1 Department of Integrative Zoology, Institute of Evolutionary and Ecological Sciences, University of Leiden, Van der Klaauw Laboratories, 2300 RA Leiden; and 2 Department of Molecular Pharmacology, University of Groningen, 9713 AV Groningen, The Netherlands
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ABSTRACT |
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The regulation of triglyceride
mobilization by catecholamines was investigated in the teleost fish
Oreochromis mossambicus (tilapia) in vivo and in vitro. In
vitro experiments were carried out with adipocytes that were isolated
for the first time from fish adipose tissue. For the in vivo
experiments, cannulated tilapia were exposed to stepwise decreasing
oxygen levels (20, 10, and 5% air saturation; 3.9, 1.9, and 1.0 kPa
PO2, respectively), each level being maintained
for 2 h. Blood samples were taken at timed intervals and analyzed
for plasma lactate, glucose, free fatty acids, epinephrine,
norepinephrine, and cortisol. Hypoxia exposure did not change plasma
epinephrine levels. In contrast, the plasma norepinephrine
concentration markedly increased at all hypoxia levels. Over the same
period, plasma free fatty acid levels showed a significant continuous
decrease, suggesting that norepinephrine is responsible for the reduced
plasma free fatty acid concentration, presumably through inhibition of
lipolysis in adipose tissue. To elucidate the mechanism, adipocytes
were isolated from mesenteric adipose tissue of tilapia and incubated
with 1) norepinephrine, 2) norepinephrine + phentolamine (1,
2-antagonist),
3) isoproterenol (nonselective
-agonist), 4)
isoproterenol + timolol
(
1,
2-antagonist), 5)
norepinephrine + timolol, and 6) BRL-35135A
(
3-agonist). The results demonstrate for the first time
that norepinephrine and isoproterenol suppress lipolysis in isolated
adipocytes of tilapia. The effect of norepinephrine is not mediated
through
2-adrenoceptors but, like isoproterenol, via
-adrenoceptors. Furthermore, this study provides strong indications
that
3-adrenoceptors are involved.
teleost fish; fat cells; adrenoceptors; free fatty acids; hypoxia
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INTRODUCTION |
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IN TELEOST FISH, catecholamines play an important role in the regulation of energy metabolism during stressful conditions like hypoxia (6, 32, 46, 51). In these vertebrates, lipids dominate metabolism, whereas carbohydrates play a minor role in energy production since low amounts of carbohydrates are ingested and stored (38, 50). Nevertheless, most information on the regulation of energy metabolism by catecholamines deals with carbohydrates (9, 19, 25, 30, 53), whereas relatively little information is known about the regulation of lipid metabolism by these neurohormones (34).
Exposure of carp to deep hypoxia (46) resulted in a strong
increase of circulating norepinephrine (NE) accompanied with a marked
decrease of plasma free fatty acid (FFA) levels. In rainbow trout, on
the other hand, both epinephrine (Epi) and NE increased modestly,
whereas the plasma FFA concentration ([FFA])
showed only a minor reduction. From these results, it was
suggested that the Epi-to-NE ratio is important for the effect on the
plasma [FFA]. Infusion experiments with catecholamines in carp
(45) demonstrated that infusion of Epi results in elevated
plasma FFA levels, whereas NE infusion, on the contrary, causes a
marked decrease of plasma FFA levels. So far, in mammals, both Epi and NE have been shown to stimulate lipolysis via -adrenoceptors, resulting in increased plasma [FFA] (33, 54).
Accumulation of these amphiphilic compounds may be deleterious to
membrane structure and membrane-dependent functions (17,
21). Therefore, the observation that an increase in FFA levels
does not occur in fish, showing even a marked reduction instead, may be
considered as an adaptive strategy to survive hypoxic conditions.
