A homeostatic gut-to-brain insulin antagonist restrains neuronally stimulated fat loss

Liu, Chung-Chih; Khan, Ayub; Seban, Nicolas; Littlejohn, Nicole; Shah, Aayushi; Srinivasan, Supriya

doi:10.1038/s41467-024-51077-3

A homeostatic gut-to-brain insulin antagonist restrains neuronally stimulated fat loss

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Published: 11 August 2024

Volume 15, article number 6869, (2024)
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A homeostatic gut-to-brain insulin antagonist restrains neuronally stimulated fat loss

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Abstract

In C. elegans mechanisms by which peripheral organs relay internal state information to the nervous system remain unknown, although strong evidence suggests that such signals do exist. Here we report the discovery of a peptide of the ancestral insulin superfamily called INS-7 that functions as an enteroendocrine peptide and is secreted from specialized cells of the intestine. INS-7 secretion is stimulated by food withdrawal, increases during fasting and acts as a bona fide gut-to-brain peptide that attenuates the release of a neuropeptide that drives fat loss in the periphery. Thus, INS-7 functions as a homeostatic signal from the intestine that gates the neuronal drive to stimulate fat loss during food shortage. Mechanistically, INS-7 functions as an antagonist at the canonical DAF-2 receptor and functions via FOXO and AMPK signaling in ASI neurons. Phylogenetic analysis suggests that INS-7 bears greater resemblance to members of the broad insulin/relaxin superfamily than to conventional mammalian insulin and IGF peptides. The discovery of an endogenous insulin antagonist secreted by specialized intestinal cells with enteroendocrine functions suggests unexpected and important properties of the intestine and its role in directing neuronal functions.

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Introduction

The central nervous system is known to play a major role in governing systemic lipid homeostasis across species^1,2,3. It is also now understood that endocrine hormones signal from organs in the periphery to relay fed- and fasted state information across the body^4,5, including to the nervous system^6,7. For example, the intestine has emerged as a preeminent sensory and metabolic organ that relays internal state information via gut hormones to the brain and other important organs in the body^8,9,10,11. It is plausible that many additional endocrine signals relaying interoceptive information to the nervous system remain undiscovered. In the roundworm C. elegans, the nervous system plays important roles in regulating fat homeostasis in the intestine¹², the predominant organ in which lipids are stored and metabolized¹³. Several sensory neurons and the circuits in which they operate are known to regulate the rate and extent of fat utilization. Because these neurons detect and respond to distinct sources of sensory information from the environment, it has become possible to ascribe roles for individual sensory modalities in regulating lipid homeostasis. Previous work from our group has shown that oxygen-sensing (in the normoxic range) via the URX and BAG neurons¹⁴, population density-sensing via the ADL neurons¹⁵, and bacterial food sensing via the serotonergic ADF neurons strongly influence lipid stores¹⁶. These and other studies^17,18 have shown that C. elegans calibrates lipid metabolism to the sensory environment in order to optimize behavior, physiology, and lifespan.

An interesting feature of the C. elegans nervous system is that sensory neurons do not directly innervate the intestine^16,19 although some neurons do access the coelomic fluid, allowing a conduit by which information can transfer across tissues. To address the question of how sensory information from the nervous system is relayed to the intestine, in previous efforts we had conducted a genetic screen that uncovered a brain-to-gut neuroendocrine peptide called FLP-7, the ancestral ortholog of the mammalian tachykinin family of peptides. The FLP-7 neuropeptide is secreted from neurons and is detected by the G protein-coupled receptor NPR-22, ortholog of the mammalian Neurokinin 2 Receptor (NK2R), which resides in the intestine in which it is necessary and sufficient to trigger fat loss²⁰. We developed an assay to measure FLP-7 secretion in living animals and showed that FLP-7 is released by neurosecretory cells called ASI in proportion to fluctuations in the functions of serotonergic and octopaminergic neurons²⁰. Additionally, FLP-7 secretion is also modulated by the oxygen-sensing URX neurons and the population density-sensing ADL neurons (Supplementary Fig. 1a–d). Thus, the FLP-7/NPR-22 neuroendocrine axis represents the final common brain-to-gut pathway by which the sensory nervous system relays information to the intestine for the regulation of lipid metabolism¹².

Over the course of these studies, we contemplated the possibility that the intestine relayed metabolic information back to the nervous system. Tantalizing evidence from us and others suggested that the C. elegans intestine communicates with distinct organs including regulation of pathogen avoidance²¹, avoidance of aversive stimuli^22,23, regulation of rhythmic behaviors²⁴, serotonin production and signaling²⁵, and oxygen sensing²⁶. However, no clear gut-to-brain molecules regulating metabolic functions had been found, and it was not clear whether the C. elegans gut itself secreted enteroendocrine peptides. One barrier to identifying such molecules was the lack of readouts of neuronal function and output that are amenable to genetic screens. To test whether the FLP-7 secretion assay could prove to be a metabolically relevant experimental handle that could circumvent these limitations, we measured the extent to which FLP-7 secretion from ASI neurons (hereafter called FLP-7^ASI) could be modulated by depleting intestinal fat stores. Inactivation of genes across distinct lipid synthesis pathways previously shown to deplete intestinal fats^26,27,28 (pod-2, acyl CoA Carboxylase; sbp-1, Sterol Regulatory Element Binding Protein; elo-2, Fatty Acid Elongase) resulted in increased steady-state FLP-7^ASI secretion (Supplementary Fig. 1e). Prompted by this observation, we undertook an investigation to identify and define the molecular features underlying gut-to-brain information relay.

