Camp. Bio&vn.
Pergamon
Phwiol. Vol. I I IA. No. 3, pp. 421 428. 1995
Copyright ,I;. 1995 Elsevier Science Ltd
Printed in Great Britain. All rights reserved
0300-9629195 $9.50 + 0.00
0300-%29(%)00035-6
Influence of the relative proportions of energy
yielding nutrients on liver intermediary metabolism
of the European eel
M. D. Sk-ez, M. C. Hida .lgo, M. Garcia Gallego, A. Sanz and
M. de la Higuera
Dpto de Biologia Animal
Universidad
de Granada,
y Ecologia, Unidad de Fisiologia
18071 Granada,
Spain
Animal,
Facultad
de Ciencias,
The influence of dietary protein, lipid and carbohydrate proportions on liver glutamate dehydrogenase
(GDH), fructose l&bisphosphatase
(FBPase), glucose&phosphate
dehydrogenase (G6PDH) and
pyruvate kinase (PK) activities was investigated in the European eel (Anguilla anguillu). The highest
weight-gain (“A) and feed-efficiency indices were obtained using diets with the highest energy content
and, for the same energetic content, those with the greatest carbohydrate level. GDH activity increased
in fish fed diets with the highest protein:energy ratio with respect to diets either with lower protein
or with higher total energy contents. FBPase showed an increased activity in the liver of fish fed on
diets with the lowest carbohydrate content and the highest total lipids concentrations. G6PDH activity
correlated positively with dietary carbohydrate concentrations and negatively with dietary lipids levels.
The increase of carbohydrates and energy levels in the diet increased liver PK activity. In general
terms, the European eel showed a certain capacity to adapt intermediary metabolism to changes in
diet composition. Amino acid catabolism increased in response to a higher dietary protein:energy
ratio. High carbohydrate levels enhanced glucose utilization to yield energy and lipid formation.
Gkconeogenesis increased in response to a lower dietary carbohydrate content. High dietary lipid
concentrations reduced gluconeogenesis and lipogenesis from carbohydrates.
Key words: Eel; Fish culture;
Comp. Biochem.
Physiol.
Nutrition;
I I lA, 421-428,
Metabolism;
Growth;
Diet; Enzymes;
Energy.
1995.
Introduction
Many studies on fish nutrition
have recently
been devoted to evaluating
the protein-saving
effects of dietary lipids and digestible carbohydrates on food conversion, growth, metabolic
response and macronutrient
utilization
in fish.
Dietary lipids as energy-yielding
nutrients have
proved capable
of reducing
dietary
protein
levels and of improving nitrogen retention in the
Correspondence
10: M. De la Higuera, Dpto de Biologia
Animal y Ecologia, Unidad de Fisiologia Animal, Facultad de Ciencias,
Universidad
de Granada,
18071
Granada,
Spain.
Received 19 July 1994; revised 5 January 1995; accepted IO
January
1995.
421
European
eel (Garcia Gallego et al., 1993),
Japanese eel (Watanabe,
1980) and other fish
species (de la Higuera et al., 1977; Viola and
Rappaport,
1979). Eels fed on high-fat diets
(20-30% total lipids) showed high growth rates
associated with increased body-fat stores (Degani et al., 1986; Dosoretz and Degani, 1987).
The capacity of eels to accumulate
energy as
body fat (Gallagher et af., 1984; Degani et al.,
1986) should be considered a characteristic
of
migratory fish and, in fact, the amount of stored
fat has been recently considered as a triggering
mechanism in migrations (Larsson et al., 1990).
