Metabolic compensation via gluconeogenesis explains the non-essentiality of glycogen phosphorylase as an insecticidal target in Plutella xylostella

Reviewer #1 (Public review):

Summary:

In this study, the authors investigate whether glycogen phosphorylase is a potential molecular target of benzoylphenylurea insecticides and examine the physiological consequences of inhibiting glycogen breakdown in the diamondback moth Plutella xylostella. The authors express and characterize recombinant glycogen phosphorylase, test its inhibition by a mammalian glycogen phosphorylase inhibitor and by the insecticide diflubenzuron, and assess the physiological effects of glycogen phosphorylase inhibition through chemical exposure and RNA interference. Based on these experiments, the authors conclude that benzoylphenylurea insecticides do not target glycogen phosphorylase and propose that insects compensate for glycogen phosphorylase inhibition through activation of gluconeogenesis, allowing them to maintain glucose homeostasis and complete development despite strong suppression of the enzyme.

Strengths:

The study addresses an interesting and long-standing question in insect toxicology regarding the mechanism of action of benzoylphenylurea insecticides. The authors combine several complementary approaches, including recombinant enzyme characterization, inhibitor assays, RNA interference, gene expression analyses, and metabolite measurements. The biochemical characterization of the recombinant glycogen phosphorylase and the demonstration that the tested glycogen phosphorylase inhibitor can strongly inhibit enzyme activity represent important technical strengths. In addition, the study integrates biochemical and physiological observations to explore how insects might compensate for disruptions in central carbohydrate metabolism.

Weaknesses:

Several aspects of the central conclusions rely on indirect evidence and would benefit from additional validation. The proposed compensatory mechanism (gluconeogenesis supported by amino acid mobilization) is inferred primarily from transcriptional changes in gluconeogenic genes, reduced protein levels, and changes in metabolite concentrations. While these observations are consistent with increased gluconeogenic activity, they do not directly demonstrate metabolic flux through this pathway. Direct measurements of gluconeogenic flux would be required to confirm that carbon derived from non-carbohydrate substrates contributes to glucose production.

Some interpretations are also speculative. For example, the lack of glycogen accumulation following glycogen phosphorylase knockdown is attributed to alternative glycogen degradation pathways, such as α-amylase or glycogen debranching enzymes, but these possibilities are not experimentally examined. Measuring the expression or activity of these enzymes would help evaluate whether such pathways contribute to the observed metabolic response.

The physiological consequences of the proposed metabolic compensation are also not fully explored. If proteins are mobilized to support gluconeogenesis, this shift might be expected to affect organismal traits such as adult body size, flight capacity, or reproductive performance. Assessing these traits could provide valuable insight into whether the proposed compensatory metabolism carries fitness costs.

Finally, some conclusions extend beyond the direct evidence presented. The study shows that diflubenzuron does not inhibit glycogen phosphorylase in vitro, but broader conclusions regarding the mechanism of action of benzoylphenylurea insecticides as a class may require additional evidence. In addition, some biochemical and cell-based observations would benefit from confirmation in whole insects, given that metabolic regulation can differ substantially between isolated enzyme or cell-based systems and intact larvae, where hormonal signaling, tissue interactions, and nutrient availability influence metabolic responses.

Reviewer #2 (Public review):

(1) Significance of the findings and strength of the evidence

This manuscript evaluates the hypothesis that benzoylurea (BPU) insecticides exert their effects through inhibition of glycogen phosphorylase rather than chitin synthase (CHS). The central premise-that structural similarity among acylurea compounds implies shared molecular targets-is not supported by existing evidence.

Extensive genetic and biochemical studies, including Reference 5, demonstrate that chitin synthase is the primary insecticidal target of BPUs. In particular, amino acid substitutions at a single site in CHS confer high levels of resistance to diflubenzuron and related compounds, with causality established through CRISPR/Cas9 editing in Drosophila melanogaster. This body of evidence substantially weakens the rationale for proposing glycogen phosphorylase as an alternative primary target.

The manuscript reports that an acylurea compound previously identified as an inhibitor of mammalian glycogen phosphorylase also inhibits glycogen phosphorylase from Plutella xylostella, while diflubenzuron does not. This observation is consistent with prior work showing that glycogen phosphorylase inhibition among acylureas depends on specific side chain substitutions rather than the shared acylurea core. Consequently, the finding does not support the broader inference that acylurea structure predicts common biological function.

The manuscript further argues that inhibition of glycogen phosphorylase is not insecticidal and attributes this to metabolic compensation through alternative glucose producing pathways. While it is well established that eukaryotic cells possess multiple mechanisms for maintaining glucose availability, the evidence provided here does not fully support the broader claim that this mechanism explains the lack of insecticidal activity. In particular, the conclusion that the study "resolves" the primary hypothesis is not justified by the data presented.

Overall, while some experimental observations are sound in isolation, the overarching conclusions are not supported by the strength of the evidence. The significance of the findings is therefore limited.

