The pleiotropic role of human secretin (hSCT) validates its potential use as a therapeutic agent. Nevertheless, the structure of secretin in complex with its receptor is necessary to develop a suitable therapeutic agent. Therefore, in an effort to design a three-dimensional virtual homology model and identify a peptide agonist and/or antagonist for the human secretin receptor (hSR), the significance of the primary sequence of secretin peptides in allosteric binding and activation was elucidated using virtual docking, FRET competitive binding and assessment of the cAMP response. Secretin analogs containing various N- or C-terminal modifications were prepared based on previous findings of the role of these domains in receptor binding and activation. These analogs exhibited very low or no binding affinity in a virtual model, and were found to neither exhibit in vitro binding nor agonistic or antagonistic properties. A parallel analysis of the analogs in the virtual model and in vitro studies revealed instability of these peptide analogs to bind and activate the receptor.

Small and large intrahepatic bile ducts consist of small and large cholangiocytes, respectively, and these cholangiocytes have different morphology and functions. The gastrointestinal peptide hormone, secretin (SCT) that binds to secretin receptor (SR), is a key mediator in cholangiocyte pathophysiology. Extracellular vesicles (EVs) are membrane-bound vesicles and cell-cell EV communication is recognized as an important factor in liver pathology, although EV communication between cholangiocytes is not identified to date. Cholangiocytes secrete proinflammatory cytokines during bacterial infection leading to biliary inflammation and hyperplasia. We demonstrate that cholangiocytes stimulated with lipopolysaccharide (LPS), which is a membrane component of gram-negative bacteria, secrete more EVs than cholangiocytes incubated with vehicle. These LPS-derived EVs induce inflammatory responses in other cholangiocytes including elevated cytokine production and cell proliferation. Large but not small cholangiocytes show inflammatory responses against large but not small cholangiocyte-derived EVs. Large cholangiocytes with knocked down either SCT or SR by short hairpin RNAs show reduced EV secretion during LPS stimulation, and EVs isolated from SCT or SR knocked down cholangiocytes fail to induce inflammatory reactions in control large cholangiocytes. This study identifies cholangiocyte EV communication during LPS stimulation, and demonstrates that the SCT/SR axis may be important for this event.

At present, secretin and its receptor have only been identified in mammals, and the origin of this ligand-receptor pair in early vertebrates is unclear. In addition, the elusive similarities of secretin and orexin in terms of both structures and functions suggest a common ancestral origin early in the vertebrate lineage. In this article, with the cloning and functional characterization of secretin receptors from lungfish and X. laevis as well as frog (X. laevis and Rana rugulosa) secretins, we provide evidence that the secretin ligand-receptor pair has already diverged and become highly specific by the emergence of tetrapods. The secretin receptor-like sequence cloned from lungfish indicates that the secretin receptor was descended from a VPAC-like receptor prior the advent of sarcopterygians. To clarify the controversial relationship of secretin and orexin, orexin type-2 receptor was cloned from X. laevis. We demonstrated that, in frog, secretin and orexin could activate their mutual receptors, indicating their coordinated complementary role in mediating physiological processes in non-mammalian vertebrates. However, among the peptides in the secretin/glucagon superfamily, secretin was found to be the only peptide that could activate the orexin receptor. We therefore hypothesize that secretin and orexin are of different ancestral origins early in the vertebrate lineage.

We have previously shown that intracerebellar infusion of the neuropeptide secretin enhances the acquisition phase of eyeblink conditioning (EBC). Here, we sought to test whether endogenous secretin also regulates EBC and to test whether the effect of exogenous and endogenous secretin is specific to acquisition. In Experiment 1, rats received intracerebellar infusions of the secretin receptor antagonist 5-27 secretin or vehicle into the lobulus simplex of cerebellar cortex immediately prior to sessions 1-3 of acquisition. Antagonist-infused rats showed a reduction in the percentage of eyeblink CRs compared with vehicle-infused rats. In Experiment 2, rats received intracerebellar infusions of secretin or vehicle immediately prior to sessions 1-2 of extinction. Secretin did not significantly affect extinction performance. In Experiment 3, rats received intracerebellar infusions of 5-27 secretin or vehicle immediately prior to sessions 1-2 of extinction. The secretin antagonist did not significantly affect extinction performance. Together, our current and previous results indicate that both exogenous and endogenous cerebellar secretin modulate acquisition, but not extinction, of EBC. We have previously shown that (1) secretin reduces surface expression of the voltage-gated potassium channel α-subunit Kv1.2 in cerebellar cortex and (2) intracerebellar infusions of a Kv1.2 blocker enhance EBC acquisition, much like secretin. Kv1.2 is almost exclusively expressed in cerebellar cortex at basket cell-Purkinje cell pinceaus and Purkinje cell dendrites; we propose that EBC-induced secretin release from PCs modulates EBC acquisition by reducing surface expression of Kv1.2 at one or both of these sites.