Results

Discovery of INS-7 as a gut peptide

We reasoned that in the context of lipid metabolism, a molecule that relays internal state information might originate in the intestine since it is the major somatic depot for stored lipids in C. elegans. To find such a signal, we conducted an intestine-specific RNAi-mediated screen of the family of genes encoding small peptides for alterations in FLP-7 secretion from ASI neurons (Fig. 1a). The gene encoding a peptide called ins-7 emerged as the most potent hit from the screen (Fig. 1b, c); a mutant allele tm1907 that lacks the bulk of the gene including the F peptide domain, and our newly generated CRISPR null allele ssr1532 (Fig. 1d–f) recapitulated the effect of the RNAi: absence of ins-7 increased FLP-7^ASI secretion nearly 2-fold (Fig. 1g, h). INS-7 belongs to the broad insulin/relaxin superfamily of peptides; in C. elegans there are 40 members (10 in humans) that are thought to be distinguished by their developmental stage- and tissue-specific expression patterns^29,30. C. elegans ins-7 has been reported to be one of the few ins genes expressed selectively in adults³⁰; a second gene called ins-19 that has adult-specific expression did not yield a phenotype in the FLP-7^ASI secretion assay.

**Fig. 1: Discovery of *ins-7* as a FLP-7-regulating gene.**

An extrachromosomal ins-7::GFP reporter line and an endogenous transcriptional GFP reporter we generated revealed robust expression restricted to the first quartet of intestinal cells (Fig. 2a and Supplementary Fig. 2). Called INT1, these anatomically specialized cells define the anterior-most section of the upper intestine, just posterior to the terminal bulb of the pharynx³¹. This intriguingly restricted expression pattern is in stark contrast to the many metabolic genes reported by us and others, which are broadly expressed throughout all the cells of the intestine (INT1-9, 20 cells)^16,27,28,32. While the intestine-specific RNAi experiment suggested the necessity of ins-7 expression in INT1 cells, to determine whether ins-7 expression in INT1 cells is sufficient to rescue the aberrantly increased FLP-7^ASI secretion, we generated transgenic rescue lines in which ins-7 gene expression was restored in ins-7(ssr1532) null mutants under the ins-7 endogenous promoter, the pan-intestinal promoter Pvha-6, as well as a newly developed INT1-specific promoter, Pges-1ΔB (Fig. 2b–e). We found that heterologous ins-7 expression in INT1 cells alone was sufficient to confer complete rescue of FLP-7^ASI secretion to the same extent as restoring ins-7 expression more broadly across the intestine (Fig. 2b–e).

**Fig. 2: INS-7 functions from the first pair of intestinal cells to regulate FLP-7^ASI secretion.**

Further, INT1-specific ins-7 RNAi increased FLP-7^ASI secretion (Fig. 2f and Supplementary Fig. 1f), and overexpression of ins-7 in INT1 cells led to suppression of FLP-7^ASI secretion (Fig. 2g), suggesting that INS-7 functions as an instructive cue from the intestine that is necessary and sufficient to inhibit FLP-7^ASI secretion from neurons. INT1 cells were first defined in the context of the developmental biology of the C. elegans intestine: they have shorter microvilli than the INT2-9 conventional epithelial enterocytes, not unlike the enteroendocrine, goblet and immune M cells of the mammalian intestinal epithelium^31,33,34. Their location as the anterior-most cells of the intestine would also suggest privileged anatomic access to incoming nutrients.

