Body fluid osmolytes and urea and ammonia flux in the colon of two chondrichthyan fishes, the ratfish, Hydrolagus colliei, and spiny dogfish, Squalus acanthias

The present study has examined the role of the colon in regulating ammonia and urea nitrogen balance in two species of chondrichthyans, the ratfish, Hydrolagus colliei (a holocephalan) and the spiny dogfish, Squalus acanthias (an elasmobranch). Stripped colonic tissue from both the dogfish and ratfish was mounted in an Ussing chamber and in both species bi-directional urea flux was found to be negligible. Urea uptake by the mucosa and serosa of the isolated colonic epithelium through accumulation of ¹⁴C-urea was determined to be 2.8 and 6.2 fold greater in the mucosa of the dogfish compared to the serosa of the dogfish and the mucosa of the ratfish respectively. Furthermore, there was no difference between serosal and mucosal accumulation of ¹⁴C-urea in the ratfish. Through the addition of 2 mM NH₄Cl to the mucosal side of each preparation the potential for ammonia flux was also examined. This was again found to be negligible in both species suggesting that the colon is an extremely tight epithelium to the movement of both urea and ammonia. Plasma, chyme and bile fluid samples were also taken from the agastric ratfish and were compared with solute concentrations of equivalent body fluids in the dogfish. Finally molecular analysis revealed expression of 3 isoforms of the urea transport protein (UT) and an ammonia transport protein (Rhbg) in the gill, intestine, kidney and colon of the ratfish. Partial nucleotide sequences of the UT-1, 2 and 3 isoforms in the ratfish had 95, 95 and 92% identity to the equivalent UT isoforms recently identified in another holocephalan, the elephantfish, Callorhinchus milii. Finally, the nucleotide sequence of the Rhbg identified in the ratfish had 73% identity to the Rhbg protein recently identified in the little skate, Leucoraja erinacea.     

A glucagon-like peptide, structurally related to mammalian oxyntomodulin, from the pancreas of a holocephalan fish, Hydrolagus colliei.

The pancreatic islets of the holocephalan fishes contain, in addition to A-, B- and D-cells, X-cells, which are immunoreactive towards antisera directed against the N-terminal region of glucagon but not towards antisera directed against the C-terminal region. A 36-amino-acid-residue peptide was isolated from the pancreas of a holocephalan fish, the Pacific ratfish (Hydrolagus colliei), that shows homology (69%) to mammalian glucagon in its N-terminal region and is reactive towards an N-terminally directed antiserum. Reactivity towards C-terminally directed antisera is prevented by the presence of a 7-residue C-terminal extension to the glucagon sequence that shows limited homology to the C-terminal region of glucagon-37 (oxyntomodulin). It is proposed that this peptide represents a major storage product of the islet X-cell.     

Opossum insulin, glucagon and pancreatic polypeptide: Amino acid sequences

Pancreatic hormones have been purified from the opossum, a New World marsupial. Opossum insulin contains a Leu substitution at the N-terminus of the B-chain in place of the Phe that is generally present in mammalian insulins. In addition, there are two other amino acid substitutions in the opossum insulin A-chain (positions 8 and 18) compared to pig insulin. Opossum glucagon is identical to chicken glucagon with both differing from the usual mammalian glucagon by Ser in place of Asn at its penultimate C-terminal position. Opossum PP differs from the porcine peptide in only 3 sites (position 3, 19 and 30).     

Immunoreactive glucagons purified from dog pancreas, stomach and ileum

Previous studies have shown that pig intestine contains a 69 amino acid glucagon (glicentin) as well as a 37 amino acid glucagon (oxyntomodulin). In pig pancreas the 29 amino acid glucagon predominates. Since glucagon is thought to be expressed from a single gene in mammals, these differences in molecular forms indicate differential posttranslational processing of the glucagon precursor by different tissues. In the current study glucagon immunoreactivity (IR) was separately purified from dog pancreas, stomach mucosa and ileum mucosa. Purification and sequence analysis of the different tissue glucagons show that dog pancreas and stomach mucosa contain glucagon-29 while ileum mucosa contains glucagon-37 and glucagon-69. The latter is the major form present with glucagon-37 accounting for only 10-20% of the total ileum glucagon content. The N-terminal 32 amino acid portion of dog glucagon-69 differs at 6 sites from pig glucagon-69: RSLQDTEEKSRSFSAPQTEPLNDLDQMNEDKR... The C-terminal glucagon-37 is identical to pig oxyntomodulin.     

