History of key regulatory peptide systems and perspectives for future research

Throughout the 20th Century, regulatory peptide discovery advanced from the identification of gut hormones to the extraction and characterization of hypothalamic hypophysiotropic factors, and to the isolation and cloning of multiple brain neuropeptides. These discoveries were followed by the discovery of G‐protein‐coupled and other membrane receptors for these peptides. Subsequently, the systems physiology associated with some of these multiple regulatory peptides and receptors has been comprehensively elucidated and has led to improved therapeutics and diagnostics and their approval by the US Food and Drug Administration. In light of this wealth of information and further potential, it is truly a time of renaissance for regulatory peptides. In this perspective, we review what we have learned from the pioneers in exemplified fields of gut peptides, such as cholecystokinin, enterochromaffin‐like‐cell peptides, and glucagon, from the trailblazing studies on the key stress hormone, corticotropin‐releasing factor, as well as from more recently characterized relaxin‐family peptides and receptors. The historical viewpoints are based on our understanding of these topics in light of the earliest phases of research and on subsequent studies and the evolution of knowledge, aiming to sharpen our vision of the current state‐of‐the‐art and those studies that should be prioritized in the future.


| THE EVOLUTION OF REGULATORY PEPTIDE DISCOVERY
The history of the biannual International Symposium on Regulatory Peptides (RegPep) reflects the evolution of regulatory peptide discovery ranging from gut hormones to hypothalamic hypophysiotropic factors, to brain neuropeptides, and, finally and more recently, to studies of the encompassing systems physiology of regulatory peptides (Figure 1) (Box 1).
Notably, the milestones of regulatory peptide system discovery also include the identification, cloning and characterization of regulatory peptide receptors; the elucidation of their physiological functions and mechanisms; their involvement in pathophysiology; and the development of regulatory peptide diagnostics and therapies.These themes have been covered throughout RegPep symposia during the past 50 years.
In light of the discovery of many G-protein-coupled receptor (GPCR) structures and recent advances in therapeutics and diagnostics, with several peptides approved by the US Food and Drug Administration for treating metabolic disorders, interest in regulatory peptides has seen a renaissance.The motivation of this article, inspired by the recent RegPeg24, is to further promote the IRPS mission to make a scientific and educational contribution for public benefit.In this article, we have exemplified some research in the gut (cholecystokinin [CCK], enterochromaffin-like [ECL]-cell peptides, and glucagon), and the brain, from anatomical descriptive studies to systematic approaches, and from the early discovered peptides to the newest relaxin-family peptide developments.It was our aim to showcase some of the challenges and excitement of the field to newcomers, based on our academic and personal experiences and thoughts, particularly regarding the reflections on Viktor Mutt, Wylie Vale, and Rolf Håkanson.
"Peptidergic regulation of glucagon secretion: implications for diabetes pathophysiology and therapy".

