Mol. Cells 2021; 44(7): 529-537
Published online June 18, 2021
https://doi.org/10.14348/molcells.2021.0051
© The Korean Society for Molecular and Cellular Biology
Correspondence to : chanhee.kang@snu.ac.kr
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
We previously showed that MGL-1 and MGL-2,
Strains were maintained as described (Brenner, 1974) at 19°C. All worms were maintained and grown on
cDNA corresponding to the entire coding sequence of
Polymerase chain reaction (PCR) construction of
PCR construction of
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
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 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 14,000
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
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
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
Like mammals,
Next, we examined whether MGL-2, FLP-20, and GCY-28 are involved in starvation-induced fat degradation. Since
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
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
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
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
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.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.
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.
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
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
We previously showed that MGL-1 and MGL-2,
Strains were maintained as described (Brenner, 1974) at 19°C. All worms were maintained and grown on
cDNA corresponding to the entire coding sequence of
Polymerase chain reaction (PCR) construction of
PCR construction of
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
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 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 14,000
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
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
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
Like mammals,
Next, we examined whether MGL-2, FLP-20, and GCY-28 are involved in starvation-induced fat degradation. Since
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
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
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
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
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.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.
Jae Hyeong Lee, Sang-Ah Park, Il-Geun Park, Bo Kyung Yoon, Jung-Shin Lee, and Ji Min Lee
Mol. Cells 2023; 46(8): 476-485 https://doi.org/10.14348/molcells.2023.0011Seon Beom Song, Woosung Shim, and Eun Seong Hwang*
Mol. Cells 2023; 46(8): 486-495 https://doi.org/10.14348/molcells.2023.0019Soyoung Kim, Jaeseok Han, Young-Ho Ahn, Chang Hoon Ha, Jung Jin Hwang, Sang-Eun Lee, Jae-Joong Kim, and Nayoung Kim
Mol. Cells 2022; 45(6): 403-412 https://doi.org/10.14348/molcells.2022.2010