Gill carbohydrate metabolism of rainbow trout is modified during gradual adaptation to sea water

The levels of glycogen and glucose, and the activities of several key enzymes of glycogenolysis, glycolysis and the pentose phosphate shunt were assessed in gills of rainbow trout of two sizes (97.03 ± 3.03 g and 165.69 ± 5.01 g) during gradual transfer to sea water (0, 9, 18 and 28 ppt). The results indicated changes, mainly size-independent, in carbohydrate metabolism of gills during gradual adaptation to sea water. Glucose availability in gills increased as a result of both higher glycogenolysis and greater supply of exogeneous glucose. This glucose was mainly used for increased glycolysis, providing the ATP needed to perform the hypo-osmoregulatory work occurring in gills during adaptation to sea water.     

Lack of significant long-term effect of dietary carbohydrates on hepatic glucose-6-phosphatase expression in rainbow trout (Oncorhynchus mykiss)

Hepatic glucose-6-phosphatase (G6Pase) plays an important role in glucose metabolism because it catalyzes the release of glucose to the circulatory system in the processes of glycogenolysis and gluconeogenesis. The present study was initiated to analyze the regulation of hepatic G6Pase expression by dietary carbohydrates in rainbow trout. The first step in our study was the identification of a partial G6Pase cDNA in rainbow trout that was highly homologous to that of mammals. Hepatic G6Pase activities and mRNA levels were measured in trout fed one of the experimental diets, with or without carbohydrates. We found no significant effect of intake of dietary carbohydrates on G6Pase expression (mRNA and activity) 6 hours and 24 hours after feeding. These results suggest that there is no control of G6Pase synthesis by dietary carbohydrates in rainbow trout and that the lack of regulation of gluconeogenesis by dietary carbohydrates could at least partially explain the postprandial hyperglycemia and the low dietary glucose utilization observed in this species.     

Relative contribution of glycogenolysis and gluconeogenesis to basal, glucagon- and nerve stimulation-dependent glucose output in the perfused liver from fed and fasted rats

The relative contribution to basal, glucagon- and nerve stimulation-enhanced glucose output of glycogenolysis (glucose output in the presence of the gluconeogenic inhibitor mercaptopicolinate) and gluconeogenesis (difference in glucose output in the absence and presence of the inhibitor) was investigated in perfused livers from fed rats with high and from fasted animals with low levels of glycogen. 1) Basal glucose output in both states was due only to gluconeogenesis. 2) Glucagon-enhanced glucose output was due about equally to glycogenolysis and gluconeogenesis in the fed state, but predominantly to gluconeogenesis (80%) in the fasted state. 3) Nerve stimulation-increased glucose output was due mainly to glycogenolysis (65%) in the fed state and about equally to both processes in the fasted state. The results suggest that under basal conditions of normal demands the liver supplies glucose only via gluconeogenesis and thus spares its glycogen stores, and that in situations of enhanced demands signalled by an increase in glucagon or sympathetic tone the liver liberates glucose mainly via glycogenolysis.     

Ketogenesis in vertebrate livers.

1. The hypothesis is advanced that a gluconeogenic organ such as the liver would evolve to oxidise fatty acids as its source of ATP for gluconeogenesis. It is also argued that such an organ might, in the light of current knowledge, be expected to be ketogenic. The animals investigated were lamprey, rainbow trout, eel, toad, axolotl, lizard and rat. 2. The respiratory quotients of liver slices from all animals was close to 0.74. Ketone bodies were produced from butyrate by all livers excepting the lamprey and ketone bodies were present in all blood samples examined. 3. There was no convincing evidence that direct deacylation of acetoacetyl CoA was important in any liver. HMGCoA synthase activity could not be found in the livers of the lamprey and eel. This enzyme was present in livers of the other animals. There was a large amount of acetoacetyl CoA-succinate transferase in the livers of the rainbow trout and eel, but only small amounts in the higher animals. 4. It is suggested that, initially the transferase was the important ketogenic pathway and the HMGCoA pathway evolved later.     

