Effects of temperature on glucose release and glycogen metabolism in isolated hepatocytes from rainbow trout (Salmo gairdneri)

Endogenous glucose release and glycogen metabolism were investigated in isolated hepatocytes from rainbow trout acclimated to 10 and 20 degrees C. Thermal acclimation did not significantly affect hepatocyte glycogen contents and the rates of glucose release during substrate-free incubations. In both acclimation groups glucose production and glycogen metabolism exhibited clearly different dependencies on assay temperature. It was concluded, that there are different sources of glucose release in the lower and upper temperature range--gluconeogenesis from endogenous precursors at low temperatures and glycogenolysis at high temperatures. This conclusion was supported by experiments with 3-mercaptopicolinic acid, which stimulated glycogen breakdown especially in the low temperature range.     

Regulation of lipolysis in isolated adipocytes of rainbow trout (Oncorhynchus mykiss): the role of insulin and glucagon

In the present study, we have examined the effects of insulin and glucagon on the lipolysis of rainbow trout (Oncorhynchus mykiss). To this end, adipocytes were isolated from mesenteric fat and incubated in the absence (basal lipolysis) or presence of different concentrations of insulin and glucagon. In addition, to further elucidate the effects of these hormones in vivo on adipocyte lipolysis, both fasting and intraperitoneal glucagon injection experiments were performed. Basal lipolysis, measured as the glycerol released in the adipocyte medium, increased proportionally with cell concentration and incubation time. Cell viability was verified by measuring the release of lactate dehydrogenase (LDH) activity in the medium. Insulin (at doses of 35 and 350 nM) decreased lipolysis in isolated adipocytes of rainbow trout in vitro, while glucagon was clearly lipolytic at concentrations of 10 and 100 nM. Furthermore, hypoinsulinemia induced by fasting, as well as glucagon injection, significantly increased lipolysis in isolated adipocytes approximately 1.5- and 1.4-fold, respectively, when compared with adipocytes from control fish. Our data demonstrate that lipolysis, as measured in isolated adipocytes of rainbow trout, can be regulated by both insulin and glucagon. These results not only indicate that insulin is an important hormone in lipid deposition via its anti-lipolytic effects on rainbow trout adipocytes, but also reveal glucagon as a lipolytic hormone, as shown by both in vitro and in vivo experiments.     

Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes

This study, using ¹³C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from ¹³C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1^-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10^-8 mol·1^-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1^-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.     

Temperature dependence of lipogenesis in isolated hepatocytes from rainbow trout (Salmo gairdneri)

Temperature dependence of lipogenesis in trout liver cells was investigated in the presence of 5 mM lactate using either [14C]lactate or [3H]water. A ratio of 3H/14C-incorporation greater than one is found, irrespective of temperature. Acclimation of fish to 4, 10 or 16 degrees C affects neither the height of lipid synthesis nor its temperature sensitivity. The distribution of [14C]lactate between the main lipid classes and the capacities for cholesterol- and triacylglycerol-synthesis are correlated to the glycogen stores of the hepatocytes. A comparison of fatty acid synthesis and cholesterogenesis in livers of normal fed rat and of trout suggests a capability for lipogenesis in trout somewhat similar to that in mammals.     

The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss)

Summary To establish the energetic cost of protein synthesis, isolated trout hepatocytes were used to measure protein synthesis and respiration simultaneously at a variety of temperatures. The presence of bovine serum albumin was essential for the viability of isolated hepatocytes during isolation, but, in order to measure protein synthesis rates, oxygen consumption rates and RNA-to-protein ratios, BSA had to be washed from the cells. Isolated hepatocytes were found to be capable of protein synthesis and oxygen consumption at constant rates over a wide range of oxygen tension. Cycloheximide was used to inhibit protein synthesis. Isolated hepatocytes used on average 79.7±9.5% of their total oxygen consumption on cycloheximide-sensitive protein synthesis and 2.8±2.8% on maintaining ouabain-sensitive Na⁺/K⁺-ATPase activity. The energetic cost of protein synthesis in terms of moles of adenosine triphosphate per gram of protein synthesis decreased with increasing rates of protein synthesis at higher temperatures. It is suggested that the energetic cost consists of a fixed (independent of synthesis rate) and a variable component (dependent on synthesis rate).Abbreviations BSA bovine serum albumin - dpm disintegrations per min - k _s fractional rate of protein synthesis - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - PHE phenylalanine; PO₂ oxygen tension - PCA perchloric acid     

