Development of a synthetic relaxin-3/INSL5 chimeric peptide ligand for NanoBiT complementation binding assays.

INSL5 and relaxin-3 are relaxin family peptides with important roles in gut and brain function, respectively. They mediate their actions through the class A GPCRs RXFP4 and RXFP3. RXFP4 has been proposed to be a therapeutic target for colon motility disorders whereas RXFP3 targeting could be effective for neurological conditions such as anxiety. Validation of these targets has been limited by the lack of specific ligands and the availability of robust ligand-binding assays for their development. In this study, we have utilized NanoBiT complementation to develop a SmBiT-conjugated tracer for use with LgBiT-fused RXFP3 and RXFP4. The low affinity between LgBiT:SmBiT should result in a low non-specific luminescence signal and enable the quantification of binding without the tedious separation of non-bound ligands. We used solid-phase peptide synthesis to produce a SmBiT-labelled RXFP3/4 agonist, R3/I5, where SmBiT was conjugated to the B-chain N-terminus via a PEG12 linker. Both SmBiT-R3/I5 and R3/I5 were synthesized and purified in high purity and yield. Stable HEK293T cell lines expressing LgBiT-RXFP3 and LgBiT-RXFP4 were produced and demonstrated normal signaling in response to the synthetic R3/I5 peptide. Binding was first characterized in whole-cell binding kinetic assays validating that the SmBiT-R3/I5 bound to both cell lines with nanomolar affinity with minimal non-specific binding without bound and free SmBiT-R3/I5 separation. We then optimized membrane binding assays, demonstrating easy and robust analysis of both saturation and competition binding from frozen membranes. These assays therefore provide an appropriate rigorous binding assay for the high-throughput analysis of RXFP3 and RXFP4 ligands.

Developing insulin-like peptide 5-based antagonists for the G protein-coupled receptor, RXFP4.

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.

Body Weight Reduction by Bariatric Surgery Reduces the Plasma Levels of the Novel Orexigenic Gut Hormone Insulin-like Peptide 5 in Patients with Severe Obesity.

Insulin-like factor 5 (INSL5), a novel hormone secreted by the enteroendocrine cells of the distal colon, has been implicated in appetite and body weight regulation in animals given its orexigenic properties. We investigated basal INSL5 plasma levels in a group of morbidly obese subjects before and after laparoscopic sleeve gastrectomy. Furthermore, we analyzed the expression of INSL5 in human adipose tissue. Before bariatric surgery, obese subjects showed basal INSL5 plasma levels that were positively correlated with BMI, fat mass, and leptin plasma levels. After weight loss by laparoscopic sleeve gastrectomy, INSL5 plasma levels in obese subjects were significantly lower than those observed before surgery. Finally, we did not detect any expression of the INSL5 gene in human adipose tissue, both at the mRNA and protein levels. The present data show that subjects with obesity have INSL5 plasma levels positively correlating with adiposity markers. After bariatric surgery, INSL5 plasma levels decreased significantly, and this decrease was not directly due to the loss of adipose tissue since this tissue does not express INSL5. Considering the orexigenic properties of INSL5, the reduction of its plasma levels after bariatric surgery in obese subjects could participate in the still unclear mechanisms leading to appetite reduction that characterize bariatric surgery procedures.

Evidence that RXFP4 is located in enterochromaffin cells and can regulate production and release of serotonin.