Van Raaij et al. (45) suggested that the observed decline
of FFA levels by NE involved a direct stimulation of
2-adrenoceptors in adipocytes, resulting in inhibition
of lipolysis, whereas Epi was proposed to stimulate lipolysis by
activation of
-adrenoceptors. This concept is based on the
observations of mammalian systems in which stimulation of
2- and
-adrenoceptors results in a decrease and an
increase of cAMP, respectively, and consequently in a reduction and
enhancement of triglyceride lipase activity (36). In a
recent in vivo study, carp were infused with adrenergic agonists and antagonists (39). It was observed that yohimbine
(selective
2-adrenoceptor antagonist) delayed the
FFA-suppressing effect of NE. However, infusion of the
2-adrenoceptor agonist clonidine only partially mimicked
the lipolysis-inhibiting effect of NE followed by a strong rebound
immediately after the infusion. These results did not support a direct
involvement of
2-adrenoceptors on lipolysis.
Surprisingly, infusion of isoproterenol (Iso; a nonselective
-adrenoceptor agonist) caused an unexpected marked decline of plasma
[FFA]. This effect could be enlarged by preincubation with
ICI-118,551 (selective
2-antagonist), whereas
preincubation with atenolol (selective
1-antagonist)
resulted in increased FFA levels. From these results, it was concluded
that
1-adrenoceptors inhibit and
2-adrenoceptors stimulate lipolysis. In mammalian fat
cells
1-,
2-, and/or
3-adrenoceptors have been found only to stimulate
lipolysis (55), though in rat adipocytes
3-adrenoceptors were described to interact not only with
Gs but also with Gi proteins, thereby
restraining Gs-mediated adenylyl cyclase activation
(7). Obviously, to establish unequivocally the occurrence
of inhibitory
-adrenoceptors in fish adipose tissue, in vitro
experiments with adipocytes isolated from lipid depots have to be
carried out.
In the present study, we demonstrated that tilapia depresses its plasma
[FFA] during hypoxia. Furthermore, we were able to show that the
release of FFA by isolated adipocytes is suppressed through activation
of -adrenoceptors.
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MATERIALS AND METHODS |
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Experimental Animals
Tilapia (Oreochromis mossambicus), of both sexes, weighing 400-700 g, were obtained from the department of Animal Physiology (University of Nijmegen, Nijmegen, The Netherlands). Fish were kept in aquaria with well-aerated running local tap water, fed daily with pelleted trout food (10 g/kg body mass, Trouvit, Putten, The Netherlands), and acclimated to a 14:10-h light-dark cycle. Before the in vivo experiments were carried out, fish were acclimated for at least a month at 20°C. In vitro experiments were performed at the department of Molecular Pharmacology (University of Groningen, Groningen, The Netherlands). Before these experiments, fish were adapted to 25°C, transported and kept for at least a week in a 200-liter tank with well-aerated running water at 25°C, and fed daily with pelleted trout food (10 g/kg body mass).In Vivo Experiments
Preexperimental protocol. The experiments were performed in a recirculation system as described by Vianen et al. (47). The water of the system was kept at a temperature of 20.0 ± 0.5°C, air saturated at a normoxic level of 80-90% (15.5-17.5 kPa PO2), and pumped through experimental flow chambers at a rate of 0.8-1.0 l/min. The air saturation (AS) level of the in-flowing water was measured by an oxygen electrode, which was connected to an oxygen controller (Applikon) set to a desired AS value. When the AS level reached values below the set point (as a consequence of oxygen consumption by the fish or N2 bubbling), a magnetic valve, built in the air supply, was activated, resulting in an increase of water PO2.
