© 2000 by The Society for Integrative and Comparative Biology
The Agnathan Enteropancreatic Endocrine System: Phylogenetic and Ontogenetic Histories, Structure, and Function1
1 Department of Zoology and Division of Life Sciences, University of Toronto at Scarborough, Scarborough, Ontario M1C 1A4 Canada
 |
SYNOPSIS |
|---|
TOP
 SYNOPSIS INTRODUCTIONHAGFISHLAMPREYCONCLUSIONReferences |
|---|
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.
|
INTRODUCTION |
|---|
|
TOP
SYNOPSISINTRODUCTIONHAGFISHLAMPREYCONCLUSIONReferences |
|---|
Hagfishes and lampreys are jawless fishes with direct ancestory to a once fluorishing group of Agnatha in the Palaeozoic. Among these agnathans were the ostracoderms which fossil records suggest were among the first vertebrates. In the past, the living agnathans were referred to as cyclostomes (round mouths) and they were grouped within the vertebrate Class, the Cyclostomata, and within distinct subclasses (or orders) as Petromyzontids (the lampreys) or Myxinids (the hagfishes). More recent classification of fishes (Nelson, 1994
) has the lampreys and hagfishes within the vertebrate superclass Agnatha and as distinct classes, Cephalaspidomorphi and Myxini, respectively. Petromyzontiformes and Myxiniformes are the distinct orders. The taxanomic relationship of hagfishes and lampreys has always been controversial, for they show many structural, functional, and behavioural differences (Hardisty, 1979, 1982). Recently, lampreys were considered to be more similar to gnathostome fishes than to hagfishes (Forey and Janvier, 1994). In fact, Janvier (1986) has always questioned whether hagfishes should be included among the Vertebrata because they lack segmented vertebral elements.
Fossil evidence of early agnathans suggest that hagfishes may date back to the Cambrian period (Forey and Janvier, 1994) while lampreys originated from a naked anaspid line about 350 million years ago. Thus the ancestors to the two extant agnathan lines diverged early in craniate evolution.
The life histories of the two living agnathans are distinctly different. Hagfishes live their entire life in a marine environment, produce small batches of large yolky eggs and, following hatching, exhibit direct development to sexual maturity (Gorbman, 1997). Lamprey reproduction in freshwater yields many small eggs and, following hatching, there is a larval period of growth and a metamorphosis (indirect development) to a juvenile (Youson, 1988). Juveniles of nonparasitic species immediately commence sexual maturation in freshwater whereas those of parasitic species become predatory and feed on the blood, body fluids, and flesh of host teleosts. In some parasitic species juveniles are capable of marine osmoregulation and they migrate to the open sea for feeding. It is assumed that other species are restricted to feeding within their natal stream because of an inability to osmoregulate in hyperosmotic environments (Hardisty et al., 1989). Genetic, morphometric and meristic analyses have grouped similar parasitic and nonparasitic lamprey species as paired or satellite species with the implication that they arose from a common ancestor, which was likely parasitic. This view also implies that the nonparasitic adult life history type is more recent.
Due to their ancient history, lampreys and hagfishes have been the subject of much anatomical and physiological investigation. In many cases, the objective has been to find clues which might provide a bridge between the earliest and more modern forms of vertebrates or between vertebrates and other members of Phylum Chordata, i.e., the protochordates. Furthermore, all systems of lampreys and hagfishes have been directly compared to provide some answer to questions of their relationship to one another and to their environmental history (Hardisty, 1979, 1982; Hardisty et al., 1989). Among these comparisons are reports of the endocrine cells of the alimentary canal and the pancreas. Collectively these cells are part of a system termed in many other vertebrates, the gastroenteropancreatic (GEP) system. Included in the GEP are enteroendocrine cells of the intestine and endocrine cells of both the pancreas and stomach. Since neither the hagfish nor the lamprey has a stomach (Hardisty, 1979), the enteropancreatic (EP) system seems more appropriate for agnathans.
Hagfishes and lampreys are also distinct among other vertebrates in having their exocrine and endocrine equivalents of the vertebrate pancreas in isolation (Barrington, 1972; Youson, 1981). The exocrine elements are present within the intestinal or diverticular epithelia while the equivalent of the endocrine pancreas is an aggregate of submucosal islets, the islet organ. The presence of a compacted aggregate of zymogen cells in the diverticulum, a so-called protopancreas, in one species of lampreys is often used as evidence that a concentrated mass of pancreatic exocrine cells evolved more than once in vertebrate evolution (Epple and Brinn, 1987).
The hormones elaborated by the EP system of lampreys and hagfishes have, among other functions, an important role in intermediary metabolism. This latter fact was recognized by some of the early pioneers of comparative vertebrate endocrinology and we owe them much for their effort and their inspiration. Among these are Barrington, Epple, Falkmer, Hardisty and Plisetskaya. Falkmer and Plisetskaya have included some discussion of the hagfish and lamprey EP system in their reviews (Plisetskaya, 1990; Falkmer, 1995; Plisetskaya and Mommsen, 1996). A section on the distribution and the structure of agnathan EP can be found in the most recent review of the GEP systems of fishes (Youson and Al-Mahrouki, 1999) This essay will review some of the early work on the ontogenetic and phylogenetic development of the agnathan EP system and the function of the hormones elaborated by this system. Descriptions will be confined to hormones which are shared in common by the islet organ and the endocrine cells of the gut, namely, peptides of the insulin, glucagon, pancreatic polypeptide, and somatostatin families. Ultimately, the description will lead to the present state of affairs in these systems in lampreys and hagfishes, which were last specifically reviewed in 1990 (Van Noorden, 1990; Youson and Cheung, 1990), and to the directions of future research.
