C. elegans insulin-like peptides

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In contrast to the numerous INS ligands in C. elegans, there is only one insulin-like receptor called DAF-2 (Abnormal Dauer Formation protein 2).Upon activation of the DAF-2/IR, it recruits the insulin receptor substrate IST-1/IRS leading to the activation of AGE-1/PI3K and subsequently triggering a cascade of kinase activities including PDK-1 and AKT-1/2 that eventually leads to the phosphorylation of the transcription factor DAF-16/FOXO, preventing it from entering the nucleus, thus blocking cellular process regulated by DAF-16/FOXO (Fig. 1) (Murphy, 2006;Murphy et al., 2003;Tullet, 2015;Yen et al., 2011).If the IIS pathway is inactive, DAF-16/FOXO localizes to the nucleus and target various genes, including but not limited to the antioxidant gene, small heat shock protein genes, metabolic genes, antibacterial genes (Yen et al., 2011).In addition, DAF-16/FOXO may auto-regulate some INS genes, see section 4.4 INS feedback regulation.
Within C. elegans there are eight isoforms of DAF-16, labeled A through H. Isoforms DAF-16 E and DAF-16G lack a DNA binding domain and their functions are currently unknown.The remaining six isoforms can be categorized into three groups based on their distinct tissue distribution.DAF-16 A and DAF-16D/F/H are present in most tissues that function to suppress the dauer-constitutive phenotype and extend lifespan.In contrast, DAF-16 B is found exclusively in the pharyngeal nervous system, and somatic gonad, and it has minimal impact on lifespan extension and dauer formation (Murphy, 2013).
Beyond DAF-16/FOXO, the IIS may also regulate other pathways such as SKN-1, TOR, MAPK, among others.Our emphasis on DAF-16/ FOXO is due to its prominent role as the canonical and major target of IIS, which is central to our understanding of the system.
The evolutionary conservation of insulin and INS, along with their signaling pathways, across diverse organisms from C. elegans to D. melanogaster and humans underscores the fundamental importance of these systems (Barbieri et al., 2003;Viola et al., 2023).This conservation has established C. elegans as an invaluable model for studying the IIS pathways in relation to humans.Utilizing C. elegans for research presents an opportunity to conduct comprehensive experiments and therapeutic trials with fewer ethical concerns.Characterized by its short lifespan of up to 21 days, C. elegans undergoes an embryonic stage, four larval stages (L1-L4) and an adult stage.This life cycle makes the nematode worm model particularly suitable for longevity studies (Fig. 2) (Zheng et al., 2018a).Additionally, C. elegans have well characterized genes and neurons, making it an ideal subject for neurological studies and allowing for convenient comparison with their human counterparts (White et al., 1986).Moreover, C. elegans can suspend its development under unfavourable environmental conditions and enter an alternative life stage, namely L1 arrest and dauer arrest (Fig. 2).Both stages are controlled by the IIS pathway in C. elegans.
Here, we will dissect the multifaceted roles of the C. elegans insulinlike peptides, offering insight into their structural intricacies and regulatory mechanics.Beyond structures, we will delve into the profound impact these peptides have across a spectrum of physiological processes in C. elegans from metabolic regulation to development and survival strategies under environmental stress.Through this exploration, we aim to illustrate the parallels and distinctions between insulin signaling in humans and C. elegans, thus enriching our understanding of endocrinology from a broader, cross-species perspective.