(2) Interpretation in the context of existing literature

The introduction states that the molecular target of BPU insecticides remains a major unresolved controversy. However, multiple prior studies, including References 1, 4, and 5, provide strong genetic evidence that CHS is the primary and essential target of BPUs. These results demonstrate causality rather than simple correlation, particularly through targeted gene editing approaches.

The manuscript further claims that biochemical studies have failed to demonstrate CHS inhibition by BPUs in cell free assays. However, the cited references (6-9) did not express CHS in such assays and therefore do not directly address this question. As a result, the suggested discrepancy between genetic and enzymatic evidence is not well founded.
Structural analysis of acylurea compounds indicates that biological activity depends on side chain composition rather than the conserved acylurea core. Prior screening studies (Reference 11) show substantial variability in glycogen phosphorylase inhibition among acylureas despite a shared core structure. This undermines the proposal that the acylurea moiety itself constitutes a meaningful clue to a shared molecular mechanism.

Regarding implications for pesticide design, targeting chitin synthesis remains an attractive strategy because chitin is essential for arthropods and absent in mammals, providing both efficacy and specificity. By contrast, metabolic enzymes such as glycogen phosphorylase are widely conserved, making them less suitable targets from a toxicological and safety perspective.

(3) Specific technical comments

The manuscript uses the term "dataology," which is neither defined nor contextualized within the text. As currently used, the term appears unrelated to the subject matter and may be confusing to readers. Clarification or removal would improve clarity.

Author response:

Public Reviews:

Reviewer #1 (Public review):

(1) The proposed compensatory mechanism is inferred primarily from transcriptional changes and metabolite levels; direct measurements of gluconeogenic flux are lacking.

We agree that isotopic tracer experiments would provide the most direct evidence for gluconeogenic flux. While such experiments are beyond the scope of the current revision, we will explicitly acknowledge this as a key limitation and clearly state it as an important direction for future research. We note, however, that the convergent evidence from multiple independent lines, transcriptional upregulation of PEPCK and G-6-Pase, declining protein levels, altered amino acid profiles, and maintained trehalose levels, collectively supports gluconeogenic activation, even though each individual line is indirect. In the revised manuscript, we will present this evidence more cautiously, framing it as “consistent with gluconeogenic compensation” rather than definitively establishing metabolic flux.

(2) Alternative glycogen degradation pathways (α-amylase, glycogen debranching enzymes) are proposed but not experimentally examined.

We have now directly addressed this by measuring, via RT-qPCR, the expression of glycogen branching enzyme (GBE) and α-amylase following PxGP knockdown. Our preliminary results reveal a striking and informative pattern:

GBE was significantly upregulated at 24 h (+29.24%), 48 h (+16.78%), and 96 h (+44.46%) post-injection, indicating transcriptional activation of an alternative glycogen-metabolizing enzyme in response to GP suppression.

α-Amylase showed no significant change at any time point, suggesting that the compensatory response is pathway-specific rather than a generalized upregulation of all starch/glycogen-degrading enzymes.

This differential response, GBE up while α-amylase unchanged, provides the first direct evidence that P. xylostella selectively activates specific alternative glycogen catabolic pathways when GP function is compromised. These data will be incorporated into the revised manuscript as a new figure panel.

(3) Physiological consequences of the proposed metabolic compensation (fitness costs) are not explored.

We have now assessed fitness consequences of PxGP knockdown by measuring feeding rate, larval body weight, and pupal weight. The results reveal a transient but significant fitness cost:

Feeding rate: no significant difference between dsGP and dsGFP groups across all time points (24–120 h), indicating that the observed metabolic changes are not attributable to reduced food intake.

Larval weight: significantly reduced at 24 h (−29.10%) and 48 h (−25.38%) in the dsGP group, demonstrating that metabolic compensation carries a measurable short-term cost.

Pupal weight: no significant difference, indicating that larvae recover from the transient weight deficit before pupation.

This pattern, transient larval weight loss with full pupal recovery, is consistent with our proposed model: GP suppression triggers protein catabolism to fuel gluconeogenesis (explaining the weight loss), but the compensatory mechanism is sufficiently effective to restore metabolic homeostasis before the pupal transition. Adult wing area and female fecundity measurements are currently in progress and will be included in the revised manuscript.

(4) Enzyme activity is not measured in RNAi-treated insects; only transcript-level knockdown is reported.

We have now measured GP enzyme activity (GPa) in crude extracts from RNAi-treated larvae using the coupled-enzyme spectrophotometric assay. The results provide important new insights:

Per-larva GP activity was significantly reduced at 24 h (−27.57%) and 48 h (−29.28%), confirming that RNAi-mediated transcript suppression translates to reduced enzyme function in vivo.

Per-protein GP activity showed a significant reduction only at 48 h (−10.35%). This apparent discrepancy is explained by a substantial decrease in total protein concentration at 24 h (−44.48%), which then gradually recovered. When enzyme activity is normalized to a declining protein pool, the per-protein reduction appears smaller.

Importantly, the 44.48% decline in total protein at 24 h provides independent biochemical confirmation of our proposed protein catabolism: it is consistent with the mobilization of protein stores to supply amino acids for gluconeogenesis, directly supporting the compensatory mechanism described in our manuscript.