Adenylate cyclase activity in fish gills in relation to salt adaptation

The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of [alpha-32p]-ATP into [alpha-32p]-cAMP) was investigated in gill plasma membranes of rainbow trout (Salmo gairdneri) adapted to various salinities (deionized water, DW; fresh water, FW; 3/4 sea water, 3/4 SW; sea water, SW) and in sea water adapted-mullet (Mugil sp.). Basal activity declined by a factor of 2 in trout with increasing external salinity (pmoles cAMP/mg protein/10 min: 530 in DW, 440 in FW, 340 in 3/4 SW; 250 in SW) and was very low in SW adapted-mullet: 35. The Km for ATP was similar (0.5 mM) in both FW adapted- and SW adapted- trout in either the absence (basal activity) or in the presence of stimulating agents (isoproterenol; NaF) while the Vm varied. Analysis of stimulation ratios with respect to basal levels of the enzyme showed that hormones (glucagon, VIP) and pharmacological substances (isoproterenol, NaF) display a greater potency in high salt than in low salt adapted- fish gills. In contrast, salt adaptation did not have any effect on the regulation of adenylate cyclase by PGE1. These results are interpreted in relation to the general process of osmoregulation.     

Effect of verapamil on glycogenolysis and gluconeogenesis in the perfused rat liver

In perfused livers from fed rats, rates of glucose production (glycogenolysis) were 133 +/- 12 mumol/g/hr. Infusion of 2 microM verapamil into these livers decreased the rates of glucose production significantly to 97 +/- 15 mumol/g/hr within 10 min. Conversely, rates of production of lactate plus pyruvate (glycolysis) of 64 +/- 6 mumol/g/hr were not significantly altered by verapamil (60 +/- 3 mumol/g/hr). When 50 microM verapamil was infused, however, rates of both glycogenolysis and glycolysis were diminished to 56 +/- 11 and 43 +/- 5 mumol/g/hr, respectively. In perfused livers from fasted rats, infusion of 20 mM fructose increased the rates of production of glucose (gluconeogenesis) significantly from 11 +/- 7 to 121 +/- 17 mumol/g/hr. These rates reached 138 +/- 7 mumol/g/hr upon the simultaneous infusion of verapamil (2 microM). In these livers, fructose also increased rates of production of lactate from 6 +/- 2 to 132 +/- 11 mumol/g/hr, which were further increased to 143 +/- 8 mumol/g/hr when 2 microM verapamil was infused. The results show that calcium-dependent processes involved in hepatic carbohydrate metabolism respond differently to the calcium channel blocker verapamil. Low concentrations of verapamil inhibited glycogenolysis significantly while having no effect on either glycolysis or gluconeogenesis. These data suggest that these two processes have different sensitivities to changes in intracellular calcium concentrations and/or different sources of regulatory calcium.     

Effects of salinity on vitamin B6 deficiency in rainbow trout (Salmo gairdneri Richardson)

1. The link between vitamin B6 deficiency and sea water adaptation in rainbow trout was investigated. 2. A plasma-hyperosmotic salinity (20%) exacerbates the effect of vitamin B6 deficiency in rainbow trout. 3. Na/K-ATPase activity in the gill and kidney of the rainbow trout is not affected by vitamin B6 deficiency.     

Metabolic activity in the saccus vasculosus of the rainbow trout,salmo gairdneri (richardson)

Summary Labelling experiments to test mucopolysaccharide synthesis in the saccus vasculosus of the rainbow trout gave negative results and provoked analysis of succinic dehydrogenase activity in the coronet cells. Such activity was almost absent in the fresh-water living rainbow trout but was intense in sea water adapted individuals as well as in a sea water teleost, the flounder. Conversely, in sea water adapted rainbow trout which had been transferred back to fresh-water, activity had again decreased. In the sea water adapted rainbow trout high succinic dehydrogenase activity was paralleled by strongly positive staining with alcian blue, indicating the presence of mucopolysaccharides. The latter fishes also showed incorporation of N-acetyl-³H-glucosamine. Our findings indicate a role for saccus vasculosus in adaptation for osmoregulation in sea water and favour the hypothesis of a secretory function for the coronet cells.This work has been supported by grants from the Royal Physiographical Society of Lund and the Faculty of Natural Sciences at the University of Lund. We are greatly indebted to Mrs. Lena Eriksson, Mrs. Kristine Fogelström and Mrs. Lena Svenre for excellent technical assistance.     

