Regulation of glucose production in rainbow trout: role of epinephrine in vivo and in isolated hepatocytes

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1 Am J Physiol Regulatory Integrative Comp Physiol 278: R956 R963, Regulation of glucose production in rainbow trout: role of epinephrine in vivo and in isolated hepatocytes JEAN-MICHEL WEBER AND DEENA S. SHANGHAVI Biology Department, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5 Weber, Jean-Michel, and Deena S. Shanghavi. Regulation of glucose production in rainbow trout: role of epinephrine in vivo and in isolated hepatocytes. Am J Physiol Regulatory Integrative Comp Physiol 278: R956 R963, The rate of hepatic glucose production (R a glucose) of rainbow trout (Oncorhynchus mykiss) was measured in vivo by continuous infusion of [6-3 H]glucose and in vitro on isolated hepatocytes to examine the role of epinephrine (Epi) in its regulation. By elevating Epi concentration and/or blocking -adrenoreceptors with propranolol (Prop), our goals were to investigate the mechanism for Epi-induced hyperglycemia to determine the possible role played by basal Epi concentration in maintaining resting R a glucose and to assess indirect effects of Epi in the intact animal. In vivo infusion of Epi caused hyperglycemia ( to mm) and a twofold increase in R a glucose ( to µmol kg 1 min 1, n 7), whereas Prop infusion decreased R a from to µmol kg 1 min 1 (n 10). Isolated hepatocytes increased glucose production when treated with Epi, and this response was abolished in the presence of Prop. We conclude that Epi-induced trout hyperglycemia is entirely caused by an increase in R a glucose, because the decrease in the rate of glucose disappearance normally seen in mammals does not occur in trout. Basal circulating levels of Epi are involved in maintaining resting R a glucose. Epi stimulates in vitro glucose production in a dose-dependent manner, and its effects are mainly mediated by -adrenoreceptors. Isolated trout hepatocytes produce glucose at one-half the basal rate measured in vivo, even when diet, temperature, and body size are standardized, and basal circulating Epi is responsible for part of this discrepancy. The relative increase in R a glucose after Epi stimulation is similar in vivo and in vitro, suggesting that indirect in vivo effects of Epi, such as changes in hepatic blood flow or in other circulating hormones, do not play an important role in the regulation of glucose production in trout. glucose kinetics; hormonal control of hepatic glucose production; circulating catecholamines; fish carbohydrate metabolism The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. THE HORMONAL REGULATION OF fish carbohydrate metabolism has received a lot of attention over the last two decades (16, 20, 25), and numerous in vitro studies have investigated the specific role of catecholamines in the mobilization of glucose (5). Epinephrine (Epi), norepinephrine, glucagon, and cortisol have all been shown to increase glucose production in isolated hepatocytes from a variety of teleosts (5, 13, 19, 27). At the whole organism level, stresses such as exhaustive swimming (11) and environmental hypoxia (18) stimulate Epi release, and the administration of exogenous Epi is known to cause hyperglycemia (5). This elevation in blood glucose could be due to an increase in the rate of appearance (R a glucose), a decrease in the rate of disappearance (R d glucose), or a combination of both as in mammals (2). Recent experiments on rainbow trout show that prolonged exercise causes a parallel decline in R a glucose and plasma Epi below normal resting levels (29), suggesting that this hormone may be involved in maintaining basal R a glucose in fish. Circulating Epi does not play such a role in mammals, because the administration of - or -adrenergic antagonists fails to reduce basal glucose production (2). The effects of Epi on fish hepatocytes are thought to be primarily mediated through -adrenoreceptors, because they are abolished in the presence of the -blocker propranolol (Prop) (5). The role of basal or elevated plasma Epi levels on hepatic glucose production has never been examined in vivo in teleosts. In this study, we ran parallel experiments on intact fish and on isolated hepatocytes from the same batch of animals for several reasons. Comparing values of basal R a glucose previously measured in vivo (8, 9, 29) and in vitro (4, 12, 17) reveals that rainbow trout hepatocytes seem to produce glucose more rapidly in the intact animal than after isolation. The significance of