Top

Research Article

Split Viewer

Mol. Cells 2021; 44(7): 529-537

Published online July 31, 2021

https://doi.org/10.14348/molcells.2021.0051

© The Korean Society for Molecular and Cellular Biology

The FMRFamide Neuropeptide FLP-20 Acts as a Systemic Signal for Starvation Responses in Caenorhabditis elegans

Chanhee Kang1,* and Leon Avery2

1School of Biological Sciences, Seoul National University, Seoul 08826, Korea, 2Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA

Correspondence to : chanhee.kang@snu.ac.kr

Received: March 1, 2021; Revised: March 26, 2021; Accepted: April 8, 2021

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Most animals face frequent periods of starvation throughout their entire life and thus need to appropriately adjust their behavior and metabolism during starvation for their survival. Such adaptive responses are regulated by a complex set of systemic signals, including hormones and neuropeptides. While much progress has been made in identifying pathways that regulate nutrient-excessive states, it is still incompletely understood how animals systemically signal their nutrient-deficient states. Here, we showed that the FMRFamide neuropeptide FLP-20 modulates a systemic starvation response in Caenorhabditis elegans. We found that mutation of flp-20 rescued the starvation hypersensitivity of the G protein β-subunit gpb-2 mutants by suppressing excessive autophagy. FLP-20 acted in AIB neurons, where the metabotropic glutamate receptor MGL-2 also functions to modulate a systemic starvation response. Furthermore, FLP-20 modulated starvation-induced fat degradation in a manner dependent on the receptor-type guanylate cyclase GCY-28. Collectively, our results reveal a circuit that senses and signals nutrient-deficient states to modulate a systemic starvation response in multicellular organisms.

Keywords autophagy, metabotropic glutamate receptor, neuropeptide, nutrient-deficient states, receptor-type guanylate cyclase, starvation response, systemic regulation

Since nutrient availability in the environment dramatically fluctuates over time, most animals face frequent periods of starvation throughout their entire life. Animals have evolved a complex layer of regulation in response to starvation, adjusting their behavior and metabolism for survival. Behavioral adjustments include changes in foraging behaviors, which increase the chance of finding new food sources (Douglas et al., 2005; Kniazeva et al., 2015; Wang et al., 2006). Metabolic adjustments are mainly related to the conservation and generation of metabolic substrates and energy, thereby maintaining essential cellular activity for survival (Finn and Dice, 2006; Mizushima, 2007; Wang et al., 2006). These adaptive responses are regulated in both cell-autonomous and non-cell-autonomous manners. The individual cell decreases anabolic pathways, such as general transcription and translation, as well as increasing catabolic pathways, such as protein degradation through the ubiquitin-proteasome system and lysosomal degradation pathways, preserving intracellular metabolites to support its basic functions (Anding and Baehrecke, 2017; Kim et al., 2018; Lee et al., 2020; Miller and Thorburn, 2021; Sebastian and Zorzano, 2020; Shin and Zoncu, 2020). Animals decrease basal metabolic rate by lowering energy-demanding processes, such as reproduction (Gerisch et al., 2020). Furthermore, animals allocate their stored energy to several tissues by increasing lipolysis, which in turn generates internal energy sources to maintain their viability (Olsen et al., 2021; Shin, 2020; Texada et al., 2019). These starvation responses can be coordinated systemically through the highly complex endocrine system which consists of various hormones and neuropeptides (Douglas et al., 2005; Wang et al., 2006).

During the past decade, the molecular mechanisms by which hormones and neuropeptides regulate behavioral and metabolic responses to internal nutrient and energy availability have been extensively studied (Berthoud and Morrison, 2008). For example, the insulin-PI3K and leptin pathways are among the best-characterized endocrine mechanisms by which animals sense and signal their nutrient-excessive states (Davis et al., 2010; Konner and Bruning, 2012). By contrast, no particular hormones or neuropeptides were assigned for signaling nutrient-deficient states until recently. Instead, reductions in the levels of hormones and neuropeptides that modulate energy-excessive states were considered a starvation cue, as best exemplified by the leptin-dependent regulation of feeding during starvation (Chan and Mantzoros, 2005). However, starvation responses might be regulated in a more directed manner by specific hormones or neuropeptides whose expression is increased in response to nutrient-deficient states. Consistent with this notion, recent studies suggest a role for the fibroblast growth factor 21 (FGF21), a hormone secreted from the liver upon starvation, in systemically coordinating starvation responses, including gluconeogenesis, fatty acid oxidation, ketogenesis, lipid degradation, and autophagy (Badman et al., 2007; Byun et al., 2020; Fazeli et al., 2015; Inagaki et al., 2008). However, it is still incompletely understood whether additional factors can act as a systemic signal for starvation and, if so, how they work.

The neuropeptide FMRFamides are the first member of the family of RFamide neuropeptides to be recognized and have been shown to regulate various biological processes, such as cardiovascular function, muscle contraction, locomotor activity, neuroendocrine and neuromodulatory activities (Bechtold and Luckman, 2007; Chen et al., 2016; Cohen et al., 2009; Park et al., 2019). In addition, FMRFamide neuropeptides were suggested to act as part of the nutrient-sensing mechanism in worms, birds, and mice. For example, starvation increases the expression of the FMRFamide-related neuropeptide QRFP in mice, which in turn modulates feeding behavior and energy expenditure (Bechtold and Luckman, 2007; Takayasu et al., 2006). The Caenorhabditis elegans FMRFamide neuropeptide FLP-18 activates two G protein-coupled receptors of the NPY/RFamide family, NPR-4 and NPR-5 to modulate fat metabolism and foraging behavior, suggesting that FLP-18 acts as a nutrient-excessive signal (Cohen et al., 2009). However, it is currently unclear whether other FMRFamide neuropeptides are also involved in the nutrient-sensing mechanism and, if so, whether they can act as a starvation signal in a systemic manner.

We previously showed that MGL-1 and MGL-2, C. elegans homologs of metabotropic glutamate receptors, coordinate a systemic starvation response, such as the lysosomal degradation pathway autophagy, by modulating the activity of AIY and AIB interneurons, respectively (Kang and Avery, 2009a; 2009b). While AIY neurons inhibit a systemic starvation response, AIB neurons stimulate it, suggesting that AIB neurons might secrete a systemic signal, potentially FMRFamide neuropeptides, to modulate starvation responses. However, the identity and in vivo relevance of such a systemic signal, possibly hormones or neuropeptides, were not characterized. Here, using the starvation-hypersensitive gpb-2 mutants in which heterotrimeric Gq protein activity is constitutively active (You et al., 2006), we show that the FMRFamide neuropeptide FLP-20 systemically regulates starvation responses, including starvation survival, autophagy, and starvation-induced fat degradation. FLP-20 is released by AIB neurons and acts on the receptor-type guanylate cyclase GCY-28 to modulate fat degradation. Together, these findings suggest that FLP-20 functions as a systemic signal that directly modulates starvation responses.

Strains

Strains were maintained as described (Brenner, 1974) at 19°C. All worms were maintained and grown on Escherichia coli HB101 bacteria. The following strains were generated using standard genetic procedures: gpb-2(ad541) I, flp-20(pk1596) X, gcy-28(ky713) I, gcy-28(tm2411) I, mgl-2(tm355) I, flp-18(db99) X, gpb-2(ad541) I; mgl-1(tm1811) X, gpb-2(ad541) mgl-2(tm355) I, gpb-2(ad541) I; flp-20(pk1596) X, gcy-28(ky713)gpb-2(ad541) I, gcy-28(tm2411) gpb-2(ad541) I, gpb-2(ad541) I; flp-18(db99) X, gcy-28(tm2411) gpb-2(ad541) I; flp-20(pk1596) X, gcy-28(tm2411) gpb-2(ad541) I; mgl-1(tm1811), gcy-28(tm2411) gpb-2(ad541) mgl-2(tm355) I, gpb-2(ad541) mgl-2(tm355) I; flp-20(pk1596) X, gpb-2(ad541) I; flp-18(db99) mgl-1(tm1811) X, gpb-2(ad541) I; adIs2122[GFP::lgg-1 rol-6(d)], gpb-2(ad541) I; flp-20(pk1596); adIs2122[GFP::lgg-1 rol-6(d)], gcy-28(tm2411) gpb-2(ad541) I; adIs2122[GFP::lgg-1 rol-6(d)], gpb-2(ad541) mgl-2(tm355) I; flp-20(pk1596) X; adIs2122[GFP::lgg-1 rol-6(d)], gpb-2(ad541) I; flp-20(pk1596) X; [pord-2b::flp-20 rol-6(d)], gpb-2(ad541) I; flp-20(pk1596) X; [pnmr-2::flp-20 rol-6(d)].

Molecular biology

cDNA corresponding to the entire coding sequence of flp-20 was amplified and cloned under cell-specific promoters as indicated. Expression in AIB was achieved using the odr-2b promoters, from Cornelia Bargmann (The Rockefeller University, USA).

Polymerase chain reaction (PCR) construction of flp-20 driven by the odr-2b promoter was achieved as follows: the odr-2b promoter region was amplified from a Podr-2b:: mod-1 cDNA::GFP plasmid (from Cornelia Bargmann, The Rockefeller University) using primers 5’GTCTAGTCAGCATTTCACCCTG’ and 5’TTGAGTGTAACCCAACATATTCTGTCTGAAATATAAATGT’ (PCR#1). cDNA corresponding to the entire coding sequence of flp-20 was amplified from yk782e04 (from Yuji Kohara, National Institute of Genetics, Japan) using primers 5’TTATATTTCAGACAGAATATGTTGGGTTACACTCAATCTC’ and 5’AGGGAAGGAAAGTTATAATCTCTAG3’ (PCR#2). PCR#1 and #2 were fused using primers 5’GTCTAGTCAGCATTTCACCCTG’ and 5’AGGGAAGGAAAGTTATAATCTCTAG3’.

PCR construction of flp-20 driven by the nmr-2 promoter was achieved as follows: the nmr-2 promoter region was amplified from Pka155 (from Kaveh Ashrafi, University of California, USA) using primers 5’GAGCCATCAGAATTTATTTGAATTTTC’ and 5’TTGAGTGTAACCCAACATTGATTTTTCGAATAACTTCCTT’ (PCR#1). cDNA corresponding to the entire coding sequence of flp-20 was amplified from yk782e04 (from Yuji Kohara, National Institute of Genetics) using primers 5’AAGTTATTCGAAAAATCAATGTTGGGTTACACTCAATCTC’ and 5’AGGGAAGGAAAGTTATAATCTCTAG3’ (PCR#2). PCR#1 and #2 were fused using primers 5’GAGCCATCAGAATTTATTTGAATTTTC’ and 5’AGGGAAGGAAAGTTATAATCTCTAG3’.

Starvation survival analyses

Starvation survival analyses were performed as described (You et al., 2006) with a few modifications. After collection of L1 worms from synchronization by egg preparation, we incubated them in 3 ml of sterilized M9 buffer for the time indicated in the figures at 19°C. At each time point, an aliquot from each sample tube was placed on a plate seeded with E. coli HB101. The number of worms surviving to L4 or adulthood was determined after 3 days of further growth at 19°C. The number from day 1 of starvation was used as control and as the denominator to calculate the percentage of worms recovering after starvation. Since starvation survival was influenced by assay conditions, all relevant experimental data were examined and compared within the same experiment.