| WYLIE W. VALE JR AND THE DISCOVERY AND CHARACTERIZATION OF CORTICOTROPIN-RELEASING FACTOR
Corticotropin-releasing factor (CRF)/hormone (CRH) is a 41 amino acid peptide synthesized in a set of parvicellular neuroendocrine neurons located in the paraventricular hypothalamic nucleus, as well as many other locations in the brain and in some peripheral organs, including the gut. 34,35The most well-known function of CRFand from where it derives its nameis to stimulate adrenocorticotropic hormone (ACTH) secretion from corticotrophs that drives glucocorticoid secretion from the adrenal cortex.CRF thereby accounts for the glucocorticoid component of the stress response first described by Hans Selye. 36Given that glucocorticoid receptors are expressed by a multitude of cell types throughout the body, this elusive factor was also likely to play an important role in controlling energy metabolism, immune responses, and many other functions.
A series of important discoveries that began in the 1930s eventually culminated in the discovery and characterization of CRF in 1981 by Wylie Vale (1941-2012) (Figure 3) and his colleagues at the Salk Institute (La Jolla, CA, USA).This led to the identification of a family of four ligands, two receptors, and a binding protein.With their widespread expression in the brain and elsewhere, members of this peptide family are involved in a much wider range of functions than just ACTH secretion.These include brain functions related to autonomic and behavioural stress responses, anxiety, and energy metabolism, as well as actions in other organs including the gut and heart.
The following sections describe the historical context for Wylie Vale's discovery and characterization of the CRF family members in 1981 and the years that followed.
The search that eventually identified hypothalamic neuropeptides, including CRF, started with two key findings and two central hypotheses.The 1930s was a period of intense debate about the mechanisms responsible for controlling all parts of the pituitary gland.
At this time, there were many false starts, and many paths followed that led to dead ends. 38e two findings were the existence of a portal vasculature between the median eminence and the pituitary gland 39,40 and the presence of axons of the neurohypophysial tract in the pituitary stalk, which had been recognized since the days of Ram on y Cajal.
The first hypothesis was formulated by Hinsey and Markee. 41ey proposed that a humoral route was responsible for conveying hypothalamically processed information about ovulation to the pituitary gland, which then stimulated ovulation via hormone release.This hypothesis eventually proved to be correct and laid the foundations for what we now know as neuroendocrinology.The second hypothesis involved the neurocrine concept ('neurocrinie'), which was first advanced by Roussy, Mosinger, and Collin in France, but was eventually disproved. 38They proposed that chemical signals were synthesized in the pituitary gland and then moved back up the stalk to be released within the hypothalamus.Incidentally, this same group was also the first to coin the term 'neuroendocrinology' with the publication of their substantial 'Traite de Neuro-Endocrinologie' in 1946. 38e neurocrine mechanism proposed by Collin and others bore some resemblance to the original idea of neurosecretion developed in the 1930s by Ernst and Bertha Scharrer and others.They proposed that specialized hypothalamic neurosecretory neurons released factors directly into the hypothalamic neuropil, 42 but, similar to neurocrinie, neurosecretion in its original formulation also proved to be incorrect. 38til about 1950, combining mechanistic hypotheses with the emerging structural findings was impeded by the prevailing view that humoral and neural information flowed from the pituitary to the hypothalamus, and not in the opposite direction. 38 Photograph reproduced with permission. 374][45] It was during this same period that efforts to isolate and characterize hypothalamic neuropeptides began to accelerate.The first two of these peptides were vasopressin and oxytocin, which were characterized by Vincent du Vigneaud in the 1950s.For a number of years, vasopressin was considered a significant contributor to the control of ACTH secretion, and perhaps even the principal CRF. 46,47 the late 1960s and early 1970s, the groups of Andrew Schally (Veterans Administration Hospital, New Orleans) and Roger Guillemin (Baylor College, Houston, and the Salk Institute) were the first to characterize thyrotropin-releasing hormone (TRH), somatostatin, and luteinizing hormone releasing factor (LRF).Wylie Vale was an important contributor to Guillemin's group, particularly with regard to the bioassays that were critical for assessing the activity of purified compounds.46,48  By 1980, the most significant pituitary hormone releasing factor still to be characterized was the one that stimulates the release of ACTH from corticotrophs.Despite more than 25 years of effort by many different groups, including that of Roger Guillemin, 46 the exact nature of this factor was still unknown.Its great physiological relevance was obvious, given the many roles of glucocorticoids in energy metabolism and other bodily functions.However, there was also the potential for understanding the underlying causes of anxiety and stress-related disorders.
By the time Vale had moved with Guillemin to the Salk Institute in 1970, he had already played a key role in Guillemin's efforts to characterize TRH, somatostatin, and LRF, although Schally's group were the first to report the identity of this third peptide, more widely known today as gonadotropin-releasing hormone (GnRH).But once Vale established an independent lab at the Salk in 1978, he turned his attention and that of his group to isolating and characterizing CRF.It was an arduous task involving many daunting technical issues that had proved less problematic for peptides identified in the previous decade. 48ree years later, in 1981, Vale's group had solved the CRF puzzle. 49Impressively, not only did Vale's group identify the 41-amino acid peptide that was the primary peptide to stimulate ACTH secretion, but also, during the next 20 years, they went on to identify three additional CRF family members, their gene structures, two CRF receptors (including the receptor expressed by corticotrophs), and a CRF binding protein (Table 1).1][52][53][54][55][56] Collectively, this was a remarkable achievement that provided the foundation of a great many major discoveries. 57