RXFP4 is a G protein-coupled receptor (GPCR) in the relaxin family. It has recently been recognised that this receptor and its cognate ligand INSL5 may have a role in the regulation of food intake, gut motility, and other functions relevant to metabolic health and disease. Recent data from reporter-mice showed co-location of Rxfp4 and serotonin (5-HT) in the lower gut. We used human single-cell RNA sequence data (scRNASeq) to show that RXFP4 is in a subset of gut enterochromaffin cells that produce 5-HT in humans. We also used RNAScope to show co-location of Rxfp4 mRNA and 5-HT in mouse colon, confirming prior findings. To understand whether RXFP4 might regulate serotonin production, we developed a cell model using Colo320, a human gut-derived immortalised cell line that produces and releases serotonin. Overexpression of RXFP4 in these cells resulted in a constitutive decrease in cAMP levels in both the basal state and in cells treated with forskolin. Treatment of cells with two RXFP4 agonists, INSL5 derived peptide INSL5-A13 and small molecule compound-4, further reduced cAMP levels. This was paralleled by a reduction in expression of mRNA for TPH1, the enzyme controlling the rate limiting step in the production of serotonin. Overexpression of RXFP4 also attenuated the cAMP-induced release of serotonin from Colo320 cells. Together this demonstrates that serotonin producing enterochromaffin cells are the major site of RXFP4 expression in the gut and that RXFP4 can have inhibitory functional impacts on cAMP production as well as TPH1 expression and serotonin release.

Relaxin/insulin-like family peptide receptor 4 (Rxfp4) expressing hypothalamic neurons modulate food intake and preference in mice.

OBJECTIVE: Insulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations. METHODS: We generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing. RESULTS: Rxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM. CONCLUSION: These findings identify RXFP4VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

Expression of the relaxin family peptide 4 receptor by enterochromaffin cells of the mouse large intestine.

The gastrointestinal hormone, insulin-like peptide 5 (INSL5), is found in large intestinal enteroendocrine cells (EEC). One of its functions is to stimulate nerve circuits that increase propulsive activity of the colon through its receptor, the relaxin family peptide 4 receptor (RXFP4). To investigate the mechanisms that link INSL5 to stimulation of propulsion, we have determined the localisation of cells expressing Rxfp4 in the mouse colon, using a reporter mouse to locate cells expressing the gene. The fluorescent signal indicating the location of Rxfp4 expression was in EEC, the greatest overlap of Rxfp4-dependent labelling being with cells containing 5-HT. In fact, > 90% of 5-HT cells were positive for Rxfp4 labelling. A small proportion of cells with Rxfp4-dependent labelling was 5-HT-negative, 11-15% in the distal colon and rectum, and 35% in the proximal colon. Of these, some were identified as L-cells by immunoreactivity for oxyntomodulin. Rxfp4-dependent fluorescence was also found in a sparse population of nerve endings, where it was colocalised with CGRP. We used the RXFP4 agonist, INSL5-A13, to activate the receptor and probe the role of the 5-HT cells in which it is expressed. INSL5-A13 administered by i.p. injection to conscious mice caused an increase in colorectal propulsion that was antagonised by the 5-HT3 receptor blocker, alosetron, also given i.p. We conclude that stimuli that excite INSL5-containing colonic L-cells release INSL5 that, through RXFP4, excites 5-HT release from neighbouring endocrine cells, which in turn acts on 5-HT3 receptors of enteric sensory neurons to elicit propulsive reflexes.

Reviewing the physiological roles of the novel hormone-receptor pair INSL5-RXFP4: a protective energy sensor?

There is no common consensus on the physiological role of insulin-like peptide 5 (INSL5) and its cognate receptor, relaxin family peptide receptor 4 (RXFP4). The experimental data for INSL5-RXFP4 expression and function point to a potential role of the peptide hormone and receptor pair in linking energy availability, homeostasis, and inflammation. In this review, we summarize studies on the INSL5-RXFP4 system and propose that the current findings from diverse experimental settings point broadly to a role as a protective energy sensor (PES). Specifically, we review the evidence that (1) INSL5-RXFP4 could regulate immune response by decreasing the production of proinflammatory cytokines and may be involved in the stress response via the HPA axis; (2) INSL5-RXFP4 may signal through sensory neurons on the vagus nerve, transmitting signals to the CNS; and (3) INSL5-RXFP4 could have local autocrine/paracrine roles within the intestinal tract and immune cells. Further investigation and clarification of these proposed roles of INSL5-RXFP4 may prove a greater physiological relevance for the pair and add to existing evidence of INSL5-RXFP4 role as a PES.