We wished to define additional phenotypes of ins-7. In previous studies, we had shown that stimulating FLP-7 secretion from ASI neurons activates its cognate receptor NPR-22 in the intestine, whose activity governs fat loss and increases energy expenditure in the intestine via transcriptional activation of the triglyceride lipase ATGL-1^16,20,35. Thus, any condition that increases FLP-7^ASI secretion would be predicted to trigger the hydrolysis of fat stored in the intestine via ATGL-1, accompanied by increased energy expenditure. Accordingly, we found that ins-7 null mutants that display increased FLP-7^ASI secretion (Fig. 1g, h) had significantly decreased fat stores (Fig. 3a and Supplementary Fig. 3a). This decrease in fat stores was fully dependent on the presence of the flp-7 gene, because ins-7;flp-7 mutants suppress the effect of ins-7 on intestinal fat loss (Fig. 3a). Next, we selectively inactivated flp-7 in the ASI neurons via antisense inhibition and found that the fat phenotype of ins-7 mutants requires the presence of flp-7 in the ASI neurons (Fig. 3b). Further, ASI-specific flp-7 rescue in the ins-7;flp-7 double mutants was sufficient to drive fat loss to the extent in the ins-7 mutants (Fig. 3c). Thus, the fat phenotype of ins-7 mutants is entirely dependent on flp-7 from ASI neurons. The decreased fat stores in the intestine were corroborated by the expected increase in maximal energy expenditure in the ins-7 mutants as judged by oxygen consumption, which was also dependent on flp-7 (Fig. 3d, e). As predicted, the decreased intestinal fat stores of ins-7 nulls were dependent on atgl-1 induction (Fig. 3f, g), and this increase in atgl-1 expression was fully dependent on the presence of the flp-7 gene (Fig. 3h). Further, RNAi-mediated inactivation of atgl-1 abrogated the ins-7 fat phenotype (Fig. 3i). The effects of ins-7 on intestinal fat stores were not dependent on food intake or locomotion, which were not altered in ins-7 mutants (Supplementary Fig. 3b–d). To corroborate a role for INT1 cells in INS-7-mediated fat loss, we generated an independent set of ins-7 transgenic rescue lines using the promoters described in Fig. 2 and measured intestinal fat. We observed complete restoration of the decreased intestinal fat stores of ins-7 null mutants in all cases, again suggesting that ins-7 functions from INT1 cells to regulate fat stores across the broader intestine (Fig. 3j–l). Finally, overexpression of ins-7 in INT1 cells resulted in a small but significant increase in fat storage throughout the intestine (Fig. 3m).

**Fig. 3: Role of INS-7 in intestinal fat metabolism.**

Mechanism of INS-7 action

Our data support a model in which ins-7 functions in the INT1 cells of the intestine to negatively regulate FLP-7^ASI secretion from neurons, to regulate fat storage throughout the intestine (Fig. 4a). This model of interorgan communication led us to consider the mechanism of INS-7 action. Phylogenetic analysis of the ins-7 gene suggests that it belongs to an entirely different clade than that of the conventional mammalian insulin genes, IGF1 and IGF2, which encode proteins that function as agonist ligands for the insulin and IGF receptors³⁶ (Fig. 4b). To investigate the mechanism of ins-7 action, we examined its relationship to daf-2, the sole insulin receptor of C. elegans³⁷ which single-cell RNAseq efforts reveal to be clearly expression in ASI neurons³⁸. Using the canonical e1370 allele, we found that daf-2 mutants had a dramatic reduction in FLP-7^ASI secretion, a phenotype in opposition to that of ins-7 and ins-7;daf-2 mutants phenocopied daf-2 (Fig. 4c, d). Absence of DAF-16/FOXO, the major downstream target of insulin signaling, did not appreciably alter FLP-7^ASI secretion, however it completely suppressed the effect of the daf-2 mutation (Fig. 4c, d). It was possible that the daf-2 phenotype of reduced FLP-7^ASI secretion resulted from daf-2 expression elsewhere in the nervous system. To address this concern, we conducted antisense inhibition of daf-2 in ASI neurons alone, which phenocopied the global daf-2 mutation (Fig. 4e, f). To prevent the RNAi from spreading to other neurons³⁹, we also performed the daf-2 antisense experiments in a sid-1(qt9) background and found a similar phenotype to that observed in the wild-type background (Fig. 4g). Thus, the loss of daf-2^ASI reduced FLP-7^ASI secretion. In addition, ASI-specific transgenic rescue of daf-2 restored FLP-7 secretion to wild-type levels (Fig. 4h, i). Together, these data were most consistent with ins-7 functioning in opposition to daf-2, that is, as an endogenous antagonist to the DAF-2 receptor in ASI neurons. Previous studies of local effects of INS-7 in the nervous system have also defined its action at DAF-2 as antagonistic⁴⁰, although it may function as an agonist in the periphery^41,42 (see “Discussion” section).

**Fig. 4: INS-7 functions in opposition to DAF-2.**

As suggested by our data, we tested the hypothesis that INS-7 acts as an antagonist for neuronal DAF-2 by conducting a direct test of DAF-2 function: we examined DAF-16 localization within the ASI neurons in various genetic contexts. DAF-16 activity as measured by the ratio of its cytoplasmic-to-nuclear localization (abbreviated as C:N ratio) has long been established as an accurate and sensitive hallmark of DAF-2 function^43,44,45. In wild-type well-fed animals (in which ins-7 and daf-2 are present), DAF-16 resides predominantly in the cytoplasm, such that the DAF-16 C:N ratio is approximately 1.2 (Fig. 5a, b). In daf-2(e1370) mutants in which insulin signaling is diminished, as expected, DAF-16/FOXO translocates to the nucleus such that the C:N ratio drops to 0.5 (Fig. 5a, b). However, in the ins-7(ssr1532) and ins-7^OX worms, we also did not observe any deviation in the C:N ratio of DAF-16 localization relative to wild-type animals (Fig. 5a, b).