this discrepancy is not clear because the animals used for these various measurements were fed different diets, they were kept at different temperatures, and they had different body sizes (in vivo experiments typically requiring larger fish to perform catheterizations). Moreover, in vivo Epi and Prop treatments are known to influence the levels of several other hormones and to affect the respiratory and cardiovascular systems, at least in mammals (23). A dual in vivo-in vitro approach on the same batch of animals would allow standardization of experimental conditions (diet, temperature, and body size) and separation of the direct and indirect effects of Epi and Prop. Therefore, the goal of this study was to quantify the role of Epi in the regulation of hepatic glucose production in rainbow trout and to test the following hypotheses by elevating Epi concentration and/or blocking its -adrenergic effects in intact animals or in isolated hepatocytes: 1) elevated Epi levels cause hyperglycemia by increasing R a glucose and decreasing R d glucose, and 2) basal plasma Epi is not involved in maintaining resting R a glucose. We also R /00 $5.00 Copyright 2000 the American Physiological Society

2 R957 examined whether trout hepatocytes can produce glucose at similar rates in vivo and in vitro when diet, temperature, and body size are standardized. This comparison was made to determine the integrated effect of the cell-isolation procedure [e.g., destruction of membrane integrity, abnormal stimulation of glycogen breakdown (17), and elimination of neural input], to evaluate how relevant in vitro measurements of glucose production are to the intact organism. METHODS Animals Adult rainbow trout, Oncorhynchus mykiss (Walbaum), of both sexes were purchased in September from Linwood Acres Trout Farm (Campbellcroft, Ontario, Canada) and held in a 1,300-liter flow-through tank at 13 C. They were kept in dechloraminated, well-oxygenated water under a 12:12-h light-dark photoperiod. The animals were fed low-fat trout chow (11% lipids, 42% protein, 20% carbohydrate) until satiation three times a week. They were acclimated to these conditions for 6 wk before the first experiment and were randomly divided into two groups for the in vivo and in vitro experiments, respectively. In Vivo Experiments Catheterizations. Double cannulation of the dorsal aorta was performed under anaesthesia (0.1 g/l ethyl-n-aminobenzoate sulphonic acid, MS-222, buffered with 0.2 g/l sodium bicarbonate) (8). Cannulated animals were allowed to recover for at least 36 h in opaque acrylic boxes ( cm) with a weak water current to ensure adequate oxygenation. Fish with hematocrits 20% after recovery from surgery were not used. During the experiments, the acrylic boxes were fitted with a tight lid, preventing exchange with atmospheric air to allow the measurement of oxygen consumption (ṀO 2 ; see Refs. 9 and 31 for details). Measurement of glucose kinetics. R a glucose was measured by continuous infusion of 6-3 H glucose as described and validated previously (7, 8). The infusate was prepared daily by drying the isotope under N 2 and resuspending in Cortland saline (36). A priming dose equivalent to 90 min of infusion was injected as a bolus before starting the tracer infusion at 1 ml/h using a calibrated syringe pump (Harvard Apparatus, South Natick, MA). Tracer infusion was started 1 h before treatment with vehicle saline (control), Epi, or Prop to quantify baseline glucose kinetics. Infusions of saline, Epi, and Prop. One hour after starting the tracer infusion, a second syringe pump was activated to infuse vehicle saline (n 3, mean body mass g), Epi (n 7, g), or Prop (n 10, g) at 1 ml/h. Epi was infused for 35 min at a rate of 1.34 nmol kg 1 min 1. These values were selected after preliminary experiments showed that they were adequate to reach final plasma Epi concentrations of 500 nm, thereby simulating normal levels reached after exhaustive swimming. Prop was infused for 1 h at a rate of µmol kg 1 min 1 to reach a final plasma concentration of 50 µm. Preliminary measurements showed that this amount of Prop was sufficient to block -adrenoreceptors completely, because the rapid decline in arterial blood ph normally caused by the injection of Epi was completely abolished by this Prop treatment. Finally, tracer infusion was continued for 2 h after stopping the administration of saline, Epi, or Prop to monitor recovery. The infusions were performed under red, low-intensity light with the catheters covered by an opaque plastic sheet to avoid light-induced