Glucagon and Glucagon-like Peptides in Fishes

Glucagon and Glucagon-like Peptides in Fishes Erika M. Pbetskaya* and Thomas P. Mommsent *School of Fisheries, University of Washington, Seattle, Washington 98195 and

?Department of Biochemistry and Microbiology, University of Victoria, Victoria, B. C. Canada V8W 3P6

Glucagon and glucagon-like peptides (GLPs) are coencoded in the vertebrate proglucagon gene. Large differences exist between fishes and other vertebrates in gene structure, peptide expression, peptide chemistry, and function of the hormones produced. Here we review selected aspects of glucagon and glucagon-like peptides in vertebrates with special focus on the contributions made by analysis of piscine systems. Our topics range from the history of discovery to gene structure and expression, through primary structures and regulation of plasma concentrations to physiological effects and message transduction. In fishes, the pancreas synthesizes glucagon and GLP-1, while the intestine may contribute oxyntomodulin, glucagon, GLP-1, and GLPB. The pancreatic gene is short and lacks the sequence for GLP-2. GLP-1, which is produced exclusively in its biologically active form, is a potent metabolic hormone involved in regulation of liver glycogenolysis and gluconeogenesis. The responsiveness of isolated hepatocytes to glucagon is limited to high concentrations, while physiological concentrations of GLP-1 effectively regulate hepatic metabolism. Plasma concentrations of GLP-1 are higher than those of glucagon, and liver is identified as the major site of removal of both hormones from fish plasma. Ultimately, GLP-1 and glucagon exert effects on glucose metabolism that directly and indirectly oppose several key actions of insulin. Both glucagon and GLP-1 show very weak insulinotropic activity, if any, when tested on fish pancreas. Intracellular message transduction for glucagon, especially at slightly supraphysiological concentrations, involves CAMPand protein kinase A, while pathways for GLP are largely unknown and may involve a multitude of messengers, including CAMP. In spite of fundamental differences in GLP-1 function between fishes and mammals, fish GLP-1 is as powerful an insulinotropin for mammalian B-cells as mammalian GLP-1 is a metabolic hormone if tested on piscine liver. Internarional Review of Cyrology, Vol. 168

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E. M. PUSETSKAYA AND T. P. MOMMSEN

KEY WORDS: Fishes, Brockmann body, Glucagon, Glucagon-like peptides, Metabolic regulation, lnsulinotropic action, Receptors, Message transduction.

1. Introduction As major players directing metabolic traffic toward catabolism, glucagon and glucagon-related peptides have attracted much attention by physiologists, biochemists, and, in recent years, molecular biologists alike. As a result, substantial knowledge has been gained at least on glucagon-the best-known member of a large superfamily of peptides. This superfamily has grown steadily and now includes secretin, vasoactive intestinal peptide (VIP); gastric inhibitory peptide (GIP, also called glucose-dependent insulinotropic peptide), growth hormone-releasing hormone (GHRH); parathyroid hormone (PTH); peptide histidine methionine, helospectine, helodermin, exendin, pituitary adenylyl cyclase-activating polypeptides (PACAP), PACAP-related peptide and, of course, glucagon-related peptides derived from posttranslational processing of the proglucagon gene. Numerous articles have been published covering various aspects of gene structure, expression, chemistry and functions of glucagon in mammalian systems. In addition, the morphology and physiology of glucagon-producing cells in the endocrine pancreas, stomach and intestine of mammals and birds have been reviewed.’ In contrast, other vertebrates have been largely ignored. Mentioning the high degree of conservation of proglucagon genes and glucagon sequences in the course of evolution, Foa and FOB (1991) nevertheless commented that they deliberately excluded publications “dealing with phylogenetic, evolutionary and comparative studies.” Thus we are left with only two sources dealing with nonmammalian and nonavian vertebrates: a review by Falkmer and Van Noorden (1983), written more than 10 years ago, covering ontogeny and phylogeny of the glucagon cells, while other valuable information can be retrieved from the book by Epple and Brinn (1987) with its emphasis on comparative physiology of the endocrine pancreas and related hormones. Since our own laboratories have been involved in studies of pancreatic hormones and their physiological functions in fish for a long time, we have in the past highlighted varying aspects of research conducted in this field (Plisetskaya, l975,1989,1990a,b,c; Mommsen and Moon, 1990,1994;Plisetskaya and Duguay, 1993;Mommsen and Plisetskaya, l991,1993a,b; Duguay and Mommsen, 1994). In this review, we intend to present a more integrated

’ (FOB, 1985; FOBand FOB, 1991; Lefkbvre, 1995)

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picture of the involvement of glucagon and glucagon-like peptide-1 in piscine physiology and intermediary metabolism. Although a more detailed discussion on fish endocrine pancreas will follow in Section II,C, it is important to point out from the onset why fish always have been, and continue to be, highly suited and valued experimental models for studies of pancreatic hormones. The major reason is the unique arrangement of the pancreas in many teleostean fishes, namely, an almost complete separation of endocrine from exocrine tissues. The largest accumulation of endocrine tissue in the fish pancreas, surrounded by only a thin rim of exocrine tissue, is called “the principal islet.” Several such islets form the so-called Brockmann body, named after Heinrich Brockmann, who first discovered this structure in sculpin (Cottus scorpius) and cod (Gadus cullarius). In his doctoral dissertation of 1846,Brockmann describes a little “corpus glandulosum” without an external duct, containing some milky substance. Brockmann bodies are discernible by the naked eye as whitish or pinkish oval or round structures. These are either close to, or in the vicinity of, the gall bladder duct (Fig. 1). Such a unique anatomical arrangement greatly facilitated isolation of fish pancreatic hormones and ensured a high yield of peptides of interest. Contamination of preparations with exocrine pancreatic tissue and its inherent high concentration of powerful hydrolytic enzymes is minimal compared with mammalian systems. The anatomical and biochemical characteristics of the piscine endocrine pancreas also simplified the design of in situ and in vitro physiological experiments on perfused, dispersed or cultured pancreatic endocrine cells, including the so-called A-cells which produce pancreatic glucagon and glucagon-like peptide 1 (see Sections II,B,1,2 and IV,A,B). It also has rendered assemblages of relatively pure endocrine cells of interest for xenografts (Wright et al., 1992; Morsiani et al., 1995).

FIG. 1 Schematic representation of the Brockmann body of coho salmon (Oncorhynchus

kisutch). An incision was made from the right side. L, liver; GB, gall bladder; PC, pyloric caecum; I, intestine; OV, ovary; arrow, Brockmann body.

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However, with specific regard to glucagon, the pancreatic A-cells are not the only cells expressing and processing the glucagon gene. In recent years, an impressive body of literature has accumulated dealing with the many diverse aspects of the so-called enteroglucagon, encoded, processed, and released from the L-cells located along the intestine (Lund et al., 1993; Holst and (drskov, 1994). Enteroglucagon appears to be also present in fishes, since the peptide has been successfully isolated from the intestine of an elasmobranch (Conlon et aZ., 1994) and a lamprey (Conlon et al., 1993a). Unfortunately, much less attention has been devoted to either its gene(s) or processing in fishes. The same discouragingstate of affairs seems to apply to other glucagon-related peptides expressed in fish intestine (see Section 11). Although we have attempted to keep our focus sternly on the situation in fishes, throughout this review we have to refer to mammalian models. We do this deliberately, not only to point out similarities and differences, but also to delineate an integrated picture of trends in the evolution, biochemistry, and regulation of glucagon and some of its related peptides.

II. Glucagon Family Peptides

A. Discovery of Glucagon It is generally believed that glucagon, the second important pancreatic hormone, was discovered much later than insulin. Actually, this assumption is inaccurate since these two “active principles” were discovered almost simultaneously, although it did not become clear immediately that researchers were dealing with a biologically active substance different from insulin, rather than with some contamination of pancreatic extracts. From their early experiments, Banting and Best seemed to have been aware that injection of pancreatic extracts into rabbits caused, in some cases, a transient hyperglycemia followed by a prolonged drop in blood sugar (C. H. Best, Letter to P. P. FOB, c$ FOB, 1985). Confirming this observation, Collip (1923) guessed that this hyperglycemic effect was dependent on the method used for the extraction of the major “active principle,” insulin. However, the fact that his extracts were prepared from a mollusk, Mya sp., makes his results less convincing for a contemporary reader. In late December of 1922, Murlin and collaborators (Murlin et al., 1923; Kimball and Murlin, 1923),testing extracts of mammalian pancreas on dogs and rabbits, correctly concluded that the clear, albeit transient, hyperglycemic effect even if caused by some “impurity” in pancreatic preparations of insulin, was independent of it. The authors coined the term “glucagon” for this hyperglyce-

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mic substance or called it “glucose release stimulating principle.” In the following year, Macleod and Orr (1924) reported that after injection of insulin preparations, “A slight rise in blood glucose occurred in the first 10 min in all animals except those that had been rendered aglycogenic by epinephrine. . . .” The astuteness of Kimball and Murlin’s conclusion was again confirmed 4 years later when Abel and co-workers (1927) prepared the first pure, crystalline insulin: this preparation was devoid of any hyperglycemic effect. Nevertheless, the term “glucagon” did not enter general usage until much later. At the time, glucagon was still referred to as “hyperglycemic factor” (see Burger and Brandt, 1935). Since this “factor” persisted in the pancreas of alloxan-treated animals (i.e., after the destruction of B-cells), pancreatic a (A)-cells and similar argentophile cells in the upper two-thirds of the gastric mucosa of the dog were correctly suggested as a place of glucagon’s origin (Sutherland and de Duve, 1948). Several years passed before Staub and colleagues (1953) prepared the first pure crystalline glucagon (from pig), and 4 years later Bromer and colleagues (1957) reported its primary amino acid structure. It took much less time to accumulate information on some key glucagonrelated peptides. In particular, the history of two new members of the glucagon family, glucagon-like peptides (GLPs) 1 and 2, which are coencoded in the proglucagon gene, was very different from that of glucagon. As is often the case today, the primary structures of these peptides were deduced from cDNA sequences before any physiological function for them was discovered (see Sections II,B,1,2 and IV). In fact, 10 years after its detection and sequencing, the physiologicalroles of GLP-2 still remain enigmatic. Although fish glucagon entered into the research arena relatively recently, fish and their Brockmann bodies have made significant contributions to our knowledge of glucagon and other pancreatic peptides. The first piscine (tuna, Thunnus germo, and anglerfish, Lophius piscatorius) hyperglycemic factors were discovered concurrently by Mialhe (1952) and Audy and Kerly (1952). Planas and Lluch (1956) and Lluch and Planas (1956) confirmed the hyperglycemic action of extracts of the Brockmann bodies from tuna (Thunnus thymus) and linked this action with certainty to the presence of glucagon. During the early 1970s, the first piscine (Lophius americanus) proglucagon was isolated and partially characterized (Trakatellis et al., 1973) and the exceptionally large Brockmann bodies of anglerfish became the organ of choice for studies of glucagon biosynthesis and a pivotal model for the analysis of the processing of peptide hormones via prohormones (Noe and Bauer, 1971, 1973). In addition, Brockmann bodies had served as an excellent model for investigating the metabolism of endocrine pancreas (Hellman and Larsson, 1961; Humbel and Renold, 1963). In the 1980s,

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Lophius sp. was used for studies of the structure of the proglucagon gene (Lund et ul., 1981,1982,1983). In the same period, several techniques were established for the perfusion of isolated Brockmann bodies from different species and these techniques made significant contributions toward an analysis of hormone release (Ronner and Scarpa, 1987). The first native GLP-1 to be isolated from any vertebrate came from Brockmann bodies of the channel catfish, Zctulurus punctutus (Andrews and Ronner, 1985). A detailed account of the structures and biological activities of piscine GLPs compared with their mammalian counterparts is presented in Sections II,C and IV. 6. Gene Structure and Expression

Like many other biologically active peptides, glucagon and related peptides are coencoded in, and subsequently proteolytically derived from, a larger precursor molecule. In humans, the proglucagon gene contains six exons separated by five introns. Coencoded in the mammalian proglucagon, starting from its N-terminal, are glicentin-related polypeptide (GRPP), glicentin, glucagon (corresponding to amino acid residues 33-61), oxyntomodulin (a C-terminally extended glucagon with a distinct function, residues 33-70), and two glucagon-like peptides (residues 72-108 and 126-159). The two glucagon-like peptides (GLPs) are separated by a 16-residue intervening peptide. Glucagon and oxyntomodulin are encoded by exon 3, whereas GLP-1 and GLP-2 are covered by exons 4 and 5, respectively (White and Saunders, 1986). Using chromatographic analysis of gene products and specificradioimmunoassays, George and co-workers (1985) noted that the human pancreas processes preproglucagon into small amounts of N-terminally extended GLP-1, with the major product being a large peptide comprising GLP-1, GLP-2, and the intervening peptide (Patzelt and Schiltz, 1984), as well as smaller amounts of GLP-2. This observation led the authors to postulate that there is additional processing or degradation of GLP-2, resulting in a form of the peptide that does not cross-react with antibodies raised against the full-length peptide. 1. Pancreas

In one key year, 1983, the primary structures of anglerfish, hamster, ox, human, and rat proglucagons were deduced from cDNA sequences (reviewed by Plisetskaya et al., 1986). In all four mammalian species, a single gene codes for glucagon plus the two distinct glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). These peptides show strong sequence conservation

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and most probably arose from two independent duplications of an ancestral glucagon gene (Lopez et al., 1984). For the human peptides, sequence conservation between the 31-residue GLP-1 and glucagon is 48%, with 14 of 29 positions being identical (Tables I and II), while the 34-residue GLP2 reveals 19 substitutions compared with glucagon (10 conserved) and the same number compared with GLP-1 (11 conserved). In contrast, the Brockmann body of the anglerfish, L. americanus, expresses two nonallelic proglucagon genes coding for two proglucagons (proglucagon I and 11). Each of these proglucagons, in turn, contains the sequence of glucagon and one GLP. Interestingly, sequences corresponding to oxyntomodulin (i.e. a C-terminally extended glucagon) and as discussed in detail below, to GLP-2, are absent from the primary transcript in the endocrine pancreas of fishes. The single expressed GLP is largely homologous to mammalian GLP-1 (Table 11). Besides the anglerfish, a number of unrelated fishes, such as alligator gar, paddlefish, daddy sculpin, American eel, and flounder possess two proglucagon genes. However, it should be pointed out that to date at least as many fish species have been found to express only a single proglucagon gene (Table 1) or, in cases where peptide analyses rather than molecular techniques were used, only one glucagon could be isolated with ease. The general structures of piscine and mammalian proglucagon genes and their main products are presented in Fig. 2. In the anglerfish, a single mRNA with 650 bases codes for glucagon-I, whereas two other transcripts of 630 and 670 bases code for glucagon I1 (Fig. 2) (Lund et al., 1983). It is not known whether the two latter transcripts belong to two different genes, are products of differential splicing of the mRNA, or reflect different degrees of polyadenylation of the 3’ end of the mRNAs (Duguay and Mommsen, 1994). At around 650 bases, the proglucagon transcripts from the fish pancreas are much smaller than their mammalian counterparts, which contain approximately 1300 bases. This difference occurs largely because fish pancreas cDNAs for proglucagon genes I and I1 contain stop codons after the sequence of a single GLP. Thus, the entire sequence of GLP-2 and the region coding for the peptide between GLP-1 and GLP-2 are absent. This particular difference between fish and mammals is discussed in more detail elsewhere (Duguay and Mommsen, 1994). In the pancreata of all cartilaginous or bony fishes whose proglucagon genes have been analyzed, the peptide corresponding to mammalian GLP2 was absent. Incidentally, the same observation holds for avian pancreas (Hasegawa et al., 1990), if the situation in the chicken can be extrapolated to other birds. In contrast to fishes and the chicken, both GLP-1 and GLP2 were successfully located and isolated from the pancreas of the bullfrog,

TABLE I Primary Structures of Glucagon in Fishes Compared with Selected Other Vertebrates

