The fish endocrine pancreas: review, new data, and future research directions in ontogeny and phylogeny.

The literature on the ontogeny and phylogeny of the endocrine pancreas of ray-finned fishes is summarized since the latest review in fish [Youson, J.H., Al-Mahrouki, A.A., 1999. Review. Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fishes. Gen. Comp. Endocrinol. 116, 303-335]. A basic description and a demonstration of the diversity of the fish islet organ is provided through new immunohistochemical data on islet tissue from a basal teleost, an osteoglossomorph, and a more derived teleost, a perciforme. Unlike the previous review, the present report provides a review and discussion of the utility of sequence data of insulin, somatostatin, and NPY- and glucagon-family peptides in phylogenetic analyses of jawed and jawless fishes. The present study also provides the first comparative analysis of sequences of preprohormones of endocrine peptides from closely related basal teleost species. Some nucleotide and deduced amino acid sequence data for preprosomatostatins (PPSS-I and/or -II) are compared for four species of bonytongues, Osteoglossomorpha, and with PPSSs of the white sucker, Catostomus commersoni, representing Cypriniformes, a more generalized teleost order. Phylogenetic analysis of deduced amino acid sequences of the PPSSs of these species and others from databases indicates good support for the monophyly of Osteoglossomorpha and some support for the present taxonomic grouping of the osteoglossomorphs examined, and also the white sucker. However, PPSS may have limited phylogenetic utility due to the relative short sequence, particularly in resolving relationships among lineages that diverged over a short period of time. Since in the few fish species examined we have just touched the surface in describing the diversity of structure of the islet organ, and likely the nature of the products of its cells, this report promotes the continued study of this organ.

Patterns of proopiomelanotropin and proopiocortin gene expression and of immunohistochemistry for gonadotropin-releasing hormones (lGnRH-I and III) during the life cycle of a nonparasitic lamprey: relationship to this adult life history type.

There are two adult life history types among lamprey species, nonparasitic and parasitic, with the former commencing the final interval of sexual maturation immediately after metamorphosis. There are no extensive studies that directly compare hormone profiles during the life cycles of nonparasitic and parasitic lamprey species, yet such data may explain differences in development, reproductive maturation, and feeding status. The present study uses immunohistochemistry to show the life cycle profiles for gonadotropin-releasing hormones (GnRH-I and -III) in the brain of the nonparasitic species, the American brook lamprey, Lampetra appendix, for comparison with the extensive, published, immunohistochemical data on these hormones in the parasitic species, the sea lamprey, Petromyzon marinus. The complete cDNAs for the two lamprey prohormones, proopiocortin (POC), and proopiomelanotropin (POM), were cloned for L. appendix and both nucleotide and deduced amino acid sequences were compared with those previously published for P. marinus. The POC and POM cDNAs for both species were used in expression studies, with Northern blotting, throughout their life cycles. Although GnRH-I and -III immunohistochemistry revealed a similar distribution of immunoreactive cells and fibers in the two species during the life cycles, a qualitative evaluation of staining intensity in L. appendix, implied early activity in the brains of metamorphosis of this species, particularly in GnRH-I. GnRH-III seems to be important in larval life and early metamorphosis in both species. A novel feature of this immunohistochemical study is the monthly observations of the distribution and relative intensity of the two GnRHs during the critical period of final sexual maturation that lead to spawning and then the spent animal. L. appendix POC and POM nucleotide sequences had 92.9 and 94.6% identity, respectively, with P. marinus POC and POM and there was an earlier increase in their expression during metamorphosis and postmetamorphic life. Since there was some correlation between the timing of metamorphic development, gonad maturation, and brain irGnRH intensity with POC and POM expression in L. appendix, it was concluded that these prohormones yield posttranslational products that likely play a substantial role in development and maturation events that lead to the nonparasitic adult life history of this species.

What is metamorphosis?

