This paper is the forty-eighth consecutive installment of the annual anthological review of research concerning the endogenous opioid system, summarizing articles published during 2025 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides and receptors as well as effects of opioid/opiate agonists and antagonists. The review is subdivided into the following specific topics: molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors (1), the roles of these opioid peptides and receptors in pain and analgesia in animals (2) and humans (3), opioid-sensitive and opioid-insensitive effects of nonopioid analgesics (4), opioid peptide and receptor involvement in tolerance and dependence (5), stress and social status (6), learning and memory (7), eating and drinking (8), drug abuse and alcohol (9), sexual activity and hormones, pregnancy, development and endocrinology (10), mental illness and mood (11), seizures and neurologic disorders (12), electrical-related activity and neurophysiology (13), general activity and locomotion (14), gastrointestinal, renal and hepatic functions (15), cardiovascular responses (16), respiration and thermoregulation (17), and immunological responses (18).

Sulfakinin (SK) peptides are the insect members of the ancient cholecystokinin (CCK)/gastrin superfamily, defined by a sulfated tyrosine near the C-terminus and a C-terminally amidated heptapeptide core, the modifications essential for receptor activity. Although SK peptides share C-terminal similarity with vertebrate CCK-8 and gastrin, this homology is pharmacologically incomplete: both Drosophila sulfakinin receptors cluster in an arthropod-specific clade absent from vertebrate receptor families, and cross-species bioassays confirm receptor-level incompatibility. This review synthesizes current knowledge of SK structure, receptor biology, signal transduction, and physiological function across insect orders to evaluate the SK-receptor axis with emphasis on factors that may guide future pest-management applications. SKR complement ranges from one gene in Hymenoptera and Lepidoptera to two paralogous receptors in several Diptera and Coleoptera, arising from independent lineage-specific duplications. Both Ca[2+] and cAMP second messenger arms are engaged in species-dependent combinations governed by G-protein coupling selectivity. SK peptides regulate meal termination, fat body lipid and glycogen metabolism, ecdysteroid-mediated moulting, reproductive behaviour, and agonistic signalling, with these functions partitioned among specific receptor subtypes, neural circuits, and peripheral tissues. In laboratory studies of economically significant pest species, RNAi-mediated SK-receptor silencing disrupts feeding, development, and fecundity, whereas biostable peptidomimetics represent a promising but still exploratory chemical approach. Receptor coupling identity in phytophagous crop pests, validated field-compatible dsRNA delivery, and ecological safety remain major unresolved barriers to practical application.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a class B1 G protein-coupled receptor (GPCR) that promotes glucose-dependent insulin secretion upon activation by GIP. Dual agonism of GIPR and glucagon-like peptide-1 receptor (GLP-1R) has emerged as a breakthrough therapeutic strategy for type 2 diabetes and obesity, improving glycaemic control and promoting weight loss. Although GIPR is widely expressed in the central nervous system, endogenous GIP expression in the brain is controversial, and peripherally-administered fluorescent GIPR/GLP-1R agonists predominantly localise to circumventricular organs, suggesting a lack of GIP-ligand for receptors shielded by the blood-brain barrier. Here, we considered the existence of alternative endogenous ligands for GIPR, potentially modulated by receptor activity-modifying proteins (RAMPs). Ligand activity was profiled at heterologously expressed human GIPR using cAMP accumulation, calcium mobilisation, and cAMP inhibition assays. Among the 42 ligands tested, glicentin was the only non-proGIP-derived peptide to elicit a cAMP response at GIPR (EC50 = 877 nM). No ligand-induced calcium mobilisation or cAMP inhibition was observed. Glicentin also activated human GLP-1R and glucagon receptor (GCGR), with receptor-specific antibody blockade reducing potency by 10-20-fold at all three receptors. Glicentin's activity was conserved at rodent GIPRs, albeit with reduced potency. Co-expression with RAMP2 or RAMP3 attenuated cAMP responses to GIPR-active ligands, including glicentin, but did not affect other ligands tested. These findings show that the GIPR is highly selective and identifies glicentin as a low-potency, cross-reactive agonist at GIPR, GLP-1R and GCGR, although its high EC50 makes it unlikely to act as a physiological ligand under basal conditions.