**Fig. 5: Subcellular DAF-16 localization reveals role for INS-7 as a fasting-induced antagonist.**

This result prompted a deeper investigation into the conditions in which ins-7 normally functions. Conventional insulin, IGF1 and IGF2 are peptides secreted during abundant food availability and during growth and development. Because the phylogenetic tree suggested that INS-7 belongs to a different clade (Fig. 4b), and the functional role of INS-7 was in opposition to DAF-2, we postulated that as a physiological antagonist to the insulin receptor, INS-7 may function preferentially in the fasted state, and that its temporal functions may not be fully revealed in well-fed animals. Thus, we tested the effects of a 3-h fast (a duration sufficient to deplete about 80% of the fat stores in the intestine²⁶) on DAF-16 localization. Interestingly, in the fasted condition DAF-16 localization in the ASI neurons did not shift from the previously noted fed state ratios between the cytoplasm and the nucleus in wild-types, daf-2(e1370) or ins-7(ssr1532) mutants. However, in the fasted state, worms bearing transgenic overexpression of ins-7 from the INT1 cells (INS-7^OX) showed a significant shift in DAF-16 localization to the nucleus (C:N ratio 0.8; Fig. 5c, d). This result resembles the effect of the daf-2(e1370)-mediated reduction C:N DAF-16 localization (0.5). Furthermore, we found the colocalization of endogenous DAF-2GFP and INT1-expressed INS-7mCherry on the cell surface of ASI neurons in wild-type animals fasted for 3 h, suggesting a direct interaction between the ligand and the receptor under these conditions (Fig. 5e–g). Together, these experiments show that INS-7 functions in an antagonistic capacity in the context of regulating ASI function and output via DAF-2, preferentially in the fasted state.

Multiple lines of evidence show that hundreds of genes containing DAF-16 cis-binding sites function as effectors of the insulin signaling pathway^{42,46,47,48,49,50}. Of these, the AMP-activated protein kinase (AMPK) occupies a central role^51,52, and are also expressed in ASI neurons based on single-cell RNAseq studies³⁸. As noted in our previous work on the role of ASI neurons in regulating FLP-7 secretion²⁰, we found that AMPK α subunit aak-2(ok524) mutants had significantly increased FLP-7^ASI secretion (Fig. 6a, b). To exclude the possibility that the increased FLP-7^ASI secretion in aak-2 mutants resulted from its absence elsewhere in the nervous system, we used antisense inhibition of aak-2 solely in the ASI neurons. Antisense inhibition of ASI-specific aak-2 resembled the global aak-2 mutation (Fig. 6c, d), that is, loss of aak-2^ASI increased FLP-7^ASI secretion in a manner indistinguishable from the global absence of aak-2. Additionally, ASI-specific antisense inhibition of aak-2 increased FLP-7^ASI secretion in daf-2(e1370) mutants to the same extent as it did in the wild-type animals (Fig. 6c, d), thus establishing a mechanistic link between DAF-2, DAF-16 and AMPK in the ASI neurons for the modulation of FLP-7^ASI secretion (Fig. 6e). Together, our results show that INS-7 from specialized INT1 intestinal cells inhibits the functional output of ASI neurons to dampen FLP-7 secretion, which drives fat loss throughout the intestine (INT2-9). This effect occurs via INS-7-mediated antagonism of DAF-2 and its downstream actions via DAF-16/FOXO and AMPK signaling in ASI neurons.

Fig. 6: FLP-7^ASI secretion dynamics during fasting and re-feeding are controlled by *ins-7.*

Dynamics of INS-7 function

The experiments delineating a role for INS-7 as a gut-derived signal were designed to reveal steady-state differences in homeostatic mechanisms in well-fed animals. However, our results from the DAF-16 localization experiment suggested that INS-7^INT1 may regulate FLP-7^ASI differentially in the fed and fasted states. Additionally, a role for INS-7 as an antagonist might indicate a function in opposition to canonical insulin which is a peptide secreted in the post-prandial state⁵³. To test the idea that INS-7 may regulate FLP-7 secretion differentially in the fed and fasted states, we measured the dynamics of FLP-7 secretion in the presence and absence of ins-7. In wild-type animals subjected to food deprivation, an increase in FLP-7 secretion is not discernable until 180 min (3 h; Fig. 6f and Supplementary Fig. 4b), a time point at which greater than 80% of the intestinal fat stores of the animal have been depleted²⁶. Re-exposure to food after 3 h of fasting restores FLP-7 secretion back to baseline levels (Fig. 6g), suggesting that the rise in FLP-7 secretion during the fasting regimen is reset by re-feeding. Notably, in ins-7 null animals (Fig. 6h), the feeding-state-dependent regulation of FLP-7 is abrogated such that FLP-7 secretion levels are chronically high, regardless of feeding or fasting status (Fig. 6h,i) as per the original observation for ins-7 nulls (Fig. 1). Thus, gut-derived INS-7 provides a brake on FLP-7^ASI secretion to inhibit the neuronal drive to trigger fat loss during food shortage.