C. elegans autophagy analysis

Autophagy analysis was performed as described (Kang et al., 2007). For light microscopic analysis of autophagy, starved L1 animals carrying an integrated transgene that expresses a GFP::LGG-1 fusion were collected at 3 days of starvation. GFP-positive punctate regions were visualized in the pharyngeal muscles of L1 animals using a Zeiss Axioplan 2 compound microscope (Zeiss, Germany).

Body fat assessment

Body fat analysis using Oil-Red-O staining was performed as described (Soukas et al., 2009) with a few modifications. Briefly, synchronized animals were collected and washed three times with M9 buffer. We incubated them in M9 buffer for the time indicated in the figures at 19°C. At each time point, using an aliquot from each sample tube, Oil-Red-O staining was conducted. The worms were resuspended and washed twice with phosphate-buffered saline (PBS) and then suspended in PBS to which an equal volume of 2× MRWB buffer containing 2% paraformaldehyde was added. The worms were taken through three freeze-thaw cycles between liquid nitrogen and warm water, followed by spinning at 14,000g, washing once in PBS, resuspension in 60% isopropanol, and addition of 60% Oil-Red-O stain for 1 h. To quantify the amount of Oil-Red-O staining, Adobe Photoshop CS5 software was used. For approximate quantification of the levels of Oil-Red-O staining, we categorized stained worms into 3 groups: 1) Full – worms showing strong staining throughout the intestine, 2) Partial – worms showing only staining around the tail region, and 3) None – worms showing no staining.

Statistical analysis was performed using the GraphPad PRISM 9 (GraphPad Software, USA).

To search for the systemic signal secreted by AIB neurons to modulate a systemic starvation response, we checked the expression pattern of neuropeptides with a focus on FMRFamide-related peptides (Kim and Li, 2004; Li and Kim, 2008). FLP-20 is the only one reported to be expressed in AIB neurons. To test whether FLP-20 was involved in the modulation of starvation responses, we examined whether mutation of flp-20 could suppress the starvation-hypersensitivity of gpb-2 mutants in which starvation responses, such as autophagy, are overactivated (Kang et al., 2007; You et al., 2006). Suppression of AIB activity by mutation of mgl-2 suppressed the starvation-induced death of gpb-2 mutants, as previously demonstrated (Kang and Avery, 2009b). Mutation of flp-20 also rescued the death of gpb-2 mutants during starvation, yet did not further suppress the starvation-induced death of gpb-2 mgl-2 mutants (Fig. 1A). These data suggest that FLP-20 acts in the same pathway as MGL-2 to modulate starvation responses. Consistent with this interpretation, AIB-specific expression of flp-20 driven by the promoter of ord-2b, but not AVA, AVB, AVG, RIM, PVC, AVD, and AVE expression of flp-20 (driven by the promoter of nmr-2), restored the starvation sensitivity of gpb-2; flp-20 mutants (Fig. 1B). Mutation of flp-18, which is expressed in AIY neurons that have been also shown to modulate a systemic starvation response (Kang and Avery, 2009b), did not rescue the starvation-survival of gpb-2 mutants, indicating the specificity of FLP-20 as a starvation-regulating FMRFamide-related peptide (Fig. 1C). Together, these data suggest that FLP-20 may act as a systemic signal from AIB neurons downstream of MGL-2 to positively regulate starvation responses.

MGL-1 and MGL-2 sense several amino acids, including leucine, as a food signal to modulate AIY and AIB neurons, respectively. Treatment with leucine suppresses MGL-2, which in turn reduces the activity of AIB neurons and thus decreases a systemic starvation response, including autophagy in the pharyngeal muscles (Kang and Avery, 2009b). While searching for additional regulators of these amino acid responses, we found that mutation of gcy-28, a receptor-type guanylate cyclase (Tsunozaki et al., 2008), abolished the positive effect of leucine on the starvation survival of gpb-2 mutants (Fig. 1D). GCY-28 is known to be expressed in many head neurons, including AWC and AIA neurons that are upstream of AIB neurons, as well as in peripheral tissues, including body-wall muscles, hypodermis, and intestine, which are potential downstream targets of AIB neurons during starvation responses (Ortiz et al., 2006). Thus, we hypothesized that GCY-28 is involved in starvation responses, and may act either upstream or downstream of the MGL-2 and FLP-20 signaling pathway. To test this, we generated gcy-28 gpb-2 mgl-2 and gcy-28 gpb-2; flp-20 mutants. Mutation of gcy-28 restored the starvation sensitivity of gpb-2 mgl-2 and gpb-2; flp-20 mutants, suggesting that GCY-28 acts downstream, but not upstream, of MGL-2 and FLP-20 to modulate starvation responses (Fig. 1E).

To corroborate our finding that FLP-20 and GCY-28 modulate a systemic starvation response, we further focused on their roles in modulating autophagy and fat mobilization, two well-described starvation responses. The main cause of death in gpb-2 mutants during starvation is excessive autophagy in their pharyngeal muscles, and MGL-2 modulates such autophagy in a systemic manner (Kang and Avery, 2008; 2009a; 2009b; Kang et al., 2007). Thus, we asked whether FLP-20 and GCY-28 are also involved in autophagic regulation of the pharyngeal muscles during starvation. We generated mutant strains carrying an integrated LGG-1::GFP transgene, a specific marker for autophagy in C. elegans (Kang et al., 2007; Klionsky et al., 2016; Melendez et al., 2003; Zhang et al., 2015), and found that mutation of flp-20 decreased excessive levels of autophagy in gpb-2 mutants during starvation (Fig. 2). Double mutation of flp-20 and mgl-2 did not show an additive effect on suppressing excessive autophagy in gpb-2 mutants, suggesting that FLP-20 and MGL-2 act in the same pathway to modulate autophagy, consistent with their effects on starvation survival. By contrast, mutation of gcy-28 did not aggravate excessive levels of autophagy (Fig. 2). This could be due to the fact that GCY-28 is not expressed in the pharyngeal muscles (Ortiz et al., 2006). Alternatively, GCY-28 could only abolish the suppressive effect of amino acids on excessive autophagy, as was the case for fat degradation.

Like mammals, C. elegans can store surplus energy as fat and consumes stored fat during starvation to maintain energy homeostasis (Jo et al., 2009; McKay et al., 2003; Van Gilst et al., 2005). Indeed, we found that starvation decreased the levels of fat in wild-type worms, and gpb-2 mutants had an increased rate of fat depletion, as assessed by the Oil-Red-O staining (Fig. 3), a histochemical method for measuring stored fat (Soukas et al., 2009). In addition, gpb-2 mutants already lower their fat deposition under control conditions, compared to wild-type worms, which is consistent with previous observations that they are in a perceived state of starvation (You et al., 2006). These results are consistent with the finding that starvation signaling is overactivated in gpb-2 mutants.

Next, we examined whether MGL-2, FLP-20, and GCY-28 are involved in starvation-induced fat degradation. Since gpb-2 mutation provides a sensitive genetic background to readily observe starvation-induced fat degradation (Fig. 3), we first examine the effect of each mutation on fat degradation during starvation in gpb-2 mutants. We found that mutation of either mgl-2 or flp-20 reduced the rate of fat degradation during starvation, and these effects were not additive (Fig. 4A). These data suggest that MGL-2 and FLP-20 positively regulate starvation-induced fat degradation in the same pathway, as was the case for starvation survival and autophagy regulation (Figs. 1A and Fig. 2). By contrast, mutation of gcy-28 accelerated the rate of fat degradation, and completely reversed the effects of mgl-2 and flp-20 mutations on fat degradation (Fig. 4A). These data suggest that GCY-28 acts downstream of MGL-2 and FLP-20 to modulate starvation-induced fat degradation, consistent with its role in starvation survival (Fig. 1E). Next, we examined whether such regulation also operates in a wild-type genetic background. As wild-type worms are less sensitive to starvation-induced fat degradation, we starved worms for 24 h to assess their fat contents (Fig. 4B). Mutation of gcy-28 exacerbated starvation-induced fat degradation, suggesting that GCY-28 is generally involved in fat regulation during starvation. Together with our previous studies, these data suggest that MGL-2 positively regulates the activity of AIB neurons, which in turn secrets FLP-20 that acts on GCY-28 in peripheral tissues to modulate a systemic starvation response, including starvation survival, autophagy, and fat degradation (Fig. 4C).

Although physiological and behavioral effects of the starvation response have been widely studied (Douglas et al., 2005; Finn and Dice, 2006; Wang et al., 2006), little is known about the systemic signals that directly modulate starvation responses in multicellular organisms. Previously, it has been proposed that reduction in the levels of leptin, in response to nutrient-excessive states, is a signal of nutrient-deficiency and induces starvation responses, including increased levels of food intake (Ahima et al., 1996; Chan and Mantzoros, 2005; Chan et al., 2003). However, leptin-deficient ob/ob mice did not perfectly mimic the starvation state, as exemplified by their normal levels of proteolysis in skeletal muscles (Turpin et al., 2009). Thus, it is reasonable to propose that specific hormones or neuropeptides that increase their expression under nutrient-deficient conditions positively regulate starvation responses, including autophagy that is key in maintaining cellular homeostasis during stress responses including starvation (Cho et al., 2020; Kim et al., 2018; 2021; Kwon et al., 2017; Lee et al., 2021; Molinari, 2021; Sebastian and Zorzano, 2020; Zachari et al., 2019). In fact, recent studies suggest that FGF21 is a starvation-regulating hormone, which is induced under nutrient-deficient conditions and positively regulates a collection of starvation responses, such as growth inhibition, tissue breakdown, autophagy, and lipid degradation (Badman et al., 2007; Byun et al., 2020; Fazeli et al., 2015; Inagaki et al., 2008). We identified the FMRFamide neuropeptide FLP-20 as a systemic starvation signal in C. elegans that positively regulates starvation responses, including starvation survival, autophagy, and starvation-induced fat degradation, echoing the discovery of FGF21 in mammals. Since, however, mutation of flp-20 could not fully rescue starvation-induced responses, it is likely that FLP-20 acts in parallel with other systemic signals, potentially originating from AIY neurons that have been previously shown to modulate starvation responses together with AIB neurons (Kang and Avery, 2009a; 2009b).

GCY-28 is a receptor-type guanylate cyclase, which has been previously shown to modulate chemotaxis (Tsunozaki et al., 2008). GCY-28 is expressed in various tissues, including neurons, body-wall muscles, hypodermis, and intestine, suggesting that its ligand(s) should be a cue of general interest to many tissues. However, the ligand(s) of GCY-28 has not been identified yet (Ortiz et al., 2006; Tsunozaki et al., 2008). Previous studies suggested that gcy-28 mutants seem to exist in a state of perceived starvation, based on the phenotype of avoidance of AWCon-sensed odors (Tsunozaki et al., 2008). Our results showing that mutation of gcy-28 is epistatic to mutation of flp-20 in starvation survival and starvation-induced fat degradation suggest the possibility that FLP-20 may be a ligand for GCY-28, possibly acting as an antagonist. It will be interesting to test this possibility using a heterologous expression system. In addition, further experiments are necessary to elucidate where and how GCY-28 acts to modulate starvation responses. Our starvation hypersensitive model system might be helpful to elucidate such molecular mechanisms.