breakdown of Epi. Blood sampling and analysis. In each experiment, blood samples ( ml each) were taken before, during, and after the administration of vehicle saline, Epi, or Prop, and they were centrifuged immediately. Plasma was stored at 80 C before measuring glucose concentration spectrophotometrically (Beckman DU 640) and glucose activity by scintillation counting (Packard Tri-Carb 2500). Activity was quantified by drying 30 µl of plasma under N 2, resuspending in 1 ml water, and counting in Safety-Solve scintillant (Research Products International, Chicago, IL). In the Epi infusion experiments, plasma catecholamines were measured on alumina-extracted plasma, using HPLC with electrochemical detection (37) and 3,4-dihydroxybensalamine hydrobromide as internal standard. In Vitro Experiments Hepatocyte isolation. The fish were killed by a blow to the head (n 22, mean body mass g), and a midventral incision was made to expose the liver. Hepatocytes were isolated according to Moon et al. (21). Media A, B, and C, described in detail by these authors, were used with the following modifications: the liver was digested with medium C, containing 30 mg/100 ml collagenase (type IV from Clostridium histolyticum, 125 collagen digestion units/mg solid), and cell incubations were carried out in medium B added with 2 mm alanine and 1.5 mm lactate. The final pellet was resuspended in medium B and left on ice for 1 h before the cells were counted and their viability assessed with the trypan blue exclusion method. Cell preparations with viability 80% were not used in experiments. Hepatocyte incubations. The rate of glucose production was measured by monitoring the accumulation of glucose in the incubation medium. For each treatment, 50 mg of cells were incubated for min at 13 C with medium B alone (control) (21) or added with Epi, Prop, or both. Incubations with Epi were performed at low (5 nm) or high (500 nm) concentrations to simulate normal levels at rest (low Epi) or after exhaustive exercise (high Epi). Incubations with Prop were performed at a concentration of 50 µm. In experiments in which both Prop and Epi were used, Prop was added 15 min before Epi to ensure that all -adrenoreceptors were blocked. All incubations were carried out in the dark and stopped by adding perchloric acid. Two sets of experiments were performed. The first set consisted of control, Prop, and high-epi treatments. The second set consisted of control, low-epi, high-epi, low-epi Prop, and high-epi Prop treatments. Calculations and statistics. All in vivo R a glucose values reported in this paper were calculated with the steady-state equation of Steele (30), because they were never significantly different from R d glucose when both rates were calculated separately with the non-steady-state equation. To allow meaningful comparisons with in vivo R a glucose, in vitro rates of glucose production measured in micromoles of glucose produced per gram of hepatocytes were converted to micromoles per kilogram per minute as follows: in vitro R a glucose [glucose produced (µmol/g hepatocytes) 0.9 liver mass (g)]/[body mass (g) incubation time (min)]. Liver mass was multiplied by 0.9 because 90% of all liver cells are hepatocytes (15). For all the comparisons between rates of glucose production in vivo and in vitro, measured in vivo R a values were corrected for renal glucose production by subtracting 10.5% (7). For the in vivo experiments, changes in ṀO 2, glucose concentration, glucose specific activity, and R a glucose were assessed by two-way ANOVA and Dunnett s or Tukey s test. Differences between in vitro treatments and compari-

3 R958 sons between R a values measured in vivo and in vitro were tested with one-way ANOVA or Kruskal-Wallis one-way ANOVA on ranks when normality tests failed. Percentages were converted to the arcsine of their square root before analyses, and all the values presented are means SE. RESULTS In Vivo Experiments Control saline infusions. Infusion of vehicle saline had no effect on ṀO 2 (Fig. 1), plasma glucose concentration (Fig. 2A), glucose specific activity (Fig. 2B), or R a glucose (Fig. 2C; P 0.05). Throughout the saline infusion experiments, ṀO 2 averaged µmol O 2 kg 1 min 1 (n 6), plasma glucose concentration mm (n 13) and R a glucose µmol kg 1 min 1 (n 13). These values were not different from mean baseline levels measured before starting the administration of Epi or Prop (see Figs. 1, 4, and 5; P 0.05). Mean hematocrit did not change significantly during the saline infusion experiments and averaged % (n 3). Epi