Group

Sequence 1

Cyclostomes (lampreys) Petromyzon Lampetra Elasmobranchs Dogfish Dogfish" p Dogfish Ray Ratfish Elephantfish

Source

30

20

10

HSEGT HSQGS

FTSDY FTSDY

SKYLE SKYLD

NKQAK SKQAK

DFVRW DFVIW

LMNA LMNT

Intestine Intestine

HSEGT HSEGT HSEGT HSEGT HTDGI HSEGT

FTSDY FTSDY FTSDY FTSDY FSSDY FSSDY

SKYMD SKYMD SKYMD SKYLD SKYLD SKYLD

NRRAK NRRAK NRRAK NRRAK NRRTK SRRAK

DFVQW DFVQW DFVQW DFVQW DFVQW DFVQW

LMST LMSTK RNG LMNT LMNT LLSTK RNGAN T LMST

Pancreas Pancreas Intestine Pancreas Pancreas Pancreas

Actinopterygians Bowfin Gar Paddlefish I Paddlefish I1

HSQGT HSQGT HSQGM HSQGM

FTNDY FTNDY FTNDY FTNDY

SKYQD SKYLD SKYLE SKYLE

TRRAQ TRRAQ EKRAK EKSAK

DFVQW DFVQW EFVEW EFVEW

LMST LMST LKNGK S LKNGK S

Pancreas Pancreas Pancreas Pancreas

Teleosts Anglerfish I Anglerfish I1 Catfish Coho salmon Rainbow trout I Rainbow trout I1

HSEGT HSEGT HSEGT HSEGT HSEGT QSEGT

FSNDY FSNDY FSNDY FSNDY FSNDY FSNYY

SKYLE SKYLE SKYLE SKYQE SKYQE SKYQE

DRKAQ TRRAQ TRRAQ ERMAQ ERMAQ ERMAR

EFVRW DFVQW DFVQW DFVQW DFVQW DFVQW

LMNN LKNS LMNS LMNS LMNS LMNS

cDNA cDNA Pancreas Pancreas cDNA cDNA

4

Eel I Eel I1 Flounder Sculpin I sculpin I1 Tuna

HSQGT HSQGT HSEGT HSEGT HSEGT HSEGT

FTNDY FlWDY FSNDY FSNDY FSNDY FSNDY

SKYLE SKYQE SKYLE SKYLE SKYLE SKYLE

TRRAQ MKQAQ TRRAQ DRKAQ TRRAQ TRRAQ

DFLHW DFVQW DFVQW DFVQW DFVQW DFVQW

LMNS LMNSK RNGSS LKNS LMNS LKNS LKNS

Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas

Amphibians Bullfrog Salamander

HSQGT HSQGT

FTSDY FTSDY

SKYLD SKYLD

SRRAQ NRRAQ

DFVQW DFIQW

LMST LMST

Pancreas Pancreas

Mammals Human

HSQGT

FTSDY

SKYLD

SRRAQ

DFVQW

LMNT

Pancreas

SE T H--GTQ M

TS F--DY SN

LD SKY-QE

RR Q #--AKQ K

D QW -FV-E ET

MNT L--KSS

(12 residues of 29)

Invariant or one major alternate Most abundant Invariant co Alternate A

Different post-translational processing of the same gene. References: Marine lamprey (Petromyzon marinus)(Conlon et al., 1993a); river lamprey (LampetraJuviatilis) (Conlon et al., 1995) dogfish (Scyliorhinus canicula) (Conlon et al., 1994); dogfish gut peptide (Conlon et al., 1987d); ray (Torpedo marmorata) (Conlon and Thim, 1985); ratfish (Hydrolagus colliei) (Conlon et al., 1987a); elephantfish (Callorhynchus milii) (Berks e l aL, 1989); bowfin (Amia calva) (Conlon et al., 1993b); alligator gar (Lepisosteus spatula) (Pollock et af., 1988a); paddlefish (Polyodon spathula) (Raufman et al., 1992); anglerfish (Lophius americanus) I (Lund et al., 1982); I1 (Lund et al., 1983), channel catfish (Ictalurus punctatus) (Andrews and Ronner, 1985); coho salmon (Oncorhynchuskisutch) (Plisetskaya et al., 1986); rainbow trout (0.mykiss) (Irwin and Wong, 1995); European eel (Anguilla anguilla) (Conlon et al., 1988); flounder (PlatichthysJesus) (Conlon et al., 1987b); daddy sculpin (Cottus scorpius) I (Cutfield and Cutfield, 1993), I1 (Conlon et al., 1987~); bigeye tuna (Thunnus obesus) (Navarro et al., 1991);bullfrog (Rana catesbeiana) (Pollock et al., 1988b); salamander (Amphiuma tridactylum) (Cavanaugh et al., 1996); # Amino acid residue variable (see text).

TABLE II Primary Structures of Glucagon-like Peptide 1 in Fishes Compared with Selected Other Vertebratess

Group

Sequence 1

Cyclostomes Lamprey Elasmobranchs Dogfish Ratfish Ratfishb Actinopterygians Bowfin Gar Paddlefish Teleosts Anglerfish I Anglerfish I1 Catfish Coho salmon

10

Source

20

30

HADGT

ETNDM

TSYLD

AKAAR

DFVSW

LARSD KS

Intestine

HAEGT HADGI HADGI

YTSDV YTSDV YTSDV

DSLSD ASLTD ASLTD

YFKAK YLKSK YLKSK

RFVDS RFVES RFVES

LKSY LSNYN RKQND LSNYN RKQND RRM

Pancreas Pancreas Pancreas

YADAP HADGT HADGT

YISDV YTSDV YTSDA

YSYLQ SSYLQ SSFLQ

DQVAK DQAAK EQAAR

K---W' KFVTW DFISW

LKSGQ DRRE LKQGQ DRRE LKKGQ

Pancreas Pancreas Pancreas

HADGT HADGT HADGT HADGT

YTSDV FTSDV YTSDV YTSDV

SSYLQ SSYLK SSYLQ STYLQ

DQAAK DQAIK DQAAK DQAAK

DFVSW DFVDR DFITW DFVSW

LKAGR LKAGQ LKSGQ LKSGR

cDNA cDNA Pancreas Pancreas

GRRE VRRE PKPE A

Rainbow trout I Rainbow trout I1 American eeld European eeld Sculpin

A

HADGT HADGT HAEGT HAEGT HADGT

YTSDV YTSDV YTSDV YTSDV FTSDV

STYLQ STYLQ SSYLQ SSYLQ SSYLN

DQAAK DQAAK DQAAK DQAAK DQA I K

DFVSW DFVSW EFVSW EFVSW DFVAK

LKSGR A LKSGP A LKTGR LKTGR LKSGK V

cDNA cDNA Pancreas Pancreas Pancreas

Amphibians Bullfrog Salamander

HADGT HADGT

FTSDM LTSDI

SSYLE SSFLE

E KAAK KQATK

EFVDW EFIAW

LIKGR PK LVSGR G(RRQ)

Pancreas Pancreas

Mammals Human

HAEGT

FTSDV

SSYLE

GQAAK

EFIAW

LVKGR G

Intestine

a For species and references see Table I, except: ratfish (Hydrolagus colliei) (Conlon et al., 1989); American eel (Anguilla rostrata); and European eel (A. anguilla) (Conlon et al., 1991). Same gene, different post-translational processing. '--- Deletions. Carboxyl-terminal is arginine-amide.

198

E. M. PLISETSKAYA AND T. P. MOMMSEN

Rana catesbeiana (Pollock et al., 1988b) and a salamander, Arnphiurna tridactylurn (Cavanaugh et al., 1996), setting up room for some interesting speculation on the specific evolution and function of pancreatic GLP-2 in the different groups of vertebrates. Although channel catfish GLP-1 was the first native GLP isolated from any vertebrate animal (Andrews and Ronner, 1985), attempts to isolate piscine peptides corresponding to GLP2 remained unsuccessful. The occurrence of a stop codon following GLP1 together with the inability to isolate GLP-2-like substances from fish tissues, led researchers to the well-founded, if mistaken (see later discussion), conclusion that GLP-2 is not encoded or processed in piscine tissues. 2. Extrapancreatic Tissues

In addition to endocrine pancreas, the proglucagon gene is expressed in a number of extrapancreatic tissues. In mammals, transcription of the proglucagon gene results in identical mRNAs, irrespective of tissue of origin. However, the further processing of the primary transcript in pancreas, stomach, gut, brain stem and hypothalamic neurons is tissue-specific at the post-translational level (Mojsov et al., 1986; Orskov et al., 1987; Holst and Orskov, 1994). Transcription of the proglucagon gene in fish appears to be fundamentally different, as is already clear from the two tissues analyzed, namely, pancreas and intestine. While the pancreatic proglucagon cDNAs contain stop codons immediately following GLP-1, it was recently discovered that both piscine and avian proglucagon genes from gut contain no such stop codon. Thus, mRNAs from trout and chicken pancreas and intestine have different 3' ends. While the pancreatic mRNAs terminate between exons 4 and 5 ,

FIG. 2 Structures of mammalian and piscine proglucagon genes and their products. Glucagon, stippled; GLP-1, horizontal stripes; IP, intervening peptides; UT, untranslated regions. Dibasic processing sites (RR or KR) are indicated by bold vertical lines, monobasic processing sites by thin vertical lines. RR (mammal) or RK (anglerfish) within the glucagon sequence indicates intraglucagon processing sites that can lead to the production of miniglucagon (see text). (A) Mammalian proglucagon gene and mRNA. Modified from Bell (1986) and White and Saunders (1986). t indicates the 6-amino acid sequence which is truncated off the N-terminal end of GLP-1 (1-37) to yield the biologically active GLP-1 (GLP-17-37 or GLP-l7-=amide). The following peptides are encoded: Signal peptide (1-23), glucagon (33-61), oxyntomodulin (33-69); GLP-ll-37(72-108); GLP-17-37(78-108); GLP-2 (126-159). (B) Anglerfish (Lophius americanus) proglucagon mRNA. The following peptides are encoded: Signal peptide (1-Zl), glucagon (52-SO), GLP-1 (89-122). [Modified from Lund et al. (1983) and Andrews and Ronner (1985)l. (C) Translational products of the proglucagon gene in mammalian and piscine pancreas and intestine. See text for references.

199

GLUCAGON FAMILY PEPTIDES IN FISHES

A

Exon 1 II ..

Exon

Exon 3 II

2

II

,. ..

Exon 4 II

, .

. . .. ..

..

.

.

...

. ... -. - . .... .

. . ..

...

. ....

.. .

.

:

.

...

.

...

.:

...

Exon

Exon

5

6

..I.! .1 , : : , : .. .. .. ..: ... ...

...

1... G e n e ( l 0 k b )

..

... ... ... : ..

..

:

3’ (1150b) Signal peptie

Glucagon Pl

B

GLP-1

P Z

RK

Signal

peptide

GLP-2

UT

3’ mRNA(650 b)

Glucagon GLP-1 IP

C

Mammal

Oxyntomodulin N-extended GLP-1 GLP- 1 GLP- 1-1P2 GLP-2

Glucagon GRPP Glicentin GLP-1-IPZ GLP-2

(GLP-I)

Pancreas

Intestine Glucagon GLP- 1 Oxyntomodulin GLP-2

Glucagon GLP- 1 Oxyntomodulin

Fish

200

E. M. PLISETSKAYA AND T.

P. MOMMSEN

the intestinal mRNA is alternatively spliced to one or more exons, encoding GLP-2. Therefore, extrapolating from one (basic) teleost and one bird to their respective groups, it can be surmised that expression of the proglucagon gene in piscine and avian intestines may be regulated at the level of mRNA splicing (Irwin and Wong, 1995). Other fish tissues (brain?) may contain additional surprises. For example, due to unique, tissue-specific, post-translational processing of the single proglucagon mRNA transcript, fetal rat hypothalamus produces glicentin and oxyntomodulin, and quantitatively less impressive amounts of glucagon (Lui et aL, 1990). Keeping this in mind, we can assume now that intestinal L-cells in mammals process and release GLP-1, GLP-2, glicentin and oxyntomodulin, while pancreatic A-cells produce glucagon and a large fragment spanning GLP-1, an intervening peptide as well as GLP-2 (Fig. 2C) (Patzelt and Schiltz, 1984; Weir et al., 1989) and smaller amount of an N-terminally extended GLP-1 (Holst et al., 1994). In contrast, piscine pancreatic A-cells produce glucagon, GLP-1 and in some fish oxyntomodulin (Conlon, 1989; Plisetskaya, 1990a) while intestinal L-cells probably process GLP-1, GLP2 and, at least in some primitive fishes, glucagon (Fig. 2C) or a sequence homologous to oxyntomodulin (Conlon, 1989); however, it is questionable whether the name oxyntomodulin is justified for such a peptide, considering the widespread absence of true oxyntic cells in teleostean fishes (see also Section I1,C).

C. Primary Structures and Localization 1. Primary Structures

a. Glucugon The primary structures of piscine glucagons and glucagonlike peptides are presented in Tables I and 11. Lampreys, belonging to the agnathans, the most ancient extant vertebrates, express glucagon and GLP1 only in the intestine while their islet organ appears to be devoid of Acells (Falkmer and Van Noorden, 1983). It was not until recently that glucagon and GLP were isolated from lamprey intestine (Conlon et al., 1993a) and it is not known yet whether lampreys produce intestinal GLP2. Because, as mentioned in the preceding section, a number of fishes possess two proglucagon genes, several fish species express two closely related glucagons and two GLPs (Tables I and 11). Interestingly, glucagon sequences of the primitive fishes such as dogfish and ray have greater similarities to mammalian (human) glucagon than do glucagons of teleost fishes, which represent a “dead-end side branch” of the accepted evolutionary tree. Using structural similarities of glucagons within the fishes and between elasmobranchs and mammals, Cutfield and Cutfield (1993) com-

GLUCAGON FAMILY PEPTIDES IN FISHES

201

posed an unrooted phylogenetic tree of various glucagon sequences. The authors agree with Conlon and Thim (1985) that the greater divergence of pancreatic glucagons from teleostean fishes may be linked with the transformation of the pancreas into concentrated endocrine islet tissue (i.e. principal islets and the Brockmann bodies). At first impression, fish glucagons appear fairly variable in sequence. However, on closer inspection, a surprisingly uniform pattern becomes apparent. Twelve key amino acids are invariant in all species and another fourteen or so have one predominant amino acid with one major alternate. Often these alternates represent single base changes and result in neutral changes in the physicochemistry of a particular residue [i.e., Thr substituting for Ser (positions 2,6,29), Asp substituting for Glu (positions 14,21) or Lys for Arg (position 17)]. The only really variable position is 16 (designated XX in the bottom of Table I), where six different residues, varying widely in chemical properties, can be found. These are, in decreasing abundance, Thr, Asn, Ser, Glu, Asp, and Met. Two positions in the glucagon molecule have been identified as playing critical roles in receptor binding and in the activation of adenylyl cyclase. Although, as pointed out below, the activation of adenylyl cyclase may not always constitute the main route of message transduction for glucagon, assay of adenylyl cyclase activity in dependence on hormone concentration is a common tool to assess the biological effectiveness of glucagon in in vitro systems such as isolated hepatic plasma membranes. Undoubtedly, His’ is a prerequisite to the biological activity of the peptide (Unson et al., 1993). Almost as essential is Asp’. In this case, side chain length appears to be more crucial to activation of adenylyl cyclase than charge, since simultaneous replacement with Glu’ and deletion of His’ generates an antagonist peptide with unaltered receptor binding, but compromised ability to activate adenylyl cyclase of rat liver membrane preparations (Unson et al., 1991). Although both amino acids are flanked by very conserved regions in the piscine peptides, des-His’-G1u’-glucagon behaved differently in a piscine setting. Instead of delivering the expected inhibition of adenylyl cyclase, the mammalian antagonist functioned as a weak agonist of fish hepatocyte glycogenolysis (T. P. Mommsen, unpublished results). Admittedly, the test systems-rat liver membranes vs fish hepatocytes-are not directly comparable, but these results already indicate that alleged antagonists cannot always be directly used effectively across vertebrate systems, even in such a “conserved” case as vertebrate glucagon. Native piscine glucagons and GLP-1 amino acid sequences, known now for a number of fish species, may reveal a similarity to either products of anglerfish proglucagon gene I or proglucagon gene 11. For example, pancreatic glucagon and GLP-1 isolated from endocrine pancreas of catfish, salmon and flounder are closer to the products of anglerfish proglucagon-I1 than

