Volatile diacetyl triggers rapid fmo-2 expression through DHAP-glycerol shunt induction

Reviewer #1 (Public review):

Short overview:

This work demonstrates that exposure to the odor diacetyl in C. elegans first induces the expression of genes that act in the DHAP (dihydroxyacetone phosphate) - glycerol shunt, followed by induction of the flavin-containing monooxygenase fmo-2. This diacetyl exposure also increases the survival of starved animals. However, the mechanism through which diacetyl is sensed by the animal does not involve any of the known receptors of diacetyl, and it is unknown whether any sensory neurons are involved in these responses.

Summary:

This manuscript shows that diacetyl exposure induces metabolic remodeling in C. elegans, where genes in the DHAP (dihydroxyacetone phosphate) - glycerol shunt pathway are activated. This leads to a secondary activation of the flavin-containing monooxygenase fmo-2, which is a longevity-promoting gene upon dietary restriction. The activation of fmo-2 is consistent with the authors' observations that diacetyl exposure increases the survival of starved animals, but not of fed animals. The authors also show that diacetyl exposure improves the animals' resistance to hyperosmotic stress, which is consistent with an increase in glycerol and glycerolipids in these animals. Together, the authors show that a volatile odor or odors can induce gene expression changes that promote metabolic changes and survival under certain environmental conditions.

Strengths:

Through transcriptomics, metabolomics and lipidomics of wild type, with or without diacetyl, and phenotypic analyses of mutant or RNAi-treated animals, the authors delineate a mechanism through which an odor or odors remodel metabolism and increase survival. They show that genes of the DHAP-glycerol shunt are activated during diacetyl exposure, and that these in turn activate fmo-2, which is known to promote longevity under different stressors, like food deprivation. The authors also show that an upstream transcription factor, the co-activator MDT-15, is required for the diacetyl-mediated initial activation of the DHAP-glycerol shunt, and thereby of fmo-2. The involvement of MDT-15 and, to a lesser extent, one of its partner nuclear hormone receptors (NHRs), NHR-49, might also explain the lipidomic changes that accompany diacetyl exposure.

Weaknesses:

The authors tested the involvement of the ODR-10 receptor, which is the known receptor for attractive concentrations of diacetyl, the same concentrations used in this study. Surprisingly, ODR-10 is not required for any of the DHAP-glycerol shunt or fmo-2 induction. In addition, they found that loss of sensory cilia (in the background mutation of the NHR daf-12) enhances diacetyl-mediated fmo-2 induction, which suggests that wild-type sensory cilia inhibit fmo-2 and likely synergizes with the DAF-12 receptor. Considering that chemosensory receptors, like ODR-10, are normally localized to the ciliary endings, their observations suggest a different mechanism through which the animals process this odor. There are at least two possibilities.

One, diacetyl might affect the animals by permeating their cuticles. However, the authors did not test if any neurons are involved in their phenotypes. The AWA neuron, where ODR-10 is expressed, is required to sense attractive concentrations of diacetyl. Thus, what happens when the AWA neurons are ablated or lost?

Two, diacetyl is both light- and temperature-sensitive and can break down into two other odors, acetoin and 2, 3-butanediol. Acetoin is sensed by the chemoattractive AWA neurons (Siddiqui et al, eLife 2024, 101936.1), but not by ODR-10 (Zhang et al., PNAS 1997, vol 94, pp 12162-12167). Thus, this odor would have a different receptor. Although C. elegans chemosensory receptors have been found within the cilia, there is a formal possibility that some of the sensory receptors will also be found on other parts of some sensory neurons, like the dendrites, as in Drosophila (Joseph and Carlson, Trends Genet 2016, vol 31, pp 683-695). Regarding the other diacetyl derivative, 2, 3-butanediol, it has not been shown to elicit any chemosensory responses in C. elegans (Siddiqui et al. eLife 2024, vol 13, RP101936), but 2, 3-butanediol has been linked to the microbiome of fmo knockout mice (Said et al., Metabolomics 2025, vol 21, 170). Thus, it is possible that it is the breakdown products of diacetyl that elicit the responses the authors see.