Metamorphosis (Gr. meta- "change" + morphe "form") as a biological process is generally attributed to a subset of animals: most famously insects and amphibians, but some fish and many marine invertebrates as well. We held a symposium at the 2006 Society for Integrative and Comparative Biology (SICB) annual meeting in Orlando, FL (USA) to discuss metamorphosis in a comparative context. Specifically, we considered the possibility that the term "metamorphosis" could be rightly applied to non-animals as well, including fungi, flowering plants, and some marine algae. Clearly, the answer depends upon how metamorphosis is defined. As we participants differed (sometimes quite substantially) in how we defined the term, we decided to present each of our conceptions of metamorphosis in 1 place, rather than attempting to agree on a single consensus definition. Herein we have gathered together our various definitions of metamorphosis, and offer an analysis that highlights some of the main similarities and differences among them. We present this article not only as an introduction to this symposium volume, but also as a reference tool that can be used by others interested in metamorphosis. Ultimately, we hope that this article-and the volume as a whole-will represent a springboard for further investigations into the surprisingly deep mechanistic similarities among independently evolved life cycle transitions across kingdoms.

Response by the corpuscles of Stannius to hypotensive stimuli in three divergent ray-finned fishes (Amia calva, Anguilla rostrata, and Catastomus commersoni): cardiovascular and morphological changes.

In accordance with their vital role in cardiovascular physiology () corpuscles of Stannius (CS) from two teleosts and an holostean species showed marked and consistent degranulation and exocytotic responses to hypotensive stimuli. In eels (Anguilla rostrata LeSueur) acute blood withdrawal (hypovolemic hypotension) was followed by a prompt decrease in cardiac output (CO) and dorsal aortic pressure (P(DA)), a compensatory tachycardic response and an increase in systemic vascular resistance (R(SYS)). Isovolemic hypotension induced by papaverine i.v., led to a similar, but more prolonged, decrease in P(DA) but the heart rate (HR) continued to accelerate, thereby counterbalancing the severe and persistent decrease in R(SYS). Both hypovolemic and isovolemic hypotension were followed by a significant depletion of cytoplasmic granules from eel CS even though plasma concentrations of Ca, Mg, Na(+), and K(+) were normal. In an ancient holostean fish, the bowfin, Amia calva and a generalized teleost fish, Catastomus commersoni, the number of cytoplasmic granules decreased by 39% and 54%, respectively, 120 min after the acute withdrawal of 8 ml kg bw(-1) of blood. These findings suggest that a primary role of the CS is to release cytoplasmic granules containing renin or isorenin into the blood circulation, in response to hypotension and/or hypovolemia.

The endocrine cells in the gastroenteropancreatic system of the bowfin, Amia calva L.: an immunohistochemical, ultrastructural, and immunocytochemical analysis.

The gastroenteropancreatic (GEP) endocrine system of bowfin (Amia calva) was described using light and electron microscopy and immunological methods. The islet organ (endocrine pancreas) consists of diffusely scattered, mostly small islets and isolated patches of cells among and within the exocrine acini. The islets are composed of abundant, centrally located B cells immunoreactive to bovine and lamprey insulin antisera and D cells showing a widespread distribution and specificity to somatostatin antibodies. A and F cells are present at the very periphery of the islets and are immunoreactive with antisera against glucagon (and glucagon-like peptide) and several peptides of the pancreatic polypeptide (PP)-family, respectively. The peptides of the two families usually collocates within the same peripheral islet cells and are the most common immunoreactive peptides present in the extra-islet tissue. Immunocytochemistry and fine structural observations characterised the granule morphology for B and D cells and identified two cell types with granules immunoreactive to glucagon antisera. These two putative A cells had similar granules, which were distinct from either B or D cells, but one of the cells had rod-shaped cytoplasmic inclusions within cisternae of what appeared to be rough endoplasmic reticulum. The inclusions were not immunoreactive to either insulin or glucagon antisera. Only small numbers of cells in the stomach and intestine immunoreacted to antisera against somatostatin, glucagon, and PP-family peptides. The paucity of these cells was reflected in the low concentrations of these peptides in intestinal extracts. The GEP system of bowfin is not unlike that of other actinopterygian fishes, but there are some marked differences that may reflect the antiquity of this system and/or may be a consequence of the ontogeny of this system in this species.