202

E. M. PLISETSKAYA AND T. P. MOMMSEN

to proglucagon-I (Conlon et al., 1987b; Pollock et al., 1988a). Glucagonrelated peptides isolated from anglerfish Brockmann body by Noe and Andrews (1986) also appeared to be metabolic cleavage products of proglucagon 11. Thus it seems likely that processing of proglucagon I1 predominates in fishes, with gene I1 products more conserved in structure. In a mammalian context with pancreatic glucagon and intestinal oxyntomodulin as primary proglucagon gene products, two rather unusual findings have been reported for some ancient fishes. The first is the presence of a pancreas-type glucagon in the gut of an elasmobranch, the common dogfish (Conlon et al., 1987d). The structure of this peptide (Table I), with its conserved N-terminal region, its tell-tale dibasic site in positions 17/18 and the 29-residue chain length, clearly makes this peptide a true glucagon, albeit matured in the “wrong” tissue. The second surprising finding is the presence of a peptide structurally resembling oxyntomodulin in the endocrine pancreas of ratfish (Conlon et al., 1987a) and alligator gar (Pollock et al., 1988a). The question remains of whether the described situations are partial to these species. Unique as this situation of an oxyntomodulin in the pancreas may be, it is not without counterpart in mammals. The guinea pig, for instance, is known to express oxyntomodulin in the pancreas. However, among the mammals, the guinea pig is clearly unusual, with pancreatic insulin and glucagon also differing substantially from their pancreatic equivalents in other mammalian species (Conlon et al., 1985b), although guinea pig GLP-1 is sequence identical to other mammals (Seino et al., 1988). Although alligator gar, ratfish, and eel gene I1 pancreatic glucagons possess 6-8 amino acid extensions at the carboxyl end, they could hardly be regarded as typical oxyntomodulins. These peptides have very little resemblance in the sequence of their C-terminal extensions with the Cterminal region of mammalian intestinal oxyntomodulin (three matches). In spite of the fact that these extended peptides are more abundant in the fish pancreas than glucagon, it remains to be tested whether they possess any oxyntomodulin-like biological activity. For reasons detailed by Conlon and co-workers, the 36-residue C-extended glucagon from the European eel (Table I) is more likely a storage form of the 29-residue glucagon, rather than an oxyntomodulin-like peptide with a distinct biological activity (Conlon et al., 1988). Unfortunately, it has not been analyzed to what degree C-terminal extension will affect biological activity or potency of the peptides compared with glucagon. We are not aware of any evidence for the existence or production of a glicentin-like peptide in either pancreas or gut of fish. Mammalian glicentin can serve as a substrate for CAMP-dependent protein kinase (protein kinase A) (Conlon et d., 1984), although no change in physiological function of the resulting phosphorylated hormone has been reported yet.

GLUCAGON FAMILY PEPTIDES IN FISHES

203

The possibility of glucagon being processed into shorter bioactive peptides has only been pursued in rats. The almost invariant mammalian glucagon contains an arginine doublet in position 17-18, potentially a dibasic processing site. In fact, it has been shown that at least in the rat, liver glucagon is processed by a specific endopeptidase into a so-called miniglucagon ( g l u c a g ~ n ( ~ ~(Blache - ~ ~ , ) et al., 1994). Evidence has been accumulating that this minipeptide displays its own discrete biological activities (Lotersztajn et al., 1990), including selective inhibition of the hepatic calcium pump (Mallat et aZ., 1987). Oxyntomodulin, which possesses the same potential processing site, is also N-terminally truncated into a biologically active peptide. In this case, however, the truncated peptide reveals activity identical with the full-length oxyntomodulin (Jarrousse et al., 1993). Obviously, the variability in sequence between species delivers an extremely powerful tool for the analysis of a peptide sequence vis-h-vis its biological activity. This type of approach makes it possible to identify key regions of a peptide that set this peptide apart from other, similar hormones and might provide indicators for specific biological traits beyond physical chemistry. Ultimately, such an analysis might also deliver insights into the evolution of hormone receptors and mechanisms of receptor recognition. For a comparative analysis with a clear evolutionary bent, glucagon is an excellent example, not only for pointing out how limited and ultimately frustrating an analysis of mammalian sequences can be. Glucagon is sequence identical in all species of mammals analyzed to date, with the exception of the guinea pig, which contains five substitutions in the Cterminal region (Conlon et aZ., 1985b; Huang et al., 1986). As seen in Table I, the dogfish glucagon and little more than half of the teleostean glucagons sequenced to date contain the same Arg-Arg processing site, while 15 out of 20 fish species possess two basic residues in positions 17 and 18, and thus may be subject to similar processing into miniglucagon. It may be a coincidence that two of the remaining, diverging, fish glucagons are gene I1 products of species also expressing a glucagon with the “common” dibasic site. The question arises of whether expression of two glucagons may lead to functional divergence of the two resulting peptides, with one leading to the production of “miniglucagon” functionally separated from the unprocessed full-length glucagon.

b. Glucagon-like Peptide As function has somewhat superseded peptide chemistry, the nomenclature of mammalian GLP has changed over the years. Initially some confusion had arisen due to the existence, in mammals, of active and inactive forms of the hormone and tissue-specific processing. The full-length peptide, which represents an intestinal gene product, is biologically largely inactive and is now referred to as the N-terminally extended form GLP-l(l-37).When the peptide is truncated at a monobasic

204

E. M. PLISETSKAYA AND T. P. MOMMSEN

site (Arg), it yields one of the two biologically active forms of GLP-1 with an N-terminal His. Thus proglucagon 78-107-amide is processed posttranslationally into GLP-1(7-36)amide,while proglucagon 78-107 is processed into GLP-l(7-37).The term “GLP-1” is now used to designate these two biologically active forms with 30 (or 31) residues. When the first native piscine GLPs were isolated from endocrine pancreas (Andrews and Ronner, 1985), it immediately became evident that they corresponded to GLP-1 of mammals, and also that sequences corresponding to the amino-terminally extended GLP of mammals were absent (Table 11). Remarkably, it is the removal of these additional six residues from the full-length mammalian peptide which transforms a peptide largely devoid of hormonal activity into a very powerful insulinotropin (see Section IV,B). Mojsov and co-workers (1990) demonstrated that the processing of proglucagon in the rat intestine, and to a lesser extent in the rat pancreas, results in the appearance of at least three different circulating forms of GLP-1-the biologically inactive full-length GLP-1(1-3vand two truncated forms. One of these has 31 residues (GLP-1) with a C-terminal glycine, and the other has 30 residues, terminating in an amidated arginine. Both shorter peptides possess identical biological activity as insulinotropins. In contrast, fish tissues encode only the active short forms, while N-terminally extended forms could be located neither from the cDNAs nor in any of the peptide sequencing studies. Nevertheless, some piscine GLPs-1, with more than 31 amino acid residues (Table 11) have been described. In noted contrast to their mammalian counterparts, these are C-terminally extended forms, the result of different processing at two adjacent arginine residues at the carboxyl-terminal. Just like the 30-residue mammalian GLP-1, some fish GLPs possess a glycine residue at the C-terminal and are released in amidated forms (Andrews et al., 1986), which has been interpreted as a general key feature of hormonally active peptides (Bradbury el al., 1982). With few exceptions (see later discussion) all fish GLP-1 has been isolated and purified from endocrine pancreatic tissue. The two forms of GLP-1 found by Conlon et al. (1989) in the ratfish that differ only in their C-terminal sequence (R-R-M vs R-K-Q-N-D) arise from a single proglucagon gene through differential post-translational processing (Table 11). Comparing the primary structures of intestinal GLP-1 of agnathans and pancreatic GLP-1 from gnathostomian fish, Conlon et al. (1993a, 1994) suggested that evolutionary pressure to conserve primary structures of glucagon is much stronger than the pressure to conserve the structures of GLPs. This suggestion will be probed as more amino acid sequences of GLP-1, and, it is hoped, GLP-2 become available. We already know, however, that in some related fish species such as American (A. rostrata) and European (A. anguilla) eels, glucagon-like peptides-l can reveal highly conserved primary amino acid sequences (Conlon et al., 1991).

GLUCAGON FAMILY PEPTIDES IN FISHES

205

In several analyses of GLP-1 structure and function, a number of key residues have been identified. Gallwitz et al. (1994), singly substituting every GLP-1 position with alanine, documented that replacement of the following specific residues exerted negative effects on GLP binding and activation of adenylyl cyclase in an insulinoma cell line: His1, Glf, Phe6, Phe22,and Ile23. At the same time, residues in position 5,8, 10-12, 14, 16-21, and 25-30 were deemed less crucial to GLP-1 functions since their substitution with alanine failed to change receptor affinity for the appropriate synthetic GLPs (Gallwitz et al., 1994). Using an identical approach, but a different test system, Adelhorst and colleagues (1994) consider His', Gly4,and Phe6, but also Thr' and Asp' as essential to receptor interaction, while positions 22 (Phe) and 23 (Ile) are thought to play crucial conformational roles in receptor recognition. Previous studies using selective amino acid deletions, rather than substitutions, had identified His' as a key residue for biological actions and, in hindsight rightfully, dismissed large parts of the C-terminal regions as inconsequential to GLP-1 action (Suzuki etal., 1989).Not surprisingly, then, these key residues are conserved in mammalian GLP-1. This point finds additional support in a recently discovered peptide which functions as an excellent agonist for GLP, but which has a 39-residue amino acid backbone (Raufman et al., 1992). This peptide, the so-called exendin4, was first isolated from the poison gland of a reptile (the gila monster) and clearly belongs to the glucagon family peptides (see Section IV). Incidentally, GLP-1 shares the key amino-terminal His' and Asp' with glucagon, exendin, and peptide histidine isoleucine (PHI). Using the evolutionary playing field and especially the wide diversity of fishes and their pancreatic peptides, instead of a peptide synthesizer, we can come to very similar conclusions about fish GLPs (and incidentally also glucagon) and perhaps even get an inkling of additional residues that should be considered when designing as yet elusive GLP-1 agonists and powerful antagonists. To this end, we present a list of fish GLP sequences published to date in Table 11. If we exclude, for reasons listed below, the peculiar GLP of the bowfin from this analysis, a number of invariant residues are apparent in the fishes. As expected from the above analysis, these residues include His', Glf, an aromatic amino acid (Phe or Tyr) in position 6, Thr7, Asp' as well as PheZ2.The importance of conserving an aliphatic side chain of the amino acid in position 23 is underlined by the use of Val or Ile in different species of fishes. However, a comprehensive analysis of GLP-1 variability in the fishes does not stop there. Other key features, which were not unearthed in the synthetic approach, can be detected. Among these are strict conservation of residue 2 (Ala), an acidic residue in position 3 (Glu or Asp), Ser*and Thr'O. In fact, the entire amino-terminal half of the peptide is highly conserved, while fewer constraints appear to mold the C-terminal region. However, going beyond the chemical approach,

206

E. M. PLISETSKAYA AND T. P. MOMMSEN

basic residues in position 20 and an invariant Leu in position 26, preferentially followed by a LysZ7,appear essential to biological activity. What may initially have appeared as an exercise in stamp collecting and a patchwork approach to GLP-1 sequences, in fact has led to an instructive quilt. Clearly, the comparative use of naturally occurring variations in different species, covering an enormous range in life histories, habitats, and evolutionary trends, promotes much deeper insight into biochemical constraints on hormone design than the synthetic approach. Bowfin GLP was excluded from this comparative analysis for a number of reasons. Although the categorizing of the peptide as a GLP-1 seems justified by the overall chromatographic behavior and sequence of the peptide (Conlon et al., 1993b), a number of unique features seem to put the peptide apart from other GLP-1s and locate it somewhere in the area between GIP and GLP (Table 111). For instance, His' is replaced by Tyr', a trait of GIP, and other key GLP features are lacking, including Gly4 (replacement: Ala), Ths (replacement: Pro), Thr' (replacement: Ile), as well as the entire three residue sequence Phezz,Val/Ilez3,Xyz". Residues close to the N-terminal are invariant in all other fish GLPs (Table 11) and had been identified as important to activation of adenylyl cyclase and insulinotropin action in mammals while positions 22 and 23 had been assigned crucial roles in receptor binding (Adelhorst et al., 1994). Considering these many noteworthy deviations from the GLP theme, it is not unexpected that bowfin "GLP" fails to function as a true GLP in a piscine test system (Conlon et al., 1993b; see also Section IV). This unusual GLP may also serve as an excellent test system when assessing potential insulinotropic actions of GIP in fishes. It also raises the question of whether a true, functionally glucagon-like GLP is encoded and expressed in the bowfin but was missed during the isolation of the unique product. It is often found that glycine residues preceding basic processing sites in prohormones tend to donate an amide function to the adjacent amino acid, leading to C-terminal amidation of the liberated peptide (Bradbury et al., 1982). C-terminal amidation, in turn, is a key characteristic of bioactive neuropeptide and peptide hormones, and indeed, in the case of mammalian GLP, the final processing product may be C-terminally amidated. However, no difference has been noted in the insulinotropic powers between the 31residue GLP-1 (C-terminal Gly-Arg-Gly), a synthetic glycine amide, and the native 30-residue arginine-amide (Suzuki et al., 1989). The situation in the fishes is quite different. Of all piscine GLPs, only gene product I1 of the anglerfish contains a glycine adjacent to a dibasic processing site, thus resulting in the production of an amidated C-terminal (Andrews et al., 1986). In all fishes, the C-terminal appears to be in the carboxyl form, although it should be noted that many fish GLPs do contain additional potential dibasic and monobasic processing sites at the C-

TABLE 111 Glucagon-like Peptide 1 in the Holostean Bowfin (Amia @ha) Compared with “Fish” GLP-1 and Mammalian GIPa

20 GQAAK DQAAK

Mammal GLP-1 Anglerfish GLP-I

1 HAEGT HADGT

10 FTSDV YTSDV

SSYLE SSYLQ a nn

?Jan

Bowfin GLP-lb Mammal GIP“

YADAP YAEGT

YISDV FISDY

YSYLQ SIAMD

DQVAK KIRQQ

L1

*****

****

*

EFIAW DFVSW

30 LVKGR LKAGR

K---WE DFVNW

LKSGQ LLAKG

**

40 G GRRE

*U

DRRE KKSDW

IHNIT Q

Stars indicate key positions, either invariant in fish GLPs or identified by replacement studies in mammalian GLP-1 (bold) or both (see text).

n denotes invariant residues for the bony fishes studied to date.

Bowfin (Arnia calva) GLP-1 (Conlon et al., 1993b).

--- deletions.

In mammalian GIP, only positions 23 and 24 are variable. Bovine GIP is listed.