|
HAGFISH |
|---|
|
TOP
SYNOPSISINTRODUCTIONHAGFISHLAMPREYCONCLUSIONReferences |
|---|
Distribution and structure of the EP
Barrington (1945)
was intrigued by the report by Maas (1896) of a "pancreas-like organ" in the Myxiniformes with hollow lobules of cells closely associated with the bile duct. Subsequently, Barrington (1945) described in the Atlantic hagfish, Myxine glutinosa, unusual collections of cords of cells in a white, ovoid swelling on the distal end of the bile duct, near where it enters the intestine. He emphasized the cord-like arrangement, the extensive connective tissue beween cords, the paucity of blood vessels, the lumina in the cords, and the origin of cords from the bile duct. Schirner (1963) provided another detailed description of the glands and stimulated twelve years of light and electron microscopic description of B cells arranged as follicles within M. glutinosa (Falkmer and Winbladh, 1964; Thomas and Östberg, 1972; Boquist and Östberg, 1975; Winbladh and Horstedt, 1975). However, Winbladh (1976) reported that islet tissue follicles are sparse or absent in two other hagfishes, Epatretus burgeri and Eptatretus stouti. A common feature of the islet tissue of the hagfishes was the absence of any A cells, that is, an equivalent to the glucagon-producing cells of most gnathostomes. This conclusion, which was initially based on morphology, was confirmed by immunohistochemistry using antisera against mammalian insulin which immunostained approximately 99% of the cells (Östberg et al., 1975; Falkmer, 1995). Cells which did not react with the insulin antibody and had different secretory granules than those in B cells (Thomas et al., 1973; Boquist and Östberg, 1975) were later identified as D cells due to their immunostaining with anti-somatostatin serum (Van Noorden et al., 1977). It was also noteworthy that cells of the bile duct also immunostain with anti-insulin and anti-somatostatin (Östberg et al., 1975, 1976b; Van Noorden et al., 1977), for the islets originate as cell clusters from this duct (Östberg et al., 1976a). B and D cells of both the islet tissue and the bile ducts possess prominent crystalline inclusions within rough endoplasmic reticulum (Boquist and Östberg, 1975). Crystalline arrangement in these inclusions differs in the two cell types (Raska et al., 1982). The intestinal epithelium contains somatostatin cells and cells which immunoreact with antisera against pancreatic polypeptide and glucagon but not anti-insulin. Immunoreactivity to antisera against insulin-like growth factor 1 (IGF-1) is ubiquitous in the islet tissue and is found in cells throughout the length of the intestinal mucosa (Reinecke et al., 1991). Since no receptors for IGF-1 appear in the intestinal tract, the action of this hormone is not likely to be autocrine or paracrine but more systemic (Drakenberg et al., 1993).
Sture Falkmer (Falkmer and Patent, 1972; Falkmer et al., 1973; Falkmer, 1985, 1995) has long emphasized that the hagfish islet tissue represents the most phylogenetically original islet organ. That is, an islet organ with an almost total population of B (insulin-containing) cells; most of the other cell types (A, D and F cells) that are found in higher vertebrate islet tissue still reside in the hagfish intestinal mucosa. Furthermore, the islet tissue buds from the bile duct epithelium. It is presumed that Falkmer's statement on originality of the hagfish islet tissue as a two-hormone gland (Falkmer, 1995) is meant to be compared with definitive (adult) islet organs in vertebrates, for it will be seen below that the endocrine pancreas of larval lamprey possesses an even simpler design. Insulin-like (Thorndyke et al., 1989) and IGF-1 (Reinecke et al., 1991) immunoreactivity has been detected in the brain of hagfish, in addition to its presence in the EP system. Falkmer (1995) interprets this distribution as an example of a primitive organism that first demonstrates the complete stepwise sequence of a neuroendocrine system from neuronal cells to disseminated mucosal endocrine cells to an isolated islet tissue. The most primitive condition among chordates is seen in the protochordates where there is no islet tissue but neuroendocrine elements in both the central nervous system and in the mucosal epithelium of the alimentary canal (Reinecke, 1981; Conlon et al., 1988b; Reinecke et al., 1993). It has been suggested that the protochordate, Branchiostoma lanceolatum (Amphioxus) and the hagfish may share a common feature of possessing an ancestral insulin/IGF-1 molecule (Reinecke et al., 1991, 1993). A hybrid insulin/IGF cDNA has been isolated from B. lanceolatum (Chan et al., 1990).
Bioactivity of EP peptides
Hardisty (1979) and Plisetskaya (1985) provided comprehensive reviews of early investigations of insulin and carbohydrate metabolism in hagfish. Hagfishes share with lampreys the feature of slow reactions to both insulin injections and glucose loading (Falkmer and Matty, 1966). However, there appears to be marked interspecific variation among hagfishes in the response to insulin; E. stouti requires a smaller dosage and the hypoglycemic response is more prolonged than in M. glutinosa (Matty and Falkmer, 1965; Inui and Gorbman, 1977). Insulin also seems to be important in protein synthesis in E. stouti (Inui and Gorbman, 1978).
Structure of EP peptides
Hagfish insulin was the first EP peptide to be isolated from an agnathan (Peterson et al., 1974; Cutfield et al., 1979). Although the hagfish insulin molecule contains the putative receptor binding region noted in porcine insulin, amino acid substitutions in part of the B chain may explain a reduced biological potency relative to the mammalian molecule (Cutfield et al., 1979). Coincidentally, hagfish and lamprey somatostatins were described in the same year by independent groups. Conlon et al. (1988a) isolated somatostatins with 34 and 14 amino acids from the islet tissue of M. glutinosa. These same two somatostatins are also present in the intestine (Conlon and Falkmer, 1989). The smaller somatostatin was the invariant form present in all other vertebrate islet tissue but lampreys. The biological activity of hagfish somatostatins has not been examined but, since there are relatively few cells, there may be a paracrine role for this hormone in islet and intestinal tissues. The primary structures of members of the glucagon and the pancreatic polypeptide families have not been provided.
|
LAMPREY |
|---|
|
TOP
SYNOPSISINTRODUCTIONHAGFISHLAMPREYCONCLUSIONReferences |
|---|
Distribution and structure of the EP
Unlike the hagfish where knowledge of embryogenesis (Gorbman, 1997
) and subsequent growth is scanty, the life cycle of lampreys from the zygote to time of death after spawning has been well documented. During many of the intervening life cycle periods organogenesis has been examined (Youson, 1985). These studies have included descriptions of the EP system from embryogenesis to the upstream migration period (Youson and Elliott, 1989; Youson and Cheung, 1990). During this time the system undergoes significant changes which are important to permit differences in behaviour between individuals of the life cycle periods. The following is a summary of the EP system in embryos, larvae, metamorphosing individuals, juveniles, and upstream-migrant (prespawning) adults.
