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[Image: see text] Insulin-like peptide 5 (INSL5), the natural ligand for the relaxin family peptide receptor 4 (RXFP4), is a gut hormone that is exclusively produced by colonic L-cells. We have recently developed an analogue of INSL5, INSL5-A13, that acts as an RXFP4 agonist in vitro and stimulates colorectal propulsion in wild-type mice but not in RXFP4-knockout mice. These results suggest that INSL5 may have a physiological role in the control of colorectal motility. To investigate this possibility, in this study we designed and developed a novel INSL5 analogue, INSL5-A13NR. This compound is a potent antagonist, without significant agonist activity, in two in vitro assays. We report here for the first time that this novel antagonist peptide blocks agonist-induced increase in colon motility in mice that express RXFP4. Our data also show that colorectal propulsion induced by intracolonic administration of bacterial products (short-chain fatty acids, SCFAs) is antagonized by INSL5-A13NR. Therefore, INSL5-A13NR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrheas.

Insulin-like peptides (ILPs) including insulin, insulin-like growth factor (IGF) and relaxin are evolutionarily conserved hormones in metazoans, and they are involved in diverse physiological processes. The migratory brown planthopper (BPH), Nilaparvata lugens, encodes four ILP genes (Nlilp1, Nlilp2, Nlilp3 and Nlilp4) but their physiological roles are largely unknown. Sequence analysis showed that NlILP1 contained a relaxin-specific G protein-coupled receptor-binding motif and a variant motif of cysteine residues, and NlILP2 and NlILP4 resembled vertebrate IGFs. RNA interference (RNAi)-mediated gene silencing showed that depletion of each of Nlilp1, 2 and 3 significantly delayed the developmental duration of nymphs, and this effect could be exacerbated by double or triple gene depletion. Depletion of Nlilp1, Nlilp2 or Nlilp3 induces the accumulation of glucose, trehalose and glycogen, which is contradictory to depletion of the insulin receptor (NlInR1) in the BPH. Depletion of Nlilp1 significantly enhanced starvation resistance in both females and males although its extent was smaller than NlInR1 depletion. A parental RNAi assay showed that depletion of each of Nlilp1-4 dramatically impaired female fecundity. These findings indicate that NlILP1-4 have redundant and distinct roles in physiological processes in the BPH, thereby enhancing our understanding of the contribution of each NlILP to the ecological success of this species in natural habitats.

The role of insulin and insulin-like peptides (ILPs) in vertebrate animals is well studied. Numerous ILPs are also found in invertebrates, although there is uncertainty as to the function and role of many of these peptides. We have identified transcripts with similarity to the insulin family in the tentacle transcriptomes of the sea anemone Oulactis sp. (Actiniaria: Actiniidae). The translated transcripts showed that these insulin-like peptides have highly conserved A- and B-chains among individuals of this species, as well as other Anthozoa. An Oulactis sp. ILP sequence (IlO1_i1) was synthesized using Fmoc solid-phase peptide synthesis of the individual chains, followed by regioselective disulfide bond formation of the intra-A and two interchain disulfide bonds. Bioactivity studies of IlO1_i1 were conducted on human insulin and insulin-like growth factor receptors, and on voltage-gated potassium, sodium, and calcium channels. IlO1_i1 did not bind to the insulin or insulin-like growth factor receptors, but showed weak activity against KV1.2, 1.3, 3.1, and 11.1 (hERG) channels, as well as NaV1.4 channels. Further functional studies are required to determine the role of this peptide in the sea anemone. ispartof: BIOMOLECULES vol:11 issue:12 ispartof: location:Switzerland status: published

In insects, trehalose accumulation is associated with the insulin/insulin-like growth factor signalling (IIS) pathway. However, whether insulin-like peptide is involved in the regulation of the trehalose metabolism during diapause termination remains largely unknown. This study assessed whether insulin-like peptide (ApILP) enhances the trehalose catabolism in the pupae of Antheraea pernyi during their diapause termination process. Injection of 10 μg of bovine insulin triggered diapause termination and synchronous adult eclosion in diapausing pupae. Moreover, treatment with bovine insulin increased the expression of trehalase 1A (ApTre-1A) and trehalase 2 (ApTre-2), as well as the activity of soluble and membrane-bound trehalase, resulting in a decline in trehalose levels in the haemolymph. Silencing ApILP via RNA interference significantly suppressed the expression of ApTre-1A and ApTre-2, thus leading to an increase in the trehalose concentration during diapause termination. However, neither injection with bovine insulin nor ApILP knockdown directly affected trehalase 1B (ApTre-1B) expression. Moreover, overexpression of the transcription factor forkhead box O (ApFoxO) induced an increase in trehalose levels during diapause termination, however, depletion of ApFoxO accelerated the breakdown of trehalose in diapausing pupae by increasing the expression of ApTre-1A and ApTre-2. The results of this study help to understand the contributions of ApILP and ApFoxO to the trehalose metabolism during diapause termination.

Most insect species encode multiple insulin-like peptides (ILPs) that exhibit functional overlaps in mediating physiological processes such as development and reproduction. Why do they need multiple ILPs? To address this question, we tested a hypothesis of the requirement of multiple ILPs by generating mutants lacking individual ILP genes using the CRISPR/Cas9 technology. Two ILPs (ILP1 and ILP2) in the legume pod borer, Maruca vitrata, mediate similar physiological processes such as hemolymph sugar level, larval development, and adult reproduction. Individual knock-out mutants (ΔILP1 and ΔILP2) were generated. They showed successful development from larvae to adults. However, they suffered from high hemolymph sugar levels by enhancing trehalose titers in the hemolymph. The hyperglycemic effect was more evident in ΔILP2 mutants than in ΔILP1 mutants. Both mutants showed increased expression of trehalose-6-phosphate synthase but suppressed expression of trehalase. These mutants also showed altered expression patterns of insulin signaling components. Expression levels of insulin receptor and Akt genes were upregulated, while those of FOXO and Target of rapamycin genes were downregulated in these mutants. These alterations of signal components resulted in significant retardation of immature development and reduced body sizes. ΔILP1 or ΔILP2 females exhibited poor oocyte development. Bromo-uridine incorporation was much reduced at the germarium of ovarioles of these mutants compared with wild females. Expression of the vitellogenin gene was also reduced in these mutants. Furthermore, males of these deletion mutants showed impaired reproductive activities when they mated with wild-type females. These results suggest that both ILPs are required for mediating larval development and adult reproduction in M. vitrata.

Culex pipiens form pipiens and Cx. pipiens form molestus differ in their ability to produce eggs without a bloodmeal. Autogenous mosquitoes, such as the molestus bioform of Cx. pipiens, depend on nutrition acquired as larvae instead of a bloodmeal to fuel the energy intensive process of vitellogenesis, which requires abundant production of yolk proteins. In anautogenous mosquito systems, ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs) transduce nutritional signals and trigger egg maturation in response to a bloodmeal. It is unclear to what extent the process is conserved in autogenous mosquitoes and how the bloodmeal trigger has been replaced by teneral reserves. Here, we measured the effects of a series of nutritional regimens on autogeny, time to pupation, and survival in Cx. pipiens form molestus and form pipiens. We find that abundant nutrients never result in autogenous form pipiens and extremely poor food availability rarely eliminates autogeny from form molestus. However, the number of autogenous eggs generated increases with nutrient availability. Similarly, using qPCR to quantify gene expression, we find several differences in the expression levels of ilps between bioforms that are reduced and delayed by poor nutrition, but not extinguished. Changes in OEH expression do not explain bioform-specific differences in autogeny. Surprisingly, the source of most of the gene expression differences correlated with autogeny is the abdomen, not the brain. Overall, our results suggest that autogeny is modulated by nutritional availability, but the trait is encoded by genetic differences between forms and these impact the expression of ILPs.