C. elegans insulin-like peptides
Although most C. elegans INS peptides lack sequence identity to that of human insulin and IGFs (Pierce et al., 2001), they still retain the   (Hua et al., 2003).The IIS pathway is conserved between humans and C. elegans, but there are some differences (Piñero González et al., 2009).In humans, there are 10 insulin/insulin-like peptides, while C. elegans have 40 INS peptides (INS 1-39 and DAF-28) (Duret et al., 1998;Pierce et al., 2001).All the human insulin/insulin-like peptides are agonists that bind and activate to their receptors to trigger the IIS pathway, while C. elegans uniquely include antagonistic INS peptides, thus providing a complex mechanism for modulating IIS (Chen et al., 2013;Cornils et al., 2011;Hung et al., 2014;Matsunaga et al., 2017;Matsunaga et al., 2018;Zheng et al., 2018a).In contrast to the vast range of ligands, C. elegans only has one insulin receptor: DAF-2/IR, through which all INS peptides are thought to signal.
Structurally, INS peptides are divided into three classes-α, β, and γ-based on the number and location of disulfide bridges (Duret et al., 1998).The γ type INS peptides have the same disulfide bridges as human insulin and IGF-1/2, with the A and B chains linked together by two disulfide bonds, and an additional disulfide bond within the A chain.The α type INS peptides, in contrast, contains an additional disulfide bond between the A and B chains but lacks the disulfide bond within the A chain.This bond is replaced by an interaction between amino acids with aromatic side chains (Fig. 3).
The functional redundancy among the 40 INS peptides in C. elegans are evident, as the knockout of any individual peptide does not fully recapitulate the daf-2 loss of function phenotype.Zheng et al. (2018a) adopted an overexpression approach to categorize these peptides based on phenotypes associated with IIS activation, including L1 arrest survival, L1 arrest cell divisions, dauer formation and fat metabolism.weak antagonists.Interestingly, depending on their context  could have both agonist and antagonistic roles.did not exhibit significant IIS phenotypes, and like humans and D. melanogaster, it is conceivable that these INS peptides could signal through receptors independent of DAF-2/IR (Zheng et al., 2018a).
Why do some C. elegans INS peptide behave as antagonists?Using a molecular modeling approach to understand the differences between antagonistic and agonistic INS peptides, Matsunaga et al. (2018) suggested that the antagonistic INS peptides, differ in their electrostatic surface potential.The differences in their electrostatic surface potential can be partially attributed to the insertion of amino acids around the conserved cysteine residues in the B chain that forms disulfide bonds with the A chain.This is thought to alter the receptor binding surface, and combined with its impact on the shape and electrostatic potential, may contribute to the differentiation between agonistic and antagonistic peptides (Matsunaga et al., 2018).

Processing and secretion of C. elegans INS
The precursor of all INS peptides upon the initial translation is called the preproinsulin which consists of a signal peptide, an A Chain, and a B Chain (Li et al., 2003).INS-1 and INS-18 precursors contain an additional C peptide between the A and B chain, like that of human insulin.Peptides  and DAF-28 have an additional F peptide located between the signal peptide and B peptide, resembling human IGF-1 and IGF-2, which also contains additional D and E peptides at the carboxyl ends (Fig. 3) (Duret et al., 1998;Pierce et al., 2001;Zheng et al., 2018a).The F peptide is important for the agonistic activity of INS peptides, potentially enhancing their binding affinity and function for the DAF-2 insulin receptor, similar to the proposed role of the D and E peptides in IGFs binding to the insulin receptor and IGFRs (Bayne et al., 1989;Mayer et al., 2007;Pfeffer et al., 2009).
The preproinsulin signal peptide directs the polypeptide into the endoplasmic reticulum (ER), facilitating disulfide linkages between the A and B chains, and the cleavage of the signal sequence by signal peptidase (Davidson, 2004;Vasiljević et al., 2020).Once the signal peptide is cleaved, proinsulin is formed and packaged into secretory granules containing the prohormone convertase enzymes PC-1 and PC-2, known as KPC-1 and EGL-3, respectively, in C. elegans.KPC-1/PC-1 cleaves between the B and C chains, while EGL-3/PC-2 cleaves between the C and A chains (Hung et al., 2014).In human IGF-1 and IGF-2, the E peptide is cleaved off through an unknown proteolytic process, while the D peptide remains uncleaved.The C peptide is removed when processing human insulin, INS-18, and INS-1, but it remains intact in IGF-1 and IGF-2 (O' Dell and Day, 1998;Philippou et al., 2014;Saltiel et al., 2007) , DAF-28 also needs to be cleaved, as evidenced by the loss of function in the egl-3 proprotein convertase mutant (Fig. 3) (Hung et al., 2014;Pierce et al., 2001;Zheng et al., 2018a).After these cleavages, proinsulin retains carboxyterminal basic amino acids, which are then removed by EGL-21/carboxypeptidase E (Husson et al., 2007).Structurally, of all the C. elegans INS peptides, INS-1 and INS-18 are most similar to human insulin as they possess a C peptide between the B and A chains.The rest of the C. elegans INS peptides are more akin to human IGF-1 and IGF-2, being single-chain peptides (Fig. 3).Following cleavage, mature insulins are stored within the dense-core vesicles, released for secretion via exocytosis in response to environmental stimuli or neuronal signals (Tsuboi et al., 2004).