Embryos.
Organogenesis during embryological development in lampreys has been described (Piavis, 1971) but it is still unclear when the EP system first appears. Von Kupffer (1893) described the pancreas as a dorsal diverticulum first arising from the anterior intestine, separate from ventral diverticulum of the liver, near its junction with the oesophagus in a 3.3 mm Ammocoetes (Lampetra) planeri. The size of this specimen certainly suggests that he was describing an embryo. However, it is not clear whether he was describing an intestinal diverticulum which persists into larvae or the first islets of the islet organ. Brachet (1897) questioned this observation of von Kupffer and preferred earlier observations, e.g., Langerhans (1873) and Schneider (1879), that follicles of Langerhans developed as epithelial proliferations of the intestine "sans participation de la cavité íntestinale" (p. 633). These follicles were described in animals of 25–30 mm size which were well beyond the embryo period. To date, there has not been a thorough description of the appearance of the EP system in embryos of any lamprey species. That this investigation is both long overdue and highly warranted is illustrated in Figures 1A and B figures depict anti-insulin immunoreactivity in a 35-day old lamprey with insulin-positive cells located within the gut epithelium, in the subepithelial cell clusters, and in the bile duct. It is noteworthy that this is the youngest, post-fertilization lamprey tissue to be examined by immunohistochemistry and it is the first time that insulin-positive cells have been noted in the bile duct prior to metamorphosis. The origin of islets in this manner from bile ducts is reminiscent of that observed in adult hagfish. The position taken by Falkmer (1995) is that the entire EP system in the alimentary canal is the intermediate step in the evolutionary development of the endocrine pancreas from the central nervous system. This intermediate is represented in the adult protochordates (Reinecke, 1981). The nature of the EP system in embryos of lampreys is an important link to our understanding of the ontogenetic and phylogenetic development of the endocrine pancreas in vertebrates. As noted above in a 35-day old animal, immunohistochemistry will be a useful tool in this type of study.
|
Larvae.
As noted above, the recognition of submucosal follicles near the junction of the anterior intestine and the oesophagus of larval lampreys as pancreatic tissue dates to at least 125 years ago. The suggestion of their endocrine nature can be initially traced to Brachet (1897)
but histological descriptions of Cotronei (1927) and Boenig (1929) influenced Barrington (1936. 1942, 1945) to conduct his classical histological and physiological investigations. These latter studies confirmed the endocrine and follicular (cells surrounding a lumen) natures of the larval tissue and the existence of B-cell equivalents involved in carbohydrate metabolism, i.e., the cells show features of secretory activity when hyperglycemia is created with glucose injections. Barrington (1945) concluded that the follicles represent a primitive stage in the evolution of the vertebrate endocrine pancreas, and that they should appropriately be referred to as "follicles of Langerhans." Barrington (1945) also reviewed the early literature that speculated on the presence of "light" and "dark" cells and the existence of the higher vertebrate equivalent of 
and ß cells, now commonly referred to a A and B cells, respectively. Histological examination after glucose injections allowed Barrrington (1942) to conclude that only one cell type exists in the larval follicles (islets) and this is equivalent to the B cell in the islets of Langerhans of higher vertebrates.
It was left to Morris and Islam (1969a) to prove through histochemical means that cells of the follicles of Langerhans of larvae of L. planeri contain no A-like cells and that the only cell type present secretes insulin or an insulin-like substance. In the same year, Titlbach and Kern (1969) provided the first fine structural observation of the cells in the follicles of Langerhans from a larval lamprey (L. planeri). They concluded that clusters of only B-cells bud from the basal epithelium of intestinal epithelium and ultimately reside in the submucosal connective tissue. Definitive confirmation of insulin-like material within the follicles of Lampetra fluviatilis and L. planeri was provided by an immunofluorescent investigation with guinea pig anti-insulin (Van Noorden et al., 1972), but no immunoreactivity was found with mammalian glucagon antisera in either the follicles or the intestinal epithelium. Newly formed islets of larval L. fluviatilis showed no somatostatin immunoreactivity (Van Noorden et al., 1977). Other early immunohistochemical data on larval intestine of these species are difficult to interpret but seem to imply that there is a cell type immunoreactive with antiserum against caerulein, perhaps indicating gastrin-cholesystokinin-secretin activity (Van Noorden and Pearse, 1974).