Finally, the authors' data contradict the observations made by Park et al (Aging Cell 2021, vol 20, e13300). Park et al previously showed that diacetyl exposure reduces the survival of food-deprived animals, although this phenotype is also independent of ODR-10 or of the SRI-14 receptor for aversive concentrations of diacetyl.

Reviewer #2 (Public review):

Short overview:

This study presents potentially important findings showing that DHAP-glycerol shunt involved in energy balance is regulated by food availability in a widely used C. elegans model. The genetic evidence supporting this conclusion is solid and is based on an extensive set of experiments; however, key metabolic measurements and comprehensive metabolic profiling are not provided, limiting the strength of the conclusions about the underlying metabolic and redox changes.

Comments:

Giorda and colleagues report interesting findings demonstrating that the DHAP-Gro3P shuttle is modulated by food availability in C. elegans. Although the authors provide multiple interesting observations in worms, supported by an extensive number of experiments, the metabolic aspect of the study requires additional development. It appears that targeted lipidomics and metabolomics analyses were performed, but the corresponding datasets are largely absent from the manuscript. Only a very limited subset of lipid species is presented in Fig. 2D. What about triglycerides? It would be highly informative to include comprehensive lipidomic profiles covering major lipid classes. A similar concern applies to the metabolomics data. Where are the measurements of Gro3P, DHAP, and glycerol? The authors state that their LC-MS method was unsuccessful and that glycerol levels were ultimately measured using a commercial kit. Given that glycerol production and excretion appear to be major output across many of the experiments presented, this approach is not entirely satisfactory. Reliable GC-MS based methods are available for the quantification of all major components of this pathway, including Gro3P, DHAP, and glycerol (derivatization helps to preserve these species, especially glycerol).

Furthermore, comprehensive LC-MS/GC-MS-based metabolic profiling should be included. Metabolites reported and organized by pathway (e.g., glycolysis, TCA cycle, pentose phosphate pathway) would provide a broader understanding of the metabolic consequences of DHAP-glycerol shunt activation.

Finally, because the DHAP-glycerol shunt is closely linked to cellular redox homeostasis, it would be important to determine how its activation affects intracellular pyridine nucleotide pools, and measurements of NAD+, NADH, NADPH, NADP+ would substantially strengthen the mechanistic conclusions and provide direct evidence for alterations in cellular redox state.

Reviewer #3 (Public review):

Short overview:

This study asks whether the perception of a volatile and attractive cue, diacetyl, leads to changes in metabolic nutrient-responsive programs in C. elegans. Using multi-omic and genetic evidence, they connect the transcriptional response to diacetyl to early activation of the DHAP-glycerol shunt, suggesting worms may activate this metabolic pathway in preparation for food intake. This work also identifies transcription factors involved, and while it does not test whether this response is diacetyl-specific, could identify a conserved pathway of food intake preparation.

Summary:

This study asks whether C. elegans can use a volatile food cue alone, in the absence of ingestion, to anticipate nutrient availability. Using the attractive odorant diacetyl, the authors show that fasting worms rapidly induce the DHAP-Glycerol shunt, a metabolic pathway normally associated with glucotoxicity and hyperosmotic stress, and that this response depends on the transcription factor MDT-15. This drives measurable metabolic rewiring (glycerol and phosphatidylglycerol accumulation) and confers protection against subsequent hyperosmotic stress. Prolonged diacetyl exposure further triggers a second, HLH-30-dependent wave of fmo-2 expression linked to depleted NTP levels, resulting in enhanced heat tolerance and food-seeking behavior upon refeeding. Together, the authors build a testable model for a pathway linking sensory cue detection to gene expression, metabolism, and physiological changes.

