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AMER. ZOOL., 40:179–199 (2000)

The Agnathan Enteropancreatic Endocrine System: Phylogenetic andOntogenetic Histories, Structure, and Function1

JOHN H. YOUSON2

Department of Zoology and Division of Life Sciences, University of Toronto at Scarborough,Scarborough, Ontario M1C 1A4 Canada

SYNOPSIS. The extant jawless fishes (Agnatha) include the hagfishes and lampreyswhose ancestry can be traced through a conserved evolution to the earliest ofvertebrates. This review traces the study of the enteropancreatic (EP), endocrinecells and their products in hagfishes and lampreys over the past two centuries.Erika Plisetskaya is one of several prominent comparative endocrinologists whostudied the development, distribution or function of the agnathan EP system. Herphysiological studies in Russia laid the foundation for her subsequent isolation inNorth America of the first lamprey EP peptides (insulin and somatostatin) andproviding the first homologous radioimmunoassay for agnathan (lamprey) insulin.This review also emphasizes the nature and the method of development of theagnathan endocrine pancreas (islet organ), for it reflects the earliest vertebrateendocrine pancreas originating from intestinal and/or bile-duct epithelia. The lam-prey life cycle includes a protracted larval period and a metamorphosis when theadult EP system develops. Differences in morphogenesis during metamorphosis ofsouthern- and northern-hemisphere lampreys dictate that a single cranial mass(islet organ) appear in the former and both a cranial and a caudal principal isletcomprises most of the islet organ in holarctic species. There are differences indistribution of cell types and in the primary structure of the peptides in the defin-itive islet organ of hagfishes and lampreys. The primary structures of insulin, so-matostatins, glucagons, glucagon-like peptide, and peptide tyrosine tyrosine arenow available for three lamprey species representing three genera and two of thethree families. Differences in structure of peptides within, and between, families isproviding support for earlier views on the time of divergence of the families andthe different genera. It is concluded that due to the ancient lineage and successfulhabitation of lampreys and hagfishes, and the importance of the EP system to theirsurvival, that their EP systems should be a research focus well into the next cen-tury.

INTRODUCTION

Hagfishes and lampreys are jawless fish-es with direct ancestory to a once fluorish-ing group of Agnatha in the Palaeozoic.Among these agnathans were the ostraco-derms which fossil records suggest wereamong the first vertebrates. In the past, theliving agnathans were referred to as cyclo-stomes (round mouths) and they weregrouped within the vertebrate Class, the Cy-clostomata, and within distinct subclasses

1 From the symposium A Tribute to Erika M. Pli-setskaya: New Insights on the Function and Evolutionof Gastroenteropancreatic Hormones presented at theAnnual Meeting of the Society for Integrative andComparative Biology, 6–10 January 1999, at Denver,Colorado.

2 E-mail: [email protected]

(or orders) as Petromyzontids (the lam-preys) or Myxinids (the hagfishes). Morerecent classification of fishes (Nelson,1994) has the lampreys and hagfishes with-in the vertebrate superclass Agnatha and asdistinct classes, Cephalaspidomorphi andMyxini, respectively. Petromyzontiformesand Myxiniformes are the distinct orders.The taxanomic relationship of hagfishes andlampreys has always been controversial, forthey show many structural, functional, andbehavioural differences (Hardisty, 1979,1982). Recently, lampreys were consideredto be more similar to gnathostome fishesthan to hagfishes (Forey and Janvier, 1994).In fact, Janvier (1986) has always ques-tioned whether hagfishes should be includ-ed among the Vertebrata because they lacksegmented vertebral elements.

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Fossil evidence of early agnathans sug-gest that hagfishes may date back to theCambrian period (Forey and Janvier, 1994)while lampreys originated from a naked an-aspid line about 350 million years ago.Thus the ancestors to the two extant agna-than lines diverged early in craniate evo-lution.

The life histories of the two living ag-nathans are distinctly different. Hagfisheslive their entire life in a marine environ-ment, produce small batches of large yolkyeggs and, following hatching, exhibit directdevelopment to sexual maturity (Gorbman,1997). Lamprey reproduction in freshwateryields many small eggs and, followinghatching, there is a larval period of growthand a metamorphosis (indirect develop-ment) to a juvenile (Youson, 1988). Juve-niles of nonparasitic species immediatelycommence sexual maturation in freshwaterwhereas those of parasitic species becomepredatory and feed on the blood, body flu-ids, and flesh of host teleosts. In some par-asitic species juveniles are capable of ma-rine osmoregulation and they migrate to theopen sea for feeding. It is assumed that oth-er species are restricted to feeding withintheir natal stream because of an inability toosmoregulate in hyperosmotic environ-ments (Hardisty et al., 1989). Genetic, mor-phometric and meristic analyses havegrouped similar parasitic and nonparasiticlamprey species as paired or satellite spe-cies with the implication that they arosefrom a common ancestor, which was likelyparasitic. This view also implies that thenonparasitic adult life history type is morerecent.

Due to their ancient history, lampreysand hagfishes have been the subject ofmuch anatomical and physiological inves-tigation. In many cases, the objective hasbeen to find clues which might provide abridge between the earliest and more mod-ern forms of vertebrates or between verte-brates and other members of Phylum Chor-data, i.e., the protochordates. Furthermore,all systems of lampreys and hagfishes havebeen directly compared to provide some an-swer to questions of their relationship toone another and to their environmental his-tory (Hardisty, 1979, 1982; Hardisty et al.,

1989). Among these comparisons are re-ports of the endocrine cells of the alimen-tary canal and the pancreas. Collectivelythese cells are part of a system termed inmany other vertebrates, the gastroentero-pancreatic (GEP) system. Included in theGEP are enteroendocrine cells of the intes-tine and endocrine cells of both the pancre-as and stomach. Since neither the hagfishnor the lamprey has a stomach (Hardisty,1979), the enteropancreatic (EP) systemseems more appropriate for agnathans.

Hagfishes and lampreys are also distinctamong other vertebrates in having theirexocrine and endocrine equivalents of thevertebrate pancreas in isolation (Barrington,1972; Youson, 1981). The exocrine ele-ments are present within the intestinal ordiverticular epithelia while the equivalentof the endocrine pancreas is an aggregateof submucosal islets, the islet organ. Thepresence of a compacted aggregate of zy-mogen cells in the diverticulum, a so-calledprotopancreas, in one species of lampreysis often used as evidence that a concentrat-ed mass of pancreatic exocrine cellsevolved more than once in vertebrate evo-lution (Epple and Brinn, 1987).

The hormones elaborated by the EP sys-tem of lampreys and hagfishes have, amongother functions, an important role in inter-mediary metabolism. This latter fact wasrecognized by some of the early pioneers ofcomparative vertebrate endocrinology andwe owe them much for their effort and theirinspiration. Among these are Barrington,Epple, Falkmer, Hardisty and Plisetskaya.Falkmer and Plisetskaya have includedsome discussion of the hagfish and lampreyEP system in their reviews (Plisetskaya,1990; Falkmer, 1995; Plisetskaya andMommsen, 1996). A section on the distri-bution and the structure of agnathan EP canbe found in the most recent review of theGEP systems of fishes (Youson and Al-Mahrouki, 1999) This essay will reviewsome of the early work on the ontogeneticand phylogenetic development of the ag-nathan EP system and the function of thehormones elaborated by this system. De-scriptions will be confined to hormoneswhich are shared in common by the isletorgan and the endocrine cells of the gut,

181AGNATHAN ENTEROPANCREATIC ENDOCRINE SYSTEM

namely, peptides of the insulin, glucagon,pancreatic polypeptide, and somatostatinfamilies. Ultimately, the description willlead to the present state of affairs in thesesystems in lampreys and hagfishes, whichwere last specifically reviewed in 1990(Van Noorden, 1990; Youson and Cheung,1990), and to the directions of future re-search.