Maskell (1930) introduced the subject of the diversity of the pancreas among lampreys through a description of the bile duct-intestinal diverticular association in larvae of the southern hemisphere lamprey, Geotria australis. However, it was left for Hilliard et al. (1985) to describe the specific location and arrangement of larval "islet follicles" in this species using histochemical means. A single type of cell (B cell) was present in follicles (only a few with lumina) which were restricted to a region of connective tissue between the oesophagus and right and left, cranially directed, intestinal diverticula. The cell type stained poorly with aldehyde fuchsin and small islet buds were present within the diverticular epithelia. Later, Youson and Potter (1993) showed that the islets of larval Mordacia mordax, another southern hemisphere species, are more closely associated with the anterior intestine rather than with its single intestinal diverticulum, the so-called protopancreas (Epple and Brinn, 1987)
Confirmation of the distribution of follicles (with lumina) at the intestinal-oesophageal junction for holarctic larvae was provided through a description of the sea lamprey (Petromyzon marinus) using antisera directed against mammalian insulin (Elliott and Youson, 1986). This study confimed the absence of D cells in the follicles but was the first to show cells immunoreactive to anti-somatostatin-14 (anti-SS-14) in the intestinal epithelium; this latter cell type was even noted in a 79-day old larva (Youson and Cheung, 1990). Yui et al. (1988) used various heterologous antisera to identify three types of cells most abundant in the upper intestine of larval Lampetra japonica. One type was immunoreactive for somatostatin, a second cell immunoreactive for gastrin/cholecystokinin, and the most numerous third cell was simultaneously immunoreactive for glucagon, pancreatic polypeptide, and FMRFamide. Later studies used homologous antisera directed against lamprey insulin and lamprey somatostatin-34 (SS-34) isolated from upstream-migrant P. marinus (Youson and Elliott, 1989; Youson and Cheung, 1990; Cheung et al., 1991b). No comparisons of immunoreactive intensity between bovine-insulin and lamprey-insulin antisera were conducted, but the latter antibody showed a ubiquitous distribution of intensely immunoreactive follicles in both intraepithelial and submucosal locations. The new finding from these studies was that some of the intraepithelial clusters of cells were immunoreactive to antisera directed against members of the pancreatic polypeptide (PP) family but not to lamprey insulin or to glucagon-like peptide (GLP) antisera. The follicular buds (cell clusters still connected to the epithelium) and isolated follicles immunostained for only insulin like that seen in Figure 1A. The physiological and/or ontogenetic significance of these PP family-immunoreactive cell clusters is unknown. However, perhaps they are precursors to cells that eventually reside in the adult islets as F cells (containing PP-family peptides) or precursor cells which eventually yield the insulin-containing follicles of larvae.
Peptides of the PP-family, namely peptide tyrosine tyrosine (PYY), are the first to appear in endocrine cell differentiation of mouse pancreas and colon and later coexist with other regulatory peptides, eg. glucagon, insulin and somatostatin, in A, B, and D cells, respectively (Upchurch et al., 1994, 1996). If B cells in lamprey larvae arise from a similar progenitor cell, to date there is no evidence of a step in cell differentiation where peptides co-exist. Future studies should address this question in lampreys but should also include the role of adrenomedullin during the differentiation (Martinéz et al., 1998). This multifunctional peptide appears early in development of rat pancreas (Montuenga et al., 1997) and also is involved in the modulation of insulin secretion (Martinéz et al., 1996).
In addition to some variation in distribution of larval islet follicles relative to intestinal diverticula, immunohistochemistry of islet tissue in southern hemisphere species suggested that there might be some interspecific differences in the nature of the peptides among the three lamprey families (Youson and Potter, 1993). Islets of larvae of M. mordax showed strong immunoreactivity to anti-bovine- and anti-lamprey-insulin serum but islets from larval G. australis only weakly immunostained with antiserum against bovine insulin and not with anti-lamprey insulin. A small number of intestinal cells immunostained with either anti-SS-14 or anti-SS-34 sera in Geotria larva but, surprisingly, no cells stained with these antisera in Mordacia larvae. Scattered insulin-positive cells, independent of intraepithelial cell clusters, were present in the Mordacia intestine. The intestine of both species possessed cells immunoreactive to PP-family antisera but not to antisera against either salmon glucagon or salmon GLP. These immunohistochemical data of southern hemisphere larvae and those from their corresponding adults (description to follow) served as a stimulus for comparative peptide sequencing among members of the three lamprey families.
Metamorphosis.
The relationship of liver transformation and the postembryonic ontogeny of the islet organ in lampreys has intrigued biologists for over 100 years (Bujor, 1891). One of the classical features of metamorphosis in all lamprey species studied to date, is the loss of the entire biliary tree. Included are the gall bladder, bile canaliculi, bile ductules, and the intrahepatic and extrahepatic common bile ducts. The events of this regressive process and the potential consequences of the absence of a method of eliminating bile in a postmetamorphic lamprey are of biomedical interest (Youson, 1993, 1999). It was first noted by Keibel (1927) and Boenig (1928) that in L. planeri, a northern hemisphere nonparasitic species, that the extrahepatic duct, and occasionally some intrahepatic ducts, transform into an endocrine pancreatic mass, now referred to as the caudal principal islet (Youson and Al-Mahrouki, 1999). Furthermore, during metamorphosis modifications of the alimentary canal result in a new junction of the oesophagus and the anterior intestine close to the pericardial cartilage (Youson, 1981). At the same time, a mass of islet tissue comes to reside in the submucosal connective tissue at this junction. This more anterior mass is called the cranial principal islet and may be connected to the caudal principal islet by a continous or discontinuous cord of islet tissue, the intermediate cord or secondary islet (Youson and Al-Mahrouki, 1999). The extent of development of these three components of the islet organ varies with the species (Youson and Elliott, 1989; Youson and Cheung, 1990) but also intraspecfic variances are observed (Youson et al., 1988).
Early speculations were that the cranial principal islet developed through direct expansion of larval islets (Barrington, 1945, 1972 quoting studies of Keibel, 1927 and Boenig, 1928). Autoradiographic studies using in vivo administration of 3H-thymidine to P. marinus (Elliott and Youson, 1993a) showed that the cranial principal islet arises through budding of cell clusters from the diverticular epithelium and some proliferation of the resulting islet cells. This study also confirmed (Keibel, 1927) that the caudal principal islet is a direct result of proliferation of cell clusters arising from the extrahepatic, and some intrahepatic, epithelial cells of the common bile duct. Figures 1B and C show immunohistochemistry for insulin and somatostatin at the time of the development of the caudal principal islet from the bile duct during metamorphosis of Lampetra appendix. It is noted that the transforming bile duct of this species contains more cells reactive with anti-SS-14 than with anti-insulin (Figs. 1B, C).
