Vol. 278, Issue 4, R956-R963, April 2000
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
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ABSTRACT |
The rate of hepatic glucose production
(Ra glucose) of rainbow trout (Oncorhynchus mykiss)
was measured in vivo by continuous infusion of
[6-3H]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 Ra glucose and to
assess indirect effects of Epi in the intact animal. In vivo infusion of Epi caused hyperglycemia (3.75 ± 0.16 to 8.75 ± 0.54 mM) and a
twofold increase in Ra glucose (6.57 ± 0.79 to 13.30 ± 1.78 µmol · kg
1 · min1,
n = 7), whereas Prop infusion decreased Ra from
7.65 ± 0.92 to 4.10 ± 0.56 µmol · kg1 · min1
(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 Ra 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 Ra 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 Ra 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
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INTRODUCTION |
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 (Ra glucose), a decrease
in the rate of disappearance (Rd glucose), or a combination
of both as in mammals (2). Recent experiments on rainbow trout show
that prolonged exercise causes a parallel decline in Ra
glucose and plasma Epi below normal resting levels (29), suggesting
that this hormone may be involved in maintaining basal Ra
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 Ra 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
Ra glucose and decreasing Rd glucose, and
2) basal plasma Epi is not involved in maintaining resting
Ra glucose. We also 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.
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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 (60 × 16 × 18 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 (
O2; see Refs. 9
and 31 for details).
Measurement of glucose kinetics. Ra glucose was
measured by continuous infusion of 6-3H glucose as
described and validated previously (7, 8). The infusate was prepared
daily by drying the isotope under N2 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 566 ± 19 g), Epi (n =
7, 694 ± 58 g), or Prop (n = 10, 611 ± 63 g) at 1 ml/h. Epi
was infused for 35 min at a rate of 1.34 nmol · kg1 · min1.
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 0.113 µmol · kg1 · min1
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, 12-13
blood samples (0.1-0.2 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 N2, 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 715 ± 47 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 15-60 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 Ra glucose
values reported in this paper were calculated with the steady-state
equation of Steele (30), because they were never significantly
different from Rd glucose when both rates were calculated
separately with the non-steady-state equation. To allow meaningful
comparisons with in vivo Ra 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 Ra 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 Ra values were corrected for renal glucose
production by subtracting 10.5% (7). For the in vivo experiments,
changes in O2, glucose
concentration, glucose specific activity, and Ra glucose
were assessed by two-way ANOVA and Dunnett's or Tukey's test.
Differences between in vitro treatments and comparisons between
Ra 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.
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RESULTS |
In Vivo Experiments
Control saline infusions. Infusion of vehicle saline had no
effect on O2 (Fig.
1), plasma glucose concentration (Fig.
2A), glucose specific activity
(Fig. 2B), or Ra glucose (Fig. 2C;
P > 0.05). Throughout the saline infusion experiments,
O2 averaged 38.69 ± 0.36 µmol
O2 · kg1 · min1
(n = 6), plasma glucose concentration 5.44 ± 0.06 mM (n
= 13) and Ra glucose 7.95 ± 0.08 µmol · kg1 · min1
(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 24.9 ± 1.6% (n = 3).
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Fig. 1.
Rates of oxygen consumption
(O2) 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
O2 before infusion; b,
different from mean O2 during
epinephrine infusion; c, different from mean
O2 during propranolol
infusion.
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Fig. 2.
Effects of vehicle saline infusion on plasma glucose concentration
(A), glucose specific activity (B), and glucose rate of
appearance (Ra glucose; C) in rainbow trout. Shaded
area indicates saline infusion, and values are means ± SE (n
= 3).
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Epi infusions. The infusion of Epi caused an increase in
O2 from a mean baseline value
of 40.67 ± 0.39 to 61.36 ± 2.67 µmol O2 · kg1 · min1
(n = 7, P < 0.05), and
O2 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 2.36 ± 0.72 nM (n = 7) before starting the
exogenous administration of Epi and increased to 545 ± 23.91 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 19.47 ± 5.34 nM (n = 8). The effects of Epi
infusion on glucose concentration, glucose specific activity, and
Ra glucose are presented in Fig.
4. Before Epi infusion, plasma glucose
concentration and Ra glucose averaged 3.75 ± 0.16 mM and
6.57 ± 0.79 µmol · kg1 · min1
(n = 7), respectively. Glucose concentration increased to a
maximal value of 8.51 ± 0.54 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 Ra glucose to a maximal value of 13.30 ± 1.78 µmol · kg1 · min1
(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 5.29 ± 0.43 µmol · kg1 · min1
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 24.5 ± 0.9% (n = 7).