INS peptide secretion
In humans, insulin is secreted from dense-core vesicles in response to increased blood glucose levels, a process acutely regulated by nutrient availability (Saltiel and Pessin, 2007).This suggests a similar regulatory mechanism in C. elegans, where environmental factors like nutrient availability crucially influence the release of INS peptides.This release relies on UNC-31/CAP, a calcium-dependent activator essential for the exocytosis of neuropeptides from dense-core vesicles.Hence, the availability of INS peptides in C. elegans, contingent on the effective functioning of UNC-31/CAP, align with the worm's nutritional environment (Baugh and Sternberg, 2006;Lee and Ashrafi, 2008;Leinwand and Chalasani, 2014).The secretion process in the intestine of C. elegans also requires asna-1, which encodes a conserved ATPase for this secretion (Kao et al., 2007).Moreover, INS-1's role in neural development involves the secretory protein HEN-1, facilitating nonautonomous signaling (Kodama et al., 2006).

C. elegans INS peptide regulation
The regulation of INS peptides is complex, influenced by spatialtemporal expression patterns and the specific nature of each peptide.This section will delve into various examples highlighting how INS peptides are regulated and localized within tissues.
These peptides relay the organism's nutrient status to various organs, coordinating growth and metabolic responses to environmental conditions.Serotonin, a critical neurotransmitter involved in feeding and locomotion, can trigger the secretion of INS peptides from secretory neurons, thereby facilitating reproduction and development.The underlying mechanism, though not fully understood, appears to involve serotonin's suppression of the IIS pathway, resulting in the accumulation of DAF-16/FOXO (Fielenbach and Antebi, 2008;Kaplan et al., 2008;Schwartz et al., 2021;Srinivasan et al., 2008).

Temporal regulation
Using an mRNA expression approach Baugh et al. (2011) provided an overview of insulin-like mRNA expression during the C. elegans life cycle.Their research revealed varied expression patterns of insulin like genes, suggesting complex regulation and specific functional roles.While most INS peptides are expressed during all developmental stages, certain INS peptides such as ins-2 and ins-34 are exclusive to embryonic stages, hinting at a crucial role in embryogenesis (Baugh et al., 2011).The expression of the daf-28 gene peaks at the end of L1 development, aligning with its role in L1 arrest recovery.Ins-15 expression appears specific to the dauer stage, underscoring the nuanced regulation of INS gene expression (Baugh et al., 2011).

Signaling pathways that regulate INS gene expression
Several signaling pathways, including G-protein coupled receptors (GPCR) signaling, are thought to regulate INS gene expression.DAF-11, a transmembrane guanylyl cyclase of the cGMP signaling pathway, reportedly downregulates DAF-28 expression in sensory neurons, influencing IIS activity (Li et al., 2003;Pierce et al., 2001).cGMP can trigger serotonin release, affecting the production of TGF-β and INS peptides from neurons (Fielenbach and Antebi, 2008).TGF-β, a key signaling molecule for cell growth and differentiation, responds to various stimuli in C. elegans, like nutrient shifts, high temperature, and population density.These stimuli impact INS peptide expression, modulating IIS (Savage-Dunn and Padgett, 2017).Microarray studies identified several INS peptides, including all under the influence of TGF-β signaling (Liu et al., 2004).Additionally, INS-3 and INS-4 are directly regulated by TGF-β signaling pathways (Zheng et al., 2018b).
The MAPK pathway, central to stress response and cell survival, stimulates the transcription of INS-4, INS-11, INS-39, and DAF-28.This pathway is activated in C. elegans by various stress stimuli, such as pathogen or oxidative stress, playing a pivotal role in insulin-mediated communication between neurons and the intestine (Lee and Mylonakis, 2017;Qu et al., 2020).Through these mechanisms, both TGF-β and MAPK pathway contribute to the complex regulation of INS expression in response to environmental cues and internal signals in C. elegans.(Harris et al., 2011).
In addition to INS-to-INS signaling regulation, DAF-16, a downstream transcription factor that accumulates in absence of IIS activation can induce a positive feedback loop regulating the expression of INS-7, an antagonistic INS peptide that would amplify IIS pathway inactivation (Murphy et al., 2003).This mechanism allows DAF-16/FOXO activation in one tissue to trigger the same DAF-16/FOXO activation in another, promoting "FOXO-to-FOXO" for longevity (Murphy et al., 2007).This result explains how DAF-2/IR inactivation in the intestine can sufficiently increase the organism's lifespan (Roy et al., 2022).
DAF  (Kaplan et al., 2019).These INS peptides play a role in modulating the IIS pathway, with their expression influenced by life stage and environmental conditions, like nutrient availability or temperature shifts (Kaplan et al., 2019).Feeding behaviour activates agonistic INS peptides and suppresses antagonist ones in the worm's neuron and the intestine, while starvation reverses this pattern (Fig. 4) (Chen and Baugh, 2014).