| ROLF HÅKANSON AND THE DISCOVERY OF THE ECL CELL AND ITS RAMIFICATIONS
In 1952, V. Erspamer and B. Asero identified 5-hydroxytryptamine (5-HT) in enterochromaffin (EC) cells. 72[75] Professor Rolf Håkanson from Lund University was a pioneer in the field of regulatory peptides, such as gastrointestinal hormones, NPY, and VIP.He was the Editor of Regulatory Peptides from 1983 to 1998.
Peptic ulcer disease was once known to cause a great deal of pain and discomfort to a great number of patients.It was speculated quite early on that gastrin and histamine were somehow jointly responsible for stimulating acid secretion from the parietal cells, and gastric hyperacidity was thought to explain the symptoms.Bleeding from the stomach or the upper duodenum was known to cause discomfort and even death in a number of these patients.Indeed, ulcer bleeding was so common that a generation of surgeons spent their careers endeavouring to develop methods to alleviate pain and prevent haemorrhage, and ultimately to eliminate the cause of peptic ulcer.Diverse diet restrictions combined with refined gastric surgery and vagotomy were introduced and became very popularalthough not very effective.In late 1970, James Black (Nobel Laureate 1988) and coworkers described and introduced the first histamine H 2 -receptor blocker, which was effective in alleviating symptoms of the disease, suggesting and supporting the view that gastric histamine had a key role to play.
These agents were promptly introduced into the clinic, followed soon after (1989) by the more effective class of proton pump inhibitors (PPIs). 76The PPIs were uniquely effective in eliminating pain and discomfort in peptic ulcer patients.
In the ECL cells, histamine occurs in three distinct cellular compartments: releasable histamine (together with releasable pancreastatin) in granules/vesicles, free amine in the cytosol (in a pool not related to pancreastatin), and histamine associated with proteins within microvesicles.Histamine is processed differently from pancreastatin and can be mobilized differently from pancreastatin. 77,78e role of ECL-cell histamine in the granules/vesicles is well known Corticotropin-releasing factor Organization of ovine corticotrophin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study 58 Potter E et al.
Corticotropin-releasing factor binding protein Cloning and characterization of the cDNAs for human and rat corticotrophin releasing-binding proteins 59 Potter E et al.
Corticotropin-releasing factor binding protein The central distribution of a corticotropin-releasing factor (CRF)-binding protein predicts multiple sites and modes of interaction with CRF 60 Chen R et al.
Corticotropin-releasing factor receptor 1 Expression cloning of a human corticotrophin-releasing-factor receptor 61 Behan DP et al.
Corticotropin-releasing factor binding protein Cloning and structure of the human corticotrophin releasing factor-binding protein gene (CRHBP) 62 Potter E et al.
The ECL cells have been studied in rats with intact oxyntic mucosa, 97 by local microdialysis of the oxyntic mucosa 98 and in cell culture following their isolation from the stomach. 99From studies of isolated ECL cells, it is clear that they express gastrin receptors, receptors for the neuropeptides pituitary adenylate-cyclase-activating polypeptide (PACAP) and VIP, and β-adrenergic receptors, all of which stimulate histamine mobilization.In addition, isolated ECL cells have receptors for somatostatin, galanin, and misoprostol to inhibit gastrin-induced histamine mobilization. 93,99,100Studies of ECL cells subjected to local microdialysis reveal that endothelin (or ischemia) is capable of mobilizing extremely large amounts of histamine (no pancreastatin), which appears to derive from an unidentified source within the ECL cells. 101rgical removal of the stomach (i.e., gastrectomy [GX]), has long been linked to a greatly reduced trabecular bone volume and a reduced amount of mineralized bone. 102,103The size of the bone fluid compartment was reduced following GX and rats with GX responded with exaggerated hypercalcemia to an oral and intravenous calcium load. 104Fed sham-operated rats did not respond unfavourably to oral calcium, whereas GX rats tolerated excess calcium poorly and responded to calcium with a huge (potentially fatal) increase in blood calcium. 104Thus, the stomach is important for maintaining normal bone, and potential osteotropic hormones released from the ECL cells are considered to control calcium homeostasis by promoting the transfer of calcium from blood to bone. 105kanson and his colleagues demonstrated that gastrin released a blood calcium-lowering peptide from the acid-producing part of the rat stomach, presumably from ECL cells, and named it gastrocalcin. 106ter, they reported that extracts of ECL-cell granules/vesicles and of isolated ECL cells from rat oxyntic mucosa evoked a Ca 2+ -second messenger response in osteoblastic cells through a pertussis toxininsensitive mechanism leading to activation of phospholipase C (PLC) and production of inositol 1,4,5-trisphosphate (IP 3 ). 107,108Furthermore, global gene expression analysis of mouse stomach, particularly in connection with ECL-cell genes encoding histidine decarboxylase (HDC), CGA, vesicular monoamine transporter 2, synaptophysin, and CCK 2 receptor, revealed parathyroid hormone-like hormone (Pthlh) or parathyroid hormone-related protein (PTHrP) as a candidate ECL-cell peptide hormone. 109Gene expression and immunohistochemical analyses of stomachs from CCK 1 and/or CCK 2 receptor gene knockout (KO) mice revealed that Pthlh was expressed in the ECL cells and ECL-cell genes including Pthlh were downregulated in CCK 2 receptor KO and CCK 1+2 receptor KO mice. 110Pthlh modulates calcium homeostasis as a hormone via the PTH/PTHrP receptor and cellular growth or differentiation as a local (paracrine) factor. 111,112Recently, scRNA-seq and in situ RNA hybridization analyses revealed that ECL cells express luteinizing hormone (LH). 113 should also be noted that an impaired acidification in the stomach was claimed to have a negative effect on calcium homeostasis, leading to bone loss.This claim was based on the bone phenotype of CCK 2 receptor KO mice. 114,115However, this was at odds with the bone phenotype of HDC KO mice that displayed the same impaired acidification as CCK 2 receptor KO mice but had an increased bone formation and reduced bone resorption. 116 Oldberg, who identified the function and named the hormonal activity. 118The main function was originally considered to be stimulation of hepatic bile secretion and regulation of gallbladder contraction (hence the name).Subsequent physiological studies, however, added further gastrointestinal activities such as release of digestive enzymes from the pancreas; increase of intestinal motility and enzyme secretion; growth stimulation of the pancreas and intestinal mucosa; inhibition of gastric acid secretion and gastric emptying; and early satiety signalling to the brain via afferent vagal fibers.
A decisive milestone was reached in the late 1960s, when Mutt and Jorpes purified and identified the structure of CCK to be a carboxyamidated polypeptide of 33 amino acid residues, including a crucial O-sulfated tyrosyl residue. 16,119The structure analyses revealed that the C-terminal heptapeptide sequence is the receptorbound epitope of the peptide and that it is strongly homologous to the active site sequence in the gastrins. 120,121These pioneering studies provided a firm experimental base for future studies that have led to today's highly complex picture of the physiology and pathophysiology what is now sometimes referred to as the CCK messenger system. 122-124