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Fig. 3.
Plasma concentration of epinephrine ( ) and norepinephrine ( ) in
rainbow trout during exogenous infusion of epinephrine (shaded area).
Values are means ± SE (n = 6). * Significant differences
from baseline levels (P < 0.05).
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Fig. 4.
Effects of exogenous epinephrine infusion on plasma glucose
concentration (A), glucose specific activity (B), and
Ra 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).
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Prop infusions. One-hour infusion of the -adrenoreceptor
blocker Prop caused a decline in
O2 from a baseline value of
41.06 ± 0.96 to 32.80 ± 1.37 µmol
O2 · kg1 · min1
(n = 7, P < 0.05). A further decrease to 29.36 ± 1.47 µmol
O2 · kg1 · min1
was observed after the end of the Prop infusion (Fig. 1). Changes in
glucose concentration, glucose specific activity, and Ra
glucose during and after Prop infusion are plotted in Fig.
5. Baseline glucose concentration and
Ra glucose before Prop infusion were 4.98 ± 0.49 mM and
7.65 ± 0.92 µmol · kg1 · min1
(n = 10), respectively. Plasma glucose concentration decreased progressively to reach values significantly lower than baseline in the
recovery period (Fig. 5A). Ra glucose declined
progressively to 5.17 ± 0.54 µmol · kg1 · min1
(n = 10, P < 0.05) during Prop infusion and reached a
minimal value of 4.10 ± 0.56 µmol · kg1 · min1
(P < 0.05) by the end of recovery (Fig. 5C).
Hematocrit did not change significantly during the Prop-infusion
experiments and averaged 23.9 ± 0.8% (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 Ra glucose are quantified.
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Fig. 5.
Effects of propranolol infusion on plasma glucose concentration
(A), glucose specific activity (B), and Ra
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).
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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).
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In Vitro Experiments
Effects of Epi and Prop on glucose production by
hepatocytes. Mean glucose production rates of isolated 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).
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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.
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Comparison of In Vivo and In Vitro Glucose Production Rates
Figure 8 shows a comparison of
Ra 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 Ra glucose measured in vivo that averaged 7.31 ± 0.54 µmol · kg1 · min1
(n = 20) after correction for renal glucose production (see
METHODS). Mean in vitro Ra glucose was 3.13 ± 0.09 µmol · kg1 · min1
(n = 22) and increased to 3.83 ± 0.16 (n = 11) and
5.92 ± 0.19 µmol · kg1 · min1
(n = 22) in the presence of low (5 nM) and high (500 nM) Epi concentration, respectively (P < 0.05). The relative 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 Ra glucose by
102% and 89%, respectively. Prop alone caused a 46% decline in
Ra glucose in vivo but had no significant effect in vitro
(+1.8%).
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Fig. 8.
Rates of glucose production (Ra 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.
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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).
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DISCUSSION |
Earlier in vivo measurements of fish substrate kinetics had revealed
high basal rates of hepatic glucose production and a limited ability to
increase Ra 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 Ra 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 Ra 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 Ra and
Rd, the difference between these rates could not be
detected statistically, because Rd increased almost
simultaneously with Ra. Therefore, hyperglycemia resulted
from a rapid increase in Ra, whereas the Epi-induced
reduction in Rd known to occur in mammals (2) did not play
a role in fish. The increase in trout Ra 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 Ra 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 Ra 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 Ra 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. 7.31 µmol · kg1 · min1;
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 Ra 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 Ra 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 · kg1 · min1)
falls within the range of values previously obtained by others for
trout hepatocytes (1-5
µmol · kg1 · min1,
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 Ra 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 Ra 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 Ra 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 (Ra) and glucose
disposal (Rd) were never statistically different, all the
figures showing changes in Ra also depict how
Rd was affected. Arterial Epi infusion caused
Rd 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 Rd 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 Rd 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 Ra glucose, but the concomitant reduction in
Rd glucose observed in mammals does not occur in trout.
2) Basal levels of circulating Epi are partly responsible for
maintaining resting Ra glucose in rainbow trout. 3)
Epi stimulates hepatic glucose production in a dose-dependent 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 Ra
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.
| |
ACKNOWLEDGEMENTS |
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).
| |
FOOTNOTES |
The work was supported by National Sciences and Engineering Research
Council research and equipment grants to J.-M. Weber.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J.-M. Weber,
Biology Dept., Univ. of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada
K1N 6N5 (E-mail: jmweber{at}science.uottawa.ca).
Received 20 May 1999; accepted in final form 29 September 1999.
| |
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