Sequestration of INS peptides by the DAF-2 receptor isoform, DAF-2B
In both D. melanogaster and humans, there are insulin-binding proteins that regulate insulin availability by preventing its binding to the insulin receptor (Allard and Duan, 2018;Okamoto et al., 2013).Unlike these species, C. elegans lack these insulin-binding protein, which might explain the evolution of antagonistic INS peptides in these worms.In addition to antagonistic INS peptides, C. elegans employs another significant insulin signaling control mechanism through the DAF-2B isoform of the insulin receptor.daf-2b encodes the extracellular ligand binding domain but lacks the intracellular signaling domain.By binding to free INS peptides, DAF-2B receptor effectively reduces their availability, thereby diminishing DAF-2/IR triggered IIS activation.This decoy receptor mechanism is crucial for modulating insulin signaling in response to environmental cues (Martinez et al., 2020).

INS peptide tissue-specific expression
To map the distribution of INS peptides, researchers have utilized transcriptional GFP (Green Fluorescent Protein), identifying six major tissue types of their expression: the nervous system, muscle, reproductive tissue, epithelia, the alimentary system, and coelomocytes.Most tissues express multiple INS peptides (Li et al., 2003;Pierce et al., 2001;Ritter et al., 2013).While the nervous system is a common site for INS peptide expression, certain peptides such as INS-2, INS-27, and INS-33, do not express in the nervous system (Table 1).

Physiological functions of C. elegans INS
The IIS pathway regulates a multitude of biological processes including cell division, metabolism, growth and development, lifespan, and learning and memory.This section highlights the crucial roles of specific C. elegans INS peptides in various biological processes.

Metabolism
In humans, insulin primarily regulates blood glucose levels.Similarly, in C. elegans, excessive glucose intake leads to increased INS-1 expression in the BAG neuron, activating the IIS pathway to metabolize intestinal fat.Conversely, worms with ins-1 mutations show more fat storage when fed excessive glucose (Handley et al., 2022).High glucose conditions also cause INS-7 to induce mitochondrial-induced ROS and glycation, resulting in neuronal damage, impaired motility, and reduced lifespan (Lee et al., 2009;Mendler et al., 2017).Adapted from (Kaplan et al., 2015;Chen and Baugh, 2014).

L 1 arrest survival
When C. elegans hatches in the absence of food, it enters a dormant state known as L1 arrest.Typically, these L1 arrested worms can survive up to 21 days, resuming their developmental cycle upon food availability (Fig. 2).The downregulation of the IIS pathway is crucial for L1 arrest survival.For example, loss of function mutations in daf-18, a gene analogous to the human PTEN tumor suppressor, results in a constitutively active IIS pathway, reducing L1 survival to about 3-5 days.A study by Zheng et al. (2018a)

Table 1
Expression patterns of INS peptides in C. elegans across six major tissue types: nervous system, muscle, reproductive, epithelia, alimentary system, and coelomocytes (Li et al., 2003;Pierce et al., 2001;Ritter et al., 2013).providing an alternative explanation for the observed differences.
While the approaches of Zheng et al. (2018a) and Chen and Baugh (2014) identified potential INS peptide agonists and antagonists, the challenge is to identify which of these are the natural peptides that regulate the L1 arrest survival (Fig. 5).INS peptides identified in both approaches gives more confidence for future tests.INS-4 and DAF-28 were both identified as agonists by both approaches and are endogenous peptides as they play redundant roles as the ins-4; daf-28 double mutant causes a significant increase in L1 arrest survival, in contrast single mutants had no effect on L1 arrest survival (Chen and Baugh, 2014).

