-16 signaling pathway mediates feeding state-dependent modulation of ascr#3 avoidance by regulating synaptic outputs of ADL. Click here to expand this figure. Figure EV3. ascr#3 avoidance behaviors of daf-16 mutants and of skn-1 mutants A, B. Fraction reversing of daf-16 mutants (A) and skn-1 mutants (B) in response to 100, 200, and 400 nM ascr#3 under fed conditions. (A) n = 60, *P < 0.05 (Bonferroni's test). (B) n = 40–50. Data information: All error bars represent ± SEM. Download figure Download PowerPoint Intestinal INS-18 insulin-like peptide modulates ascr#3 avoidance behavior by inhibiting DAF-2 signaling in ADL Next, we sought to identify how feeding status regulates DAF-2 signaling in the ADL sensory neurons. We first searched for ligands for DAF-2 insulin-like receptor by screening a subset of insulin-like peptide (ILPs) mutants including ins-1, ins-7, ins-18, ins-22, ins-32, ins-35, and daf-28 in well-fed conditions. We found that ins-1 and ins-18 mutants showed decreased and increased ascr#3 avoidance, respectively, but other mutants did not exhibit altered ascr#3 avoidance (Fig 5A). We then generated daf-16;ins-1 double mutants and found that these double mutants showed decreased ascr#3 avoidance, comparable to that of ins-1 mutants (Fig 5B), indicating that ins-1 may function in parallel to or downstream of daf-2/daf-16 to regulate ascr#3 avoidance. We next tested daf-2;ins-18 double mutants and found that daf-2 mutation suppressed the increase of ascr#3 avoidance in ins-18 mutants (Fig 5C). In addition, ins-18 mutants showed increased SNB-1 level in ADL, and daf-2 mutation abolished increased SNB-1 level in ins-18 mutants (Fig 5D and E). These results suggest that INS-18 acts upstream of DAF-2 signaling and that INS-18 antagonizes the DAF-2 function. Figure 5. INS-18, which is secreted in the intestine, inhibits DAF-2 signaling in ADL Fraction reversing of insulin-like peptide mutants, ins-1, ins-7, ins-18, ins-22, ins-32, ins-35, and daf-28 in response to ascr#3. n = 40–170. *P < 0.05 and ***P < 0.001 (Dunnett's test). Fraction rever
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