Gut peptide regulation of food intake - evidence for the modulation of hedonic feeding.

The number of people living with obesity has tripled worldwide since 1975 with serious implications for public health, as obesity is linked to a significantly higher chance of early death from associated comorbidities (metabolic syndrome, type 2 diabetes, cardiovascular disease and cancer). As obesity is a consequence of food intake exceeding the demands of energy expenditure, efforts are being made to better understand the homeostatic and hedonic mechanisms governing food intake. Gastrointestinal peptides are secreted from enteroendocrine cells in response to nutrient and energy intake, and modulate food intake either via afferent nerves, including the vagus nerve, or directly within the central nervous system, predominantly gaining access at circumventricular organs. Enteroendocrine hormones modulate homeostatic control centres at hypothalamic nuclei and the dorso-vagal complex. Additional roles of these peptides in modulating hedonic food intake and/or preference via the neural systems of reward are starting to be elucidated, with both peripheral and central peptide sources potentially contributing to central receptor activation. Pharmacological interventions and gastric bypass surgery for the treatment of type 2 diabetes and obesity elevate enteroendocrine hormone levels and also alter food preference. Hence, understanding of the hedonic mechanisms mediated by gut peptide action could advance development of potential therapeutic strategies for the treatment of obesity and its comorbidities.

Identification and verification of HCAR3 and INSL5 as new potential therapeutic targets of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers of the gastrointestinal tract and ranks third in cancer-related deaths worldwide. This study was conducted to identify novel biomarkers related to the pathogenesis of CRC based upon a bioinformatics analysis, and further verify the biomarkers in clinical tumor samples and CRC cell lines. METHODS: A series of bioinformatics analyses were performed using datasets from NCBI-GEO and constructed a protein-protein interaction (PPI) network. This analysis enabled the identification of Hub genes, for which the mRNA expression and overall survival of CRC patients data distribution was explored in The Cancer Genome Atlas (TCGA) colon cancer and rectal cancer (COADREAD) database. Furthermore, the differential expression of HCAR3 and INLS5 was validated in clinical tumor samples by Real-time quantitative PCR analysis, western blotting analysis, and immunohistochemistry analysis. Finally, CRC cells over-expressing INSL5 were constructed and used for CCK8, cell cycle, and cell apoptosis validation assays in vitro. RESULTS: A total of 286 differentially expressed genes (DEGs) were screened, including 64 genes with increased expression and 143 genes with decreased expression in 2 CRC database, from which 10 key genes were identified: CXCL1, HCAR3, CXCL6, CXCL8, CXCL2, CXCL5, PPY, SST, INSL5, and NPY1R. Among these genes, HCAR3 and INSL5 had not previously been explored and were further verified in vitro. CONCLUSIONS: HCAR3 expression was higher in CRC tissues and associated with better overall survival of CRC patients. INSL5 expression in normal tissue was higher than that in tumor tissue and its high expression was associated with a better prognosis for CRC. The overexpression of INSL5 significantly inhibited the proliferation and promoted the shearing of PARP of CRC cells. This integrated bioinformatics study presented 10 key hub genes associated with CRC. HCAR3 and INSL5 were expressed in tumor tissue and these were associated with poor survival and warrant further studies as potential therapeutic targets.

Autocrine INSL5 promotes tumor progression and glycolysis via activation of STAT5 signaling.

Metabolic reprogramming plays important roles in development and progression of nasopharyngeal carcinoma (NPC), but the underlying mechanism has not been completely defined. In this work, we found INSL5 was elevated in NPC tumor tissue and the plasma of NPC patients. Plasma INSL5 could serve as a novel diagnostic marker for NPC, especially for serum VCA-IgA-negative patients. Moreover, higher plasma INSL5 level was associated with poor disease outcome. Functionally, INSL5 overexpression increased, whereas knockdown of its receptor GPCR142 or inhibition of INSL5 reduced cell proliferation, colony formation, and cell invasion in vitro and tumorigenicity in vivo. Mechanistically, INSL5 enhanced phosphorylation and nuclear translocation of STAT5 and promoted glycolytic gene expression, leading to induced glycolysis in cancer cells. Pharmaceutical inhibition of glycolysis by 2-DG or blockade of INSL5 by a neutralizing antibody reversed INSL5-induced proliferation and invasion, indicating that INSL5 can be a potential therapeutic target in NPC. In conclusion, INSL5 enhances NPC progression by regulating cancer cell metabolic reprogramming and is a potential diagnostic and prognostic marker as well as a therapeutic target for NPC.

