Flight of Winter Moths Near 0{degrees}C.

Some noctuid winter moths fly at near 0 degrees C by maintaining an elevated(30 degrees to 35 degrees C) thoracic muscle temperature. Geometrid winter moths sustain themselves in free flight at subzero muscle temperatures. However, the temperature characteristics of citrate synthase and pyruvate kinase from both of these different kinds of moths and from a sphinx moth that flies with a muscles temperature of 40 degrees C are nearly identical. Furthermore, mass-specific rates of energy expenditure of both kinds of winter moths are also similar at given thoracic temperature (near 0 degrees C). The geometrids that are able to fly with a thoracic temperature near 0 degrees C do so largely because of unusually low wing-loading, which permits a low energetic cost of flight.

Evolution of urea synthesis in vertebrates: the piscine connection.

Elasmobranch fishes, the coelacanth, estivating lungfish, amphibians, and mammals synthesize urea by the ornithine-urea cycle; by comparison, urea synthetic activity is generally insignificant in teleostean fishes. It is reported here that isolated liver cells of two teleost toadfishes, Opsanus beta and Opsansus tau, synthesize urea by the ornithine-urea cycle at substantial rates. Because toadfish excrete ammonia, do not use urea as an osmolyte, and have substantial levels of urease in their digestive systems, urea may serve as a transient nitrogen store, forming the basis of a nitrogen conservation shuttle system between liver and gut as in ruminants and hibernators. Toadfish synthesize urea using enzymes and subcellular distributions similar to those of elasmobranchs: glutamine-dependent carbamoyl phosphate synthethase (CPS III) and mitochondrial arginase. In contrast, mammals have CPS I (ammonia-dependent) and cytosolic arginase. Data on CPS and arginases in other fishes, including lungfishes and the coelacanth, support the hypothesis that the ornithine-urea cycle, a monophyletic trait in the vertebrates, underwent two key changes before the evolution of the extant lungfishes: a switch from CPS III to CPS I and replacement of mitochondrial arginase by a cytosolic equivalent.

Protons and anaerobiosis.

During oxygen limitation in animals, glucose can be fermented via several metabolic pathways varying in energetic efficiency and leading to various end products (such as lactate, alanopine, octopine, succinate, or propionate). Because of opposite pH dependencies of proton production by fermentation and by hydrolysis of adenosine triphosphate formed in the fermentation, the total number of moles of protons generated is always two per mole of the fermentable substrate. However, two and three times more adenosine triphosphate can be turned over per mole of protons produced in succinate and propionate fermentations, respectively, than in lactate fermentation.

Pollen feeding in an orb-weaving spider.

Juvenile orb-weaving spiders appear in spring, when insect prey are scarce but when aerial plankton, such as pollen and fungus spores, is abundant. Microscopic organic matter may be the main food of orb-weaving spiderlings, with insects providing only a dietary supplement. Pollen, which is caught on the sticky spirals of Araneus diadematus orb webs, doubles the life expectancy of spiderlings and alters their web-spinning behavior, so that they spin more frequently than do fasting controls. Fungus spores do not have the same nutritional value as pollen and may be deleterious to the spiderlings.

Glucagon and glucagon-like peptides in fishes.

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 GLP-2. 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 cAMP and 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.

Stimulation of estrogen receptor accumulation by estradiol in primary cultures of salmon hepatocytes.

Hepatocytes of Salmo salar in primary culture form confluent monolayers and can be maintained at 11 degrees centigrade in serum-free medium for 8 days with minimal cell loss. Cultured hepatocytes from immature male salmon contain estrogen receptor both in nuclear and cytosol fractions (2000 and 2400 cites/cell, respectively). A single addition of estradiol results in an increase in the nuclear receptor to a level of 23 000 sites/cell after 24 h. This nuclear receptor concentration is similar to that in liver of estrogen-treated salmon in vivo, and is much higher than has been found for any other egg-laying vertebrate. The cultured salmon hepatocytes thus represent a highly sensitive system for the study of estrogen receptor dynamics and vitellogenesis in vitro.

Glucagon-like peptides activate hepatic gluconeogenesis.

