May 7, 1998 - in muscle and brain of shallow-living (Scorpaena guttata). 432. and deep-living (Sebastolobus alascanus) scorpaenid fishes. Keppler D. and K.
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Glucose, Lactate, and b-Hydroxybutyrate Utilization by Rainbow Trout Brain: Changes during Food Deprivation Jose´ L. Soengas1 Elaine F. Strong2 M. Dolores Andre´s2,* 1 Laboratorio de FisioloxıB a Animal, Facultade de Ciencias, Universidade de Vigo, 36200 Vigo, Spain; 2Laboratorio de FisioloxıB a Animal, Facultade de BioloxıB a, Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain Accepted by C.P.M. 11/13/97
ABSTRACT In order to evaluate the normal (fed conditions) substrate utilization rates of rainbow trout (Oncorhynchus mykiss) brain, CO2 production from glucose, lactate, and b-hydroxybutyrate was tested in pooled brains. Oxidation rates, as well as the capacity for metabolism of carbohydrate and ketone bodies, were also evaluated in brain of rainbow trout that were food-deprived for 14 d. Under normal (fed) conditions, rainbow trout brain oxidized glucose and lactate at rates higher than those described for mammals; oxidation rates of b-hydroxybutyrate were lower in rainbow trout brain than those observed for lactate and glucose, and also lower than those described for mammals. Under food-deprivation conditions, glucose and lactate oxidation rates decreased in brains, suggesting the existence of brain metabolic depression, and bhydroxybutyrate oxidation rates sharply increased, suggesting increased utilization of ketone bodies.
Introduction Glucose is assumed to be a required brain fuel for all vertebrates (Clarke et al. 1989). The mammalian brain has small glycogen reserves and also uses ketones to an appreciable extent, and ketones become the major and possibly the primary fuels during food deprivation. One-half or more of the energy for the mammalian brain may be provided by ketones during starvation (Hamprecht and Dringen 1995). Lactate is also an important metabolic substrate for the brain during the early neo-
*To whom correspondence should be addressed; E-mail: fsandres@ uscmail.usc.es. Physiological Zoology 71(3):285 – 293. 1998. q 1998 by The University of Chicago. All rights reserved. 0031-935X/98/7103-9749$03.00
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natal period in several mammalian species, with utilization rates being higher than those of glucose and b-hydroxybutyrate (Vicario et al. 1991). In fish, brain metabolism probably accounts for only a small percentage of the overall metabolic demand (Heath 1988). There are very few data in the literature regarding fuel transport (Washburn et al. 1992; Blasco et al. 1996) or utilization (Yang and Somero 1993) in fish brain. In elasmobranchs, DeRoos (1994) demonstrated that the spiny dogfish brain uses both glucose and ketone bodies as energy sources, but he did not address the relative contribution made by each fuel to the brain. Glucose and lactate utilization were measured in lamprey brain by Foster et al. (1993), and it was demonstrated that lamprey brain pieces are capable of metabolizing glucose and lactate. There are discrepancies regarding the utilization of ketone bodies in teleost fish (Zammit and Newsholme 1979; LeBlanc and Ballantyne 1993), although utilization rates have not been evaluated to date. Furthermore, no studies have been performed to date in which utilization rates of different fuels were tested in a teleost brain, although Heath (1988) described changes in oxygen consumption rates of rainbow trout minced brains after incubation with glucose. The effects of food deprivation in fish carbohydrate metabolism have been thoroughly studied in recent years (Sheridan and Mommsen 1991; Vijayan et al. 1993), describing changes in glycogen and glucose levels, gluconeogenic enzyme activities, and hormone levels. However, little attention has been paid to the effects of food deprivation on the metabolism of brain carbohydrate and ketone bodies. In a previous study, we demonstrated the existence of changes in the metabolism of brain carbohydrate and ketone bodies in food-deprived Atlantic salmon (Soengas et al. 1996) and suggested an increased importance of ketone bodies metabolism as well as decreased use of exogenous glucose under food-deprivation conditions. However, in that study, no measurements of oxidation rates were provided to confirm the hypothesis suggested. Therefore, the aim of the present study, using the rainbow trout as a model, was twofold: to evaluate the utilization rates of those fuels (glucose, lactate, and b-hydroxybutyrate) theoretically capable of being used in fish brain; and to determine whether or not the utilization rates of the different fuels assessed changed due to food deprivation. Material and Methods Experiment 1: Characterization of Normal Rates of Substrate Utilization in Rainbow Trout Brain Immature rainbow trout, weighing 102 { 6 g, were obtained in December 1996 from a fish farm in Soutorredondo (Noia,