Before the start of each experiment, three fishes were captured and placed individually in the experimental flow chambers. During the experimental period, the animals were deprived of food. After 3 days of acclimation, each fish was anesthetized in the flow chamber with MS-222 (150 mg/l tricaine methanesulfonate), which was buffered by the carbonate in the water. After cessation of breathing movements, the animal was placed on an operating table, which permitted continuous irrigation of the gills with aerated water containing 100 mg/l MS-222. In all fish, a polyurethane catheter (~10 cm), which was connected to 50 cm of polyethylene tubing (PE-50; Rubber, Hilversum, The Netherlands), was inserted occlusively in the third afferent gill artery to permit withdrawal of blood. The cannula was filled with heparinized (200 IU/ml) saline and was secured to the skin with sutures after implantation. After surgery, the animals were placed in the flow chambers again and allowed to recover for 2 days before the experiments were carried out. This 5-day preexperimental protocol has been shown to minimize the effects of handling, anesthesia, and surgery (46). Two times per day each cannula was flushed with heparinized saline (200 IU/ml) and filled with a viscose solution of polyvinylpyrrolidone (PVP; 1 g/ml saline) containing 500 IU/ml heparin. With the use of this procedure, the diffusion of heparin in the circulation is negligible.Experiments. Before the in vivo experiments were started, one control blood sample was taken (between 0900 and 0930) for measuring the initial values of plasma substrates and stress hormones. Directly after this blood sample, tilapia (n = 6) were exposed to a controlled linear PO2 decline (over 1 h) from 80-90% AS to 20% AS (~17-3.9 kPa PO2). This decline was followed by a stepwise hypoxic load of successively 20, 10, and 5% AS (3.9, 1.9, and 1.0 kPa PO2, respectively), each level being maintained for 2 h. Blood samples were taken before and during the stepwise hypoxia exposure for measuring the substrates at the following time points (in h): 0 (control); 1.5 and 3 (20% AS); 3.75 and 5.25 (10% AS); 6 and 7.5 (5% AS). Samples for measuring the stress hormones were taken at the following time points (in h): 0 (control), 3 (20% AS), 5.25 (10% AS), and 7.5 (5% AS). Control experiments with tilapia (n = 6) were carried out at constant AS levels of 80-90% AS (15.5-17.5 kPa PO2), and blood samples were taken at the same time points.
Withdrawal of blood (300 or 450 µl) was carried out with ice-cooled, heparin-flushed (10,000 IU/ml) microliter syringes (500 µl; Hamilton) that were placed immediately on ice. Thereafter, the volume of blood was replaced by Ringer saline (52), and the cannula was refilled with PVP. The blood samples were centrifuged for 5 min at 10,000 g (Eppendorf model 5415), and plasma was separated directly. For analysis of lactate and glucose, 100 µl plasma were added to 400 µl of TCA solution (6% vol/vol) to precipitate plasma proteins. After being mixed and incubated (minimal 20 min), these samples were centrifuged, and two aliquots of 200 µl supernatant were stored atIn Vitro Experiments
Adipose tissue collection and incubation procedure. Adipocytes were isolated and incubated according to the procedure in rats described by Hollenga et al. (18) with some modifications to isolate fat cells from fish adipose tissue. Tilapia were killed by a sharp blow on the head followed by spinal transection at the cervical level. Samples of mesenteric adipose tissue were removed rapidly, obtaining portions of 8-10 g/fish, which were placed immediately into a petri dish with Krebs-Henseleit buffer [at 25°C (in mmol/l): 117.5 NaCl, 5.6 KCl, 1.18 MgSO4, 2.52 CaCl2, 1.28 NaH2PO4, 25.0 NaHCO3, and 5.5 D-glucose] pregassed with 5% CO2 in O2, pH 7.4. The portions of adipose tissue were chopped with a McIIwain tissue chopper, obtaining slices of 1 mm2. Cells were isolated by incubation at 25°C in a shaking water bath in a Teflon vessel containing 20 ml of Krebs-Henseleit buffer per portion with the addition of 1% BSA (fraction V) and collagenase (type II, 130 U/ml) under an atmosphere of 5% CO2 in O2, pH 7.4. After 1.5 h, the suspension of adipocytes was filtered through a nylon cloth and washed three times with Krebs-Henseleit buffer (1% BSA). In a shaking water bath equilibrated at 5% CO2 in O2, portions of 100 µl of adipocyte suspension (~3 × 105 cells) were incubated in Teflon vessels for 5 h at 25°C in a total volume of 3 ml Krebs-Henseleit buffer containing 2% BSA with or without (controls) the addition of adrenoceptor (ant)agonist. Control measurements were performed in triplicate and experimental measurements in duplicate. When both an agonist and antagonist were used, the adipocytes were preincubated with the antagonist 15 min before the agonist was added. The incubation period was terminated by adding the content of each vessel to 3 ml of extraction medium [1-propanol-n-heptane-1 N H2SO4 (40:20:1)]. The tubes were mixed for 60 s and centrifuged for 5 min at 2,000 g in a Hettich Rotixa/KS centrifuge. From the upper layer, 400 µl were used for FFA determination.