Proteins immunoreactive with antibody against a human leptin fragment are found in serum and tissues of the sea lamprey, Petromyzon marinus L.

An affinity-purified, polyclonal antibody raised against a peptide corresponding to amino acids 137-156 at the carboxy terminus of human leptin (16 kD) was used to search for immunoreactive protein(s) in the lamprey, Petromyzon marinus. Immunoblots of serum from different phases of the life cycle showed the presence of a 65-kD immunoreactive protein in the larvae and all stages of metamorphosis but not in feeding juvenile and upstream migrant adults. Extracts of tissues known to store fat were also examined using the same antibody. Muscle and fat column from all phases tested (larvae, stage 2 and 4 metamorphosing animals, feeding juveniles and upstream migrants) showed 100- and 50-kD immunoreactive proteins. Extracts of nephric fold, the primary site of fat storage during metamorphosis, lacked the 100-kD protein but had the 50 kD; they also had a 16 kD immunoreactive protein not found in the other tissues. The immunoreactivity of the proteins of both serum and tissue extracts was blocked by pretreatment of the antibody with the leptin-derived antigen. The results indicate that P. marinus has proteins that share at least one epitope with mammalian leptin.

Theory on the evolutionary history of lamprey metamorphosis: role of reproductive and thyroid axes.

Metamorphosis is a developmental strategy used by only a small number of extant fishes and little is known about its phylogenetic development during the evolution history of this large group of vertebrates. The present report provides a putative evolutionary history of metamorphosis in the lamprey, an extant agnathan with direct descendancy from some of the oldest known vertebrates. The study reviews recent data on the role of the thyroid gland and its hormones in metamorphosis, summarizes some recent views on the evolution of the endostyle/follicular thyroid in lampreys, and provides new data on the content of two gonadotropin-releasing hormones (GnRH-I and -III) in brain during goitrogen-stimulated, precocious metamorphosis. These new data support an earlier viewpoint of a relationship between thyroid and reproductive axes during metamorphosis. It is proposed that the earliest lampreys were paedomorphic larvae and they lived in a marine environment; as such, they resembled in many ways the larvae from which the ancient protochordates, Larvacea, are derived. The iodide-concentrating efficiency of the endostyle was a critical factor in the evolution of metamorphosis and this gland was replaced by a follicular thyroid, for postmetamorphic animals needed to store iodine following their invasion of freshwater. Larval growth and postmetamorphic reproduction in freshwater became fixtures in the lamprey life cycle; a non-parasitic adult life-history type appeared later. The presence among extant lampreys of two different adult life-history types, and examples of the lability of the timing of sexual maturation in some species, imply that there has been a complex interplay between the thyroid and reproductive axes during the evolution of metamorphosis in lampreys. This proposal is consistent with what we know of interplay of these axes in extant adult lampreys and with the long-held viewpoint that thyroid function and sexual maturation are an association with an ancient history.

Molecular cloning of preproinsulin cDNAs from several osteoglossomorphs and a cyprinid.

Several preproinsulin cDNAs were isolated and characterized from four members of the Osteoglossomorpha (an ancient teleost group); Osteoglossum bicirrhosum (arawana), Pantodon buchholzi (butterfly fish), Notopterus chitala (feather fin knife fish), Hiodon alosoides (goldeye) and Gnathonemus petersii (elephantnose). In addition, we isolated and characterized the preproinsulin cDNA from Catostomus commersoni (white sucker, as a representative of a generalized teleost). The comparative analysis of the sequences revealed conservation of the cystine residues known to be involved in the formation of the disulfide bridges, as well as residues involved in the hexamer formation, except for B-17 in the butterfly fish, the arawana and the goldeye. However, the N-terminus of the B-chain was very weakly conserved among the species studied. Residues known to be significant for maintaining receptor-binding conformation and those known to comprise the receptor-binding domain were all conserved, except for a conservative substitution at B13, aspartate substituted glutamate in the arawana, goldeye, butterfly fish and white sucker, and at B16, phenylalanine substituted tyrosine in the elephantnose. Phylogenetic analysis of the sequences revealed a monophyletic grouping of the osteoglossomorphs, and showed that they were not the most basal living teleost. Comparative sequence analysis of preproinsulins among the osteoglossomorphs was useful in assessment of intergroup relationship, relating elephantnose with the feather fin knife fish and the arawana, butterfly fish, and goldeye. This arrangement of species is consistent with relationships based on other more classical parameters, except for the goldeye which was assessed as being sister to all the osteoglossomorphs. The white sucker was grouped with the common carp and both are cyprinids.