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286 J. L. Soengas, E. F. Strong, and M. D. Andre´s Galicia, Spain). Fish were acclimatized (14 fish per tank) for 4 wk under laboratory conditions; during that period they were maintained in 27 tanks (125 L) supplied with constantly running and aerated well water at 157C (daily temperature change õ 0.27C), at 7.2 { 0.03 pH, and under an artificial 12L : 12D light regime (lights on at 0800 hours). The fish were fed daily in the morning (1130 hours), before the experiments, with commercial dry pellets (Sterling Silver Cup, Spain; proximate analysis: 50% crude protein, 20% fat, 21% carbohydrate, and 9% ash in the dry matter), with a whole ration equivalent to 1.5% body weight d01, and were food-deprived 24 h before sampling. These experiments were performed four times (N Å 4) per substrate using, in each experiment, brains pooled from 10 – 12 fish. Fish were anesthetized with MS-222 (75 mg L01) buffered to pH 7.4 with sodium bicarbonate. Brains were dissected from the fish and minced on a petri dish, and the resultant pooled tissue was suspended in Cortland saline. Analysis of CO2 production of the suspension from labeled substrates was carried out according to Foster et al. (1993) and Soengas and Moon (1995). Labeled glucose (D-[U-14C] glucose; Amersham England, Ltd.) had a specific activity of 10.8 Gbq mmol01. Labeled lactate (L-[U-14C] lactate; Amersham England, Ltd.) had a specific activity of 5.62 Gbq mmol01. Labeled b-hydroxybutyrate (b-[1-14C] hydroxybutyrate; American Radiolabelled Chemicals, St. Louis) had a specific activity of 2.05 Gbq mmol01. Incubations were performed in triplicate. In each experiment, 20-mL glass vials contained 0.5 mL of suspension, which contained 80 mg of minced tissue (pooled from 10 – 12 fish) and unlabeled substrates with concentrations from 0.125 to 10 mmol L01. The time course of substrate oxidation rates were previously determined (data not shown), describing linear rates up to 3 h for glucose and lactate and up to 2 h for bhydroxybutyrate. Therefore, in the determination of the concentration dependence, the incubation times chosen were 2 h for glucose and lactate and 1 h for b-hydroxybutyrate. The vials were gassed with a 99.5% O2/0.5% CO2 mixture for 3 min and then sealed with a rubber septum, through which was suspended a center well containing a glass microfiber filter (GF/A, Whatman, Maidstone). After the vials were preincubated, the experiment was initiated by the addition of 0.5 mL of Cortland saline containing the labeled substrate (0.5 mCi per vial). Then the vials were incubated at 157C for 2 h (glucose and lactate) or 1 h (b-hydroxybutyrate). The vials were shaken during the incubation period (157C). After incubation, 0.1 mL of 2-methoxyethylamine was injected through the rubber septum, onto the filter in the center well, and the minced tissue was lysed with 0.1 mL of 70% (v/v) perchloric acid to release the CO2 and terminate the incubation. The sealed vials were shaken for a further 2-h period (157C) to ensure the collection of CO2 onto the filter. The radioactivity trapped on the filter was determined by liquid scintillation counting (BCS-NA, Amersham) in a Beckman counter, with internal standard quench correction. The CO2 production rate was calculated from the specific activity