The peripheral blood concentrations of insulin-like peptide 3 (INSL3) have been detected in many mammalian species, but the level of INSL3 in horse remains unknown. The objectives were to develop a time-resolved fluorescence immunoassay (TRFIA) to detect INSL3 concentrations from horse blood as well as to determine the age-related and seasonal changes of plasma concentrations of INSL3 and testosterone from birth to early-puberty in Thoroughbred male horse (n = 11). Monthly blood sample and measurement of body weight, height, chest and cannon bone size were done from birth until 16 mo. The TRFIA and EIA were used to measure plasma concentrations of INSL3 and testosterone, respectively. An increase in mean body weight, height, chest and cannon bone size was observed throughout the study. The monthly blood sampling revealed an increase in mean plasma INSL3 concentrations up to 2 mo, followed by a decreasing and increasing pattern until the end of experiment at 16 mo. A high testosterone level was detected at birth followed by a sharp decrease to basal level within 1 mo, maintained low level up to10 mo before a gradual rise until 16 mo. In case of seasonality, there was no difference in mean plasma INSL3 concentrations between breeding (March to September) and non-breeding (October to February) seasons, whereas a higher (P 0.001) mean plasma testosterone concentrations in the second breeding season compared to non-breeding season was observed. In age categorized group, an increase (P 0.01) in mean plasma INSL3 concentrations was noticed at pre-puberty (1-12 mo) and early-puberty (13-16 mo) compared to birth, but a lower (P 0.001) mean plasma testosterone concentrations was observed at pre-puberty compared to birth and early-puberty. In conclusion, a TRFIA was developed to measure INSL3 levels in horse. An increase in plasma concentrations of INSL3 and testosterone were observed with the advancement of age, whereas for testosterone a very lower level was detected at the non-breeding season than in the second breeding season after birth in Thoroughbred male horse. The INSL3 secretions seemed independent of seasonal influence, at least before puberty.

In insects, the insulin receptor (InR) pathway is involved in regulating key physiological processes, including juvenile hormone (JH) synthesis, vitellogenin production, and oocyte growth. This raises the question about which ligand (or ligands) binds to InR to trigger the above effects. We have cloned seven insulin-like peptides (BgILP1 to 7) from female Blattella germanica cockroaches and found that the brain expresses BgILP1 to 6, the fat body BgILP7, and the ovary BgILP2. Starvation induces the reduction of BgILP3, 5, and 6 mRNA levels in the brain, and the various BgILPs are differentially expressed during the gonadotrophic cycle. In addition, by knocking down the BgILPs we were able to identify compensatory regulation at transcriptional level between the different BgILPs, although none of the BgILP knockdown assays, including the knockdown of the seven BgILPs, produced the same phenotypes that we achieved by depleting InR. Taken together, the results indicate that B. germanica ILPs are differentially expressed in tissues and in response to physiological conditions, and that they are affected by compensatory regulation., This work was supported by grants BFU2010-15906 (Spanish Ministry of Science and Innovation (MICINN)), CGL2016-76011-R (Spanish Ministry of Economy and Competitiveness (MINECO) and FEDER) and 2014-SGR-619 (Secretaria d’Universitats i Recerca, Catalan Goverment) to J.L.M.

The relaxin family peptide receptors have been implicated in numerous physiological processes including energy homeostasis, cardiac function, wound healing, and reproductive function. Two family members, RXFP3 and RXFP4, are class A GPCRs with endogenous peptide ligands (relaxin-3 and insulin-like peptide 5 (INSL5), respectively). Polymorphisms in relaxin-3 and RXFP3 have been associated with obesity, diabetes, and hypercholesterolemia. Moreover, central administration of relaxin-3 in rats has been shown to increase food intake, leading to body weight gain. Reported RXFP3 and RXFP4 ligands have been restricted to peptides (both endogenous and synthetic) as well as a low molecular weight positive allosteric modulator requiring a non-endogenous orthosteric ligand. Described here is the discovery of the first potent low molecular weight dual agonists of RXFP3/4. The scaffold identified is competitive with a chimeric relaxin-3/INSL5 peptide for RXFP3 binding, elicits similar downstream signaling as relaxin-3, and increases food intake in rats following acute central administration. This is the first report of small molecule RXFP3/4 agonism.

The present study attempted to: (1) simplify the existing enzyme immunoassay (EIA) for the measurement of insulin-like peptide 3 (INSL3) concentrations in goats (2) to measure circulating INSL3 and testosterone in pre-pubertal Jamnapari X local crossbred goats (3) to examine the relationships among INSL3 concentration, testosterone concentration, scrotal circumference and body weight during the pre-pubertal age. Serial blood samples were collected from normal pre-pubertal male Jamnapari X local crossbred goats (n = 6) at the ages of 19 to 28 weeks. Serum INSL3 was measured using a recently reported EIA with modifications to the original procedure. The detection ranges of the INSL3 and testosterone assays were 0.08 to 80 ng/mL and 0.01 to 40 ng/mL, respectively. The intra-assay coefficient of variations were 3.79% for INSL3 (n = 6) and 3.72% (n = 6) for testosterone. Serum INSL3 concentrations ranged from 13.62 ± 3.25 to 22.45 ± 6.09 ng/mL (mean ± SEM) in pre-pubertal goats. Those concentrations increased (p < 0.05) from 20 (13.62 ± 3.25 ng/mL) to 22 (22.45 ± 6.09 ng/mL) weeks of age. Testosterone concentrations ranged from 0.30 ± 0.07 to 1.22 ± 0.43 ng/mL in pre-pubertal goats. A significant drop was observed in testosterone concentrations at 23 weeks of age. INSL3 was correlated (r = 0.58; p < 0.05) with scrotal circumference while no significant correlation was observed among other tested parameters. A rapid, sensitive EIA system was simplified to quantify INSL3 in goats, by simplifying the existing procedure. Different serum INSL3 and testosterone dynamics were found from 19 to 28 weeks of age of the goats. Compared with testosterone, INSL3 dynamics seemed to be more consistent with the age of pre-pubertal goats and showed a relationship with the testicular growth. Asian J. Med. Biol. Res. December 2018, 4(4): 383-388

The involvement of insulin/insulin-like growth factor signaling (IIS) pathways in the growth regulation of marine invertebrates remains largely unexplored. In this study, we used a fast-growing Pacific oyster (Crassostrea gigas) variety “Haida No.1” as the material with which to unravel the role of IIS systems in growth regulation in oysters. Systematic bioinformatics analyses allowed us to identify major components of the IIS signaling pathway and insulin-like peptide receptor (ILPR)-mediated signaling pathways, including PI3K-AKT, RAS-MAPK, and TOR, in C. gigas. The expression levels of the major genes in IIS and its downstream signaling pathways were significantly higher in “Haida No.1” than in wild oysters, suggesting their involvement in the growth regulation of C. gigas. The expression profiles of IIS and its downstream signaling pathway genes were significantly altered by nutrient abundance and culture temperature. These results suggest that the IIS signaling pathway coupled with the ILPR-mediated signaling pathways orchestrate the regulation of energy metabolism to control growth in Pacific oysters.