Variable effects of goitrogens in inducing precocious metamorphosis in sea lampreys (Petromyzon marinus).

The ability of different goitrogens (anti-thyroid agents) to induce precocious metamorphosis in larval sea lampreys (Petromyzon marinus) was assessed in four separate experiments. Two of these goitrogens (propylthiouracil [PTU] and methimazole [MMI]) are inhibitors of thyroid peroxidase-catalyzed iodination, and three (potassium perchlorate [KClO(4)], potassium thiocyanate [KSCN], and sodium perchlorate [NaClO(4)]) are anionic competitors of iodide uptake. Because, theoretically, all of these goitrogens prevent thyroid hormone (TH) synthesis, we also measured their influence on serum concentrations of thyroxine and triiodothyronine. All goitrogens except PTU significantly lowered serum TH concentrations and induced metamorphosis in some larvae. The incidence of metamorphosis appeared to be correlated with these lowered TH concentrations in that KClO(4), NaClO(4), and MMI treatments resulted in the lowest serum TH concentrations and the highest incidence of metamorphosis in sea lampreys. Moreover, fewer larvae metamorphosed in the KSCN and low-KClO(4) treatment groups and their serum TH concentrations tended to be greater than the values in the aforementioned groups. MMI treatment at the concentrations used (0.087 and 0.87 mM) was toxic to 55% of the exposed sea lampreys within 6 weeks. The potassium ion administered as KCl did not alter serum TH concentrations or induce metamorphosis. On the basis of the results of these experiments, we have made the following conclusions: (i) In general, most goitrogens other than PTU can induce metamorphosis in larval sea lampreys, and this induction is coincident with a decline in serum TH concentrations. (ii) The method by which a goitrogen prevents TH synthesis is not directly relevant to the induction of metamorphosis. (iii) PTU has variable effects on TH synthesis and metamorphosis among lamprey species. (iv) Unlike in protochordates, potassium ions do not induce metamorphosis in sea lampreys and are not a factor in the stimulation of this event.

The structure and organization of lamprin genes: multiple-copy genes with alternative splicing and convergent evolution with insect structural proteins.

Lamprin is a unique structural protein which forms the extracellular matrix of several cartilaginous structures found in the lamprey. Lamprin is noncollagenous in nature but shows sequence similarities to elastins and to insect structural proteins. Here, we characterize the structure and organization of lamprin genes, demonstrating the presence of multiple similar but not identical copies of the lamprin gene in the genome of the lamprey. In at least one species of lamprey, Lampetra richardsoni, the multiple gene copies are arranged in tandem in the genome in a head-to-tail orientation. Lamprin genes from Petromyzon marinus contain either seven or eight exons, with exon 4 being alternatively spliced in all genes, resulting in a total of six different lamprin transcripts. All exon junctions are of class 1,1. An unusual feature of the lamprin gene structure is the distribution of the 3' untranslated region sequence among multiple exons. A TATA box and cap sequence have been identified in upstream sequences in close proximity to the transcription start site, but no CAAT box could be identified. Sequence and gene structure comparisons between lamprins, elastins, and insect structural proteins suggest that the regions of sequence similarity are the result of a process of convergent evolution.

An albumin-like protein in the serum of non-parasitic brook lamprey (Lampetra appendix) is restricted to preadult phases of the life cycle in contrast to the parasitic species Petromyzon marinus.