Starvation also induces behavioral changes in animals (e.g., changes in foraging behavior), which contribute to increasing the chance of locating new food sources (Douglas et al., 2005; Wang et al., 2006). Foraging behaviors are often coordinately regulated with metabolic changes in various organisms, including C. elegans (Cohen et al., 2009; Greer et al., 2008; Srinivasan et al., 2008), suggesting the possibility that FLP-20 and GCY-28 may regulate behavioral changes in response to starvation as well. Indeed, we found that mutations of flp-20 and gcy-28 oppositely affect feeding rates of worms during prolonged starvation (Fig. 4D). Further experiments are needed to elucidate the circuit regulating this starvation-regulated feeding behavior.

Interestingly, after 24 h starvation, a portion of wild-type worms (~36%; Fig. 4B) still retained the high levels of fat. It was recently reported that starvation induces adult reproductive diapause in approximately 34% of worms (Angelo and Van Gilst, 2009). Given the fact that fat accumulation is important for another developmental diapause, dauer entry in C. elegans (Fielenbach and Antebi, 2008), our observation leads to the intriguing possibility that fat retention during starvation is critical for adult reproductive diapause. It would be interesting to test whether gcy-28 mutants, very few of which retained high levels of fat during starvation (Fig. 4B), show defects in entering adult reproductive diapause.

In summary, our data suggest that the FMRFamide neuropeptide FLP-20, probably secreted from AIB in an MGL-2 dependent manner, acts as a systemic signal for starvation responses through the receptor-type guanylate cyclase GCY-28. Thus, this study strongly indicates a specific neuroendocrine circuit that senses and signals nutrient-deficient states to modulate a systemic starvation response in multicellular organisms.

We thank Scott Cameron, Beth Levine, Melanie Cobb, Alexander Soukas, Merav Cohen, Niels Ringstad, members of the Avery and Cameron laboratories for helpful discussions, Cori Bargmann, Kevin Ashrafi, Maureen Barr, Mario De Bono, Merav Cohen for providing strains and plasmids, the C. elegans Genetics Center and S. Mitani (National Bioresource Project) for providing strains, Yuji Kohara (National Institute of Genetics, Japan) for providing flp-20 cDNA. We are indebted to Cori Bargmann for the discussion about GCY-28 and for sharing unpublished data. This work was supported by grants from the POSCO Science Fellowship of POSCO TJ Park Foundation, the SNU invitation program for the distinguished scholar, and the U.S. Public Health Service (HL46154).

C.K. and L.A. conceived and designed the project. C.K. performed all experiments, and C.K. and L.A. analyzed the data. C.K. and L.A. wrote the manuscript.

The authors have no potential conflicts of interest to disclose.