Neuroendocrine hormones such as dopamine and insulin/insulin-like peptides play indispensable roles in growth regulation of animals, while the interplay between dopamine and insulin signaling pathways remains largely unknown in invertebrates. In the present study, we showed that tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis, was highly expressed in all tissues of the fast-growing oysters, and gradually increased with the development, which indicated the potential role of dopamine in growth regulation. Incubated with dopamine hydrochloride and insulin-like peptide recombinant proteins in vitro induced the expression of TH, suggesting a mutual regulatory relationship between insulin and dopamine signaling. Fasting and re-feeding experiments confirmed the role of TH in food intake regulation, also provide a clue about the potential regulatory relationship between the FoxO and TH. Further luciferase assay experiment confirmed that FoxO was involved in transcriptional regulation of TH gene through binding to its specific promoter region. This work provided insights into the crosstalk between dopamine and insulin signaling in growth control of mollusks.

The involvement of insulin/insulin-like growth factor (IIS) signaling pathway in growth regulation of marine invertebrates remains largely unexplored. In this study, we used a fast-growing Pacific oyster (Crassostrea gigas) variety “Haida No.1” as material to unravel the role of IIS system in growth regulation in oysters. Systematic bioinformatics analyses allowed to identify major components of IIS signaling pathway and insulin-like peptide receptor (ILPR) mediated signaling pathways, including PI3K-AKT, RAS-MAPK, and TOR, in C. gigas. Expression levels of the major genes in IIS and its downstream signaling pathways were significantly higher in “Haida No.1” than wild oysters, suggesting their involvement in growth regulation of C. gigas. Expression profiles of IIS and its downstream signaling pathway genes were significantly altered by nutrient abundance and culture temperature. These results suggested that IIS signaling pathway coupled with the ILPR mediated signaling pathways orchestrated energy homeostasis to regulate growth in the Pacific oyster.Research HighlightsILPR, IRS, IGFBPRP, and IGFALS genes were characterized in the C. gigas.Major genes of IIS signaling pathway were highly expressed in fast-growing C. gigas.IIS and downstream pathways participates in energy homeostasis of oysters.ILPR mediated signaling pathways orchestrate growth regulation in oysters.

Leucokinins (LKs) constitute a family of neuropeptides identified in numerous insects and many other invertebrates. LKs act on G-protein-coupled receptors that display only distant relations to other known receptors. In adult Drosophila, 26 neurons/neurosecretory cells of three main types express LK. The four brain interneurons are of two types, and these are implicated in several important functions in the fly’s behavior and physiology, including feeding, sleep–metabolism interactions, state-dependent memory formation, as well as modulation of gustatory sensitivity and nociception. The 22 neurosecretory cells (abdominal LK neurons, ABLKs) of the abdominal neuromeres co-express LK and a diuretic hormone (DH44), and together, these regulate water and ion homeostasis and associated stress as well as food intake. In Drosophila larvae, LK neurons modulate locomotion, escape responses and aspects of ecdysis behavior. A set of lateral neurosecretory cells, ALKs (anterior LK neurons), in the brain express LK in larvae, but inconsistently so in adults. These ALKs co-express three other neuropeptides and regulate water and ion homeostasis, feeding, and drinking, but the specific role of LK is not yet known. This review summarizes Drosophila data on embryonic lineages of LK neurons, functional roles of individual LK neuron types, interactions with other peptidergic systems, and orchestrating functions of LK.

IntroductionInsulin-like peptide 5 (INSL5) is a peptide hormone with proposed actions in glucose homeostasis and appetite regulation via its cognate receptor, relaxin family peptide receptor 4 (RXFP4). Here, we look for evidence for their involvement in the immune system using a mouse model.MethodsIn silico analyses: we queried public databases for evidence of expression of INSL5-RXFP4 in immune system tissues/cells (NCBI’s SRA and GeoProfiles) and disorders (EMBO-EBI) and performed phylogenetic footprinting to look for evidence that they are regulated by immune-associated transcription factors (TFs). Experimental analyses: We characterized the expression and correlation of INSL5/RXFP4 and other immune system markers in central and peripheral immune organs from C57/bl6 mice in seven cohorts. We tested whether fluctuations in circulating INSL5 induce an immune response, by injecting mice with 30 μg/kg of INSL5 peptide in the peritoneum, and examining levels of immune markers and metabolic peptides in plasma. Lastly, we quantified the expression of Rxfp4 in T-cells, dendritic cells and cell lines derived from human and mouse and tested the hypothesis that co-incubation of ANA-1 cells in INSL5 and LPS alters cytokine expression.ResultsWe find Insl5 expression only in thymus (in addition to colon) where its expression was highly correlated with Il-7, a marker of thymocyte development. This result is consistent with our in silico findings that Insl5 is highly expressed in thymic DP, DN thymocytes and cortical TEC’s, and with evidence that it is regulated by thymocyte-associated TF’s. We find Rxfp4 expression in all immune organs, and moderately high levels in DCs, particularly splenic DCs, and evidence that it is regulated by immune-associated TF’s, such as STAT’s and GATA. Systemic effects: We observed significantly elevated concentrations of blood GLP-1, GIP, GCG and PYY following intraperitoneal injection of INSL5, and significantly altered expression of cytokines IL-5, IL-7, M-CSF, IL-15, IL-27 and MIP-2. Immune cell effects: Incubation of ANA-1 cells with INSL5 impeded cell growth and led to a transient elevation of IL-15 and sustained reduction in IL-1β, IL-6 and TNFα.ConclusionWe propose that INSL5-RXFP4 play a novel role in both central and peripheral immune cell signaling.

The insulin family consists of insulin, IGFs, relaxins, and insulin-like peptides, and is one of the most phylogenetically ancient groups of hormones in animals. Mature peptides are the heterodimers comprising the A- and B-chains. The cysteine motif (Cys–Cys–3X–Cys–8X–Cys) in the A-chain has been termed the insulin signature. The insulin family has a broad spectrum of functions, including roles in nutrient metabolism, cell and organism growth, and reproduction. The receptors are classified into three structurally different types, the heterotetramer type with a tyrosine kinase domain, the GPCR type, and the cation-independent mannose-6-phosphate receptor, indicating that the receptors are phylogenetically polyphyletic. The various functions of the insulin family members are results of co-evolution of ligands and receptors encountered at several different times throughout evolution.

[Image: see text] The receptor for the neuropeptide relaxin 3, relaxin family peptide 3 (RXFP3) receptor, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviors. Several structure–activity relationship studies on both human relaxin 3 (containing 3 disulfide bonds) and its analogue A2 (two disulfide bonds) suggest that the C-terminal carboxylic acid of the tryptophan residue in the B-chain is important for RXFP3 activity. In this study, we have added amide, alcohol, carbamate, and ester functionalities to the C-terminus of A2 and compared their structures and functions. As expected, the C-terminal amide form of A2 showed lower binding affinity for RXFP3 while ester and alcohol substitutions also demonstrated lower affinity. However, while these analogues showed slightly lower binding affinity, there was no significant difference in activation of RXFP3 compared to A2 bearing a C-terminal carboxylic acid, suggesting the binding pocket is able to accommodate additional atoms.