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of the added labeled substrate, the amount of protein used, and the length of incubation, after correction for the CO2 released from control vials (without minced tissue). The brain protein levels were determined in triplicate in an aliquot of pooled brains using the method of Bradford (1976). Experiment 2: Effect of Food Deprivation on CO2 Production Rates and on Several Biochemical Parameters in Rainbow Trout Brain Immature rainbow trout were obtained in January 1997 from a fish farm in Soutorredondo (Noia, Galicia, Spain). Fish were acclimatized and fed under conditions similar to those described in experiment 1. After acclimatization, on day 0 of the experiment, 15 fish from three tanks were killed (denoted time zero) to perform three different experiments with each substrate. Tanks were randomly allocated to one of two treatment groups (fed or food-deprived). From that date onward, one group of fish (12 tanks) remained on the feeding regime described in experiment 1 (denoted as fed), while the other fish (12 tanks) were deprived of all food (denoted as food-deprived). The next samplings were performed on days 1, 4, 7, and 14 after the start of the experiment. On each of those sampling dates, six pools of five fish each were taken from six tanks (3 replicates 1 2 treatments) at 1100 hours (before delivering food to the fish in the fed groups). On each sampling, fish were removed quickly from the holding tank with a dipnet and anesthetized with MS-222 (75 mg L01) buffered to pH 7.4 with sodium bicarbonate. The brain was quickly dissected and prepared for CO2 production studies exactly as described above. Therefore, on each sampling date, three different experiments of CO2 production were performed per treatment. An aliquot of the pooled brains (six per sampling date) was frozen on dry ice and stored at 0807C until further assay of metabolites and enzyme activities. Blood was obtained with ammonium-heparinized syringes from the caudal peduncle. Plasma samples pooled from each experiment were obtained after centrifugation of blood (10 min at 2,000 g; Kubota KM 15200) and were immediately deproteinized (using 6% perchloric acid) and neutralized (using 1 mol L01 sodium bicarbonate) before being frozen on dry ice and stored at 0807C until further assay. The oxidation rates of each substrate (glucose, lactate, and b-hydroxybutyrate) were analyzed in triplicate for each sampling date using pools of brain tissue obtained from five fish. These experiments were performed three times for each substrate, that is, using three pools of five fish each per substrate on each sampling date. In each experiment, 20-mL glass vials contained 0.5 mL of suspension, which contained 80 mg of minced tissue (pooled from five fish) and unlabeled substrate with final concentrations of 5 mmol L01, 2 mmol L01, and 5 mmol L01, for glucose, lactate, and b-hydroxybutyrate, respectively. The tissues were incubated for 2 h (glucose and lactate) or 1 h (b-hydroxybutyrate) as described in experiment 1.
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Metabolite Utilization by Trout Brain 287 The aliquots of minced brain previously frozen were homogenized using a Potter-Elvejhem teflon-in-glass homogenizer held on ice with 10 vol of ice-cold stopping buffer containing 50 mmol L01 imidazole-HCl (pH 7.5), 15 mmol L01 2-mercaptoethanol, 100 mmol L01 potassium fluoride, 5 mmol L01 EDTA, 5 mmol L01 EGTA, and 0.1 mmol L01 phenylmethylsulphonyl fluoride (the last added as dry crystals immediately before homogenization). The homogenate was centrifuged (2 min at 9,000 g; Kubota microcentrifuge KM 15200), and the supernatant was used in enzyme assays. Aliquots of the supernatant were deproteinized and neutralized (with 6% perchloric acid and 1 mol L01 sodium bicarbonate, respectively) to assay tissue metabolites. Plasma and brain lactate levels were determined using the enzymatic method of Guttman and Wahlefeld (1974). Brain glycogen levels were assessed using the method of Keppler and Decker (1974). Glucose obtained after glycogen breakdown and plasma glucose levels were determined with a glucose oxidase-peroxidase method (Spinreact, Spain). Brain enzyme activities were determined using a Uvikon 930 spectrophotometer. Reaction rates of enzymes were determined by increase or decrease in the absorbance of NADPH or NADH at 340 nm. The reactions were started by the addition of brain homogenates (0.05 mL) at a preestablished protein concentration, omitting the substrate in control cuvettes (final volume, 1.1 mL), and allowing the reactions to proceed at 207C for preestablished times (data not shown). Protein was assayed per triplicate in homogenates as detailed by Bradford (1976), using bovine seroalbumin (Sigma, U.S.A.) as standard. Enzymatic analyses were all carried out at maximum rates in each tissue, with the reaction mixtures set up in preliminary tests to render optimal activities. The specific conditions for enzyme assays were described previously (Soengas et al. 1996) and