We investigated whether INS-7 itself is a secreted peptide by generating a transgenic line expressing an INS-7mCherry fusion protein solely from INT1 (pseudocolored yellow; Fig. 7a, b). To our interest, we found that INT1-expressed INS-7 also accumulates in the coelomocytes, the characteristic hallmark of a secreted peptide in C. elegans^20,54,55. Importantly, INS-7 accumulation in coelomocytes does not correlate with their GFP intensity, suggesting that these two parameters are independent of one another (Fig. 7c). Extensive validation of the coelomocyte secretion assay by us and others^20,54 has shown that it faithfully represents the steady-state levels of a given secreted peptide under a broad range of genetic and physiological conditions. Intestine-specific RNAi of aex-4, an intestinal SNARE protein required for vesicular protein secretion⁵⁶ greatly diminished INS-7 secretion, as did asna-1, an ATP hydrolase known to specifically regulate insulin secretion⁵⁷ (Fig. 7d, e). Next, to test whether INS-7, as an antagonist, is secreted under conditions in opposition to canonical insulin, we measured the dynamics of INS-7 secretion in wild-type animals subjected to food deprivation. INS-7 secretion increased within 30 min off food and further increased until 3 h off food (Fig. 7f). Re-feeding restores INS-7 secretion back to baseline levels (Fig. 7g). Thus, INS-7 is a bona fide gut peptide secreted by the INT1 cells of the intestine and acts as a homeostatic signal to restrict FLP-7^ASI secretion during food deprivation. Together, our results define a novel gut-brain pathway that relays intestinal state information during acute food shortage.

**Fig. 7: INS-7^INT1 secretion level is induced by fasting.**

Discussion

Here, we report that a peptide of the insulin superfamily called INS-7 is released from specialized INT1 enteroendocrine cells of the C. elegans intestine. INS-7 functions as an antagonist at the canonical DAF-2 receptor in the ASI neurons to inhibit FLP-7 secretion. Because FLP-7^ASI secretion is a signal that integrates neuronal information to promote fat loss, our results show that the INS-7 gut-to-brain inhibitory peptide serves to limit this signal in the absence of incoming food to the intestine. Work presented here describes the molecular features of our previously postulated hypothesis that homeostatic mechanisms must exist to ensure that neural signals that stimulate fat loss in the intestine are only deployed when there are sufficient fat reserves to do so²⁶. These findings uncover a hitherto unknown mechanism of gut-to-brain homeostatic communication in which C. elegans lipid metabolism balances between external sensory cues and internal metabolic states.

The identification of INT1 cells as specialized secretory cells of the intestine is intriguing. As the foremost cells of the intestine immediately adjoining the pharynx in the alimentary canal³¹, they have privileged access to incoming nutrient information before food absorption has begun. Sulston et al. showed that INT1 cells have shorter microvilli than the rest of the intestinal cells (INT2-9)³¹ and form a collapsible conical reservoir at the intestine anterior, suggesting that INT1 cells may possess unique properties in the context of enteroendocrine INS-7 secretion that remain to be discovered. Based on our results, we propose that the INT1 quartet functions as enteroendocrine cells that receive information not only from the lumen but perhaps also from the rest of the intestine.

Amongst the many peptides we screened for gut-to-brain signals, INS-7 emerged as the primary suppressor of FLP-7^ASI secretion. Initial reports describing a role for INS-7 in longevity regulation had suggested an agonist function at the DAF-2 receptor^41,42, whereas later reports suggested antagonistic functions in olfactory learning^40,58. It is possible that an explanation lies within the expression features of the various DAF-2 isoforms. In the C. elegans nervous system, mutually exclusive expression of DAF-2a and DAF-2c isoforms has been noted. Multiple lines of evidence show that neuronal DAF-2c expression in sensory neurons including the ASI neurons is further induced by fasting and drives the antagonistic effects of DAF-2 signaling in the nervous system^59,60,61. Our results indicate a clear role for INS-7 as an antagonist for DAF-2 in ASI neurons; we postulate that agonism and antagonism of insulin peptides at the DAF-2 receptor is determined in a cell-type-specific manner. In contrast to gut-derived INS-7, neuronally-derived INS-7 has been implicated in innate immunity and pathogen avoidance⁶².

The increase in INS-7 secretion upon acute food withdrawal and fasting is of great interest. During short-term fasting, two additional molecular events occur: the colocalization of INS-7mCherry and DAF-2GFP in ASI neurons and the nuclear translocation of DAF-16GFP in the ASI neurons. These observations suggest that the action of INS-7 on DAF-2 in ASI occurs preferentially in the short-term fasted state. In this way, INS-7 secretion from the intestine could be the origin of the fascinating FOXO-to-FOXO signaling paradigm described in the literature^41,63,64. In this scenario, we would postulate that the origin of the signaling loop would be the secretion event of INS-7 from INT1 cells upon food deprivation. Further experimentation is needed to delve into the effects of INS-7 secretion across the nervous system and the rest of the body, particularly with respect to the opposite effects of DAF-16 and PQM-1 and their key target genes⁶⁵ and across time scales⁶⁶.