Previous work showed that the parasitic sea lamprey, Petromyzon marinus, has two different albumin-like serum proteins during the course of its life cycle. One of these, AS, is the predominant protein in the serum of larval and metamorphosing intervals but is absent in the upstream migration phase of the life cycle; the other, SDS-1, is found at its highest level in the upstream migration phase and is the predominant serum protein in this phase. The present investigation examines the nonparasitic brook lamprey Lampetra appendix for the presence of albumins. Using electrophoresis and antisera monospecific for serum proteins, it was found that L. appendix has a serum protein that is antigenically similar to the AS protein of P. marinus. This protein (LAS) makes up about 70% of the total serum protein of the Lampetra larva, is present in metamorphic stages 1,2,3,4 and 5 but is virtually absent from the remaining metamorphic stages and the adult. Furthermore, the adult of L. appendix has no serum protein that is antigenically similar to SDS-1 of the adult P. marinus; our study shows that, unlike P. marinus, there is no single protein that represents a major portion of the total serum protein content in the L. appendix adult.

Deiodination activity in extrathyroidal tissues of the atlantic hagfish, Myxine glutinosa.

We measured low substrate (<1 nM) thyroid hormone (TH) deiodination activities in liver, muscle, intestine, and brain microsomes of Atlantic hagfish fasted for 2 weeks and found extremely low thyroxine (T(4)) outer-ring deiodination (T(4)ORD) and inner-ring deiodination (T(4)IRD) as well as 3,5,3'-triiodothyronine (T(3)) IRD activities. T(3)ORD, 3',5'-triiodothyronine (rT(3)) ORD and rT(3)IRD activities were undetectable. Hagfish deiodinating pathways resembled those of teleosts in requiring a thiol cofactor (dithiothreitol, DTT) and in their inhibition by established deiodinase inhibitors and by TH analogues. However, under optimal pH and DTT conditions intestinal T(4)ORD activity exceeded that of liver about 10-fold. This contrasts with the situation in teleosts but resembles that reported recently in larval and adult lampreys, suggesting the intestine as a primary site of TH deiodination in lower craniates.

Changes in intestinal and hepatic thyroid hormone deiodination during spontaneous metamorphosis of the sea lamprey, Petromyzon marinus.

We measured microsomal low-K(m) outer-ring deiodination (ORD) and inner-ring deiodination (IRD) activities for thyroxine (T(4)) and 3, 5,3'-triiodothyronine (T(3)) in intestine and liver in nonmetamorphosing (undersized) larvae, immediately premetamorphic larvae, animals in stages 1-7 of metamorphosis, and immediately postmetamorphic sea lampreys (Petromyzon marinus). For intestine: T(4)ORD activity was relatively low in nonmetamorphosing larvae, increased in premetamorphic individuals, was highest in stages 1 and 2 and was very low during stages 3-7; T(4)IRD activity was negligible until stage 3 but increased 4.7-fold through stages 3 to 7 such that T(4)IRD activity was 14 times T(4)ORD activity at stage 6; T(3)ORD activity was undetectable; T(3)IRD activity was not measured through stages 3-7 but correlated with T(4)IRD activity at other stages. For liver: deiodination was only measured up to stage 2 and in postmetamorphic animals; in contrast to intestine, T(4)ORD activity fell to low levels at stage 2 and was low during postmetamorphosis; T(4)IRD and T(3)IRD activities were very low and uninfluenced by developmental stage; T(3)ORD activity was undetectable. We conclude that (1) deiodination activity is usually much higher in intestine than in liver, (2) intestinal ORD and IRD activities change reciprocally so that ORD predominates in early metamorphosis but IRD predominates in mid and late metamorphosis, and (3) changes in intestinal deiodination may contribute to the characteristic depression of plasma T(4) and T(3) levels during spontaneous metamorphosis. J. Exp. Zool. 286:305-312, 2000.

Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fish.