Piscine (anglerfish, catfish, coho salmon) glucagon-like peptides (GLPs), applied at 3.5 nM, stimulate (1.1-1.9-fold) flux through gluconeogenesis above control levels in isolated trout and salmon hepatocytes. Human GLP-1 and GLP-2 also activate gluconeogenesis, but to a lesser degree than their piscine counterparts. Minor increases of substrate oxidation are noticed at times of peak gluconeogenic activation through GLPs. These hormones, which are derived from the same precursor peptide as glucagon are more potent activators of gluconeogenesis than glucagon when applied at equimolar concentrations, and do not appear to employ cAMP or cGMP as the intracellular messenger in hepatic tissue.

Divergence of insulin-like growth factors I and II in the elasmobranch, Squalus acanthias.

Recent studies have shown that vertebrates, including teleostean fishes, amphibians, birds and mammals, contain two distinct insulin-like growth factor (IGF) genes. In contrast agnathans, represented by hagfish, apparently have only one IGF that has features characteristic of both IGF-I and IGF-II. Between these groups the elasmobranchs occupy a critical position in terms of the phylogeny of IGFs. We sought to determine if gene duplication and divergence of IGF-I and IGF-II occurred before or after divergence of elasmobranchs from other vertebrates by cloning IGF-like molecules from Squalus acanthias. Our analysis shows that Squalus liver produces two distinct IGF-like molecules. One has greater sequence identity to, and conserved features characteristic of, known IGF-I molecules, while the other is more IGF-II like. These results suggest that the prototypical IGF molecule duplicated and diverged in an ancestor of the extant gnathostomes.

Molecular characterization of glycosomal NAD(+)-dependent glycerol 3-phosphate dehydrogenase from Trypanosoma brucei rhodesiense.

The primary structure of a 38-kDa protein isolated from membrane preparations of African trypanosomes was determined by protein and DNA sequencing. Searching of the protein database with the trypanosome translated amino acid sequence identified glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) from various prokaryotic and eukaryotic organisms as the optimal scoring protein. Surprisingly, the eukaryotic trypanosome enzyme showed the highest degree of sequence identity with the corresponding enzyme from the prokaryote Escherichia coli. The trypanosome molecule was expressed in Escherichia coli and found to be enzymatically active, thus confirming the identity of the molecule as an NAD(+)-dependent glycerol 3-phosphate dehydrogenase. A monoclonal antibody specific for the 38-kDa protein was used to localize the enzyme to glycosomes. Immunoblotting showed that the monoclonal antibody bound to a 38-kDa protein in African trypanosomes but not in T. cruzi, Leishmania or Crithidia. The enzyme has a pI of 9.1, a net charge of +17 and contains the peroxisome-like targeting tripeptide SKM at its C-terminus, all characteristic of glycosomal enzymes. Amino acids predicted to be involved in the NAD(+)-dependent glycerol 3-phosphate dehydrogenase active site have diverged from those of the mammalian enzyme. Kinetic analyses of the trypanosome GPD and GPD from rabbit muscle showed that the Km values of the two enzymes are different. The data suggest that the trypanosome protein may be a candidate target for rational drug design.

Metabolic response of teleost hepatocytes to glucagon-like peptide and glucagon.

Salmon glucagon-like peptide (GLP), bovine glucagon (B-glucagon) and anglerfish glucagon (AF-glucagon), all activate glucose production in teleost hepatocytes through gluconeogenesis and glycogenolysis, but notable species differences exist in their respective effectiveness. In trout hepatocytes, gluconeogenesis appears to be the main target of hormone action. In eel cells, sampled in November, glycogenolysis was activated threefold, while gluconeogenesis was increased by 12% only. In March, glycogenolytic activation was 1.7-fold, while gluconeogenesis was increased by about 1.7-fold after exposure to B-glucagon. In brown bullhead cells, increases in glycogenolysis from seven- (GLP) to tenfold (B- and AF-glucagon) were noted, while activation of gluconeogenesis was slight. Fragments of two AF-glucagons (19-29) revealed only insignificant metabolic activity. Treatment of eel cells with B-glucagon led to large (up to 20-fold) increases in intracellular cyclic AMP (cAMP) concentrations, while exposure to GLP was accompanied by a modest (less than twofold) increase in cAMP, although metabolic effectiveness (gluconeogenesis and glycogenolysis) was similar for the two treatments. Under identical conditions, brown bullhead cellular cAMP responded poorly. Levels of cAMP peaked within 15 min following hormone application. The results imply that no simple or direct relationship exists between the amount of intracellular cAMP and the metabolic action of the glucagon family of hormones. It can further be concluded that GLPs are important regulators of hepatic metabolism, influencing identical targets as glucagon, while the mechanisms of action seem to differ.