Secretin (Sct), a classical gut hormone, is now known to play pleiotropic functions in the body including osmoregulation, digestion, and feeding control. As Sct has long been implicated to regulate metabolism, in this report, we have investigated a potential lipolytic action of Sct. In our preliminary studies, both Sct levels in circulation and Sct receptor (SctR) transcripts in adipose tissue were upregulated during fasting, suggesting a potential physiological relevance of Sct in regulating lipolysis. Using SctR knockout and Sct knockout mice as controls, we show that Sct is able to stimulate lipolysis in vitro in isolated adipocytes dose- and time-dependently, as well as acute lipolysis in vivo. H-89, a protein kinase A (PKA) inhibitor, was found to attenuate lipolytic effects of 1 μM Sct in vitro, while a significant increase in PKA activity upon Sct injection was observed in the adipose tissue in vivo. Sct was also found to stimulate phosphorylation at 660(ser) of hormone sensitive lipase (HSL) and to bring about the translocation of HSL from cytosol to the lipid droplet. In summary, our data demonstrate for the first time the in vivo and in vitro lipolytic effects of Sct, and that this function is mediated by PKA and HSL.

Activation of the secretin (Sct)/secretin receptor (SR) axis stimulates ductular reaction and liver fibrosis, which are hallmarks of cholangiopathies. Our aim was to define the role of Sct-regulated cellular senescence, and we demonstrated that both ductular reaction and liver fibrosis are significantly reduced in Sct-/-, SR-/-, and Sct-/-/SR-/- bile duct ligated (BDL) mice compared with BDL wild-type mice. The reduction in hepatic fibrosis in Sct-/-, SR-/-, and Sct-/-/SR-/- BDL mice was accompanied by reduced transforming growth factor-β1 levels in serum and cholangiocyte supernatant, as well as decreased expression of markers of cellular senescence in cholangiocytes in contrast to enhanced cellular senescence in hepatic stellate cells compared with BDL wild-type mice. Secretin directly stimulated the senescence of cholangiocytes and regulated, by a paracrine mechanism, the senescence of hepatic stellate cells and liver fibrosis via modulation of transforming growth factor-β1 biliary secretion. Targeting senescent cholangiocytes may represent a novel therapeutic approach for ameliorating hepatic fibrosis during cholestatic liver injury.

The secretin family is a pleotropic group of brain-gut peptides with affinity for class 2 G-protein coupled receptors (secretin family GPCRs) proposed to have emerged early in the metazoan radiation via gene or genome duplications. In human, 10 members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. Secretin-like family GPCR homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic.

1. This study aims (1) to determine whether secretin is synthesized centrally, specifically by the HPA axis and (2) to discuss, on the basis of the findings in this and previous studies, secretin's possible neuroregulatory role in autism. 2. An immunocytochemical technique with single-cell resolution was performed in 12 age/weight-matched male rats pretreated with stereotaxic microinjection of colchicine (0.6 microg/kg) or vehicle into the lateral ventricle. Following 2-day survival, rats were anesthetized and perfused for immunocytochemistry. Brain segments were blocked and alternate frozen 30-microm sections incubated in rabbit antibodies against secretin, vasoactive intestinal peptide, glucagon, or pituitary-adenylate-cyclase-activating peptide. Adjacent sections were processed for Nissl stain. Preadsorption studies were performed with members of the secretin peptide family to demonstrate primary antibody specificity. 3. Specificity of secretin immunoreactivity (ir) was verified by clear-cut preadsorption control data and relatively high concentrations and distinct topographic localization of secretin ir to paraventricular/supraoptic and intercalated hypothalamic nuclei. Secretin levels were upregulated by colchicine, an exemplar of homeostatic stressors, as compared with low constitutive expression in untreated rats. 4. This study provides the first direct immunocytochemical demonstration of secretinergic immunoreactivity in the forebrain and offers evidence that the hypothalamus, like the gut, is capable of synthesizing secretin. Secretin's dual expression by gut and brain secretin cells, as well as its overlapping central distribution with other stress-adaptation neurohormones, especially oxytocin, indicates that it is stress-sensitive. A neuroregulatory relationship between the peripheral and central stress response systems is suggested, as is a dual role for secretin in conditioning both of those stress-adaptation systems. Colchicine-induced upregulation of secretin indicates that secretin may be synthesized on demand in response to stress, a possible mechanism of action that may underlie secretin's role in autism.