| Extraintestinal expression
The biological spectrum of CCK expanded and changed entirely in the 1980s and 1990s.6][127] In quantitative terms, the mammalian expression of CCK peptides in neuronal tissue is higher and phylogenetically older than that of the endocrine CCK production in the intestine.Moreover, CCK is the most abundant and widespread peptide neurotransmitter system in the brain in comparison with other neuropeptides. 127,128dditionally, CCK is expressed in several other non-neuronal extraintestinal cells such as cardiac myocytes, 129 corticotropic and melanotropic cells in the pituitary, 130 thyroid C-cells, 131 and medullary adrenal cells, 132 as well as being occasionally expressed in pancreatic islet cells, 133 male germ cells, 134 and in several immune cells. 135e widespread cellular expression is characterized by differentiated post-translational processing of proCCK.Recognition of this cellspecific maturation is essential for understanding the physiological significance of the prohormone products.For example, the enteroendocrine I-cells secrete a mixture of long carboxyamidated CCK polypeptides (CCK-58, CCK-33, and CCK-22) and, to a lesser extent, the short CCK-8 and CCK-5. 136Moreover, of the intestinal CCK peptides, 20%-25% are not sulfated 137 and consequently not agonists for the CCK-A (or CCK 1 ) receptor, but only for the CCK-B (or CCK 2 ) receptor.
In humans and pigs, sulfated CCK-33 appears to be the most abundant form in intestinal tissue and in blood, and by far most of the CCK in circulation is derived from enteroendocrine cells. 138,139By contrast, the short sulfated CCK-8 (in particular) and non-sulfated CCK-5 constitute most of the CCK peptides released as transmitters from CCK neurons. 140,141The thyroid C-cells also synthetize mainly CCK-8, but only in unsulfated form. 131An entirely different processing pattern, however, is seen in cardiac myocytes.The myocytes contain only trace amounts of the known bioactive, carboxyamidated CCK peptides, whereas the post-translational main product is a long nonamidated N-terminally truncated and triple O-sulfated fragment 25-94 of pro-CCK. 1293][144] The CCK-A (or CCK 1 ) receptor (the alimentary receptor) is expressed mainly in the gastrointestinal tract, including the gallbladder, pancreas, and afferent vagal neurons, but only sporadically in the brain (area postrema, nucleus tractus solitarius, and the posterior hypothalamus).The CCK-A receptor binds the agonists with high affinity and specificity.It only binds O-sulfated and carboxyamidated CCK peptides.The CCK-B or CCK 2 receptor (the brain receptor) is expressed not only mainly in cerebral regions, but also on ECL-cells in the stomach, throughout the intestinal mucosa, 145 and in pancreatic islet cells. 146In comparison with the CCK-A receptor, its is rather promiscuous because it binds not only sulfated and unsulfated CCK peptides, but also all of the gastrin peptides.Notably, the CCK-B receptor was originally identified in the stomach as the gastrin receptor. 143

| Clinical significance
Exogenous CCK peptidesmainly as sulfated CCK-8 and the nonsulfated CCK-4have been used for decades for examination and occasionally for therapy of dysfunctions and disease in different organs; for example, for gallbladder emptying in biliary dyskinesia 147 ; for pancreatic enzyme secretion in exocrine pancreatic disease 148,149 ; 5][156][157][158][159][160][161][162] Generally, the tumor cell level and secretion have been too low to significantly increase the plasma concentrations of CCK.Accordingly, CCK-containing tumors have not provoked specific symptoms and measurements in plasma have been of little diagnostic use.There are, however, two exceptions.The first is Ewing sarcomas that express and release unprocessed proCCK in significant amounts. 159Hence, the plasma concentrations of proCCK are sufficiently high to monitor development and therapy of these sarcomas with simple blood tests. 159The other exception is the endocrine pancreatic CCKoma that releases bioactive CCK in amounts sufficient to cause a specific clinical syndrome. 156The symptoms are diarrhoea, weight loss, duodenal ulcers (as seen in the gastrinoma syndrome, but with normal concentrations of gastrin in plasma), and, more specifically, occurrence of gallstones. 156,1635][166][167] The plasma concentrations of bioactive CCK in normal lean and obese subjects are rather similar, both in the fasting state and after meals. 168A modest increase of CCK in plasma has been observed following 2 years of diet and exercise-induced weight loss. 169Gastric bypass surgery for obesity also results in increased postprandial CCK secretion. 170,171Along these lines, basal and mealstimulated CCK concentrations in plasma are decreased in morbidly obese women. 172| GLUCAGON AND THE CONTROL OF THE ENDOCRINE PANCREAS

| Discovery of glucagon
In 1921, Banting and Best (together with Macleod and Collip) discovered insulin and correctly concluded that the pancreatic islets (islets of Langerhans) are the endocrine part of the pancreas. 173Banting and Best observed that transient hyperglycemia preceded a drop in plasma glucose upon administration of pancreatic extracts.In 1923, Murlin proposed that this effect reflected a 'contaminant' with glucogenic capacity and named this substance 'glucagon' (glucose agonist). 174In 1948, it was demonstrated by de Duve and Sutherland that glucagon is secreted by the alpha-cells of the pancreatic islets. 175

| Pancreatic islets and glucagon
The pancreatic islets of Langerhans are the endocrine part of the pancreas and scattered in the exocrine parenchyma lie the small islets of endocrine cells.A human pancreas contains approximately one million islets, each consisting of (on average) 200 cells. 176Their total weight is about 1 g, approximately 1% of the pancreas.The pancreatic islets secrete two peptide hormones that play a central role in systemic glucose homeostasis: (1) insulin is secreted by the beta-cells (75% of the islet cells) and is the body's only hormone capable of lowering blood glucose and (2) glucagon is secreted by the alpha-cells (15%-20% of the islet cells) and is one of the body's principal blood glucose-increasing hormones. 177Whereas the role of insufficient insulin secretion in diabetes is widely recognized, the significance of dysregulated glucagon secretion is less well-known.However, diabetes is now increasingly regarded as a bihormonal disorder where the metabolic impact of the lack of insulin is compounded by oversecretion of glucagon. 178