Superoxide dismutases, which catalytically remove intracellular superoxide radicals by the disproportionation of molecular oxygen and hydrogen peroxide, are encoded by the sod-1 to -5 genes in the nematode C. elegans. Expression of the sod genes is mutually compensatory for the modulation of intracellular oxidative stress during aging. Interestingly, several-fold higher expression of the sod-1 to -4 was induced in a sod-5 deletion mutant, despite the low expression levels of sod-5 in wild-type animals. Consequently, this molecular compensation facilitated recovery of lifespan in the sod-5 mutant. In previous reports, two transcription factors DAF-16 and SKN-1 are associated with the compensatory expression of sod genes, which are downstream targets of the ins/IGF-1 and p38 MAPK signaling pathways activated under oxidative and heavy metal stresses, respectively. Here, we show that p38 MAPK signaling regulates induction of not only the direct expression of sod-1, -2 and -4 but also the indirect modulation of DAF-16 targets, such as sod-3 and -5 genes. Moreover, a SKN-1 target, the insulin peptide gene ins-5, partially mediates the expression of DAF-16 targets via p38 MAPK signaling. These findings suggest that the interaction of ins/IGF-1 and p38 MAPK signaling pathways plays an important role in the fine-tuning of molecular compensation among sod genes to protect against mitochondrial oxidative damage during aging., Highlights • Mitochondrial ROS is removed by SODs during aging. • Expression of sod genes in C. elegans related to lifespan maintenance. • Interaction of the ins/IGF-1 and p38 MAPK signalings regulates the fine-tuning of sod genes expression. • ins-5 of SKN-1 target encodes an agonist of ins/IGF-1 signaling.

InDrosophilaeight insulin-like peptides (DILP1-8) are encoded on separate genes. These DILPs are characterized by unique spatial and temporal expression patterns during the lifecycle. Whereas functions of several of the DILPs have been extensively investigated at different developmental stages, the role of DILP8 signaling is primarily known from larvae and pupae where it couples organ growth and developmental transitions. In adult female flies, a study showed that a specific set of neurons that express the DILP8 receptor, Lgr3, is involved in regulation of reproductive behavior. Here, we further investigated the expression ofdilp8/DILP8 andLgr3in adult female flies and the functional role of DILP8 signaling. The only site where we found bothdilp8expression and DILP8 immunolabeling was in follicle cells of mature ovaries.Lgr3expression was detected in numerous neurons in the brain and ventral nerve cord, a small set of peripheral neurons innervating the abdominal heart, as well as in a set of follicle cells close to the oviduct. Ovulation was affected indilp8mutants as well as afterdilp8-RNAi usingdilp8and follicle cell Gal4 drivers. More eggs were retained in the ovaries and fewer were laid, indicating that DILP8 is important for ovulation. Our data suggest that DILP8 signals locally toLgr3expressing follicle cells as well as systemically toLgr3expressing efferent neurons in abdominal ganglia that innervate oviduct muscle. Thus, DILP8 may act at two targets to regulate ovulation: follicle cell rupture and oviduct contractions. Furthermore, we could show that manipulations ofdilp8expression affect food intake and starvation resistance. Possibly this reflects a feedback signaling between ovaries and the CNS that ensures nutrients for ovary development. In summary, it seems that DILP8 signaling in regulation of reproduction is an ancient function, conserved in relaxin signaling in mammals.

Invertebrate insulin-like peptide-binding proteins (ILPBPs) are structurally homologous to vertebrate insulin-like growth factor binding protein 7 (IGFBP7). One of the invertebrate ILPBPs is considered as a potential binding protein of insulin-like androgenic gland factor (IAG) in males of crayfish, Cherax quadricarinatus. However, the ILPBP expression is not limited in the androgenic gland and found in most examined tissues, implicating that ILPBP may have additional functions in crustaceans. Here, the full-length cDNA sequence of ILPBP (termed ChqILPBP) is isolated from the ovary of the red deep-sea crab, Chaceon quinquedens. ChqILPBP transcripts are present in the various tissues, as similar to other crab species. The crustacean ILPBPs have their putative amino acid sequences conserved much less than vertebrate IGFBP7s. To understand if ChqILPBP is involved in ovarian development, examined are levels of ChqILPBP, together with vitellogenin (ChqVTG) in the same ovary and hepatopancreas of adult females at the different ovarian stages: 2, 3, and 5. Chaceon hepatopancreas exhibits as the primary VTG synthesis site, while VTG transcript levels do not differ by the ovarian stages. The ovary contains ChqILPBP transcripts ~10-fold higher than hepatopancreas that changes significantly from stage 2 to 3. Such an expression pattern mirrors that of ovarian ChqVTG. In hepatopancreas, ChqILPBP transcripts are similar at stages 2 and 3 and increase significantly at stage 5. The data indicate that ovarian ILPBP may function differently from that of the hepatopancreas and may play a role in ovarian development. ChqAK transcripts are ~six folds higher in the ovary than the hepatopancreas. While they do not differ by ovarian stages, suggesting that AK may not be involved in vitellogenesis of the cold water crustacean species.

Establishing appropriate sizes and shapes of dendritic arbors is critical for proper wiring of the central nervous system. Here we report that Insulin-like Peptide 2 (DILP2) locally activates transiently expressed insulin receptors in the central dendrites of Drosophila Dm8 amacrine neurons to positively regulate dendritic field elaboration. We found DILP2 was expressed in L5 lamina neurons, which have axonal terminals abutting Dm8 dendrites. Proper Dm8 dendrite morphogenesis and synapse formation required insulin signaling through TOR (target of rapamycin) and SREBP (sterol regulatory element-binding protein), acting in parallel with previously identified negative regulation by Activin signaling to provide robust control of Dm8 dendrite elaboration. A simulation of dendritic growth revealed trade-offs between dendritic field size and robustness when branching and terminating kinetic parameters were constant, but dynamic modulation of the parameters could mitigate these trade-offs. We suggest that antagonistic DILP2 and Activin signals from different afferents appropriately size Dm8 dendritic fields.

Insulin-like peptide 3 (INSL3) is an important hormone for testicular descent during embryonic development and a factor for assessing functional status of Leydig cells of testes, but there is limited number of equine studies. Anti-Müllerian hormone (AMH) is a useful diagnostic marker for cryptorchidism in horses. This study aimed to compare serum concentrations and testicular expression intensity of INSL3 and AMH in intact and cryptorchid male horses. Serum INSL3 concentrations in intact (n = 9; mean ± SEM, 19.9 ± 5.9 ng/mL) and noncastrated unilateral cryptorchid (UC) male horses (n = 16; mean ± SEM, 16.8 ± 4.1 ng/mL) were higher compared with hemicastrated unilateral cryptorchid (HCUC) male horses (n = 9; mean ± SEM, 3.8 ± 0.7 ng/mL) (P 0.05). And serum INSL3 in bilateral cryptorchid (BC) male horses (n = 4; 1.9 ± 0.4; mean ± SEM, ng/mL) were lower compared with intact male horses (P 0.05). Serum AMH concentrations in BC male horses (n = 3; mean ± SEM, 30.6 ± 4.8 ng/mL) were higher compared with intact male horses (n = 5; mean ± SEM, 12.2 ± 3.9 ng/mL) (P 0.05). Immunostaining of scrotal and cryptorchid testis showed that Sertoli cells were positive for AMH, and Leydig cells were positive for INSL3. Staining intensity of AMH was higher in cryptorchid testis than in scrotal testis (P 0.05). Furthermore, AMH expression intensity was higher in abdominal testis than in inguinal testis (P 0.05). Immunostaining intensity of INSL3 in the testis was positively correlated with serum INSL3 (r, 0.7; P 0.01), seminiferous tubule area (r, 0.727; P 0.01), and Johnsen score for spermatogenesis (r, 0.604; P 0.05), whereas immunostaining intensity of AMH in the testis was negatively correlated with seminiferous tubule area (r, -0.814; P 0.01) and Johnsen score for spermatogenesis (r, -0.807; P 0.01). Our findings suggested that AMH is a good biomarker for diagnosing cryptorchidism in male horses, in addition to INSL3 values to assess the testis of intact and cryptorchid male horses.