These enzyme activity data will be presented alongside the existing transcript-level data in the revised manuscript, providing a complete picture from gene expression through enzyme function.

(5) Conclusions regarding BPU class may require testing additional compounds beyond diflubenzuron.

We agree and will explicitly limit our conclusion to diflubenzuron in the revised manuscript. The relevant text will be revised to state that “DFB does not inhibit PxGP” rather than making broader claims about the BPU class as a whole.

(6) Structural evidence that GPI can bind PxGP in a comparable manner to its mammalian target is lacking.

We have performed molecular docking and binding free energy analysis to address this concern directly. The PxGP homodimer structure was modeled using SWISS-MODEL with the rabbit muscle GP–acyl urea co-crystal structure (PDB: 2ATI; Klabunde et al., 2005) as the template. Molecular docking and MM/GBSA calculations were performed using Cresset Flare V11.

Key findings:

GPI exhibited substantially stronger binding to PxGP (ΔG = −34.63 kcal/mol) compared to DFB (ΔG = −29.29 kcal/mol), with a ΔΔG of −5.34 kcal/mol.

Energy decomposition revealed that van der Waals interactions are the primary driver of selectivity (ΔG_VDW = −11.49 kcal/mol), reflecting superior shape complementarity of GPI within the binding pocket.

GPI was predicted to bind at the allosteric site at the dimer interface, engaging seven residues across both subunits (Asn44 and Val45 from chain A; Trp67, Gln71, Tyr75, Arg193, and Asp227 from chain B), a binding mode consistent with the experimentally determined site of acyl urea inhibitors in mammalian GP.

DFB contacted only six residues, primarily from a single subunit, and its difluorobenzoyl moiety remained entirely solvent-exposed without productive protein contacts, explaining its inability to achieve effective target engagement.

These structural data, together with the biochemical inhibition data (IC₅₀ = 2.96 nM for GPI; no inhibition by DFB), provide a comprehensive molecular explanation for the observed selectivity. The results will be presented as a new figure and table in the revised manuscript.

(7) Dietary carbohydrates could mask the metabolic effects of GP inhibition.

Our new data showing no difference in feeding rate between dsGP and dsGFP groups addresses this concern from one angle: the metabolic changes we observe are not attributable to altered food intake. We will also add a discussion of the potential contribution of dietary carbohydrates to glucose homeostasis and acknowledge this as a caveat in interpreting the metabolite data.

Minor points: All terminology errors (“gluconeogenolysis” → “gluconeogenesis”), typographical errors (“over over four decades”), and formatting inconsistencies will be corrected. We will clarify the metabolite normalization approach and improve figure labeling and pathway schematics.

Reviewer #2 (Public review):

(1) The central premise — that structural similarity among acylurea compounds implies shared molecular targets — is not supported by existing evidence.

We agree that the original manuscript overstated the significance of the shared acylurea core as a predictor of common biological activity. In the revised manuscript, we will substantially restructure the Introduction to:

(1) Explicitly acknowledge the compelling genetic evidence from CRISPR/Cas9 experiments (Reference 5) establishing CHS as the primary site conferring BPU resistance.

(2) Reframe the study’s objective: rather than proposing to “resolve” the BPU target controversy, the revised manuscript will focus on the systematic evaluation of GP as an independent insecticidal target and the discovery of a gluconeogenic compensation mechanism, questions that have scientific value independent of the BPU mechanism debate.

(3) Remove the claim that the study “resolves the primary hypothesis.” The conclusion will instead state that our biochemical data demonstrate DFB does not inhibit PxGP, adding enzyme-level evidence to the existing genetic framework.

(2) Target selectivity among acylurea compounds is determined by side-chain composition, not the shared core.

We fully agree, and our new structural data now provide a molecular explanation for this principle at the atomic level. Molecular docking reveals that both GPI and DFB anchor to PxGP through their common acylurea carbonyl groups (forming hydrogen bonds with Arg193), but diverge dramatically in their side-chain engagement: GPI’s methoxyphenyl-methylurea moiety engages five additional residues across the dimer interface, while DFB’s difluorobenzoyl group remains entirely solvent-exposed. The van der Waals energy difference (ΔΔG_VDW = −11.49 kcal/mol) quantitatively reflects this differential shape complementarity. These data directly support Reviewer 2’s point and will be presented as new evidence in the revised manuscript.

(3) References 6–9 did not express CHS in cell-free assays.

We will revise the relevant passage for greater precision. Our revised text will distinguish between (a) the absence of direct biochemical evidence for BPU-mediated CHS inhibition in cell-free systems and (b) the technical challenge of expressing and purifying functional CHS for such assays. This distinction will be stated more carefully to avoid any mischaracterization of the cited literature.

(4) The term “dataology” is non-standard.

This term has been removed and replaced with “data.” In accordance with eLife’s policy on the use of AI tools and technology, we will include a statement in the Materials and Methods section declaring that AI-based language editing tools were used for English grammar and style refinement. All scientific content was generated entirely by the authors.

Author response table 1.

We are confident that the substantial new experimental data and restructured narrative will meaningfully strengthen the manuscript.