were as follows. Glycogen phosphorylase (EC 2.4.1.1; GPase) was assayed with the following specific conditions: 50 mmol L01 phosphate buffer (pH 7.0); 0.5 mmol L01 NADP; 5 mmol L01 glucose 1,6-biphosphate; 2.5 mmol L01 AMP; excess phosphoglucomutase; excess glucose 6-phosphate dehydrogenase (from baker’s yeast); and 10 mg mL01 of glycogen (omitted for control). GPase a activities were measured with 10 mmol L01 caffeine present, and total GPase activities were estimated without caffeine. The ratio of GPase activities with and without caffeine represents the percentage of total GPase (a / b) in the active form. 6-Phosphofructo 1-kinase (EC 2.7.1.11; PFK) was assessed using 50 mmol L01 imidazole-HCl (pH 7.8), 175 mmol L01 KCl, 0.25 mmol L01 NADH, 2 mmol L01 ATP, 17.5 mmol L01 MgCl2, excess aldolase, excess triose phosphate isomerase, and excess a-glycerol phosphate dehydrogenase. Activities were determined at low (0.1 mmol L01) and high (10 mmol L01) fructose 6-phosphate concentrations (omitted for control). An activity ratio was calculated as the activity at low [fructose 6phosphate] divided by the activity at high [fructose 6-phosphate]. Similarly, a fructose 2,6-bisphosphate activation ratio
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was determined using low (1 mmol L01) and high (5 mmol L01) fructose 2,6-bisphosphate concentrations, and a 0.1 mmol L01 fructose 6-phosphate concentration. Hexokinase (EC 2.7.1.1; HK) was assessed using 50 mmol L01 imidazole-HCl (pH 8), 0.16 mmol L01 NADP, 5 mmol L01 MgCl2, 1 mmol L01 ATP, excess glucose 6-phosphate dehydrogenase (from baker’s yeast), and 5 mmol L01 glucose (omitted for control). b-Hydroxybutyrate dehydrogenase (EC 1.1.1.30; b-HBDH) was assessed using 50 mmol L01 phosphate buffer (pH 6.8), 0.15 mmol L01 NADH, and 1 mmol L01 acetoacetate (omitted for control).
Data Analyses The normal distribution of variables was tested using the Kolmogorov-Smirnov test, and group variance homogeneity was assessed using Cochran’s C-test. Logarithmic transformations of the data were made where necessary to fulfill the conditions of the ANOVA, but data are shown in their decimal values for simplicity. Statistical differences were tested using a two-way ANOVA, with treatment (fed and food-deprived) and time (0, 1, 4, 7, and 14 d) being the main factors. In those cases where a significant effect for a factor was obtained in the ANOVA, values were compared using a one-way ANOVA followed by a Student-Newman-Keuls multiple range test. The differences were considered statistically significant at P õ 0.05.
Results Figure 1 shows the rates of glucose oxidation at increasing glucose concentration. The oxidation of glucose increased with its concentration, reaching saturation at about 5 mol L01 glucose. The rate of lactate oxidation (Fig. 2) increased with its concentration, reaching saturation at lactate concentrations of about 2 mmol L01. The rate of b-hydroxybutyrate oxidation (Fig. 3) increased with its concentration, reaching saturation at about 5 mmol L01 b-hydroxybutyrate. Eadie-Hoffstee transformations of these results (insets in Figs. 1 – 3) allow the calculation of the apparent maximum velocity (Vmax) and Michaelis constant (Km) values of oxidation rates (Table 1). The estimates obtained for the Vmax of b-hydroxybutyrate oxidation rates showed that they were approximately 100 times lower than those of glucose and lactate. The oxidation rates of substrates assayed under optimal conditions were assessed after food deprivation, and the results are shown in Figures 4 – 6. The oxidation rate of glucose significantly decreased in food-deprived fish from day 4 of food deprivation onward compared with fed fish (Fig. 4). In contrast, no significant changes were noticed in lactate oxidation rates of lactate in brains of food-deprived fish compared with controls (Fig. 5). b-hydroxybutyrate oxidation rate sharply increased in brains of food-deprived fish, with the rates obtained
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288 J. L. Soengas, E. F. Strong, and M. D. Andre´s
Figure 1. Dependence on substrate concentration of oxidation from glucose in brain of rainbow trout. Minced brains obtained from 10–12 fish were pooled and incubated in the presence of labeled and unlabeled glucose. The labeled substrate was 0.5 mCi mL01 D-[U-14C] glucose. Results are the means { SEM of four experiments. The inset represents the Eadie-Hoffstee transformation of original parameters.