infusions. The infusion of Epi caused an increase in ṀO 2 from a mean baseline value of to µmol O 2 kg 1 min 1 (n 7, P 0.05), and ṀO 2 was still significantly higher than baseline 1 h after the end of Epi infusion (P 0.05, Fig. 1). Figure 3 shows plasma catecholamine concentrations before, during, and after Epi infusion. Plasma Epi was nm (n 7) before starting the exogenous administration of Epi and increased to 545 Fig. 2. Effects of vehicle saline infusion on plasma glucose concentration (A), glucose specific activity (B), and glucose rate of appearance (R a glucose; C) in rainbow trout. Shaded area indicates saline infusion, and values are means SE (n 3) nm (n 6; P 0.001) after 30 min of exogenous Epi infusion. Plasma Epi concentration returned to baseline 25 min after the end of infusion and remained at basal levels until the end of the measurements. Plasma norepinephrine concentration did not change significantly throughout the experiments (P 0.05) and averaged nm (n 8). The effects of Epi infusion on glucose concentration, glucose specific activity, and R a glucose are presented in Fig. 4. Before Epi infusion, plasma glucose concentration and R a Fig. 1. Rates of oxygen consumption (ṀO 2 ) before, during, and after infusion of vehicle saline (A), epinephrine (B), or propranolol (C). Shaded areas indicate infusion times. Values are means SE. Significant differences (P 0.05) are indicated as follows: a, different from mean ṀO 2 before infusion; b, different from mean ṀO 2 during epinephrine infusion; c, different from mean ṀO 2 during propranolol infusion. Fig. 3. Plasma concentration of epinephrine (r) and norepinephrine (s) in rainbow trout during exogenous infusion of epinephrine (shaded area). Values are means SE (n 6). *Significant differences from baseline levels (P 0.05).

4 R959 Fig. 4. Effects of exogenous epinephrine infusion on plasma glucose concentration (A), glucose specific activity (B), and R a glucose (C) in rainbow trout. Shaded area indicates infusion time for epinephrine, and values are means SE (n 7). *Significant differences from baseline (P 0.05). glucose averaged mm and µmol kg 1 min 1 (n 7), respectively. Glucose concentration increased to a maximal value of mm (P 0.001) after 20 min of Epi infusion but had returned to baseline within 1 h after the end of Epi infusion (Fig. 4A). The administration of Epi caused a twofold increase in R a glucose to a maximal value of µmol kg 1 min 1 (P 0.001, Fig. 4C). Glucose production declined progressively after the end of Epi infusion. It reached baseline values after 1 h of recovery and was µmol kg 1 min 1 after 2 h of recovery, a significantly lower rate than before Epi infusion (P 0.05). Mean hematocrit did not change significantly during the Epi infusion experiments and averaged % (n 7). Prop infusions. One-hour infusion of the -adrenoreceptor blocker Prop caused a decline in ṀO 2 from a baseline value of to µmol O 2 kg 1 min 1 (n 7, P 0.05). A further decrease to µmol O 2 kg 1 min 1 was observed after the end of the Prop infusion (Fig. 1). Changes in glucose concentration, glucose specific activity, and R a glucose during and after Prop infusion are plotted in Fig. 5. Baseline glucose concentration and R a glucose before Prop infusion were mm and µmol kg 1 min 1 (n 10), respectively. Plasma glucose concentration decreased progressively to reach values significantly lower than baseline in the recovery period (Fig. 5A). R a glucose declined progressively to µmol kg 1 min 1 (n 10, P 0.05) during Prop infusion and reached a minimal value of µmol kg 1 min 1 (P 0.05) by the end of recovery Fig. 5. Effects of propranolol infusion on plasma glucose concentration (A), glucose specific activity (B), and R a glucose (C) in rainbow trout. Shaded area indicates infusion time of propranolol, and values are means SE (n 10). *Significant differences from baseline (P 0.05). (Fig. 5C). Hematocrit did not change significantly during the Prop-infusion experiments and averaged % (n 10). A comparison of the relative effects of vehicle saline, Epi, and Prop infusions is presented in Fig. 6, in which percent changes in R a glucose are quantified. In Vitro Experiments Effects of Epi and Prop on glucose production by hepatocytes. Mean glucose production rates of isolated Fig. 6. Relative effects of vehicle saline (control), epinephrine, or propranolol infusion on hepatic glucose production of rainbow trout in vivo. Solid bars indicate infusion times for epinephrine and propranolol. Values are means SE (controls n 3; epinephrine n 7; propranolol n 10).