208

E. M. PLISETSKAYA AND T. P. MOMMSEN

terminal end. In many cases, these are preceded by a glycine residue. In addition, it should be recalled that the C-terminal residues are deemed largely irrelevant to biological activity of the peptide: replacement studies on mammalian GLP identified positions 22 and 23 as essential, but noted that residues beyond these numbers were quite dispensable (Adelhorst et al., 1994;Gallwitz et al., 1994), although deletion of the C-terminal arginine amide may slightly right-shift the dose-response curve (Suzuki et al., 1989). Critical analysis of fish GLP-1 sequences leads to the conclusion that the invariant Leuz6 plus the hardly variant LysZ7must have important roles in GLP function. Altogether it is therefore not implausible to postulate additional processing of the piscine GLPs into shorter, possibly amidated peptides. In the evolution of hormones, conservation of functionality is the central tenet, while sequence diversity is likely to reflect evolutionary history and functional constraints only. The striking difference between fish GLP-1s and their mammalian counterpart, then, is the GLP gene itself, its processing and, not least, the site of processing and target tissues. Fish GLPs are processed in pancreas from a short gene and released in their active form with liver as a major target, while mammalian GLP-1s are produced in intestine, and processed and released from an N-terminally extended precursor molecule with the fl cells of the endocrine pancreas as a primary target. This clear distinction in processing site and target appears to be muddied by the situation in amphibians. To date, only two species of amphibians-a frog (Pollock et al., 1988b) and a salamander (Cavanaugh et al., 1996)have been analyzed, but it is interesting to note that these two express GLP-1 in pancreas. In contrast to the situation in fishes, frog (Xenupus and Rana) liver does not appear to be a target for GLP action (Mommsen and Moon, 1994). To complicate the evolutionary relationships between the glucagon gene and GLP processing even further, it should be noted that amphibian pancreas also expresses and produces GLP-2, a situation that sets amphibians apart from the fish and mammalian pancreas. As summarized in Table IV, fish pancreas is devoid of GLP-2 (stop codon) and in mammalian pancreas, GLP-2 is expressed, but processed only as part of a much larger peptide (Fig. 2). Although GLP-2s from comparatively fewer species have been sequenced to date, it is already clear that the peptide is less stringently conserved than either glucagon or GLP-1, especially toward the Cterminal region (Table IV). His' and Asp' are highly conserved and position specific as in glucagon and GLP. Of the first 15 amino acids of GLP-2, only two positions are fairly variable (positions 10 and 13), while the other variant positions conserve either chemical nature (Ser for Thr) or charge (Glu for Asp).

TABLE IV

Primary Structures of Glucagon-like Peptide 2 in Vertebratesa Gro w

Sequence 1

10

Source

20

30

Teleost Rainbow trout Amphibians Bullfrog Salamander

HVDGS

FTSDV

NKVLD

SLAAK

EYLLW

VMTSK

TSG

cDNA/Intestine

HADGS HADGS

FTSDF FTSDI

NKALD NKVLD

IKAAQ TIAAK

EFLDW EFLNW

IINTF' LISTK

VKE VTE

Pancreas Pancreas

Bird Chicken Invariant (ex mammal)

HADGT H-DG-

FTSDI FTSD-

NKILD NK-LD

DMAAK --AA-

EFLKW E-L-W

LINTK

VTQ

-----

---

cDNA/Intestine 16 of 33

Mammal Human

HADGS

FSDEM

NTILD

NLAAR

DFINW

LIQTK

ITD

cDNAlIntestine

a References: Rainbow trout (Oncorhynchus mykiss) and chicken (Callus domesticus) (Irwin and Wong, 1995); bullfrog (Rana catesbeiana) (Pollock et al., 1988b); salamander (Amphiuma tridactylum) (Cavanaugh et al., 1996).

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E. M. PLISETSKAYA AND T. P. MOMMSEN

2. Localization

In different vertebrate animals, the distribution of pancreatic cell types producing insulin, glucagon, GLPs, somatostatin, and pancreatic polypeptide is not uniform, and distinct pancreatic cell types may predominate. For example, in reptilian and avian pancreas, glucagon-producing cells outnumber other cell types in the splenic lobe of the organ, while in mammals and amphibians, the insulin-producing B-cells tend to occur in highest abundance (Epple and Brinn, 1987). As mentioned (see Section II,C,l), neither glucagon immunoreactive cells, nor the corresponding extractable peptides were found in the islet organ of lamprey and hagfish . However, glucagon-immunoreactive endocrine cells of an open type exist in the gut mucosa of the river lamprey, Lampetra fluviatilis and the Atlantic hagfish, Myxine glutinosa (Falkmer and Van Noorden, 1983) and GLP-1 immunopositive cells were localized in the intestine of juvenile and upstream migrant marine lampreys, Petromyzon marinus (Cheung et al., 1991). A true endocrine pancreas evolved first in holocephalans. Stefan and colleagues (1981) observed that the endocrine pancreas of the ratfish contains two populations of cells with different glucagon immunoreactivity. Some cells could be immunostained only with antibodies raised against the amino-terminal section of (mammalian) glucagon, while another population of cells immunoreacted with C- and N-terminally directed antiglucagon sera. These results strongly imply the presence in pancreas (and in gut) of two different glucagon family peptides-one close to glucagon and another conceivably close to oxyntomodulin. Even reactivity with GLP-1, which was not yet isolated when Stefan et al. published their study (1981), cannot be excluded, although the relatively large species differences observed for the C-terminal region of GLP-1 would argue against this reinterpretation. In all other fishes, including elasmobranchs and actinopterygians, the endocrine pancreas matures both glucagon and GLP-1 (see Tables I and 11). Figures 3 and 4 present pictures of the Brockmann body and intestine of rainbow trout (0.mykiss) or coho salmon (0.kisutch) immunostained with antibodies generated against salmon glucagon and GLP-1. It is apparent that glucagon and GLP-1 immunoreactivities are colocalized in all Acells from the Brockmann bodies of both salmonid fishes, and a similar

FIG. 3 Two successive sections of pyloric caecum (A) and the Brockmann body (B) of the rainbow trout (Oncorhynchus mykiss) stained differentiallywith antisalmon glucagon (a) and antisalmonglucagon-likepeptide (b). Identicalcells (arrows) are immunoreactiveto both glucagon and glucagon-like peptide. [From Cell Tkue Res. Colocalization of glucagon-like peptides and glucagon imunoreactivities in pancreatic islets and intestine of salmonids, Nozaki et al. 253,371-375, figs. 2 and 4 (1988a) 0 Springer-Verlag with permission.]

GLUCAGON FAMILY PEPTIDES IN FISHES

21 1

212

E. M. PLISETSKAYA AND T. P. MOMMSEN

FIG. 4 Two successive sections of coho salmon (Oncorhynchus kisutch) pancreas stained differentially with antisalmon somatostatin-25 (A) or anti-salmon glucagon (B). Note the close association of glucagon-positive cells and SST-25 producing cells. [From Nozaki et al. (1988b) with permission.]

colocalization is found in open-type endocrine cells in mucosae of the small intestine-including the pyloric caeca-and specific parts of the large intestine. Glucagon-producing cells could not be found in the mucosal cells

GLUCAGON FAMILY PEPTIDES IN FISHES

213

of the body of the stomach. Interestingly, glucagon/GLP-positive cells are located at the outer rim of the Brockmann body and always in close proximity to the D-cells expressing somatostatin-25 (SST-25), a product of a second SST gene, differing from SRIF (SST-14). In contrast, insulin cells are located in the central part of the pancreas in close apposition to the SST-14 cells. Similar topographical patterns have been reported for other fish species, including anglerfish, trout, salmon, and sea bass (McDonald et al., 1987; Nozaki et al., 1988b; Abad et al., 1988). The physiological meaning of this particular spatial arrangement remains obscure. By comparison, the microcirculation in the endocrine pancreas of mammals, such as dog or rat, is directed from insulin-producing B-cells via glucagon-producing A-cells, to D-cells expressing SST-14. Thus, the products of the D-cells are unable to directly affect glucagon secretion through the intraislet vasculature (Samols and Stagner, 1990).The microcirculation pattern is different inside piscine Brockmann bodies (Lange, 1984; Syed Ali, 1985). Here, glucagon and SST-25 cells are adjacent to each other on the periphery of the islet. Therefore, it might be anticipated that SST14 as well as SST-25 could inhibit the secretion of glucagon and GLP through paracrine mechanisms. Cell-cell interactions may be achieved by yet another route, namely through gap junctions linking cells in a kind of functional syncytium (Unger and Orci, 1981). The exact ontogenetic stage at which glucagon starts to be processed in the piscine pancreas is not well defined. For example, in the sea bass, the endocrine pancreas appears at the prelarval stage, while the immunopositive reaction with glucagon is observed only at the beginning of the juvenile stage, 3 months after hatching (Beccaria et al., 1990). However, this differential timing may reflect technical limitations rather than developmental stages. It is conceivable that with continuing development of more sensitive and precise techniques, the first appearance of glucagon/GLP-1 may shift to the earlier developmental stages, similar to what has been described for insulin (PCrez-Villamil et al., 1994). Glucagon-positive immunoreactivity was found in the nervous system of the protochordate ascidian Styela plicatu (Pestarino, 1990) and in the brain of lamprey, hagfish (Falkmer and Van Noorden, 1983), and shark (Conlon and Thim, 1985). These data support previous reports on the localization of glucagon in fetal and adult rat hypothalamus and some other parts of the brain (Tager et al., 1980; Salazar and Vaillant, 1990; Lui et al., 1990). In the brains of frogs and turtles, the glucagon-like immunoreactivity is restricted to a small population of nerve cells, while no such immunoreactivity was detected in the brain of the axolotl, Ambystoma rnexicanum (Conlon et al., 1985a). GlucagodGLP immunoreactivity in piscine pancreatic and intestinal endocrine cells may be colocalized with immunoreactivity to some other peptides, most often belonging to the pancreatic polypeptide family

214

E. M. PLISETSKAYA AND T. P. MOMMSEN

of hormones (Abad et al., 1992; Cheung et al., 1991). In this respect the piscine system resembles other vertebrates, since such colocalization has been shown to exist in all other major groups of vertebrates, namely, amphibians, reptiles, birds, and mammals (Epple and Brinn, 1987).

111. Systemic Levels and Their Regulation in Fish A. Secretion Circulating levels of any biologically active peptide or hormone in plasma are set through the integration of a number of processes, such as the rate of peptide synthesis, secretion, binding and uptake by the target tissues and clearance. Experiments with perfused fish endocrine pancreas made it possible to separate synthesis and secretion from the other competing events. Ince and So (1984) perfused in situ a preparation consisting of combined pancreas and intestine of European silver eel, while Ronner and Scarpa (1987) used the isolated Brockmann body of the channel catfish for perfusion studies. Both preparations increased glucagon secretion after stimulation with glucose and arginine. These results were corroborated by Harmon et al. (1991), who found hyperglucagonemia in glucose-injected rainbow trout. In v i m , pronounced release of glucagon can be induced also by high concentrations (25 mEqlliter) of KCl (Ronner and Scarpa, 1987). Interestingly, none of these compounds represent specific glucagonotropes, since all of the mentioned treatments also lead to the concurrent release of insulin (Fig. 5B). The situation in fishes is distinctly different from that in mammals. In the mammals, amino acids such as arginine appear to exert their specific actions indirectly, since selective treatment with antibodies to glucagon (Tan et al., 1985a,b) decreases or even abolishes arginine’s insulinotropic actions. If the same immunoneutralization experiment with antiglucagon antibodies is performed in a piscine setting, no decrease in insulinotropic effect is noticed (Plisetskaya et al., 1991), implying that secretion of insulin in fish is relatively independent from glucagon family peptides. Obviously, arginine is a powerful insulinotropin in its own right. In fact, it is more powerful at inducing insulin release than glucose, matched only by other amino acids. However, arginine is not a specific insulinotropin since it also causes a substantial release of other hormones from the Brockmann bodies, including glucagon and GLP-1. To sum up, arginine may merely function as a general activator of peptide release from fish endocrine pancreas. Surprisingly, the same statement applies to the role of glucose in fish pancreatic performance, and thus the

21 5

GLUCAGON FAMILY PEPTIDES IN FISHES

500 W salmon GLP-1 (7-37)

400

300 200

100 0

Glucagon-like peptide 1 200

*

(nM)

100

0

0 0.1 1.010100 Glucagon-like Arginine peptide 1 (nM)

FIG. 5 (A) Mammalian and salmon GLPs-1 in the presence of physiological concentrations of glucose stimulate insulin secretion from monolayer cell cultures of rat embryonic pancreas. Asterisks indicate significant difference from control cells incubated in hormone-free medium. [From Plisetskaya and Duguay (1993) with permission.] (B) Lack of stimulation of insulin secretion by GLP-1 in the presence of physiological concentrations of glucose in dispersed cells of coho salmon (Oncorhynchus kisutch) Brockmann body. L-Arginine functions as an effective insulin secretagogue (See also Table VII).

famous glucose dependence of many insulinotropins may not apply to the piscine situation at all. As a result, the circulating hormone ratios, sometimes considered to be the real regulators of metabolic output, are somewhat altered by arginine or glucose treatment, but are not necessarily skewed in favor of insulin. Besides, in fishes, pancreatic GLP-1 must be viewed as

216

E. M. PLISETSKAYA AND T. P. MOMMSEN

a true glucagon-like hormone (i.e. a potential antagonist of insulin action) and as a result, the sum of glucagon and GLP-1 should be considered metabolically. With respect to the release of GLP-1 from fish pancreas, an even smaller data base exists, while no information is available on mechanisms controlling production and release of GLP-1 from intestine. It should be noted that in the mammals, postprandial glucose, GIP, catecholamines, bombesin family peptides, and CGRP have the potential to increase the rate of GLP1 release from the intestine (Roberge and Brubaker, 1993; Herrmann et al., 1993); while amino acids, fat, and other peptide hormones [VIP (vasointestinal inhibitory peptide), NPY (neuropeptide), TRH (thyroid releasing hormone)] are without effect. In contrast to insulin and some other piscine hormones, fish glucagon can be measured in mammalian-based radioimmunoassays (RIAs) (GutiCrrez et al., 1986; Navarro et al., 1995), although species-specific RIAs or enzymelinked assays for glucagon are preferable whenever possible (Plisetskaya et al., 1989a; Sundby et al., 1991). The immunoreactivity of piscine GLP-1 in mammalian RIA systems remains to be explored. To date, all GLP-1 measurements in fishes have been performed using a salmonid-specific kisutch) (Plisetskaya et al., 1989a; RIA, based on coho salmon GLP-1 (0. Sundby et al., 1991). Not surprisingly, plasma concentrations of glucagon and GLP-1 depend on the physiological condition of the experimental animals (Sundby el al., 1991). In Tables V and VI, we have collated glucagon and GLP-1 levels measured in the peripheral blood of fishes, usually representing values for the caudal blood vessels; overall, concentrations in fishes are well above those commonly found in mammals. Concentrations of GLP1 in these peripheral vessels generally exceed those for glucagon. While the reasons for this discrepancy are not immediately apparent, it should be kept in mind that in the fishes, intestine as well as pancreas can contribute to the pool of GLP-1 and, as shown below, hepatic removal of GLP-1 does not appear as efficient as that for glucagon. Titers of all pancreatic peptides, including insulin, are highest in the portal vein, collecting blood from the gut and endocrine pancreas. For all three important pancreatic hormones, the biggest drop in blood concentration occurs on passage through the liver, with nonhepatic tissues playing only subordinate roles. As is apparent from the data presented in Table VII, under numerous physiological conditions, removal by the liver may account completely for the glucagon lost, while extrahepatic tissue may make a minor contribution to the removal of GLP-1 (Table VIIB). Binding of glucagon and GLP-1 to their target tissues is discussed in Section V,A.