Secretin activates brown adipose tissue (BAT) and induces satiation in both mice and humans. However, the exact brain mechanism of this satiety inducing, secretin-mediated gut-BAT-brain axis is largely unknown.

Receptor activity-modifying proteins (RAMPs) have been shown to modulate the functions of several G protein-coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

To elucidate and categorize the murine placental hormones expressed across gestation, including the expression of hormones with previously undescribed roles.

Comparative approaches using protostome and deuterostome data have greatly contributed to understanding gene function and organismal complexity. The family 2 G-protein coupled receptors (GPCRs) are one of the largest and best studied hormone and neuropeptide receptor families. They are suggested to have arisen from a single ancestral gene via duplication events. Despite the recent identification of receptor members in protostome and early deuterostome genomes, relatively little is known about their function or origin during metazoan divergence. In this study a comprehensive description of family 2 GPCR evolution is given based on in silico and expression analyses of the invertebrate receptor genes.

In mammals, the suprachiasmatic nucleus (SCN) is a principal circadian pacemaker that optimizes the timing of behavioral rhythms and physiological events. Normally, circadian behavioral rhythms are entrained by the environmental light-dark (LD) cycle via the SCN. However, daily rhythms of other synchronizing signals, such as food availability, also emerge. When food availability is restricted to a single recurring daytime meal in nocturnal rodents, they exhibit increased activity during the hours immediately preceding feeding time; this is called food anticipatory activity (FAA). Many reports suggest that FAA is mediated by the food-entrainable oscillator (FEO) with circadian properties, but not the SCN. However, the neural locus and timekeeping mechanisms of the FEO, including its relationship with gastrointestinal hormone signaling, remain unclear. Herein, to examine whether secretin receptor signaling is necessary for the FEO, the effect of daily food restriction was studied in secretin receptor-deficient (Sctr-/-) mice. Adult wild-type (WT) and Sctr-/- mice were housed in separate cages containing a running wheel, with ad libitum food access and in a LD cycle (12 hours:12 hours) for at least 2 weeks. After acclimation to the condition, food access times were gradually restricted and 4-hour restricted feeding lasted over 10 days. Subsequently, mice had ad libitum food access for 2 days and then fasted for 2 days. Thereafter, robust FAAs were observed in both WT and Sctr-/- mice during restricted feeding and subsequent fasting. These results indicate that secretin receptor signaling is not essential for the timekeeping mechanism of FEO.

Previous studies demonstrated that secretin could modulate synaptic transmission in the rat cerebellum. In the present report, we provide evidence for the endogenous release of secretin in the cerebellum and further characterize the actions of secretin in this brain area. First, to show that secretin is released endogenously, blocks of freshly dissected cerebella were challenged with a high concentration of KCl. Incubation with KCl almost doubled the rate of secretin release. This KCl-induced release was sensitive to tetrodotoxin and cadmium suggesting the involvement of voltage-gated sodium and calcium channels. The use of specific channel blockers further revealed that L-type and P/Q-type calcium channels underlie both basal and KCl-evoked secretin release. In support of this, depolarization of Purkinje neurons in the presence of NMDA, group II mGluR and cannabinoid CB1 receptor blockers resulted in increased inhibitory postsynaptic current frequency. Second, we found that the previously reported facilitatory action of secretin on GABAergic inputs to Purkinje neurons is partly dependent on the release of endogenous glutamate. In the presence of CNQX, an AMPA/kainate receptor antagonist, the facilitatory effect of secretin on GABA release was significantly reduced. In support of this idea, application of AMPA, but not kainate receptor agonist, facilitated GABA release from inhibitory terminals, an action that was sensitive to AMPA receptor antagonists. These data indicate that a direct and an indirect pathway mediate the action of secretin in the basket cell-Purkinje neuron synapse. The results provide further and more solid evidence for the role of secretin as a neuropeptide in the mammalian CNS.