5 R960 Table 1. Amount of glucose released by isolated hepatocytes incubated in different media 15-min Control 15 min 30 min 45 min 60 min Experiment 1 Control Prop High Epi * Experiment 2 Control Low Epi * Low Epi Prop High Epi * High Epi Prop Values are means SE in µmol/g cells; n 11 fish. First column (15-min control) shows glucose release for a subsample of each batch of cells used in both experiments, but without the addition of epinephrine (Epi) or propranolol (Prop). Low Epi (5 nm); high Epi (500 nm); Prop (50 µm). Symbols indicate significant differences between treatments after 60 min of incubation (P 0.05): *, different from control;, different from Prop;, different from low Epi; and, different from high Epi. hepatocytes incubated for 60 min with or without Epi and/or Prop are summarized in Table 1. In each experiment, all treatment groups measured before starting the incubations (time 0) produced glucose at the same rate (experiment 1, P 0.63; experiment 2, P 0.95). Over time, the control groups of experiments 1 and 2 were not different from each other (P 0.05). Low and high Epi concentrations stimulated glucose production in a dose-dependent manner, but these effects were abolished by Prop (Table 1). Figure 7 summarizes the relative changes in glucose production measured after 60 min of incubation. Low Epi caused a 23% increase in glucose release (P 0.05; Fig. 7B), whereas high Epi stimulated glucose output by 110% and 133% in experiments 1 and 2, respectively (P 0.001; Fig. 7, A and B). Prop had no effect in the absence of Epi (Fig. 7A), and it prevented both low and high Epi from stimulating glucose production (Fig. 7B). Comparison of In Vivo and In Vitro Glucose Production Rates Figure 8 shows a comparison of R a glucose measured in the whole organism and in isolated hepatocytes. Rates of glucose production measured in vitro in micromoles per gram of hepatocytes (Table 1) were converted to micromoles per minute per kilogram body mass to compare them with baseline R a glucose measured in vivo that averaged µmol kg 1 min 1 (n 20) after correction for renal glucose production (see METHODS). Mean in vitro R a glucose was µmol kg 1 min 1 (n 22) and increased to (n 11) and µmol kg 1 min 1 (n 22) in the presence of low (5 nm) and high (500 nm) Epi concentration, respectively (P 0.05). The relative Fig. 7. Relative changes in total glucose release from isolated rainbow trout hepatocytes after 60 min of incubation with 50 µm propranolol (P), 5 nm epinephrine [low epinephrine (LE)], or 500 nm epinephrine [high epinephrine (HE)]. Two series of incubations were carried out: experiment 1 (A) had control, P- and HE-treated cells; experiment 2 (B) had control, LE, LE P, HE, and HE P treated cells. Significant differences (P 0.05) are indicated by letters: a, different from control; b, different from P; c, different from LE; and d, different from HE. Fig. 8. Rates of glucose production (R a glucose) of rainbow trout in micromoles per minute per kilogram body mass measured in vivo and in isolated hepatocytes. Values are means SE. C, control in vitro values (without epinephrine or propranolol in incubation medium) and all other abbreviations as in Fig. 7. Significant differences (P 0.05) are indicated by letters: a, different from in vivo; b, different from in vitro control; c, different from LE; d, different from LE P; and e, different from HE.