Strengths:

The central finding that smell alone without ingestion activates a metabolic-stress adaptation pathway is highly interesting and supported by a convergence of methods including RNA-seq, transcriptional reporters, metabolomics, and functional behavior assays. The transcriptomic time course distinguishes two temporally separable gene expression waves (the shunt at 30 min and fmo-2 at 90 min), and epistasis experiments comparing food status and osmotic stress (Fig. 1i-j) convincingly argue diacetyl acts as a food-predictive cue rather than mimicking osmotic stress. A particular strength is the test of the relationship between the two pathways identified in the study. RNAi knockdown of DHAP-Glycerol shunt enzymes block fmo-2 induction, and pretreatment with salt then switching to food deprivation shows that it is the shunt's activation state that drives fmo-2 expression. This is further reinforced by depletion of energetic mechanism (NTP/ATP). Figure 5 extends this upstream to MDT-15 validated through gene expression, metabolomics, and functional readouts. Throughout the study, transcriptional and metabolic findings are paired with functional outcomes such as hyperosmotic protection, heat stress resistance, or survival, strengthening the paper's model. Together, the authors largely achieve their aim of establishing that a food-related olfactory cue is sufficient to trigger an anticipatory metabolic and transcriptional program. The core claim that diacetyl sensing activates the shunt and subsequent fmo-2 induction and physiological protection is well supported by convergent genetic and biochemical experiments.

Weaknesses:

The authors show that diacetyl-induced fmo-2 induction does not require canonical olfactory sensing since neither diacetyl receptor mutants (odr-10, sri-14) nor a cilia-deficient strain (daf-19; daf-12) blocked the response. However, the pathway characterized here is defined almost entirely through diacetyl, and it remains unclear whether the anticipatory response reflects general food-predictive olfaction or a diacetyl-specific effect. This distinction is important because the authors' central hypothesis is whether volatile food cues in general can be used to anticipate nutrient availability. Testing a limited number of additional attractive odorants, ideally sensed through distinct chemosensory receptors, for a limited number of phenotypes, would establish whether this pathway is generalized to food-predictive smells or only diacetyl. This would increase the impact of the work. Identifying the mechanism(s) for diacetyl perception would as well, but is much more challenging and less likely to be feasible in this work.

The RNA-seq and reporter data disagree on when fmo-2 induction begins, and thus do not fully support that there are two temporally distinct waves. RNA-seq (sampled at 5, 15, 30, and 90 min) shows fmo-2 is not a significantly differentially expressed gene at 30 min and only reaches significance at 90 min, while the fmo-2 transcriptional reporter (Fig.S3a) shows detectable induction as early as 30 minutes. Other readouts in the paper use the reporter for fmo-2 but qPCR for shunt genes, further muddling the timing since mRNA should precede reporter visualization. Since reporter signal generally lags transcript level detection, this discrepancy could be further clarified through a time-course qPCR (like what was tested for the shunt genes) between 30 and 90 minutes. This would help establish whether the two waves are separated by time or whether this reflects a difference in detection method.

Minor weaknesses:

In Figure 1i, gpdh-1 induction is compared between control and 200 mM NaCl (3 hr exposure), with food present or absent. While this directly tests food status, a 3-hour exposure approaches the ~5-6-hour window previously shown to be sufficient for salt-food associative learning to form (worms move toward the high-salt side of a gradient plate if pretreated with high salt and food). This raises the possibility that, in the food-present condition, part of the measured gpdh-1 response could reflect an emerging learned association between salt and food forming during the assay itself, rather than purely reflecting an interaction between nutrient status and osmotic stress signaling. Testing a shorter exposure window (e.g., under 1 hour, as used for the diacetyl exposure in panel j) would help clarify this.

