Fish-hunting cone snail disrupts prey’s glucose homeostasis with weaponized mimetics of somatostatin and insulin

Venomous animals have evolved sophisticated molecular mechanisms to subdue prey and defend against predators. Most venoms target the nervous, locomotor, and cardiovascular systems or cause direct tissue damage. However, the discovery of fish-hunting cone snails using weaponized insulins to induce hypoglycemia in prey reveals a novel venomous strategy. This study investigates whether *Conus geographus*, a highly venomous snail, uses additional toxins to further disrupt prey glucose homeostasis. Normal glycemia is maintained by a balance between insulin and glucagon. Insulin promotes glucose uptake, while glucagon stimulates glucose release. Somatostatin (SST), another pancreatic hormone, inhibits glucagon secretion. Homologous peptides and receptors in fish also play critical roles in regulating glycemia. Previous research indicated the presence of SST-like toxins in *C. geographus* venom. This study explores the hypothesis that *C. geographus* utilizes weaponized somatostatins, in conjunction with insulins, to induce and sustain dangerously low blood glucose levels in prey, thereby facilitating prey capture. The study focuses on characterizing the molecular mechanisms and evolutionary origins of these toxins and their combined effects on glucose homeostasis.

Extensive research on cone snail venoms has uncovered a diverse array of neurotoxins targeting various ion channels and receptors. The 'motor cabal' of toxins disrupts neuromuscular transmission, while 'doppleganger peptides' mimic endogenous hormones to manipulate prey physiology. Previous work established that *C. geographus* employs venom insulins (con-insulins) to induce hypoglycemia. These con-insulins originate from a conserved insulin gene in the snail's nervous system, experiencing adaptive diversification to target prey insulin receptors. The study builds upon this foundation, investigating the role of somatostatin-like toxins (consomatins) in enhancing the hypoglycemic effect of con-insulins.

The research employed a multi-faceted approach including transcriptomic analysis of the *C. geographus* venom gland to identify co-expressed toxins. RNA sequencing was performed on four segments of the venom gland to determine the expression patterns of con-insulins and consomatins. Synthetic peptides, including Consomatin pG1 (a predicted mature peptide), were synthesized and functionally evaluated using various assays. PRESTO-Tango β-arrestin recruitment assays were used to assess the activation of human and fish somatostatin receptors (SSTRs). G protein dissociation assays were conducted to determine the G protein signaling profiles of Consomatin pG1 and other peptides. Glucagon secretion assays using isolated mouse islets and perfused rat pancreas investigated the effects of Consomatin pG1 on glucagon secretion. Activity-guided fractionation of *C. geographus* venom, employing RP-HPLC, was used to isolate and identify the native Consomatin G1. Mass spectrometry (MS), MS/MS (HCD and ETD), and Edman sequencing were utilized for peptide sequencing and characterization, particularly focusing on glycosylation patterns. Phylogenetic analyses explored the evolutionary origins of Consomatin G1 and its relationship to endogenous somatostatin-like peptides. Molecular evolutionary analysis was conducted to examine the evolution of Conostatin G1 from an endogenous somatostatin-like signaling gene.

The study found that con-insulins and consomatins are co-expressed in the distal region of the *C. geographus* venom gland, suggesting coordinated release to disrupt glucose homeostasis. Consomatin pG1, a synthetic analog, was found to be a potent and selective agonist of human SSTR2, effectively inhibiting glucagon secretion. However, Consomatin pG1 showed selectivity for only one of the two zebrafish SSTR2 isoforms, indicating potential species-specific target selection or receptor evolution. Activity-guided fractionation identified the native Consomatin G1, revealing a unique structure with a heavily glycosylated N-terminal tail. This glycosylated tail is crucial for its activity, closely mimicking a glycosylated somatostatin from fish pancreas. Phylogenetic analysis indicated that Consomatin G1 evolved from an endogenous somatostatin-like peptide gene, acquiring a novel exon encoding the glycosylated N-terminal region. A synthetic analog of Conostatin nG1 lacking O-glycans displayed significantly reduced activity, highlighting the importance of glycosylation for biological function. Consomatin G1 effectively suppressed glucagon secretion in both rodent and fish SSTRs.

The findings demonstrate that *C. geographus* employs a sophisticated combinatorial venomous strategy targeting glucose homeostasis. The coordinated action of con-insulins and consomatins creates a sustained hypoglycemic state in prey, impairing locomotion and facilitating prey capture. The discovery of a venom peptide mimicking a synthetic somatostatin drug analog underscores the potential of natural compounds for drug design. The glycosylation of Consomatin G1 plays a crucial role in receptor binding and activation, highlighting the importance of post-translational modifications in venom toxin evolution. The observed species-specific differences in SSTR2 activation suggest potential variations in target selection or receptor evolution among different fish species. Future research will focus on examining these variations. The research also highlights the use of venom toxins as templates for drug design, as exemplified by the development of monomeric insulin analogs based on cone snail toxins.

This study provides compelling evidence for the combinatorial use of weaponized insulin and somatostatin mimetics by *Conus geographus* to disrupt prey glucose homeostasis. The findings highlight the remarkable evolutionary adaptations of venom toxins and their potential applications in drug discovery. Future research should explore the full range of toxins involved in this intricate strategy, and investigate the precise mechanisms of action at the cellular and organismal levels, while further examining species-specific target variations.

The study primarily focused on in vitro and ex vivo assays, limiting the direct assessment of the combinatorial action of con-insulins and consomatins in live prey. The complex glycosylation of Consomatin G1 presented challenges in fully characterizing the native structure and its precise effects. Further investigation is needed to fully elucidate the exact composition and linkages of the glycans.