| Regulation of glucagon secretion
Both alpha-and beta-cells are typical endocrine cells, 178 and their hormones are stored in secretory granules pending release into the blood stream.The islets are extensively vascularized and the endocrine cells are in contact with a least one blood vessel. 179There are approximately 10,000 secretory granules per cell, but only a few hundred of these are in direct contact (docked) with the plasma membrane. 180wever, the release rate is very low (typically 0.1% per h). 181Thus, the docked pool of granules is sufficient for a day's requirement.
Similar to the beta-cells, glucagon-secreting alpha-cells are electrically excitable.They express many of the ion channels found in neurons, including voltage-gated, tetrodotoxin (TTX)-sensitive Na +channels (Nav1.3) 182and multiple types of voltage-gated, Ca 2+ -channel.The Ca 2+ -channel subtype that is linked to glucagon secretion at low glucose is of the P/Q subtype (Cav2.1),which is sensitive to the toxin omega-agatoxin. 183These Ca 2+ -channels are tightly linked to the release competent secretory granules and the delay between opening of the channel and exocytosis is only approximately 10 ms.
Depolarization-evoked exocytosis is resistant to intracellular Ca 2+chelators such as ethylene glycol tetraacetic acid (EGTA).These characteristics suggest an arrangement similar to that found in the active zones of nerve terminals.
Glucagon secretion is high at low glucose and most of the alphacells (unlike the beta-cells) are electrically active under these conditions.This electrical activity consists of overshooting (i.e., they go beyond zero mV) action potentials that start from a membrane potential of -55 to -50 mV.Interestingly, the alpha-cells are equipped with ATP-regulated K + -channels of exactly the same type as those found in beta-cells.In beta-cells, these channels are highly active at low glucose, which accounts for the negative membrane potential and low rate of insulin secretion under these conditions.High glucose, via its metabolism, elevates the intracellular ATP/ADP ratio and this leads to K ATP channel closure, membrane depolarization, initiation of beta-cell electrical activity, and insulin secretion.Exactly how high glucose inhibits glucagon secretion in alpha-cells is still debated, but it has been proposed that it involves closure of K ATP channels leading to stronger membrane depolarization and voltage-dependent inactivation of the Na + -channels involved in action potential firing.This culminates in lowered action potential amplitude, reduced Ca 2+ entry, and suppression of glucagon secretion. 183 important question that remains unresolved is how K ATP channel activity is sufficiently reduced at low glucose (or even in the complete absence of glucose) to allow action potential firing to proceed.It is not because KATP channel density in alpha-cells is lower; indeed, experiments involving the washout of intracellular ATP to maximally activate the KATP channels suggest that the channel density, if anything, is higher in alpha-cells than in beta-cells.There is some evidence suggesting that alpha-cells maintain high intracellular ATP levels in the absence of glucose by beta-oxidation of fatty acids. 184In addition to glucose and fatty acids, glucagon secretion is also stimulated by amino acids and it has been proposed that their effect on glucagon secretion might be more important than that of glucose. 185wever, it is notable that whereas a fall in glucose leads to a sustained stimulation of glucagon secretion, the stimulatory effect of amino acids tends to be transient. 186

| Neuronal and hormonal regulation of glucagon secretion
Alpha-cells and glucagon secretion are also under extensive control by circulating hormones, locally released peptides and circulating neurotransmitters. 187For example, the gut peptides, GIP and glucagon-like peptide (GLP)-1, stimulate and inhibit glucagon secretion, 188 respectively.Somatostatin released by the delta-cells in the pancreatic islets inhibits glucagon secretion by a paracrine effect.There is some evidence that the well-known glucagonostatic effect of insulin is mediated by insulin-induced stimulation of somatostatin secretion. 189radrenaline, released by nerve endings within the islet, stimulates glucagon secretion by activation of β-adrenergic receptors on the alpha-cells. 190Finally, vasopressin is released by the posterior pituitary in response to a fall in plasma glucose and is a potent stimulus of glucagon secretion. 191Its release is impaired in insulin-dependent type-1 diabetes, which might explain why glucagon secretion in response to a fall in plasma glucose becomes impaired in patients with diabetes, an effect that might increase the risk of (fatal) hypoglycemia. 192