Background Insulin-like peptide 5 (INSL5) is a hormone stored in colonic enteroendocrine cells that also contain the unrelated hormones, GLP-1 and PYY. It acts at the relaxin family peptide 4, RXFP4, receptor. RXFP4 is expressed by enteric neurons in the colon, and it has been speculated that INSL5, through its action on enteric neurons, might be involved in the control of colonic contractions. Similar to insulin and relaxin, INSL5 consists of A and B peptide chains linked by three disulfide bonds, two between the chains and one intrinsic to the A chain. Because of its complex structure, it is difficult to synthesize and to prepare peptide analogues to investigate its roles. We have recently developed a potent simplified peptide analogue, INSL5-A13 (INSL5 analogue 13). Methods In the present work, we have investigated the actions of INSL5-A13 in mice. We investigated the ability of INSL5-A13 to increase the speed of emptying of a bead from the colon, after expulsion had been slowed by the peripherally restricted opioid agonist, loperamide (1 mg/kg). Key results INSL5-A13 was a full agonist at the mouse RXFP4 expressed in HEK cells, with an EC50 of ~9 nmol/L. INSL5-A13 caused an acceleration of colorectal bead propulsion in mice constipated by loperamide in the dose range 0.2 to 60 µg/kg, with an EC50 of ~6 µg/kg in vivo. It also accelerated bead propulsion in untreated mice. Bead expulsion was not accelerated in RXFP4-/- mice. Conclusion and inferences Our data suggest that RXFP4 agonists could be useful in the treatment of constipation.

AIM The appendix testis (AT) is a vestigial remnant of Muller's paramesonephric duct. Insulin-like 3 hormone (INSL3) is produced in the Leydig cells of the testis. We investigated the possible correlation between AT length and plasma INSL3 concentrations in patients with congenital cryptorchidism (CCO) and patients with hydrocele, who served as controls. METHODS A total of 40 patients with CCO and 34 patients with hydrocele and orthotopic testes were investigated. Sixteen patients presented high cryptorchidism and 24 low cryptorchidism. During surgery, AT was identified in 34 patients with CCO (high cryptorchidism:15, low cryptorchidism:19) and 28 controls. Plasma INSL3 levels were measured with a spectrophotometry enzyme immunoassay Elisa sandwich technique. RESULTS AT was present in 85.0% of the boys with CCO and 82.4% of the controls. A significant positive correlation was found between the AT length and INSL3 concentrations in CCO patients. CONCLUSIONS A longer AT may reflect better testicular function in boys with CCO, since it is correlated with higher INSL3 concentrations.

Insulin-like peptide 2 is an important gene in the insulin/insulin-like signaling pathway. The insulin-like peptide 2 gene (ILP-2) appears to play a key role in metabolism, growth, reproduction, stress resistance and aging. In this study, we isolated and characterized an ILP-2 gene from Apis cerana cerana known as AccILP-2. The full-length AccILP-2 cDNA is 494 bp and contains a 222-bp open reading frame (ORF) encoding a protein of 73 amino acids with a calculated molecular weight of 8.706 kDa and an isoelectric point of 9.02. Multiple sequence alignment revealed that AccILP-2 shares high identity with ILP genes from Apis mellifera, Apis florea, Bombus terrestris, Eufriesea mexicana and Dufourea novaeangliae and contains a typical IlGF-insulin-bombyxin-like domain. Quantitative PCR analysis indicated that the greatest expression of AccILP-2 was observed in the muscles of adult workers. Expression analysis results also indicated that AccILP-2 expression was greatest during the dark-eye developmental period. Furthermore, expression of the AccILP-2 transcript was induced by environmental stressors, including exposure to 4 °C, ultraviolet light, H2O2, heavy metals (HgCl2 and CdCl2) and pesticides (dichlorvos, paraquat and cyhalothrin). However, AccILP-2 expression was downregulated at 44 °C. In conclusion, these results suggested that AccILP-2 might play an important role in the response to abiotic stress. We expect that our study will promote future research on the function of the insulin/insulin-like signaling pathway.

Many insulin-like peptides (ILPs) have been identified in insects, yet only a few were isolated in their native form for structural and functional studies. Antiserum produced to ILP3 in Aedes aegypti was used in a radioimmunoassay to monitor the purification of an ILP from heads of adult An. stephensi and recognized the ILP in other immunoassays. The structure of the purified peptide matched that predicted for the ILP3 in this species. The native form stimulated ecdysteroid production by ovaries isolated from non-blood fed females. Synthetic forms of An. stephensi ILP3 and ILP4 similarly activated this process in a dose responsive manner. This function was first established for ILP3 and ILP4 homologs in Aedes aegypti, thus suggesting their structural and functional conservation in mosquitoes. We tested the extent of conservation by treating ovaries of An. gambiae, Ae. aegypti, and Culex quinquefasciatus with the An. stephensi ILPs, and both the native and synthetic ILP3 were stimulatory, as was the ILP4. Taken together, these results offer the first evidence for ILP functional conservation across the Anophelinae and Culicinae subfamilies.

The endocrine stress response in Drosophila includes catecholamines, juvenile hormone (JH), 20-hydroxyecdysone (20E) and the insulin/insulin-like growth factor signalling pathway (IIS). Several changes in the IIS and hormonal status that occur under unfavourable conditions are universal and do not depend on the nature of stress exposure. The reviewed studies on the impact of different element of the Drosophila IIS, such as insulin-like receptor, the homologue of its substrate, CHICO, the transcription factor dFOXO and insulin like peptide 6, on the hormonal status suggest that the IIS controls catecholamine metabolism indirectly via JH, and there is a feedback loop in the interaction of JH and IIS. Moreover, at least one of the ways in which the IIS is involved in the control of stress resistance is mediated through JH/dopamine signalling.

Insulin and insulin-like peptides (ILPs) are conserved in living organisms to modulate homeostasis by functioning as ligands. For understanding of molecular mechanisms regulated by the ligands, the nematode Caenorhabditis elegans is a good model since: 1) the C. elegans genome size is small with over 40% homology to the human genome, 2) numerous genetic methods are available, and 3) the worms are transparent throughout the life cycle, so that the secretion of peptide hormones can be followed at cellular level in living preparations by GFP-tagged peptides. In this review, we describe the general appearance of the insulin/insulin-like growth factor (IGF)-1 signaling (IIS), and then focus on physiological functions, secretion, and transcriptional regulation of the C. elegans ILPs.