HAGFISH

Distribution and structure of the EP

Barrington (1945) was intrigued by thereport by Maas (1896) of a ‘‘pancreas-likeorgan’’ in the Myxiniformes with hollowlobules of cells closely associated with thebile duct. Subsequently, Barrington (1945)described in the Atlantic hagfish, Myxineglutinosa, unusual collections of cords ofcells in a white, ovoid swelling on the distalend of the bile duct, near where it entersthe intestine. He emphasized the cord-likearrangement, the extensive connective tis-sue beween cords, the paucity of blood ves-sels, the lumina in the cords, and the originof cords from the bile duct. Schirner (1963)provided another detailed description of theglands and stimulated twelve years of lightand electron microscopic description of Bcells arranged as follicles within M. gluti-nosa (Falkmer and Winbladh, 1964; Thom-as and Ostberg, 1972; Boquist and Ostberg,1975; Winbladh and Horstedt, 1975). How-ever, Winbladh (1976) reported that islettissue follicles are sparse or absent in twoother hagfishes, Epatretus burgeri and Ep-tatretus stouti. A common feature of the is-let tissue of the hagfishes was the absenceof any A cells, that is, an equivalent to theglucagon-producing cells of most gnathos-tomes. This conclusion, which was initiallybased on morphology, was confirmed byimmunohistochemistry using antiseraagainst mammalian insulin which immu-nostained approximately 99% of the cells(Ostberg et al., 1975; Falkmer, 1995). Cellswhich did not react with the insulin anti-body and had different secretory granulesthan those in B cells (Thomas et al., 1973;Boquist and Ostberg, 1975) were later iden-tified as D cells due to their immunostain-ing with anti-somatostatin serum (Van

Noorden et al., 1977). It was also notewor-thy that cells of the bile duct also immu-nostain with anti-insulin and anti-somato-statin (Ostberg et al., 1975, 1976b; VanNoorden et al., 1977), for the islets origi-nate as cell clusters from this duct (Ostberget al., 1976a). B and D cells of both theislet tissue and the bile ducts possess prom-inent crystalline inclusions within rough en-doplasmic reticulum (Boquist and Ostberg,1975). Crystalline arrangement in these in-clusions differs in the two cell types (Raskaet al., 1982). The intestinal epithelium con-tains somatostatin cells and cells which im-munoreact with antisera against pancreaticpolypeptide and glucagon but not anti-in-sulin. Immunoreactivity to antisera againstinsulin-like growth factor 1 (IGF-1) is ubiq-uitous in the islet tissue and is found in cellsthroughout the length of the intestinal mu-cosa (Reinecke et al., 1991). Since no re-ceptors for IGF-1 appear in the intestinaltract, the action of this hormone is not like-ly to be autocrine or paracrine but moresystemic (Drakenberg et al., 1993).

Sture Falkmer (Falkmer and Patent,1972; Falkmer et al., 1973; Falkmer, 1985,1995) has long emphasized that the hagfishislet tissue represents the most phylogenet-ically original islet organ. That is, an isletorgan with an almost total population of B(insulin-containing) cells; most of the othercell types (A, D and F cells) that are foundin higher vertebrate islet tissue still residein the hagfish intestinal mucosa. Further-more, the islet tissue buds from the bileduct epithelium. It is presumed that Falk-mer’s statement on originality of the hagfishislet tissue as a two-hormone gland (Falk-mer, 1995) is meant to be compared withdefinitive (adult) islet organs in vertebrates,for it will be seen below that the endocrinepancreas of larval lamprey possesses aneven simpler design. Insulin-like (Thorn-dyke et al., 1989) and IGF-1 (Reinecke etal., 1991) immunoreactivity has been de-tected in the brain of hagfish, in addition toits presence in the EP system. Falkmer(1995) interprets this distribution as an ex-ample of a primitive organism that firstdemonstrates the complete stepwise se-quence of a neuroendocrine system fromneuronal cells to disseminated mucosal en-

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docrine cells to an isolated islet tissue. Themost primitive condition among chordatesis seen in the protochordates where there isno islet tissue but neuroendocrine elementsin both the central nervous system and inthe mucosal epithelium of the alimentarycanal (Reinecke, 1981; Conlon et al.,1988b; Reinecke et al., 1993). It has beensuggested that the protochordate, Branchi-ostoma lanceolatum (Amphioxus) and thehagfish may share a common feature ofpossessing an ancestral insulin/IGF-1 mol-ecule (Reinecke et al., 1991, 1993). A hy-brid insulin/IGF cDNA has been isolatedfrom B. lanceolatum (Chan et al., 1990).

Bioactivity of EP peptides

Hardisty (1979) and Plisetskaya (1985)provided comprehensive reviews of earlyinvestigations of insulin and carbohydratemetabolism in hagfish. Hagfishes share withlampreys the feature of slow reactions toboth insulin injections and glucose loading(Falkmer and Matty, 1966). However, thereappears to be marked interspecific variationamong hagfishes in the response to insulin;E. stouti requires a smaller dosage and thehypoglycemic response is more prolongedthan in M. glutinosa (Matty and Falkmer,1965; Inui and Gorbman, 1977). Insulinalso seems to be important in protein syn-thesis in E. stouti (Inui and Gorbman,1978).

Structure of EP peptides

Hagfish insulin was the first EP peptideto be isolated from an agnathan (Petersonet al., 1974; Cutfield et al., 1979). Althoughthe hagfish insulin molecule contains theputative receptor binding region noted inporcine insulin, amino acid substitutions inpart of the B chain may explain a reducedbiological potency relative to the mamma-lian molecule (Cutfield et al., 1979). Coin-cidentally, hagfish and lamprey somatostat-ins were described in the same year by in-dependent groups. Conlon et al. (1988a)isolated somatostatins with 34 and 14 ami-no acids from the islet tissue of M. gluti-nosa. These same two somatostatins arealso present in the intestine (Conlon andFalkmer, 1989). The smaller somatostatinwas the invariant form present in all other

vertebrate islet tissue but lampreys. The bi-ological activity of hagfish somatostatinshas not been examined but, since there arerelatively few cells, there may be a para-crine role for this hormone in islet and in-testinal tissues. The primary structures ofmembers of the glucagon and the pancreaticpolypeptide families have not been provid-ed.

LAMPREY

Distribution and structure of the EP

Unlike the hagfish where knowledge ofembryogenesis (Gorbman, 1997) and sub-sequent growth is scanty, the life cycle oflampreys from the zygote to time of deathafter spawning has been well documented.During many of the intervening life cycleperiods organogenesis has been examined(Youson, 1985). These studies have includ-ed descriptions of the EP system from em-bryogenesis to the upstream migration pe-riod (Youson and Elliott, 1989; Youson andCheung, 1990). During this time the systemundergoes significant changes which areimportant to permit differences in behav-iour between individuals of the life cycleperiods. The following is a summary of theEP system in embryos, larvae, metamor-phosing individuals, juveniles, and up-stream-migrant (prespawning) adults.