Fig. 1. FLP-20 and GCY-28 modulate the starvation hypersensitivity of gpb-2 mutants. (A) FLP-20 and MGL-2 modulate starvation responses in the same pathway. Mutation of flp-20 and mgl-2 rescued the starvation-induced death of gpb-2 mutants (n = 3, mean ± SEM, one-way ANOVA test). n.s., not significant. (B) FLP-20 mainly acts in AIB neurons. AIB specific expression of flp-20 with the promoter of ord-2b restored the starvation-sensitivity of gpb-2; flp-20 (n = 3, mean ± SEM, two-way ANOVA test). (C) Mutation of flp-18 did not rescue the starvation hypersensitivity of gpb-2 and gpb-2; mgl-1 mutants (n = 2, mean ± SEM, one-way ANOVA test). (D) GCY-28 is involved in the amino acid sensing pathway. Mutation of gcy-28 abolished the rescue effect of leucine on the starvation hypersensitivity of gpb-2 mutants (n = 4-6, mean ± SEM, two-way ANOVA test). (E) GCY-28 acts downstream of MGL-2 and FLP-20 to modulate starvation responses. Mutation of gcy-28 restored the starvation sensitivity of gpb-2 mgl-2 and gpb-2; flp-20 mutants (n = 3-4, mean ± SEM, one-way ANOVA test).
Fig. 2. FLP-20 modulates autophagy in the pharyngeal muscle of gpb-2 mutants during starvation. (A) Representative images of the indicated genotype after 3 days of starvation. The arrows show representative GFP::LGG-1 positive punctate structures. GFP::LGG-1 labels pre-autophagosomal and autophagosomal structures. In the inset, the area marked by the box is magnified. (B) Quantification of autophagy in the pharyngeal muscle of worms of the indicated genotype after 3 days of starvation (n = 116-147, chi-square test). n.s., not significant.
Fig. 3. gpb-2 mutants exhibit an increased rate of fat degradation during starvation. (A) Representative images of Oil-Red-O staining in N2 (wild-type) worms and gpb-2 mutants (top). Violin plot analysis of Oil-Red-O staining (bottom, the horizontal solid line shows the median and the horizontal dot lines show the 1st and 3rd quartiles, one-way ANOVA test). n.s., not significant. (B) Quantification of Oil-Red-O staining. (Full) worms showing strong staining throughout the intestine; (Partial) worms showing staining only around the tail region; (None) worms showing no staining (n = 62-370).
Fig. 4. FLP-20, MGL-2, and GCY-28 modulate starvation-induced fat degradation. (A) Quantification of Oil-Red-O staining of the indicated genotype with or without 4 h starvation. Mutation of either flp-20 or mgl-2 decreased the rate of starvation-induced fat degradation, and mutation of gcy-28 reversed it (left, n = 214-446 for gpb-2, n = 243-265 for gpb-2; flp-20, n = 53-71 for gpb-2 mgl-2, n = 88-142 for gpb-2 mgl-2; flp-20, n = 27-39 for gcy-28 gpb-2, n = 109-156 for gcy-28 gpb-2; flp-20, and n = 16-20 for gcy-28 gpb-2 mgl-2, chi-square test). n.s., not significant. (B) Quantification of Oil-Red-O staining of the indicated genotype with or without 24 h starvation. gcy-28 mutants increased the rate of starvation-induced fat degradation (right, n = 171-407 for N2, n = 221-389 for flp-20, n = 196-229 for mgl-2, and n = 248-365 for gcy-28, chi-square test). (C) Model of the systemic regulation of starvation responses by the MGL-2–FLP-20–GCY-28 axis. Amino acids or food signals modulates the activity of AIB neurons through MGL-2, which in turn stimulates the secretion of FLP-20. FLP-20 acts on GCY-28 that suppresses starvation responses, including autophagy and fat degradation in the peripheral tissues. (D) Pumping rates of the indicated genotype with or without starvation. Unstarved or 4-day starved L1 animals were examined on food. Each dot represents an individual worm. Horizontal lines represent the average (line) and SEM (error bars).
  1. Ahima R.S., Prabakaran D., Mantzoros C., Qu D., Lowell B., Maratos-Flier E., and Flier J.S. (1996). Role of leptin in the neuroendocrine response to fasting. Nature 382, 250-252.
    Pubmed CrossRef
  2. Anding A.L. and Baehrecke E.H. (2017). Cleaning house: selective autophagy of organelles. Dev. Cell 41, 10-22.
    Pubmed KoreaMed CrossRef
  3. Angelo G. and Van Gilst M.R. (2009). Starvation protects germline stem cells and extends reproductive longevity in C. elegans. Science 326, 954-958.
    CrossRef
  4. Badman M.K., Pissios P., Kennedy A.R., Koukos G., Flier J.S., and Maratos-Flier E. (2007). Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states. Cell Metab. 5, 426-437.
    Pubmed CrossRef
  5. Bechtold D.A. and Luckman S.M. (2007). The role of RFamide peptides in feeding. J. Endocrinol. 192, 3-15.
    Pubmed CrossRef
  6. Berthoud H.R. and Morrison C. (2008). The brain, appetite, and obesity. Annu. Rev. Psychol. 59, 55-92.
    Pubmed CrossRef
  7. Brenner S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.
    CrossRef
  8. Byun S., Seok S., Kim Y.C., Zhang Y., Yau P., Iwamori N., Xu H.E., Ma J., Kemper B., and Kemper J.K. (2020). Fasting-induced FGF21 signaling activates hepatic autophagy and lipid degradation via JMJD3 histone demethylase. Nat. Commun. 11, 807.
    Pubmed KoreaMed CrossRef
  9. Chan J.L., Heist K., DePaoli A.M., Veldhuis J.D., and Mantzoros C.S. (2003). The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men. J. Clin. Invest. 111, 1409-1421.
    Pubmed KoreaMed CrossRef
  10. Chan J.L. and Mantzoros C.S. (2005). Role of leptin in energy-deprivation states: normal human physiology and clinical implications for hypothalamic amenorrhoea and anorexia nervosa. Lancet 366, 74-85.
    Pubmed CrossRef
  11. Chen Y.C., Chen H.J., Tseng W.C., Hsu J.M., Huang T.T., Chen C.H., and Pan C.L. (2016). A C. elegans thermosensory circuit regulates longevity through crh-1/CREB-dependent flp-6 neuropeptide signaling. Dev. Cell 39, 209-223.
    Pubmed CrossRef
  12. Cho D.H., Kim J.K., and Jo E.K. (2020). Mitophagy and innate immunity in infection. Mol. Cells 43, 10-22.
    Pubmed KoreaMed CrossRef
  13. Cohen M., Reale V., Olofsson B., Knights A., Evans P., and de Bono M. (2009). Coordinated regulation of foraging and metabolism in C. elegans by RFamide neuropeptide signaling. Cell Metab. 9, 375-385.
    Pubmed CrossRef
  14. Davis J.F., Choi D.L., and Benoit S.C. (2010). Insulin, leptin and reward. Trends Endocrinol. Metab. 21, 68-74.
    Pubmed KoreaMed CrossRef
  15. Douglas S.J., Dawson-Scully K., and Sokolowski M.B. (2005). The neurogenetics and evolution of food-related behaviour. Trends Neurosci. 28, 644-652.
    Pubmed CrossRef
  16. Fazeli P.K., Lun M., Kim S.M., Bredella M.A., Wright S., Zhang Y., Lee H., Catana C., Klibanski A., and Patwari P., et al. (2015). FGF21 and the late adaptive response to starvation in humans. J. Clin. Invest. 125, 4601-4611.
    Pubmed KoreaMed CrossRef
  17. Fielenbach N. and Antebi A. (2008). C. elegans dauer formation and the molecular basis of plasticity. Genes Dev. 22, 2149-2165.
    CrossRef
  18. Finn P.F. and Dice J.F. (2006). Proteolytic and lipolytic responses to starvation. Nutrition 22, 830-844.
    Pubmed CrossRef
  19. Gerisch B., Tharyan R.G., Mak J., Denzel S.I., Popkes-van Oepen T., Henn N., and Antebi A. (2020). HLH-30/TFEB is a master regulator of reproductive quiescence. Dev. Cell 53, 316-329.e5.
    Pubmed CrossRef
  20. Greer E.R., Perez C.L., Van Gilst M.R., Lee B.H., and Ashrafi K. (2008). Neural and molecular dissection of a C. elegans sensory circuit that regulates fat and feeding. Cell Metab. 8, 118-131.
    Pubmed KoreaMed CrossRef
  21. Inagaki T., Lin V.Y., Goetz R., Mohammadi M., Mangelsdorf D.J., and Kliewer S.A. (2008). Inhibition of growth hormone signaling by the fasting-induced hormone FGF21. Cell Metab. 8, 77-83.
    Pubmed KoreaMed CrossRef
  22. Jo H., Shim J., Lee J.H., Lee J., and Kim J.B. (2009). IRE-1 and HSP-4 contribute to energy homeostasis via fasting-induced lipases in C. elegans. Cell Metab. 9, 440-448.
  23. Kang C. and Avery L. (2008). To be or not to be, the level of autophagy is the question: dual roles of autophagy in the survival response to starvation. Autophagy 4, 82-84.
    Pubmed KoreaMed CrossRef
  24. Kang C. and Avery L. (2009a). Systemic regulation of autophagy in Caenorhabditis elegans. Autophagy 5, 565-566.
    CrossRef
  25. Kang C. and Avery L. (2009b). Systemic regulation of starvation response in Caenorhabditis elegans. Genes Dev. 23, 12-17.
    CrossRef
  26. Kang C., You Y.J., and Avery L. (2007). Dual roles of autophagy in the survival of Caenorhabditis elegans during starvation. Genes Dev. 21, 2161-2171.
    Pubmed KoreaMed CrossRef
  27. Kim J., Lee Y., Roh K., Kim M.S., and Kang C. (2021). Targeting the stress support network regulated by autophagy and senescence for cancer treatment. Adv. Cancer Res. 150, 75-112.
    Pubmed CrossRef
  28. Kim J., Lim Y.M., and Lee M.S. (2018). The role of autophagy in systemic metabolism and human-type diabetes. Mol. Cells 41, 11-17.
    Pubmed KoreaMed CrossRef
  29. Kim K. and Li C. (2004). Expression and regulation of an FMRFamide-related neuropeptide gene family in Caenorhabditis elegans. J. Comp. Neurol. 475, 540-550.
    CrossRef
  30. Klionsky D.J., Abdelmohsen K., Abe A., Abedin M.J., Abeliovich H., Acevedo Arozena A., Adachi H., Adams C.M., Adams P.D., and Adeli K., et al. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1-222.
    Pubmed KoreaMed CrossRef
  31. Kniazeva M., Zhu H., Sewell A.K., and Han M. (2015). A lipid-TORC1 pathway promotes neuronal development and foraging behavior under both fed and fasted conditions in C. elegans. Dev. Cell 33, 260-271.
    CrossRef
  32. Konner A.C. and Bruning J.C. (2012). Selective insulin and leptin resistance in metabolic disorders. Cell Metab. 16, 144-152.
    Pubmed CrossRef
  33. Kwon Y., Kim J.W., Jeoung J.A., Kim M.S., and Kang C. (2017). Autophagy is pro-senescence when seen in close-up, but anti-senescence in long-shot. Mol. Cells 40, 607-612.
    Pubmed KoreaMed CrossRef
  34. Lee C., Lamech L., Johns E., and Overholtzer M. (2020). Selective lysosome membrane turnover is induced by nutrient starvation. Dev. Cell 55, 289-297.e4.
    Pubmed CrossRef
  35. Lee Y., Kim J., Kim M.S., Kwon Y., Shin S., Yi H., Kim H., Chang M.J., Chang C.B., and Kang S.B., et al. (2021). Coordinate regulation of the senescent state by selective autophagy. Dev. Cell 56, 1512-1525.e7.
    Pubmed CrossRef
  36. Li C. and Kim K. (2008). Neuropeptides. . https://doi.org/10.1895/wormbook.1.142.1.
    CrossRef
  37. McKay R.M., McKay J.P., Avery L., and Graff J.M. (2003). C elegans: a model for exploring the genetics of fat storage. Dev. Cell 4, 131-142.
    CrossRef
  38. Melendez A., Talloczy Z., Seaman M., Eskelinen E.L., Hall D.H., and Levine B. (2003). Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301, 1387-1391.
    CrossRef
  39. Miller D.R. and Thorburn A. (2021). Autophagy and organelle homeostasis in cancer. Dev. Cell 56, 906-918.
    Pubmed KoreaMed CrossRef
  40. Mizushima N. (2007). Autophagy: process and function. Genes Dev. 21, 2861-2873.
    Pubmed CrossRef
  41. Molinari M. (2021). ER-phagy responses in yeast, plants, and mammalian cells and their crosstalk with UPR and ERAD. Dev. Cell 56, 949-966.
    Pubmed CrossRef
  42. Olsen L., Thum E., and Rohner N. (2021). Lipid metabolism in adaptation to extreme nutritional challenges. Dev. Cell . 2021 Mar 9 [Epub]. https://doi.org/10.1016/j.devcel.2021.02.024.
    Pubmed CrossRef
  43. Ortiz C.O., Etchberger J.F., Posy S.L., Frokjaer-Jensen C., Lockery S., Honig B., and Hobert O. (2006). Searching for neuronal left/right asymmetry: genomewide analysis of nematode receptor-type guanylyl cyclases. Genetics 173, 131-149.
    Pubmed KoreaMed CrossRef
  44. Park J., Choi W., Dar A.R., Butcher R.A., and Kim K. (2019). Neuropeptide signaling regulates pheromone-mediated gene expression of a chemoreceptor gene in C. elegans. Mol. Cells 42, 28-35.
  45. Sebastian D. and Zorzano A. (2020). Self-eating for muscle fitness: autophagy in the control of energy metabolism. Dev. Cell 54, 268-281.
    Pubmed CrossRef
  46. Shin D.W. (2020). Lipophagy: molecular mechanisms and implications in metabolic disorders. Mol. Cells 43, 686-693.
    Pubmed KoreaMed CrossRef
  47. Shin H.R. and Zoncu R. (2020). The lysosome at the intersection of cellular growth and destruction. Dev. Cell 54, 226-238.
    Pubmed KoreaMed CrossRef
  48. Soukas A.A., Kane E.A., Carr C.E., Melo J.A., and Ruvkun G. (2009). Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes Dev. 23, 496-511.
    CrossRef
  49. Srinivasan S., Sadegh L., Elle I.C., Christensen A.G., Faergeman N.J., and Ashrafi K. (2008). Serotonin regulates C. elegans fat and feeding through independent molecular mechanisms. Cell Metab. 7, 533-544.
    Pubmed KoreaMed CrossRef
  50. Takayasu S., Sakurai T., Iwasaki S., Teranishi H., Yamanaka A., Williams S.C., Iguchi H., Kawasawa Y.I., Ikeda Y., and Sakakibara I., et al. (2006). A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 7438-7443.
    Pubmed KoreaMed CrossRef
  51. Texada M.J., Malita A., Christensen C.F., Dall K.B., Faergeman N.J., Nagy S., Halberg K.A., and Rewitz K. (2019). Autophagy-mediated cholesterol trafficking controls steroid production. Dev. Cell 48, 659-671.e4.
    Pubmed CrossRef
  52. Tsunozaki M., Chalasani S.H., and Bargmann C.I. (2008). A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans. Neuron 59, 959-971.
    CrossRef
  53. Turpin S.M., Ryall J.G., Southgate R., Darby I., Hevener A.L., Febbraio M.A., Kemp B.E., Lynch G.S., and Watt M.J. (2009). Examination of 'lipotoxicity' in skeletal muscle of high-fat fed and ob/ob mice. J. Physiol. 587, 1593-1605.
    Pubmed KoreaMed CrossRef
  54. Van Gilst M.R., Hadjivassiliou H., and Yamamoto K.R. (2005). A Caenorhabditis elegans nutrient response system partially dependent on nuclear receptor NHR-49. Proc. Natl. Acad. Sci. U. S. A. 102, 13496-13501.
    Pubmed KoreaMed CrossRef
  55. Wang T., Hung C.C., and Randall D.J. (2006). The comparative physiology of food deprivation: from feast to famine. Annu. Rev. Physiol. 68, 223-251.
    Pubmed CrossRef
  56. You Y.J., Kim J., Cobb M., and Avery L. (2006). Starvation activates MAP kinase through the muscarinic acetylcholine pathway in Caenorhabditis elegans pharynx. Cell Metab. 3, 237-245.
    Pubmed KoreaMed CrossRef
  57. Zachari M., Gudmundsson S.R., Li Z., Manifava M., Cugliandolo F., Shah R., Smith M., Stronge J., Karanasios E., and Piunti C., et al. (2019). Selective autophagy of mitochondria on a ubiquitin-endoplasmic-reticulum platform. Dev. Cell 50, 627-643.e5.
    Pubmed KoreaMed CrossRef
  58. Zhang H., Chang J.T., Guo B., Hansen M., Jia K., Kovacs A.L., Kumsta C., Lapierre L.R., Legouis R., and Lin L., et al. (2015). Guidelines for monitoring autophagy in Caenorhabditis elegans. Autophagy 11, 9-27.

Article

Research Article

Mol. Cells 2021; 44(7): 529-537

Published online July 31, 2021 https://doi.org/10.14348/molcells.2021.0051

Copyright © The Korean Society for Molecular and Cellular Biology.

The FMRFamide Neuropeptide FLP-20 Acts as a Systemic Signal for Starvation Responses in Caenorhabditis elegans

Chanhee Kang1,* and Leon Avery2

1School of Biological Sciences, Seoul National University, Seoul 08826, Korea, 2Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA

Correspondence to:chanhee.kang@snu.ac.kr

Received: March 1, 2021; Revised: March 26, 2021; Accepted: April 8, 2021

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Abstract

Most animals face frequent periods of starvation throughout their entire life and thus need to appropriately adjust their behavior and metabolism during starvation for their survival. Such adaptive responses are regulated by a complex set of systemic signals, including hormones and neuropeptides. While much progress has been made in identifying pathways that regulate nutrient-excessive states, it is still incompletely understood how animals systemically signal their nutrient-deficient states. Here, we showed that the FMRFamide neuropeptide FLP-20 modulates a systemic starvation response in Caenorhabditis elegans. We found that mutation of flp-20 rescued the starvation hypersensitivity of the G protein β-subunit gpb-2 mutants by suppressing excessive autophagy. FLP-20 acted in AIB neurons, where the metabotropic glutamate receptor MGL-2 also functions to modulate a systemic starvation response. Furthermore, FLP-20 modulated starvation-induced fat degradation in a manner dependent on the receptor-type guanylate cyclase GCY-28. Collectively, our results reveal a circuit that senses and signals nutrient-deficient states to modulate a systemic starvation response in multicellular organisms.

Keywords: autophagy, metabotropic glutamate receptor, neuropeptide, nutrient-deficient states, receptor-type guanylate cyclase, starvation response, systemic regulation

INTRODUCTION

Since nutrient availability in the environment dramatically fluctuates over time, most animals face frequent periods of starvation throughout their entire life. Animals have evolved a complex layer of regulation in response to starvation, adjusting their behavior and metabolism for survival. Behavioral adjustments include changes in foraging behaviors, which increase the chance of finding new food sources (Douglas et al., 2005; Kniazeva et al., 2015; Wang et al., 2006). Metabolic adjustments are mainly related to the conservation and generation of metabolic substrates and energy, thereby maintaining essential cellular activity for survival (Finn and Dice, 2006; Mizushima, 2007; Wang et al., 2006). These adaptive responses are regulated in both cell-autonomous and non-cell-autonomous manners. The individual cell decreases anabolic pathways, such as general transcription and translation, as well as increasing catabolic pathways, such as protein degradation through the ubiquitin-proteasome system and lysosomal degradation pathways, preserving intracellular metabolites to support its basic functions (Anding and Baehrecke, 2017; Kim et al., 2018; Lee et al., 2020; Miller and Thorburn, 2021; Sebastian and Zorzano, 2020; Shin and Zoncu, 2020). Animals decrease basal metabolic rate by lowering energy-demanding processes, such as reproduction (Gerisch et al., 2020). Furthermore, animals allocate their stored energy to several tissues by increasing lipolysis, which in turn generates internal energy sources to maintain their viability (Olsen et al., 2021; Shin, 2020; Texada et al., 2019). These starvation responses can be coordinated systemically through the highly complex endocrine system which consists of various hormones and neuropeptides (Douglas et al., 2005; Wang et al., 2006).