Figure 3. Dependence on substrate concentration of oxidation from b-hydroxybutyrate in brain of rainbow trout. Minced brains obtained from 10–12 fish were pooled and incubated in the presence of labeled and unlabeled b-hydroxybutyrate. The labeled substrate was 0.5 mCi mL01 b-[1-14C] hydroxybutyrate. Results are the means { SEM of four experiments. The inset represents the Eadie-Hoffstee transformation of original parameters.
being significantly higher after 7 and 14 d of food deprivation (Fig. 6). The remaining biochemical parameters were assessed to compare with previous data obtained in Atlantic salmon (Soengas et al. 1996). As expected, glycemia decreased in plasma of
food-deprived fish from day 4 of food deprivation onward, with levels remaining constant until the end of the experiment (Fig. 7). Data for the remaining parameters are shown in Table 2. A significant increase was observed in brain GPase activity, particularly in the percentage of GPase in the active form, which was significantly higher in food-deprived than in fed fish from day 4 of the experiment onward. Brain PFK activity of food-deprived fish was lower than that of fed fish, considering both the optimal activity as well as the activity ratio of the enzyme, from day 4 of food deprivation onward. However, no significant changes were observed in the fructose 2,6-bisphosphate activation ratio of the enzyme. Brain HK activity was lower in food-deprived than in fed fish throughout the experiment, with the differences being significant on days 4 and 14. b-HBDH activity in brains of food-deprived fish was significantly higher from day 4 of experiment onward. Brain glycogen levels were lower in food-deprived than in fed fish throughout the experiment. Finally, lactate levels displayed few changes when comparing fed and food-deprived fish, showing a significant increase only in brain of food-deprived fish after 1 d of the experiment.
Figure 2. Dependence on substrate concentration of oxidation from lactate in brain of rainbow trout. Minced brains obtained from 10–12 fish were pooled and incubated in the presence of labeled and unlabeled lactate. The labeled substrate was 0.5 mCi mL01 L-[U-14C] lactate. Results are the means { SEM of four experiments. The inset represents the Eadie-Hoffstee transformation of original parameters.
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Discussion In addition to glucose, mammalian brain uses ketone bodies as a source of both energy and carbon skeletons for its needs (Hamprecht and Dringen 1995). In fish, studies of substrate utilization rates in elasmobranchs (DeRoos 1994) and cyclostomes (Foster et al. 1993) examined the use of glucose, lactate,
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Metabolite Utilization by Trout Brain 289 Table 1: Vmax and Km values for oxidation rates of glucose, lactate, and b-hydroxybutyrate in minced brains pooled from 10 – 12 rainbow trout and incubated in the presence of labeled and unlabeled substrate
Substrate
Vmax (nmol CO2 mg01 protein h01)
Glucose ...................... 129.5 { 10.5 Lactate ........................ 120.2 { 9.5 b-hydroxybutyrate .... 1.423 { .001
Km (mmol L01)
Minimal Saturation Concentration (mmol L01)
.66 { .09 .51 { .08 .25 { .01
2 2 5
Note. These parameters were calculated by Eadie-Hoffstee transformations of the results depicted in Figures 1 – 3. The minimal saturation concentration was defined as the concentration of substrate from which no further statistically significant increase in substrate oxidation was found.
and b-hydroxybutyrate as energy supplies, demonstrating that brain is capable of metabolizing them at different rates. However, this is the first study, as far as we are aware, in which oxidation rates of different substrates were measured in a teleost brain. In normal (fed) rainbow trout brain, production of CO2 from different substrates clearly indicated that the best substrates for oxidation are glucose and lactate, whereas b-hydroxybutyrate was oxidized at rates lower than 1% of those of glucose and lactate. Production of CO2 from all substrates was
Figure 4. Changes in the oxidation rate from glucose in brain of fed (closed circles) and food-deprived (open circles) rainbow trout. The period of food deprivation is shown on the x-axis. The labeled substrate was 0.5 mCi mL01 D-[U-14C] glucose, and the unlabeled substrate was 5 mmol L01 D-glucose. Data are shown as mean { SEM of three experiments per treatment and date (each experiment was performed with brains pooled from five fish) and were analyzed by two-way ANOVA. An asterisk indicates a significant difference (P õ 0.05) from the fed fish sampled at the same time. a, Significantly different (P õ 0.05) from the day 0 sampling.
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linear with time up to 3 h in a way similar to that described in mammals (Edmond et al. 1987). Moreover, when comparing the oxidation rates of glucose and lactate in rainbow trout brain pieces with those of mammalian brain (Edmond et al. 1987; Tildon et al. 1993), we found that rates were slightly higher in rainbow trout brain than in mammals, for both glucose (130 and 3 – 30 nmol CO2 h01 mg01 protein for rainbow trout and mammals, respectively) and lactate (120 and 18 nmol CO2 h01 mg01 protein for rainbow trout and mammals, respec-
Figure 5. Changes in the oxidation rate from lactate in brain of fed (closed circles) and food-deprived (open circles) rainbow trout. The period of food deprivation is shown on the x-axis. The labeled substrate was 0.5 mCi mL01 L-[U-14C] lactate and the unlabeled substrate was 2 mmol L01 L-lactate. Data are shown as mean { SEM of three experiments per treatment and date (each experiment was performed with brains pooled from five fish) and were analyzed by two-way ANOVA. An asterisk indicates a significant difference (P õ 0.05) from the fed fish sampled at the same time. b, Significantly different (P õ 0.05) from the food-deprived fish sampled on day 1.