Germline development
Although INS peptides are predominantly expressed in the nervous system, they can exert cell non-autonomous effects on germline development (Zheng et al., 2018a).Activation of the IIS pathway is known to increase both the size and number of germline cells (Jorgensen and Tyers, 2004).Specifically, the agonistic INS peptides: INS-3 and INS-33, primarily expressed in neurons and the epidermis respectively, are required in the soma to promote germline development.Diminished activity of either INS-3 or INS-33 lead to similar phenotypes in germline proliferation, and reducing the activity of both does not further exacerbate the phenotype.This observation suggests that INS-3 and INS-33 may not function redundantly, but rather work in conjunction, sequentially, or potentially interact with receptors other than DAF-2/IR (Michaelson et al., 2010).
Additionally, the agonistic INS-6 peptide is implicated in germline development.Chemosensory neurons are thought to promote the early onset of oogenesis by increasing IIS via INS-6 expression.This mechanism indicates that C. elegans may integrate sensory responses to food signal and relay these through INS-6 signaling to trigger oogenesis (Mishra et al., 2023).Therefore, in C. elegans, INS peptides play a significant role in linking environmental cues to physiological processes like germline development.

Dauer formation
Dauer formation in C. elegans represents a distinct developmental phase that is triggered by unfavourable environmental conditions, such as high temperatures, high population density, and low food availability (Fig. 2) (Cassada and Russell, 1975).In this alternative life stage, the nematode arrests its development, and reallocate resources towards survival instead of development.A unique INS mutant, daf-28 (sa191), can induce dauer arrest even under fed conditions, like the daf-2 insulin receptor mutants (Hung et al., 2014;Malone and Thomas, 1994).The daf-28 (sa191) allele encodes a missense mutation, DAF-28 R37C, that disrupts a proteolytic cleavage site necessary for processing DAF-28 proinsulin into mature insulin (Li et al., 2003).Among the other INS peptides in C. elegans, INS-4 and INS-6 are the most like DAF-28 and suggest they could function similarly to DAF-28.Indeed, high doses of DAF-28, INS-4 or INS-6, can suppress the daf-28 (sa191) R37C-induced dauer arrest, suggesting that DAF-28 R37C acts as a dominant negative, that competes with the activity of DAF-28, INS-4 and INS-6 (Li et al., 2003).
While no single INS mutation fully recapitulates the daf-2 loss of function receptor phenotype, the combined removal of INS-4, INS-6, and DAF-28 leads to a fully penetrant constitutive dauer formation, akin to the loss of DAF-2/IR.This suggests that INS-4, INS-6, and DAF-28 are the main agonistic INS peptides preventing dauer formation (Hung et al., 2014).Conversely, simultaneous deletion of two antagonistic INS peptides, INS-1 and INS-18, impedes dauer formation, indicating their involvement in regulating dauer entry (Hung et al., 2014)  as an antagonist for DAF-2/IR (Fernandes De Abreu et al., 2014;Matsunaga et al., 2012).
Using an overexpression approach, 19 INS peptides (INS-1, INS-2,  significantly reduced dauer formation, aligning with their roles as IIS agonists for dauer exit (Zheng et al., 2018a).Conversely, Twelve INS peptides  led to higher dauer penetrance than wildtype consistent with these INS peptides acting as an IIS antagonist (Zheng et al., 2018a).