Influence of cAMP and calcium on epinephrine-mediated hepatic glycogenolysis in rainbow trout,Oncorhynchus mykiss

1. The role of cAMP and of calcium in mediating epinephrine-stimulated glycogenolysis was studied by incubating rainbow trout liver in vitro.2. Epinephrine significantly stimulates glucose release from liver pieces incubated in either calciumcontaining or calcium-free medium. However, the development of the glycogenolytic profile occurred more rapidly in the presence of calcium.3. The β-antagonist, propranolol, inhibited epinephrine-stimulated glucose release from liver pieces incubated in either calcium-containing or calcium-free medium.4. Calcium ionophore, A3187, stimulated glucose release from liver pieces incubated in calciumcontaining medium. Verapamil, a putative calcium channel blocker, had no effect on A23187-stimulated glycogenolysis. However, verapamil completely inhibited epinephrine-stimulated glycogenolysis.5. Dibutyryl cAMP and IBMX, singly or together, stimulated glucose release from liver pieces. cAMP-mediated glycogenolysis was more pronounced in liver pieces incubated in calcium-containing medium.6. These results indicate that epinephrine-stimulated hepatic glycogenolysis in rainbow trout proceeds through the activation of β-adrenergic receptors and that both cAMP and calcium are involved in the post-receptor signal transduction process.     

Influence of salinity and ratio of lipid to protein in diets on certain enzyme activities in rainbow trout (Salmo gairdneri Richardson)

The connection between metabolic and sea water adaptation of the rainbow trout was investigated. The rainbow trout were kept in fresh water and diluted sea water of 8 and 20 0/00 S at 16 degrees C and fed on three different diets for 51 days. Hyperosmotic salinity (20 0/00) tends to inhibit growth in rainbow trout by reducing the food conversion efficiency. A higher protein concentration in the diet can partly compensate for this effect. The liver IDH, G6PDH and 6PGDH activities of the rainbow trout are influenced only by food quality, whereas the liver G1DH, AspT and A1T activities, like the muscle A1T, are also affected by salinity. The salinity had no significant effect on the activities of the kidney enzymes we investigated (Na/K-ATPase, G1DH, A1T, AspT) or of the muscle AspT in these experiments.     

Salinity tolerance of the mosquito fish, Gambusia affinis (Baird and Girard)

Experiments were conducted to determine the adaptability of mosquito fish to various salt concentrations. Mosquito fish, Gambusia affinis , (total length (T.L.) 27–37 mm) were subjected to abrupt and gradual changes from fresh water to various salinities (S). Low mortality (10%) occurred when the fish were transferred from fresh water (S = 0·5‰) to 50% sea water (S = 19·5‰ Through gradual adaptation 65·0% of the fish were able to tolerate 100% sea water for 7 days. After a 7 -day stay in sea water, fish were readapted to fresh water during a 3-h period. The results indicate that they were well able to tolerate the abrupt change. Through a gradual adaptation fish were also able to tolerate salinities ranging from 39·0‰ (100% sea water) to 58·8‰     

Retinoid X receptor agonists inhibit phorbol-12-myristate-13-acetate (PMA)-induced differentiation of monocytic THP-1 cells into macrophages

Although metformin has been used to treat type 2 diabetes for several decades, the mechanism of its action on glucose metabolism remains controversial. To further assess the effect of metformin on glucose metabolism this work was undertaken to investigate the acute actions of metformin on glycogenolysis, glycolysis, gluconeogenesis, and ureogenesis in perfused rat livers. Metformin (5 mM) inhibited oxygen consumption and increased glycolysis and glycogenolysis in livers from fed rats. In perfused livers of fasted rats, the drug (concentrations higher than 1.0 mM) inhibited oxygen consumption and glucose production from lactate and pyruvate. Gluconeogenesis and ureogenesis from alanine were also inhibited. The cellular levels of ATP were decreased by metformin whereas the AMP levels of livers from fasted rats were increased. Taken together our results indicate that the energy status of the cell is probably compromised by metformin. The antihyperglycemic effect of metformin seems to be the result of a reduced oxidative phosphorylation without direct inhibition of key enzymatic activities of the gluconeogenic pathway. The AMP-activated protein kinase cascade could also be a probable target for metformin, which switches on catabolic pathways such as glycogenolysis and glycolysis, while switches off ATP consuming processes.     

Glucagon effects on brain carbohydrate and ketone body metabolism of rainbow trout.