217

GLUCAGON FAMILY PEPTIDES IN FISHES TABLE V Plasma Glucagon in the Peripheral Vessels of Fish

Glucagon Species and RIA system

(ndd)"

Dogfish shark (Scyliorhinus canicula) Mammalian 0.02-0.21 Brown trout (Salmo trutta) 1.1 t 0.2 Mammalian 1.6 ? 0.1 1.7 t 0.3 3.8 t 0.2 R. trout (Oncorhynchus mykiss) Mammalian Salmon Salmon

Salmon Salmon Coho salmon (0.kisutch) Salmon Salmon Salmon

0.2 t 0.06 0.8 t 0.1 0.2 t 0.1 0.2

0.1 0.05 0.08 1.6 ? 0.1 1.2 t 0.2 0.01-2.0

0.4-2.3

0.05-0.06

Reference Gutitrrez et al. (1986)

Feeding, spring; Carneiro et al. (1993) Fasting Autumn Autumn, arginine injection GutiCrrez et al. (1986) Fed Moon et ul. (1989) Fasted 6 weeks Fed for 6 weeks: Storebakken et al. 2% body weight (1991) 1% body weight 0.3% body weight Fasted Fed Plisetskaya (199Oc) Fasted 6 weeks Harmon et ul. (1991)

0.01-2.0 0.05 ? 0.01 1.0 year old

(0.1-0.2 Chinook salmon (0.tshawytschu) Salmon Atlantic salmon (Salmo salur) Salmon Salmon Mammalian Carp (Cyprinus curpio) Goldfish (Carassius auratus) Perch (Percu fIuviatilis) Catfish (Ictalurus punctutus) Tilapia (Oreochromis mossambicus) Barbus (Barbus comiza) Rudd (Scurdinius erythrophthalmus)

Comments

Juveniles Winter-spring Juveniles Summer Fasting

0.13-0.40 Juveniles 0.14-3.8 3.5 years old 0.65 t 0.19

Plisetskaya (1990~) Plisetskaya et al. (1989a) Plisetskaya (1990~) Moon et al. (1989)

Plisetskaya (1990~) Plisetskaya et ul. (1994) Sundby et al. (1991) Gutitrrez et al. (1986)

0.66 t 0.16 0.16 2 0.03 0.34 ? 0.36 0.58 t 0.19 0.56 ? 0.13 0.35 t 0.08 (Continues)

218

E. M. PLISETSKAYA AND T. P. MOMMSEN

TABLE V (continued) Glucagon Species and RIA system

(ndd)"

Largemouth bass ( M . salmoides) Sea bass (Dicentrarchus labrux) Gilthead seabream (Sparus aurata) Human

0.29 2 0.16 0.44 ? 0.06 1.12 2 0.17

Gutikrrez et al. (1986)

0.024-0.33'

Ensinck (1983)

a

Mean

?

Comments

Reference

S.E.M.

'Differences in the molecular mass of fish (coho salmon = 3547) and mammalian glucagon

(human = 3481) are negligible; concentrations in fishes range from about 23 to 450 picomolesl liter; values are about 3 to 5 times higher than in mammals.

TABLE VI Plasma Glucagon-like Peptide 1 in the Peripheral Vessels of Fish Species and RIA system

GLP-I (ng/ml)"

Comments

Rainbow trout (Oncorhynchus mykiss) Salmon 0.6 ? 0.1 Fed 0.3 ? 0.04 Fasted 6 weeks Salmon 1.25 Fed 2% body weight for 6 weeks 1.09 Fed 1%body weight 0.95 Fed 0.3% body weight Fasted 0.85 Salmon 1.9 2 0.3 Fed Fasted 6 weeks 0.4 2 0.02 Salmon 0.1-2.1 Coho salmon Salmon Salmon Salmon

(0.kisutch) 0.10-2.3 2.8 2 0.17

1.0 year old 0.8-2.0 Winter-spring juveniles Chinook salmon (0. tshawytscha) Salmon 0.2-2.8 Fasting Atlantic salmon (Salmo salar) 3.5 years old Salmon 0.65 2 0.14 Salmon 0.05-1.9 Human Human 0.02-0.15'

Reference Moon et al. (1989) Storebakken ef al. (1991)

Plisetskaya (1990~)

Plisetskaya (1990~) Plisetskaya et al. (1989a) Plisetskaya, (1990~) Plisetskaya (1990~) Sundby ef al. (1991) Plisetskaya (1990~) 0rskov et al. (1987); Hvidberg ef al. (1994)

'Mean ? S.E.M. Differences in the molecular mass of coho salmon (3436) and mammalian (3354) GLP1 are negligible; systemic concentrations are considerably higher in fishes (30-850 pM) than in mammals (6-45 pM).

'

GLUCAGON FAMILY PEPTIDES IN FISHES

219

6.Clearance Glucagon and GLP are cleared from vertebrate circulatory systems by the same routes and sites as other biologically active peptides, with liver and kidney identified as playing key roles. In addition, a putative ability of brain to inactivate glucagon with an insulin-glucagon-specific proteinase system has been reported (Dorn et al., 1983). Of course, in addition to specific proteolytic systems, any tissue with substantial hormone receptor activity has the potential to take up and subsequently degrade the hormone in question, if receptor activity involves receptor-mediated endocytosis of the hormone. Unfortunately, at least in the case of GLP, attention has usually been focused on message transduction rather than receptormediated endocytosis of the hormone. At any rate, as detailed below, GLP1receptors have been identified in mammalian pancreatic B-cells, lung, and brain, and tentatively in adipocytes. Thus all these tissues could conceivably contribute to the removal of GLP-1 from the mammalian circulatory system.

1. Liver In mammals, the half-disappearance time (tlI2)assessed by bolus injection varies from 5 to 15 min for GLP-1 (Ruiz-Grande et al., 1993) to 47.7 min (Oshima et al., 1988) for the N-terminally extended form. Use of a constant infusion of GLP(1-37)results in an estimate of tlR of approximately 39.5 min (Oshima et al., 1988). Disappearance times for glucagon are much shorter at 3.3 min after bolus injection and about 5.8 rnin after constant infusion. Vice versa, metabolic clearance rates for GLP are 27.4 and 18.6 ml kg-' min-' compared with 83.1 and 46.7 ml kg-l min-' for glucagon, respectively (Oshima et al., 1988). The authors suggest that one explanation of the slower disappearance of GLP-1 from the system, compared with glucagon, may be the absence of GLP-1 receptors in mammalian liver. Neither presence nor absence of GLP-1 receptors could be confirmed in fish liver yet, although strong inference leads the authors to the conclusion that the liver may indeed represent one of the major targets for GLP-1. First, as detailed below, fish liver responds quickly and sensitively to GLP1when the hormone is added at physiological concentrations. Second, both GLP-1 and glucagon are removed during liver passage, amounting to about 59% for GLP and 47 to 89% for glucagon in a single pass, hinting at an active turnover of both peptides and a relatively short half-life for GLPl-certainly shorter than in mammals (Plisetskaya and Sullivan, 1989; Carneiro et al., 1993). This situation is in marked contrast to mammals, where GLP-1 fails to bind to hepatocytes and does not decrease measurably while transiting the liver. In the rainbow trout, all nonhepatic tissues together remove much smaller amounts and percentages of both GLP-1 or

TABLE VII Levels of Pancreatic Hormones in Different Vessels of Salmonid Fish A. Insulin, glucagon, and GLP-1 in different vessels of rainbow trout (Oncorhynchusmykiss)a ~

Insulin (pmoles liter-l) Hepatic portal vein Heart Caudal vein Hormone removed (pmoles ml-') totalb

1057 ? 158 243 2 12 303 ? 25 754 (71%)

Glucagon (pmoles liter-') 124 ? 23 14 ? 3 17 ? 3 107 (86%)

GLP-1 (pmoles liter-') 157 2 15 64?9

44?9 113 (72%)

B. Insulin and glucagon in different vessels of feeding and fasting brown trout (Salmo trutta fario) under various experimental conditionsc Feeding

Insulin (pmoles liter-') Portal vein Hepatic vein Caudal vessel Total removedb

Fasting

Fasting

Control

Glucose-injected

Control

Glucose-injected

Control

Arginine-injected

1720 f 53 1223 t 71 1223 t 53 497 (29%)

1542 t 71 1064 2 89 1010 2 71 532 (35%)

691 t 230 355 f 177 319 f 106 372 (54%)

1436 t 195 780 t 177 709 t 160 727 (51%)

1223 f 355 514 t 124 425 t 106 798 (65%)

3918 2 106 3865 ? 160 3794 -+ 71 124 (3%)

90%

90%

90%

89%

dad

592 t 85 338 2 56 282 2 28 310 (52%)

987 t 56 479 ? 28 451 f 28 536 (54%)

902 t 85 395 t 85 395 2 85 507 (56%)

789 t 113 479 t 85 479 2 85 310 (39%)

1128 2 56 1071 ? 28 1071 -+ 28 57 (5%)

89%

100%

100%

Hepatic contribution to insulin removal (% of total removed) 100%

ru

Y

Glucagon (pmoles liter-') Portal vein 1635 t 197 Hepatic vein 338 t 56 310 f 56 Caudal vessel Total removed" 1325 (81%)

Hepatic contribution to glucagon removal (% of total removed) 98%

~

82%

dad

~

Recalculated from the data of Plisetskaya and Sullivan (1989). Percentage of hormone concentration found for portal vein in brackets. Recalculated from the data of Carneiro et al. (1993). Plasma was sampled 3 hr after intraperitoneal injection of glucose (1.67 mmoYkg weight) or arginine-HC1 (6.6 mmollkg), respectively. Not applicable, since hormone concentrations in these vessels are not significantly different. a

222

E. M. PLISETSKAYA AND T. P. MOMMSEN

glucagon. In fact, in the study by Plisetskaya and Sullivan (1989), no significant differences were detected in the concentrations of GLP-1 and glucagon between heart and caudal vein. Means were slightly decreased for GLP-1 (by 13% of total) between the two sampling sites, and paired re-analysis of data may reveal minor uptake by tissues other than liver. The observation that liver has the ability to remove larger fractions of glucagon than of GLP-1 might explain why GLP-1 usually is found in fish plasma at higher concentrations than glucagon (Sundby et aL, 1991). Interestingly, isolation of pancreatic peptides normally leads to higher yields in GLP-1 than glucagon. One possible explanation for the disproportional yield of glucagon and GLP-1 is that the slightly smaller and more hydrophobic glucagon is precipitated less effectively during the purification of the crude acid-alcohol extract of the Brockmann bodies (Plisetskaya et al., 1986). Of course, to complete this discussion, the potential contribution of these hormones, especially GLP-1, from intestinal sources must be considered. The new discovery that in fishes GLP-2 can only be contributed by intestine might lead to a coarse estimate of intestinal contributions to GLP1 levels, with the underlying assumption that intestine produces and releases equimolar amounts of these two peptides. However, at this point, plasma concentrations of GLP-2 are unknown. 2. Kidney

Unfortunately, because of the anatomical arrangement of the fish kidney, it is inherently difficult to determine arteriovenous differences across this particular tissue, and thus the few studies done on hormone removal (Table VII) fail to provide insight into the renal potential. For this reason, we have to turn our attention to existing experiments in mammals. In rats, kidney evidently contributes about 30% to overall GLP-1 clearance. The process may include the action of the brush border-associated peptidases and the removal of truncated GLP-1 from the peripheral circulation by a mechanism involving glomerular filtration and tubular metabolism (RuizGrande et al., 1993).

IV. Physiological Effects A. Mammals The effects of glucagon in mammals have been investigated in great detail and thoroughly reviewed (FOBand FOB,1991; Lefkbvre, 1995). Besides its well-known involvement in glycogenolytic and gluconeogenic pathways

GLUCAGON FAMILY PEPTIDES

IN FISHES

223

(Stalmans, 1983), glucagon participates in the control of lipid, urea, and amino acid metabolism (Cahill et al., 1983; Lefbbvre, 1983,1985), although its role in the regulation of adipose tissue lipolysis remains controversial (Jensen and Miles, 1993). Glucagon is known to suppress feeding behavior (Morley, 1987) by accelerating postprandial satiety (Geary et al., 1993). Secondary processing of glucagon into the truncated g l u c a g ~ n ( ~raises ~-~~) its potency to inhibit the Ca2+pumpin liver plasma membranes by three orders of magnitude (Mallat et al., 1987), an action mediated by G-proteins (Lotersztajn et al., 1990). M i n i g l u c a g ~ n ( ~may ~ - ~also ~ ) be a component of the positive inotropic effect of glucagon (Pavoine et al., 1991), a conclusion based on stimulation of the cardiac Ca2+current by activation of adenylyl cyclase and inhibition of phosphodiesterase (MCry et al., 1990). The recent description of appreciable amounts of glucagon receptor mRNA in nontraditional target tissues, such as spleen, thymus, thyroid, adrenal gland, ovary, and skeletal muscle (Hansen et al., 1995), has opened up a new window on glucagon action for mammals. As mentioned earlier, glucagon has been shown to exert insulinotropic actions in mammals, albeit only at higher concentrations than GLP-1 or GIP (Suzuki et al., 1992b), and contradictory reports are available on the existence of glucagon receptors on B-cells (Kawai et al., 1995; Kofod et al., 1993; Hansen et al., 1995). At any rate, glucagon exerts negative feedback on the secretion of other pancreatic compounds and decreases the release of peptides with GLP-1 like immunoreactivity from perfused canine and rat pancreata to about 60 to 70% of control levels (Kawai et al., 1989). A similar negative effect is exerted by GLP-1 on pancreatic glucagon release (Kawai et al., 1989; 0rskov et al., 1988). GLP-1 also suppresses the amounts of glucagon contained in pancreas without affecting glucagon gene transcription (Yamato et al., 1990). Thus the interesting situation exists in which two products of the same gene, although most likely derived from different locales and through different routes governed by post-translational processing, clearly oppose each others’ actions, while potentially cooperating in the activation of insulin release. The best-known and predominant physiological effect of mammalian (truncated) GLP-1 is its insulinotropic activity. Being released preferentially from the distal gut after a meal, when the level of plasma glucose is elevated, GLP-1 stimulates insulin excretion from pancreatic B-cells in a glucosedependent manner (Mojsov et al., 1987;Holz et al., 1993) while concurrently regulating the rate of insulin synthesis by B-cells and suppressing glucagon synthesis and release in pancreatic A-cells (D’Alessio et al., 1989). GLP-1 also increases the amount of insulin mRNA transcripts in a rat islet cell line coupled with increases in the rate of adenylyl cyclase (Drucker et al., 1987). Through its effect on insulin, GLP-1 may control hepatic glucose production and glucose clearance, thus favoring its use in the treatment of

224

E. M. PLISETSKAYA AND T. P. MOMMSEN

type I1 diabetes (Hvidberg et al., 1994). In its insulinotropic action, GLP1 is about 100-fold more potent than glucagon. Whenever analyzed, the two short forms of GLP-1 are interchangeable with no differences in their apparent biological potency. In addition, GLP-1(7-36)-amideexerts a number of other actions. These include inhibition of gastric acid secretion in humans (O’Halloran et al., 1990), stimulation of cAMP production in isolated gastric glands (Hansen etal., 1988),and activation of SSTsecretion in the perfused isolated pancreas (0rskov et al., 1988), as well as repression of the pentagastrin-induced secretion of gastric acid in humans (Schjoldager et al., 1989). Interestingly, the peptide fails to activate adipose tissue lipoprotein lipase, while GIP, which shares GLP-1’s powerful insulinotropic features, does (Suzuki et al., 1992a;Knapper et al., 1995). In lung, the hormone is involved in the regulation of macromolecule biosynthesis and relaxation of the pulmonary artery (Richter et al., 1993). The glucagonostatic action of GLP-1 (i.e., its potent inhibitory action on glucagon secretion) is independent of glucose concentrations, while its insulinotropic actions are not (Komatsu et al., 1989). Intriguingly, the removal of two amino acid residues from the C-terminal end of the peptide somewhat decreases its insulinotropic effectiveness but obliterates its glucagonostatic action (Suzuki et al., 1989), implying indirectly that different intracellular routes of message transduction govern these two processes. As in vivo, the most important effect of GLP-1 in mammals in vitro is GLP-1’sstrong insulinotropic activity,discovered simultaneously in isolated perfused pancreas of pigs (Holst et al., 1987) and rats (Mojsov et al., 1987). Subsequent studies have elucidated the mechanism of insulinotropic action of GLP-1(7-36)and GLP-1(7-37)(Holst and Brskov, 1994).The GLP-1 agonist exendin-4 can replace GLP-1 in its insulinotropic (Goke et al., 1993a) and acid secretory action (Danger et al., 1991) in mammals. It seems that in mammals GLP-1 is devoid of any direct metabolic activity, especially targeting the liver (Ghiglione et al., 1985; Shimizu et al., 1986; Murayama, et al., 1990), and any potential metabolic effects are mediated indirectly by GLP-1-dependent release of insulin (Hvidberg et al., 1994; Egan et al., 1994). The only research group that found any hepatic effects of GLP-1 (Valverde et al., 1994) reported that GLP-lp36) amide in physiological concentrations stimulates the formation of glycogen from glucose in isolated rat hepatocytes. This effect is accompanied by the stimulation of glycogen synthase “a” activity, a decrease in cAMP content, and can be abolished by glucagon. These results seem to be contradicted by a large body of independent evidence on mammalian models, in which insulinindependent metabolic effects of GLP-1 on the liver seem to be absent (Ghiglione et al., 1985; Murayama et al., 1990; Blackmore et al., 1991), as is

GLUCAGON FAMILY PEPTIDES IN FISHES

225

binding of GLP-1 to hepatocytes or liver membranes (0rskov and Poulsen, 1991), or expression of GLP-1 receptor (Wei and Mojsov, 1995) in this tissue. Besides, in those mammalian tissues where GLP-1 is thought to exert a direct physiological function (3-cells, lung, brain, gastric mucosa), the hormone mediates its action through increases, not decreases, in CAMP. The latter property is clearly observed in all instances where GLP receptors have been transfected into mammalian cell lines. An earlier report on GLP-1-dependent glycogenesis in mammalian muscle cells (VillanuevaPeiiacarrillo et al., 1994) has recently been refuted quite convincingly (Fiirnsinn et aL, 1995). To date, GLP-2 has remained a somewhat cryptic peptide. Unlike its two counterparts, glucagon and GLP-1 coencoded in the same gene, neither GLP-2 nor the intervening peptide located between GLP-1 and GLP-2 sequences displays any insulinotropic potency in mammals (Schmidt et al., 1985; Komatsu et al., 1989; Weir et aZ., 1989). In addition, GLP-2 has no effect on the activity of adenylyl cyclase of rat liver membranes. Although initially human GLP-2 had been described to increase pituitary and hypothalamic adenylyl cyclase at concentrations in the midpicomolar range (Hoosein and Gurd, 1984), a single clear-cut physiological function in brain remains to be correlated with this peptide. Of the two remaining products of the proglucagon gene, glicentin seems to display low biological activity, if any (Bataille et al., 1986, 1988), while oxyntomodulin is highly active in stimulating gastric adenylyl cyclase and in inhibiting acid secretion by gastric oxyntic glands previously stimulated by pentagastrin (Moody and Thim, 1983; Bataille et al., 1988).