Learning and memory
C. elegans serve as excellent model organisms for studying the impact of INS peptides on neurodevelopment due to its simple nervous system, comprising of only 302 neurons with a complete connectivity map (White et al., 1986).C. elegans INS peptides are predominantly expressed and regulated in sensory neurons and interneurons, and plays a robust role in modulating neural plasticity, affecting the animal's learning, and memory capabilities (Pierce et al., 2001;Ritter et al., 2013).
C. elegans can learn to avoid harmful environments such as pathogenic bacteria.The INS-11 peptide, expressed in the intestine, negatively regulates aversive learning behaviour by inhibiting abnormal activation of IIS in neurons that stimulate this behaviour, helping the nematodes to adjust their behaviour in the presence of pathogenic and non-pathogenic microbes (Lee and Mylonakis, 2017).Additionally, pathogen avoidance involves a delicate balance of IIS between INS-16, expressed in the pheromone-sensing Amphid Dual Ciliated Ending L (ADL) neuron, and INS-4, expressed in the chemosensory Amphid Wing A (AWA) neuron.In crowded conditions, C. elegans releases a pheromone mixture that activates the ADL neuron, increasing INS-16 expression.The upregulation initiates an "INS-to-INS" regulation to antagonize AWA neuron activity and reduce INS-4 expression, thereby impairing learning processes and increasing the likelihood of pathogen ingestion.This repression of aversion behaviour is also associated with enhanced pathogen resistance, indicating an alternative adaptive strategy in scenarios where pathogen avoidance is not feasible.Meanwhile INS-4 may also inhibit INS-16 to promote learning, creating a delicate balance between the two INS peptides.The research suggest that INS-4 and INS-16 here have both agonistic and antagonistic effect on daf-2, depending on their expression level.The balance is complicated as deleting either ins-16 or ins-4 disrupts learning, deleting both rescues learning defect to the wildtype level.Similarly, increasing either of the INS peptide would also disrupt learning, and overexpressing both would restore learning to wildtype level.The critical balance between INS-16 and INS-4 demonstrates a complex mechanism regulating learning in pathogenic avoidance behaviour (Wu et al., 2019).
Research has highlighted the critical roles of INS-6 and INS-7 in neural plasticity regarding pathogen avoidance learning.INS-6, an agonist of IIS is produced in the environment-sensing neuron (Amphid Single Cilium I (ASI) neuron), may facilitate learning by antagonizing INS-7 transcription in the Unknown Receptor, not Ciliated X (URX) neuron through a paracrine dependent mechanism.Reduced INS-7 production in the URX neuron is essential for facilitating learning, as INS-7 may hinder learning by antagonizing DAF-2/IR in the Ring Interneuron A (RIA) postsynaptic neuron that is responsible for learning aversive behaviour triggered smell.When the IIS pathway is inhibited by INS-7, it suppresses developmental and neural plasticity (Chen et al., 2013).Sometimes inhibition of C. elegans' aversive learning is necessary, to prevent the worm from dying of starvation.Conversely, when enhanced learning is required, INS-6 represses INS-7 expression, activating the IIS in RIA interneuron to induce neuroplasticity.(Chen et al., 2013).
C. elegans has been instrumental in understanding memory mechanisms.They can undergo classical conditioning for learning and memory, as they can learn to associate salt, with the absence of food.After prolonged exposure to NaCl in absence of food, C. elegans would show aversive behaviour from NaCl.INS-1, secreted from the Anterior Interneuron A (AIA), plays a crucial role in this associative memory by antagonizing the DAF-2/IR in the salt sensing neuron: Amphid Single Cilium E Right (ASER) (Oda et al., 2011).Additionally, INS-1 may regulate taste-associated avoidance learning by binding to DAF-2 isoforms (DAF-2A, DAF-2C) and can act as both an antagonist for DAF-2A or an agonists with DAF-2C to regulate behaviour plasticity independent of DAF-16/FOXO (Tomioka et al., 2022).Furthermore, INS-1 is essential for thermotactic plasticity in adult worms, playing a key role in memory associated with food and temperature conditioning.In this experiment, a well-nourished worm within a thermal gradient would move towards a temperature at which it was previously conditioned with food.INS-1 plays a crucial role in regulating this memory by inhibiting IIS activity (Kodama et al., 2006).