Characterization of insulin and atypically processed proglucagon-derived peptides from the surinam toad Pipa pipa (Anura:Pipidae)

Electrospray mass spectrometry was used to identify insulin, glucagon and two peptides related to glucagon-like peptide-1 (GLP-1) in an extract of the pancreas of the Surinam toad, Pipa pipa, a species belonging to the same family as the African clawed frog, Xenopus laevis. Purification and characterization of the peptides established the primary structure of Pipa insulin as A-chain: GIVEQCCHSS(10)CTLLQLETYC(20) N and B-Chain: FSNQR LCGSH(10) LVEALHLVCG(20) DRGFFYYPKA(30). This amino acid sequence contains several substitutions (B5 His --> Arg, B16 Tyr --> His, A12 Ser --> Thr, A14 Tyr--> Leu, A18Asn --> Thr) of residues that have otherwise been quite strongly conserved during vertebrate evolution. Pipa glucagon comprises 37 amino acid residues (HSQGTFTSDY(10) SKYLDSRRAQ(20) DFVQWLMNTK(30)QSGGLSS) and the 29 amino-acid-residue peptide was not identified in the extract. In Xenopus and mammalian preproglucagons, the glucagon-29 sequence is followed by Lys-Arg which functions as a recognition site for a prohormone convertase. We propose that a point mutation in the gene encoding Pipa preproglucagon has transformed the Lys(30)-Arg(31) processing site into Lys-Gln with the result that the site in no longer recognized by the processing enzyme. In contrast, Pipa GLP-32 and GLP-37 are of the same molecular size as the corresponding peptides from Xenopus.     

Cloning of complementary DNAs encoding islet amyloid polypeptide, insulin, and glucagon precursors from a New World rodent, the degu, Octodon degus

The degu, Octodon degus, is a South American hystricomorph rodent that is of interest because it develops spontaneous diabetes mellitus and has been found to have islet amyloidosis. To help clarify these problems we have cloned cDNAs encoding islet amyloid polypeptide (IAPP), insulin, and glucagon precursors from this species. The predicted amino acid sequence of degu IAPP is very similar to that of nonamyloid-forming guinea pig IAPP. In contrast, degu insulin and the C-terminal region of degu glucagon are highly divergent from those of other mammals, as is also the case in the guinea pig, suggesting the existence of some form of positive evolutionary pressure on these hormones of carbohydrate metabolism in the hystricomorph rodents.     

Metabolic organization of the spotted ratfish, Hydrolagus colliei (Holocephali: Chimaeriformes): insight into the evolution of energy metabolism in the chondrichthyan fishes

The metabolic organization of a holocephalan, the spotted ratfish (Hydrolagus colliei), was assessed using measurements of key enzymes of several metabolic pathways in four tissues and plasma concentrations of free amino acids (FAA) and non-esterified fatty acids (NEFA) to ascertain if the Holocephali differ metabolically from the Elasmobranchii since these groups diverged ca. 400 Mya. Activities of carnitine palmitoyl transferase indicate that fatty acid oxidation occurs in liver and kidney but not in heart or white muscle. This result mirrors the well-established absence of lipid oxidation in elasmobranch muscle, and more recent studies showing that elasmobranch kidney possesses a capacity for lipid oxidation. High activities in oxidative tissues of enzymes of ketone body metabolism, including D-beta-hydroxybutyrate dehydrogenase, indicate that, like elasmobranchs, ketone bodies are of central importance in spotted ratfish. Like many carnivorous fishes, enzyme activities demonstrate that amino acids are metabolically important, although the concentration of plasma FAA was relatively low. NEFA concentrations are lower than in teleosts, but higher than in most elasmobranchs and similar to that in some "primitive" ray-finned fishes. NEFA composition is comparable to other marine temperate fishes, including high levels of n-6 and especially n-3 polyunsaturated fatty acids. The metabolic organization of the spotted ratfish is similar to that of elasmobranchs: a reduced capacity for lipid oxidation in muscle, lower plasma NEFA levels, and an emphasis on ketone bodies as oxidative fuel. This metabolic strategy was likely present in the common chondrichthyan ancestor, and may be similar to the ancestral metabolic state of fishes.     

Purification, structure, and characterization of caltrin proteins from seminal vesicle of the rat and mouse.