The morphology of the gastroenteropancreatic (GEP) system of fish was reviewed with the objective of providing the phylogenetic and ontogenetic development of the system in this vertebrate group, which includes agnathans and gnathostome cartilaginous, actinoptyerygian, and sarcopterygian fish. Particular emphasis is placed on the fish homolog of the endocrine pancreas of other vertebrates, which is referred to as the islet organ. The one-hormone islet organ (B cells) of larval lampreys is the most basic pattern seen among a free-living vertebrate, with the two-hormone islet organ (B and D cells) of hagfish and the three-hormone islet organ (B, D, and F cells) of adult lampreys implying a phylogenetic trend toward the classic four-hormone islet tissue (B, D, F, and A cells) in most other fish. An earlier stage in the development of this phylogenetic sequence in vertebrates may have been the restriction of islet-type hormones to the alimentary canal, like that seen in protochordates. The relationship of the islet organ to exocrine pancreatic tissue, or its equivalent, is variable among bony, cartilaginous, and agnathan fishes and is likely a manifestation of the early divergence of these piscine groups. Variations in pancreatic morphology between individuals of subgroups within both the lamprey and chondrichthyan taxa are consistent with their evolutionary distance. A comparison of the distribution and degree of concentration of the components of the islet organ among teleosts indicates a diffuse distribution of relatively small islets in the generalized euteleosts and the tendency for the concentration into Brockmann bodies of large (principal) islets (with or without secondary islets) in the more derived forms. The holostean actinopterygians (Amiiformes and Semiontiformes) share with the basal teleosts (osteoglossomorphs, elopomorphs) the diffuse arrangement of the components of the islet organ that is seen in generalized euteleosts. Since principal islets are also present in adult lampreys the question arises whether principal islets are a derived or a generalized feature among teleosts. There is a paucity of studies on the ontogeny of the GEP system in fish but it has been noted that the timing of the appearance of the islet cell types parallels the time that they appear during phylogeny; the theory of recapitulation has been revisited. It is stressed that the lamprey life cycle provides a good opportunity for studying the development of the GEP system. There are now several markers of cell differentiation in the mammalian endocrine pancreas which would be useful for investigating the development of the islet organ and cells of the remaining GEP system in fish.

Ultrastructure and immunocytochemistry of the islet organ of osteoglossomorpha (Teleostei).

Both routine electron microscopy and immunocytochemistry with protein A-gold were used to identify the cell types within the islet organs of four species of teleosts (Osteoglossum bicirrhosum, Pantodon buchholzi, Notopterus chitala, and Gnathonemus petersii) within Osteoglossomorpha, a subdivision with an ancient lineage. Four primary endocrine cell types, A, B, D, and F, were identified within the islets of the four species examined. The B- and D-cells were located mainly in the central core of the islet in the four species. In general, the A-cells were located at the islet periphery in all of the four species but in P. buchholzi and N. chitala they were also differently distributed toward the islet core. F-cells were present only at the islet periphery. Granules of B-cells in three species had a relatively homogeneous shape of the matrix core, but in O. bicirrhosum, the shape varied greatly. Variation in matrix shape of B-cell granules may indicate a different conformation of insulin molecules among at least some species of osteoglossomorphs, and this observation may have some taxonomic significance. Two somatostatin-containing (SST) D-cell types (D1 and DX) with granules of different shape were observed in all four species of osteoglossomorphs. The granules of the two D-cells immunostained either with anti-SST-25 and anti-SST-14 (D1-cells) or with anti-SST-34 (DX-cells). Immunocytochemistry confirmed that A-cells, containing glucagon-family peptides, and F-cells, containing peptides of the pancreatic polypeptide family, had granules of different shape. The cells of the islet organs of these osteoglossomorphs are more similar to those in more derived teleosts than they are to those of nonteleost actinopterygians.

Lamprey proglucagon and the origin of glucagon-like peptides.