Embryos. Organogenesis during embry-ological development in lampreys has beendescribed (Piavis, 1971) but it is still un-clear when the EP system first appears. VonKupffer (1893) described the pancreas as adorsal diverticulum first arising from theanterior intestine, separate from ventral di-verticulum of the liver, near its junctionwith the oesophagus in a 3.3 mm Ammo-coetes (Lampetra) planeri. The size of thisspecimen certainly suggests that he was de-scribing an embryo. However, it is not clearwhether he was describing an intestinal di-verticulum which persists into larvae or thefirst islets of the islet organ. Brachet (1897)questioned this observation of von Kupfferand preferred earlier observations, e.g.,Langerhans (1873) and Schneider (1879),that follicles of Langerhans developed asepithelial proliferations of the intestine‘‘sans participation de la cavite ıntestinale’’


FIG. 1. Light micrographs of lamprey pancreatic islet tissue immunostained with either insulin or somatostatin(SS) antisera. Sections are not counterstained and the peroxidase-anti-peroxidase was used. A. A 35-day oldlarval Petromyzon marinus shows insulin-immunoreactive cells in a bile duct (arrowhead), in the intestinalepithelium (thick arrow), and the submucosa connective tissue (thin arrow). *Pigment. Bar 5 20 mm Inset:Higher magnfication of the cell in the bile duct and other cells in the submucosa. Bar 5 10 mm B. Somatostatin-immunoreactivity in cells within the bile duct epithelium (arrowhead) and in the surrounding tissue (arrow) inmetamorphosing Lampetra appendix. Bar 5 10 mm C. A close to adjacent section to Figure 1B shows insulin-immunoreactivity in cells near (arrow) the transforming bile duct, but not in the clumps of cells (arrowhead)within the duct. Bar 5 10 mm.

(p. 633). These follicles were described inanimals of 25–30 mm size which were wellbeyond the embryo period. To date, therehas not been a thorough description of theappearance of the EP system in embryos ofany lamprey species. That this investigationis both long overdue and highly warrantedis illustrated in Figures 1A and B. Thesefigures depict anti-insulin immunoreactivityin a 35-day old lamprey with insulin-posi-tive cells located within the gut epithelium,in the subepithelial cell clusters, and in thebile duct. It is noteworthy that this is theyoungest, post-fertilization lamprey tissueto be examined by immunohistochemistryand it is the first time that insulin-positivecells have been noted in the bile duct prior

to metamorphosis. The origin of islets inthis manner from bile ducts is reminiscentof that observed in adult hagfish. The po-sition taken by Falkmer (1995) is that theentire EP system in the alimentary canal isthe intermediate step in the evolutionarydevelopment of the endocrine pancreasfrom the central nervous system. This in-termediate is represented in the adult pro-tochordates (Reinecke, 1981). The nature ofthe EP system in embryos of lampreys isan important link to our understanding ofthe ontogenetic and phylogenetic develop-ment of the endocrine pancreas in verte-brates. As noted above in a 35-day old an-imal, immunohistochemistry will be a use-ful tool in this type of study.

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Larvae. As noted above, the recognitionof submucosal follicles near the junction ofthe anterior intestine and the oesophagus oflarval lampreys as pancreatic tissue dates toat least 125 years ago. The suggestion oftheir endocrine nature can be initially tracedto Brachet (1897) but histological descrip-tions of Cotronei (1927) and Boenig (1929)influenced Barrington (1936. 1942, 1945)to conduct his classical histological andphysiological investigations. These latterstudies confirmed the endocrine and follic-ular (cells surrounding a lumen) natures ofthe larval tissue and the existence of B-cellequivalents involved in carbohydrate me-tabolism, i.e., the cells show features of se-cretory activity when hyperglycemia is cre-ated with glucose injections. Barrington(1945) concluded that the follicles representa primitive stage in the evolution of the ver-tebrate endocrine pancreas, and that theyshould appropriately be referred to as ‘‘fol-licles of Langerhans.’’ Barrington (1945)also reviewed the early literature that spec-ulated on the presence of ‘‘light’’ and‘‘dark’’ cells and the existence of the highervertebrate equivalent of a and b cells, nowcommonly referred to a A and B cells, re-spectively. Histological examination afterglucose injections allowed Barrrington(1942) to conclude that only one cell typeexists in the larval follicles (islets) and thisis equivalent to the B cell in the islets ofLangerhans of higher vertebrates.

It was left to Morris and Islam (1969a)to prove through histochemical means thatcells of the follicles of Langerhans of larvaeof L. planeri contain no A-like cells and thatthe only cell type present secretes insulin oran insulin-like substance. In the same year,Titlbach and Kern (1969) provided the firstfine structural observation of the cells in thefollicles of Langerhans from a larval lam-prey (L. planeri). They concluded that clus-ters of only B-cells bud from the basal epi-thelium of intestinal epithelium and ulti-mately reside in the submucosal connectivetissue. Definitive confirmation of insulin-likematerial within the follicles of Lampetra flu-viatilis and L. planeri was provided by animmunofluorescent investigation with guin-ea pig anti-insulin (Van Noorden et al.,1972), but no immunoreactivity was found

with mammalian glucagon antisera in eitherthe follicles or the intestinal epithelium.Newly formed islets of larval L. fluviatilisshowed no somatostatin immunoreactivity(Van Noorden et al., 1977). Other early im-munohistochemical data on larval intestineof these species are difficult to interpret butseem to imply that there is a cell type im-munoreactive with antiserum against caeru-lein, perhaps indicating gastrin-cholesysto-kinin-secretin activity (Van Noorden andPearse, 1974).

Maskell (1930) introduced the subject ofthe diversity of the pancreas among lam-preys through a description of the bile duct-intestinal diverticular association in larvaeof the southern hemisphere lamprey, Geo-tria australis. However, it was left for Hil-liard et al. (1985) to describe the specificlocation and arrangement of larval ‘‘isletfollicles’’ in this species using histochemi-cal means. A single type of cell (B cell) waspresent in follicles (only a few with lumina)which were restricted to a region of con-nective tissue between the oesophagus andright and left, cranially directed, intestinaldiverticula. The cell type stained poorlywith aldehyde fuchsin and small islet budswere present within the diverticular epithe-lia. Later, Youson and Potter (1993) showedthat the islets of larval Mordacia mordax,another southern hemisphere species, aremore closely associated with the anteriorintestine rather than with its single intestinaldiverticulum, the so-called protopancreas(Epple and Brinn, 1987)

Confirmation of the distribution of folli-cles (with lumina) at the intestinal-oesoph-ageal junction for holarctic larvae was pro-vided through a description of the sea lam-prey (Petromyzon marinus) using antiseradirected against mammalian insulin (Elliottand Youson, 1986). This study confimedthe absence of D cells in the follicles butwas the first to show cells immunoreactiveto anti-somatostatin-14 (anti-SS-14) in theintestinal epithelium; this latter cell typewas even noted in a 79-day old larva (You-son and Cheung, 1990). Yui et al. (1988)used various heterologous antisera to iden-tify three types of cells most abundant inthe upper intestine of larval Lampetra ja-ponica. One type was immunoreactive for


somatostatin, a second cell immunoreactivefor gastrin/cholecystokinin, and the mostnumerous third cell was simultaneously im-munoreactive for glucagon, pancreaticpolypeptide, and FMRFamide. Later studiesused homologous antisera directed againstlamprey insulin and lamprey somatostatin-34 (SS-34) isolated from upstream-migrantP. marinus (Youson and Elliott, 1989; You-son and Cheung, 1990; Cheung et al.,1991b). No comparisons of immunoreac-tive intensity between bovine-insulin andlamprey-insulin antisera were conducted,but the latter antibody showed a ubiquitousdistribution of intensely immunoreactivefollicles in both intraepithelial and submu-cosal locations. The new finding from thesestudies was that some of the intraepithelialclusters of cells were immunoreactive to an-tisera directed against members of the pan-creatic polypeptide (PP) family but not tolamprey insulin or to glucagon-like peptide(GLP) antisera. The follicular buds (cellclusters still connected to the epithelium)and isolated follicles immunostained foronly insulin like that seen in Figure 1A. Thephysiological and/or ontogenetic signifi-cance of these PP family-immunoreactivecell clusters is unknown. However, perhapsthey are precursors to cells that eventuallyreside in the adult islets as F cells (contain-ing PP-family peptides) or precursor cellswhich eventually yield the insulin-contain-ing follicles of larvae.