Insulin-like peptide 3 (INSL3) is a member of the relaxin/insulin superfamily and is expressed in testicular Leydig cells. Essential for fetal testis descent, INSL3 has been implicated in testicular and sperm function in adult males via interaction with relaxin/insulin-like family peptide receptor 2 (RXFP2). The INSL3 is typically prepared using chemical synthesis or overexpression in Escherichia coli followed by oxidative refolding and proteolysis. Here, we expressed and purified full-length porcine INSL3 (pINSL3) using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system. Biophysical measurements and proteomic analysis revealed that this recombinant pINSL3 exhibited the correct conformation, with the three critical disulfide bonds observed in native pINSL3, although partial cleavage occurred. In cAMP stimulation assays using RXFP2-expressing HEK293 cells, the recombinant pINSL3 possessed full biological activity. This is the first report concerning the production of fully active pINSL3 without post-expression treatments and provides an efficient production platform for expressing relaxin/insulin superfamily peptides.

Objective: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. Materials and methods: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. Results: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. Conclusion: The CCAP, corazonin and ETH neuropeptides are potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

Insulin-like peptide 5 (INSL5) is a member of the insulin superfamily peptide that interacts with the relaxin family peptide receptor 4 (RXFP4). Numerous recent studies have focused on the functional effects of INSL5 on fat and glucose metabolism. Although there is no evidence that the human sperm may be a candidate target of INSL5, it has been detected in mice testis and sperm. Therefore, the present study sought to analyze the localization and expression of RXFP4 on human sperm and determine the efficiency of INSL5 in human sperm. Normal semen samples were incubated in different doses and exposure time periods of INSL5. We analyzed sperm motility by computer-assisted sperm analysis (CASA) and ROS levels by flow cytometry using the MitoSOX™ Red probe. Localization and expression of RXFP4 were assayed by immunofluorescence and RT-PCR, respectively. The results confirmed the presence of RXFP4 in human spermatozoa, which localized in the neck and midpiece of sperm. Nested PCR showed the expression of RXFP4 in human sperm. INSL5 could attenuate generation of mitochondrial ROS at the 1, 10, 30, and 100nmol/L doses. This result was particularly noted in the 30nmol/L treated samples after 4h incubation. Total motility of sperm was significantly preserved in the 100nmol/L after 2h and in 30nmol/L after 4h incubation period. This study, for the first time, clarified the expression and localization of RXFP4 on human sperm and revealed the role of INSL5 in sperm motility and mitochondrial ROS generation in a dose-dependent manner.

Objective Natural sources of molecular diversity remain of utmost importance as a reservoir of proteins and peptides with unique biological functions. We recently identified such a family of viral insulin-like peptides (VILPs). We sought to advance the chemical methods in synthesis to explore the structure-function relationship within these VILPs, and the molecular basis for differential biological activities relative to human IGF-1 and insulin. Methods Optimized chemical methods in synthesis were established for a set of VILPs and related analogs. These modified forms included the substitution of select VILP chains with those derived from human insulin and IGF-1. Each peptide was assessed in vitro for agonism and antagonism at the human insulin and the human insulin-like growth factor 1 receptor (IGF-1R). Results We report here that one of these VILPs, lymphocystis disease virus-1 (LCDV1)-VILP, has the unique property to be a potent and full antagonist of the IGF-1R. We demonstrate the coordinated importance of the B- and C-chains of the VILP in regulating this activity. Moreover, mutation of the glycine following the first cysteine in the B-chain of IGF-1 to serine, in concert with substitution to the connecting peptide of LCDV1-VILP, converted native IGF-1 to a high potency antagonist. Conclusions The results reveal novel aspects in ligand–receptor interactions at the IGF-1 receptor and identify a set of antagonists of potential medicinal importance., Graphical abstract Image 1, Highlights • Improved chemical synthesis of single-chain and two-chain analogs of insulin-like peptides derived from viral sources. • Characterization of virus-1 (LCDV1)-VILP as a potent and selective IGF-1 antagonist. • Molecular identification of the amino acid changes in the B- and C-domains that define IGF-1 agonism versus antagonism. • Correlation of a (LCDV1)-VILP residue essential to IGF-1 receptor antagonism with a proinsulin mutation linked with diabetes.

Insulin-like androgenic gland factor (IAG) that is produced by the male androgenic gland (AG), plays a role in sexual differentiation and maintenance of male secondary sex characteristics in decapod crustaceans. With an earlier finding of IAG expression in a female Callinectes sapidus ovary, we aimed to examine a putative role of IAG during the ovarian development of this species. To this end, the full-length cDNA sequence of the ovarian CasIAG (termed CasIAG-ova) has been isolated. The predicted mature peptide sequence of CasIAG-ova is identical to that of the IAG from the AG, except in their signal peptide regions. The CasIAG-ova contains an alternative initiation codon (UUG) as the start codon, which suggests that the translational regulation of CasIAG-ova may differ from that of the IAG from AG. To define the function of CasIAG-ova, the expressions of CasIAG-ova as well as its putative binding protein, insulin-like peptide binding protein (ILPBP), are measured in the ovaries at various developmental stages obtained from different seasons. Season affects both CasIAG and ILPBP expression in the ovary. Overall, summer females at earlier ovarian stages contain high levels of CasIAG and ILPBP than spring or fall females. These findings indicate that CasIAG-ova and CasILPBP may be involved in the ovarian development. When comparing the levels of CasIAG and CasILPBP in the ovary, the latter are much higher (∼10–10000 fold) than the former. Expression patterns of CasILPBP differ from those of CasIAG-ova during ovarian development and by season, suggesting that ILPBP may have an additional role in ovarian development rather than a function of a putative binding protein of IAG.

This report presents an entirely chemical, general strategy for the synthesis of relaxin-2 and insulin-like peptide 5. Historically, these two peptides have represented two of the more synthetically challenging members of the insulin superfamily. The key synthetic steps involve two sequential oxime ligations to covalently link the individual A-chain and B-chain, followed by disulfide bond formation under aqueous, redox conditions. This is followed by two chemical reactions that employ diketopiperazine cyclization-mediated cleavage and ester hydrolysis to liberate the connecting peptide and the heterodimeric product. This approach avoids the conventional iodine-mediated disulfide bond formation and enzyme-assisted proteolysis to generate biologically active two-chain peptides. This novel synthetic strategy is ideally suited for peptides such as relaxin and insulin-like peptide 5 as they possess methionine and tryptophan that are labile under strong oxidative conditions. Additionally, these peptides possess multiple arginine and lysine residues that preclude the use of trypsin-like enzymes to obtain biologically active hormones. This synthetic methodology is conceivably applicable to other two-chain peptides that contain multiple disulfide bonds. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Background Previous studies have shown that insulin receptors in schistosomes, triggered by host insulin, play an important role in parasite growth, development and fecundity by regulating glucose metabolism. However, limited information is available on the recently identified endogenous insulin-like peptide (ILP) in blood flukes. Results We isolated ILPs from Schistosoma japonicum (SjILP) and S. recognised (SmILP) and present results of their molecular and structural analysis. SjILP shares 63% amino acid identity with SmILP, but only 18% identity with human insulin. There is high cross immunological reactivity between the S. japonicum and S. mansoni ILPs as observed in western blots using an anti-SjILP polyclonal antibody. ADP binding/hydrolysis ability was observed in both SjILP and SmILP, but not in human insulin, suggesting a parasite-specific role for ILP compared with host insulin. Protein binding assays using the Octet-RED system showed SjILP binds S. japonicum IRs (SjIR1 and SjIR2) strongly. An anti-phospho antibody against extracellular signal-regulated kinase (Erk) recognised a 44-kDa target band in an extract of adult worms after stimulation by rSjILP in vitro, suggesting an important role for SjILP in activating SjIRs and in regulating downstream signal transduction. Immunolocalisation showed SjILP is located on the tegument and the underlying musculature, similar to that observed for SjIR1, but it is also present throughout the parenchyma of males and in the vitelline cells of females, the same locations as SjIR2 described in an earlier published study of ours. The same localisation of SjILP and the SjIRs is suggestive of an interaction between the insulin-like peptide and the IRs. In addition to binding host insulin, schistosomes also can express their own endogenous ILPs, which can activate the parasite insulin signal pathway, thereby playing a critical role in worm growth, development and fertility. Conclusions These findings shed new light on ILPs in schistosomes, providing further insight into the distinct and specialised functions of SjIR1 and 2 in S. japonicum and their interaction with host insulin. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2095-7) contains supplementary material, which is available to authorized users.