Phylogenetic analysis shows that INS-7 may be more orthologous to the mammalian insulin-like peptides and to the relaxin superfamily, rather than the canonical insulin/IGF peptides. Most of the mammalian insulin superfamily members remain poorly characterized, however the insulin-like peptide INSL5 is secreted by L cells of the colon and is a gut hormone thought to be secreted in response to dietary fat intake, and modulates food intake via the hypothalamus^67,68,69. In mammals, gut hormones that play critical roles in the regulation of energy and glucose metabolism or in the modulation of food intake via the gut-brain axis have been identified^70,71. For example, glucagon-like peptide 1 (GLP-1) and cholecystokinin (CCK) are produced in enteroendocrine cells in the intestine and released after nutrient ingestion⁷² and target various brain regions to reduce food intake^73,74,75; long-acting GLP-1 variants are now the basis of medications that effect substantial fat loss via the suppression of food intake and restoring glucose homeostasis in humans^76,77.

In Drosophila, gut-secreted peptide CCHa1 modulates the activity of dopaminergic neurons to suppress the responsiveness to vibration during sleep⁷⁸, and in addition, gut-secreted Hedgehog (Hh) targets taste sensory neurons and suppresses sweet sensation and preference⁷⁹. Interestingly, most gut peptides to date do not directly target fat oxidation in the periphery but instead regulate animal behavior such as food intake, thus indirectly affecting fat metabolism. Instead, we report here that INS-7 acts as a gut-to-brain signal to suppress neuronally stimulated fat loss by inhibiting the secretion of FLP-7 peptide without modulating feeding behaviors. The discovery of intestinal INS-7 as an insulin antagonist that targets neurons that directly regulate fat metabolism defines a new mode of interorgan communication regulated by insulin signaling.

The many uncharacterized insulin family peptides across species suggest that new features of interorgan communication regulated by insulin signaling remain to be discovered. For example, an insulin antagonist ortholog to INS-7 may be secreted and function primarily in the fasted state, in opposition to canonical insulin which signals the fed state. We speculate that such a ‘food absence’ signal from the intestine may help animals optimize their physiology while retaining energy reserves during short-term fasts or between meals. Finally, the discovery of a specialized intestinal cell with enteroendocrine functions suggests that much remains to be learned about the C. elegans intestine and its role in directing neuronal functions.

Methods

C. elegans maintenance and strains

Worms were cultured on nematode growth medium (NGM) agar plates with Escherichia coli strain OP50 at 20 °C as described⁸⁰. The N2 Bristol strain was obtained from the Caenorhabditis Genetic Center (CGC) and used as wild-type. All mutant and transgenic strains used in this study are listed in Supplementary Table 1. Worms were synchronized for Oil Red O staining and oxygen consumption by hypochlorite treatment, after which hatched L1 larvae were seeded on plates with the appropriate bacteria; worms were synchronized for neuropeptide secretion assays and behavioral assays by letting gravid adult worms lay eggs on plates with the appropriate bacteria for 1.5 h. For fasting experiments, young gravid adult worms were transferred with platinum wire without bacteria, to intermediate unseeded NGM plates first after which they were transferred to new unseeded NGM plates for the fasted conditions. All experiments were performed on Day 1 adults. C. elegans studies did not require IRB or IACUC approvals at The Scripps Research Institute.

Cloning and transgenic strain construction

Promoters and cDNAs were amplified using standard PCR techniques from N2 genomic DNA or cDNA and subcloned into expression vectors using Gateway Cloning Technology (Life Technologies). Primers used for cloning in this study are listed in Supplementary Table 2. All transgenic rescue constructs were generated using polycistronic GFP. Transgenic strains were generated by injecting plasmids into the germline of wild-type or mutant worms followed by visual selection of co-injection markers (Punc-122::GFP or Plin-44::GFP) under the fluorescence microscope. To inhibit the expression of daf-2 or aak-2 specifically in ASI neurons, we generated plasmids for antisense-mediated inhibition⁸¹ with the str-3 promoter. Sense and antisense sequences targeting daf-2 or aak-2 were amplified from N2 lysates and subcloned into donor vectors using Gateway Cloning Technology (Life Technologies). The final plasmids for the sense and antisense expression of daf-2 or aak-2 under the str-3 promoter were generated using Gateway Cloning Technology (Life Technologies) and injected into FLP-7 secretion line (SSR1164) at 5 ng/μL (daf-2) or 1 ng/μL (aak-2) each. The FLP-7 secretion line was previously developed and validated²⁰.

For the INS-7 secretion line, N2 worms were injected with 10 ng/μL of the INT1::ins-7mCherry plasmid, 15 ng/μL of a Punc-122::GFP plasmid, and 75 ng/μL of an empty vector to bring the final concentration of injection mix to 100 ng/μL. A transgenic line with high transmission rate and consistent expression was integrated using the Stratalinker UV Crosslinker 2400 (Stratagene) and backcrossed six times before experimentation. For other microinjections, we injected the animals with 5–25 ng/μL of the desired plasmid, 25 ng/μL of Punc-122::GFP or 10 ng/μL of Plin-44::GFP and empty vector to maintain a final injection mix concentration of 100 ng/μL. Two lines were selected for experimentation based on the transmission rate and consistency of expression.