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290 J. L. Soengas, E. F. Strong, and M. D. Andre´s
Figure 6. Changes in the oxidation rate from b-hydroxybutyrate in brain of fed (closed circles) and food-deprived (open circles) rainbow trout. The labeled substrate was 0.5 mCi mL01 b-[1-14C] hydroxybutyrate and the unlabeled substrate was 5 mmol L01 bhydroxybutyrate. The period of food deprivation is shown on the x-axis. Data are shown as mean { SEM of three experiments per treatment and date (each experiment was performed with brains pooled from five fish) and were analyzed by two-way ANOVA. An asterisk indicates a significant difference (P õ 0.05) from the fed fish sampled at the same time. a, Significantly different (P õ 0.05) from the day 0 sampling. b, c, d, Significantly different (P õ 0.05) from the food-deprived fish sampled on days 1, 4, and 7, respectively.
tively). These results are comparable to those obtained by Foster et al. (1993) in lamprey brain, thus suggesting increased metabolic rates of glucose and lactate in fish compared with mammals. In contrast, b-hydroxybutyrate utilization rates were much lower in rainbow trout brain than in mammals (1.4 and 9 – 30 nmol CO2 h01 mg01 protein for rainbow trout and mammals, respectively). Km values for glucose utilization in mammals are much lower (1 mmol L01) than those observed for the transport of glucose in neurons, suggesting that transport through plasma membrane is not the limiting step in glucose metabolism (Vicario et al. 1991). Because most glucose-utilizing tissues lack glucose 6-phosphatase activity, phosphorylation is an irreversible or committed step in glucose metabolism. Consequently, rates of glucose utilization can only be affected directly by HK or the preceding transport processes (Furler et al. 1991). In rainbow trout brain, Km values for glucose utilization were even lower, with values of 0.66 mmol L01. However, in the few studies performed to date analyzing glucose uptake into fish brain (Washburn et al. 1992; Blasco et al. 1996), no estimates of kinetic constants were provided, and therefore we cannot speculate about the possible limitations of transport on metabolism. Moreover, the apparent Km of HK activity in
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rainbow trout brain is around 0.2 mmol L01, which allows us to suggest that glucose utilization may be more limited by phosphorylation in rainbow trout brain than it is in mammals. CO2 production from lactate operated at almost the same rate as that of glucose in brains of rainbow trout. In contrast, Foster et al. (1993) observed in lamprey brain higher rates of lactate oxidation compared with glucose, which is similar to the trend observed in mammals. Lactate flux to glycogen is low relative to flux to CO2 (2%) in lamprey brain (Foster et al. 1993), and therefore its importance as a metabolic fuel may be related to a possible role as a major fuel for specific brain cells in a way similar to that described in mammals (Dringen et al. 1993). Furthermore, it seems that the importance of ketone body utilization is different when comparing normal (fed) rainbow trout with mammalian brain. In mammals, ketone body utilization occurs at rates comparable with those of glucose and lactate, and even ketone bodies are seven times more effective than is glucose as a substrate for respiratory energy in neurons and astrocytes (Tildon et al. 1993). In contrast, b-hydroxybutyrate utilization in rainbow trout brain occurred at only 1% of the rates found for glucose and lactate. Since the circulating levels of ketones in fish plasma are very low compared to those in mammals, it is not surprising that the utilization rates of b-hydroxybutyrate are also low. In mammals, ketone body utilization by tissues depends strictly on their availability in
Figure 7. Changes in the levels of glucose in plasma of fed (closed circles) and food-deprived (open circles) rainbow trout. The period of food deprivation is shown on the x-axis. Data are shown as mean { SEM of three experiments per treatment and date (each experiment was performed with brains pooled from five fish) and were analyzed by two-way ANOVA. An asterisk indicates a significant difference (P õ 0.05) from the fed fish sampled at the same time. a, Significantly different (P õ 0.05) from the day 0 sampling. b, Significantly different (P õ 0.05) from the food-deprived fish sampled on day 1.