Restructuring of plasma membrane phospholipids in isolated hepatocytes of rainbow trout during brief in vitro cold exposure

Adaptive changes in membrane physical properties in response to changing environmental temperature (e.g., inereased fluidity at low growth temperatures) are well known in poikilotherms; however, the timecourse of this response has received little attention. In this study the plasma membrane lipids of hepatocytes prepared from 20°C-acclimated trout were analyzed for phospholipid class and molecular species composition and metabolism after the cells were exposed to 5°C for 6 hours. Proportions of phosphatidylethanolamine and phosphatidylcholine were not altered by in vitro incubation at either 5 or 20°C. Molecular species analysis revealed that proportions of 18:1/20:5-phosphatidylcholine were significantly lower in plasma membranes of 5°C incubated cells, while decreases in 16:0/20:4-phosphatidylcholine, an unidentified phosphatidylcholine species, and 16:0/16:0-phosphatidylethanolamine as well as increases in 16:0/16:1-phosphatidylethanolamine as well as increases in 16:0/16:1-phosphatidylcholine bordered on significance. Exogenous radiolabeled molecular species of phosphatidylcholine (16:0/16:0-phosphatidylcholine and 16:0/18:1-phosphatidylcholine) were converted into other species at both temperatures, and the formation of some was influenced by incubation temperature. For example, cells exposed to 5°C convert significantly more 16:0/16:0-phosphatidylcholine into 16:0/20:4-phosphatidylcholine and 18:0/16:1-phosphatidylcholine and less into 18:1/18:1-phosphatidylcholine and 16:0/22:6-phosphatidylcholine than cells incubated at 20°C. In addition, cells at 5°C metabolized 16:0/18:1-phosphatidylcholine to a lesser extent than those at 20°C. The profile of conversion products indicates that deacylation/reacylation, elongation and desaturation reactions all participate in this early membrane restructuring. It is concluded that the plasma membrane of trout hepatocytes is a highly dynamic structure characterized by continuous lipid restructuring/turnover which can be rapidly altered upon acute cold exposure to adjust membrane phospholipid molecular species composition to the prevailing thermal environment.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesnlphonic acid) - HELC high-performance liquid chromatography - HVA homeoviscous adaptation - MS molecular species - MS-222 2-aminobenzoic acid ethyl ester (methanesulphonate salt) - RRT relative reteption time - PC phosphatidylcholine - PE phosphatidylethanolamine - TLC thin-layer chromatography - TRIS tris(hydroxymethyl)aminoethane - T _a ambient temperature     

Palmitate and oleate metabolism of rainbow trout in vivo

The turnover rates of palmitate and oleate were measured in vivo by continuous infusion of 1-[14C]palmitate and 9,10-[3H]oleate in rainbow trout. Our goals were: (1) to quantify the incorporation of a saturated and of a monounsaturated fatty acid into other classes of plasma lipids (neutral lipids, NL, and phospholipids, PL); and (2) to determine whether they could both be used as tracers to quantify fluxes of total non-esterified fatty acids (NEFA). We found that both acids play very different physiological roles because palmitate is preferentially channeled towards plasma PL, whereas oleate is mainly incorporated in circulating NL. Consequently, palmitate is predominantly involved in membrane PL turnover and oleate in the metabolism of circulating NL that may be used to shuttle oxidative fuel in teleosts. Despite this striking difference in their metabolism, palmitate and oleate have flux rates that are proportional to their relative abundance in plasma NEFA (i.e. they have the same fractional turnover rate). They can therefore both be used as reliable tracers to quantify the kinetics of total NEFA.     