Selective stimulation of colonic L cells improves metabolic outcomes in mice.

AIMS/HYPOTHESIS: Insulin-like peptide-5 (INSL5) is found only in distal colonic L cells, which co-express glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). GLP-1 is a well-known insulin secretagogue, and GLP-1 and PYY are anorexigenic, whereas INSL5 is considered orexigenic. We aimed to clarify the metabolic impact of selective stimulation of distal colonic L cells in mice. METHODS: Insl5 promoter-driven expression of Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (DREADD) was employed to activate distal colonic L cells (LdistalDq). IPGTT and food intake were assessed with and without DREADD activation. RESULTS: LdistalDq cell stimulation with clozapine N-oxide (CNO; 0.3 mg/kg i.p.) increased plasma GLP-1 and PYY (2.67- and 3.31-fold, respectively); INSL5 was not measurable in plasma but was co-secreted with GLP-1 and PYY in vitro. IPGTT (2 g/kg body weight) revealed significantly improved glucose tolerance following CNO injection. CNO-treated mice also exhibited reduced food intake and body weight after 24 h, and increased defecation, the latter being sensitive to 5-hydroxytryptamine (5-HT) receptor 3 inhibition. Pre-treatment with a GLP1 receptor-blocking antibody neutralised the CNO-dependent improvement in glucose tolerance but did not affect the reduction in food intake, and an independent group of animals pair-fed to the CNO-treatment group demonstrated attenuated weight loss. Pre-treatment with JNJ-31020028, a neuropeptide Y receptor type 2 antagonist, abolished the CNO-dependent effect on food intake. Assessment of whole body physiology in metabolic cages revealed LdistalDq cell stimulation increased energy expenditure and increased activity. Acute CNO-induced food intake and glucose homeostasis outcomes were maintained after 2 weeks on a high-fat diet. CONCLUSIONS/INTERPRETATION: This proof-of-concept study demonstrates that selective distal colonic L cell stimulation has beneficial metabolic outcomes. Graphical abstract.

High-throughput screening campaign identifies a small molecule agonist of the relaxin family peptide receptor 4.

Relaxin/insulin-like family peptide receptor 4 (RXFP4) is a class A G protein-coupled receptor (GPCR), and insulin-like peptide 5 (INSL5) is its endogenous ligand. Although the precise physiological role of INSL5/RXFP4 remains elusive, a number of studies have suggested it to be a potential therapeutic target for obesity and other metabolic disorders. Since selective agonists of RXFP4 are scarcely available and peptidic analogs of INSL5 are hard to make, we conducted a high-throughput screening campaign against 52,000 synthetic and natural compounds targeting RXFP4. Of the 109 initial hits discovered, only 3 compounds were confirmed in secondary screening, with JK0621-D008 displaying the best agonism at human RXFP4. Its S-configuration stereoisomer (JK1) was subsequently isolated and validated by a series of bioassays, demonstrating a consistent agonistic effect in cells overexpressing RXFP4. This scaffold may provide a valuable tool to further explore the biological functions of RXFP4.

Colokinetic effect of an insulin-like peptide 5-related agonist of the RXFP4 receptor.

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.

Single cell transcriptomic profiling of large intestinal enteroendocrine cells in mice - Identification of selective stimuli for insulin-like peptide-5 and glucagon-like peptide-1 co-expressing cells.