Chemicals and analytical procedures.
MS-222 (tricaine methanesulfonate), heparin, BSA (fraction V),
collagenase (type II), ()-isoproterenol hydrochloride, and (
)-norepinephrine hydrochloride were obtained from Sigma (St. Louis,
MO). Phentolamine was obtained from Novartis (Arnhem, The Netherlands)
and polyvinylpyrrolidone from Merck (Darmstadt, Germany). Timolol was a
gift from Merck Sharpe & Dohme (Haarlem, The Netherlands), and
BRL-35135A was donated by SmithKline Beecham (Welwyn, UK).
Presentation and Data Analysis
The data of the in vivo experiments are presented in Table 1 and in Fig. 1, A-C, as means ± SE for both the normoxia (n = 6) and hypoxia (n = 6) groups. Statistical differences between each time point and the initial value at time (t) = 0 h were analyzed with SigmaStat with the nonparametric Friedman test (Friedman repeated-measures ANOVA on ranks); multiple comparisons were made by the Dunnett's test, and the level of significance was set to P < 0.05. Statistical differences between the normoxia group and the hypoxia group were determined with the Mann-Whitney rank sum test.
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The results of the in vitro experiments were normalized to the mean
value of the controls [without (ant)agonist] and are presented in
Figs. 2-4 as means ± SE, representing 5-8 measurements
for each concentration. Statistically significant differences
(P < 0.05) between a treatment and the control value
were determined using one-way ANOVA followed by the
multiple-comparisons Dunnett's test. Differences between a treatment
with antagonist and without antagonist were tested for statistical
significance using the paired Student's two-tailed t-test
(P < 0.05).
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RESULTS |
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In Vivo Experiments
Substrates. The initial plasma lactate and glucose concentrations (Table 1) in tilapia were 0.36 ± 0.08 and 3.62 ± 0.29 mmol/l, respectively, for the normoxia group and 0.24 ± 0.08 and 3.46 ± 0.44 mmol/l, respectively, for the hypoxia group. In the control group, both plasma glucose and lactate remained at these levels during the whole experimental period. Exposure to 20% AS (3.9 kPa PO2) did not change plasma lactate levels. After transition and 0.5-h exposure to 10% AS (1.9 kPa PO2), the lactate concentration ([lactate]) was significantly elevated at t = 3.75 h followed by a further increase up to 2.18 ± 0.75 mmol/l at the end of 10% AS. Also, exposure to 5% AS (1.0 kPa PO2) caused a continuous increase of plasma lactate levels, reaching a value of 5.65 ± 1.05 mmol/l at the end of the experiment. The continuous accumulation of lactate was accompanied with a hyperglycemia during the same period. During exposure to 10% AS, plasma glucose levels increased to 6.36 ± 0.75 mmol/l at the end of this period. This was followed by a further increase during the 5% AS period to a concentration of 7.79 ± 0.70 mmol/l at t = 7.5 h. The initial level of plasma FFA was 0.47 ± 0.05 and 0.38 ± 0.05 mmol/l for the normoxia and hypoxia groups, respectively (Fig. 1A). During subsequent normoxia exposure, plasma FFA levels fluctuated around the initial value. In the hypoxia group, plasma FFA levels showed a continuous depression that was significant at 10 and 5% AS, reaching a value of 0.22 ± 0.02 mmol/l at t = 7.5 h.
(Neuro)hormones. The initial plasma concentrations of cortisol (Table 1), NE (Fig. 1B), and Epi (Fig. 1C) were 55.0 ± 9.2, 0.05 ± 0.01, and 0.15 ± 0.07 ng/ml, respectively, for the control group and 41.0 ± 12.2, 0.07 ± 0.01, and 0.21 ± 0.07 ng/ml, respectively, for the hypoxia group. In the control group, these values did not change during normoxic conditions. In the hypoxia group, plasma Epi concentration ([Epi]) fluctuated around basal levels during the whole experimental period. In contrast, both plasma NE and cortisol increased significantly at all hypoxia levels. At 20% AS, these hormones were elevated to 0.22 ± 0.05 ng/ml for plasma NE and 99.0 ± 12.9 ng/ml for plasma cortisol. Both during 10 and 5% AS, there was a further increase to 2.47 ± 0.51 ng/ml for NE and 317.2 ± 93.1 ng/ml for cortisol at t = 7.5 h.