The levels of glycogen in brain, lactate and acetoacetate in brain and plasma, glucose in plasma and the activities of brain key enzymes of glycogen metabolism (glycogen phosphorylase, GPase, glycogen synthetase, GSase), gluconeogenesis (fructose 1,6-bisphosphatase, FBPase), and glycolysis (6-phosphofructo 1-kinase, PFK) were evaluated in rainbow trout, Oncorhynchus mykiss, from 0.5 to 3 hr after intraperitoneal injection of 1 ml/kg(-1) body weight of saline alone (controls) or containing bovine glucagon at three different doses: 10, 50, and 100 ng/g(-1) body weight. The results obtained demonstrate, for the first time in a teleost fish, the existence of changes in brain carbohydrate and ketone body metabolism following peripheral glucagon treatment. A clear stimulation of brain glycogenolytic potential was observed after glucagon treatment, as judged by the time- and dose-dependent changes observed in brain glycogen levels (up to 88% decrease), and GPase (up to 30% increase) and GSase (up to 42% decrease) activities. In addition, clear time- and dose-dependent increased and decreased levels were observed in brain of glucagon-treated rainbow trout for lactate (up to 60% increase) and acetoacetate (up to 67% decrease), respectively. In contrast, no significant changes were observed after glucagon treatment in those parameters related to glycolytic/gluconeogenic capacity of rainbow trout brain. Altogether, these in vivo results suggest that glucagon may play a role (direct or indirect) in the regulation of carbohydrate and ketone body metabolism in brain of rainbow trout.     

Salinity tolerance of the guppy, Poecilia reticulata Peters

Experiments were conducted to determine the adaptability of the guppy to various salt concentrations. The guppy, Poecilia reticulata , (total length 11–40 mm) were subjected to abrupt and gradual changes from fresh water (salinity=0.1%) to various salinities (%). No mortality occurred when the fish were transferred from fresh water to 50% sea water (19.5%). Through gradual adaptation from 50% sea water to 80% of the fish were able to tolerate 100% (39%) sea water for 7 days. After a 7 day stay in sea water, fish were readapted to fresh water during a 3 h period. Through gradual adaptation fish were also able to tolerate salinities ranging from 39.0% (100% sea water) to 58.5%. After a 30 day stay in 150% sea water (58.5%), fish were readapted to fresh water over a 5 h period. The results indicate that they were well able to tolerate the abrupt change from 100 or 150% sea water to fresh water. Females that stayed in 150% sea water (58.5%) for 30 days had embryos in their gonads.     

Glycogen metabolism in neonatal liver of the rat

Prior to birth the fetus of the rat accumulates large quantities of hepatic glycogen, with these stores mobilized as glucose in the early postnatal period to sustain the newborn until the onset of suckling and gluconeogenesis. The liver acts to mobilize glycogen in the early neonatal period and gradually adjusts to the alternating supply of nutrients that results from the onset of a feeding cycle. Early postnatal glycogen mobilization is reflected in the decreased active form of glycogen synthase (GS), the rate-limiting enzyme of glycogenesis, and increased activation of glycogen phosphorylase (GP), the rate-limiting enzyme of glycogenolysis. Levels of smooth endoplasmic reticulum (SER)-associated synthase phosphatase and phosphorylase phosphatase activities are diminished from high prenatal levels, contributing to these changes in activation of GS and GP. With the onset of suckling at 1-4 h after birth the liver again accumulates small quantities of glycogen. The period of 6 to 12 h after birth is characterized by large scale glycogenolysis. Glycogen levels are again increased at 24 h after birth, reflecting hepatic adaptation to the onset of meal feeding.     

Substrate mobilization and hormonal changes in rainbow trout (Oncorhynchus mykiss,L.) and common carp (Cyprinus carpio,L.) during deep hypoxia and subsequent recovery

Common carp (at 20°C) and rainbow trout (at 15°C) were fitted with an indwelling cannula in the dorsal aorta. The fish were exposed to a controlled decline of waterpO₂ followed by 90 min deep hypoxia at 0.3 kPa (carp) or 4.8 kPa (trout). Thereafter, normoxic recovery was monitored in both species for 48 h. At regular intervals blood samples were analysed for glucose, lactate, free fatty acids, adrenaline, noradrenaline and cortisol. The oxygen restriction was maximal in both species and resulted in a significant increase of plasma lactate levels. In carp, adrenaline, noradrenaline and cortisol levels increased to 2, 50, and 753 ng·ml^-1 respectively during anoxia, whereas in trout these hormones increased to 12, 8 and 735 ng·ml^-1 respectively during hypoxia. In hypoxic trout, the plasma levels of glucose (3 mol·l^-1) were increased modestly whereas levels of free fatty acids (0.25 mmol·l^-1) were decreased to 0.15 mmol·l^-1. In carp, however, a marked and prolonged hyperglycaemia (from 5 to 10 mmol·l^-1) and a significant continuous depression of plasma levels of free fatty acids (from 0.4 to 0.2 mmol·l^-1) were observed indicating a difference in metabolic organization. It is suggested that hyperglycaemia is likely to be the result of hepatic glycogenolysis, stimulated by circulating catecholamines and a stimulation of gluconeogenesis by cortisol during recovery. The mechanism for the decline of plasma levels of free fatty acids is most probably a reduction of lipolytic activity, which appears to be an adaptation to hypoxia.     