Peptides of the PP-family, namely pep-tide tyrosine tyrosine (PYY), are the first toappear in endocrine cell differentiation ofmouse pancreas and colon and later coexistwith other regulatory peptides, eg. gluca-gon, insulin and somatostatin, in A, B, andD cells, respectively (Upchurch et al., 1994,1996). If B cells in lamprey larvae arisefrom a similar progenitor cell, to date thereis no evidence of a step in cell differentia-tion where peptides co-exist. Future studiesshould address this question in lampreysbut should also include the role of adreno-medullin during the differentiation (Marti-nez et al., 1998). This multifunctional pep-tide appears early in development of ratpancreas (Montuenga et al., 1997) and alsois involved in the modulation of insulin se-cretion (Martinez et al., 1996).

In addition to some variation in distri-bution of larval islet follicles relative to in-testinal diverticula, immunohistochemistryof islet tissue in southern hemisphere spe-cies suggested that there might be some in-terspecific differences in the nature of thepeptides among the three lamprey families(Youson and Potter, 1993). Islets of larvaeof M. mordax showed strong immunoreac-tivity to anti-bovine- and anti-lamprey-in-sulin serum but islets from larval G. aus-tralis only weakly immunostained with an-tiserum against bovine insulin and not withanti-lamprey insulin. A small number of in-testinal cells immunostained with eitheranti-SS-14 or anti-SS-34 sera in Geotrialarva but, surprisingly, no cells stained withthese antisera in Mordacia larvae. Scatteredinsulin-positive cells, independent of intra-epithelial cell clusters, were present in theMordacia intestine. The intestine of bothspecies possessed cells immunoreactive toPP-family antisera but not to antiseraagainst either salmon glucagon or salmonGLP. These immunohistochemical data ofsouthern hemisphere larvae and those fromtheir corresponding adults (description tofollow) served as a stimulus for compara-tive peptide sequencing among members ofthe three lamprey families.

Metamorphosis. The relationship of livertransformation and the postembryonic on-togeny of the islet organ in lampreys hasintrigued biologists for over 100 years (Bu-jor, 1891). One of the classical features ofmetamorphosis in all lamprey species stud-ied to date, is the loss of the entire biliarytree. Included are the gall bladder, bile can-aliculi, bile ductules, and the intrahepaticand extrahepatic common bile ducts. Theevents of this regressive process and the po-tential consequences of the absence of amethod of eliminating bile in a postmeta-morphic lamprey are of biomedical interest(Youson, 1993, 1999). It was first noted byKeibel (1927) and Boenig (1928) that in L.planeri, a northern hemisphere nonparasiticspecies, that the extrahepatic duct, and oc-casionally some intrahepatic ducts, trans-form into an endocrine pancreatic mass,now referred to as the caudal principal islet(Youson and Al-Mahrouki, 1999). Further-more, during metamorphosis modifications

186 JOHN H. YOUSON

of the alimentary canal result in a new junc-tion of the oesophagus and the anterior in-testine close to the pericardial cartilage(Youson, 1981). At the same time, a massof islet tissue comes to reside in the sub-mucosal connective tissue at this junction.This more anterior mass is called the cranialprincipal islet and may be connected to thecaudal principal islet by a continous or dis-continuous cord of islet tissue, the inter-mediate cord or secondary islet (Yousonand Al-Mahrouki, 1999). The extent of de-velopment of these three components of theislet organ varies with the species (Yousonand Elliott, 1989; Youson and Cheung,1990) but also intraspecfic variances are ob-served (Youson et al., 1988).

Early speculations were that the cranialprincipal islet developed through direct ex-pansion of larval islets (Barrington, 1945,1972 quoting studies of Keibel, 1927 andBoenig, 1928). Autoradiographic studiesusing in vivo administration of 3H-thymi-dine to P. marinus (Elliott and Youson,1993a) showed that the cranial principal is-let arises through budding of cell clustersfrom the diverticular epithelium and someproliferation of the resulting islet cells. Thisstudy also confirmed (Keibel, 1927) that thecaudal principal islet is a direct result ofproliferation of cell clusters arising from theextrahepatic, and some intrahepatic, epithe-lial cells of the common bile duct. Figures1B and C show immunohistochemistry forinsulin and somatostatin at the time of thedevelopment of the caudal principal isletfrom the bile duct during metamorphosis ofLampetra appendix. It is noted that thetransforming bile duct of this species con-tains more cells reactive with anti-SS-14than with anti-insulin (Figs. 1B, C).

Immunohistochemistry (Elliott and You-son, 1987) and a combined fine structuraland immunocytochemical investigation (El-liott and Youson, 1993b) on P. marinus re-vealed that, despite their different origins,insulin-positive cells appear first withinboth principal islets. In the caudal principalislet, these B cells develop through a pro-cess of transdifferentiation (dedifferentia-tion/redifferentiation) from the bile ductcells (Elliott and Youson, 1993b). Anothercell type with a distinct granular fine struc-

ture appears next but the identity of this cellis not known. It is perhaps noteworthy thatthe unknown cell type decreases in fre-quency during later metamorphosis, for itmay represent a step in the differentiationof D cells or perhaps be a progenitor cellfrom which all other types arise (see dis-cussion of PP-family cells as progenitorcells in larval islets). An anti-SS-14 im-munoreactive cell, the D cell, was not iden-tified through immunocytochemistry (Elli-ott and Youson, 1993b) until late (stage 7)metamorphosis, yet histochemical observa-tions (Elliott and Youson, 1987) depicted Dcells arising as early as midmetamorphosis(stage 4).

Maskell (1931) described the progessiveloss of the extrahepatic common bile ductduring metamorphosis of Geotria. Accord-ing to Hilliard et al. (1985), because thisduct enters into the cephalac end of the leftdiverticulum in larva, it does not contributeto the formation of the adult islet organ. Forthis reason, the islet organ in adult Geotriaconsists of only a cranial principal isletwhich is believed to arise from proliferationof larval islets that migrated to the cardiacregion during metamorphosis. Immunolog-ical and autoradiographic studies describedabove for transforming P. marinus have notbeen carried out with Geotria and it is pos-sible that the cranial principal islet of thelatter arises from the intestinal epitheliumas in the former species. Detailed descrip-tions of development of the islet organ inadult Mordacia have not been conductedbut rationale for the single principal isletfollows that of Geotria (Potter, 1986).

Adult. Rathke in 1826 (see Youson,1981) was one of the first to be associatedwith the islet organ in adult lampreys eventhough he doubted the existence of the tis-sue. Langerhans (1873) described glandulartissue around the anterior intestine and be-lieved it to be the pancreas. The ductlessnature of the tissue led subsequent investi-gators to assume that it was of endocrinenature (e.g., Cotronei, 1927). It was left toBarrington (1945) to describe in L. fluvia-tilis two main collections of what he calledcranial (‘‘in the dorsal wall of the alimen-tary canal close behind the pharynx andabove the heart’’) and caudal (‘‘in the con-


nective tissue which joins the intestine tothe anterior portion of the liver’’) cords.Small groups of intermediate cords werepresent between the two main cords. As ithas now been shown for other lamprey spe-cies, the cranial cords (now referred to asthe cranial principal islet) envelope the tipof the small caecum or diverticulum (You-son 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 holarcticlampreys which usually have the two prin-cipal islets. In L. fluviatilis, the caudal prin-cipal islet has twice the mass of the cranialprincipal islet, but in L. planeri they are ofsimilar size (Hardisty et al., 1975). Theonly holarctic lamprey to lack a distinct cra-nial principal islet is the unique variety,marifuga, of nonparasitic Lampetra ri-chardsoni (Youson et al., 1988; Youson andBeamish, 1991).