B. Metabolic Effects of Glucagon and GLP in Fish While the primary structures of GLP-1s in fishes and mammals differ substantially, these peptides are surprisingly similar, if not identical, in their biological actions. On the one hand, the metabolic effects of mammalian GLP-1 (31 residues) or the amidated 30-residue form are indistinguishable from those of fish GLP-1 when tested with a piscine system. On the other hand, fish GLPs are effective insulinotropins in mammalian test systems (Plisetskaya and Duguay, 1993), with potencies very similar to those of mammalian GLP-1 (Fig. 5A). As in many other aspects mentioned earlier, fish constitute a very challenging and different model for physiological studies, including glucagon and GLP-1. While the specific actions of glucagon are fairly similar across all vertebrates, with minor variations on the theme, the same cannot be said about GLP-1. First, all fish studied to date encode and synthesize a GLP1 sequence in the endocrine pancreas (Table 11), albeit at times from

226

E. M. PLISETSKAYA AND T. P. MOMMSEN

two different genes. Consequently, the peptide should be entering the circulatory system along with pancreatic glucagon and intestinal GLP-1. Second, GLP-1 is indeed released from perifused Brockmann bodies of coho salmon (Oncorhynchus kisutch) (E. M. Plisetskaya, unpublished results). Third, the higher levels of GLP-1 in fish compared with mammalian plasma (Table VI) might not only be a result of slower clearance rates at lower ambient temperatures, but also of more abundant secretion of the peptide from two different sources. Fourth, native fish GLPs-1 do not possess the six amino acid N-terminal extensions characteristic of mammalian GLP-l(l-37).Native fish GLP-ls released from the pancreas (and intestine?) are thus identical to the processed-truncated-mammalian GLP-1 released by the intestine. Finally, the physiological effects of GLPs in fish appear very different from the effects of the same peptides in mammals. As mentioned above, these physiological effects are independent of the origin of GLP-ls, and fish and mammalian peptides are freely interchangeable, without differences in biological activity (Fig. 6).

: 1E-09

1E-08

I€-07

1E-06

1E-05

Hormone Concentration (M)

1E-04

FIG. 6 Stimulation of glucose release from isolated fish hepatocytes by mammalian glucagonlike peptide(7-37, (GLP-l,.) compared with bovine glucagon (V)andepinephrine (0).Hepatocytes were incubated at the given hormone concentration for 30 min in the absence of added carbon sources. Glucose released from endogenous glycogen into the surrounding medium was measured enzymatically. Values are expressed as a percentage of the maximum response achievedwith6.6 X 10-7MGLP-1.Cells wereisolatedfromoneadultredIrishlord(Hemi1epidotus hemilepidotus, Teleostek Cottidae) according to data of Danulat and Mommsen, unpublished.

GLUCAGON FAMILY PEPTIDES IN FISHES

227

Glucagon is involved in multiple actions in fish, although the effects are not totally consistent among species. The majority of relatively short-term (up to 6 hr) experiments in which glucagon was injected into fish resulted in hyperglycemia and depletion of liver glycogen. Experiments published before 1975 were reviewed by one of us (Plisetskaya, 1975). Results of the administration of glucagon to eels (Ince and Thorpe, 1977;Inui and Yokote, 1977; Chan and Woo, 1978), carp (Murat and Plisetskaya, 1977),Pimelodus maculatus-a Brazilian freshwater teleost-(Carneiro and Amaral, 1983), rainbow trout (de la Higuera and Cardenas, 1986), black bullhead catfish (Ottolenghi et al., 1988b), and coho and chinook salmon (Plisetskaya et al., 1989a) suggest involvement of this hormone in the regulation of plasma glucose, liver glycogen, and some hepatic enzymes. Affected are fluxes through glycogenolysis (Mommsen and Moon, 1989, 1990; Mommsen et al., 1991a,b; Conlon et al., 1993b; Nguyen et al., 1994; Ottolenghi et al., 1994b), gluconeogenesis (Mommsen et al., 1991a; Suarez and Mommsen, 1987) and glycolysis. In the in vivo experiments of Plisetskaya and co-workers (1989a), glucagon and GLP-1 were both glycogenolyticand lipolytic. In addition, glucagon stimulates amino acid transport into the liver and other tissues of the Japanese eel (A. japonica) (Inui and Yokote, 1977; Inui and Ishioka, 1983a,b)and increases ammonia excretion by the ureogenic toadfish, Opsanus beta (Mommsen etal., 1992).With the exception of a report on increased amino acid oxidation in sea raven (Hemitripterus americanus) hepatocytes (Foster and Moon, 1987), the overall metabolic rate or oxidation of other substrates appears to be unaffected by glucagon treatment (Mommsen et al., 1987). In contrast to the situation in mammalian liver (Jackson et al., 1986), the hormone does not regulate the production of urea in fish (0. beta) liver cells. However, one of the ancillary enzymes of urea synthesisnamely, glutamine synthetase-is increased after injection of mammalian glucagon (Mommsen et al., 1992). Hepatic glutamine synthetase is the unique feeder enzyme for ammonia nitrogen in the piscine urea cycle (Mommsen and Walsh, 1991a)while glutaminase and glutamate dehydrogenase assume a similar function in mammalian liver. The involvement of glucagon in regulation of lipid metabolism-mostly by its lipolytic action through stimulation of triacylglycerol lipase-in fish and other ectotherm vertebrates-has been recently reviewed by Sheridan (1994). Glucagon alters the rate of somatostatin-25 and SST-14 release from Brockmann bodies (Eilertson et al., 1995) but exerts only a very weak insulinotropic action in fishes (Plisetskaya et al., 1989a,b). A number of enzymes have been shown to be regulated by glucagon, often through reversible phosphorylation. These enzymes include glycogen phosphorylase (Brighenti et al., 1991; Foster and Moon, 1990), pyruvate kinase (Petersen et al., 1987), triglyceride lipase (Harmon et al., 1993),

228

E. M. PLISETSKAYA AND T.

P. MOMMSEN

and adenylyl cyclase (Ottolenghi et al., 1988a). In the case of glycogen phosphorylase, the hormone appears to alter the amount of total assayable enzyme (Umminger and Benziger, 1975), as well as the percentage of enzyme present in the active (phosphorylated) a form (Vernier and Sire, 1978; Brighenti et al., 1991). Further, glucagon-dependent increases have been documented for the activity of aspartate aminotransferase, alanine aminotransferase, and malic enzyme (Chan and Woo, 1978; Gerhard et al., 1988; Mommsen et al., 1992). Enzyme activities negatively affected by glucagon include pyruvate kinase (Petersen et al., 1987), glycogen synthase (Murat and Plisetskaya, 1977), phosphofructokinase (Foster et al., 1989), glucose 6-phosphate dehydrogenase, and hydroxyacyl coenzyme A dehydrogenase (Mommsen et aL, 1992). In the temperate zone fishes analyzed to date, the metabolic effects of glucagon and GLP-1 are strongly dependent on nutritional status and feeding. Effects tend to be enhanced in summer compared with spring and autumn (Plisetskaya, 1975). In contrast to fasting mammals and birds, in which glucagon levels are usually elevated when plasma insulin declines (Hazelwood, 1984; Unger, 1985), such increases in fish are only transient or are absent altogether (Navarro et al., 1992).During periods of fasting, the plasma levels of all three peptides-insulin, glucagon, and GLP-1-decline (Moon et aL, 1989). However, the rates of decline are not uniform. Insulin levels decline faster than those of glucagon and GLP, shifting the ratio of (glucagon+GLP-1) over insulin. The resulting increase in the ratio in favor of glucagon and GLP-1, in turn, may trigger gluconeogenic flux through activation of gluconeogenic enzymes in fasting fish (Moon et al., 1989). This feature may be an important weapon in the metabolic arsenal, since periods of self-imposed fasting form a fundamental part of the life cycles of many fishes. The experiments of Storebakken and co-workers (1991) illustrate this point. Three groups of rainbow trout received an identical diet, at doses totaling 2, 1, and 0.3% of their body weight per day, respectively, while a fourth group of fish was deprived of food. At the end of the experiments, plasma insulin levels differed by an order of magnitude and ranged from 22.0 ng/ml, 12.8 ng/ml, 4.8 ng/ml to 2.2 ng/ml (3.9 nM to 0.39 nM) in the four groups of fish, respectively. Glucagon levels also decreased in the suboptimally fed groups and in the fasting fish, but declined only from 0.23 to 0.08 nglml (0.07-0.02 nM), while GLP-1 levels experienced a smaller drop from 1.3 to 0.9 ng/ml (0.4-0.26 nM) in the extreme groups. As a result, the above ratio is skewed in favor of glucagon gene family peptides over insulin. In concert with some other hormonal changes, this may serve to direct metabolic flux toward lipolysis, depleting lipid depots in the liver and fat stores in the white and red skeletal muscles (Storebakken et al., 1991).

GLUCAGON FAMILY PEPTIDES IN FISHES

229

Acute deficiency in either glucagon or GLP resulting from administration of antisalmon glucagon or antisalmon GLP-1 sera led to comparatively smaller metabolic changes than deficiency in insulin or somatostatin. As expected, and in line with the postulated glycogenolyticand gluconeogenic role of glucagon in fishes, immunoneutralization of glucagon results in increased liver glycogen content and conversion of pyruvate kinase into the dephosphorylated (more active) form (Plisetskaya et al., 1989b). Again, in contrast to the mammalian pattern (Tan et al., 1985a,b), acute insufficiency in either glucagon or GLP-1 or both peptides at the same time failed to affect the arginine-stimulated release of insulin from the fish endocrine pancreas (Plisetskaya et al., 1991). Implicit in these results is that secretion of insulin in fish is relatively independent from glucagon family peptides. In the initial experiments on fish, glucagon was used in very high, generally pharmacological concentrations. Although this approach may be reasonable in identifying potential target tissues, pathways, and message transduction systems, it does not allow any conclusions with respect to the in vivo situation. Also, tissues responding rapidly with receptor desensitization may be erroneously excluded from the list of potential targets. More recently, when researchers became aware of actual levels of glucagon family peptides in piscine plasma, doses of glucagon and GLP-1 were adjusted to be more reasonable physiologically. There are numerous studies of the in vitro effects of glucagon and GLP1 in fish. Liver slices and dispersed hepatocytes, either in static incubation or immobilized in perfusion columns, have served as important tools for such studies. Anatomical and spatial relationships in piscine liver differ substantially from those in mammals and thus fish lack the physiological diversity of mammalian liver (Tosh et aL, 1988; Jungermann and Katz, 1989). Fish liver is composed almost exclusively (>95%) of parenchym:-1 cells and is characterized by small diffusion distances and a comparatively small extent of metabolic zonation; as a result, the metabolic effects of glucagon and GLP-1 are almost identical in hepatocytes from periportal and perivenous locations (Mommsen et aL, 1991a ; Ottolenghi et al., 1991; Mommsen and Walsh, 1991b). Mirroring the in vivo response, in vitro experiments using glucagon caused depletion of glycogen and increased glucose release, and stimulated phosphorylase activity in liver slices of various fish such as brown bullhead (Umminger and Benziger, 1975); scorpion fish (Plisetskaya, 1975), killifish (Umminger and Bair, 1976), rainbow trout (Morata et al., 1982; Brighenti et al., 1991), catfish (Ottolenghi et al., 1988b, 1989), carp (Janssens and Waterman, 1988), and coho salmon (Plisetskaya et aL, 1989a). Phosphorylase activity and glycogen content in white and red skeletal muscle slices remained unaffected (Ottolenghi et al., 1988b). Glucagon also increased gluconeogenic rates from lactate in carp with lower hepatic glycogen content

230

E. M. PLISETSKAYA AND T. P. MOMMSEN

(Janssens and Waterman, 1988) and stimulated the entry of amino acids into eel liver slices independently of protein synthesis (Inui and Ishioka, 1983a,b). The rate of gluconeogenesis is increased by glucagon through increasing lactate and alanine fluxes to glycogen in trout (Mommsen and Suarez, 1984;Petersen et al., 1987),sea raven (Foster and Moon, 1987), and American eel (Foster and Moon, 1989). Further, the hormone participates together with insulin in the regulation of 6-phosphofructo-1-kinaseactivity (Foster et al., 1989). Glucagon applied in somewhat pharmacological doses increased the rate of lactate production from endogenous glycogen in the gas gland of bluegill sunfish (Lepornis rnacrochirus) (Deck, 1970).Although this tissue is thought to rely largely on exogenous glucose and not endogenous glycogen for its metabolism, it is likely that this effect indicates a hormonal effect on glycolytic flux rather than the usual effect on the phosphorylation status of glycogen phosphorylase. Needless to say, experiments should be done to identify the true target in gas gland tissue and to analyze the potential effects of the hormone on acid secretion and other outputs of the gas gland cells so intricately linked to their metabolic behavior (Pelster, 1995). The nutritional state of the experimental fish used as a source of hepatocytes or liver slices modulated hormone-mediated glycogenolysis in coho (Sheridan and Mommsen, 1991) and chinook salmon (Klee et al., 1990). While glucagon stimulated the release of glucose from liver slices from fed fish and fish that had fasted for 1 week, this effect disappeared in fish starved for 3 weeks. Epinephrine, in contrast, retained its glycogenolytic potency on the liver slices from fish fasted for 3 weeks (Klee et al., 1990). When applied in equimolar concentrations, epinephrine appeared to be more potent in causing glycogenolysis than glucagon (Klee et al., 1990; Ottolenghi et af., 1991). However, in our own studies on glycogenolysis in perfused and statically incubated hepatocytes of different species of marine teleostean fishes from four different families, glucagon was always a more potent and more effective glycogenolytic agent than the catecholamine (Fig. 6). The nutritional condition of the hepatocyte donor fish also influences the effects of glucagon on lipid metabolism. Glucagon directly stimulated lipid breakdown in both liver slices and adipose tissue, manifested by enhanced fatty acid and glycerol release into the culture medium. The intracellular target was triacylglycerol lipase whose phosphorylation status was changed in a hormone-dependent manner (Harmon et al., 1993). Glucagonstimulated lipolysis was more pronounced in livers sampled from fish fasted for 4 weeks than in the liver from fed fish, although, curiously, 2 additional weeks of fasting did not affect lipolytic rate in response to glucagon (Harmon and Sheridan, 1992).

