During the past two decades there have been rapid advances in our knowledge of the structure and function of the protein hormones in the brain and gastroenteropancreatic system (GEP). Many published articles have highlighted the superfamily of hormonal peptides, specifically, the mechanisms and control of peptide synthesis in neural and non-neural tissues, and gene structure. Here we present an analysis of the annual trends, between 1980 and 1997, of research emphasis on six protein/peptide hormones, as reflected by their individual frequency of publication per year. Although this symposium is focused on the GEP hormones, we provide herein a perspective on the level of research activity of the hormones insulin, glucagon, cholecystokinin, insulin-like growth factor-I and -II, neuropeptide Y and somatostatin in the brain/gut systems throughout the vertebrates and invertebrates. Many publications deal with the evolution of these peptides and their superfamilies, yet as noted in this review, there are relatively few references to these peptides in invertebrates and non-mammalian species. Typically in invertebrates, the number of citations is low and mostly focused on three phyla, the arthropods, mollusks and helminths. Generally, in the vertebrates the smallest number of citations is in the cyclostomes and elasmobranchs. Because most groups of invertebrates and vertebrates have received scant attention, phylogenetic comparisons are limited. Evolutionary information concerning important groups of animals, such as helminths, mollusks, protochordates and cyclostomes, is essential to establish the phylogenetic histories of the hormonal peptides. The challenge to comparative endocrinologists is to examine species in key evolutionary positions in order to gain an understanding of the diversity and function of the hormones and to determine the molecular features that form clues to their phyletic interrelationships and progression.

The extant jawless fishes (Agnatha) include the hagfishes and lampreys whose ancestry can be traced through a conserved evolution to the earliest of vertebrates. This review traces the study of the enteropancreatic (EP), endocrine cells and their products in hagfishes and lampreys over the past two centuries. Erika Plisetskaya is one of several prominent comparative endocrinologists who studied the development, distribution or function of the agnathan EP system. Her physiological studies in Russia laid the foundation for her subsequent isolation in North America of the first lamprey EP peptides (insulin and somatostatin) and providing the first homologous radioimmunoassay for agnathan (lamprey) insulin. This review also emphasizes the nature and the method of development of the agnathan endocrine pancreas (islet organ), for it reflects the earliest vertebrate endocrine pancreas originating from intestinal and/or bile-duct epithelia. The lamprey life cycle includes a protracted larval period and a metamorphosis when the adult EP system develops. Differences in morphogenesis during metamorphosis of southern- and northern-hemisphere lampreys dictate that a single cranial mass (islet organ) appear in the former and both a cranial and a caudal principal islet comprises most of the islet organ in holarctic species. There are differences in distribution of cell types and in the primary structure of the peptides in the definitive islet organ of hagfishes and lampreys. The primary structures of insulin, somatostatins, glucagons, glucagon-like peptide, and peptide tyrosine tyrosine are now available for three lamprey species representing three genera and two of the three families. Differences in structure of peptides within, and between, families is providing support for earlier views on the time of divergence of the families and the different genera. It is concluded that due to the ancient lineage and successful habitation of lampreys and hagfishes, and the importance of the EP system to their survival, that their EP systems should be a research focus well into the next century.

The traditional view, based primarily on X-ray crystallographic data, is that the amino acid residues at positions B12, B16, B23-B26, A1-A5, A19 and A21 in the insulin molecule comprise the receptor-binding domain. More recently, however, it has been proposed that the conformation adopted by insulin in the crystal structure is an inactive one. The results of alanine-scanning mutagenesis studies suggest that GlyB23, PheB24, IleA2, ValA3, and TyrA19 interact directly with the receptor with LeuB6, GlyB8, LeuB11, GluB13 and PheB25, although not part of the binding epitope, being important in maintaining the receptor-binding conformation. A comparison of the primary structures of insulins from a wide range of non-mammalian vertebrates, from hagfish to birds, provides support for this revised view by demonstrating that strong evolutionary pressure has acted to conserve those amino acids postulated to be important in the biologically active conformation. In addition to the cysteine residues, the amino acids at B6, B8, B11, B23, B24, A2, A3, and A19 are invariant in all species yet studied with only conservative substitutions (Glu → Asp) at B13 and (Phe → Tyr) at B25. In contrast, several insulins containing substitutions at positions B16, A5 and A21, sites of importance in maintaining the crystal structure conformation, have been identified. Although the amino acid sequences of insulin are not generally useful as molecular markers for inferring phylogenetic relationships between species, the presence of common structural features in insulins from closely related species may permit a valid inference. For example, the presence of an N-terminal pentapeptide extension to the B-chains of insulins isolated from both holarctic and southern hemisphere lampreys supports the monophyletic status of the Petromyzontiformes.