During the past decade, the molecular mechanisms by which hormones and neuropeptides regulate behavioral and metabolic responses to internal nutrient and energy availability have been extensively studied (Berthoud and Morrison, 2008). For example, the insulin-PI3K and leptin pathways are among the best-characterized endocrine mechanisms by which animals sense and signal their nutrient-excessive states (Davis et al., 2010; Konner and Bruning, 2012). By contrast, no particular hormones or neuropeptides were assigned for signaling nutrient-deficient states until recently. Instead, reductions in the levels of hormones and neuropeptides that modulate energy-excessive states were considered a starvation cue, as best exemplified by the leptin-dependent regulation of feeding during starvation (Chan and Mantzoros, 2005). However, starvation responses might be regulated in a more directed manner by specific hormones or neuropeptides whose expression is increased in response to nutrient-deficient states. Consistent with this notion, recent studies suggest a role for the fibroblast growth factor 21 (FGF21), a hormone secreted from the liver upon starvation, in systemically coordinating starvation responses, including gluconeogenesis, fatty acid oxidation, ketogenesis, lipid degradation, and autophagy (Badman et al., 2007; Byun et al., 2020; Fazeli et al., 2015; Inagaki et al., 2008). However, it is still incompletely understood whether additional factors can act as a systemic signal for starvation and, if so, how they work.

The neuropeptide FMRFamides are the first member of the family of RFamide neuropeptides to be recognized and have been shown to regulate various biological processes, such as cardiovascular function, muscle contraction, locomotor activity, neuroendocrine and neuromodulatory activities (Bechtold and Luckman, 2007; Chen et al., 2016; Cohen et al., 2009; Park et al., 2019). In addition, FMRFamide neuropeptides were suggested to act as part of the nutrient-sensing mechanism in worms, birds, and mice. For example, starvation increases the expression of the FMRFamide-related neuropeptide QRFP in mice, which in turn modulates feeding behavior and energy expenditure (Bechtold and Luckman, 2007; Takayasu et al., 2006). The Caenorhabditis elegans FMRFamide neuropeptide FLP-18 activates two G protein-coupled receptors of the NPY/RFamide family, NPR-4 and NPR-5 to modulate fat metabolism and foraging behavior, suggesting that FLP-18 acts as a nutrient-excessive signal (Cohen et al., 2009). However, it is currently unclear whether other FMRFamide neuropeptides are also involved in the nutrient-sensing mechanism and, if so, whether they can act as a starvation signal in a systemic manner.

We previously showed that MGL-1 and MGL-2, C. elegans homologs of metabotropic glutamate receptors, coordinate a systemic starvation response, such as the lysosomal degradation pathway autophagy, by modulating the activity of AIY and AIB interneurons, respectively (Kang and Avery, 2009a; 2009b). While AIY neurons inhibit a systemic starvation response, AIB neurons stimulate it, suggesting that AIB neurons might secrete a systemic signal, potentially FMRFamide neuropeptides, to modulate starvation responses. However, the identity and in vivo relevance of such a systemic signal, possibly hormones or neuropeptides, were not characterized. Here, using the starvation-hypersensitive gpb-2 mutants in which heterotrimeric Gq protein activity is constitutively active (You et al., 2006), we show that the FMRFamide neuropeptide FLP-20 systemically regulates starvation responses, including starvation survival, autophagy, and starvation-induced fat degradation. FLP-20 is released by AIB neurons and acts on the receptor-type guanylate cyclase GCY-28 to modulate fat degradation. Together, these findings suggest that FLP-20 functions as a systemic signal that directly modulates starvation responses.

MATERIALS AND METHODS

Strains

Strains were maintained as described (Brenner, 1974) at 19°C. All worms were maintained and grown on Escherichia coli HB101 bacteria. The following strains were generated using standard genetic procedures: gpb-2(ad541) I, flp-20(pk1596) X, gcy-28(ky713) I, gcy-28(tm2411) I, mgl-2(tm355) I, flp-18(db99) X, gpb-2(ad541) I; mgl-1(tm1811) X, gpb-2(ad541) mgl-2(tm355) I, gpb-2(ad541) I; flp-20(pk1596) X, gcy-28(ky713)gpb-2(ad541) I, gcy-28(tm2411) gpb-2(ad541) I, gpb-2(ad541) I; flp-18(db99) X, gcy-28(tm2411) gpb-2(ad541) I; flp-20(pk1596) X, gcy-28(tm2411) gpb-2(ad541) I; mgl-1(tm1811), gcy-28(tm2411) gpb-2(ad541) mgl-2(tm355) I, gpb-2(ad541) mgl-2(tm355) I; flp-20(pk1596) X, gpb-2(ad541) I; flp-18(db99) mgl-1(tm1811) X, gpb-2(ad541) I; adIs2122[GFP::lgg-1 rol-6(d)], gpb-2(ad541) I; flp-20(pk1596); adIs2122[GFP::lgg-1 rol-6(d)], gcy-28(tm2411) gpb-2(ad541) I; adIs2122[GFP::lgg-1 rol-6(d)], gpb-2(ad541) mgl-2(tm355) I; flp-20(pk1596) X; adIs2122[GFP::lgg-1 rol-6(d)], gpb-2(ad541) I; flp-20(pk1596) X; [pord-2b::flp-20 rol-6(d)], gpb-2(ad541) I; flp-20(pk1596) X; [pnmr-2::flp-20 rol-6(d)].

Molecular biology

cDNA corresponding to the entire coding sequence of flp-20 was amplified and cloned under cell-specific promoters as indicated. Expression in AIB was achieved using the odr-2b promoters, from Cornelia Bargmann (The Rockefeller University, USA).

Polymerase chain reaction (PCR) construction of flp-20 driven by the odr-2b promoter was achieved as follows: the odr-2b promoter region was amplified from a Podr-2b:: mod-1 cDNA::GFP plasmid (from Cornelia Bargmann, The Rockefeller University) using primers 5’GTCTAGTCAGCATTTCACCCTG’ and 5’TTGAGTGTAACCCAACATATTCTGTCTGAAATATAAATGT’ (PCR#1). cDNA corresponding to the entire coding sequence of flp-20 was amplified from yk782e04 (from Yuji Kohara, National Institute of Genetics, Japan) using primers 5’TTATATTTCAGACAGAATATGTTGGGTTACACTCAATCTC’ and 5’AGGGAAGGAAAGTTATAATCTCTAG3’ (PCR#2). PCR#1 and #2 were fused using primers 5’GTCTAGTCAGCATTTCACCCTG’ and 5’AGGGAAGGAAAGTTATAATCTCTAG3’.

PCR construction of flp-20 driven by the nmr-2 promoter was achieved as follows: the nmr-2 promoter region was amplified from Pka155 (from Kaveh Ashrafi, University of California, USA) using primers 5’GAGCCATCAGAATTTATTTGAATTTTC’ and 5’TTGAGTGTAACCCAACATTGATTTTTCGAATAACTTCCTT’ (PCR#1). cDNA corresponding to the entire coding sequence of flp-20 was amplified from yk782e04 (from Yuji Kohara, National Institute of Genetics) using primers 5’AAGTTATTCGAAAAATCAATGTTGGGTTACACTCAATCTC’ and 5’AGGGAAGGAAAGTTATAATCTCTAG3’ (PCR#2). PCR#1 and #2 were fused using primers 5’GAGCCATCAGAATTTATTTGAATTTTC’ and 5’AGGGAAGGAAAGTTATAATCTCTAG3’.

Starvation survival analyses

Starvation survival analyses were performed as described (You et al., 2006) with a few modifications. After collection of L1 worms from synchronization by egg preparation, we incubated them in 3 ml of sterilized M9 buffer for the time indicated in the figures at 19°C. At each time point, an aliquot from each sample tube was placed on a plate seeded with E. coli HB101. The number of worms surviving to L4 or adulthood was determined after 3 days of further growth at 19°C. The number from day 1 of starvation was used as control and as the denominator to calculate the percentage of worms recovering after starvation. Since starvation survival was influenced by assay conditions, all relevant experimental data were examined and compared within the same experiment.

C. elegans autophagy analysis

Autophagy analysis was performed as described (Kang et al., 2007). For light microscopic analysis of autophagy, starved L1 animals carrying an integrated transgene that expresses a GFP::LGG-1 fusion were collected at 3 days of starvation. GFP-positive punctate regions were visualized in the pharyngeal muscles of L1 animals using a Zeiss Axioplan 2 compound microscope (Zeiss, Germany).

Body fat assessment

Body fat analysis using Oil-Red-O staining was performed as described (Soukas et al., 2009) with a few modifications. Briefly, synchronized animals were collected and washed three times with M9 buffer. We incubated them in M9 buffer for the time indicated in the figures at 19°C. At each time point, using an aliquot from each sample tube, Oil-Red-O staining was conducted. The worms were resuspended and washed twice with phosphate-buffered saline (PBS) and then suspended in PBS to which an equal volume of 2× MRWB buffer containing 2% paraformaldehyde was added. The worms were taken through three freeze-thaw cycles between liquid nitrogen and warm water, followed by spinning at 14,000g, washing once in PBS, resuspension in 60% isopropanol, and addition of 60% Oil-Red-O stain for 1 h. To quantify the amount of Oil-Red-O staining, Adobe Photoshop CS5 software was used. For approximate quantification of the levels of Oil-Red-O staining, we categorized stained worms into 3 groups: 1) Full – worms showing strong staining throughout the intestine, 2) Partial – worms showing only staining around the tail region, and 3) None – worms showing no staining.

Statistical analysis was performed using the GraphPad PRISM 9 (GraphPad Software, USA).

RESULTS

To search for the systemic signal secreted by AIB neurons to modulate a systemic starvation response, we checked the expression pattern of neuropeptides with a focus on FMRFamide-related peptides (Kim and Li, 2004; Li and Kim, 2008). FLP-20 is the only one reported to be expressed in AIB neurons. To test whether FLP-20 was involved in the modulation of starvation responses, we examined whether mutation of flp-20 could suppress the starvation-hypersensitivity of gpb-2 mutants in which starvation responses, such as autophagy, are overactivated (Kang et al., 2007; You et al., 2006). Suppression of AIB activity by mutation of mgl-2 suppressed the starvation-induced death of gpb-2 mutants, as previously demonstrated (Kang and Avery, 2009b). Mutation of flp-20 also rescued the death of gpb-2 mutants during starvation, yet did not further suppress the starvation-induced death of gpb-2 mgl-2 mutants (Fig. 1A). These data suggest that FLP-20 acts in the same pathway as MGL-2 to modulate starvation responses. Consistent with this interpretation, AIB-specific expression of flp-20 driven by the promoter of ord-2b, but not AVA, AVB, AVG, RIM, PVC, AVD, and AVE expression of flp-20 (driven by the promoter of nmr-2), restored the starvation sensitivity of gpb-2; flp-20 mutants (Fig. 1B). Mutation of flp-18, which is expressed in AIY neurons that have been also shown to modulate a systemic starvation response (Kang and Avery, 2009b), did not rescue the starvation-survival of gpb-2 mutants, indicating the specificity of FLP-20 as a starvation-regulating FMRFamide-related peptide (Fig. 1C). Together, these data suggest that FLP-20 may act as a systemic signal from AIB neurons downstream of MGL-2 to positively regulate starvation responses.