Novel role for prostaglandin E2 in fish hepatocytes: regulation of glucose metabolism.

Prostaglandin E(2) (PGE(2)) potently activated glycogenolysis and gluconeogenesis in isolated rockfish (Sebastes caurinus) hepatocytes. The average degree of activation for glycogenolysis was 6.4+/-0.67-fold (mean+/-S.E.M.; n=37), and could be as much as 19-fold. Analysis of dose-concentration relationships between glycogenolytic actions and PGE(2) concentrations yielded an EC(50) around 120 nM in hepatocyte suspensions and 2 nM for hepatocytes immobilized on perifusion columns. For the activation of gluconeogenesis (1.74+/-0.14-fold; n=10), the EC(50) for suspensions was 60 nM. Intracellular targets for PGE(2) actions are adenylyl cyclase, protein kinase A and glycogen phosphorylase. Concentrations of cAMP increased with increasing concentrations of PGE(2), and peaked within 2 min of hormone application. In the presence of the phosphodiesterase inhibitor, isobutyl-3-methylxanthine, peak height was increased and peak duration extended. The protein kinase A inhibitor, Rp-cAMPS, counteracted the activation of glycogenolysis by PGE(2), implying that the adenylyl cyclase/protein kinase A pathway is the most important, if not exclusive, route of message transduction. PGE(2) activated plasma membrane adenylyl cyclase and hepatocyte glycogen phosphorylase in a dose-dependent manner. The effects were specific for PGE(2); smaller degrees of activation of glycogenolysis were noted for PGE(1), 11-deoxy PGE(1), 19-R-hydroxy-PGE(2), and prostaglandins of the A, B and Falpha-series. The selective EP(2)-receptor agonist, butaprost, was as effective as PGE(2), suggesting that rockfish liver contains prostaglandin receptors pharmacologically related to the EP(2) receptors of non-hepatic tissues of mammals. Rockfish hepatocytes quickly degraded added PGE(2) (t((1/2))=17-26 min). A similar ability to degrade PGE(2) has been noted in catfish (Ameiurus nebulosus) hepatocytes, but no glycogenolytic or gluconeogenic actions of the hormone are noted for this species. We conclude that PGE(2) is an important metabolic hormone in fish liver, with cAMP-mediated actions on glycogen and glucose metabolism, and probably other pathways regulated by cAMP and protein kinase A. The constant presence of EP(2)-like receptors is a unique feature of the fish liver, with interesting implications for function and evolution of prostaglandin receptors in vertebrates.

The purine nucleotide cycle as two temporally separated metabolic units: a study on trout muscle.

Experimental results on fast-twitch muscle of rainbow trout following exercise and during subsequent recovery lead us to a reinterpretation for the function of the components of the purine nucleotide cycle (PNC). Exhaustive exercise depletes tissue ATP by more than 90% and results in a stoichiometric gain in IMP and ammonium ions. Simultaneously, white-muscle aspartate decreases by half, but its maximum contribution can account for less than 2% of the accumulated ammonium. Of the three enzymes of the purine nucleotide cycle, AMP deaminase, adenylosuccinate synthetase and adenylosuccinate lyase, only AMP deaminase is functional during exhaustive exercise. During the slow (greater than 15 hour) recovery, AMP deaminase is effectively shut off, while the other two enzymes replenish the adenylate pool. At all times, a tight inverse correlation exists between ATP and IMP concentrations. Tissue ammonium and malate supply the required aspartate. Theoretical treatment with special attention to proton dynamics in a potentially anaerobic tissue also leads to the conclusion that rather than constituting a true cycle, distinct parts of the PNC are temporally segregated. We hypothesize that during periods of high energy demand, exclusively AMP deaminase is activated as a means (1) to push the myokinase reaction toward ATP synthesis, (2) to supply allosteric effectors, and (3) to remove some of the accumulating protons through the formation of ammonium, all at the expense of the adenylate pool. The process leading to its replenishment, which involves the production of two protons and the consumption of a high-energy phosphate, can be active during aerobic recovery only.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphibian glucagon family peptides: potent metabolic regulators in fish hepatocytes.