Insulin was discovered in 1922 as the causative factor for a human metabolic disorder (diabetes mellitus), but it was recognized early that the hormone had a broad phylogenetic distribution. By the mid 1970s, insulin had been isolated and sequenced from all classes of vertebrates, including Agnatha. Also it was discovered that the insulin gene family in vertebrates included two closely related hormones named insulin-like growth factor (IGF)-I and -II. More recently, the application of recombinant DNA techniques have identified insulin-like peptide genes in invertebrates, including insects, molluscs and nematode and these findings clearly establish that insulin is an evolutionarily ancient hormone which is present in all metazoa. Here we briefly review the structure and function of the insulin/IGF gene family in vertebrates and invertebrates. Although these studies are ongoing, it appears that in invertebrates the insulin-like peptides function predominately as mitogenic growth factors that act to promote tissue growth and development. However, in vertebrates the mitogenic growth function has been subsumed by IGF-I and -II while insulin has acquired the function of being primarily a metabolic regulatory hormone. The gene duplication and divergence events necessary for this development probably occurred early during vertebrate evolution in the transition from protochordates, represented by extant amphioxus, to primitive jawless vertebrates, represented by extant lamprey and hagfish.

In fish, the structural and functional characteristics of insulin and IGF-I receptors have been well studied. Current evidence indicates that all gnatostome animals, from fish to mammals, contain separate insulin and IGF-I molecules and specific receptors for insulin and IGF-I. However, qualitative differences in the functional aspects of insulin and IGF-I receptors among vertebrate species can account for variations in the biological activity of insulin and IGF-I. In this paper we will focus on the functional evolution of the insulin and IGF-I receptors in vertebrates and on the appearance of the unrelated IGF-II receptors.

Certain teleost fish have large anatomically discrete islet organs called Brockmann bodies (BBs). When transplanted into streptozotocin diabetic athymic nude mice, tilapia BBs provide long-term normoglycemia. This has afforded us the opportunity to examine tilapia islet in vivo function in a non-piscine environment and compare this with in vivo function in the donor species. As expected, fasting and non-fasting glycemic levels in long-term murine recipients of tilapia BBs were analogous to corresponding values in donor tilapia, but, surprisingly, tilapia BB grafts provided mammalian-like glucose tolerance profiles. Teleost fish, in general, are severely glucose intolerant. When glucose tolerance tests were performed in tilapia, the mean glucose disappearance rates were very low; however, diabetic nude mice bearing long-term tilapia BB grafts were extremely glucose responsive. This suggested a severe or absolute peripheral resistance to the glucostatic effects of insulin. Using Western blotting with polyclonal antibodies and then confirmed by Northern analysis, tilapia peripheral tissues appear to be devoid of GLUT-4, the insulin-sensitive glucose transporter responsible for the hypoglycemic effect of insulin in mammals, but not GLUT-1, the insulin independent glucose transporter. This may explain why tilapia, and possibly other teleost fish, are severely glucose intolerant after pharmacologic glucose-loading. Because tilapia do not tend to consume large quantities of glucose in the wild, it is not surprising that they have evolved without a mechanism to move glucose rapidly from the bloodstream into muscle and fat. Nevertheless, insulin still appears to play an important role in maintaining normoglycemia in tilapia; however, this is mostly likely a result of its effect on glucose uptake in the liver. We also present comparative data on tilapia beta cell function, quantification of islet cell numbers and types, islet products, insulin gene structure and expression, and beta cell sensitivity to the diabetogenic drug streptozotocin.

Glucose metabolism in mammalian species and teleost fish is controlled by different metabolic pathways. These include differences in the function of several major hormones, especially insulin and GLP-1. The major physiological role of GLP-1 in mammals is to connect the consumption of nutrients with glucose metabolism. The glucose lowering effects of GLP-1 in the postprandial state of mammals are regulated predominantly through metabolic pathways that integrate different physiological processes. These are: (i) stimulation of insulin release from the pancreatic β-cell during hyperglycemia and (ii) inhibition of nutrient absorption in the gastrointestinal tract. These effects are mediated by a same type of a highly selective GLP-1 receptor, often referred to as the “pancreatic GLP-1 receptor.” In teleost fish GLP-1 increases glucose levels through the activation of glycogenolysis and gluconeogenesis from liver. Functional characterization of the recombinant GLP-1 receptor from zebrafish, which is the first example of a recombinant fish GLP-1 receptor, demonstrated that zebrafish GLP-1 receptor has a binding specificity towards a wider range of GLP-1 structures than the mammalian GLP-1 receptor. This property of the zebrafish GLP-1 receptor, and most likely other fish GLP-1 receptors, sets apart the structure of the zebrafish GLP-1 receptor from the structures of mammalian GLP-1 receptors. These differences in the binding specificity between the zebrafish and mammalian GLP-1 receptors might reflect in part the differences in the mechanism by which GLP-1 regulates glucose metabolism in mammals and teleost fish.