Despite the use of extensive histochemi-cal methods available to him at the time,Barrington (1945) was not willing to acceptthe view of Cotronei (1927) that ‘‘dark’’and ‘‘light’’ cells represent A and B celltypes. In fact, Barrington (1945) emphati-cally stated that A cells are not part of thecords and that the staining differencesmight reflect functional states of the samecell type, the B cell. Winbladh (1966) pro-vided an ultrastructural description of isletsfrom all three components of the islet organof L. fluviatilis and found three cell typesin each region. Granule morphology and al-dehyde fuchsin staining verified that one-third of the cells were B cells. The majorityof the remaining two-thirds of the cells hadempty vesicles and were believed to be ei-ther equivalent to mammalian D cells or Bcells depleted of their secretions; a fewagranular cells were understood to be un-differentiated.

Several reviews at this time emphasizedthe varied functional state of B cells in thelamprey islet organ (Falkmer and Patent,1972; Falkmer et al., 1973). Immunohisto-chemical evidence was eventually providedfor the presence of insulin-containing Bcells and the absence of A cells in islets ofadult L. fluviatilis (Van Noorden et al.,

1972; Van Noorden and Pearse, 1974); anti-glucagon immunoreactivity was found inthe intestinal epithelium. However, Eppleand Brinn (1975) used aldehyde fuchsin-tri-chrome on principal islets of P. marinus toidentify four acidophilic cells (Types I–IV),which varied in their distribution in the cra-nial and caudal principal islets, in additionto the most numerous lobules of aldehydefuchsin-positive, B cells. Brinn and Epple(1976) later referred to the acidophilic cellsas P cells (I–IV). This implication of theexistence of endocrine cell types other thanB cells was confirmed with the identifica-tion of somatostatin-like immunoreactivityin the islets of adult L. fluviatilis (VanNoorden et al., 1977). No somatostatin im-munoreactivity was mentioned for the in-testine but this organ was reported to pos-sess cholecystokinin (CCK)-like peptides(Holmquist et al., 1979). A CCK-gastrinimmunoreactive cell type could not befound in adult L. japonica (Yui et al.,1988).

The single principal islet of G. australiscontains B cell follicles which undergo acontinual atrophy during the progression ofthe upstream migration (Hilliard et al.,1985). Although the PI cells described byHilliard et al. (1985) do not show the clas-sical histochemical features of D cells ofother vertebrates, the time of their appear-ance during metamorphosis and their num-bers are consistent with subsequent obser-vations of cells immunoreactive with so-matostatin antisera (Youson and Potter,1993). A third cell type was argyrophilic(Hilliard et al., 1985). Studies on cranialand caudal principal islets in two adult in-tervals of P. marinus confirmed that alde-hyde fuchsin-positive cells were insulin-containing B cells and that the aldehydefuchsin-negative cells were mostly somato-statin-containing D cells (Elliott and You-son, 1986); a similar description was pro-vided for L. japonica (Yui et al., 1988).

Following the isolation of somatostatinsfrom principal islets of adult P. marinus(Andrews et al., 1988), it was noted thatSS-14 and SS-34 are colocalized with Dcells of both the cranial and caudal princi-pal islets of juvenile and upstream-migrantsof this species (Cheung et al., 1990). Fine

188 JOHN H. YOUSON

structural observations indicated the pres-ence of three distinct cell types based ontheir granule morphology; two of these celltypes were identified through immunocy-tochemistry as either B or D cells (Elliottand Youson, 1988). The D cell was consid-ered equivalent to the PI cell of Brinn andEpple (1976), whereas the unknown thirdcell type was likely their PIV cell. The B andD cells, and an unknown third cell typewere also recognized by granule morphol-ogy in islet tissue of adult Lampetra ayresi(Youson et al., 1988). In both P. marinusand L. ayresi the third cell type was mostlyrestricted to the cranial principal islet.

Two subclasses of a third cell type werefound in adult islet tissue of P. marinus us-ing immunohistochemistry (Cheung et al.,1991a). A cell was either simultaneouslyimmunoreactive to antisera against the PPfamily peptides, anglerfish peptide tyrosine(aPY), neuropeptide tyrosine (NPY), andPYY, or to anti-aPY alone. Neither of thesetwo subclasses of cells immunstained withantisera against mammalian PP, a featurenoted earlier in L. japonica (Yui et al.,1988). To date, this PP family immunore-activity has not been directly associatedwith a third cell type distinquished throughfine structural observations (Elliott andYouson, 1988; Youson et al., 1988).

It is unclear why somatostatin immuno-reactive cells were not found in the intestineof adult L. japonica (Yui et al., 1988), forthis organ in P. marinus has an ubiquitousdistribution of cells immunoreactive to anti-SS-14 and/or anti-SS-34 (Cheung et al.,1990). Although immunoreactivity to thesetwo antisera was commonly found in thesame cell, many cells immunostained withonly anti-SS-34. Near the cranial principalislet, somatostatin cells were also seen asclusters both within the diverticular epithe-lium and as submucosal islets resemblingthose of larvae. It was speculated that thiswas the site of continual production of isletlobules, for the cranial principal islet andthis morphogenetic process occurs eveninto the upstream-migrant period. Thesecells immunoreactive for somatostatin weredistinguished from other cells which wereeither simultaneously immunoreactive forglucagon-like peptide (GLP), NPY, aPY,

and PYY or were only immunoreactive forGLP (Cheung et al., 1991a). The formercell type is likely equivalent to that de-scribed as co-reacting to anti-glucagon andanti-PP serum in L. japonica (Yui et al.,1988), however, no immunoreactivity wasnoted in the intestine of P. marinus wheneither anti-bovine or anti-salmon PP serumwas applied (Cheung et al., 1991a).

The EP systems of adults of two southernhemisphere species, G. australis and M.mordax, have been the most recent ones tobe examined by immunohistochemistry(Youson and Potter, 1993). As was expectedfrom histochemistry (Hilliard et al., 1985),the islet organ of Geotria possessed B andD cells, reactive to antisera against mam-malin insulin and SS-14, respectively. Sur-prising, however, was the very weak stain-ing with mammalian insulin antisera andthe absence of staining with antisera againstinsulin and SS-34 from P. marinus. Theseresults suggested some significant differ-ences in the insulin and somatostatin mol-ecules of Geotria and Petromyzon; this factwas later demonstrated following peptideisolation and amino acid sequencing (Con-lon et al., 1995b). The Mordacia islet organimmunostained well with serum against ei-ther anti-mammalian or anti-lamprey insu-lin, but like Geotria, D cells stained withanti-SS-14 but not with anti-SS-34. The PPfamily antisera used to immunostain F cells,mainly in the cranial pancreas, of P. mari-nus (Cheung et al., 1991a) did not stain anycells in the islet organ of either Geotria orMordacia. Four cell types of similar im-munoreactivity were present in the intestineof adults of both southern hemishere spe-cies. These were immunoreactive for eitheraPY and NPY, glucagon and GLP, SS-14and SS-34 or solely for SS-34 and werecalled types 1 to 4, respectively. Mordaciaintestine had an additional EP cell, type 5,which immunostained with both anti-mam-malian and anti-lamprey insulin sera. Iso-lated clumps of islet tissue in the intestinalsubmucosa of Mordacia only immuno-stained for SS-14.