Effects of epinephrine on branchial and renal calcium handling in the rainbow trout

Acute exposure of rainbow trout (Salmo gairdneri) to low external calcium (25 microM) caused an immediate but transient increase in plasma epinephrine concentration that may have been related to a concomitant depression of blood pH. Intra-arterial infusion of epinephrine at normal ambient calcium levels (0.35 mM) for 4 h caused circulating levels of epinephrine to rise from 2.9 X 10(-9) to 8.0 X 10(-8) M but did not affect norepinephrine levels, or branchial unidirectional calcium fluxes. Active (ATP-dependent) calcium transport across basolateral plasma membranes prepared from gill epithelial cells was not affected by pretreatment of fish with epinephrine or by direct application of epinephrine or cAMP, in vitro. Epinephrine infusion elevated urine flow rate, decreased urine pH, and increased urine phosphate levels significantly. Net renal calcium efflux increased significantly as a result of the increased urine flow rate. It is concluded that epinephrine does not stimulate branchial calcium uptake or renal conservation of calcium in rainbow trout at normal external calcium levels and therefore we cautiously suggest that epinephrine is unlikely to be involved in calcium balance during periods of exposure to low external calcium. Instead, epinephrine may play a role in compensating the acid-base disturbances and the increased branchial water influx that are associated with exposure to low ambient calcium.     

Viability control and oxygen consumption of isolated hepatocytes from thermally acclimated rainbow trout (Salmo gairdnerii)

Hepatocytes were isolated by collagenase perfusion of the liver from rainbow trout acclimated to 10 and 20 degrees C. The suitability of the stimulation of cellular respiration by succinate as criterion of viability was examined and discussed. Endogenous respiration rates of the hepatocytes were a function of cell size to the power of 0.8. Specific oxygen consumption of the hepatocytes and respiratory control ratios of the mitochondria in situ were independent of acclimation temperature.     

Application of XANES spectroscopy in understanding the metabolism of selenium in isolated rainbow trout hepatocytes: insights into selenium toxicity

Selenium (Se) is an essential element, but causes toxic effects in fish at a slightly elevated level beyond the threshold. However, the degree of Se toxicity differs depending on the chemical forms of Se (e.g., organic vs. inorganic) to which fish are exposed to. The mechanisms of Se metabolism and toxicity in fish, particularly at cellular level, are poorly understood. The present study was designed to examine the metabolic fate of different seleno-compounds, both inorganic and organic, in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) in primary culture using XANES spectroscopy. In cells exposed to 100 μM of selenate and selenite for 6-24 h, elemental Se was found to be the primary metabolite. Whereas, selenocystine appeared to be the major metabolite in cells exposed to 100 μM seleno-L-methionine for 6-24 h. Interestingly, we recorded L-methionine-γ-lyase activity in S9 fraction of cell lysate-an enzyme that directly catalyzes selenomethionine into methylselenol. We also found concurrent reduction of glutathione (GSH) concentration following reaction of seleno-L-methionine with cellular S9 fraction. Moreover, we observed a rapid increase in cellular reactive oxygen species (ROS) generation with increasing seleno-L-methionine exposure dose (100-1000 μM). These findings indicated the rapid cellular metabolism of seleno-L-methionine into methylselenol at higher exposure dose (≥100 μM), and the occurrence of GSH mediated redox cycling of methylselenol--a process that is known to produce reactive oxygen species (ROS). Overall, our results suggest that inorganic and organic selenium are metabolized through different metabolic pathways in rainbow trout hepatocytes. The findings of our study have important implications for understanding the chemical species-specific differences in Se toxicity to fish.     

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.     