The incretin hormone glucagon-like peptide-1 (GLP-1), coencoded and expressed in the proglucagon gene in intestine and endocrine pancreas of all vertebrates, is an important regulator of insulin secretion in the postprandial state of mammals. Additionally, the hormone acts in concert with insulin to remove glucose from the plasma. In mammalian B cells, lung, intestine and brain, GLP-1 receptors activate the adenylyl cyclase/cAMP system of message transduction, with ancillary involvement of calcium and inositoltrisphosphate. While the peptide is fairly conserved in vertebrates, the fishes show dramatic biochemical and physiological differences to the situation in mammals and an incretin function in fishes is questionable. Fish GLP-1 acts preferentially on the liver, and recently enterocytes and brain membranes have been shown to be potential targets. GLP-1 actions generally oppose those of insulin and supplant or supplement those of glucagon by activating glycogenolysis, gluconeogenesis and lipolysis in liver and by accelerating glucose transport and curtailing glucose oxidation in enterocytes. In brain and enterocytes, GLP-1 targets adenylyl cyclase, while in the liver adenylyl cyclase and cAMP play subordinate roles only. Although phospholipase C had been implicated in GLP-1 action, the prevalent route of message transduction in fish liver needs to be elucidated. The unique functional switch of GLP-1 from a hyperglycemic hormone in fish to a glucostatic incretin in mammals remains a matter of conjecture.

Somatostatins are a diverse family of peptide hormones that regulate a vast array of biological processes in vertebrates, including the modulation of growth, development, and metabolism. The multi-functional nature of the somatostatin family arises from the an elaborate, multi-faceted signaling system consisting of somatostatin signaling molecules, G-protein-coupled receptors, and cellular effector pathways. A striking aspect of this signaling system is the substantial diversity at every level. The signal molecules themselves display considerable structural heterogeneity. This molecular heterogeneity results from tissue-specific differential processing of a single large precursor protein (preprosomatostatin) as well as from the existence of multiple somatostatin genes, each giving rise to different precursors. In addition, numerous SS receptor subtypes have been characterized (five in mammals), some of which exhibit preferential binding to one ligand form over another. Propagation of the signal results from linkage of the receptors via numerous types of G-proteins to several different cellular effector pathways, including adenylyl cyclase, various protein kinases, numerous ion channels, and phospholipase C/inositol-3-phosphate. Ultimately, a particular response in a given target cell may be determined by structural interactions between and among the various elements of the signaling system.

The intestinal hormone, cholecystokinin (CCK), and the stomach hormone, gastrin, form a simple two member family of peptides with much to offer students of hormone and receptor evolution. They share a common carboxyl-terminal tetrapeptide sequence, which is the bioactive site of each peptide and is also antigenic, making heterologous biological and immunological assays feasible. Current evidence indicates that CCK evolved in chordate ancestors and that gastrin-like peptides that separately regulate stomach functions evolved from an ancestral CCK at the level of the divergence of tetrapods from fish. This tentative conclusion may require modification when the two separate CCK- and gastrin-like peptides recently identified in the dogfish shark are characterized further. The CCK-X receptor appears to be ancestral to the CCK-A and CCK-B receptors identified in amniotes. The evolution of gastrin and of CCK-A and -B receptors may have played roles in the evolution of the stomach and the evolution of endothermy in vertebrate phylogeny.

Recent abundant studies report that in rodents starvation induces increased neuropeptide Y (NPY) mRNA expression and peptide secretion in the hypothalamus which reduces autonomic nervous activity and promotes food intake, and intracerebroventricular (ICV) injection of NPY has potent orexigenic effects. Conversely, the effect of insulin in the central nervous system is to inhibit food intake and NPY biosynthesis and secretion. In mammals body fatness is regulated and insulin acts as one intake inhibitory signal related to fatness. In salmon (Oncorhynchus sp.) we have demonstrated a rise in NPY-like mRNA expression and a coincident decrease in plasma insulin levels during 2 to 3 weeks of starvation. Additionally, experimentally manipulating body fatness with high and low fat diets has demonstrated that body fatness affects food intake in teleost fishes, raising the possibility that NPY and insulin act to regulate their food intake. Therefore, we hypothesized that as in rodents, ICV treatment with NPY would stimulate food intake while ICV insulin would reduce food intake. Preliminary results suggest that ICV NPY administration does stimulate food intake in channel catfish (Ictalurus punctatus), but central injection of insulin has no effect. Results of treatments with the sulfated octapeptide of cholecystokinin and the recombinant fragment of rat leptin 22–56 are also discussed.