MGL-1 and MGL-2 sense several amino acids, including leucine, as a food signal to modulate AIY and AIB neurons, respectively. Treatment with leucine suppresses MGL-2, which in turn reduces the activity of AIB neurons and thus decreases a systemic starvation response, including autophagy in the pharyngeal muscles (Kang and Avery, 2009b). While searching for additional regulators of these amino acid responses, we found that mutation of gcy-28, a receptor-type guanylate cyclase (Tsunozaki et al., 2008), abolished the positive effect of leucine on the starvation survival of gpb-2 mutants (Fig. 1D). GCY-28 is known to be expressed in many head neurons, including AWC and AIA neurons that are upstream of AIB neurons, as well as in peripheral tissues, including body-wall muscles, hypodermis, and intestine, which are potential downstream targets of AIB neurons during starvation responses (Ortiz et al., 2006). Thus, we hypothesized that GCY-28 is involved in starvation responses, and may act either upstream or downstream of the MGL-2 and FLP-20 signaling pathway. To test this, we generated gcy-28 gpb-2 mgl-2 and gcy-28 gpb-2; flp-20 mutants. Mutation of gcy-28 restored the starvation sensitivity of gpb-2 mgl-2 and gpb-2; flp-20 mutants, suggesting that GCY-28 acts downstream, but not upstream, of MGL-2 and FLP-20 to modulate starvation responses (Fig. 1E).

To corroborate our finding that FLP-20 and GCY-28 modulate a systemic starvation response, we further focused on their roles in modulating autophagy and fat mobilization, two well-described starvation responses. The main cause of death in gpb-2 mutants during starvation is excessive autophagy in their pharyngeal muscles, and MGL-2 modulates such autophagy in a systemic manner (Kang and Avery, 2008; 2009a; 2009b; Kang et al., 2007). Thus, we asked whether FLP-20 and GCY-28 are also involved in autophagic regulation of the pharyngeal muscles during starvation. We generated mutant strains carrying an integrated LGG-1::GFP transgene, a specific marker for autophagy in C. elegans (Kang et al., 2007; Klionsky et al., 2016; Melendez et al., 2003; Zhang et al., 2015), and found that mutation of flp-20 decreased excessive levels of autophagy in gpb-2 mutants during starvation (Fig. 2). Double mutation of flp-20 and mgl-2 did not show an additive effect on suppressing excessive autophagy in gpb-2 mutants, suggesting that FLP-20 and MGL-2 act in the same pathway to modulate autophagy, consistent with their effects on starvation survival. By contrast, mutation of gcy-28 did not aggravate excessive levels of autophagy (Fig. 2). This could be due to the fact that GCY-28 is not expressed in the pharyngeal muscles (Ortiz et al., 2006). Alternatively, GCY-28 could only abolish the suppressive effect of amino acids on excessive autophagy, as was the case for fat degradation.

Like mammals, C. elegans can store surplus energy as fat and consumes stored fat during starvation to maintain energy homeostasis (Jo et al., 2009; McKay et al., 2003; Van Gilst et al., 2005). Indeed, we found that starvation decreased the levels of fat in wild-type worms, and gpb-2 mutants had an increased rate of fat depletion, as assessed by the Oil-Red-O staining (Fig. 3), a histochemical method for measuring stored fat (Soukas et al., 2009). In addition, gpb-2 mutants already lower their fat deposition under control conditions, compared to wild-type worms, which is consistent with previous observations that they are in a perceived state of starvation (You et al., 2006). These results are consistent with the finding that starvation signaling is overactivated in gpb-2 mutants.

Next, we examined whether MGL-2, FLP-20, and GCY-28 are involved in starvation-induced fat degradation. Since gpb-2 mutation provides a sensitive genetic background to readily observe starvation-induced fat degradation (Fig. 3), we first examine the effect of each mutation on fat degradation during starvation in gpb-2 mutants. We found that mutation of either mgl-2 or flp-20 reduced the rate of fat degradation during starvation, and these effects were not additive (Fig. 4A). These data suggest that MGL-2 and FLP-20 positively regulate starvation-induced fat degradation in the same pathway, as was the case for starvation survival and autophagy regulation (Figs. 1A and Fig. 2). By contrast, mutation of gcy-28 accelerated the rate of fat degradation, and completely reversed the effects of mgl-2 and flp-20 mutations on fat degradation (Fig. 4A). These data suggest that GCY-28 acts downstream of MGL-2 and FLP-20 to modulate starvation-induced fat degradation, consistent with its role in starvation survival (Fig. 1E). Next, we examined whether such regulation also operates in a wild-type genetic background. As wild-type worms are less sensitive to starvation-induced fat degradation, we starved worms for 24 h to assess their fat contents (Fig. 4B). Mutation of gcy-28 exacerbated starvation-induced fat degradation, suggesting that GCY-28 is generally involved in fat regulation during starvation. Together with our previous studies, these data suggest that MGL-2 positively regulates the activity of AIB neurons, which in turn secrets FLP-20 that acts on GCY-28 in peripheral tissues to modulate a systemic starvation response, including starvation survival, autophagy, and fat degradation (Fig. 4C).

DISCUSSION

Although physiological and behavioral effects of the starvation response have been widely studied (Douglas et al., 2005; Finn and Dice, 2006; Wang et al., 2006), little is known about the systemic signals that directly modulate starvation responses in multicellular organisms. Previously, it has been proposed that reduction in the levels of leptin, in response to nutrient-excessive states, is a signal of nutrient-deficiency and induces starvation responses, including increased levels of food intake (Ahima et al., 1996; Chan and Mantzoros, 2005; Chan et al., 2003). However, leptin-deficient ob/ob mice did not perfectly mimic the starvation state, as exemplified by their normal levels of proteolysis in skeletal muscles (Turpin et al., 2009). Thus, it is reasonable to propose that specific hormones or neuropeptides that increase their expression under nutrient-deficient conditions positively regulate starvation responses, including autophagy that is key in maintaining cellular homeostasis during stress responses including starvation (Cho et al., 2020; Kim et al., 2018; 2021; Kwon et al., 2017; Lee et al., 2021; Molinari, 2021; Sebastian and Zorzano, 2020; Zachari et al., 2019). In fact, recent studies suggest that FGF21 is a starvation-regulating hormone, which is induced under nutrient-deficient conditions and positively regulates a collection of starvation responses, such as growth inhibition, tissue breakdown, autophagy, and lipid degradation (Badman et al., 2007; Byun et al., 2020; Fazeli et al., 2015; Inagaki et al., 2008). We identified the FMRFamide neuropeptide FLP-20 as a systemic starvation signal in C. elegans that positively regulates starvation responses, including starvation survival, autophagy, and starvation-induced fat degradation, echoing the discovery of FGF21 in mammals. Since, however, mutation of flp-20 could not fully rescue starvation-induced responses, it is likely that FLP-20 acts in parallel with other systemic signals, potentially originating from AIY neurons that have been previously shown to modulate starvation responses together with AIB neurons (Kang and Avery, 2009a; 2009b).

GCY-28 is a receptor-type guanylate cyclase, which has been previously shown to modulate chemotaxis (Tsunozaki et al., 2008). GCY-28 is expressed in various tissues, including neurons, body-wall muscles, hypodermis, and intestine, suggesting that its ligand(s) should be a cue of general interest to many tissues. However, the ligand(s) of GCY-28 has not been identified yet (Ortiz et al., 2006; Tsunozaki et al., 2008). Previous studies suggested that gcy-28 mutants seem to exist in a state of perceived starvation, based on the phenotype of avoidance of AWCon-sensed odors (Tsunozaki et al., 2008). Our results showing that mutation of gcy-28 is epistatic to mutation of flp-20 in starvation survival and starvation-induced fat degradation suggest the possibility that FLP-20 may be a ligand for GCY-28, possibly acting as an antagonist. It will be interesting to test this possibility using a heterologous expression system. In addition, further experiments are necessary to elucidate where and how GCY-28 acts to modulate starvation responses. Our starvation hypersensitive model system might be helpful to elucidate such molecular mechanisms.

Starvation also induces behavioral changes in animals (e.g., changes in foraging behavior), which contribute to increasing the chance of locating new food sources (Douglas et al., 2005; Wang et al., 2006). Foraging behaviors are often coordinately regulated with metabolic changes in various organisms, including C. elegans (Cohen et al., 2009; Greer et al., 2008; Srinivasan et al., 2008), suggesting the possibility that FLP-20 and GCY-28 may regulate behavioral changes in response to starvation as well. Indeed, we found that mutations of flp-20 and gcy-28 oppositely affect feeding rates of worms during prolonged starvation (Fig. 4D). Further experiments are needed to elucidate the circuit regulating this starvation-regulated feeding behavior.

Interestingly, after 24 h starvation, a portion of wild-type worms (~36%; Fig. 4B) still retained the high levels of fat. It was recently reported that starvation induces adult reproductive diapause in approximately 34% of worms (Angelo and Van Gilst, 2009). Given the fact that fat accumulation is important for another developmental diapause, dauer entry in C. elegans (Fielenbach and Antebi, 2008), our observation leads to the intriguing possibility that fat retention during starvation is critical for adult reproductive diapause. It would be interesting to test whether gcy-28 mutants, very few of which retained high levels of fat during starvation (Fig. 4B), show defects in entering adult reproductive diapause.

In summary, our data suggest that the FMRFamide neuropeptide FLP-20, probably secreted from AIB in an MGL-2 dependent manner, acts as a systemic signal for starvation responses through the receptor-type guanylate cyclase GCY-28. Thus, this study strongly indicates a specific neuroendocrine circuit that senses and signals nutrient-deficient states to modulate a systemic starvation response in multicellular organisms.

ACKNOWLEDGMENTS

We thank Scott Cameron, Beth Levine, Melanie Cobb, Alexander Soukas, Merav Cohen, Niels Ringstad, members of the Avery and Cameron laboratories for helpful discussions, Cori Bargmann, Kevin Ashrafi, Maureen Barr, Mario De Bono, Merav Cohen for providing strains and plasmids, the C. elegans Genetics Center and S. Mitani (National Bioresource Project) for providing strains, Yuji Kohara (National Institute of Genetics, Japan) for providing flp-20 cDNA. We are indebted to Cori Bargmann for the discussion about GCY-28 and for sharing unpublished data. This work was supported by grants from the POSCO Science Fellowship of POSCO TJ Park Foundation, the SNU invitation program for the distinguished scholar, and the U.S. Public Health Service (HL46154).