Effects of temperature, salinity, and feeding on aminotransferase activity in the liver and white muscle of rainbow trout (Salmo gairdneri Richardson).

1. The liver-somatic index of rainbow trout is governed by temperature and salinity, and by the interaction of these two factors. 2. The overall liver-alanine aminotransferase activity (in units/100 g body weight) increases slightly with increasing salinity of the surroundings in the case of rainbow trout. 3. The overall liver-aspartate aminotransferase activity (in units/100 g body weight) in rainbow trout depends on their food and the temperature at which they are kept. 4. Salinity adaptation leads to reductions in the specific alanine and aspartate aminotransferase activity in the liver of rainbow trout. 5. The specific alanine aminotransferase activity in the muscle of starving rainbow trout kept in diluted seawater (580 mOsm/l, 18 degrees C) is clearly higher than in control animals kept in tapwater.     

Growth hormone and prolactin actions on osmoregulation and energy metabolism of gilthead sea bream (Sparus auratus)

The gilthead sea bream (Sparus auratus) is an euryhaline fish where prolactin (PRL) and growth hormone (GH) play a role in the adaptation to different environmental salinities. To find out the role of these pituitary hormones in osmoregulation and energy metabolism, fish were implanted with slow release implants of ovine GH (oGH, 5 microg g(-1) body mass) or ovine prolactin (oPRL, 5 microg g(-1) body mass), and sampled 7 days after the start of the treatment. GH increased branchial Na(+),K(+)-ATPase activity and decreased sodium levels in line with its predicted hypoosmoregulatory action. GH had metabolic effects as indicated by lowered plasma protein and lactate levels, while glucose, triglycerides and plasma cortisol levels were not affected. Also, GH changed liver glucose and lipid metabolism, stimulated branchial and renal glucose metabolism and glycolytic activity, and enhanced glycogenolysis in brain. PRL induced hypernatremia. Furthermore, this hormone decreased liver lipid oxidation potential, and increased glucose availability in kidney and brain. Both hormones have opposite osmoregulatory effects and different metabolic effects. These metabolic changes may support a role for both hormones in the control of energy metabolism in fish that could be related to the metabolic changes occurring during osmotic acclimation.     

Inhibitory effect of anaesthesia with 2-phenoxyethanol as compared to MS222 on glucose release in isolated hepatocytes from rainbow trout (Salmo gairdneri)

1. Glucose production by freshly isolated hepatocytes from rainbow trout was studied after anaesthesia of the animals with 2-phenoxy ethanol (2PE) or tricaine methanesulphonate (MS222). 2. At the end of the procedure, hepatic contents of glycogen, glucose, lactate, ATP, ADP, AMP, were not significantly different between the two treatments. 3. Glucose production was considerably lower for 2PE than for MS222 anaesthetized trouts. This discrepancy results probably from an inhibition of glycogenolysis, suggesting that 2PE anaesthetized animals were less stressed than MS222 anaesthetized ones.     

Zymosan-induced changes in glucose release and fatty acid oxidation in the perfused rat liver

The aim of the present study was to investigate the actions of zymosan on glucose release and fatty acid oxidation in perfused rat livers and to determine if Kupffer cells and Ca2+ ions are implicated in these actions. Zymosan caused stimulation of glycogenolysis in livers from fed rats. In livers from fasted rats zymosan caused gradual inhibition of glucose production and oxygen consumption from lactate plus pyruvate. Ketogenesis, oxygen consumption, and [14C-]-CO2 production were inhibited by zymosan when the [1-14C]-palmitate was supplied exogenously. However, ketogenesis and oxygen consumption from endogenous sources were not inhibited. An interference with substrate-uptake by the liver may be the cause of the changes in gluconeogenesis and oxidation of fatty acids from exogenous sources. The pretreatment of the rats with gadolinium chloride and the removal of Ca2+ ions did not suppress the effects of zymosan on glucose release, a finding that argues against the participation of Kupffer cells or Ca2+ ions in the liver responses. The hepatic metabolic changes caused by zymosan could play a role in the systemic metabolic alterations reported to occur after in vivo zymosan administration.