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Table 2: Changes in the activities of potential regulatory enzymes assayed in brain, brain glycogen and lactate levels, and plasma lactate levels of fed and food-deprived rainbow trout Time (d) Parameter and Treatment
0
GPase: Total activity (U mg01 protein): Fed ................................. 1.42 { .03 Food deprived ............... GPase a (%):a Fed ................................. 70.4 { 2.1 Food deprived ............... PFK: Optimal activity (U mg01 protein): Fed ................................. .74 { .01 Food deprived ............... Activity ratio:b Fed ................................. .65 { .02 Food deprived ............... Fructose 2,6-bisphosphate activation ratio:c Fed ................................. .43 { .03 Food deprived ............... HK activity (U mg01 protein): Fed ................................. .29 { .03 Food deprived ............... b-HBDH activity (mU mg01 protein): Fed ................................. 13.4 { 2.01 Food deprived ............... Brain glycogen (mg g01 wet wt): Fed ................................. 40.0 { 3.2 Food deprived ............... Brain lactate (mmol g01 wet wt): Fed ................................. 4.76 { .15 Food deprived ............... Plasma lactate (mmol mL01): Fed ................................. 1.92 { .13 Food deprived ...............
1
4
1.38 { .09 1.55 { .09 73.4 { 4.6 83.9 { 1.4e
7
1.45 { .10 1.70 { .01 72.2 { 1.4 96.9 { .05d,e,f
14
1.34 { .03 1.74 { .03d
1.45 { .03 1.73 { .09d
74.5 { 1.3 95.7 { 1.5d,e,f
75.3 { 1.1 93.5 { .06d,e,f
.74 { .02 .71 { .05
.73 { .02 .63 { .02d,e
.76 { .03 .61 { .02d,e
.74 { .02 .60 { .02d,e
.61 { .07 .56 { .06
.67 { .02 .49 { .03d
.63 { .01 .47 { .03d
.62 { .09 .46 { .08
.41 { .16 .40 { .14
.41 { .08 .64 { .18
.52 { .08 .72 { .12
.54 { .01 .54 { .12
.30 { .03 .24 { .08
.28 { .03 .20 { .01d
.27 { .02 .15 { .03
.26 { .01 .17 { .01d
14.3 { .84 22.5 { .44d,e
13.9 { .59 23.7 { 1.22d,e
14.3 { 1.31 24.7 { .52d,e
15.6 { .83 25.5 { 1.05d,e
41.0 { 2.2 16.6 { 1.9d,e
43.2 { 4.2 12.1 { 5.9e
42.0 { 5.5 15.3 { 6.1d,e
48.8 { 9.4 16.4 { 5.6d,e
4.55 { .22 6.21 { .66d
4.96 { .39 5.40 { .40
4.97 { .31 5.29 { .33
4.84 { .22 5.10 { .39
1.99 { .07 1.80 { .18
2.01 { .07 2.07 { .11
2.07 { .18 2.27 { .12
1.98 { .22 2.24 { .27
Note. Data are shown as mean { SEM and were analyzed by two-way ANOVA (N Å 3; each sample comes from five pooled fish). One unit of enzyme activity is defined for the different enzymes as: GPase, that which produces 1 mmol NADPH min01; PFK, that which utilizes 1 mmol fructose 6-phosphate min01; HK, that which utilizes 1 mmol glucose min01; and b-HBDH, that which utilizes 1 mmol acetoacetate min01. a Percentage of total glycogen phosphorylase (a / b) in the active form. b The activity ratio of PFK is defined as activity at low (0.1 mmol L01)/high (10 mmol L01) substrate (fructose 6-phosphate) concentration. c Fructose 2,6-bisphosphate activation ratio was determined using low (1 mmol L01) and high (5 mmol L01) fructose 2,6-bisphosphate concentrations, and 0.1 mmol L01 fructose 6-phosphate concentrations. d Significantly different (P õ 0.05) from the fed fish sampled at the same time. e Significantly different (P õ 0.05) from the day 0 sampling. f Significantly different (P õ 0.05) from the food-deprived fish sampled on day 1 of experiment.