GLUCAGON FAMILY PEPTIDES IN FISHES

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Isolated dispersed hepatocytes, especially immobilized in miniperifusion columns, proved to be an ideally suited model for investigation of biological effects of glucagon and GLP-1 (Ottolenghi et al., 1994a,b;Mommsen et al., 1994), although one of the limitations of this sensitive technique is the inability to concurrently analyze the mechanisms of intracellular message transduction. By and large, these studies confirmed previous results on statically incubated hepatocytes, but also showed that isolated cells were generally much more sensitive to added hormones, thus bringing in vitro conditions close to the in vivo situation. As expected, exendin-4 can activate glycogenolysis in isolated fish liver cells, with a dose-response relationship that is indistinguishable from the one obtained for piscine or mammalian GLP-1 (T. P. Mommsen and E. R. Busby, unpublished results). Soon after the discovery of GLP-1, we initiated studies on the effects of salmon, and later mammalian GLP-1, on metabolism of fish hepatocytes. It became apparent immediately that piscine (anglerfish, catfish, and coho salmon) as well as mammalian GLP-1s could function as powerful metabolic hormones. Without exception and with surprising uniformity, the peptides stimulated flux through gluconeogenesis from lactate (Mommsen et al., 1987) and activated the rate of glycogenolysis (Ottolenghi et al., 1989; Mommsen and Moon, 1990). These experiments on isolated hepatocytes validated the notion that the hyperglycemia observed in in vivo experiments arises from either enhanced glycogen breakdown or faster gluconeogenesis or a combination of both pathways, with ensuing stepped-up hepatic production of glucose. Studies on hepatocytes also confirmed that the Nterminally extended version of GLP-1(1-37)-a peptide truly foreign to fishes-possesses only a fraction of the metabolic activity of the short GLP-1. We initially showed that when applied in equimolar concentrations, GLP1s were more potent than glucagon, but stimulated CAMP production to a much smaller degree than the latter peptide (Mommsen et al., 1987). These primary findings were confirmed in many studies that followed (Mommsen and Moon, 1990). In a recent study, Ottolenghi et al. (1994a) compared the glycogenolytic effects of catfish GLP-1, catfish glucagon, anglerfish glucagon 11, and synthetic fragments (19-29) of anglerfish glucagons derived either from proglucagon I or 11. The highest rate of glucose release from perfused liver cells at a dose of 1 nM was initiated by catfish GLP-1. Derivatives of glucagon I and especially glucagon 11(19-29) were less potent than GLP-1, but did not differ significantly between each other or from catfish and anglerfish glucagons. When the same doses of peptides were applied in static incubation, only catfish GLP-1 and anglerfish glucagon 1(19-29) stimulated glucose release. The results of this study corroborate reports by Mommsen and Moon (1990), who noticed that glucagon 11(19-29)

232

E. M. PLISETSKAYA AND T. P. MOMMSEN

was without effect on glycogenolysis in statically incubated hepatocytes from three species of teleosts. In several fish species (Mommsen and Moon, 1989,1990;Mommsen et al., 1991b)and especially in Pacific rockfishes of the genus Sebastes (Danulat and Mommsen, unpublished results) and some marine cottids, including the red Irish lord (Hemilepidotus hemilepidotus), the discrepancy in the relative effectiveness of GLP-1 and glucagon in activating glycogenolysisis easily noticed. GLP-1 is persistently more effective by a factor of five at activating this pathway than glucagon (Fig. 6). This phenomenon is independent of the sources of GLP (piscine or mammalian) or glucagon (coho salmon, catfish, bovine). To put the efficacy of these two peptide hormones into perspective and to validate the test system, Fig. 6 also includes data for epinephrine, a known and relatively powerful glycogenolytichormone in fishes (Hanke and Janssens, 1983;Mommsen et al., 1988,Ottolenghiet al., 1989;Fabbri et al., 1992). Since most of these experiments were done in static assays and using incubation times of up to 1hr, the possibility of an experimental artifact cannot be dismissed offhand. As described in Section III,B,l, fish liver displays an outstanding, if differential, ability for uptake (degradation?) of glucagon and GLP-1. In feeding fish, 77-86% of glucagon (Plisetskaya and Sullivan, 1989; Carneiro et al., 1993) and 59% of GLP-1 (Plisetskaya and Sullivan,1989)were removed from the circulation in a single pass through the liver. Therefore, different amounts of intact peptide remaining in the media at the end of the incubation period could skew the dose-response curves for the two peptides. To eliminate such possibility, we have determined the sensitivity of glycogenolysis to both GLP-1 and glucagon by hepatocytes of Sebastes caurinus immobilized in a carrier and constantly infused with fresh peptides. Under these conditions, the media were not recirculated and thus hormone internalization and degradation should have no effect on the actual hormone concentrations encountered by the cells. The sensitivity of glycogenolysisto GLP1found in this experiment was still much higher than to glucagon (Mommsen and Plisetskaya, unpublished results). All the studies described imply that, in contrast to the situation in mammals where liver has been dismissed as a target organ for GLP-1 (Section IV,A), in fish GLP-1 is a more potent metabolic hormone than glucagon, proving that GLP-1 is “glucagon-like,” not just in amino acid structure and name, but also in function. Activation of glycogenolysis and/or gluconeogenesis by GLP-1 has been described for each of twenty species of teleostean fish analyzed to date. Unfortunately, other fishes with fewer derived traits, such as agnathans, elasmobranchs, or even holosteans have not been analyzed yet. Amazingly, the clear-cut glycogenolytic effect of GLP-1 on the liver is widespread among the teleostean fishes, but apparently restricted to this group of vertebrates. Research in our laboratories has shown that GLP-1 exerts no comparative metabolic effects in hepatocytes of amphibi-

233

GLUCAGON FAMILY PEPTIDES IN FISHES

ans (Rana sp., Xenopus laevis) or a reptile (Pseudernys scripla), although the hepatocytes were fully responsive to glucagon and epinephrine (Mommsen and Moon, 1994). Note that fully functional (i.e., short) GLP-1s have been isolated from the pancreas of two amphibians, as has GLP-2 (Pollock ef ul., 1988b). Thus, the amphibians appear to display another variation on the GLP theme, possessing pancreatic GLP-1 and pancreatic GLP-2, juxtaposed to an apparent lack of hepatic response to GLP-1, thus occupying some unexplored middle ground between fishes and mammals. In contrast to being “superglucagon” in its metabolic potencies, GLP1s of mammalian or piscine origin are particularly poor insulinotropins in fishes. Insulinotropic effects of glucagon or GLP-1 administered in fish in different doses were either nonexistent or very weak. Only during the spring, when juvenile coho salmon undergo smoltification, did GLP-1 slightly elevate the plasma levels of insulin (Plisetskaya et al., 1989a). When added to the media perfusing whole Brockmann bodies, GLP-1 caused a transient insulin release only at concentrations exceeding M (i.e., well beyond its physiological range) and in a range where the peptide begins to displace glucagon from its receptor (Navarro and Moon, 1994) (Fig. 7). Irrespective of origin (mammalian or piscine), GLP-1s exerted no insulinotropic effect when added to the incubation media of dispersed pancreatic cells of copper rockfish (Sebasfescuurinus) (Fig. 5B) (Mommsen and Pliset-

100 M

.-a

0

’

90

80 70 .

.-.6

i r(

60

50 . 40 -

V

2

2

30 20 .- 0 GLP 10 0

.r

GLUCAGON

3 -12

-11

-10

-9

-8

-7

-6

-5

pM

log Ihormoncl

FIG. 7 Displacement of ‘251-labeledglucagon bound to freshly isolated eel (AnguiZZu rostruratu) hepatocytes by mammalian GLP-1(7-37). [From Navarro and Moon (1994) with permission.]

234

E. M. PLISETSKAYA AND T.

P. MOMMSEN

skaya, 1993b), but gave the expected potent insulinotropic response in dispersed mammalian cells (FigSA). Although a systematic study of the glucose dependence of this effect in fishes remains to be done, the experiments with GLP-1 immunoneutralization-which failed to affect insulin levels (Plisetskaya et al., 1989b)already hint that surprises are remote. Considering the general glucose intolerance of fishes, together with their, at least by mammalian standards, high insulin titers in plasma (Table VII), it is unlikely that glucose will play an overriding role in regulation of insulin release from endocrine pancreas. In fact, it has already been shown that glucose is a relatively poor insulinotropic compound in fishes, certainly much less effective than insulinotropic amino acids such as arginine or lysine. However, it has to be kept in mind that most of these studies have been done on carnivorous (rainbow trout, pike) or omnivorous (eel, catfish) species which have a comparatively low dietary glucose intake. In future, it would be interesting to focus attention on the glucose dependence of pancreatic hormone output either in species that are known to tightly regulate plasma glucose at “mammalian” levels, such as some tunas, or in vegetarian species (tilapia, grass carp). A unique position may be filled by some teleosts of the South American rainforests, such as Colossorna species, some of which feed preferentially on glucose/ fructose-rich fruit, or lipid-rich nuts. In the fishes, even less work has been done with GLP-2, largely because until the recent report of GLP-2 expression in rainbow trout intestine (Irwin and Wong, 1995), the hormone had been considered absent in fishes. In contrast to GLP-1, which has pronounced effects on fish liver metabolism, application of human GLP-2 failed to alter the rate of gluconeogenesis or glycogenolysis in isolated teleostean hepatocytes (Mommsen et al., 1987). We have recently confirmed this negative result with synthetic trout GLP2 for different species of fish (T. P. Mommsen and D. M. Irwin, unpublished results).

V. Signal Transduction

A. Glucagon In the fishes, glucagon appears to tie preferentially into the adenylyl cyclase system of message transduction, mediated through stimulatory G-proteins. Although glucagon-dependent increases in intracellular cyclic adenosine monophosphate (CAMP) concentrations have been reported numerous times, especially in the presence of isobutyl-3-methylxanthine (IBMX) or other phosphodiesterase inhibitors, little attention has been devoted to the

GLUCAGON FAMILY PEPTIDES IN FISHES

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nature of the G-proteins involved or to possible alternative systems. One major drawback of the existing studies on glucagon is the fact that fish liver is fairly insensitive to added glucagon, and generally clearly supraphysiological concentrations have been employed to activate key metabolic pathways and to record concurrent increases in intracellular CAMP.At this pharmacological rather than physiological level of peptide, it is not surprising that glucagon-dependent increases in cAMP can be detected, although a relatively poor correlation between hormone concentration and the level of pathway activation exists in the liver (Mommsen and Moon, 1990; Foster and Moon, 1990). Thus, the question of message transduction systems at low concentrations of the peptide is left open. In a study on catfish (I. rnelas) hepatocytes, for instance, epinephrine and native salmon glucagon, both applied at 30 nM, stimulated glycogenolysis to equal degrees (about threefold). Yet, although cAMP concentrations were significantly increased above control levels in both treatment groups, cAMP levels in epinephrinetreated cells exceeded those attained in response to glucagon by at least five times (Ottolenghi et aL, 1991). Glucagon was much less effective in stimulating glycogenolysisin toadfish (0. beta) hepatocytes than GLP-1, reflected in a right shift of the doseresponse curve by a factor of five, while both peptides were equipotent at increasing the rate of glucose synthesis from lactate (Mommsen et al., 1991b). The fact that both peptides activate different pathways with different efficiencies implies that selective postreceptor events in the activation of glycogenolysis somehow bridle the response to glucagon. Considering the seemingly direct route by which glycogen phosphorylase is normally activated, namely, by the well-known CAMP-dependent protein kinase cascade, necessary interactions with other pathways (e.g. Ca2' mobilization) may be compromised. Surprisingly, the generally low responsiveness to glucagon (Ottolenghi et al., 1988b) (Fig. 6 ) contrasts with the results of a detailed analysis of glucagon binding to the receptors of fish liver membranes (Fig. 7). Apparent dissociation constants (K,) for high-affinity binding sites fall into the 2 nM range (2.7 for eel, 2.0 for catfish), indirectly confirming the idea of selective attenuation within the pathway controlling glycogen phosphorylase. While these concentrations are about ten times higher than those commonly determined for mammalian hepatic glucagon receptors, it should be considered that comparatively higher glucagon concentrations reach the fish liver. Apart from this discrepancy between fish and mammalian glucagon receptors in liver, numbers of high-affinity binding sites for glucagon per liver cell are on the same order of magnitude for these two groups of vertebrates (Table VIII). In addition to the high-affinity glucagon binding sites referred to above and characterized in Table VIII, both brown bullhead and eel liver cells also display low-affinity binding sites, with KD values ranging around

236

E. M. PLISETSKAYA AND T. P. MOMMSEN

TABLE Vlll High-Affinity Binding Sites for Mammalian Glucagon in Hepatocytesof Vertebrates

KD

Temperature ("C)

3,800

2.68 1.97

Rat

5,200 14,000 14,000 19,800

1.25 0.88" 0.12 0.20

Dog

8,300

0.18

12 12 37 37 37 37 37

Species

Binding sites per cell

American eel Bullhead catfish

10,400

(nM)

Reference Navarro and Moon (1994) Navarro and Moon (1994) Kashiwagi et al. (1985) Horwitz et al. (1985) Honvitz et al. (1985) Horwitz and Gurd (1988) Bharucha and Tager (1990)

Competition assay.

20 nM(Navarro and Moon, 1994). Although already beyond the physiological limits for the systemic levels of the peptide, intriguingly this is the concentration range for in vitro half-maximum stimulation of glycogenolysis in hepatocytes of many piscine species. However, as mentioned, this is still only the lower concentration range of glucagon, resulting in reproducible increases in intracellular CAMP concentrations (Ottolenghi et d.,1989). In fish liver, glucagon is internalized to a similar degree as in rat liver cells (Horwitz and Gurd, 1988), and, just as in dog hepatocytes (Hagopian and Tager, 1984), only a small fraction of the internalized hormone is degraded. A certain degree of negative feedback exists between glucagon exposure and the number of high affinity glucagon binding sites in fish: preincubation of eel or bullhead hepatocytes with 100 nM mammalian glucagon diminished the number of binding sites to about 57% of normal in both species, without affecting the binding constant of the remaining binding sites (Navarro and Moon, 1994). This resembles the situation in mammals where homologous downregulation of glucagon receptors has been documented. It is also worth mentioning that intracellular binding sites for full-length glucagon have been found in mammalian liver associated with the Golgi apparatus and functioning independently of adenylyl cyclase (Lipson et aZ.,1986). Except for some studies analyzing changes in enzyme activity, little attention has been devoted to the longer term effects of glucagon in fishes, and in no case have the mechanisms underlying these changes been analyzed. With CAMPsupposedly assuming a central role in glucagon action, it is surprising that not a single study in fishes has analyzed the involvement of CAMP-responsive elements and their binding proteins (Meyer and Habener, 1993) in hormone action in general. In spite of the apparent close relationship between vertebrate glucagons and their sequence conservation, subtle differences in function do occur.