Insulin-like peptide 5 (INSL5) is a gut peptide hormone belonging to the insulin/relaxin superfamily. It is implicated in the regulation of food intake and glucose homeostasis by activating relaxin family peptide receptor 4 (RXFP4). Previous studies have suggested that the B-chain is important for INSL5 activity against RXFP4. However, functionalities of the B-chain residues have not yet been systematically studied. In the present work, we conducted alanine-scanning mutagenesis of the B-chain residues of human INSL5 to obtain an overview of their contributions. Binding and activation assays of these INSL5 mutants with human RXFP4 identified two essential exposed B-chain C-terminal residues (B23Arg and B24Trp) and one important exposed central B-chain residue (B16Ile). These three determinant residues together with the C-terminal carboxylate moiety probably constitute a central receptor-binding patch that forms critical hydrophobic and electrostatic interactions with RXFP4 during INSL5 binding. Some other exposed residues, including B10Glu, B12Ile, B13Arg, B17Tyr, B21Ser, and B22Ser, made minor contributions to INSL5 function. These auxiliary residues are scattered around the edge of the central receptor-binding patch, and thus form a peripheral receptor-binding patch on the surface of INSL5. Our present work provides new insights into the interaction mechanism of INSL5 with its receptor RXFP4.

Phosphorylation of the phytosulfokine peptide receptor PSKR1 differentially affects auto- and transphosphorylation activity, binding to Ca2+-calmodulin, and has differential effects on shoot and root growth., The phytosulfokine peptide receptor PSKR1 is modified by phosphorylation of its cytoplasmic kinase domain. We analyzed defined phosphorylation sites by site-directed mutagenesis with regard to kinase activity in vitro and receptor activity in planta. S696 and S698 in the juxtamembrane (JM) domain are phosphorylated in planta. The phosphomimetic S696D/S698D replacements resulted in reduced transphosphorylation activity of PSKR1 kinase in vitro but did not reduce autophosphorylation activity. Growth-promoting activity of the PSKR1(S696D/S698D) receptor isoform was impaired in the shoot but not in the root. The JM domain thus seems to be important for phosphorylation of a target protein required for shoot growth promotion. The phosphomimetic replacement T998D at the C-terminus (CT) abolished kinase activity in vitro but not receptor function in planta, indicating that additional levels of regulation exist in planta. A possible mode of receptor regulation is the interaction with regulatory proteins such as the calcium sensor calmodulin (CaM). We show that the previously reported binding of CaM2 to PSKR1 is calcium-dependent, occurs predominately to the hypophosphorylated soluble PSKR1 kinase, and does not significantly change PSKR1 kinase activity. In conclusion, our results show that peptide signaling of growth by PSKR1 is regulated by differential phosphorylation of the juxtamembrane and C-terminal domains of the intracellular receptor part and suggest that interaction of PSKR1 with CaM serves a function other than the regulation of kinase activity.

In a number of works, it was demonstrated that insulin/insulin-like growth factor signaling pathway in the Drosophila melanogaster can be involved in the control of the organism reaction to stress. However, it remains unclear which of eight insulin-like peptides (ligands of insulin/insulin-like growth factor signaling pathway) known in the D. melanogaster are involved in the response to different types of stress. We conducted immunohistochemical analysis of the expression of two insulin-like peptide genes (DILP2 and DILP3) in insulin-producing cells of the brain in adult D. melanogaster females after heat stress. We for the first time found that the DILP3 is one of the components of the response to heat stress, while the DILP2 is apparently not involved in the organism response to heat stress.

Evolutionarily conserved insulin/insulin-like growth factor (IGF) signaling (IIS) has been identified as a major physiological mechanism underlying the nutrient-dependent regulation of sexually selected weapon growth in animals. However, the molecular mechanisms that couple nutritional state with weapon growth remain largely unknown. Here, we show that one specific subtype of insulin-like peptide (ILP) responds to nutrient status and thereby regulates weapon size in the broad-horned flour beetle Gnatocerus cornutus. By using transcriptome information, we identified five G. cornutus ILP (GcorILP1–5) and two G. cornutus insulin-like receptor (GcorInR1, -2) genes in the G. cornutus genome. RNA interference (RNAi)-mediated gene silencing revealed that a certain subtype of ILP, GcorILP2, specifically regulated weapon size. Importantly, GcorILP2 was highly and specifically expressed in the fat body in a condition-dependent manner. We further found that GcorInR1 and GcorInR2 are functionally redundant but that the latter is partially specialized for regulating weapon growth. These results strongly suggest that GcorILP2 is an important component of the developmental mechanism that couples nutritional state to weapon growth in G. cornutus. We propose that the duplication and subsequent diversification of IIS genes played a pivotal role in the evolution of the complex growth regulation of secondary sexual traits., Animals can evolve sexually selected weapons and ornaments as the exaggerations of specific body parts, often linked to nutritional status. This study shows that in the broad-horned flour beetle, one insulin-like peptide (among the five encoded by their genome) was found to specifically regulate weapon growth. Functional diversification of ligand peptides may account for the ecological divergence of sexually selected exaggerated traits.

Dietary restriction (DR) is the most robust means to extend lifespan and delay age-related diseases across species. An underlying assumption in the aging field is that DR enhances both lifespan and physical activity through similar mechanisms, but this has not been rigorously tested in different genetic backgrounds. Furthermore, nutrient response genes responsible for lifespan extension or age-related decline in functionality remain underexplored in natural populations. To address this, we measured nutrient-dependent changes in lifespan and age-related decline in climbing ability in the Drosophila Genetic Reference Panel fly strains. On average, DR extended lifespan and delayed decline in climbing ability, but there was a lack of correlation between these traits across individual strains, suggesting that distinct genetic factors modulate these traits independently and that genotype determines response to diet. Only 50% of strains showed positive response to DR for both lifespan and climbing ability, 14% showed a negative response for one trait but not both, and 35% showed no change in one or both traits. Through GWAS, we uncovered a number of genes previously not known to be diet responsive nor to influence lifespan or climbing ability. We validated decima as a gene that alters lifespan and daedalus as one that influences age-related decline in climbing ability. We found that decima influences insulin-like peptide transcription in the GABA receptor neurons downstream of short neuropeptide F precursor (sNPF) signaling. Modulating these genes produced independent effects on lifespan and physical activity decline, which suggests that these age-related traits can be regulated through distinct mechanisms.