Caltrins, small basic proteins that inhibit calcium uptake by epididymal spermatozoa, have been purified from seminal vesicle content of the mouse and rat. Mouse caltrin (M(r) 8,476) contains 75 amino acid residues, 14 basic, 5 acidic, and 7 cysteines while rat caltrin (M(r) 6,217) has 56 residues, 10 basic, 5 acidic, and 6 cysteines; their pI values are 10.2 and 9.3, respectively. The proteins did not react with Ellman's reagent unless the cystine residues were previously reduced. The primary structures were determined by sequencing fragments generated by trypsin, clostripain, and endoproteinase Lys-C digestion. The sequences were ordered to give the total structural formula. The two molecules have no sequence similarity and are different from those of the bull and guinea pig previously reported. Only rat caltrin has a sequence of 13 residues nearly identical to that in guinea pig caltrin I. Both rat and mouse caltrin react with antibodies against bovine and guinea pig caltrins. Reduction and alkylation of cysteine residues suppressed the immunologic response of mouse caltrin; however, modified rat caltrin retained partially its immunoreactivity with the antiserum against guinea pig caltrin I. The same treatment abolished the calcium transport inhibitory activity of mouse caltrin and greatly reduced that of rat caltrin. It is likely that rat and mouse caltrins have the same physiological function as proposed for bovine caltrin; namely, to regulate the development of the Ca(2+)-dependent processes that "capacitate" sperm for fertilization.     

Novel organization and processing of the guinea pig pancreatic polypeptide precursor

Studies on New World hystricomorph rodents have revealed interesting structural divergences in the peptide hormones of the islets of Langerhans, particularly with respect to insulin and glucagon. Herein we report the isolation and sequencing of a cDNA encoding the precursor of pancreatic polypeptide (PP) from a guinea pig pancreas cDNA library. The 126-residue precursor sequence is predicted to include a 26-residue NH2-terminal signal peptide followed by the 36-amino acid PP hormonal sequence and a large COOH-terminal extension. The sequence identity between guinea pig and human PP is 89% (32/36 residues), and the predicted sequence is in agreement with that reported by Eng et al. (Eng, J., Huang, C.-G., Pan, Y.-C. E., Hulmes, J. D., and Yalow, R. S. (1987) Peptides 8, 165-168). In contrast, the icosapeptide domain in the guinea pig precursor exhibits only 40% (8/20) identity with the corresponding human precursor domain, and the COOH-terminal extension differs greatly in both sequence and size. The guinea pig precursor lacks the monobasic processing site (Pro-Arg) found at the COOH terminus of the icosapeptide domain in human, ovine, canine, and feline proPP. An icosapeptide is thus not likely to be liberated as such from this precursor. Of particular interest in guinea pig proPP is the substitution of serine for arginine at the dibasic amino acid processing site on the COOH-terminal side of the PP domain. Results of radioimmunoassays of gel-filtered protein fractions from a guinea pig pancreas extract indicate that efficient proteolytic cleavage takes place at this Lys-Ser site and that mature guinea pig PP is normally carboxyamidated.     

In vitro toxicity of alloxan for guinea pig B cells: comparison with rat B cells

Previous studies have shown that guinea pigs are resistant to the in vivo diabetogenic action of alloxan and that this resistance may be accompanied by a regeneration of B cells in the initial days following administration of the drug. In the studies reported here, we used the measurement of insulin and glucagon released over a 7-day culture period as indices of islet cell viability and examined effects of in vitro exposure to alloxan upon subsequent release of insulin and glucagon from guinea pig (alloxan-resistant) and rat (alloxan-sensitive) islet cell cultures. An alloxan dose-dependent decrease in subsequent insulin release was found. However, whereas the lowest concentration of the drug (1 mM) produced a significant depression in insulin release in rat islet cultures, with maximal depression occurring after exposure to 5 mM alloxan, insulin release from guinea pig cultures was not significantly depressed by 1 or 2 mM alloxan, and 5 mM alloxan treatment produced a submaximal depression. Furthermore, insulin release from guinea pig but not rat cultures increased transiently at between 6 and 18 hr during the first day following exposure to all doses of alloxan. Treatment with high doses of the drug (40 mM or greater) caused the same maximal chronic depression of insulin release for both species. In contrast, glucagon release from cultures of both species was not affected significantly following alloxan treatment. Thus, guinea pig B cells are more resistant than those of the rat to the action of alloxan, but this resistance can be overcome by employing high doses of the drug. Other factions unidentified by the present studies may also be involved in the failure of guinea pigs to develop diabetes following in vivo treatment with alloxan.     