Appetite regulation in vertebrates involves central neuropeptides and peripheral metabolic and endocrine signals that act together to maintain energy homeostasis. These include orexigenic (e.g., neuropeptide Y, orexin, ghrelin) and anorexigenic (e.g., cocaine- and amphetamine-regulated transcript, corticotropin-releasing factor, cholecystokinin, leptin) factors coordinate feeding behavior. Intelectin-1 (ITLN1), a protein implicated in innate immunity and metabolic signaling in mammals, has not yet been investigated in the context of fish feeding physiology. This study examined the potential role of ITLN1 in appetite regulation and energy metabolism in goldfish (Carassius auratus). We first determined tissue distribution of itln1, which showed highest mRNA levels in spleen and liver and lower expression in brain, intestine, kidney, and gonads. Short-term fasting reduced itln1 expression in the hypothalamus and intestine, but not in liver. Peripheral administration of recombinant human ITLN1 (250-500 ng/g) significantly reduced food intake without altering blood glucose. At the highest dose (500 ng/g), ITLN1 increased brain expression of anorexigenic neuropeptides cart1, crf, trh, cck and lep2, intestinal expression of cck and inflammatory markers (tnfa, il1b) and hepatic lep2 and glycogen synthase (gs) expressions, with no effect on orexigenic peptides or glucose transporters. These findings suggest that ITLN1 might act as a peripheral satiety signal in goldfish, modulating the expression of anorexigenic neuropeptides, gut hormones, and hepatic metabolic genes. This study provides initial evidence that ITLN1 may contribute to appetite regulation in a teleost, suggesting a potential role in coordinating immunity, nutrient metabolism, and central pathways involved in feeding.

The cocaine- and amphetamine-regulated transcript (CART) peptide is widely expressed throughout the mammalian central nervous system. CART was first identified during sequencing of a hypothalamic peptide whose biological function was unknown at the time. Subsequent studies used immunohistochemistry to detect CART peptides and in situ hybridization to localize the corresponding messenger ribonucleic acid (mRNA) in several species, including rats, mice, non-human primates, and humans. Additional investigations have examined the neurotransmitters that are co-localized with CART peptides. Accumulated evidence indicates that CART peptides participate in diverse physiological processes, including addiction, reward, pain modulation, memory, sleep, hormonal regulation and the energy homeostasis. A clearer understanding of the widespread distribution of CART peptides is therefore essential for elucidating their functional roles. Accordingly, this review summarizes recent findings on the distribution of CART peptides in the aim of this review is to summarize recent findings regarding the distribution of CART peptides within the central nervous system of rodents, non-human primates, and humans, with particular emphasis on interspecies differences as abasis for identifying future research priorities. Accordingly, this review summarizes recent findings on the distribution of CART peptides within the central nervous system of rodents, non-human primates, and humans, with particular emphasis on interspecies differences as a basis for identifying future research priorities.

Currently, few pharmacological treatments are available for Alzheimer's disease (AD). However, gut-brain peptides, especially pancreatic polypeptide (PP) analogues, have shown promise. PP analogues have been reported to cross the blood-brain barrier and activate neuropeptide Y4 receptor (NPY4R) in the brain, thereby ameliorating AD-related cognitive deficits. P1642-1 is a novel PP analogue, but its role and mechanism in AD remain unexplored. This study utilized 5 ×FAD mice as a model to assess the effects of P1642-1 on cognitive dysfunction and its underlying mechanisms, while an Aβ25-35-induced cellular model was used to provide complementary mechanistic support. The findings revealed that administration of P1642-1 significantly ameliorated cognitive deficits, alleviated neuronal injury, decreased β-amyloid (Aβ) accumulation, and attenuated mitochondrial damage in the hippocampus of 5 ×FAD mice. These improvements were accompanied by enhanced mitophagy, as evidenced by upregulation of the PINK1/Parkin axis, increased LC3-II, and decreased p62 levels. In the Aβ25-35-induced cellular AD model, P1642-1 also exerted neuroprotective effects and was associated with the regulation of PINK1/Parkin-related mitophagy. Molecular dynamics simulation suggested that P1642-1 may interact with NPY4R, although receptor expression in hippocampal neurons was not directly verified in the present study. In conclusion, our study suggests that the novel PP analogue P1642-1 ameliorates cognitive impairment in 5 ×FAD mice and is associated with enhanced PINK1/Parkin-related mitophagy. These findings provide experimental support for further investigation of P1642-1 as a potential therapeutic candidate for AD.

The regulation of pro-opiomelanocortin (Pomc) expression by the testicular orphan receptor 4 (TR4) constitutes a critical mechanism underlying the pathogenesis of Cushing's disease (CD). Although the endogenous repressor juxtaposed with another zinc finger gene 1 (JAZF1) inhibits TR4 activity, its relatively low binding affinity (KD = 2246 nM) limits its therapeutic potential. In this study, we employed an iterative structure-based lead optimization strategy to enhance the JAZF1 scaffold. Utilizing Discovery Studio for virtual screening, we conducted successive rounds of in silico saturation mutagenesis, ranging from single point to multi-site combinatorial substitutions, which were subsequently validated through incremental experimental binding assays. This recursive optimization process led to the identification of two triple-mutant JAZF1 peptides (V56L/A68Y/A69R and D67L/A68Y/A69R) exhibiting more than a 2,200-fold enhancement in TR4 binding affinity. These engineered peptides, along with high-affinity small molecules such as nilotinib (KD = 4.83 nM), effectively downregulated Pomc expression and inhibited proliferation of AtT-20 tumor cells. Taken together, these findings suggest that the JAZF1-TR4-Pomc axis may serve as a potential therapeutic target for modulating adrenocorticotropic hormone (ACTH) hypersecretion in CD.