OBJECTIVE: Enteroendocrine cells (EECs) of the large intestine, found scattered in the epithelial layer, are known to express different hormones, with at least partial co-expression of different hormones in the same cell. Here we aimed to categorize colonic EECs and to identify possible targets for selective recruitment of hormones. METHODS: Single cell RNA-sequencing of sorted enteroendocrine cells, using NeuroD1-Cre x Rosa26-EYFP mice, was used to cluster EECs from the colon and rectum according to their transcriptome. G-protein coupled receptors differentially expressed across clusters were identified, and, as a proof of principle, agonists of Agtr1a and Avpr1b were tested as candidate EEC secretagogues in vitro and in vivo. RESULTS: EECs from the large intestine separated into 7 clear clusters, 4 expressing higher levels of Tph1 (enzyme required for serotonin (5-HT) synthesis; enterochromaffin cells), 2 enriched for Gcg (encoding glucagon-like peptide-1, GLP-1, L-cells), and the 7th expressing somatostatin (D-cells). Restricted analysis of L-cells identified 4 L-cell sub-clusters, exhibiting differential expression of Gcg, Pyy (Peptide YY), Nts (neurotensin), Insl5 (insulin-like peptide 5), Cck (cholecystokinin), and Sct (secretin). Expression profiles of L- and enterochromaffin cells revealed the clustering to represent gradients along the crypt-surface (cell maturation) and proximal-distal gut axes. Distal colonic/rectal L-cells differentially expressed Agtr1a and the ligand angiotensin II was shown to selectively increase GLP-1 and PYY release in vitro and GLP-1 in vivo. CONCLUSION: EECs in the large intestine exhibit differential expression gradients along the crypt-surface and proximal-distal axes. Distal L-cells can be differentially stimulated by targeting receptors such as Agtr1a.

Insulin-like peptide 5 fails to improve metabolism or body weight in obese mice.

Insulin-like peptide 5 (INSL5) is a member of the insulin-like family of peptides. It has been reported to be orexigenic in rodent models of obesity with impaired glucose metabolism. We attempted to confirm this property as a first step in establishing the ability of INSL5 to successfully integrate with other agents more proven in their ability to reverse obesity and improve metabolism. INSL5 was chemically synthesized by two alternative methods to a native form and one that was site-specifically conjugated to a 20 KDa polyethylene glycol (PEG) polymer. The pharmacology of each peptide was assessed by high-dose chronic administration in normal and obese mice. INSL5 failed to produce pharmacologically relevant effects on food intake, body weight or glucose control indicative of a negligible role of the peptide in the control of feeding and glucose metabolism.

Co-storage and release of insulin-like peptide-5, glucagon-like peptide-1 and peptideYY from murine and human colonic enteroendocrine cells.

OBJECTIVE: Insulin-like peptide-5 (INSL5) is an orexigenic gut hormone found in a subset of colonic and rectal enteroendocrine L-cells together with the anorexigenic hormones glucagon-like peptide-1 (GLP-1) and peptideYY (PYY). Unlike GLP-1 and PYY, INSL5 levels are elevated by calorie restriction, raising questions about how these hormones respond to different stimuli when they arise from the same cell type. The aim of the current study was to identify whether and how INSL5, GLP-1 and PYY are co-secreted or differentially secreted from colonic L-cells. METHODS: An inducible reporter mouse (Insl5-rtTA) was created to enable selective characterisation of Insl5-expressing cells. Expression profiling and Ca2+-dynamics were assessed using TET-reporter mice. Secretion of INSL5, PYY, and GLP-1 from murine and human colonic crypt cultures was quantified by tandem mass spectrometry. Vesicular co-localisation of the three hormones was analysed in 3D-SIM images of immunofluorescently-labelled murine colonic primary cultures and tissue sections. RESULTS: INSL5-producing cells expressed a range of G-protein coupled receptors previously identified in GLP-1 expressing L-cells, including Ffar1, Gpbar1, and Agtr1a. Pharmacological or physiological agonists for these receptors triggered Ca2+ transients in INSL5-producing cells and stimulated INSL5 secretion. INSL5 secretory responses strongly correlated with those of PYY and GLP-1 across a range of stimuli. The majority (>80%) of secretory vesicles co-labelled for INSL5, PYY and GLP-1. CONCLUSIONS: INSL5 is largely co-stored with PYY and GLP-1 and all three hormones are co-secreted when INSL5-positive cells are stimulated. Opposing hormonal profiles observed in vivo likely reflect differential stimulation of L-cells in the proximal and distal gut.