In Vitro Experiments
Figure 2A shows the effect of increasing concentrations of NE in the absence and presence of theExposure of the adipocytes to Iso also resulted in a concentration-dependent inhibition of FFA release, as shown in Fig. 3. The FFA release already decreased significantly at 10 nM Iso, with maximal inhibition to 31.8 ± 5.6% FFA release being reached at 100 µM. Timolol (1 µM) completely antagonized the antilipolytic response induced by Iso concentrations up to 1 µM; the two higher Iso concentrations (10 and 100 µM) were hardly (nonsignificantly) antagonized.
Figure 4 shows the results of increasing
concentrations of the selective 3-adrenoceptor agonist
BRL-35135A. The FFA release was significantly decreased from 1 µM on
up to an almost complete inhibition of the FFA release below 10% at
100 µM BRL-35135A.
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DISCUSSION |
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In Vivo Experiments
The plasma [lactate] in cannulated tilapia was not affected during exposure to 20% AS (3.9 kPa PO2), suggesting that the anaerobic metabolism was not stimulated at this oxygen level. This is supported by the observation that tilapia is able to suppress its metabolic rate below the standard metabolic rate, which is an important strategy to survive hypoxia conditions (41, 42), revealing this fish species to be rather resistant to hypoxia. In contrast to the 20% AS period, there was a significant and continuous increase in plasma [lactate] during 10% AS (1.9 kPa) and 5% AS (1.0 kPa), which indicates that these oxygen levels are below the anaerobic threshold (47). In agreement with these findings, Van Ginneken et al. (43) have shown that the critical oxygen level with respect to the anaerobic threshold in tilapia is around 18% AS. The lactate accumulation was accompanied by hyperglycemia during the same period. Because in fish uptake rates of glucose by the peripheral tissues are low (4, 22), hyperglycemia is mainly the result of an increased hepatic glucose release to the blood via stimulation of glycogenolysis and/or gluconeogenesis (46). These processes can be stimulated by both catecholamines and cortisol (20, 27, 49, 53); catecholamines mainly stimulate glycogenolysis (27), whereas cortisol mainly stimulates gluconeogenesis (20, 40, 49). Plasma [Epi] did not increase in tilapia during the stepwise decreasing oxygen levels. In contrast, there were marked elevations in plasma [NE] and cortisol concentration, especially at 10 and 5% AS; hence, both glycogenolysis and gluconeogenesis might account for the observed hyperglycemia. However, because cortisol is a steroid hormone acting specifically via DNA transcription in its target cells, it is generally slow acting (40), inducing its hyperglycemic effect after hours (5, 24) or even days (49). On the other hand, catecholamines are able to induce their metabolic effects within minutes (9, 53); therefore, it is likely that the increase in plasma glucose in tilapia was the result of adrenergic stimulation of hepatic glycogenolysis. In fish, like in mammals, catecholamines stimulate this metabolic process via activation of glycogen phosphorylase (35, 53) through stimulation ofThe continuous reduction of plasma FFA levels in tilapia during stepwise hypoxia is in agreement with a previous study (48) in which exposure of carp (and trout; Vianen G, van den Thillart G, van Kampen M, van Heel T, and Steffens A, unpublished observations) to prolonged hypoxia resulted in a marked continuous suppression of plasma FFA levels in both fish species. This suggests a general protection mechanism of fish against accumulation of these amphiphilic compounds, which may cause damage to the membrane structure and membrane-dependent functions in mammals after ischemic and hypoxic insults (17, 21). Van Raaij et al. (45) demonstrated in carp that depression of plasma [FFA] is induced by NE, most probably via inhibition of lipolysis, which is in contrast to the situation in mammals (33, 54). In accordance with this finding, Vianen et al. (48) observed that the permanently reduced FFA levels in carp (and trout; Vianen G, van den Thillart G, van Kampen M, van Heel T, and Steffens A, unpublished observations) during prolonged hypoxia were accompanied in both species with chronically elevated NE levels (new steady state around 4 ng/ml). In these fish species, the FFA reduction became significant when [NE] was increased to ~2 ng/ml. In the present study, the significantly reduced FFA levels in tilapia were also accompanied with increased plasma NE levels. The FFA reduction became significant when the [NE] was ~0.5 ng/ml, which is lower compared with carp and trout. However, because catecholamines are rapidly (minutes) cleared from the plasma (16, 28), the turnover rate of NE may have already increased during exposure to 20% AS. Therefore, the results of the present in vivo experiments may support the idea that NE suppresses plasma FFA levels in tilapia most probably via inhibition of lipolytic activity in adipose tissue.