6 R961 Fig. 9. Relative effects of high epinephrine (HE) and propranolol (Prop) on hepatic glucose production of rainbow trout measured in vivo (solid bars) and in isolated hepatocytes (open bars). effects of Epi and Prop in vivo and in vitro are compared in Fig. 9. High Epi concentration had the same effect in vivo and in vitro, stimulating R a glucose by 102% and 89%, respectively. Prop alone caused a 46% decline in R a glucose in vivo but had no significant effect in vitro ( 1.8%). DISCUSSION Earlier in vivo measurements of fish substrate kinetics had revealed high basal rates of hepatic glucose production and a limited ability to increase R a glucose (7 9, 29). Stresses such as moderate exercise (29) and acute changes in water temperature (9) were shown to depress glucose production, suggesting that resting unstressed fish already release glucose at close to maximal rates. Results from the present study demonstrate otherwise and provide a new minimal estimate of reserve capacity for in vivo glucose production. Except for a small and transient increase in R a observed during acute hypoxia (9), this is the first example in which fish strongly stimulate hepatic glucose production in vivo, in this case, responding to a hormonal signal. Elevated plasma Epi levels (Fig. 3) caused a twofold increase in R a glucose (Figs. 4 and 6), whereas previous experiments by others showed that cortisol (1, 34) and estrogen (35) had no significant effects. Mechanisms of Epi Action on Hepatic Glucose Production Arterial infusion of Epi caused hyperglycemia (Fig. 4A) as noted previously in the same species (22), in other teleosts (24, 33), and in humans (28). Although the observed increase in blood glucose had to be caused by a temporary mismatch between R a and R d, the difference between these rates could not be detected statistically, because R d increased almost simultaneously with R a. Therefore, hyperglycemia resulted from a rapid increase in R a, whereas the Epi-induced reduction in R d known to occur in mammals (2) did not play a role in fish. The increase in trout R a glucose could be mediated through the direct activation of glycogenolysis and gluconeogenesis, and via indirect mechanisms involving changes in hepatic blood flow or in the levels of other circulating hormones such as glucagon, insulin, and cortisol. The respective contributions of these different factors cannot be determined from our results and further work will be needed to evaluate their potential impact separately. In mammals, the initial increase in R a glucose after Epi administration is caused by the immediate stimulation of glycogenolysis, whereas gluconeogenesis is only activated later when glycogen stores are somewhat depleted (3, 28, 32). In vitro experiments on various species of fish show that Epi increases glycogenolysis by activating glycogen phosphorylase (10, 39). In isolated fish hepatocytes, glucose production is supported almost exclusively through the breakdown of glycogen, whereas gluconeogenesis only plays a minor role. However, this overwhelming dominance of glycogenolysis may be an artifact of the cell isolation procedure and gluconeogenesis may be more significant in vivo (17). High Epi levels are known to depress pyruvate kinase activity in fish liver, thereby causing a reciprocal increase in gluconeogenesis and decrease in glycolysis (17, 39). Furthermore, Epi may cause the peripheral mobilization of several gluconeogenic precursors and promote their uptake by the liver, as in mammals (28, 32). Role of Epi in Maintaining Basal Glucose Production Resting Epi levels are not responsible for sustaining basal glucose production in mammals (2), but our study shows that they can play a significant role in fish. When the -adrenergic effects of resting Epi were blocked by the infusion of Prop in vivo, plasma glucose concentration and R a glucose of trout decreased by 14% and 46%, respectively (Figs. 5 and 6). Furthermore, glucose production by trout hepatocytes incubated at basal Epi concentrations showed a 20% decline when Prop was added to the medium (Fig. 7). Taken together, these results suggest that, in the resting state, Epi is present at higher concentrations in the portal circulation of fish than in mammals, and the observation that teleosts have much higher resting arterial Epi levels than mammals (26) supports this view. The absence of a decrease in the R a glucose of mammals after the administration of - or -blockers has been attributed to the fact that their portal concentrations of catecholamines are negligible under basal conditions (2). Glucose Production Rates: In Vivo vs. In Vitro After diet, temperature, and body size were standardized, isolated trout hepatocytes incubated without hormones still produce glucose at less than one-half the basal rate observed in vivo (3.13 vs µmol kg 1 min 1 ; Fig. 8). This difference is due to a combination of factors that may include hormonal and neural effects only acting in vivo as well as structural and/or functional damage incurred by the cells during isolation. Resting arterial Epi levels are responsible for a fraction of the difference, because the addition of 5 nm Epi to the incubation medium increases glucose production in vitro (Figs. 7B and 8), and Prop causes a decrease in R a