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292 J. L. Soengas, E. F. Strong, and M. D. Andre´s the blood (Robinson and Williamson 1980). Km of b-HBDH in brain of mammals (2 mmol L01) is similar to that observed for oxidation (Vicario et al. 1991), suggesting that oxidation may be limited by b-HBDH activity. Our estimates of apparent Km of b-HBDH activity (0.15 mmol L01) and apparent Km of b-hydroxybutyrate utilization (1.4 mmol L01) also suggest that the oxidation of ketone bodies in rainbow trout brain may be limited by b-HBDH activity. The utilization of glucose by brain during food deprivation in mammals is spared in favor of more readily oxidized metabolites that can directly supply acetyl-CoA (such as ketone bodies), with glucose being used primarily to support other activities (hexose monophosphate shunt) that cannot be supported by other substrates. In a previous study (Soengas et al. 1996), we evaluated changes in several enzyme activities and metabolite levels in brain of food-deprived Atlantic salmon. Our results suggested increased glycogen mobilization, decreased glycolytic capacity, and decreased HK activity, as well as an increased importance of ketone bodies. In the present study, we have evaluated some of those parameters in order to compare results obtained in rainbow trout with those of Atlantic salmon. Rainbow trout used here in experiment 2 clearly displayed hypoglycemia as a result of food deprivation, which was maintained throughout the time period assessed, in agreement with other studies performed in rainbow trout (Tranulis et al. 1991; Holloway et al. 1994). This hypoglycemia was reflected in brain by several changes, including: (1) increased glycogen mobilization as reflected by decreased glycogen levels and increased GPase activity; (2) decreased HK activity; (3) decreased glycolytic capacity as reflected by decreased PFK optimal activity and activity ratio; and (4) increased b-HBDH activity. In general, these results coincide with those obtained previously in Atlantic salmon (Soengas et al. 1996), and therefore we may test in the present study the main hypothesis previously suggested regarding a decreased use of exogenous glucose as well as an increased importance of ketone bodies in brain of food-deprived fish. The comparisons obtained between substrate oxidation rates in fed and food-deprived fish were unequivocal in demonstrating that, after 14 d of food deprivation: (1) a 40% decrease occurred in oxidation rates of glucose in food-deprived fish compared with fed fish; (2) a 1,700% increase occurred in oxidation rates of b-hydroxybutyrate in food-deprived fish compared with fed fish; and (3) a slight decrease (though nonsignificant) also occurred in lactate oxidation rates in fooddeprived fish compared with fed fish. The decreased oxidation rates of glucose coincide with the decline also observed in glycolytic capacity as well as in HK activity (Soengas et al. 1996; this study) and, together with the slight decrease observed in lactate oxidation rates, suggest the existence of metabolic depression in teleost brain during food deprivation. As for ketone bodies, a sharp increase occurred in oxidation rates from day 7 of food deprivation onward, corresponding
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to the increased acetoacetate levels occurring in plasma of fooddeprived Atlantic salmon (Soengas et al. 1996). Thus, b-hydroxybutyrate oxidation rates changed from 1% of those of glucose and lactate under normal conditions to approximately 17% of those of glucose and lactate at the end of the food-deprivation period assessed. Therefore, the increased qualitative and quantitative importance of ketone bodies in brain of food-deprived rainbow trout seems clear. On the other hand, the increased use of ketone bodies in rainbow trout brain occurred during a time period (from 7 d of food deprivation onward) later than that described for mammals (Hamprecht and Dringen 1995). In further studies, b-hydroxybutyrate oxidation rates should be evaluated in more prolonged food-deprivation periods to evaluate their possible further increase. Despite the relatively low rates of b-hydroxybutyrate oxidation, it seems clear that its importance increases in teleost brain during food deprivation, contrary to the hypothesis postulated by Zammit and Newsholme (1979) of a lower importance of ketone body metabolism during fasting in teleosts. In summary, the results obtained in the present study clearly demonstrate that under normal (fed) conditions, rainbow trout brain oxidized glucose and lactate at rates higher than those described for mammals, and that oxidation rates of b-hydroxybutyrate were lower in rainbow trout brain than those of lactate and glucose and also lower than those described for mammals, suggesting a lesser importance of ketone body metabolism under such conditions. On the other hand, the main results obtained after food deprivation in rainbow trout brain indicated that glucose oxidation rates decreased, suggesting brain metabolic depression, and that b-hydroxybutyrate oxidation rates sharply increased, suggesting an increased utilization of ketone bodies by brain, which is similar to the response observed in mammals, though delayed in time.
Acknowledgments This study was supported by a research grant from the Xunta de Galicia (XUGA 20001A93) to M.D.A. E.F.S. was recipient of a predoctoral fellowship from Agencia Espan˜ola de Cooperacio´n Internacional.
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