Longevity
Lifespan extension in C. elegans can be attained through the reduction of IIS, such as via loss-of-function mutations in the daf-2 insulin receptor gene (Kaletsky and Murphy, 2010;Murphy et al., 2003).Similar to mammals, C. elegans can extend their lifespan through dietary restriction, and it is likely that INS peptides play a role in this process (Kaletsky and Murphy, 2010;Van Heemst, 2010).In both in humans and C. elegans, reduced IIS has been linked to increased resistance against various stress factors, such as thermal and oxidative stress, leading to an increase in lifespan (Murphy et al., 2003).
To determine what tissues, require IIS for regulating longevity, researchers utilized an auxin-inducible system to trigger tissue-specific degradation of DAF-2/IR.It is found that the loss of IIS in neurons or the intestine is associated with lifespan extension in C. elegans.In contrast, the loss of IIS in the epidermis, germline, and muscle does not significantly affect the worm's longevity (Roy et al., 2022).This finding indicates that the nervous system and intestinal tissues plays crucial roles in the IIS pathways for C. elegans longevity.

Conclusions
In our review, we investigated the 40 INS peptides in C. elegans, shedding light on their structural characteristics, processing methods, and regulatory controls.Additionally, we have provided a variety of functional roles for C. elegans INS signaling.However, the functions of most of these INS peptides remain to be fully elucidated (Fig. 6).
A major challenge has been due to redundant functions of these INS ligands.Over-expression approaches have provided some insights to how these INS peptides might function, but there are caveats, and different approaches have led to contradictory findings.This is not too surprising as the C. elegans INS peptides can have agonistic or antagonistic roles depending on the context.Further research will be needed to identify the endogenous INS involved in the various biological processes.The current research has illuminated some aspects of the complex interplay between INS-to-INS in C. elegans, exemplified by the reciprocal inhibition observed between INS-6 and INS-7, where each peptide suppresses the expression of the other.While we have gained valuable insights into the roles of these peptides, many questions remain unanswered.For example, do these INS peptides work alone or in a combinatorial fashion?Additionally, it would be particularly intriguing to investigate the possibility of INS combinations acting synergistically or antagonistically to modulate the IIS pathway.The large number of INS peptides in C. elegans is still a mystery.One possibility is that C. elegans uses all these INS peptides to fine-tune responses to its changing environment.Alternatively, the redundancy of these INS peptides may provide a mechanism for robustness in the face of environmental stress.Furthermore, the functions of the processed peptides such as the C and F peptides remain to be fully elucidated.It is also unclear how a single insulin receptor, DAF-2/IR, can lead to differential signal outputs from these INS.What are the alternative pathways to the canonical IIS pathway triggered by INS peptide signaling?And how can an INS peptides switch its activity?For example, INS-7 and INS-18 act as agonists for L1 arrest regulation while they act as antagonists for dauer regulation (Zheng et al., 2018a).C. elegans INS peptides are also novel as they possess INS peptides that work as antagonists.Future research will shed light on how these antagonistic INS peptides function and what sequences make an antagonistic INS peptide.The information from C. elegans research will be useful to engineer novel antagonistic INS peptides.Hyperinsulinemia is associated with a range of conditions including, Type 2 diabetes, cancer and an increased risk of Alzheimer's disease (de la Monte, 2009;De La Monte and Wands, 2008;Ferreira et al., 2018;Garrido, 2002;Keowkase et al., 2010;Pinkston-Gosse and Kenyon, 2007;Suzawa and Bland, 2023).Consequently, the use of antagonistic human insulins offers a potential therapeutic intervention for diseases with increased INS signaling.In summary, there is much to be learned about the complex roles of INS peptides in C. elegans.Future research on this invertebrate model organism can help us understand IIS not only in C. elegans but also in other organisms, including humans.