Cyclic-AMP-dependent phosphorylation of glicentin

Highly purified glicentin , a 69-amino-acid-residue peptide isolated from porcine intestine that contains the full sequence of glucagon and is probably biosynthetically related to glucagon, is a substrate for cyclic-AMP-dependent protein kinase in a cell-free system, Glicentin-related pancreatic peptide (residues 1–30 of glicentin) and glucagon were not phosphorylated under the same reaction conditions. It is postulated that the serine residue at position 34 of glicentin (position 2 of glucagon), t h a t is part of the sequence Lys.Arg. His.Ser., is the probable site of phosphorylation.     

Homologous pancreastatin inhibits insulin secretion without affecting glucagon and somatostatin release in the perfused rat pancreas.

The identification of pancreastatin in pancreatic extracts prompted the investigation of its effects on islet cell function. However, in most of the investigations to date, pig pancreastatin was tested in heterologous species. Since there is great interspecies variability in the amino acid sequence of pancreastatin, we have investigated the influence of rat pancreastatin on insulin, glucagon and somatostatin secretion in a homologous animal model, namely the perfused rat pancreas. During 5.5 mM glucose infusion, pancreastatin (40 nM) inhibited insulin secretion (ca. 40%, P less than 0.025) as well as the insulin responses to 10 mM arginine (ca. 50%, P less than 0.025) and to 1 nM vasoactive intestinal polypeptide (ca. 50%; P less than 0.05). Pancreastatin failed to significantly modify glucagon or somatostatin release under any of the above experimental conditions. In addition, a lower pancreastatin concentration (15.7 nM) markedly suppressed the insulin release evoked by 11 mM glucose (ca. 85%, P less than 0.05). Our present observations reinforce the concept that pancreastatin is an effective inhibitor of insulin secretion, influencing the B-cell function directly and not through an A-cell or D-cell paracrine effect.     

Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis

Isolation and nucleotide sequencing of the complementary DNA for pig heart calpastatin have been completed. The amino acid sequence of 713 residues predicted from the nucleotide sequence contains five domains, each composed of approximately 140 amino acid residues. A unique N-terminal domain is followed by four mutually homologous domains. The best fit alignment of these four domains gives residue identities between any two domains of 22.5-36.0%. The analysis of the sequence similarities by several methods also suggests the existence of additional shorter repeats at intervals of 60-80 residues. The calculated molecular weight of pig calpastatin of 713 amino acid residues (Mr 77,122) is significantly lower than the value of purified pig heart calpastatin (Mr 107,000) estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The expression of the calpastatin genes in Escherichia coli and the detection of the translation products of 713, 366, and 140 amino acid residues by the specific anti-calpastatin antibody indicate that the products always migrate considerably slow on SDS-PAGE, giving an average of 1.53 for the ratio of the molecular weight estimated by SDS-PAGE to the value calculated from the amino acid sequences. It is most likely that the discrepancy in the molecular weight is caused by an anomalous behavior of calpastatin in SDS-PAGE.     

Differential effects of insulin, epinephrine, and glucagon on rat hepatocyte mitochondrial activity

Oxidation of [2,3-14C]succinate carbons in the mitochondrial Krebs cycle was used as a probe to investigate the effects of insulin, epinephrine, glucagon, and 2,4-dinitrophenol (2,4-DNP) on isolated rat hepatocytes. Epinephrine, glucagon, and 2,4-DNP had a far greater stimulatory effect on 14CO2 formation from [2,3-14C]succinate than insulin. Unlike insulin, epinephrine and glucagon had no significant effect on the anabolic utilization of succinate carbons for protein synthesis. Our results suggest that although epinephrine, glucagon, and 2,4-DNP enhance the movement of tracer carbons through the Krebs cycle, only insulin is capable of enhancing amphibolite utilization for protein synthesis.     