Immunohistochemistry (Elliott and Youson, 1987) and a combined fine structural and immunocytochemical investigation (Elliott and Youson, 1993b) on P. marinus revealed that, despite their different origins, insulin-positive cells appear first within both principal islets. In the caudal principal islet, these B cells develop through a process of transdifferentiation (dedifferentiation/redifferentiation) from the bile duct cells (Elliott and Youson, 1993b). Another cell type with a distinct granular fine structure appears next but the identity of this cell is not known. It is perhaps noteworthy that the unknown cell type decreases in frequency during later metamorphosis, for it may represent a step in the differentiation of D cells or perhaps be a progenitor cell from which all other types arise (see discussion of PP-family cells as progenitor cells in larval islets). An anti-SS-14 immunoreactive cell, the D cell, was not identified through immunocytochemistry (Elliott and Youson, 1993b) until late (stage 7) metamorphosis, yet histochemical observations (Elliott and Youson, 1987) depicted D cells arising as early as midmetamorphosis (stage 4).
Maskell (1931) described the progessive loss of the extrahepatic common bile duct during metamorphosis of Geotria. According to Hilliard et al. (1985), because this duct enters into the cephalac end of the left diverticulum in larva, it does not contribute to the formation of the adult islet organ. For this reason, the islet organ in adult Geotria consists of only a cranial principal islet which is believed to arise from proliferation of larval islets that migrated to the cardiac region during metamorphosis. Immunological and autoradiographic studies described above for transforming P. marinus have not been carried out with Geotria and it is possible that the cranial principal islet of the latter arises from the intestinal epithelium as in the former species. Detailed descriptions of development of the islet organ in adult Mordacia have not been conducted but rationale for the single principal islet follows that of Geotria (Potter, 1986).
Adult.
Rathke in 1826 (see Youson, 1981) was one of the first to be associated with the islet organ in adult lampreys even though he doubted the existence of the tissue. Langerhans (1873) described glandular tissue around the anterior intestine and believed it to be the pancreas. The ductless nature of the tissue led subsequent investigators to assume that it was of endocrine nature (e.g., Cotronei, 1927). It was left to Barrington (1945) to describe in L. fluviatilis two main collections of what he called cranial ("in the dorsal wall of the alimentary canal close behind the pharynx and above the heart") and caudal ("in the connective tissue which joins the intestine to the anterior portion of the liver") cords. Small groups of intermediate cords were present between the two main cords. As it has now been shown for other lamprey species, the cranial cords (now referred to as the cranial principal islet) envelope the tip of the small caecum or diverticulum (Youson and Elliott, 1989). The caudal cords (now called the caudal principal islet) of L. fluviatilis extend between the typhlosole (spiral valve) of the intestine into the liver; this feature is characteristic of holarctic lampreys which usually have the two principal islets. In L. fluviatilis, the caudal principal islet has twice the mass of the cranial principal islet, but in L. planeri they are of similar size (Hardisty et al., 1975). The only holarctic lamprey to lack a distinct cranial principal islet is the unique variety, marifuga, of nonparasitic Lampetra richardsoni (Youson et al., 1988; Youson and Beamish, 1991).
Despite the use of extensive histochemical methods available to him at the time, Barrington (1945) was not willing to accept the view of Cotronei (1927) that "dark" and "light" cells represent A and B cell types. In fact, Barrington (1945) emphatically stated that A cells are not part of the cords and that the staining differences might reflect functional states of the same cell type, the B cell. Winbladh (1966) provided an ultrastructural description of islets from all three components of the islet organ of L. fluviatilis and found three cell types in each region. Granule morphology and aldehyde fuchsin staining verified that one-third of the cells were B cells. The majority of the remaining two-thirds of the cells had empty vesicles and were believed to be either equivalent to mammalian D cells or B cells depleted of their secretions; a few agranular cells were understood to be undifferentiated.
Several reviews at this time emphasized the varied functional state of B cells in the lamprey islet organ (Falkmer and Patent, 1972; Falkmer et al., 1973). Immunohistochemical evidence was eventually provided for the presence of insulin-containing B cells and the absence of A cells in islets of adult L. fluviatilis (Van Noorden et al., 1972; Van Noorden and Pearse, 1974); anti-glucagon immunoreactivity was found in the intestinal epithelium. However, Epple and Brinn (1975) used aldehyde fuchsin-trichrome on principal islets of P. marinus to identify four acidophilic cells (Types I–IV), which varied in their distribution in the cranial and caudal principal islets, in addition to the most numerous lobules of aldehyde fuchsin-positive, B cells. Brinn and Epple (1976) later referred to the acidophilic cells as P cells (I–IV). This implication of the existence of endocrine cell types other than B cells was confirmed with the identification of somatostatin-like immunoreactivity in the islets of adult L. fluviatilis (Van Noorden et al., 1977). No somatostatin immunoreactivity was mentioned for the intestine but this organ was reported to possess cholecystokinin (CCK)-like peptides (Holmquist et al., 1979). A CCK-gastrin immunoreactive cell type could not be found in adult L. japonica (Yui et al., 1988).
The single principal islet of G. australis contains B cell follicles which undergo a continual atrophy during the progression of the upstream migration (Hilliard et al., 1985). Although the PI cells described by Hilliard et al. (1985) do not show the classical histochemical features of D cells of other vertebrates, the time of their appearance during metamorphosis and their numbers are consistent with subsequent observations of cells immunoreactive with somatostatin antisera (Youson and Potter, 1993). A third cell type was argyrophilic (Hilliard et al., 1985). Studies on cranial and caudal principal islets in two adult intervals of P. marinus confirmed that aldehyde fuchsin-positive cells were insulin-containing B cells and that the aldehyde fuchsin-negative cells were mostly somatostatin-containing D cells (Elliott and Youson, 1986); a similar description was provided for L. japonica (Yui et al., 1988).
Following the isolation of somatostatins from principal islets of adult P. marinus (Andrews et al., 1988), it was noted that SS-14 and SS-34 are colocalized with D cells of both the cranial and caudal principal islets of juvenile and upstream-migrants of this species (Cheung et al., 1990). Fine structural observations indicated the presence of three distinct cell types based on their granule morphology; two of these cell types were identified through immunocytochemistry as either B or D cells (Elliott and Youson, 1988). The D cell was considered equivalent to the PI cell of Brinn and Epple (1976), whereas the unknown third cell type was likely their PIV cell. The B and D cells, and an unknown third cell type were also recognized by granule morphology in islet tissue of adult Lampetra ayresi (Youson et al., 1988). In both P. marinus and L. ayresi the third cell type was mostly restricted to the cranial principal islet.