Declaration of generative AI and AI-assisted technologies in the writing process
During the preparation of this work the authors used OpenAI.(2024)/ChatGPT4 to improve language and readability.After using this OpenAI.(2024)/ChatGPT4, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Comparison of C. elegans, D. melanogaster and Human insulin-like peptides (INS) and signaling pathways.C. elegans have 40 INS peptides (INS 1-39 and DAF-28) that are both agonists and antagonistic peptides.The human genome encodes 10 insulin-like peptides.Insulin and IGF-1/2 bind and activate the insulin and IGF-1/2 receptors (IR, IGFR) while the other insulin-like peptides bind unique receptors.In D. melanogaster DILP 1-7 bind to and activated the insulin receptor (dInR).DILP 8 does not bind dInR and instead binds to a G-coupled receptor, LGR 3. The canonical signaling pathway is evolutionary conserved with the terminal transcription factor FOXO being inhibited.DAF-18/PTEN negatively regulate IIS by reversing the activity of AGE-1/PI3K.

Fig. 2 .
Fig. 2. C. elegans life cycle.Regulation of IIS pathway plays a crucial role in various stages of the C. elegans life cycle, including two growth suppression states: L1 arrest (an arrested life stage that occurs when C. elegans hatch in absence of food) and the dauer larva stage (an arrested life stage that occur with limited food availability, high population density, high temperature or other stresses during the late L1 to early L2 stage).In addition, IIS can inhibit adult longevity.
Fig. 3. Proinsulin domain structures of the C. elegans INS gene family.The INS peptides are divided into 3 categories -α, β, and γ-based on their disulfide bonds exhibited by (S) bracket lines.INS-1, and INS-18 are the most similar to human insulins, featuring a C peptide.INS-2 to 9, INS-19, INS-32, and DAF-28 possess an additional F peptide, potentially serving modulatory role akin to the E peptide in human IGF-1 and IGF-2.INS-31 is unique, containing multiple peptide sequences.Different proprotein convertase enzymes are involved in cleaving proinsulin, a crucial step in converting proinsulin into mature insulin.

R
. Zhu and I.D. Chin-Sang antagonists to shut down the IIS pathway.Using a temporal based analysis of mRNA expression for all 40 insulin-like genes during the initiation and recovery phases of L1 arrest.Chen and Baugh (2014) identified thirteen candidate agonists (INS-3, INS-4, INS-5, INS-6, INS-9, INS-21, INS-22, INS-26, INS-27, INS-28, INS-33, INS-35 and DAF-28) and eight candidate antagonists (INS-10, INS-11, INS-16, INS-17, INS-20, INS-24, INS-25 and INS-29) based on expression in response to nutrient availability (Fig. 5) (Chen and Baugh, 2014).These insulin-like genes are potential regulators of L1 development in C. elegans.Chen and Baugh (2014) primarily focus on the mRNA expression of 40 INS peptides during entry and recovery phases of L1 arrest, identifying potential agonistic and antagonistic candidates based on their expression patterns in response to nutrient availability.In contrast, Zheng et al. (2018a) examines a range of phenotypes, including L1 arrest, dauer formation, and fat accumulation.The distinct phenotypes analyzed by these studies could significantly contribute to the differences in their findings.Additionally, the differential expression of INS peptides in various tissue locations might lead to disparate phenotypic outcomes.Another factor to consider is the possibility of post-transcriptional and post-translational modifications of INS peptides, which might influence their function and interaction with receptors differently in the two studies.Furthermore, the distinct environmental conditions or experimental setups used in each study could lead to variations in the expression and function of these peptides, . According to previous research, INS-3, INS-5, INS-10, INS-11, INS-12, INS-14, INS-21, INS-22, INS-26, INS-33, and INS-35 also behaves as agonist of DAF-2/IR (Fernandes De Abreu et al., 2014).While INS-3, INS-12, and INS-17 act

Fig. 5 .
Fig. 5.A comparison of L1 arrest INS peptides classification between (Zheng et al., 2018a; Chen and Baugh, 2014).(A) INS agonist (B) INS antagonist.INS peptides identified using both approaches are likely to be the endogenous INS involved in L1 arrest regulation.

Fig. 6 .
Fig. 6.The involvement of INS peptides in various physiological and developmental roles in C. elegans.The colour scheme is used to indicate changes in IIS.Specifically, blue represents an increase in IIS, red denotes a decrease in IIS, and black indicates a scenario where IIS can be both increased or decreased IIS.This colour coding also extends to the INS peptides themselves: blue for agonistic INS peptides, red for antagonistic peptides, and black for peptides that exhibit diverse or mixed roles in regulating IIS.