Thrashing is used throughout the paper as the primary readout for hyperosmotic stress resistance, including the central result that diacetyl protects against subsequent hyperosmotic stress (Fig. 2g). However, the authors don't explain why this was chosen as the main stress-resistance readout.

nhr-49 knockdown reduces gpdh-1 induction in diacetyl-mediated hyperosmotic protection but has no effect on development or survival on sustained hyperosmotic stress, raising the question of how the role of nhr-49 may be distinct from mdt-15 in this context.

Author response:

We thank the Reviewing Editor and the reviewers for their highly constructive feedback and their positive assessment of our study. We plan to submit a revised manuscript that addresses these critiques through textual revisions, contextualization of our data, and explicit discussion of the study's limitations.

To address the comments from Reviewers 1 and 3 regarding the sensing mechanism and cue specificity, we agree that the precise sensor for diacetyl remains an open question. While we cannot specifically pinpoint the mechanism with our current data, we hypothesize that this response relies on a non-canonical sensing mechanism, given our multiple negative results for canonical diacetyl receptors (odr-10, sri-14), signalling pathways, and cilia-defective mutants (daf-19; daf-12). We will revise the text to suggest the mechanism could be cilia-independent or cell-autonomous. Additionally, we will explicitly frame the investigation of AWA-ablated worms, diacetyl derivatives such as acetoin, and additional volatile food cues as important future directions to establish the generalizability of this response.

Regarding the contrasting survival phenotypes observed by Park et al.24 raised by Reviewer 1, our manuscript currently discusses how chronic odour exposure represses the longevity benefits of dietary restriction48, hypothesizing that this may stem from age-dependent olfactory decline49 and the confounding variable of olfactory learning8. To make this connection clearer, we will explicitly cite Park et al. in this section to directly link their findings with our hypothesis that repeated odour exposure without a nutritional reward extinguishes its efficacy as an anticipatory cue.

In response to Reviewer 2’s feedback on our metabolic profiling, we will ensure it is clear in the text that we highlighted the lipid species most prominently affected by diacetyl, explicitly noting that triglycerides were not significantly altered. We will also better direct readers to Supplementary Table 1, which contains the comprehensive lists of all metabolites and lipids detected in our metabolomics and lipidomics, and quantifies how they are affected by the treatments in our study. As noted in our manuscript, both DHAP and Gro3P were successfully detected in our metabolomics platform but were not significantly altered by diacetyl exposure. Glycerol, however, was measured via a commercial enzymatic assay because it was not detected by our specific LC-MS platform. We will acknowledge that independently measuring the effects on cellular redox states and utilizing GC-MS for broader metabolic profiling are valuable future directions.

Finally, to address Reviewer 3’s queries regarding experimental readouts and nhr-49, we will clarify our rationale for using thrashing as our primary readout for hyperosmotic stress. Because diacetyl exposure triggers an acute induction of the DHAP-glycerol shunt, we specifically chose an acute behavioural readout to match this rapid timeline. Regarding nhr-49, we will add a new point to the discussion proposing that the distinct phenotypes may come down to expression thresholds. We hypothesize that while nhr-49 RNAi partially reduces gpdh-1 expression, this residual level of expression might still be sufficient to allow development and survival during sustained hyperosmotic stress."

References:

(8) Choi, J. I., Yoon, K., Kalichamy, S. S., Yoon, S.-S. & Lee, J. I. A natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans. ISME J. 10, 558–567 (2016).

(24) Park, S. et al. Diacetyl odor shortens longevity conferred by food deprivation in C. elegans via downregulation of DAF‐16/FOXO. Aging Cell 20, e13300 (2021).

(48) Zhang, B., Jun, H., Wu, J., Liu, J. & Xu, X. Z. S. Olfactory perception of food abundance regulates dietary restriction-mediated longevity via a brain-to-gut signal. Nat. Aging 1, 255–268 (2021).

(49) Suryawinata, N. et al. Dietary E. coli promotes age-dependent chemotaxis decline in C. elegans. Sci. Rep. 14, 5529 (2024).