We characterized two proglucagon cDNAs from the intestine of the sea lamprey Petromyzon marinus. As in other vertebrates, sea lamprey proglucagon genes encode three glucagon-like sequences, glucagon, and glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). This observation indicates that all three glucagon-like sequences encoded by the proglucagon gene originated prior to the divergence of jawed and jawless vertebrates. Estimates of the rates of evolution for the glucagon-like sequences suggest that glucagon originated first, about 1 billion years ago, while GLP-1 and GLP-2 diverged from each other about 700 MYA. The two sea lamprey intestinal proglucagon cDNAs have differing coding potential. Proglucagon I cDNA encodes the previously characterized glucagon and the glucagon-like peptide GLP-1, while proglucagon II cDNA encodes a predicted GLP-2 and, possibly, a glucagon. The existence of two proglucagon cDNAs which differ with regard to their potential to encode glucagon-like peptides suggests that the lamprey may use differential gene expression as a third mechanism, in addition to alternative proteolytic processing and mRNA splicing, to regulate the production of proglucagon-derived peptides.

Effects of insulin on lipid metabolism of larvae and metamorphosing landlocked sea lamprey, Petromyzon marinus.

This study was designed to examine the role of insulin (INS) in regulating changes in lipid metabolism of larval and metamorphosing landlocked lamprey, Petromyzon marinus. Larvae and stage 6 metamorphosing individuals were injected intraperitoneally once per day for 2 days with either saline (0.6%), bovine INS (100 ng/g body weight), or alloxan (0.2 mg/g body weight). Insulin administration resulted in depressed plasma fatty acid (FA) levels, whereas alloxan injection elevated plasma FA levels at both life cycle intervals. In larvae, INS-induced hypolipidemia was attended by increased lipid concentration in kidney and muscle, reduced rates of lipolysis in kidney, liver, and muscle (as indicated by decreased triacylglycerol lipase activity), and, to a lesser extent, by higher rates of lipogenesis in kidney and muscle (as evidenced by higher acetyl-CoA carboxylase and/or diacylglycerol acyltransferase activities). In general, the effects of alloxan were opposite of those of INS. The alloxan-induced increase in plasma FA was supported by an enhanced rate of lipolysis in the kidney, a relatively lower rate of fatty acid synthesis in kidney, liver, and muscle, and a relatively lower renal rate of TG synthesis. In stage 6 metamorphosing lamprey, the INS-induced decline in plasma FA was attended by reduced renal and hepatic rates of lipolysis and by enhanced lipogenesis, as indicated by increased renal and hepatic rates of de novo fatty acid synthesis and hepatic and muscular rates of TG synthesis. In contrast, the increase in plasma FA induced by alloxan in stage 6 animals was supported by reduced TG synthesis in liver. Immunocytochemistry revealed that alloxan was not cytotoxic to pancreatic beta cells, suggesting that the effects of alloxan were extrapancreatic in the time frame of our study. Because insulin-induced lipogenesis and antilipolysis is similar to the pattern of lipid metabolism (phase I) displayed by lamprey during their spontaneous metamorphosis, INS may play a role, possibly in concert with other factors, in coordinating metamorphosis-associated changes in lipid metabolism.

Study of the relationship between thyroid hormones and lipid metabolism during KClO4-induced metamorphosis of landlocked lamprey, Petromyzon marinus.

This study examines the role of thyroid hormones (TH) (thyroxine and triiodothyronine) in regulating lipid metabolism of landlocked larval sea lampreys, Petromyzon marinus. Larvae were treated with either thyroxine (0.5 or 1 mg l(-1) water) or triiodothyronine (0.25 or 1 mg l(-1) water) in the presence or absence of the goitrogen, potassium perchlorate (KClO4) (0.05% w/v), for 4, 8, and 16 weeks. Treatment with KClO4 alone, which induced metamorphosis after 8 weeks and lowered plasma TH levels, reduced hepatic and renal total lipid content after 8 weeks of treatment. KClO4-induced lipid depletion after the 8-week treatment was supported by an increased rate of hepatic lipolysis, as indicated by increased triacylglycerol lipase activity. Furthermore, reduced lipogenesis in the liver was indicated by decreased hepatic acetyl-CoA carboxylase and diacylglycerol acyltransferase (DGAT) activities, and by decreased renal DGAT activity following 8 weeks of KClO4 treatment. Treatment of larvae for 4 weeks with TH alone resulted in either no change or a slight increase of lipid in the liver and kidney. TH treatments in combination with KClO4 failed to induce metamorphosis and, after up to 8 weeks, several TH treatments blocked changes in lipid content and enzyme activity associated with KClO4-induced metamorphosis. These experimental results suggest that TH deficiency during metamorphosis may promote lipid catabolism, while the presence of TH tends to protect/promote lipid reserves, perhaps favoring the larval condition. The actions of TH and KClO4 on metamorphosis-associated lipid metabolism in sea lampreys may be direct, permissive, and/or indirect via other factors.