Inflammatory bowel disease (IBD) is a disorder characterized by defective intestinal barrier function, aberrant over-apoptosis of intestinal epithelial cells, and constitutive activation of pro-inflammatory signaling pathways. Glucagon-like peptide-2 (GLP-2), an endogenous gastrointestinal hormone, is known to exert protective effects against IBD by repairing the intestinal barrier and suppressing inflammatory responses. This study used molecular docking and molecular dynamics simulations to discover that [Gly[2]]-GLP-2(1-5) has a high binding affinity and strong binding stability with GLP-2R. In DSS-induced colitis mouse model, treatment with [Gly[2]]-GLP-2(1-5)]-GLP-2(1-5) can effectively reverse weight loss in mice, reduce disease activity index, increase colon length, and alleviate inflammatory damage in colon tissues; it also upregulates the expression of tight junction proteins, inhibits epithelial cell apoptosis and release of inflammatory factors, and enhances the regenerative ability of organoids. In LPS-stimulated intestinal epithelial cells, [Gly[2]]-GLP-2(1-5) can promote intestinal epithelial cell migration, maintain the integrity of tight junctions, and inhibit cell apoptosis. These findings suggest that [Gly[2]]-GLP-2(1-5) may be the shortest active short peptide of GLP-2 and is expected to be an effective peptide for the treatment of inflammatory bowel disease.

Hypothalamic kisspeptin, encoded by the Kiss1 gene, serves as an important regulator of the reproductive axis and sexual maturation. Since reproductive physiology is tightly coupled to metabolic cues, metabolic status exerts significant influence on puberty and fertility. Emerging evidence identifies kisspeptin signaling as a key determinant of central energy homeostasis. This review focuses on distinct Kiss1 neuronal populations in the arcuate nucleus of hypothalamus (Arc) and anteroventral periventricular/periventricular nucleus (AVPV/PeN) in regulating energy balance. Arc Kiss1 neurons suppress appetite via activation of POMC neurons and inhibition of AgRP/NPY neurons, while enhance energy expenditure through excitatory projections to the PVN and DMH. In contrast, AVPV/PeN Kiss1 neurons primarily exert inhibitory GABAergic regulation on PVN and DMH neurons. This indicates their suppressive modulatory function, which generally opposes the excitatory metabolic effects mediated by Arc Kiss1 neurons. Peripheral metabolic hormones, including leptin, adiponectin, insulin, and ghrelin, dynamically modulate Kiss1 neuronal activity through direct receptor or indirect POMC/AgRP pathways. By integrating these peripheral signals, Kiss1 neurons act as important modulators of metabolic homeostasis. Taken together, these findings indicate that Kiss1 neuronal pathway may be worth additional investigation in the field of metabolic disorders.

The majority of viruses considered as pandemic threats are enveloped viruses, structurally defined by lipids and integral viral envelope proteins. Scorpion venom contains an extremely diverse set of bioactive peptides, especially rich in antimicrobial agents. Here, we identified a 68-amino acid antimicrobial precursor protein Hp1412 from the venom gland of the scorpion Heterometrus petersii. Its 13-residue mature peptide was chemically synthesized and biochemically characterized. In vitro experiments demonstrated that Hp1412 exerted concentration-dependent inhibition on HCV RNA expression and exhibited low cell cytotoxicity. This peptide acted exclusively at the free virion stage of the HCV infection cycle, with optimal antiviral activity observed at 37°C. Further investigations revealed that Hp1412 possessed broad-spectrum antiviral activity against the tested enveloped viruses including IAV, HSV-1, VSV and SeV, whereas no significant inhibitory effect was observed against the non-enveloped virus EV71. These results suggested that the antiviral activity of Hp1412 was enveloped virus specific, and its underlying mechanism might involve direct interaction with free enveloped viral particles to disrupt their envelope structure. Further transmission electron microscopy analysis showed that Hp1412 inactivates IAV by directly disrupting the virion envelope. In vivo experiments showed that Hp1412 could prophylactically inhibit IAV replication in the lung tissues of mice, and significantly alleviate the excessive activation of innate immune responses and inflammatory reactions induced by IAV infection in lung tissues. Our study offers an initial clue that may inform the future development of broad-spectrum drugs for the prevention of enveloped virus infections.