RNAi and qPCR

RNAi experiments were performed as described^82,83. Carbenicillin-IPTG plates were seeded with HT115 bacteria containing the empty vector or the relevant RNAi clone and allowed to grow for four days before seeding larvae. Total RNA was extracted from Day 1 adults using TRIzol reagent (Invitrogen). Genomic DNA was isolated using an RNase-free DNase kit (QIAGEN). cDNA was prepared using iScript Reverse Transcription Supermix for RT-qPCR kit (Bio-Rad) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) following the manufacturer’s instructions. Data were normalized to actin mRNA. Primer sequences used in this study are listed in Supplementary Table 2.

CRISPR-Cas9 gene editing

Guide RNAs for generating the ins-7 null allele were designed using the CRISPR guide RNA selection tool⁸⁴. The dpy-10 guide RNA and repair template were used as reported⁸⁵. All sequences of guide RNAs and repair templates used in this study are listed in Supplementary Table 2. Steps for CRISPR-Cas9 gene editing are briefly described as follows: 0.88 μL tracrRNA (200 μM, IDT), 0.82 μL ins-7 guide RNAs (100 μM, IDT), and 0.12 μL dpy-10 guide RNA (100 μM, IDT) were mixed and incubated at 95 °C for 5 min. 2.52 μL Cas9 protein (IDT, Catalog# 1081058) was added to the mix and incubated at 25 °C for 5 min. 0.6 μL ins-7 repair template (100 μM, IDT), 0.5 μL dpy-10 repair template (10 μM, IDT), and 3.74 μL nuclease-free water were added to the mix and incubated at 25 °C for 60 min. The final injection mix was loaded using a pulled capillary needle (1B100F-4, World Precision Instruments) and injected into the germline of N2 young adults. Screening strategy for isolating ins-7 null mutants was followed as previously described for C. elegans CRISPR allele isolation⁸⁶. The final ins-7 null mutant (ins-7(ssr1532)) used for experiments was backcrossed four times before experimentation.

Guide RNAs for generating the ins-7 endogenous transcriptional GFP reporter (ins-7(ssr1732)) were designed using the CRISPR guide RNA selection tool⁸⁴. The second and third exons of ins-7 were replaced with GFP, which was inframe with the first exon. Steps for CRISPR-Cas9 gene editing are briefly described as follows: 0.88 μL tracrRNA (200 μM, IDT), 0.82 μL ins-7 guide RNAs (100 μM, IDT), and 0.12 μL dpy-10 guide RNA (100 μM, IDT) were mixed and incubated at 95 °C for 5 min. 2.52 μL Cas9 protein (IDT, Catalog# 1081058) was added to the mix and incubated at 25 °C for 5 min. 2.5 μL GFP repair template (1 μM, IDT), 0.5 μL dpy-10 repair template (10 μM, IDT), and 1.84 μL nuclease-free water were added to the mix and incubated at 25 °C for 60 min. The final injection mix was loaded using a pulled capillary needle (1B100F-4, World Precision Instruments) and injected into the germline of N2 young adults.

Oil Red O staining

Oil Red O staining was performed as described¹⁶. Briefly, worms were harvested with phosphate-buffered saline (PBS) and incubated on ice for 10 min before fixation. Worms were then stained in filtered Oil Red O (Thermo Scientific) working solution (60% Oil Red O in isopropanol: 40% water) overnight. Approximately 2000 worms were fixed and stained for all genotypes within a single experiment. For each experimental condition, we visually observed about 100 worms on slides, after which 15–20 worms were randomly chosen for imaging. All experiments were repeated at least three times.

Lipid extraction and quantification

Lipid extraction was performed as described¹⁴. For each group, 2000 worms per 10 cm plate were grown at 25 °C until the worms reached the young adult stage. After washing with PBS twice, the worms were flash-frozen in liquid nitrogen. Worms were homogenized in PBS containing 5% TritonX-100 and a protease inhibitor cocktail (Roche), and lipid was extracted using the TissueLyser II (QIAGEN) or a Dounce homogenizer. Triglyceride content was measured using the EnzyChrom Triglyceride Assay Kit (BioAssay Systems), and triglyceride levels were normalized to the total protein level determined by the Pierce BCA Protein Assay (Thermo Scientific).

Oxygen consumption

Oxygen consumption rates (OCR) were measured using the Seahorse XFe96 Analyzer (Agilent) as described¹⁴. Adult worms were washed with M9 buffer and transferred into a 96-well plate with approximately 10 worms per well. Five basal measurements were taken and then FCCP (50 mM) was injected into each well to measure maximal OCR. Lastly, sodium azide (40 mM) was injected to measure residual OCR. Values were normalized to the number of worms per well. Basal OCR was the average of all measured values prior to the addition of FCCP (50 mM); maximal OCR was the average of the first two measured values after FCCP injection.

Food intake

Food intake was measured by counting pharyngeal pumping, as described⁸³. For each worm, the rhythmic contractions of the pharyngeal bulb were counted over 10 s under a Zeiss M2 Bio Discovery microscope. For each genotype, 25 worms were assessed, and the experiment was repeated at least three times.