Two subclasses of a third cell type were found in adult islet tissue of P. marinus using immunohistochemistry (Cheung et al., 1991a). A cell was either simultaneously immunoreactive to antisera against the PP family peptides, anglerfish peptide tyrosine (aPY), neuropeptide tyrosine (NPY), and PYY, or to anti-aPY alone. Neither of these two subclasses of cells immunstained with antisera against mammalian PP, a feature noted earlier in L. japonica (Yui et al., 1988). To date, this PP family immunoreactivity has not been directly associated with a third cell type distinquished through fine structural observations (Elliott and Youson, 1988; Youson et al., 1988).
It is unclear why somatostatin immunoreactive cells were not found in the intestine of adult L. japonica (Yui et al., 1988), for this organ in P. marinus has an ubiquitous distribution of cells immunoreactive to anti-SS-14 and/or anti-SS-34 (Cheung et al., 1990). Although immunoreactivity to these two antisera was commonly found in the same cell, many cells immunostained with only anti-SS-34. Near the cranial principal islet, somatostatin cells were also seen as clusters both within the diverticular epithelium and as submucosal islets resembling those of larvae. It was speculated that this was the site of continual production of islet lobules, for the cranial principal islet and this morphogenetic process occurs even into the upstream-migrant period. These cells immunoreactive for somatostatin were distinguished from other cells which were either simultaneously immunoreactive for glucagon-like peptide (GLP), NPY, aPY, and PYY or were only immunoreactive for GLP (Cheung et al., 1991a). The former cell type is likely equivalent to that described as co-reacting to anti-glucagon and anti-PP serum in L. japonica (Yui et al., 1988), however, no immunoreactivity was noted in the intestine of P. marinus when either anti-bovine or anti-salmon PP serum was applied (Cheung et al., 1991a).
The EP systems of adults of two southern hemisphere species, G. australis and M. mordax, have been the most recent ones to be examined by immunohistochemistry (Youson and Potter, 1993). As was expected from histochemistry (Hilliard et al., 1985), the islet organ of Geotria possessed B and D cells, reactive to antisera against mammalin insulin and SS-14, respectively. Surprising, however, was the very weak staining with mammalian insulin antisera and the absence of staining with antisera against insulin and SS-34 from P. marinus. These results suggested some significant differences in the insulin and somatostatin molecules of Geotria and Petromyzon; this fact was later demonstrated following peptide isolation and amino acid sequencing (Conlon et al., 1995b). The Mordacia islet organ immunostained well with serum against either anti-mammalian or anti-lamprey insulin, but like Geotria, D cells stained with anti-SS-14 but not with anti-SS-34. The PP family antisera used to immunostain F cells, mainly in the cranial pancreas, of P. marinus (Cheung et al., 1991a) did not stain any cells in the islet organ of either Geotria or Mordacia. Four cell types of similar immunoreactivity were present in the intestine of adults of both southern hemishere species. These were immunoreactive for either aPY and NPY, glucagon and GLP, SS-14 and SS-34 or solely for SS-34 and were called types 1 to 4, respectively. Mordacia intestine had an additional EP cell, type 5, which immunostained with both anti-mammalian and anti-lamprey insulin sera. Isolated clumps of islet tissue in the intestinal submucosa of Mordacia only immunostained for SS-14.
Bioactivity of EP peptides
Larva.
The early studies of the role of islet hormones in blood-sugar regulation in lampreys have been summarized (Barrington, 1972; Hardisty,1979; Hardisty and Baker, 1982). The most notable of the studies are briefly mentioned here. Although Barrington (1936) had earlier tackled the question of the existence of exocrine and endocrine portions of the larval pancreas, his later report proved to be a classic investigation of the existence of insulin-like activity (Barrington, 1942). Glucose injection resulted in marked vacuolation of a single type of islet cell and islet cauterization raised blood sugar levels. The conclusions were that larval islet tissue can be referred to as "follicles of Langerhans" and it is involved in carbohydrate metabolism. Plisetskaya (1965) extended this view to show that adrenaline evokes prolonged hyperglycemia and insulin administration causes prolonged hypoglycemia (10 days). Variable response of liver and muscle to these hormones was discussed in a phylogenetic context. Subsequently, Leibson and Plisetskaya (1968) showed that insulin-induced hypoglycemia is prolonged at lower temperatures and increases liver, but not muscle, glycogen. Morris and Islam (1969b) induced diabetic symptoms with either glucose, glycine or alloxan injections (muscle and liver glycogen is lost; secretory activity of the gland cells is increased). The diabetic symptoms were alleviated by injections of mammalian insulin (muscle and liver glycogen increased; no secretory activity of the gland cells). Glucagon had no effect on blood glucose regulation. The view at this time was that larvae rely on insulin alone for a limited control of carbohydrate metabolism, that insulin is yielded by the only cell type in the islet, and that these features could be the primitive vertebrate condition.