7 R962 glucose in vivo (Fig. 5) as well as in isolated cells incubated with basal Epi levels (Fig. 7). Isolated cells only approached in vivo rates when they were incubated with 500 nm Epi, a concentration simulating extreme arterial conditions after exhaustive exercise (Fig. 8). Therefore, other factors than basal Epi are also responsible for the twofold difference between basal glucose production rates observed in vivo and in vitro. Resting levels of glucagon, norepinephrine, and cortisol as well as direct sympathetic stimulation may be involved in maintaining basal R a glucose in vivo. However, further experiments will be needed to clarify this issue. The mean basal rate of glucose production measured here in vitro (3.13 µmol kg 1 min 1 ) falls within the range of values previously obtained by others for trout hepatocytes (1 5 µmol kg 1 min 1, see METHODS for conversion to this unit) (4, 12, 14, 17). We could only find one in vitro study reporting glucose production rates as high as measured in vivo (22). Interestingly, these high in vitro values were obtained in experiments carried out on liver slices rather than isolated cells, suggesting that disrupting the integrity of the liver tissue impairs normal glucose production. In humans, Epi also stimulates R a glucose indirectly by increasing hepatic blood flow and by activating glucagon secretion, which enhances liver gluconeogenesis (28). Here, high Epi concentration had similar relative effects in vivo and in vitro, causing baseline R a glucose to double in both cases. These findings suggest that the indirect pathways of Epi stimulation acting in vivo in mammals do not play a significant role in trout. - Vs. -Adrenergic Effects of Epi Both in vivo and in vitro experiments show that the stimulating effect of Epi on the R a glucose of rainbow trout is eliminated in the presence of Prop. The signal transduction pathway involving the binding of Epi to -adrenoreceptors, the activation of adenyl cyclase, and the upregulation of glycogen phosphorylase A have been well characterized in fish (5). The reverse phenomenon has been observed when treatment with Prop caused a three- to fivefold decrease in glycogen phosphorylase activity (39). Recently, the presence of -adrenoreceptors has been demonstrated in the membrane of rainbow trout hepatocytes, but no clear functional link with glucose metabolism has yet been established (6). Taken together, these results show that circulating Epi stimulates trout hepatic glucose production mainly via -adrenoreceptors and that -receptors could only play a minor role if any. Effects of Epi on Glucose Disappearance Because the rates of glucose production (R a ) and glucose disposal (R d ) were never statistically different, all the figures showing changes in R a also depict how R d was affected. Arterial Epi infusion caused R d glucose to double (Fig. 4), and two possible mechanisms for this response can be proposed: 1) a simple mass action effect mediated by hyperglycemia and/or 2) the activation of the glucose transporter (GLUT-4). The large augmentation in plasma glucose levels (Fig. 4A) expanded the diffusional concentration gradient driving glucose into cells, and this mechanism must be partly responsible for the observed increase in R d glucose. However, an increase in the rate of glucose transport through the translocation of GLUT-4 to the cell membrane, as observed in mammals, is unlikely to be involved in the Epi stimulation of R d in fish. To date, every attempt to demonstrate the presence of GLUT-4 in fish tissues has failed, with the possible exception of pancreatic cells in Tilapia (38). Conclusions By combining results from our in vivo and in vitro experiments, the following conclusions can be drawn from this study: 1) the Epi-induced hyperglycemic response of rainbow trout is caused by the stimulation of R a glucose, but the concomitant reduction in R d glucose observed in mammals does not occur in trout. 2) Basal levels of circulating Epi are partly responsible for maintaining resting R a glucose in rainbow trout. 3) Epi stimulates hepatic glucose production in a dosedependent manner, and its effects are mainly mediated by -adrenoreceptors. 4) Isolated trout hepatocytes produce glucose at about one-half the basal rates measured in vivo, even when diet, temperature, and body size are standardized, and basal arterial Epi is partly responsible for this discrepancy. 5) The relative increases in R a glucose after Epi stimulation are similar in vivo and in vitro, suggesting that indirect in vivo effects of Epi, such as sympathetic stimulation, changes in hepatic blood flow, or changes in the levels of other circulating hormones, do not play an important role in trout. This study would not have been possible without the help and advice from Steve Perry (Prop infusions), Tom Moon (in vitro experiments), and Colin Montpetit (catecholamine HPLC). The work was supported by National Sciences and Engineering Research Council research and equipment grants to J.-M. Weber. Address for reprint requests and other correspondence: J.-M. Weber, Biology Dept., Univ. of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5 ( jmweber@science.uottawa.ca). Received 20 May 1999; accepted in final form 29 September REFERENCES 1. Andersen, D. E., S. D. Reid, T. W. Moon, and S. F. Perry. Metabolic effects associated with chronically elevated cortisol in rainbow trout (Oncorhynchus mykiss). Can. J. Fish. Aquat. Sci. 48: , Cherrington, A. D. The acute regulation of hepatic glucose production. In: The role of the liver in maintaining glucose homeostasis, edited by M. J. Pagliassotti, S. N. Davis, and A. D. Cherrington. Austin, TX: R. G. Landes, 1994, chap. 2, p Chu, C. 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