In Vitro Experiments
The main objective of the present study was to investigate whether in fish NE decreases lipolysis via direct activation of adipocyteThe NE-induced reduction of FFA production in tilapia adipocytes was
not affected by the addition of phentolamine, indicating that in
tilapia the effect of NE is not mediated through 2 (or
1-)-adrenoceptors. These results are in contrast to the
in vivo observations of Van den Thillart et al. (39) in
carp, which showed that yohimbine (
2-adrenoceptor
antagonist) antagonized the NE effect. However, a reduction in plasma
FFA could be mimicked only partially by the
2-adrenoceptor agonist clonidine. It may be possible
that, in carp, other lipid depot tissues like liver and muscle are
affected by NE via
2-adrenoceptors. An additional explanation may be that catecholamines are potent vasomodulators in
fish (1) like in mammals. During NE infusion in vivo, the FFA reduction in the plasma of carp may have been partly due to an
NE-induced inhibition of visceral blood flow via vasoconstriction through
2-adrenoceptors (15). This may have
resulted in a decreased release of FFA from visceral lipid stores,
which could be antagonized by yohimbine.
Van den Thillart et al. (39) observed a marked reduction
in plasma FFA after infusion of Iso in vivo in carp, indicating the
involvement of -adrenoceptors. Incubation of tilapia adipocytes with
Iso in the present study also resulted in a decline of FFA release,
which was already significant at 10 nM, indicating that Iso is more
potent (by a factor of 10-100) than NE, as is also the case for
the lipolytic effects in mammalian adipocytes (55). The
effects of the lower concentrations of Iso and NE could be inhibited by
timolol (a potent
-adrenoceptor antagonist) at 1 µM, a
concentration that does not yet antagonize
3-adrenoceptors (Obels and Zaagsma, unpublished
observations). Therefore, these results strongly suggest that the
antilipolytic effect of NE and Iso under in vivo conditions is mediated
to an important extent through
1- and/or
2-adrenoceptors.
Although -adrenoceptors generally activate Gs proteins
and thereby adenylyl cyclase activity,
1- and
3-adrenoceptors in rat adipocytes have been postulated
to interact with inhibitory Gi proteins as well. Thus
Chaudhry et al. (7) demonstrated that pertussis toxin,
which locks Gi in the GDP (inactive) form, markedly
potentiates the
3- and, to some extent, the
1-adrenoceptor-mediated cAMP accumulation by Iso. In the
present study, it is observed that at higher concentrations of Iso (10 and 100 µM) timolol (1 µM) did not significantly antagonize the Iso
effect. It is known that, in mammalian adipocytes, Iso preferentially
stimulates
1- and
2-adrenoceptors at low
concentrations, whereas higher Iso concentrations also activate
3-adrenoceptors (7, 8, 14). Therefore, the
observed reduction in FFA release at the highest Iso concentrations in
the presence of timolol (1 µM) suggests the involvement of
3-type adrenoceptors. In agreement, exposure of the
tilapia adipocytes to increasing concentrations of BRL-35135A (a
selective
3-adrenoceptor agonist) reduced the FFA
production by 90% at the highest concentration. This provides evidence
that
3-adrenoceptors are also present in fish adipose
tissue and inhibit FFA mobilization in contrast to the mammalian
situation (14, 37, 44, 55).