There was a hiatus of research on the physiology of larval EP tissue until immunoneutralization experiments were conducted on ammocoetes of P. marinus (Youson et al., 1992). This study, and later investigations, were enhanced by the isolation and sequencing of P. marinus insulin and somatostatins and production of antisera (Andrews et al., 1988; Plisetskaya et al., 1988). Injections of anti-lamprey insulin resulted in elevated plasma fatty acid levels and an accompanying reduction in total lipid content in the kidney and an increased rate of lipolysis in the liver. Lamprey SS-34 immunoneutralization provided an opposite effect; promotion of lipid deposition and lowered plasma fatty acids. These data provided some indirect evidence that the B cells of the islet tissue and the somatostatin cells of the intestine (and perhaps the brain) have a role in larval lipid metabolism. This role is particularly enhanced during metamorphosis (see below and Sheridan and Kao, 1998). An homologous radioimmunoassay for insulin in P. marinus was developed (Plisetskaya, 1994) and was used to measure serum insulin during lamprey development (Youson et al., 1994a, b). Insulin concentrations were similar in year class III larvae and older larvae, however, significantly lower serum insulin values were present in the oldest larvae kept in water at 13°C compared to those at 21°C (Fig. 2A).
|
The most recent studies on the function of EP peptides in larval lampreys have addressed their role in lipid metabolism. In particular, the emphasis has been on how injections of mammalian insulin and SS-14 in larva create their respective patterns of lipid metabolism which are found during spontaneous metamorphosis (Kao et al., 1998
; Sheridan and Kao, 1998). Spontaneous metamorphosis in P. marinus is characterized by a two phases of lipid metabolism: lipogenesis followed by lipolysis.
SS-14 injected into larvae induces hyperlipidemia (elevated plasma fatty acids) and lipolysis in the kidney and liver (elevated triacylglycerol lipase, TGL). These results are reminiscent of lipolysis during spontaneous metamorphosis and suggest that somatostatin may play a role in metamorphosis-associated lipid metabolism (Kao et al., 1998). Insulin injected into larvae induces hypolipidemia (decreased plasma fatty acids) and both reduction in lipolysis (decreased TGL) and increased lipogenesis (higher acetyl-CoA carboxylase) in the kidney. Alloxan injections provided opposite effects. Insulin-induced lipogenesis and antilipolysis in larvae are reminiscent of phase 1 lipogenesis during spontaneous metamorphosis. Insulin may play a role, in concert with other factors, in metamorphosis-associated lipid metabolism (Kao et al., 1999).
Metamorphosis.
Analyses of the activity of EP peptides during metamorphosis have been sparse and they are all quite recent. Elliott and Youson (1991) used a heterologous RIA for SS-14 to directly correlate increased tissue levels of somatostatin in intestinal-pancreatic extracts with the development of the caudal and cranial principal islets during the metamorphosis of P. marinus. A homologous RIA was used to demonstrate that serum levels of insulin in P. marinus increased significantly during later stages of metamorphosis (Youson et al., 1994b) which corresponded to the time of intense immunoreactivity for this hormone in the newly developed caudal and cranial principal islets (Fig. 2A). The importance of these hormones to the completion of metamorphosis needs to be examined. Stage 6 metamorphosing animals responded differently to SS-14 treatment than larvae in that the former displayed refractoriness to SS-14 with regard to TGL (Kao et al., 1998). Differences in insulin responsiveness at these two intervals is manifested in greater antilipolytic and lipogenic effects during metamorphosis (Kao et al., 1999). These variations may reflect differences in somatostatin and insulin receptor characters at these two developmental intervals.
Adult.
Rothwell and Fielding (1970) were among the first to identify an insulin-like factor in lampreys when homologous islet extracts caused hypoglycemia. However, there had been prior interest in carbohydrate metabolism of lampreys during their upstream spawning migration when feeding has ceased. The interest has been particularly prominent for a European species, L. fluviatilis, because during its migration it is nontrophic for up to a year before it spawns and dies. Hardisty (1979) reviewed the earlier literature which provided unequivocal documentation that insulin provided by islet B cells is important in maintaining constant levels of serum glucose and in the use and storage of carbohydrates. Classical studies are those that showed that insulin administration induces hypoglycemia (Bentley and Follett, 1965) and increases liver glycogen (Leibson and Plisetskaya, 1968). This latter study was a revelation and a review of the extensive experimental data which had been collected on carbohydrate metabolism in lampreys and other fishes by the Leningrad team. Subsequently, they showed the long-lasting hypoglycemic response to insulin in lampreys is unique among fishes (Leibson and Plisetskaya, 1969), that there is insulin-like immunoreactivity in lamprey serum, and that immunoneutralization of the insulin results in decreases in liver glucose (Plisetskaya and Leibush, 1972). Furthermore, insulin administration increases glycogen synthetase activity in the liver (Plisetskaya and Leibson, 1973). Several ingeneous studies, including insulin immunoneutralization, were undertaken to prove that the long, nontrophic period in the upstream migration is possible because of a chronic insulin insufficiency which prevents premature exhaustion of carbohydrate reserves (Plisetskaya, 1975; Plisetskaya et al., 1976).
Subtotal isletectomy (either cranial or caudal principal islets) of L. fluviatilis does not affect blood glucose levels but total isletectomy results in a 5-fold increase in blood glucose (Hardisty et al., 1975); islet tissue is essential for glucose homeostasis in lampreys. On the other hand, Larsen (1976) claimed that the hormone-induced changes in blood glucose are slow relative to other vertebrates, the physiological role of insulin in this process is unclear, and that insulin is likely important to long-term changes in carbohydrate, lipid and protein metabolism. A heterologous radioimmunoassay proved that total isletectomy (both principal islets but likely not the secondary islet tissue) causes a significant decline in serum insulin but glucose loading gives the reverse effect (Zelnik et al., 1977); these data provided further support for the important role of insulin during the nontrophic upstream migration period in the lamprey life cycle. Glucagon-like immunoreactivity was detected in intestinal and islet extracts (Zelnik et al., 1977) but the latter was not consistent with earlier immunohistochemical data (see above) and was likely contaminated with intestinal tissue. Murat and Plisetskaya (1977) then showed that mammalian glucagon has no effect on blood glucose levels; increase in glycogen synthetase activity in liver could be due to endogenous insulin release.
Following the identification of somatostatin cells in islet tissue of adult P. marinus (Elliott and Youson, 1986), some tissue concentrations of somatostatin in upstream migrants were provided through a heterologous RIA (Elliott and Youson, 1991). There were no differences in total (caudal and cranial) concentrations between early and late migrants or between individual concentrations of the two principal islets in both sexes. However, there were greater somatostatin concentrations in caudal than in cranial principal islets and in the total of the two principal islets compared to the intestine. Intestinal values in upstream migrants were less than in this organ in larvae which may be a consequence of the atrophy of the adult organ during the migration.