Isolation and structural characterization of insulin, glucagon and somatostatin from the turtle, Pseudemys scripta

The chelonians occupy an important position in phylogeny representing a very early branching from the ancestral reptile stock. Hormonal polypeptides in an extract of the pancreas of the red-eared turtle were purified to homogeneity by reversed phase HPLC and their primary structures were determined. Turtle insulin is identical to chicken insulin. Turtle glucagon differs from chicken glucagon by the substitution of a serine by a threonine residue at position 16 and from mammalian glucagon by an additional substitution of an asparagine by a serine residue at position 28. Turtle pancreatic somatostatin is identical to mammalian somatostatin-14. The crocodilians are phylogenetically much closer to the birds than are the chelonians. Alligator insulin, however, contains three amino acid substitutions relative to chicken insulin. Thus, caution is required when inferring phylogenetic relationships based upon a comparison of amino acid sequences of homologous peptides.     

Multiple molecular forms of glucagon and insulin in the kaluga sturgeon, Huso dauricus

Five molecular forms of glucagon and two molecular forms of insulin were characterized from the kaluga sturgeon. Substitutions occurred at two to thirteen internal amino acid residues among the five molecular forms of glucagons, indicating that these glucagons were encoded by five distinct genes. The amino acid sequences of two insulins from the kaluga sturgeon were identical to those of paddlefish insulin-II and Russian sturgeon insulin except that kaluga sturgeon insulin-I had an extension of five residues at the B-chain N-terminus. This is the first demonstration that more than two molecular forms of glucagon have been characterized from a single animal species.     

Multiple molecular forms of glucagon and insulin in the kaluga sturgeon, Huso dauricus

Five molecular forms of glucagon and two molecular forms of insulin were characterized from the kaluga sturgeon. Substitutions occurred at two to thirteen internal amino acid residues among the five molecular forms of glucagons, indicating that these glucagons were encoded by five distinct genes. The amino acid sequences of two insulins from the kaluga sturgeon were identical to those of paddlefish insulin-II and Russian sturgeon insulin except that kaluga sturgeon insulin-I had an extension of five residues at the B-chain N-terminus. This is the first demonstration that more than two molecular forms of glucagon have been characterized from a single animal species.     

NMR solution structure of the glucagon antagonist [desHis1, desPhe6, Glu9]glucagon amide in the presence of perdeuterated dodecylphosphocholine micelles

Glucagon, a 29-residue peptide hormone, plays an important role in glucose homeostasis and in diabetes mellitus. Several glucagon antagonists and agonists have been developed, but limited structural information is available to clarify the basis of their biological activity. The solution structure of the potent glucagon antagonist, [desHis1, desPhe6, Glu9]glucagon amide, was determined by homonuclear 2D NMR spectroscopy at pH 6.0 and 37 degrees C in perdeuterated dodecylphosphocholine micelles. The overall backbone root-mean-square deviation (rmsd) for the structured portion (residues 7-29, glucagon numbering) of the micelle-bound 27-residue peptide is 1.36 A for the 15 lowest-energy structures, after restrained molecular dynamics simulation. The structure consists of four regions (segment backbone rmsd in A): an unstructured N-terminal segment between residues 2 and 5 (1.68), an irregular helix between residues 7 and 14 (0.79), a hinge region between residues 15 and 18 (0.54), and a well-defined alpha-helix between residues 19 and 29 (0.33). The two helices form an L-shaped structure with an angle of about 90 degrees between the helix axes. There is an extended hydrophobic cluster, which runs along the inner surface of the L-structure and incorporates the side chains of the hydrophobic residues of each of the amphipathic helices. The outer surface contains the hydrophilic side chains, with two salt bridges (D15-R18 and R17-D21) implied from close approach of the charged groups. This result is the first clear indication of an overall tertiary fold for a glucagon analogue in the micelle-bound state. The relationship of the two helical structural elements may have important implications for the biological activity of the glucagon antagonist.     

Appalachian spring: variations on ancient gastro-entero-pancreatic themes in New World mammals

Studies of guinea pig genomic and/or cDNA clones encoding the gastro-entero-pancreatic (GEP) hormones--insulin, glucagon and pancreatic polypeptide--as well as portions of the insulin receptor, are described. Multiple clustered substitutions (localized rapid mutation acceptance) altering the biological properties of both insulin and glucagon have been revealed, but this does not appear to be the case with either pancreatic polypeptide or those regions of guinea pig insulin receptor cDNAs that have been examined thus far. These findings suggest that novel selective pressures operative in the New World environment, in which these animals evolved in isolation from Old World mammalian species, have led to altered solutions to problems related to the regulation of growth and carbohydrate metabolism.