Sleep is a nearly universal behavior that is regulated by diverse environmental stimuli and physiological states. A defining feature of sleep is a homeostatic rebound following deprivation, where animals compensate for lost sleep by increasing sleep duration and/or sleep depth. The fruit fly, Drosophila melanogaster, exhibits robust recovery sleep following deprivation and represents a powerful model to study neural circuits regulating sleep homeostasis. Numerous neuronal populations have been identified in modulating sleep homeostasis as well as depth, raising the possibility that the duration and quality of recovery sleep is dependent on the environmental or physiological processes that induce sleep deprivation. Here, we find that unlike most pharmacological and environmental manipulations commonly used to restrict sleep, starvation potently induces sleep loss without a subsequent rebound in sleep duration or depth. Both starvation and a sucrose-only diet result in increased sleep depth, suggesting that dietary protein is essential for normal sleep depth and homeostasis. Finally, we find that Drosophila insulin like peptide 2 (Dilp2) is acutely required for starvation-induced changes in sleep depth without regulating the duration of sleep. Flies lacking Dilp2 exhibit a compensatory sleep rebound following starvation-induced sleep deprivation, suggesting Dilp2 promotes resiliency to sleep loss. Together, these findings reveal innate resilience to starvation-induced sleep loss and identify distinct mechanisms that underlie starvation-induced changes in sleep duration and depth., Author summary Sleep is nearly universal throughout the animal kingdom and homeostatic regulation represents a defining feature of sleep, where animals compensate for lost sleep by increasing sleep over subsequent time periods. Despite the robustness of this feature, the neural mechanisms regulating recovery from different types of sleep deprivation are not fully understood. Fruit flies provide a powerful model for investigating the genetic regulation of sleep, and like mammals, display robust recovery sleep following deprivation. Here, we find that unlike most stimuli that suppress sleep, sleep deprivation by starvation does not require a homeostatic rebound. These findings are likely due to flies engaging in deeper sleep during the period of partial sleep deprivation, suggesting a natural resilience to starvation-induced sleep loss. This unique resilience to starvation-induced sleep loss is dependent on Drosophila insulin-like peptide 2, revealing a critical role for insulin signaling in regulating interactions between diet and sleep homeostasis.

We showed previously that the Schistosoma japonicum insulin-like peptide (SjILP) binds the worm insulin receptors, thereby, activating the parasite&rsquo, s insulin pathway and emphasizing its important role in regulating uptake of glucose, a nutrient essential for parasite survival. Here we show that SjILP is differentially expressed in the schistosome life cycle and is especially highly transcribed in eggs, miracidia, and adult female worms. RNA inference was employed to knockdown SjILP in adults in vitro, with suppression confirmed by significantly reduced protein production, declined adenosine diphosphate levels, and reduction in glucose consumption. Immunolocalization showed that SjILP is located to lateral gland cells of mature intra-ovular miracidia in the schistosome egg, and is distributed on the ciliated epithelium and internal cell masses of newly transformed miracidia. In schistosomula, SjILP is present on the tegument in two antero-lateral points, indicating highly polarized expression during cercarial transformation. Analysis of serum from S. japonicum-infected mice by ELISA using a recombinant form of SjILP as an antigen revealed IgG immunoreactivity to this molecule at 7 weeks post-infection indicating it is likely secreted from mature eggs into the host circulation. These findings provide further insights on ILP function in schistosomes and its essential roles in parasite survival and growth in different development stages.

Insulin signaling is a conserved pathway in all metazoans. This pathway contributed toward primordial metazoans responding to a greater diversity of environmental signals by modulating nutritional storage, reproduction, and longevity. Most of our knowledge of insulin signaling in insects comes from the vinegar fly, Drosophila melanogaster, where it has been extensively studied and shown to control several physiological processes. Mosquitoes are the most important vectors of human disease in the world and their control constitutes a significant area of research. Recent studies have shown the importance of insulin signaling in multiple physiological processes such as reproduction, innate immunity, lifespan, and vectorial capacity in mosquitoes. Although insulin-like peptides have been identified and functionally characterized from many mosquito species, a comprehensive review of this pathway in mosquitoes is needed. To fill this gap, our review provides up-to-date knowledge of this subfield.

Background Toxocara canis is quite closely related to Ascaris suum but its biology is more complex, involving a phase of arrested development (diapause or hypobiosis) in tissues as well as transplacental and transmammary transmission routes. In the present study, we explored and compared dauer-like signalling pathways of T. canis and A. suum to infer which components in these pathways might associate with, or regulate, this added complexity in T. canis. Methods Guided by information for Caenorhabditis elegans, we bioinformatically inferred and compared components of dauer-like signalling pathways in T. canis and A. suum using genomic and transcriptomic data sets. In these two ascaridoids, we also explored endogenous dafachronic acids (DAs), which are known to be critical in regulating larval developmental processes in C. elegans and other nematodes, by liquid chromatography-mass spectrometry (LC-MS). Results Orthologues of C. elegans dauer signalling genes were identified in T. canis (n = 55) and A. suum (n = 51), inferring the presence of a dauer-like signalling pathway in both species. Comparisons showed clear differences between C. elegans and these ascaridoids as well as between T. canis and A. suum, particularly in the transforming growth factor-β (TGF-β) and insulin-like signalling pathways. Specifically, in both A. suum and T. canis, there was a paucity of genes encoding SMAD transcription factor-related protein (daf-3, daf-5, daf-8 and daf-14) and insulin/insulin-like peptide (daf-28, ins-4, ins-6 and ins-7) homologues, suggesting an evolution and adaptation of the signalling pathway in these parasites. In T. canis, there were more orthologues coding for homologues of antagonist insulin-like peptides (Tc-ins-1 and Tc-ins-18), an insulin receptor substrate (Tc-ist-1) and a serine/threonine kinase (Tc-akt-1) than in A. suum, suggesting potentiated functional roles for these molecules in regulating larval diapause and reactivation. A relatively conserved machinery was proposed for DA synthesis in the two ascaridoids, and endogenous Δ4- and Δ7-DAs were detected in them by LC-MS analysis. Differential transcription analysis between T. canis and A. suum suggests that ins-17 and ins-18 homologues are specifically involved in regulating development and migration in T. canis larvae in host tissues. Conclusion The findings of this study provide a basis for functional explorations of insulin-like peptides, signalling hormones (i.e. DAs) and related nuclear receptors, proposed to link to development and/or parasite-host interactions in T. canis. Elucidating the functional roles of these molecules might contribute to the discovery of novel anthelmintic targets in ascaridoids. Electronic supplementary material The online version of this article (10.1186/s13071-018-3265-y) contains supplementary material, which is available to authorized users.

International audience; Cryptorchidism, a frequent genital malformation in male newborn, remains in most cases idiopathic. On the basis of experimental, epidemiological, and clinical data, it has been included in the testicular dysgenesis syndrome and believed to be influenced, together with genetic and anatomic factors, by maternal exposure to endocrine disrupting chemicals (EDCs). Here, we analyze how EDCs may interfere with the control of testicular descent, which is regulated by two Leydig cell hormones, testosterone, and insulin like peptide 3 (INSL3).