Molecular evolution of peptide tyrosine--tyrosine: primary structure of PYY from the lampreys Geotria australis and Lampetra fluviatilis, bichir, python and desert tortoise.

Peptide tyrosine-tyrosine (PYY) has been isolated from the intestines of two species of reptile, the desert tortoise Gopherus agassizii (Testudines) and the Burmese python Python molurus (Squamata), from the primitive Actinopterygian fish, the bichir Polypterus senegalis (Polypteriformes) and from two agnathans, the Southern-hemisphere lamprey Geotria australis (Geotriidae) and the holarctic lamprey Lampetra fluviatilis (Petromyzontidae). The primary structure of bichir PYY is identical to the proposed ancestral sequence of gnathostome PYY (YPPKPENPGE10/DAPPEELAKY20/YSALR HYINL30/ITRQRY). Tortoise and python PYY differ by six and seven residues, respectively, from the ancestral sequence consistent with the traditional view that the Testudines represent an earlier divergence from the primitive reptilian stock than the Squamates. The current views of agnathan phylogeny favor the hypothesis that the Southern-hemisphere lampreys and the holarctic lampreys arose from a common ancestral stock but their divergence is of a relatively ancient (pre-Tertiary) origin. The Geotria PYY-related peptide shows only two amino acid substitutions (Pro10-->Gln and Leu22-->Ser) compared with PYY from the holarctic lamprey Petromyzon marinus. This result was unexpected as Petromyzon PYY differs from Lampetra PYY deduced from the nucleotide sequence of a cDNA (Söderberg et al. J. Neurosci. Res. 1994;37:633-640) by 10 residues. However, a re-examination of an extract of Lampetra intestine revealed the presence of a PYY that differed in primary structure from Petromyzon PYY by only one amino acid residue (Pro10-->Ser). This result suggests that the structure of PYY has been strongly conserved during the evolution of Agnatha and that at least two genes encoding PYY-related peptides are expressed in Lampetra tissues.

Multiple forms of glucagon and somatostatin isolated from the intestine of the southern-hemisphere lamprey Geotria australis.

Current views on Agnathan phylogeny favor the hypothesis that the genera of holarctic lampreys belong to a single family (Petromyzontidae) and form an interrelated progression in which Petromyzon is near to Ichthomyzon at the base of the phylogenetic tree and Lampetra is the most derived. A stock similar to that of contemporary Ichthomyzon is considered to have given rise to the southern hemisphere lamprey Geotria australis, the sole member of the Geotriidae. In the present study, two molecular forms of glucagon were isolated from an extract of G. australis intestine that differed in structure by six amino acid residues. One form shows two amino acid substitutions (Leu14 --> Met and Ala29 --> Ser) compared with the single molecular form of glucagon isolated from the sea lamprey Petromyzon marinus and the second form shows three substitutions (Asp15 --> Glu, Ser16 --> Ala, Ile24 --> Thr) compared with the single glucagon isolated from the river lamprey Lampetra fluviatilis. As Petromyzon and Lampetra glucagons differ by six amino acid residues, the data suggest that a duplication of the glucagon gene occurred prior to or early in lamprey evolution. Although both genes are strongly expressed in G. australis, the expression of one gene predominates in P. marinus while that of the other gene predominates in L. fluviatilis. Previous work has shown that, in the islet organ of G. australis, preprosomatostatin is processed almost exclusively to somatostatin-33. However, the present study demonstrates that somatostatin-14 is the major molecular form in G. australis intestine with somatostatin-33 present only as a minor component. This result demonstrates a tissue-dependent pathway of posttranslational processing of preprosomatostatin in the Geotria enteropancreatic system.