Peptides analogous to glucagon-like peptide-1 (GLP-1) have been isolated from amphibian pancreas and intestine, and their amino acid sequences and cDNA structures elucidated. Just like their mammalian counterpart, these peptides are potent insulinotropins in mammalian pancreatic cells. We show here that these peptides also exert strong glycogenolytic actions when applied to dispersed fish hepatocytes. We compared the potencies of three synthetic GLP-1s from Xenopus laevis and two native GLP-1s from Bufo marinus in the activation of glycogenolysis in the hepatocytes of a marine rockfish (Sebastes caurinus) and two freshwater catfish (Ameiurus nebulosus and A. melas), and demonstrated their effectiveness in increasing the degree of phosphorylation of glycogen phosphorylase. We also compared the glycogenolytic potency of the peptides with those of human GLP-1 and glucagons from human and B. marinus. Sensitivity to these peptides is species-specific, with the rockfish responding at lower concentrations to GLP-1s and the two catfish reacting better to glucagons. However, the relative potency of the amphibian GLP-1s and glucagons is similar in the three species. Xenopus GLP-1C (xGLP-1C) is consistently more potent than xGLP-1B, while xGLP-1A displays the smallest activation of glycogenolysis. Similarly, Bufo GLP-1(32)-the peptide with the highest amino acid sequence identity to xGLP-1C-always shows a higher potency than Bufo GLP-1(37), which is closely related to xGLP-1B. The relative hierarchy of these glycogenolytic GLP-1s differs from their ranking as insulinotropins in mammalian beta-cells. In the rockfish system, Bufo glucagon-36, a C-terminally extended glucagon, is more potent than the shorter bovine glucagon and Bufo glucagon-29 in the activation of glycogenolysis; when tested in A. nebulosus hepatocytes, bovine and amphibian glucagons are equipotent. Amphibian GLP-1s and glucagons activate glycogenolysis in fish hepatocytes through increased phosphorylation of glycogen phosphorylase, implying involvement of the adenylyl cyclase/protein kinase A system in signal transduction. We conclude that the broad physiological effectiveness of GLP-1 has been retained throughout vertebrate evolution, and that both insulinotropic activity and glycogenolytic actions belong to the repertoire of GLP-1.

Paradigms of growth in fish.

Most fish are indeterminate growers with white muscle making up the majority of the acquired bulk. Within the muscle, the myofibrillar fraction accounts for almost two-thirds of the protein synthetic activity, implying that it is accretion of myofibrillar proteins that makes the single most important contribution to fish growth. Fish muscle growth itself is not linear and occurs through a combination of hyperplasia and hypertrophy in post-juvenile stages. Superimposed on periodicity of growth in length and mass can be other phases governed by lunar, reproductive or circannual cycles. Data on fish growth are discussed in the framework of site-specific muscle abundance, metabolic and functional zonation of muscle, proliferation and differentiation of satellite cells and the contribution of myofibrillar proteins. Hormonal control of muscle growth is described against the backdrop of plasma availability of myogens (insulin, IGF-I, growth hormone), distribution and dynamics of their respective receptors, and their interactions. Important contributions of the 'supply side' are discussed with hormones regulating amino acid resorption from the intestine, intestinal growth, liver processing and amino acid uptake by the muscle. Data are also interpreted from metabolic angles, to explain lipolytic and nitrogen-sparing effects of growth hormones, and lipogenic effects of insulin and high protein diets. Finally, special attention is devoted to the multifaceted roles of arginine in fish growth, as precursor, intermediate and hormone secretagogue.

Glucagon-like peptide-1 activates the adenylyl cyclase system in rockfish enterocytes and brain membranes.

Glucagon-like peptide (GLP) exerts important physiological functions in fish liver, but extrahepatic sites of action and physiological roles have been largely ignored. We show here that GLP activates adenylyl cyclase in isolated brain and enterocyte membranes and increases cellular cyclic adenosine monophosphate (cAMP) levels in isolated enterocytes of rockfish (Sebastes caurinus). Following exposure to synthetic zebrafish GLP (zf-GLP) (1 nM-1 microM), a concentration-dependent increase in enterocyte cAMP is noted. The maximum increase in cAMP levels is observed at 1 microM zf-GLP, and represents a 30% increase above control values. Exendin-4, a GLP receptor agonist in mammals, elicits a similar concentration-dependent increase in enterocyte cAMP. In contrast, norepinephrine or prostaglandin E2 (at 1 microM) increased cAMP levels by 2 and 4-fold, respectively. Brain membrane adenylyl cyclase is activated 20-40% by zf-GLP, and to a smaller extent by zf-glucagon, while exendin-4 is as effective as zf-GLP at a dose of 100 nM. These results suggest potential physiological roles of GLP in brain and intestine in piscine systems analogous to GLP-1 functions in these tissues described for mammals.