Bioactivity of EP peptides

Larva. The early studies of the role ofislet hormones in blood-sugar regulation in


lampreys have been summarized (Barring-ton, 1972; Hardisty,1979; Hardisty andBaker, 1982). The most notable of the stud-ies are briefly mentioned here. AlthoughBarrington (1936) had earlier tackled thequestion of the existence of exocrine andendocrine portions of the larval pancreas,his later report proved to be a classic in-vestigation of the existence of insulin-likeactivity (Barrington, 1942). Glucose injec-tion resulted in marked vacuolation of a sin-gle type of islet cell and islet cauterizationraised blood sugar levels. The conclusionswere that larval islet tissue can be referredto as ‘‘follicles of Langerhans’’ and it is in-volved in carbohydrate metabolism. Pliset-skaya (1965) extended this view to showthat adrenaline evokes prolonged hypergly-cemia and insulin administration causesprolonged hypoglycemia (10 days). Vari-able response of liver and muscle to thesehormones was discussed in a phylogeneticcontext. Subsequently, Leibson and Pliset-skaya (1968) showed that insulin-inducedhypoglycemia is prolonged at lower tem-peratures and increases liver, but not mus-cle, glycogen. Morris and Islam (1969b) in-duced diabetic symptoms with either glu-cose, glycine or alloxan injections (muscleand liver glycogen is lost; secretory activityof the gland cells is increased). The diabeticsymptoms were alleviated by injections ofmammalian insulin (muscle and liver gly-cogen increased; no secretory activity of thegland cells). Glucagon had no effect onblood glucose regulation. The view at thistime was that larvae rely on insulin alonefor a limited control of carbohydrate me-tabolism, that insulin is yielded by the onlycell type in the islet, and that these featurescould be the primitive vertebrate condition.

There was a hiatus of research on thephysiology of larval EP tissue until immu-noneutralization experiments were conduct-ed on ammocoetes of P. marinus (Yousonet al., 1992). This study, and later investi-gations, were enhanced by the isolation andsequencing of P. marinus insulin and so-matostatins and production of antisera (An-drews et al., 1988; Plisetskaya et al., 1988).Injections of anti-lamprey insulin resultedin elevated plasma fatty acid levels and anaccompanying reduction in total lipid con-

tent in the kidney and an increased rate oflipolysis in the liver. Lamprey SS-34 im-munoneutralization provided an oppositeeffect; promotion of lipid deposition andlowered plasma fatty acids. These data pro-vided some indirect evidence that the Bcells of the islet tissue and the somatostatincells of the intestine (and perhaps the brain)have a role in larval lipid metabolism. Thisrole is particularly enhanced during meta-morphosis (see below and Sheridan andKao, 1998). An homologous radioimmu-noassay for insulin in P. marinus was de-veloped (Plisetskaya, 1994) and was usedto measure serum insulin during lampreydevelopment (Youson et al., 1994a, b). In-sulin concentrations were similar in yearclass III larvae and older larvae, however,significantly lower serum insulin valueswere present in the oldest larvae kept inwater at 138C compared to those at 218C(Fig. 2A).

The most recent studies on the functionof EP peptides in larval lampreys have ad-dressed their role in lipid metabolism. Inparticular, the emphasis has been on howinjections of mammalian insulin and SS-14in larva create their respective patterns oflipid metabolism which are found duringspontaneous metamorphosis (Kao et al.,1998; Sheridan and Kao, 1998). Spontane-ous metamorphosis in P. marinus is char-acterized by a two phases of lipid metabo-lism: lipogenesis followed by lipolysis.

SS-14 injected into larvae induces hy-perlipidemia (elevated plasma fatty acids)and lipolysis in the kidney and liver (ele-vated triacylglycerol lipase, TGL). Theseresults are reminiscent of lipolysis duringspontaneous metamorphosis and suggestthat somatostatin may play a role in meta-morphosis-associated lipid metabolism(Kao et al., 1998). Insulin injected into lar-vae induces hypolipidemia (decreased plas-ma fatty acids) and both reduction in lipol-ysis (decreased TGL) and increased lipo-genesis (higher acetyl-CoA carboxylase) inthe kidney. Alloxan injections provided op-posite effects. Insulin-induced lipogenesisand antilipolysis in larvae are reminiscentof phase 1 lipogenesis during spontaneousmetamorphosis. Insulin may play a role, inconcert with other factors, in metamorpho-

190 JOHN H. YOUSON

FIG. 2. Serum insulin concentrations (mean 6 SE) inPetromyzon marinus at different periods of the life cy-cle as measured by an homologous radioimmunoassay(Plisetskaya, 1994). A. Insulin levels are significantlylower (***) at 13 compared to 218C in larvae and risesignficantly (*) from larval levels by stage 6 of meta-morphosis. The value in feeding adults is significantlyhigher (**) than that of larvae. The number of animalsused at each interval is indicated. Data are from You-son et al., 1994b. B. Serum insulin in feeding wasmeasured over a 4-month period but no correlationcould be found between feeding status (fullness of gut)within each month. There was a significantly lowerserum insulin in May (*) compared to July and in Junethe values in prespawning, upstream migrants (**) wassignificantly lower than that of feeding adults. Data arefrom Youson et al., 1994a

sis-associated lipid metabolism (Kao et al.,1999).

Metamorphosis. Analyses of the activityof EP peptides during metamorphosis havebeen sparse and they are all quite recent.Elliott and Youson (1991) used a heterolo-gous RIA for SS-14 to directly correlate in-creased tissue levels of somatostatin in in-testinal-pancreatic extracts with the devel-opment of the caudal and cranial principalislets during the metamorphosis of P. mar-

inus. A homologous RIA was used to dem-onstrate that serum levels of insulin in P.marinus increased significantly during laterstages of metamorphosis (Youson et al.,1994b) which corresponded to the time ofintense immunoreactivity for this hormonein the newly developed caudal and cranialprincipal islets (Fig. 2A). The importanceof these hormones to the completion ofmetamorphosis needs to be examined.Stage 6 metamorphosing animals respondeddifferently to SS-14 treatment than larvaein that the former displayed refractorinessto SS-14 with regard to TGL (Kao et al.,1998). Differences in insulin responsive-ness at these two intervals is manifested ingreater antilipolytic and lipogenic effectsduring metamorphosis (Kao et al., 1999).These variations may reflect differences insomatostatin and insulin receptor charactersat these two developmental intervals.

Adult. Rothwell and Fielding (1970)were among the first to identify an insulin-like factor in lampreys when homologousislet extracts caused hypoglycemia. How-ever, there had been prior interest in car-bohydrate metabolism of lampreys duringtheir upstream spawning migration whenfeeding has ceased. The interest has beenparticularly prominent for a European spe-cies, L. fluviatilis, because during its migra-tion it is nontrophic for up to a year beforeit spawns and dies. Hardisty (1979) re-viewed the earlier literature which providedunequivocal documentation that insulinprovided by islet B cells is important inmaintaining constant levels of serum glu-cose and in the use and storage of carbo-hydrates. Classical studies are those thatshowed that insulin administration induceshypoglycemia (Bentley and Follett, 1965)and increases liver glycogen (Leibson andPlisetskaya, 1968). This latter study was arevelation and a review of the extensive ex-perimental data which had been collectedon carbohydrate metabolism in lampreysand other fishes by the Leningrad team.Subsequently, they showed the long-lastinghypoglycemic response to insulin in lam-preys is unique among fishes (Leibson andPlisetskaya, 1969), that there is insulin-likeimmunoreactivity in lamprey serum, andthat immunoneutralization of the insulin re-


sults in decreases in liver glucose (Pliset-skaya and Leibush, 1972). Furthermore, in-sulin administration increases glycogensynthetase activity in the liver (Plisetskayaand Leibson, 1973). Several ingeneousstudies, including insulin immunoneutrali-zation, were undertaken to prove that thelong, nontrophic period in the upstream mi-gration is possible because of a chronic in-sulin insufficiency which prevents prema-ture exhaustion of carbohydrate reserves(Plisetskaya, 1975; Plisetskaya et al., 1976).