| History of ligand and receptor discovery
Relaxin was first characterized by Frederick Hisaw in 1926, who observed that injection of serum from pregnant guinea pigs caused the pubic symphysis of virgin guinea pigs to relax. 193Almost 60 years later, two human genes encoding complex relaxin peptides (RLN1 and RLN2) were among the first genes to be cloned. 194Subsequently, two relaxin genes were also identified in higher primates, 195 and a single gene was identified in other mammals (Rln1), including rodents and pigs, 196,197 whereas, notably, the relaxin gene is deleted or truncated in cattle, sheep and related species. 198,1995][206] Ironically, although the human, mouse and rat RLN3 genes were the last to be identified using homology searching of the Celera human genome database by teams at the Florey Institute in Australia and elsewhere, [204][205][206] all of the insulin-like/relaxin-family members were subsequently identified as having originated from an RLN3-like ancestor via different rounds of whole-genome and genespecific duplications during vertebrate evolution. 207,208 pite the earlier development of cloning techniques and homology screening, it was not until the early 2000s that various socalled "orphan" receptors were finally identified as receptors for the relaxin-family ligands by Hse and colleagues 209 and scientists at Johnson & Johnson. 210,211A significant advance was the recognition that the receptors for relaxin and its family analogues were GPCRs, rather than tyrosine kinases that signal for the structurally similar insulin peptides. 209Initially the complex leucine-rich-repeat containing GPCRs, LGR7 and LGR8, were identified as receptors for relaxin and INSL3, 209,212 followed by the identification of GPCR135 and GPCR142 as receptors for relaxin-3 and INSL5. 210,211Following a standardization of nomenclature for these relaxin-family peptide (RXFP) receptors by an IUPHAR committee, the cognate receptors for RLN, INSL3, RLN3, and INSL5 are known as RXFP1, RXFP2, RXFP3, and RXFP4, respectively. 213multaneously with the identification of these ligand-receptor pairings and thereafter, research began to characterize their biology, including studies of their chemistry, cellular anatomy, biochemistry, and physiology.[235][236][237][238]

| Recent advances and current anatomical and functional knowledge
Over the last 20 years, since the early landmark discoveries were made and the initial related research occurred, a small number of research teams have devoted their efforts to obtaining a better understanding of the different ligand-receptor pairings in the context of health and disease.0][241][242][243] Thus, it is well established that relaxin is a pleiotropic hormone with paracrine, autocrine, and endocrine roles that differ between species, but its common mechanistic actions include tissue (extracellular matrix) remodelling, as well as cardiovascular vasodilation effects and anti-fibrotic, organ protection actions. 213,244ese numerous effects of relaxin are consistent with the expression pattern of its cognate receptor, RXFP1. 239,245,246 N[251][252] The INSL3/RXFP2 system was initially identified as having a clear role in male reproduction, specifically in the development of the gubernaculum and testicular descent, because INSL3 is highly expressed in testicular Leydig cells in animals and human 253 and both 9][260] In addition, INSL3 has been implicated in regulation of the skeletomuscular system, including bone metabolism in males, where low levels are associated with an increased risk of osteopenia and osteoporosis, 261 and RXFP2 is implicated in kidney function in rats, 216 as well as in horn and antler development in different species. 262,263laxin-3 is considered a neuropeptide in mammals because relaxin-3 mRNA/peptide is highly expressed in GABAergic neurons in the midline pontine tegmental region (nucleus incertus) and in some adjacent areas, including the pontine raphe nucleus, the ventral, and lateral periaqueductal grey, as well as the deep mesencephalic area dorsal to the substantia nigra, in non-human primate, rat, and mouse brain. 205,206,210,217,220,264RXFP3 is widely expressed in brain areas including the cerebral cortex, hippocampus, septum, thalamus, hypothalamus, and brainstem, and the pattern of expression largely overlaps the efferent projection zones of the nucleus incertus in rat, mouse, and non-human primate. 214,217,220,265[268][269][270][271][272][273][274][275][276][277] In earlier studies, and more recently, comprehensive investigations have characterized receptors for various transmitters/peptides that are present on relaxin-3 neurons within the nucleus incertus [278][279][280][281] and further details of the anatomical networks utilizing relaxin-3/RXFP3 signaling in rat and mouse, 282 including those in the medial septum, 283 hippocampus, and piriform cortex. 284,285further aspect of relaxin-3/RXFP3 signaling recently reported was an association with the signaling of cellular aging.Using in vivo co-regulation analyses Maudsley and colleagues 286,287 observed that It should be noted that the structural similarities of relaxin-3 and relaxin, allow relaxin-3 to be tested pharmacologically as a likely RXFP1 agonist in various models, 288 and several reports describe its actions under these conditions. 289,290e expression of Insl5 and Rxfp4 mRNA has been detected in the pancreas, thymus and in the gastrointestinal tract, 202,291 particularly in the L-cells of distal gut [292][293][294] ; INSL5 has been shown to function as an orexigenic hormone, 291 and to influence colonic propulsion, 232,295 immune cell function, 296 and even human sperm motility. 297Finally, a recent study identified a potential role for INSL5 (or relaxin-3) signaling via ventral hypothalamic RXFP4 in the control of high-fat and high-protein diet consumption in mice. 298Therefore, the INSL5/RXFP4 system is being proposed to act as a protective energy sensor, linking energy availability, homeostasis, and inflammation, via actions to decrease proinflammatory cytokines, signal through sensory neurons and the vagus nerve to the central nervous system, as well as via local autocrine/paracrine effects within the intestinal tract and immune cells. 299