Endothelin-1 (ET-1) is known not only as a vasoconstrictor, but also as a neuromodulator. It can reset the circadian clock and we hypothesized that it modulates neuronal excitability and network activity in histaminergic neurons (HN). HN, located in the tuberomammillary nucleus (TMN) of the caudal hypothalamus (cHPT), fire spontaneously during waking and promote arousal. Previous studies have shown that ET-1 reduces firing frequency of hippocampal pyramidal neurons, whereas its effects on electrophysiological properties of HN remain unknown. We now studied the expression of ET-receptors (ETA and ETB) in the hippocampus (HPC) and caudal hypothalamus by single-cell RT-PCR and qPCR. The responses to ET-1 were studied in HN and dentate gyrus granule cells (DGgc) of brain slices using patch-clamp recordings and primary cultures of cHPT and HPC using Microelectrode Arrays (MEAs). ET-1 significantly decreased firing frequency in HPC and increased it in cHPT cultures with prevailing inhibition of individual MEA channels in HPC and excitation in cHPT. Neuronal synchrony and bursting were significantly increased in cHPT. Relative to HPC, cHPT displayed higher expression of ETB. In brain slices, ET-1 induced a delayed bosentan-sensitive increase in HN and decrease in DGgc firing frequency. HN expressed ETB (15 of 22 HN) and ETA (4 of 15 ETB positive HN), whereas only ETB was expressed in 50% of DGgc. We conclude that ET-1 differentially modulates cHPT and HPC. By exciting HN and synchronizing neuronal network activity in cHPT, ET-1 may contribute to cellular and circuit-level mechanisms in the regulation of arousal and wakefulness.

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disorder of the central nervous system (CNS), characterized by microglial activation and polarization as key drivers of disease pathogenesis. Irisin, an exercise-induced myokine, has been reported to exhibit neuroprotective effects, including anti-inflammatory activity and cognitive improvement. To investigate the therapeutic potential of irisin in the experimental autoimmune encephalomyelitis (EAE) mouse model and its effects on microglial behavior along with the underlying molecular mechanisms, we conducted the present study. Results demonstrated that irisin treatment significantly alleviated EAE severity, evidenced by reduced disease incidence, attenuated weight loss, and improved neurological scores. Histopathological analysis revealed that irisin suppressed inflammatory cell infiltration and reduced demyelination in spinal cord tissues. Furthermore, irisin inhibited microglial overactivation and promoted a phenotypic shift from the pro-inflammatory M1 to the anti-inflammatory M2 microglia. Mechanistically, immunofluorescence co-localization and Western blot analyses confirmed that these beneficial effects were mediated via suppression of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, as indicated by downregulation of STING and phosphorylated interferon regulatory factor 3 (p-IRF3) expression. Collectively, these findings indicate that irisin alleviates neuroinflammation and exerts neuroprotective effects in EAE by modulating microglial activity through inhibition of the cGAS-STING pathway, underscoring its potential as a novel therapeutic candidate for MS.

Voltage-gated potassium channels of the Kv4 subfamily (Kv4.1, Kv4.2, and Kv4.3) mediate transient A-type potassium currents that regulate neuronal excitability, dendritic integration, and cardiac repolarization. Despite their importance, no pharmacological tool has been available to selectively dissect the role of Kv4.1, as existing peptide toxins from the α-KTx15 family display broad activity across Kv4 isoforms. Here, we report the discovery and characterization of two novel scorpion toxins, BlTx1 and BlTx2, isolated from the venom gland transcriptome of Buthacus leptochelys. Both toxins were heterologously expressed in yeast and purified to homogeneity. Electrophysiological recordings from Xenopus laevis oocytes revealed that BlTx1 and BlTx2 potently inhibited Kv4.1 currents, while sparing Kv4.2 and Kv4.3. Among a panel of 20 tested potassium channels, BlTx2 exhibited clear functional selectivity for Kv4.1, with an IC₅₀ of 28 nM, whereas BlTx1 also inhibited Kv1.2. Sequence comparison with related nonselective toxins suggests that a small number of substitutions in the N-terminal half of the peptides underlie BlTx2's unique selectivity profile. This unique isoform selectivity establishes BlTx2 as, to our knowledge, the first toxin showing clear functional selectivity for Kv4.1 within the tested panel, providing a molecular probe for delineating the physiological and pathological contributions of Kv4.1. Such a tool may facilitate clarification of Kv4.1's role in neuronal firing patterns, circadian regulation, and tumor cell proliferation, while avoiding the off-target effects associated with nonselective Kv4 inhibitors. Our findings highlight scorpion venoms as a valuable source of isoform-selective ion channel ligands and open new avenues for basic research and therapeutic development.