The islet organ in G. australis provided an excellent opportunity to examine the role of the lamprey islet tissue in carbohydrate metabolism. Due to the presence of only a single principal islet, total isletectomy was possible in this species during its upstream migration. The resulting immediate hyperglycemia suggested that blood glucose homeostasis is likely dependent on insulin within the islet organ (Epple et al., 1992). The development of an homologous RIA for P. marinus insulin (Plisetskaya, 1994) coincided with a rare opportunity to measure hormones during the adult feeding phase and subsequent growth of this species during a 4-month span (Youson et al., 1994a). No correlation could be found between serum insulin concentratrations and animal length, weight or condition factor (monthly or total). Serum insulin concentration did not differ with respect to gender or nutritional status which was based on liver color and time after feeding (evaluation of intestinal fullness). Some slight differences existed between monthly serum insulin samples but these may reflect water temperature differences in the lake at the time of capture (Fig. 2B). There were significantly lower serum insulin concentrations in upstream-migrant adults compared to feeding adults in the month of June. This result was not surprising since earlier it had been shown in nontrophic, upstream-migrant, L. fluviatilis, that insulin binding to various tissues was not influenced by serum insulin levels (Leibush and Bondareva, 1987). It was surprising, however, that nutritional status and growth (i.e., during 12–18 months of adult feeding in P. marinus, length increases 60-fold and weight 200-fold from immediately postmetamorphic values) could not be correlated with changing serum insulin concentrations. Gender differences had been noted in serum insulin concentrations of upstream-migrant lampreys (Plisetskaya et al., 1976; Sower et al., 1985). The lack of gender differences in serum insulin of the feeding animals may be explained by their immaturity, for such is the case with pink and coho salmon (Plisetskaya et al., 1987). The conclusions at the present time are that systemic levels of insulin (and thyroid hormones) remain relatively constant in adult P. marinus during their adult feeding interval, they do not require or utilize insulin (and thyroid hormones) in the manner (e.g., in body growth and metabolism) that is demonstrated for feeding salmonids (Plisetskaya et al., 1986; Eales, 1988). The most recent data on insulin physiology in lampreys used isolated hepatocytes to study the regulatory mechanism of the binding of insulin to cellular insulin receptors, the internalization of insulin-receptor complexes, and the importance of temperature on the insulin receptors (Lappova and Leibush, 1995; Leibush and Lappova, 1995). Hormonal regulation of growth in adults of parasitic species of lampreys is an important future consideration.
Structure of EP peptides
Falkmer et al. (1975) deduced from comparisons of the the amino acid composition of insulins from L. fluviatilis and hagfish (M. glutinosa), that the primary structure of lamprey insulin is not likely to show large variations from the insulins of other vertebrates. The first lamprey insulin to be isolated and its amino acid sequence disclosed was from P. marinus (Plisetskaya et al., 1988). The insulin from this species has a B chain of 36 amino acids due to a unique, 5 amino-acid extension to the N-terminus (Fig. 3A). In comparison to hagfish, there are 17 substitutions among 52 amino acids in the A and B chains; a similar degree of difference was noted between lamprey insulin and insulins from teleosts and mammals. Three somatostatins were isolated from the same islet extract (Fig. 3B) with SS-34 predominating over both SS-37 and the first variant form of SS-14 ever described (Andrews et al., 1988). A SS-34 was also isolated from hagfish but it contained invariant SS-14 (Figs. 3B, 4) and, among the remaining 20 amino acids extending towards the N-terminal, there are only 2 amino acids in common with lamprey SS-34 (Conlon et al., 1988a).
|
|
Next to be isolated was PMY (a PYY) from the intestine of upstream-migrant P. marinus which has slightly higher sequence homology to pig NPY than to pig PYY (Conlon et al., 1991
). The intestinal extract of upstream migrants failed to yield members of the glucagon family, perhaps due to the atrophied state of this organ (Youson, 1981), but these were isolated and sequenced from the intestine of feeding phase P. marinus (Conlon et al., 1993). Lamprey glucagon shows only 8 amino-acid substitutions compared to human glucagon whereas glucagon-like peptide (GLP) has 16 and 15 substitutions compared to human and salmon GLP-1, respectively.
Results of the immunohistochemistry of the EP systems in larvae and adults of G. australis and M. mordax (Youson and Potter, 1993), using antisera against P. marinus SS-34 and insulin, suggested that there may be differences in the structure of these peptides between lamprey species. Falkmer et al. (1975) had also indicated that the primary structure of insulin from L. fluviatilis might not vary significantly from hagfish insulin. However, as noted above, P. marinus insulin varied markedly from hagfish insulin (Plisetskaya et al., 1988). The aforementioned facts, together with the facts that lampreys have an ancient heritage among vertebrates and that they are separated into three families which may have diverged early in lamprey evolution, were the stimulus for further peptide isolation and sequencing among lamprey species. These comparisons of the structure of EP peptides among lamprey species continue today.
The primary structures of insulin, glucagon and somatostatin from the river lamprey (Baltic lamprey), L. fluviatilis were the next to be provided (Conlon et al., 1995a). The primary structure of Lampetra insulin is identical to that of Petromyzon (Fig. 3A) and the former had equal affinity as pig insulin in binding to human insulin receptors. SS-35, with 8 substitutions and an additional residue compared to Petromyzon, was the only somatostatin isolated from Lampetra islets, but the C-terminal showed the Ser for Thr substitution noted in Petromyzon somatostatins (Fig. 4). It was noteworthy that in the brain of both P. marinus and L. fluviatilis prosomatostatin is processed to invariant SS-14, without the above substitution (Sower et al., 1994; Conlon et al., 1995a). There seems to be tissue-specific polygenic expression of SS-14 in holarctic lampreys. Glucagon from the intestine of L. fluviatilis is more similar to the human (5 substitutions) than to the Petromyzon (6 subs