| Current neurochemical-anatomicalfunctional knowledge gaps and priority research questions
By contrast to the vast knowledge of the relaxin/RXFP1 system in a range of peripheral systems, and despite some early anatomical and functional studies in rats, mice and humans, 215,219,248,249 there is still very little known about the role of the relaxin/RXFP1 system in the mammalian brain, where the receptor is highly-enriched in sensory and limbic regions, as well as hypothalamic circuits. 215Notably, recent surveys of the transcriptome of mouse and human brain has identified putative relaxin/RXFP1 circuits within cerebral cortex that warrant further investigation. 300,301Indeed, in a recent study, central administration of relaxin and a RXFP1-selective peptide, analogue, relaxin B7-33, produced an acute analgesic action in a mouse model of inflammatory pain, 251 and further studies should reveal the regional sites and mechanisms of action.Furthermore, in light of these findings, the effect of relaxin/RXFP1 signaling on other central modalities should be explored.
Similarly, although there is abundant expression of RXFP2 mRNA and [ 125 I]-INSL3 binding sites in motor, limbic, and hypothalamic circuits of rat brain, 218 there is still very little known about the role of the INSL3/RXFP2 signaling system in brain, 302 and further pharmacological, neurophysiological, and functional studies of this system are warranted, particularly of its role in the medial habenulointerpeduncular nucleus pathway, which is strongly associated with control of reward and aversive behavior. 303,304ere is little data available in the current literature regarding altered levels of relaxin-3 expression and function in disease models, and some of the reports that do exist 305,306 have failed to validate the assay used to detect the putative native relaxin-3 peptide, by using validated synthetic peptide and tissues from relaxin-3 knockout mice, as well as antiserum specificity tests.In addition, there are known cardiac and immune cell (and potentially unidentified peripheral) sources of relaxin-3, and these need to be better characterized before serum assays of the peptide can be correctly interpreted as resulting from central changes in relaxin-3 production and utilization.Furthermore, once suitable experimental assays are developed, the dynamics of RXFP3 should also be examined in pre-clinical disease models and clinical disorders, such as metabolic disease and psychiatric illness, aiming to better understand the potential role of relaxin-3/RXFP3 signaling in human health and disease.
Finally, with regard to the INSL5/RXFP4 signaling system, the Insl5 gene (and INSL5 peptide) is not expressed in the rat, which has meant that this popular preclinical model is not appropriate for further studies.Accordingly, although significant progress has been made to understand the role of INSL5/RXFP4 signaling in the mouse colon, 295,299 Insl5/INSL5 expression has not been reported in mouse brain and RXFP4 is largely absence from mouse brain.However, a recent report of RXFP4 expression in the ventromedial hypothalamus and a role in feeding and food preference 298 suggests that further studies of this system are warranted, and the newly generated RXFP4-Cre mouse line should be useful in such investigations.

| CONCLUDING REMARKS AND FUTURE PERSPECTIVES
The history of the biannual International Symposium on Regulatory Peptides reflects the evolution of regulatory peptide discovery from gut hormones to hypothalamic hypophysiotropic factors, to brain neuropeptides and their receptors, and, more recently, to studies of the encompassing systems physiology of regulatory peptides.
Although "regulatory peptides" have "come of age", their complexity and diversity are enormous, and, despite the advances made during the last 50 years, the trajectory of discovery (Figure 1) shows no sign of reaching a plateau.Regulatory peptides remain a productive and important area of preclinical and clinical research, and it is hoped that this articcle will help encourage a younger generation of investigators to take up the challenge of further exploring their biology in the future.
8.1 | How to address the current major research questions?
Novel findings regarding the actions of regulatory peptides and their receptors can still be obtained using techniques such as exogenous peptide application and assessment of neurophysiological and physiological and behavioral responses in wild-type or transgenic animals, particularly if the peptides or their analogues provide more specific receptor activation than the endogenous ligand and improved pharmacodynamics.
8][309][310] However, in line with advances in other areas of regulatory peptide biology, an ability to address critical research questions is strongly linked to the development and wide availability of additional experimental tools and methods. 311 studies of the central nervous system, this has most recently been associated with important breakthroughs, including the generation of novel transgenic mouse and rat strains used in combination with commercially available viral/genetic methods that have allowed researchers to map, control, and monitor the activity of specific neuron populations, using the now familiar methods of optogenetics, chemogenetics, and two-photon calcium imaging, as well as multielectrode array and patch-clamp recordings.However, in regard to the specific issue of regulatory peptide function, as often highlighted, there is still a need to study the peptides themselves and their receptors, rather than just manipulate populations of peptide-containing or receptor-expressing neurons and alter their release of classical amino acid and monoamine transmitters, without specifically monitoring their peptide release using appropriate traditional or newer innovative methods. 311,312Such methods include the use of peptide-receptor reporter cells and the broader development of GPCR reporter constructs that can be injected into specific target areas to monitor endogenous or exogenous peptide release and actions using twophoton immunofluorescence detection in awake animals.Methods that can accurately localize GPCR proteins rather than using proxy markers (fluorescent marker proteins, mRNAs) will be an important step towards a deeper understanding of peptide function.This heralds a fruitful area of future research for the field, once a range of validated/optimized, selective, and sensitive peptide receptor reporters are widely available.

