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

Glycogen phosphorylase (GP) catalyzes the rate-limiting step of glycogenolysis and occupies a central position in insect carbohydrate metabolism, supplying precursors for chitin biosynthesis. Acyl urea compounds structurally related to benzoylphenylurea (BPU) insecticides are potent inhibitors of mammalian GP, raising the question of whether insect GP could serve as an independent insecticidal target. Here, we systematically evaluate this possibility in the diamondback moth Plutella xylostella . We show that a mammalian GP inhibitor (GPI) potently inhibits recombinant PxGP (IC ₅₀ = 2.96 nM), while the BPU insecticide diflubenzuron (DFB) does not. Molecular docking and MM/GBSA analysis predict that this selectivity reflects differential side-chain engagement: GPI is predicted to occupy the allosteric site at the dimer interface via contacts with seven residues spanning both subunits (ΔG = −34.63 kcal/mol), whereas DFB’s difluorobenzoyl moiety is predicted to remain solvent exposed without productive protein contacts (ΔG = −29.29 kcal/mol). Despite potent in vitro inhibition, GPI exhibits no insecticidal activity, and RNAi-mediated knockdown of PxGP (confirmed by enzyme activity measurements showing ∼29% reduction in per-larva GP-a activity) does not impair development or survival. We provide evidence that insects compensate through a multi-layered metabolic response: upregulation of gluconeogenic enzymes (PEPCK, G-6-Pase), selective upregulation of glycogen branching enzyme (GBE) but not α-amylase, and changes in protein content suggestive of catabolic substrate mobilization. Fitness assessment reveals transient larval weight loss (24–48 h) with complete recovery of pupal weight, adult wing morphology, and female fecundity, indicating that the metabolic compensation response is ultimately effective in sustaining development. These findings suggest that GP is functionally non-essential under these conditions, likely through a combination of gluconeogenic compensation and the availability of alternative carbon sources, and highlight the importance of considering metabolic network redundancy in target-based insecticide design.