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For instance, catfish glucagon, which can be quantified using mammalian radioimmunoassay systems and which activates glycogenolysis in fish hepatocytes as effectively as mammalian glucagon, fails to displace mammalian glucagon from isolated rat liver membranes (Hoosein et al., 1987). Surprising as it may seem, there is still some debate about the intracellular message transduction mechanism for glucagon in mammals. The controversy arises mainly over the involvement of intracellular calcium in message transduction, with most authors acknowledgingthat at slightly supraphysiological concentrations glucagon activates adenylyl cyclase via a stimulatory Gprotein. AS a consequence, metabolic targets are regulated by phosphorylatioddephosphorylation mechanisms mediated by protein kinase A (PKA). A prime example of this immediate, covalent modification is the turning off of pyruvate kinase by glucagon, which is followed by decreases in glycolytic flux and increases in the rate of gluconeogenesis.In our own studies on rockfish hepatocytes, we noticed glucagon-dependent increases in phosphorylation of glycogen phosphorylase within 20 sec of hormone application (T. P. Mommsen, G. A. Cooper, and T. W. Moon, unpublished results). 6 . Glucagon-like Peptide 1

In the case of GLP-1, the picture emerging from studies on piscine systems is as yet incomplete. In the absence of studies on nonhepatic tissues, we have to restrict the following discussion to the parenchymal liver cells. While in some species, including the American eel and a couple of ictalurid catfishes (Ottolenghi et al., 1989; Mommsen and Moon, 1990), liver cells respond to exposure of GLP-1-from mammalian or piscine sources-with an increase in intracellular CAMP, one important fact cannot be ignored: GLP-1 concentrations leading to detectable increases in cAMP are at least an order of magnitude above those necessary to cause noticeable metabolic activation. Thus, the cAMP response is only elicited at supraphysiological concentrations of the peptide. In other species, including the rainbow trout and copper rockfish (a scorpaenid), which respond just as sensitively to GLP-1, complete absence of a cAMP response is observed, even at comparatively high concentrations of the peptide (Mommsen ef aZ., 1987;Mommsen and Moon, 1990; E. Danulat and T. P. Mommsen, unpublished results). As with glucagon, then, the actual intracellular message transduction system(s) activated at physiological concentrations of hormone is (are) yet to be determined. In spite of the information accumulated to date, it is entirely possible that cAMP plays a role in message transduction, but rather as a mediator between different pathways through crosstalk, instead of constituting the sole transduction mechanism. This view is supported by our experiments

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E. M. PLISETSKAYA AND T. P. MOMMSEN

on several species of fishes, where exposure to Rp-CAMPS, an antagonist of CAMP-dependent protein kinase (protein kinase A) diminished, but did not obliterate, the metabolic effects of GLP-1 (T. P. Mommsen, G. A. Cooper, and E. R. Busby, unpublished results). It is also possible that subtle changes in the ratio of free to bound cAMP coregulate metabolic output, but are snubbed, since common methods measure total cAMP only and few researchers bother to analyze cAMP in subfractions. Some resemblance to GLP-1 message transduction in mammalian B-cells cannot be denied. Initially, adenylyl cyclase activation and intracellular cAMP levels were de rigueur in identifying GLP-1 action in mammalian B-cells and cell lines. Recently the picture has been expanded to include GLP-1-dependent activation of phospholipase C and the mobilization of intracellular calcium stores, a conclusion reached either directly (Wheeler et al., 1993; Yada et al., 1993) or indirectly by determining the effects of membrane depolarization on the insulinotropic action of GLP-1 (MontroseRafizadeh et al., 1994). With the exception of brain (Hoosein and Gurd, 1984) and lung (Goke et al., 1993b), where GLP-1 has been shown to activate the cAMP signaling system, it is not clear by which mechanisms GLP-1 modulates cellular performance in mammalian nonpancreatic target tissues, such as kidney or heart muscle. The recent cloning of GLP-1 receptors from rat and human tissues (Thorens, 1992; Dillon et al., 1993; LankatButtgereit et al., 1994) has helped to shed some light on the issue of why often supraphysiological concentrations of GLP-1 are needed to activate the adenylyl cyclase system. Based on structure, the mammalian GLP-1 receptors belong to a receptor superfamily characterized by seven membrane-spanning domains. Considering amino acid homologies, this characterization can be narrowed down to a smaller family of G-protein-coupled receptors that do not share any sequence similarities with other known G-protein-dependent receptors. This subfamily includes the receptors for glucagon (Jelinek et al., 1993; MacNeil et al., 1994; Carruthers et al., 1994), parathyroid hormone, VIP, GHRH, and PACAP (SegrC and Goldring, 1993). All these hormones are thought to couple to adenylyl cyclase as well as to phospholipase C , resulting in the mobilization of intracellular calcium (SegrC and Goldring, 1993) and thus stimulating multiple intracellular transduction pathways. Interestingly, in COS-7 cells transfected with a GLP-1 receptor cloned from a human insulinoma, GLP-1 binding increased cAMP only, while stable transfection of the same receptor into fibroblast CHL cells resulted in augmentation of cellular cAMP as well as transient increases in intracellular Ca2+ (Van Eyll et al., 1994). While the receptor sequence may contain as-yet unidentified structural provisions needed to communicate with different transduction systems, it can be hypothesized that ultimately the activation of a specific intracellular pathway may be determined by the target tissue expressing a given receptor.

GLUCAGON

FAMILY PEPTIDES IN FISHES

239

Although it was reported that GLP-1 might act synergistically with glucose on metabolically controlled K ( A ~channels ~) of B-cells (Holz et al., 1993), additional patch-clamping experiments failed to support this concept, but found that the peptide modulated the calcium current in mouse B-cells (Britsch et al., 1995). At least as interesting as this minor effect on calcium channel inactivation might be the observation that GLP-1 induces regular oscillations in the B-cell membrane potential (Britsch et al., 1995). It has also been reported for a rat insulinoma-derived B-cell line that responds quickly and sensitively to added GLP with increases in insulin release that the number of GLP-1 receptors is under hormonal control and can be drastically reduced by the preexposure of cell cultures to dexamethasone, a synthetic glucocorticoid (Richter et al., 1989). All mammalian GLP-1 receptors cloned to date (Thorens, 1992;LankatButtgereit et al., 1994; Wei and Mojsov, 1995) possess virtually identical sequences, leading to the conclusion that the GLP-1 receptor has the same ligand specificity in all tissues. Some controversy has arisen over the actual tissues where these receptors are expressed and also concerning potential biological actions of the peptide in peripheral tissues which assume important roles in the metabolism of glucose, namely, skeletal muscle, liver, and adipose tissues. Some studies have shown metabolic effects of the peptide in mammalian liver and muscle, with glycogenic functions (VillanuevaPeiiacarrillo et al., 1994; Valverde et al., 1994) (see also Section IV,A), and in adipose tissue (Valverde et al., 1993; Egan et al., 1994), supported by localization of mRNA transcripts for the GLP receptor in these tissues (Egan et al., 1994). Yet these reports are in direct contrast to numerous other studies. Some of these have confirmed the absence of direct GLP-1 effects on mammalian liver (Ghiglione et al., 1985; Blackmore et al., 1991), the absence of activation of liver membrane adenylyl cyclase by GLP-1 (Blackmore et al., 1991), and the inability of liver to express the pancreatic type of GLP-1 receptor (Wei and Mojsov, 1995). In the face of these discussions, slowly additional physiological roles for GLP-1 in mammals are being characterized. These functions encompass reduction of plasma somatostatin and glucagon, control of glucagon release (Yamato et al., 1990), regulation of glucose utilization in diabetic mammals (Hvidberg et al., 1994), glycogenesis (Villanueva-Peiiacarrillo et al., 1994; Valverde et al., 1994), stimulation of intestinal somatostatin release (Eissele et al., 1990), inhibition of gastrin release (Eissele et al., 1994), and insulin-stimulated glucose metabolism in isolated adipocytes (Egan et af., 1994). In mammalian pancreas, the GLP-1 receptor is rapidly desensitized, possibly due to receptor phosphorylation. While similar receptor dynamics need to be analyzed for fish tissues, our own results probing GLP-1 effects on liver indicate that isolated hepatocytes decrease their response to GLP1 following a previous exposure to GLP-1. Subsequently, the cells remain

240

E. M. PLISETSKAYA AND T. P. MOMMSEN

refractory to the hormone for about 90 min before exhibiting renewed, but reduced, positive responsiveness (Mommsen and Plisetskaya, 1993a). Glucagon and GLP-1 receptors are closely related yet distinct receptors and as a rule they only interact with their proper ligand and no crossreactivity with the other hormone is observed. However, in at least one somatostatin-releasing cell line, a receptor has been described that is capable of interacting with different hormones of the glucagon family of peptides, including GLP-1, glucagon, and oxyntomodulin (Gros et al., 1993). Recently, chimaeras of mammalian glucagon-GLP-1 receptors have been successfully employed to gain insights into the receptor regions most critical to glucagon binding and to distinguish the glucagon receptor from a GLP1 receptor (Buggy et al., 1995). While studies on piscine GLP-1 receptors are still found wanting, an analysis of hepatic glucagon receptors from two teleostean fishes (American eel and brown bullhead catfish) showed that GLP-1 could displace labeled mammalian glucagon (14 pM) from the glucagon receptor, albeit not very efficiently (60%) and only at exceedingly high (10 mM) concentrations, while unlabeled glucagon leads to a 50% displacement at around 3 nM (Navarro and Moon, 1994) (Fig. 7). In mammals GLP-2 does not interact with the GLP-1 receptor and the peptide has no effect on the binding of glucagon or GLP-1 to their respective membrane receptors. There is some indication that the hormone may decrease the rate of DNA synthesis in some cell types (Lund et al., 1993), but no similar information is available for fish.

VI. Epilogue Glucagon and GLP-1 are both multifaceted and relatively plastic hormones. For glucagon, it is puzzling that the highly conserved mammalian peptide exhibits greater similarity to the peptide of elasmobranch fishes than to other piscine glucagons. The idea that this similarity might indicate a higher rate of molecular evolution of the gene in teleosts than in any other vertebrate group (Conlon and Thim, 1985) should stimulate some interesting research. Although CAMP is usually considered to be the most important intracellular messenger for glucagon, this elementary picture is under active debate for the mammals and deserves renewed attention for the fishes, not only in light of the potential actions of miniglucagons. Concurrent analysis of piscine glucagon receptors, including sequencing and tissue-specific expression under different conditions, will shed light on receptor dynamics and regulation, and elucidate the potential role of glucagon in osmoregulation and smoltification in fishes.

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Finally, the fact that GLP-1 appears to function as a “better” glucagon perhaps raises awareness about our rudimentary understanding of the role and mechanisms of glucagon in fishes and should provide food for thought about the evolutionary history of glucagon itself. Future research should also focus on a strict comparative analysis of targets, transduction mechanisms, and the interplay of glucagon and GLP-1, and analyze concurrently the many dynamic processes at the root of plasma hormone levels, namely, hormone production, processing, and release and hormone turnover at sites distant from those of gene transcription. In the case of GLP-1, the conservative nature of the peptide backbone among the vertebrates is counterpointed by a bewildering plasticity in all other facets of the hormone. Different sites of production (intestine in mammals vs pancreas and intestine in fishes), variable gene length, gene structure, unusual exon splicing, processing into bioactive forms, hormone concentration in the circulatory system, site of removal, and finally, fundamentally different targets (Table IX), all make fish GLP-1s and their mammalian counterpart an enigmatic group of peptides. Yet, in spite of all this divergence, the plethora of fish GLP-1s is freely interchangeable with the mammalian peptides and all peptides are equally powerful in their vertebrate group-specific functions. The relationship of GLP-1 to insulin may serve to illustrate this point. On one side are the fishes, where pancreatic GLP-1 appears to function as a superglucagon. Yet, substantially lower concentrations of GLP-1 than of glucagon are required to elicit tissue responses, while a priori plasma GLP-1 concentrations exceed those found for glucagon. It appears that the gene duplication that gave rise to GLP-1 has resulted in a group of glucagonlike peptides in the fishes that is more diverse in sequence than glucagon. Functionally these peptides seem to occupy a niche that at the present level of analysis is indistinguishable from glucagon. By exhibiting many of the metabolic powers attributed to glucagon, and by targeting the same tissues and pathways as glucagon, GLP-1 directly opposes many of the actions of insulin. This role is also supported by the absence of insulinotropic effects. However, this statement should include the caveat that insulin itself occupies a different, less glucosocentric position in fish metabolism (Mommsen and Plisetskaya, 1991). In this context, the weak glucose dependence of insulin secretion by pancreas should lead to comparative research on evolutionary trends in insulinotropic substances and mechanisms. It is really a shame that of the four important insulinotropins identified, only glucagon and GLP-1 have had some very limited analysis done while GIP and PACAP have been entirely ignored. We feel that in this respect, the fishes are an undervalued model system that might deliver important insights into the control of insulin release and evolutionary constraints of choice of substrates and hormones. Until more research has been done, it

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TABLE IX Characteristics of the GLP Complex in Mammalian and Piscine Systemsa

Characteristic Pancreas Proglucagon gene encodes Proglucagon mRNA transcript Glucagon-GLP-1 intervening peptide GLP-1 structure C-terminal extension N-terminal extension Intestine Proglucagon gene encodes GLP-I specifics GLP-1 contributed by Removal from circulation by Target organ (known function) (unknown function) Receptors expressed in Function Relationship with insulin Signal transduction

Fishes GLP-1, no GLP-2, stop codon after GLP-1 630-670 bases 6 or 7 amino acids No homology to truncated sequence

GLP-1 and GLP-2 ca. 1300 bases 16 amino acids 6-residue truncation

Present in some species Absent

Absent Present

Glucagon, GLP-1, GLP-2

Glucagon, GLP-1, GLP-2

Pancreas, gut liver, kidney (?) liver heart (?), brain (?)

Distal gut Kidney Pancreatic B-cells, lung Lung, brain B-cells, lung, brain Insulinotropin, Downregulates glucagon Accentuates insulin action CAMP,phospholipase C, CaZ+

???

Metabolic hormone, Glucagon-like Opposes insulin action Ca”, IP3 (?),

Secretagogues

Arginine, ?, ?

Plasma concentration (GLP-1)

30-850 pmoles/liter

a

Mammals

Glucose, GIP, catecholamines 6-45 pmoles/liter

See Fig. 2 for gene structure and tissue-specificprocessing into functional peptides.

cannot be dismissed offhand, especially considering the extended evolutionary history of fishes, that diet plays an overriding yet underappreciated role in the evolution of pancreatic hormone function. Researchers’ preoccupation with strictly carnivorous salmonids and some omnivorous species may have led to a skewed picture of the importance of glucose and consequently of insulin. On the other side we find the mammals. Here, expression of the proglucagon gene and secondary processing have reached a different dimension, resulting in a separation of sites of production for two opposing principles encoded in tandem in the same gene: the intestine contributes glucagonlike peptide-1, while pancreatic A-cells release glucagon. GLP-1 lacks overt direct metabolic actions, but its powerful insulinotropic role, its glucose

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dependence and negative modulation of glucagon expression and release have made it a prime candidate for treatment of diabetes mellitus. As far as other nonmammalian vertebrates are concerned, the picture remains fuzzy. Amphibians produce GLP-1 in their pancreas (a piscine trait), as well as GLP-2 (a unique trait), but amphibian liver does not respond directly to GLP-1 (a mammalian feature). The pancreatic proglucagon gene of birds contains GLP-1 only (a piscine trait), while intestine coencodes GLP-1 and GLP-2. Perhaps identification of a physiological role for GLP-2 will contribute to the clarification of the GLP-1 puzzle. Although GLP-1 presents itself as a highly conserved peptide, it has developed into two diametrically opposing principles regulating glucose metabolism in different groups of vertebrates. While we would like to arrive at a synthesis about how this feat has been achieved and what its consequences are, our attempts are thwarted by the limited data sets, especially those dealing with nonmammalian vertebrates. Irrespective of the “primordial” state and ultimately of function, the glucagon-like parts of the glucagon gene and its products have undergone some amazing twists in the course of vertebrate evolution. Among these are (1) the absence of GLP-2 expression in the pancreas of fishes and birds, (2) the synthesis of an inactive precursor peptide in mammals, (3) insertion (or deletion) of a truncatable intervening peptide, (4) pancreatic production of GLP-2 in amphibians only, and (5) GLP-1 maturation in intestine of mammals. A more thorough analysis of any these topics and their underlying mechanisms is likely to contribute to a better understanding of the evolution and function of this malleable, companion group of genes and their products. Acknowledgments While preparing this review, the authors were supported by National Science Foundation Grant DCB-8915935 to E. M.P., and a research grant from Natural Sciences and Engineering Research Council (Canada) to T.P.M. The authors are indebted to Dr. Elisabeth Urbinati for drawing Fig. 1.

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