Enhanced slowing response

The enhanced slowing response was measured as described⁸⁷. Day 1 adults were washed off OP50 plates with PBS, washed five times to remove bacteria, and placed on NGM agar plates without OP50. After 30 min off food, worms were collected with PBS and seeded onto NGM plates with OP50. Worms were allowed to acclimatize for 5 min, after which the number of body bends per 20 s was counted.

Thrashing assay

Thrashing rate was measured as described⁸⁸. For each worm, a movement in which the head and/or tail swung to the other side and back to the original position was counted as one thrash. 15–20 Day 1 adults were assessed for each phenotype.

DiI and DiD staining

The stock solution containing 2 mg/mL DiI or DiD was prepared in dimethylformamide. L4 animals were incubated overnight in a 1:200 dilution of DiI or DiD stock solution on the OP50 bacterial lawn. The next day, Day 1 adult worms were selected for imaging.

Image acquisition and quantitation

Oil Red O-stained worms were imaged using 10× objective on a Zeiss Axio Imager microscope. Images were acquired with the Software AxioVision (Zeiss). Lipid droplet staining over the whole body of each worm was quantified using ImageJ software (NIH). All reported results were consistent across biological replicates. Fluorescent images of reporters for FLP-7 and INS-7 secretion were acquired with the software AxioVision (Zeiss) using a 20× objective on a Zeiss Axio Imager microscope. The first pair of coelomocytes was imaged. mCherry fluorescence intensity in one of the two imaged coelomocytes was quantified and normalized to the surface area of the coelomocyte (unc-122::GFP) as previously described and validated²⁰. Within each experiment, at least 15 worms from each condition were quantified using ImageJ software (NIH). Fluorescent images of ins-7 expression pattern (Pins-7::GFP) were collected with the software AxioVision (Zeiss) using a 10× objective on a Zeiss Axio Imager microscope. Fluorescent images of DAF-16::GFP and DiI-stained neurons were collected with the software NIS-Elements (Nikon) using a 60× objective on a Nikon A1 confocal microscope. Fluorescent images of DAF-2::GFP, INS-7mCherry, and DiD-stained neurons were collected with software NIS-Elements (Nikon) using a 40× objective on a Nikon Crest X-Light spinning disk confocal microscope.

Statistics

Wild-type animals were included as controls for every experiment. Error bars represent the standard error of the mean (SEM). Student’s t-test, one-way ANOVA, and two-way ANOVA were used as indicated in the figure legends. All statistical analyses were performed using GraphPad Prism 10 (GraphPad Software). Appropriate multiple comparison corrections were used for ANOVAs. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data in the main manuscript and supplementary information are listed in the Source data file. Source data are provided with this paper.

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Acknowledgements

This work was supported by research grants to S.S. from the NIH/NIDDK (R01 DK124706) and NIH/NIA (R01 AG056648). Strains were provided by the National Bioresource Project (Japan) and the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Dr. Anthony Perez of the Srinivasan lab for his critical comments on the manuscript. C.L. was supported by a Dorris Scholar Award from the Dorris Neuroscience Center, The Scripps Research Institute. Elements of Figs. 1a, 7a and Supplementary Fig. 1a were created with BioRender.com.

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Department of Neuroscience and Dorris Neuroscience Center, The Scripps Research Institute, San Diego, CA, USA
Chung-Chih Liu, Ayub Khan, Nicolas Seban, Nicole Littlejohn, Aayushi Shah & Supriya Srinivasan
The Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, San Diego, CA, USA
Chung-Chih Liu

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Chung-Chih Liu
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Ayub Khan
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C.L., Conceptualization, Resources, Data curation, Formal analysis, Investigation, Methodology, Writing - Original Draft, Writing - Review and Editing; A.K., Formal analysis, Investigation; N.S, Resources; N.L., Formal analysis, Investigation, Methodology; A.S., Formal analysis, Investigation; S.S., Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - Original Draft, Writing - Review and Editing, Project administration. All authors read and approved the manuscript.

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Liu, CC., Khan, A., Seban, N. et al. A homeostatic gut-to-brain insulin antagonist restrains neuronally stimulated fat loss. Nat Commun 15, 6869 (2024). https://doi.org/10.1038/s41467-024-51077-3

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Received: 29 September 2023
Accepted: 29 July 2024
Published: 11 August 2024
DOI: https://doi.org/10.1038/s41467-024-51077-3
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A homeostatic gut-to-brain insulin antagonist restrains neuronally stimulated fat loss

From

Abstract

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A tachykinin-like neuroendocrine signalling axis couples central serotonin action and nutrient sensing with peripheral lipid metabolism

Context-dependent regulation of feeding behaviour by the insulin receptor, DAF-2, in Caenorhabditis elegans

Introduction

Results

Discovery of INS-7 as a gut peptide

Mechanism of INS-7 action

Dynamics of INS-7 function

Discussion

Methods

C. elegans maintenance and strains

Cloning and transgenic strain construction

RNAi and qPCR

CRISPR-Cas9 gene editing

Oil Red O staining

Lipid extraction and quantification

Oxygen consumption

Food intake

Enhanced slowing response

Thrashing assay

DiI and DiD staining

Image acquisition and quantitation

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