Subtotal isletectomy (either cranial orcaudal principal islets) of L. fluviatilis doesnot affect blood glucose levels but total is-letectomy results in a 5-fold increase inblood glucose (Hardisty et al., 1975); islettissue is essential for glucose homeostasisin lampreys. On the other hand, Larsen(1976) claimed that the hormone-inducedchanges in blood glucose are slow relativeto other vertebrates, the physiological roleof insulin in this process is unclear, and thatinsulin is likely important to long-termchanges in carbohydrate, lipid and proteinmetabolism. A heterologous radioimmuno-assay proved that total isletectomy (bothprincipal islets but likely not the secondaryislet tissue) causes a significant decline inserum insulin but glucose loading gives thereverse effect (Zelnik et al., 1977); thesedata provided further support for the im-portant role of insulin during the nontrophicupstream migration period in the lampreylife cycle. Glucagon-like immunoreactivitywas detected in intestinal and islet extracts(Zelnik et al., 1977) but the latter was notconsistent with earlier immunohistochemi-cal data (see above) and was likely contam-inated with intestinal tissue. Murat and Pli-setskaya (1977) then showed that mamma-lian glucagon has no effect on blood glu-cose levels; increase in glycogen synthetaseactivity in liver could be due to endogenousinsulin release.

Following the identification of somato-statin cells in islet tissue of adult P. marinus(Elliott and Youson, 1986), some tissueconcentrations of somatostatin in upstreammigrants were provided through a heterol-ogous RIA (Elliott and Youson, 1991).There were no differences in total (caudaland cranial) concentrations between early

and late migrants or between individualconcentrations of the two principal islets inboth sexes. However, there were greater so-matostatin concentrations in caudal than incranial principal islets and in the total of thetwo principal islets compared to the intes-tine. Intestinal values in upstream migrantswere less than in this organ in larvae whichmay be a consequence of the atrophy of theadult organ during the migration.

The islet organ in G. australis providedan excellent opportunity to examine the roleof the lamprey islet tissue in carbohydratemetabolism. Due to the presence of only asingle principal islet, total isletectomy waspossible in this species during its upstreammigration. The resulting immediate hyper-glycemia suggested that blood glucose ho-meostasis is likely dependent on insulinwithin the islet organ (Epple et al., 1992).The development of an homologous RIAfor P. marinus insulin (Plisetskaya, 1994)coincided with a rare opportunity to mea-sure hormones during the adult feedingphase and subsequent growth of this speciesduring a 4-month span (Youson et al.,1994a). No correlation could be found be-tween serum insulin concentratrations andanimal length, weight or condition factor(monthly or total). Serum insulin concen-tration did not differ with respect to genderor nutritional status which was based on liv-er color and time after feeding (evaluationof intestinal fullness). Some slight differ-ences existed between monthly serum in-sulin samples but these may reflect watertemperature differences in the lake at thetime of capture (Fig. 2B). There were sig-nificantly lower serum insulin concentra-tions in upstream-migrant adults comparedto feeding adults in the month of June. Thisresult was not surprising since earlier it hadbeen shown in nontrophic, upstream-mi-grant, L. fluviatilis, that insulin binding tovarious tissues was not influenced by seruminsulin levels (Leibush and Bondareva,1987). It was surprising, however, that nu-tritional 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 postmetamorphicvalues) could not be correlated with chang-ing serum insulin concentrations. Gender

192 JOHN H. YOUSON

FIG. 3. Comparison of amino acid sequences of in-sulin (A) and somatostatin (B) from the pancreatic islettissue of lamprey (Petromyzon marinus) and the hag-fish (Myxine glutinosa). Substituions of the hagfishmolecules are indicated by the letters. Data from lam-prey are that of Plisetskaya et al., 1988 (insulin) andAndrews et al., 1988 (somatostatin) and from hagfishare that of Peterson et al., 1974 (insulin) and Conlonet al., 1988a (somatostatin). A. The B chain of lam-prey insulin has a 5-amino acid extension of the N-terminal (left), a feature which is common in insulinsof all lampreys examined to date. B. Arrows indicatepotential cleavage sites of lamprey SS-37 and the box-es signify common amino acids between the two ag-nathans. Note however, that SS-14 (right) in lampreyis variant because of the T to S (both in bold) substi-tution. This variant SS-14 is found in the islet tissueof all lampreys examined to date.

differences had been noted in serum insulinconcentrations of upstream-migrant lam-preys (Plisetskaya et al., 1976; Sower et al.,1985). The lack of gender differences in se-rum insulin of the feeding animals may beexplained by their immaturity, for such isthe case with pink and coho salmon (Pli-setskaya et al., 1987). The conclusions atthe present time are that systemic levels ofinsulin (and thyroid hormones) remain rel-atively constant in adult P. marinus duringtheir adult feeding interval, they do not re-quire or utilize insulin (and thyroid hor-mones) in the manner (e.g., in body growthand metabolism) that is demonstrated forfeeding salmonids (Plisetskaya et al., 1986;Eales, 1988). The most recent data on in-sulin physiology in lampreys used isolatedhepatocytes to study the regulatory mech-anism of the binding of insulin to cellularinsulin receptors, the internalization of in-sulin-receptor complexes, and the impor-tance of temperature on the insulin recep-tors (Lappova and Leibush, 1995; Leibushand Lappova, 1995). Hormonal regulationof growth in adults of parasitic species oflampreys is an important future consider-ation.

Structure of EP peptides

Falkmer et al. (1975) deduced from com-parisons of the the amino acid compositionof insulins from L. fluviatilis and hagfish(M. glutinosa), that the primary structure oflamprey insulin is not likely to show largevariations from the insulins of other verte-brates. The first lamprey insulin to be iso-lated and its amino acid sequence disclosedwas from P. marinus (Plisetskaya et al.,1988). The insulin from this species has aB chain of 36 amino acids due to a unique,5 amino-acid extension to the N-terminus(Fig. 3A). In comparison to hagfish, thereare 17 substitutions among 52 amino acidsin the A and B chains; a similar degree ofdifference was noted between lamprey in-sulin and insulins from teleosts and mam-mals. Three somatostatins were isolatedfrom the same islet extract (Fig. 3B) withSS-34 predominating over both SS-37 andthe first variant form of SS-14 ever de-scribed (Andrews et al., 1988). A SS-34was also isolated from hagfish but it con-

tained invariant SS-14 (Figs. 3B, 4) and,among the remaining 20 amino acids ex-tending towards the N-terminal, there areonly 2 amino acids in common with lam-prey 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 sequencehomology to pig NPY than to pig PYY(Conlon et al., 1991). The intestinal extractof upstream migrants failed to yield mem-bers of the glucagon family, perhaps due tothe atrophied state of this organ (Youson,1981), but these were isolated and se-quenced from the intestine of feeding phaseP. marinus (Conlon et al., 1993). Lampreyglucagon shows only 8 amino-acid substi-


FIG. 4. Schematic presentation of one view of so-matostatin (SS) evolution among the Agnatha. A ver-tebrate which may have been ancestral to hagfishes andlampreys had a preprosomatostatin which contained,or was processed to produce, a SS-14 which was likethat seen in most other vertebrates (invariant form).Hagfishes retained this type of somatostatin in both thebrain and their enteropancreatic (EP) system. The dif-ferent families of lampreys (Petromyzontidae, Geotri-idae, Mordaciidae) diverged early in lamprey evolutionand yet two of these families show species with a var-iant SS-14 (Thr to Ser substitution) in the EP system;two species of Petromyzontidae have the invariantform in the brain. This latter tissue does not producethe larger forms of SS in at least one Petromyzontidae.Gene duplication of somatostatin must have occurredearly in lamprey evolution and resulted in tissue-spe-cific expression of different types of SS-14 and otherSSs. The situation in the brain of Geotriidae and thebrain and EP system of Mordaciidae (?) is not known.

tutions compared to human glucagonwhereas glucagon-like peptide (GLP) has16 and 15 substitutions compared to humanand salmon GLP-1, respectively.