AUTHOR CONTRIBUTIONS

C.K. and L.A. conceived and designed the project. C.K. performed all experiments, and C.K. and L.A. analyzed the data. C.K. and L.A. wrote the manuscript.

CONFLICT OF INTEREST

The authors have no potential conflicts of interest to disclose.

Fig. 1.FLP-20 and GCY-28 modulate the starvation hypersensitivity of gpb-2 mutants. (A) FLP-20 and MGL-2 modulate starvation responses in the same pathway. Mutation of flp-20 and mgl-2 rescued the starvation-induced death of gpb-2 mutants (n = 3, mean ± SEM, one-way ANOVA test). n.s., not significant. (B) FLP-20 mainly acts in AIB neurons. AIB specific expression of flp-20 with the promoter of ord-2b restored the starvation-sensitivity of gpb-2; flp-20 (n = 3, mean ± SEM, two-way ANOVA test). (C) Mutation of flp-18 did not rescue the starvation hypersensitivity of gpb-2 and gpb-2; mgl-1 mutants (n = 2, mean ± SEM, one-way ANOVA test). (D) GCY-28 is involved in the amino acid sensing pathway. Mutation of gcy-28 abolished the rescue effect of leucine on the starvation hypersensitivity of gpb-2 mutants (n = 4-6, mean ± SEM, two-way ANOVA test). (E) GCY-28 acts downstream of MGL-2 and FLP-20 to modulate starvation responses. Mutation of gcy-28 restored the starvation sensitivity of gpb-2 mgl-2 and gpb-2; flp-20 mutants (n = 3-4, mean ± SEM, one-way ANOVA test).
Fig. 2.FLP-20 modulates autophagy in the pharyngeal muscle of gpb-2 mutants during starvation. (A) Representative images of the indicated genotype after 3 days of starvation. The arrows show representative GFP::LGG-1 positive punctate structures. GFP::LGG-1 labels pre-autophagosomal and autophagosomal structures. In the inset, the area marked by the box is magnified. (B) Quantification of autophagy in the pharyngeal muscle of worms of the indicated genotype after 3 days of starvation (n = 116-147, chi-square test). n.s., not significant.
Fig. 3.gpb-2 mutants exhibit an increased rate of fat degradation during starvation. (A) Representative images of Oil-Red-O staining in N2 (wild-type) worms and gpb-2 mutants (top). Violin plot analysis of Oil-Red-O staining (bottom, the horizontal solid line shows the median and the horizontal dot lines show the 1st and 3rd quartiles, one-way ANOVA test). n.s., not significant. (B) Quantification of Oil-Red-O staining. (Full) worms showing strong staining throughout the intestine; (Partial) worms showing staining only around the tail region; (None) worms showing no staining (n = 62-370).
Fig. 4.FLP-20, MGL-2, and GCY-28 modulate starvation-induced fat degradation. (A) Quantification of Oil-Red-O staining of the indicated genotype with or without 4 h starvation. Mutation of either flp-20 or mgl-2 decreased the rate of starvation-induced fat degradation, and mutation of gcy-28 reversed it (left, n = 214-446 for gpb-2, n = 243-265 for gpb-2; flp-20, n = 53-71 for gpb-2 mgl-2, n = 88-142 for gpb-2 mgl-2; flp-20, n = 27-39 for gcy-28 gpb-2, n = 109-156 for gcy-28 gpb-2; flp-20, and n = 16-20 for gcy-28 gpb-2 mgl-2, chi-square test). n.s., not significant. (B) Quantification of Oil-Red-O staining of the indicated genotype with or without 24 h starvation. gcy-28 mutants increased the rate of starvation-induced fat degradation (right, n = 171-407 for N2, n = 221-389 for flp-20, n = 196-229 for mgl-2, and n = 248-365 for gcy-28, chi-square test). (C) Model of the systemic regulation of starvation responses by the MGL-2–FLP-20–GCY-28 axis. Amino acids or food signals modulates the activity of AIB neurons through MGL-2, which in turn stimulates the secretion of FLP-20. FLP-20 acts on GCY-28 that suppresses starvation responses, including autophagy and fat degradation in the peripheral tissues. (D) Pumping rates of the indicated genotype with or without starvation. Unstarved or 4-day starved L1 animals were examined on food. Each dot represents an individual worm. Horizontal lines represent the average (line) and SEM (error bars).

Fig 1.

Figure 1.FLP-20 and GCY-28 modulate the starvation hypersensitivity of gpb-2 mutants. (A) FLP-20 and MGL-2 modulate starvation responses in the same pathway. Mutation of flp-20 and mgl-2 rescued the starvation-induced death of gpb-2 mutants (n = 3, mean ± SEM, one-way ANOVA test). n.s., not significant. (B) FLP-20 mainly acts in AIB neurons. AIB specific expression of flp-20 with the promoter of ord-2b restored the starvation-sensitivity of gpb-2; flp-20 (n = 3, mean ± SEM, two-way ANOVA test). (C) Mutation of flp-18 did not rescue the starvation hypersensitivity of gpb-2 and gpb-2; mgl-1 mutants (n = 2, mean ± SEM, one-way ANOVA test). (D) GCY-28 is involved in the amino acid sensing pathway. Mutation of gcy-28 abolished the rescue effect of leucine on the starvation hypersensitivity of gpb-2 mutants (n = 4-6, mean ± SEM, two-way ANOVA test). (E) GCY-28 acts downstream of MGL-2 and FLP-20 to modulate starvation responses. Mutation of gcy-28 restored the starvation sensitivity of gpb-2 mgl-2 and gpb-2; flp-20 mutants (n = 3-4, mean ± SEM, one-way ANOVA test).
Molecules and Cells 2021; 44: 529-537https://doi.org/10.14348/molcells.2021.0051

Fig 2.

Figure 2.FLP-20 modulates autophagy in the pharyngeal muscle of gpb-2 mutants during starvation. (A) Representative images of the indicated genotype after 3 days of starvation. The arrows show representative GFP::LGG-1 positive punctate structures. GFP::LGG-1 labels pre-autophagosomal and autophagosomal structures. In the inset, the area marked by the box is magnified. (B) Quantification of autophagy in the pharyngeal muscle of worms of the indicated genotype after 3 days of starvation (n = 116-147, chi-square test). n.s., not significant.
Molecules and Cells 2021; 44: 529-537https://doi.org/10.14348/molcells.2021.0051

Fig 3.

Figure 3.gpb-2 mutants exhibit an increased rate of fat degradation during starvation. (A) Representative images of Oil-Red-O staining in N2 (wild-type) worms and gpb-2 mutants (top). Violin plot analysis of Oil-Red-O staining (bottom, the horizontal solid line shows the median and the horizontal dot lines show the 1st and 3rd quartiles, one-way ANOVA test). n.s., not significant. (B) Quantification of Oil-Red-O staining. (Full) worms showing strong staining throughout the intestine; (Partial) worms showing staining only around the tail region; (None) worms showing no staining (n = 62-370).
Molecules and Cells 2021; 44: 529-537https://doi.org/10.14348/molcells.2021.0051

Fig 4.

Figure 4.FLP-20, MGL-2, and GCY-28 modulate starvation-induced fat degradation. (A) Quantification of Oil-Red-O staining of the indicated genotype with or without 4 h starvation. Mutation of either flp-20 or mgl-2 decreased the rate of starvation-induced fat degradation, and mutation of gcy-28 reversed it (left, n = 214-446 for gpb-2, n = 243-265 for gpb-2; flp-20, n = 53-71 for gpb-2 mgl-2, n = 88-142 for gpb-2 mgl-2; flp-20, n = 27-39 for gcy-28 gpb-2, n = 109-156 for gcy-28 gpb-2; flp-20, and n = 16-20 for gcy-28 gpb-2 mgl-2, chi-square test). n.s., not significant. (B) Quantification of Oil-Red-O staining of the indicated genotype with or without 24 h starvation. gcy-28 mutants increased the rate of starvation-induced fat degradation (right, n = 171-407 for N2, n = 221-389 for flp-20, n = 196-229 for mgl-2, and n = 248-365 for gcy-28, chi-square test). (C) Model of the systemic regulation of starvation responses by the MGL-2–FLP-20–GCY-28 axis. Amino acids or food signals modulates the activity of AIB neurons through MGL-2, which in turn stimulates the secretion of FLP-20. FLP-20 acts on GCY-28 that suppresses starvation responses, including autophagy and fat degradation in the peripheral tissues. (D) Pumping rates of the indicated genotype with or without starvation. Unstarved or 4-day starved L1 animals were examined on food. Each dot represents an individual worm. Horizontal lines represent the average (line) and SEM (error bars).
Molecules and Cells 2021; 44: 529-537https://doi.org/10.14348/molcells.2021.0051