INSL5 activates multiple signalling pathways and regulates GLP-1 secretion in NCI-H716 cells.

Insulin-like peptide 5 (INSL5) is a newly discovered gut hormone expressed in colonic enteroendocrine L-cells but little is known about its biological function. Here, we show using RT-qPCR and in situ hybridisation that Insl5 mRNA is highly expressed in the mouse colonic mucosa, colocalised with proglucagon immunoreactivity. In comparison, mRNA for RXFP4 (the cognate receptor for INSL5) is expressed in various mouse tissues, including the intestinal tract. We show that the human enteroendocrine L-cell model NCI-H716 cell line, and goblet-like colorectal cell lines SW1463 and LS513 endogenously express RXFP4. Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr202/Tyr204), AKT (Thr308 and Ser473) and S6RP (Ser235/236) and inhibited cAMP production but did not stimulate Ca2+ release. Acute INSL5 treatment had no effect on GLP-1 secretion mediated by carbachol or insulin, but modestly inhibited forskolin-stimulated GLP-1 secretion in NCI-H716 cells. However, chronic INSL5 pre-treatment (18 h) increased basal GLP-1 secretion and prevented the inhibitory effect of acute INSL5 administration. LS513 cells were found to be unresponsive to INSL5 despite expressing RXFP4 Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of Rxfp4 and were unresponsive to INSL5. This study provides novel insights into possible autocrine/paracrine roles of INSL5 in the intestinal tract.

The Regulation of Peripheral Metabolism by Gut-Derived Hormones.

Enteroendocrine cells lining the gut epithelium constitute the largest endocrine organ in the body and secrete over 20 different hormones in response to cues from ingested foods and changes in nutritional status. Not only do these hormones convey signals from the gut to the brain via the gut-brain axis, they also act directly on metabolically important peripheral targets in a highly concerted fashion to maintain energy balance and glucose homeostasis. Gut-derived hormones released during fasting tend to be orexigenic and have hyperglycaemic potential. Conversely, gut hormones secreted postprandially generally promote satiety and facilitate glucose clearance. Although some of the metabolic benefits conferred by bariatric surgeries have been ascribed to changes in the secretory profiles of various gut hormones, the therapeutic potential of the enteroendocrine system as a viable target against metabolic diseases remain largely underexploited, except for incretin-mimetics. This review provides a brief overview of the physiological importance and highlights the therapeutic potential of the following gut hormones: serotonin, glucose-dependent insulinotropic peptide, glucagon-like peptide 1, oxyntomodulin, peptide YY, insulin-like peptide 5, and ghrelin.

Challenges in the design of insulin and relaxin/insulin-like peptide mimetics.

Peptidomimetics are designed to overcome the poor pharmacokinetics and pharmacodynamics associated with the native peptide or protein on which they are based. The design of peptidomimetics starts from developing structure-activity relationships of the native ligand-target pair that identify the key residues that are responsible for the biological effect of the native peptide or protein. Then minimization of the structure and introduction of constraints are applied to create the core active site that can interact with the target with high affinity and selectivity. Developing peptidomimetics is not trivial and often challenging, particularly when peptides' interaction mechanism with their target is complex. This review will discuss the challenges of developing peptidomimetics of therapeutically important insulin superfamily peptides, particularly those which have two chains (A and B) and three disulfide bonds and whose receptors are known, namely insulin, H2 relaxin, H3 relaxin, INSL3 and INSL5.

Expression and localization of relaxin family peptide receptor 4 in human spermatozoa and impact of insulin-like peptide 5 on sperm functions.

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.