1
Discovery of key regulatory peptides since 1902 and RepPep symposia during the "era of gut hormones"

BOX 1
International Regulatory Peptide Society and International Symposium on Regulatory PeptidesThe International Regulatory Peptide Society (IRPS) is a non-profit membership organization, and the only international society focused on regulatory peptides in systems biology, with goals that include enhancing diversity and opportunities for under-represented groups, promoting best practice in biomedical translational sciences, and providing networking and mentoring opportunities.The precedent society of the IRPS was a French National Association: Societe Internationale des Peptides Regulateurs, created in Toulouse, in August 2007 (Waldec/RNA: ID W313007062, www.journal-officiel.gouv.fr).During the General Assembly of the IRPS on September 25, 2018, it was resolved to constitute the IRPS as an international not-for-profit, rule governed membership society.The Statutes declared in 2007 in France and in 2019 in Mexico City served as the basis for the current Statutes, modified and approved by the General Assembly of the IRPS during the RegPep23 meeting.IRPS is an affiliate member society of the International Neuroendocrine Federation (INF), International Brain Research Organization (IBRO), and the International Union of Basic and Clinical Pharmacology (IUPHAR).The IRPS adopted the Journal of Neuroendocrinology (JNE) as its official journal in March 2020.F I G U R E 2 Viktor Mutt standing next to one of his huge Sephadex-columns used in the purification process of peptide hormones from gut extracts.It is still worth remembering how 10 mg pure secretin (necessary for sequence analysis in the 1960's) required extraction and purification from 20 km porcine jejunum.Photograph source unknown; probably from Karolinska Campus Photo Center. 6developed by Mutt and Tatemoto for identification of new carboxyamidated regulatory peptides in tissue extracts. 33Carboxyamidation is a general characteristic of approximately 50% of the bioactive, regulatory peptides.Not only did Viktor Mutt's research put gut endocrinology and neuropeptide neurobiology on a firm scientific footing, but also his generous supply of bioactive peptides to laboratories all over the world was a decisive prerequisite for a multitude of studies in gastroenterology, endocrinology, and neurobiology, as well as for several diagnostic tests of pancreatic and gastrointestinal functions.In later years, Viktor received several major European awards, and, in the USA, he received the William Beaumont and Morton Grossman lecture awards.Viktor Mutt was also nominated several times for the Nobel Prize.In view of Viktor Mutt's unique contributions to science, the steering committee of the International Symposia on Regulatory Peptides established a biannual lectureship in 1992 to commemorate Viktor Mutt's name in the future research of regulatory peptides.It is important, however, to also commemorate Viktor Mutt's unique personality.In spite of the success and significance of his scientific achievements, Viktor remained throughout his life a shy, humble, and modest person who avoided the limelight.
in connection with the neuroendocrine mechanism of gastric acid secretion (see separate article by Chen D et al. in this SI), whereas the role of histamine in the cytosol and microvesicles remains an enigma (R. Håkanson, personal communication).Gastrin and histamine interact to cause hyperacidity.The ECL cells are centrally placed, with hypergastrinemia leading to ECL cell hyperactivity.Evidence in favour of the existence of a gastrin-ECL cell-parietal cell axis has been accumulated over the years.[79][80][81]Logically, it follows that antacid treatment requires the use of proton pump inhibitors, or agents exerting histamine H 2 receptor blockade or gastrin antagonists.PPI treatment leads to anacidity and long-term hypertrophy/ hyperplasia of the oxyntic mucosa including the ECL cells (because of the ensuing hypergastrinemia).[82][83][84][85][86]H 2 receptor blockade induces similar effects (but to a lesser extent), whereas gastrin receptor blockade effectively induces low acid secretion and hypotrophy/hypoplasia of the oxyntic mucosa and ECL cells.87All three avenues may be useful in the treatment of problems related to acid hypersecretion.The most promising line of investigation is that using gastrin antagonists because they remain little studied and because they can be expected to cause the entire oxyntic mucosa to become dormant and long-term inactivation of the ECL cells can lead to unexpected consequences (R. Håkanson, personal communication).Endocrine/paracrine cells in the acid-producing area of the stomach represent an organ similar in size to the endocrine pancreas.The ECL cells make up the larger part (60%-70%) of the endocrine cells of the stomach.They are rich in pancreastatin/chromogranin (CGA)related peptides which shared with most other peptide hormoneproducing cells.78,88,89Surgical removal of the acid-producing part of the stomach eliminates most pancreastatin (70%-80%) from the blood stream, and the synthesis and release of pancreastatin from the ECL T A B L E 1 Milestones in corticotrophin-releasing factor and urocortin biology.Date Authors Molecule TitleVale Lab Spiess J et al.Corticotropin-releasing factor Primary structure of corticotrophin-releasing factor from ovine hypothalamus49 Swanson LW et al.

71 F
Other labs Furutani Y et al.Corticotropin-releasing factor Cloning and sequence analysis of cDNA for ovine corticotropin-releasing factor 70 Lovenberg TW et al.Corticotropin-releasing factor receptor 2 Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain I G U R E 4 Rolf Håkanson visited Norwegian University of Technology and Science in 2004.
RXFP3 has a close co-expression-based relationship with the ADPribosylation factor (ARF) GTPase activating protein, GIT2, which acts as a keystone protein in the aging process, and can control both DNA repair and glucose metabolism.In their studies, cellular RXFP3 expression was directly affected by DNA damage and oxidative stress and overexpression or stimulation of RXFP3 by relaxin-3 regulated the DNA damage response and repair processes.
Wylie Vale at the Salk Institute in the late 1980s.
110s, it is unlikely that a lack of gastric acid production per se causes bone loss.It was noted that neither osteopenia, nor osteoporosis was present in CCK 1 receptor KO, CCK 2 receptor KO and CCK 1+2 receptor KO mice at 3-4 months of age.110Indeed,CCK 2 receptor KO mice had impaired hista- Cholecystokinin (CCK) constitutestogether with secretin and gastrinthe classical gut hormone troika.CCK was assumed to originate from the upper intestinal mucosa as suggested in 1928 byIvy and