References

  1. Ahima R.S., Prabakaran D., Mantzoros C., Qu D., Lowell B., Maratos-Flier E., and Flier J.S. (1996). Role of leptin in the neuroendocrine response to fasting. Nature 382, 250-252.
    Pubmed CrossRef
  2. Anding A.L. and Baehrecke E.H. (2017). Cleaning house: selective autophagy of organelles. Dev. Cell 41, 10-22.
    Pubmed KoreaMed CrossRef
  3. Angelo G. and Van Gilst M.R. (2009). Starvation protects germline stem cells and extends reproductive longevity in C. elegans. Science 326, 954-958.
    CrossRef
  4. Badman M.K., Pissios P., Kennedy A.R., Koukos G., Flier J.S., and Maratos-Flier E. (2007). Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states. Cell Metab. 5, 426-437.
    Pubmed CrossRef
  5. Bechtold D.A. and Luckman S.M. (2007). The role of RFamide peptides in feeding. J. Endocrinol. 192, 3-15.
    Pubmed CrossRef
  6. Berthoud H.R. and Morrison C. (2008). The brain, appetite, and obesity. Annu. Rev. Psychol. 59, 55-92.
    Pubmed CrossRef
  7. Brenner S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.
    CrossRef
  8. Byun S., Seok S., Kim Y.C., Zhang Y., Yau P., Iwamori N., Xu H.E., Ma J., Kemper B., and Kemper J.K. (2020). Fasting-induced FGF21 signaling activates hepatic autophagy and lipid degradation via JMJD3 histone demethylase. Nat. Commun. 11, 807.
    Pubmed KoreaMed CrossRef
  9. Chan J.L., Heist K., DePaoli A.M., Veldhuis J.D., and Mantzoros C.S. (2003). The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men. J. Clin. Invest. 111, 1409-1421.
    Pubmed KoreaMed CrossRef
  10. Chan J.L. and Mantzoros C.S. (2005). Role of leptin in energy-deprivation states: normal human physiology and clinical implications for hypothalamic amenorrhoea and anorexia nervosa. Lancet 366, 74-85.
    Pubmed CrossRef
  11. Chen Y.C., Chen H.J., Tseng W.C., Hsu J.M., Huang T.T., Chen C.H., and Pan C.L. (2016). A C. elegans thermosensory circuit regulates longevity through crh-1/CREB-dependent flp-6 neuropeptide signaling. Dev. Cell 39, 209-223.
    Pubmed CrossRef
  12. Cho D.H., Kim J.K., and Jo E.K. (2020). Mitophagy and innate immunity in infection. Mol. Cells 43, 10-22.
    Pubmed KoreaMed CrossRef
  13. Cohen M., Reale V., Olofsson B., Knights A., Evans P., and de Bono M. (2009). Coordinated regulation of foraging and metabolism in C. elegans by RFamide neuropeptide signaling. Cell Metab. 9, 375-385.
    Pubmed CrossRef
  14. Davis J.F., Choi D.L., and Benoit S.C. (2010). Insulin, leptin and reward. Trends Endocrinol. Metab. 21, 68-74.
    Pubmed KoreaMed CrossRef
  15. Douglas S.J., Dawson-Scully K., and Sokolowski M.B. (2005). The neurogenetics and evolution of food-related behaviour. Trends Neurosci. 28, 644-652.
    Pubmed CrossRef
  16. Fazeli P.K., Lun M., Kim S.M., Bredella M.A., Wright S., Zhang Y., Lee H., Catana C., Klibanski A., and Patwari P., et al. (2015). FGF21 and the late adaptive response to starvation in humans. J. Clin. Invest. 125, 4601-4611.
    Pubmed KoreaMed CrossRef
  17. Fielenbach N. and Antebi A. (2008). C. elegans dauer formation and the molecular basis of plasticity. Genes Dev. 22, 2149-2165.
    CrossRef
  18. Finn P.F. and Dice J.F. (2006). Proteolytic and lipolytic responses to starvation. Nutrition 22, 830-844.
    Pubmed CrossRef
  19. Gerisch B., Tharyan R.G., Mak J., Denzel S.I., Popkes-van Oepen T., Henn N., and Antebi A. (2020). HLH-30/TFEB is a master regulator of reproductive quiescence. Dev. Cell 53, 316-329.e5.
    Pubmed CrossRef
  20. Greer E.R., Perez C.L., Van Gilst M.R., Lee B.H., and Ashrafi K. (2008). Neural and molecular dissection of a C. elegans sensory circuit that regulates fat and feeding. Cell Metab. 8, 118-131.
    Pubmed KoreaMed CrossRef
  21. Inagaki T., Lin V.Y., Goetz R., Mohammadi M., Mangelsdorf D.J., and Kliewer S.A. (2008). Inhibition of growth hormone signaling by the fasting-induced hormone FGF21. Cell Metab. 8, 77-83.
    Pubmed KoreaMed CrossRef
  22. Jo H., Shim J., Lee J.H., Lee J., and Kim J.B. (2009). IRE-1 and HSP-4 contribute to energy homeostasis via fasting-induced lipases in C. elegans. Cell Metab. 9, 440-448.
  23. Kang C. and Avery L. (2008). To be or not to be, the level of autophagy is the question: dual roles of autophagy in the survival response to starvation. Autophagy 4, 82-84.
    Pubmed KoreaMed CrossRef
  24. Kang C. and Avery L. (2009a). Systemic regulation of autophagy in Caenorhabditis elegans. Autophagy 5, 565-566.
    CrossRef
  25. Kang C. and Avery L. (2009b). Systemic regulation of starvation response in Caenorhabditis elegans. Genes Dev. 23, 12-17.
    CrossRef
  26. Kang C., You Y.J., and Avery L. (2007). Dual roles of autophagy in the survival of Caenorhabditis elegans during starvation. Genes Dev. 21, 2161-2171.
    Pubmed KoreaMed CrossRef
  27. Kim J., Lee Y., Roh K., Kim M.S., and Kang C. (2021). Targeting the stress support network regulated by autophagy and senescence for cancer treatment. Adv. Cancer Res. 150, 75-112.
    Pubmed CrossRef
  28. Kim J., Lim Y.M., and Lee M.S. (2018). The role of autophagy in systemic metabolism and human-type diabetes. Mol. Cells 41, 11-17.
    Pubmed KoreaMed CrossRef
  29. Kim K. and Li C. (2004). Expression and regulation of an FMRFamide-related neuropeptide gene family in Caenorhabditis elegans. J. Comp. Neurol. 475, 540-550.
    CrossRef
  30. Klionsky D.J., Abdelmohsen K., Abe A., Abedin M.J., Abeliovich H., Acevedo Arozena A., Adachi H., Adams C.M., Adams P.D., and Adeli K., et al. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1-222.
    Pubmed KoreaMed CrossRef
  31. Kniazeva M., Zhu H., Sewell A.K., and Han M. (2015). A lipid-TORC1 pathway promotes neuronal development and foraging behavior under both fed and fasted conditions in C. elegans. Dev. Cell 33, 260-271.
    CrossRef
  32. Konner A.C. and Bruning J.C. (2012). Selective insulin and leptin resistance in metabolic disorders. Cell Metab. 16, 144-152.
    Pubmed CrossRef
  33. Kwon Y., Kim J.W., Jeoung J.A., Kim M.S., and Kang C. (2017). Autophagy is pro-senescence when seen in close-up, but anti-senescence in long-shot. Mol. Cells 40, 607-612.
    Pubmed KoreaMed CrossRef
  34. Lee C., Lamech L., Johns E., and Overholtzer M. (2020). Selective lysosome membrane turnover is induced by nutrient starvation. Dev. Cell 55, 289-297.e4.
    Pubmed CrossRef
  35. Lee Y., Kim J., Kim M.S., Kwon Y., Shin S., Yi H., Kim H., Chang M.J., Chang C.B., and Kang S.B., et al. (2021). Coordinate regulation of the senescent state by selective autophagy. Dev. Cell 56, 1512-1525.e7.
    Pubmed CrossRef
  36. Li C. and Kim K. (2008). Neuropeptides. . https://doi.org/10.1895/wormbook.1.142.1.
    CrossRef
  37. McKay R.M., McKay J.P., Avery L., and Graff J.M. (2003). C elegans: a model for exploring the genetics of fat storage. Dev. Cell 4, 131-142.
    CrossRef
  38. Melendez A., Talloczy Z., Seaman M., Eskelinen E.L., Hall D.H., and Levine B. (2003). Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301, 1387-1391.
    CrossRef
  39. Miller D.R. and Thorburn A. (2021). Autophagy and organelle homeostasis in cancer. Dev. Cell 56, 906-918.
    Pubmed KoreaMed CrossRef
  40. Mizushima N. (2007). Autophagy: process and function. Genes Dev. 21, 2861-2873.
    Pubmed CrossRef
  41. Molinari M. (2021). ER-phagy responses in yeast, plants, and mammalian cells and their crosstalk with UPR and ERAD. Dev. Cell 56, 949-966.
    Pubmed CrossRef
  42. Olsen L., Thum E., and Rohner N. (2021). Lipid metabolism in adaptation to extreme nutritional challenges. Dev. Cell . 2021 Mar 9 [Epub]. https://doi.org/10.1016/j.devcel.2021.02.024.
    Pubmed CrossRef
  43. Ortiz C.O., Etchberger J.F., Posy S.L., Frokjaer-Jensen C., Lockery S., Honig B., and Hobert O. (2006). Searching for neuronal left/right asymmetry: genomewide analysis of nematode receptor-type guanylyl cyclases. Genetics 173, 131-149.
    Pubmed KoreaMed CrossRef
  44. Park J., Choi W., Dar A.R., Butcher R.A., and Kim K. (2019). Neuropeptide signaling regulates pheromone-mediated gene expression of a chemoreceptor gene in C. elegans. Mol. Cells 42, 28-35.
  45. Sebastian D. and Zorzano A. (2020). Self-eating for muscle fitness: autophagy in the control of energy metabolism. Dev. Cell 54, 268-281.
    Pubmed CrossRef
  46. Shin D.W. (2020). Lipophagy: molecular mechanisms and implications in metabolic disorders. Mol. Cells 43, 686-693.
    Pubmed KoreaMed CrossRef
  47. Shin H.R. and Zoncu R. (2020). The lysosome at the intersection of cellular growth and destruction. Dev. Cell 54, 226-238.
    Pubmed KoreaMed CrossRef
  48. Soukas A.A., Kane E.A., Carr C.E., Melo J.A., and Ruvkun G. (2009). Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes Dev. 23, 496-511.
    CrossRef
  49. Srinivasan S., Sadegh L., Elle I.C., Christensen A.G., Faergeman N.J., and Ashrafi K. (2008). Serotonin regulates C. elegans fat and feeding through independent molecular mechanisms. Cell Metab. 7, 533-544.
    Pubmed KoreaMed CrossRef
  50. Takayasu S., Sakurai T., Iwasaki S., Teranishi H., Yamanaka A., Williams S.C., Iguchi H., Kawasawa Y.I., Ikeda Y., and Sakakibara I., et al. (2006). A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 7438-7443.
    Pubmed KoreaMed CrossRef
  51. Texada M.J., Malita A., Christensen C.F., Dall K.B., Faergeman N.J., Nagy S., Halberg K.A., and Rewitz K. (2019). Autophagy-mediated cholesterol trafficking controls steroid production. Dev. Cell 48, 659-671.e4.
    Pubmed CrossRef
  52. Tsunozaki M., Chalasani S.H., and Bargmann C.I. (2008). A behavioral switch: cGMP and PKC signaling in olfactory neurons reverses odor preference in C. elegans. Neuron 59, 959-971.
    CrossRef
  53. Turpin S.M., Ryall J.G., Southgate R., Darby I., Hevener A.L., Febbraio M.A., Kemp B.E., Lynch G.S., and Watt M.J. (2009). Examination of 'lipotoxicity' in skeletal muscle of high-fat fed and ob/ob mice. J. Physiol. 587, 1593-1605.
    Pubmed KoreaMed CrossRef
  54. Van Gilst M.R., Hadjivassiliou H., and Yamamoto K.R. (2005). A Caenorhabditis elegans nutrient response system partially dependent on nuclear receptor NHR-49. Proc. Natl. Acad. Sci. U. S. A. 102, 13496-13501.
    Pubmed KoreaMed CrossRef
  55. Wang T., Hung C.C., and Randall D.J. (2006). The comparative physiology of food deprivation: from feast to famine. Annu. Rev. Physiol. 68, 223-251.
    Pubmed CrossRef
  56. You Y.J., Kim J., Cobb M., and Avery L. (2006). Starvation activates MAP kinase through the muscarinic acetylcholine pathway in Caenorhabditis elegans pharynx. Cell Metab. 3, 237-245.
    Pubmed KoreaMed CrossRef
  57. Zachari M., Gudmundsson S.R., Li Z., Manifava M., Cugliandolo F., Shah R., Smith M., Stronge J., Karanasios E., and Piunti C., et al. (2019). Selective autophagy of mitochondria on a ubiquitin-endoplasmic-reticulum platform. Dev. Cell 50, 627-643.e5.
    Pubmed KoreaMed CrossRef
  58. Zhang H., Chang J.T., Guo B., Hansen M., Jia K., Kovacs A.L., Kumsta C., Lapierre L.R., Legouis R., and Lin L., et al. (2015). Guidelines for monitoring autophagy in Caenorhabditis elegans. Autophagy 11, 9-27.
Mol. Cells
Nov 30, 2021 Vol.44 No.11, pp. 781~860
COVER PICTURE
3D quantitative images of the vesicular structure and the nucleolus using label free optical diffraction tomography (Kim et al., pp. 851-860).

Share this article on

  • line
  • mail

Related articles in Mol. Cells

Molecules and Cells

eISSN 0219-1032
qr-code Download