BRL-35135A is the methyl ester of BRL-37344, the prototype and most
frequently studied 3-adrenoceptor agonist. For the
present study, BRL-35135A was chosen because this compound has been
shown to relax guinea pig taenia cecum solely through
3-adrenoceptors, with an even fivefold higher potency
than BRL-37344 (23). The observation that significantly
higher concentrations of BRL-35135A are required to inhibit lipolysis
of tilapia adipocytes (present study) than to activate rodent
3-adrenoceptors (23) is reminiscent of
observations made in human adipocytes in which the parent compound BRL-37344 in stimulating lipolysis also has a low potency
(18). However, it should be mentioned that the structure
and properties not only of
3- but also of
1- and
2-type adrenoceptors in tilapia (and in fish, in general) are yet completely unknown and may deviate from those of mammalian species. Hence, the selectivity of BRL-35135A for rodent
3-adrenoceptors does not necessarily imply
selectivity for tilapia
3-adrenoceptors. Remarkably,
however, in tilapia adipocytes the maximal efficacy of BRL-35135A
exceeds that of Iso, which was not seen before in mammalian systems.
This finding would indicate that inhibition of lipid mobilization in
tilapia by activation of
3-adrenoceptors is more
efficacious than by
1- and/or
2-adrenoceptors.
It could be argued that the suppression of lipid mobilization, as
observed in the present study, may be the result of enhancement of
reesterification rather than, or in addition to, inhibition of
lipolysis. However, to our knowledge, direct activation of reesterification of FFA by stimulation of adipocyte 1-,
2-, and/or
3-adrenoceptors with
catecholamines has never been observed. Furthermore, in our previous
study in carp (39), a single injection of Iso reduced
plasma FFA levels acutely (within 30 min) and simultaneously increased
plasma glucose. Remarkably, the antilipolytic effect was significantly
accelerated and increased by pretreatment with the selective
2-adrenoceptor antagonist ICI-118,551, whereas the
glycogenolytic effect of Iso was decreased by ICI-118,551. These
results strongly indicate that reesterification of FFA does not underly
the antilipolytic effect of the catecholamines.
From the present results, it is not clear to what extent NE (released
from sympathetic nerves) exerts its effect through
3-adrenoceptors. In mammalian systems,
3-adrenoceptors have been found to be in close proximity
to sympathetic nerve terminals, leaving the possibility of high local
concentrations of NE to be attained during enhanced nerve activity
(10). Timolol at 1 µM almost completely antagonized the
reduction of FFA release up to 10 µM of NE. At the highest [NE],
however, timolol was not or at most partially effective, suggesting the
(additional) involvement of
3-adrenoceptors. It is
obvious that further research is required to characterize the
-adrenoceptor subtype in more detail, including their capacity to
couple to inhibitory Gi proteins in tilapia adipocytes.
In summary, the results of the present study indicate that, in tilapia,
endogenous (hypoxia-induced) and exogenously applied NE suppresses
lipolysis in tilapia adipose tissue, resulting in a decline of FFA
levels. Whereas in carp this effect is mediated partially
through 2-adrenoceptors, in tilapia it appeared to be
mediated solely through
-adrenoceptors. Furthermore, for the first
time, strong indications are provided that, in fish,
3-adrenoceptors are involved in the reduction of lipolysis.
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ACKNOWLEDGEMENTS |
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We thank the biology students (University of Leiden) for assistance with the in vivo experiments and Frans Brouwer and Tamara van Heel for skillful assistance with the analyses of the catecholamines and cortisol, respectively. We thank Dr. J. R. S. Arch (SmithKline Beecham, Epson, UK) for the gift of BRL-35135A, Dr. M. Axelsson for the generous gift regarding the polyurethane tube, and Dr. A. D. F. Addink for reading the manuscript critically.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. J. Vianen, Institute of Evolutionary and Ecological Sciences, Dept. of Integrative Zoology, Van der Klaauw Laboratories, PO Box 9516, 2300 RA Leiden, The Netherlands (E-mail: Vianen{at}rulsfb.leidenuniv.nl).
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.
10.1152/ajpendo.00187.2001
Received 30 April 2001; accepted in final form 20 September 2001.
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