Stimulatory effect of calcitonin on calcium uptake and glucose production in isolated rat hepatocytes

The biological effect of calcitonin (CT) was investigated in isolated rat hepatocytes. Addition of synthetic [Asu1,7] eel CT (20 and 40 ng/ml of medium with 2.5 mg dry weight of cells) into the medium containing calcium ion produced a marked elevation of calcium uptake in the hepatocytes, when calcium concentrations in the medium were monitored spectrophotometrically using arsenazo III. Intracellular calcium contents in the mitochondria and microsomes were significantly increased by CT addition. Meanwhile, glycogenolysis and gluconeogenesis in the hepatocytes were significantly stimulated by addition of CT (10, 10(2), and 10(3) ng/ml of medium with 5 mg dry weight of cells) into the medium containing calcium, although CT effects were less than those effects of 10(-7) M glucagon and 10(-5) M phenylephrine. These data clearly indicate that CT stimulates calcium uptake and glucose production in the hepatocytes, suggesting that CT has an effect in the metabolic function of rat liver cells.     

Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

Background  

Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss), which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine.     

The role of hepatic, renal and intestinal gluconeogenic enzymes in glucose homeostasis of juvenile rainbow trout

Rainbow trout is unable to utilize high levels of dietary carbohydrates and experiences hyperglycemia after consumption of carbohydrate-rich meals. Carbohydrates stimulate hepatic glycolytic activity, but gene expression of the rate-limiting gluconeogenic enzymes glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) remains high. Although there is significant mRNA expression and activity of gluconeogenic enzymes in trout intestine and kidney, the regulation of these enzymes by diet is not known. We tested the hypothesis that dietary carbohydrate modulates intestinal and renal G6Pase, FBPase and PEPCK. Fish were either fasted or fed isocaloric carbohydrate-free (CF) or high carbohydrate (HC) diets for 14 days. As expected, fish fed HC exhibited postprandial hyperglycemia and enhanced levels of hepatic glucokinase mRNA and activity. Dietary carbohydrates had no significant effect on the expression and activity of PEPCK, FBPase and G6Pase in all three organs. In contrast, fasting enhanced the activity, but not the mRNA expression of both hepatic and intestinal PEPCK, as well as intestinal FBPase. Therefore, the activity of rate-limiting gluconeogenic enzymes in trout can be modified by fasting, but not by the carbohydrate content of the diet, potentially causing hyperglycemia when fed high levels of dietary carbohydrates. In this species consuming low carbohydrate diets at infrequent intervals in the wild, fasting-induced increases in hepatic and intestinal gluconeogenic enzyme activities may be a key adaptation to prevent perturbations in blood glucose during food deprivation. Presented in part at Experimental Biology, April 2006, San Francisco, CA [Kirchner S., Panserat S., Kaushik S. and Ferraris R. FASEB-IUPS-2006 A667.6].     

Suppression of triglyceride secretion by epinephrine in isolated rat hepatocytes

The effect of epinephrine on triglyceride synthesis and secretion was examined in isolated rat hepatocytes. Epinephrine potently inhibited triglyceride secretion but did not affect cellular triglyceride content or the rate of incorporation of radiolabelled glycerol into cell triglyceride. The inhibitory effect of epinephrine was abolished by inclusion of the alpha-adrenergic antagonist prazosin but not the beta-antagonist propranolol.     

Characteristics of ouabain binding to isolated trout hepatocytes

Summary Na⁺, K⁺ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10^–4 M produced maximal inhibition (95%) of K⁺ uptake and enhanced intracellular Na⁺ accumulation, showing that active fluxes account for a very large proportion of Na⁺ and K⁺ exchanges. Inhibition of the Na–K pump by ouabain was significant at low concentrations (10^–8 M). When external K⁺ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC₅₀) of K⁺ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [³H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K⁺ concentration in the medium, the other linear and independent of external K⁺. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·10⁵ per cell) and independent of the external K⁺ concentration (7, 0.5 or 0 mM), while the dissociation constant (K_D) decreased from 4.2 M to 7.3 nM when K⁺ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min^–1), as estimated from ouabain-sensitive K⁺ flux, in comparison to those described in other cell types of higher vertebrates. At each external K⁺ concentration studied, the inhibition of K⁺ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.