The molecular mediator and functional significance of meal-associated brown fat (BAT) thermogenesis remains elusive. Here, we identified the gut hormone secretin as a non-sympathetic BAT activator mediating prandial thermogenesis, which consequentially induces satiation, thereby establishing a gut-secretin-BAT-brain axis in mammals with a physiological role of prandial thermogenesis in the control of satiation. Mechanistically, meal-associated rise in circulating secretin activates BAT thermogenesis by stimulating lipolysis upon binding to secretin receptors in brown adipocytes, which is sensed in the brain and promotes satiation. Chronic infusion of a modified human secretin transiently elevates energy expenditure in diet-induced obese mice. Clinical trials with human subjects showed that thermogenesis after a single-meal ingestion correlated with postprandial secretin levels and that secretin infusions increased glucose uptake in BAT. Collectively, our findings highlight the largely unappreciated function of BAT in the control of satiation and qualify BAT as an even more attractive target for treating obesity.

In mammals, the timing of behavior and physiological activity is controlled by the suprachiasmatic nucleus (SCN) in the hypothalamus. Incidentally, secretin is a peptide hormone that promotes digestive activities and regulates water reabsorption. In recent studies, exogenous administration of secretin has been reported to induce secretion of oxytocin in the supraoptic nucleus of the hypothalamus and modulate social behavior. These results indicate that secretin is involved in the neural network that controls social behavior and plays important roles in the central nervous system. In the present study, we investigated the effects of secretin on circadian rhythms, by assessing circadian rhythms during wheel-running behavior in secretin receptor-deficient (Sctr-/-) mice. Male adult wild-type (WT) and Sctr-/- mice were housed in separate cages containing a wheel. Every minute of the wheel-running activity was monitored during the normal light-dark (LD) cycle (12:12 h) and in constant darkness (DD). Significant differences were observed in the free-running period between the WT and Sctr-/- mice. However, no significant differences were observed in the daily wheel-running revolutions between WT and Sctr-/- mice, in the LD and DD conditions. Moreover, the ratio of the daily activity phase to the rest phase (α/ρ) was significantly smaller in Sctr-/- than that in WT mice in the DD condition. Secretin receptors were expressed in the SCN cells. These findings suggest that secretin receptors are involved in the central circadian clock in the SCN and the circadian system in general.

The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and mechanism of the TadSS is mostly unknown. This includes characterisation of the outer membrane secretin through which the pilus is channelled and recruitment of its pilotin. Here we investigate RcpA and TadD lipoprotein from Pseudomonas aeruginosa. Light microscopy reveals RcpA colocalising with TadD in P. aeruginosa and when heterologously expressed in Escherichia coli. We use cryogenic electron microscopy to determine how RcpA and TadD assemble a secretin channel with C13 and C14 symmetries. Despite low sequence homology, we show that TadD shares a similar fold to the type 4 pilus system pilotin PilF. We establish that the C-terminal four residues of RcpA bind TadD - an interaction essential for secretin formation. The binding mechanism between RcpA and TadD appears distinct from known secretin-pilotin pairings in other secretion systems.

Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.

Social recognition underlies social behavior in animals, and patients with psychiatric disorders associated with social deficits show abnormalities in social recognition. Oxytocin is implicated in social behavior and has received attention as an effective treatment for sociobehavioral deficits. Secretin receptor-deficient mice show deficits in social behavior. The relationship between oxytocin and secretin concerning social behavior remains to be determined.

The gastrointestinal functions of the 27-amino acid secretin peptide have been well established. In previous prenatal studies, secretin expression in the rat duodenum was reported after day 17 of gestation while its expression in other organs and its functions in the developing embryos are still unknown. By in situ hybridization and immunohistochemical staining, secretin transcripts and peptides were found to be widely expressed in mouse embryos. Consistent with the idea that secretin is a brain-gut peptide, its expressions are present in several developing brain regions such as cephalic mesenchyme, cerebellar primordium and choroid plexus as well as the epithelial villi lining and inner circular muscle of the developing intestine. Other than these organs, secretin was also detected in the developing heart including the ventricular epicardium and myocardium and certain structures of the developing kidney like ureteric bud, collecting duct and glomerulus. These observations strongly suggest for a functional role of secretin during mouse embryonic development.