Effects of arginine on pancreatic hormones and hepatic metabolism in rainbow trout.

Arginine (Arg), injected intraperitoneally into rainbow trout (Oncorhynchus mykiss), increases plasma concentrations of glucagon, glucagon-like peptide-1 (GLP-1), and insulin by three- to 10-fold. Resulting ratios of glucagon and GLP-1 over insulin are unchanged in 20-d food-deprived fish (saline, 1.28 vs. Arg, 0.93; not significant) while slightly increased in feeding trout (saline, 0.70 vs. Arg, 0.92; P<0.05). In food-deprived juveniles, Arg injection leads to significant decreases in plasma fatty acids (saline, 1.65 mM L(-1) vs. Arg, 1.09 mM L(-1); P<0.05) and increases in glycogen phosphorylase total activity (saline, 3.7 units g(-1) vs. Arg, 4.6 units g(-1); P<0.05) and degree of phosphorylation (saline, 1.7 units g(-1) vs. Arg, 2.33 units g(-1); P<0.05). Plasma and liver glucose and liver enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, phosphoenolpyruvate carboxykinase, lactate dehydrogenase, and malic enzyme) are unaffected. Otherwise, fish show the changes in plasma metabolites expected with food deprivation. Arg injection into feeding fish results in decreases in plasma fatty acids, liver glycogen, and glucose, while liver glucose 6-phosphate concentrations increase. Hepatocytes isolated from feeding fish injected with Arg 2 h previously show significantly lower rates of lactate oxidation than controls (85% of control), while rates of gluconeogenesis and hormonal responses to mammalian glucagon and GLP-1 remain unchanged. Rates of lactate oxidation and gluconeogenesis are significantly decreased by 5%-10% on treatment with porcine insulin. Complete immunoneutralization of insulin with rabbit antisalmon insulin serum decreases hepatic glucose 6-phosphate concentrations and abolishes the Arg-dependent effects on glycogen phosphorylase. It appears that short-term increases in pancreatic hormones cause only minor metabolic readjustments in the relatively short time frame covered in these experiments. Surprisingly, complete removal of insulin does not have immediate altering or detrimental effects on key metabolites and metabolic pathways, even if glucagon and GLP-1 concentrations are concurrently several-fold higher than usual. Our data clearly show the dual role of Arg in fish metabolism.

Concentration-response relationships and temporal patterns in hepatic gene expression of Chinook salmon (Oncorhynchus tshawytscha) exposed to sewage.

Changes in liver gene expression were examined in juvenile Chinook salmon (Oncorhynchus tshawytscha) exposed in vivo for 8d to seawater (control) or one of 5 concentrations of sewage (environmentally-relevant dilutions of 0.05%, 0.1%, and 0.7%; 2%, 5% or 10%) and subsequently transferred to clean seawater for an 8-d recovery period. Livers were sampled on days 1, 4, 8 (sewage-exposed) and 16 (8d of sewage exposure plus 8d of recovery). A custom cDNA microarray using a universal DNA reference design was used to examine trends of altered gene expression across sewage concentrations, across timepoints, and at the end of the recovery period. Alterations in gene expression followed four distinct concentration-dependent patterns: (1) concentration response (e.g. estrogen receptor alpha), (2) inverse-concentration response (e.g. insulin receptor beta), U-shaped (e.g. mineralocorticoid receptor), (3) inverse U-shaped (e.g. benzodiazepine receptor), and (4) concentration-independent responses (e.g. ubiquitin). Temporal trends included: (1) peak gene expression at one of the sewage exposure timepoints with recovery to baseline levels after the depuration phase (e.g. vitelline envelope protein beta), (2) gene expression alterations that did not recover (e.g. glucose transporter 3), and (3) delayed gene expression alterations initiated only at the recovery timepoint (e.g. insulin-like growth factor 2). In summary, patterns in gene expression changes were found across sewage concentrations and exposure timepoints. This study is the first to show gene expression trends of this nature.