Results of the immunohistochemistry ofthe EP systems in larvae and adults of G.australis and M. mordax (Youson and Pot-ter, 1993), using antisera against P. marinusSS-34 and insulin, suggested that there maybe differences in the structure of these pep-tides between lamprey species. Falkmer etal. (1975) had also indicated that the pri-mary structure of insulin from L. fluviatilismight not vary significantly from hagfishinsulin. However, as noted above, P. mari-nus insulin varied markedly from hagfishinsulin (Plisetskaya et al., 1988). The afore-mentioned facts, together with the facts thatlampreys have an ancient heritage amongvertebrates and that they are separated intothree families which may have diverged

early in lamprey evolution, were the stim-ulus for further peptide isolation and se-quencing among lamprey species. Thesecomparisons of the structure of EP peptidesamong lamprey species continue today.

The primary structures of insulin, glu-cagon and somatostatin from the river lam-prey (Baltic lamprey), L. fluviatilis were thenext to be provided (Conlon et al., 1995a).The primary structure of Lampetra insulinis identical to that of Petromyzon (Fig. 3A)and the former had equal affinity as pig in-sulin in binding to human insulin receptors.SS-35, with 8 substitutions and an addition-al residue compared to Petromyzon, was theonly somatostatin isolated from Lampetraislets, but the C-terminal showed the Ser forThr substitution noted in Petromyzon so-matostatins (Fig. 4). It was noteworthy thatin the brain of both P. marinus and L. flu-viatilis prosomatostatin is processed to in-variant SS-14, without the above substitu-tion (Sower et al., 1994; Conlon et al.,1995a). There seems to be tissue-specificpolygenic expression of SS-14 in holarcticlampreys. Glucagon from the intestine of L.fluviatilis is more similar to the human (5substitutions) than to the Petromyzon (6substitutions) peptide.

Extracts of the islets of G. australisyielded SS-33 (Fig. 4) with little N-terminalsimilarity to lamprey SS-34 or SS-35 or tohagfish SS-34 (Conlon et al., 1995b). How-ever, near the C-terminal the SS-14 domainhad the Thr to Ser substitution which seemsto be unique to lamprey EP somatostatin.These data support the view that the anti-SS-34 used in the immunohistochemicalstudy of G. australis islet tissue is likelyspecific for the N-terminal portion of thelarge somatostatin molecule (Youson andPotter, 1993). Nevertheless, the immunohis-tochemical results of the adult intestinecannot be so easily explained. Recently,variant SS-14, and not SS-33 as in the islet,was reported as the dominant form of so-matostatin in the intestine of adult G. aus-tralis (Wang et al., 1999a). There is a tis-sue-specific pathway for processing of pre-prosomatostatin in the EP system of G. aus-tralis, but so far, this tissue-dependent

194 JOHN H. YOUSON

processing for somatostatin has not beenextended to include the brain.

Proinsulin and an incompletely processedinsulin, but not insulin, were identified inan islet extract from G. australis, however,these data were sufficient to provide a viewof the primary structure of putative pro-cessed insulin in this species (Conlon et al.,1995b). There are 17 amino-acid substitu-tions in putative Geotria insulin comparedto holarctic lamprey insulin but the two in-sulins still share the feature of the 5-aminoacid residue extension at the N terminus ofthe B chain, a feature which is unique tolamprey insulin. The number of amino-acidsubstitutions relative to holarctic lampreyinsulin would explain the inability to im-munostain islet tissue of G. australis withantiserum against Petromyzon insulin (You-son and Potter, 1993). Moreover, the ten-dency for preproinsulin to produce mainlyproinsulin in G. australis might explain theweak staining of islets with antisera againstmammalian insulin. Conlon et al. (1995b)suggested that Geotria preproinsulin maypossess mutations at critical cleavage siteswhich result in incomplete posttranslationalprocessing of this prohormone.

The intestinal extract from adult G. aus-tralis yielded two molecular forms of glu-cagon differing in structure by 6 amino ac-ids (Wang et al., 1999a). One of these wassimilar to Lampetra glucagon and the otherto Petromyzon glucagon; each differed by2 amino acids from the holarctic counter-part which in turn differed by 6 amino acidsfrom one another. The suggestion of the du-plication of the glucagon gene early in lam-prey evolution (Wang et al., 1999a) is sup-ported by recent characterization of twoproglucagon cDNAs from P. marinus withdiffering coding potential (Irwin et al.,1999). Proglucagon I encodes the knownglucagon and GLP-1 while proglucagon IIencodes potentially GLP-II and possibly asecond glucagon. The presence of two glu-cagons and two GLP molecules in lampreyswas an earlier prediction (Plisetskaya andMommsen, 1996). Unlike most of the otherregulatory peptides, lamprey PYY is highlyconserved in the three species in which ithas been isolated and sequenced (Wang etal., 1999b).

CONCLUSION

There is documentation that the devel-opment and distribution of the EP endo-crine system in agnathans has been a sci-entific curiosity for close to one hundredand seventy-five years. The role of the pep-tides which are generated by this system incarbohydrate metabolism has been investi-gated by some of the most prominent work-ers in comparative vertebrate endocrinologywithin this past century. Most recently, re-search has focused on molecular evolutionof insulin, somatostatin, and peptides of theglucagon and pancreatic polypeptide fami-lies. Data from the molecular examinationsindicate some structural diversity of thepeptides among lamprey species within andbetween two extant families (Petromyzon-tidae and Geotriidae). Such comparisonsneed to made between these two familiesand the Mordaciidae, between further ex-amples of Petromyzontidae (the largestfamily), and between species of hagfish.Physiological evidence indicates that thereare marked differences in the metabolic re-sponses of two hagfish species to insulinadministration. Lamprey insulin has a high-er affinity to human insulin receptors thandoes pig insulin. The various developmen-tal intervals in the lamprey life cycle pro-vide an excellent opportunity to study theearly stages in the genesis of the definitivecell types of the EP system. There is a ma-jor question of the role of EP system pep-tides, and other hormones, in the immensegrowth phase during adult feeding of lam-preys.

The rationale for continued investiga-tions into the agnathan EP system well intothis new century is obvious. Hagfishes andlampreys have been successful through arather conserved evolution of at least 550and 350 million years, respectively. Extantagnathans have been ‘‘living right’’ throughchanging periods in our earth’s history.There is much to discover about the con-stitution of lampreys and hagfishes and,since the peptides of the EP system are fun-damental to their survival, this systemshould continue to be a prominent focus.

ACKNOWLEDGMENTS

The studies outlined in this paper inwhich the author was a part were supported


by grants from the Natural Sciences andEngineering Research Council of Canada.The author acknowledges the contributionsof former and present graduate students(Richard Cheung, Mark Elliott, Yung-hsiKao and Azza Al-Mahrouki) and many col-laborators and mentors in this research. Par-ticular appreciation is extended to PhilipAndrews, Michael Conlon, Aubrey Gorb-man, David Irwin, Jean Joss, John Leath-erland, Erika Plisetskaya, Ian Potter, MarkSheridan, and Stacia Sower.

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