The capacity
of carbohydrates
to replace
protein is more controversial,
at least in carnivorous fish. At the digestive level, physical treatments seem to improve carbohydrate
utilization
422
M. D. Suarez et al.
(Bergot and Brecque, 1983) and at the same
time improve protein and energy retention
efficiencies in trout (Kaushik and Oliva-Teles,
1985; Kaushik et al., 1989) and carp (Watanabe
et al., 1987). Dietary carbohydrates can reach
levels of about 40% without affecting growth or
nutrient retention in diets for warm-water fish
such as carp (Takeuchi et al., 1979) the tilapia
(Anderson et al., 1984) European eel (Hidalgo
et al., 1993; Sanz et al., 1993), and even freshwater carnivorous fish such as trout (Kaushik
et al., 1989). Nevertheless, metabolic studies
indicate that glucose is poorly utilized by carnivorous fish, and prolonged carbohydrate feeding
does not cause any adaptive regulation of glycaemia in trout (Cowey et al., 1977a; Hilton and
Atkinson, 1982; Kaushik et al., 1989). In any
case, a certain degree of enzyme adaptation to
changes in dietary carbohydrate content has
been observed in carnivorous fish (Cowey et al.,
1977b; Hilton and Atkinson, 1982), although
that metabolic response was less effective concerning carbohydrate utilization than that observed in omnivorous
fish such as carp
(Shimeno et al., 1981). Glycaemia levels in the
European eel did not significantly change after
the intake of a high-carbohydrate diet (Bulnheim, 1974). Furthermore, the European eel was
able to utilize efficiently cooked corn starch
at levels of 40% with good growth and food
conversion (Hidalgo et al., 1993).
The object of the present work was to study
the capacity of the European eel to adapt its
intermediary metabolism to variations in diet
concentrations of protein, lipids and carbohydrates around optimum values previously esTable
1. General
comnosition
tablished (Sanz et al., 1993). For that purpose,
liver activities of pyruvate kinase, glucose-6phosphate dehydrogenase, fructose 1,6-bisphosphatase and glutamate dehydrogenase
(as
representative enzymes involved in glycolisis,
pentose-phosphate
pathway, gluconeogenesis
and amino acids deamination, respectively)
were measured.
Materials and Methods
Animals
Eels captured in the Guadalquivir River
(southern Spain) were moved to the laboratory,
and those weighing 35-40 g were selected and
randomly divided into 27 different lots, each
composed of 37-41 individuals to achieve a
similar total initial weight per lot. An initial
control group (n = 40) was dissected for body
composition analysis.
Each lot was housed in a fibreglass tank filled
with 360 1 of dechlorinated water in an open
circuit with a renewal rate of 10% per hour. The
water was continually aerated (oxygen concentration in the tanks was constantly over 6 ppm)
and thermoregulated
(25°C). A light: dark
period of 12: 12 hr was maintained throughout
the study.
Diets and feeding
After a 30-day adaptation period to the laboratory conditions, the eels were weighed and
fed on the respective experimental diet for 60
days. Nine experimental diets were formulated
(Table l), varying in protein (supplied by white
fish meal), lipid (supplied by equal parts of corn
of the experimental
diets (ail00 a dry diet)
Diets*
Group
White fish meal
Cod liver oil
Corn oil
Gelatinized corn starch
Vitamin mixture?
Mineral mixture?
Sodium alginate
Cr&
Cellulose
Protein (X)
Fat (%)
Ash (%)
Energy (MJ/kg)S
Protein/energy
(g/MJ)
% of total energy from
protein/fat/carbohydrate§
1
Group
30/16/23
44.09
4.41
6.00
23.00
2.00
7.00
2.50
0.50
10.50
30/12/32
44.09
1.91
4.50
32.00
2.00
7.00
2.50
0.50
5.50
30/8/41
44.09
30.49
16.22
13.42
17.56
17.37
41137122
2
Group
3
2.41
41.00
2.00
7.00
2.50
0.50
0.50
25/20/23
36.74
7.84
7.50
23.00
2.00
7.00
2.50
0.50
12.92
25116132
36.14
5.34
6.00
32.00
2.00
7.00
2.50
0.50
7.92
25/12/41
36.74
2.84
4.50
41.00
2.00
7.00
2.50
0.50
2.92
30/20/23
44.09
6.91
7.50
23.00
2.00
7.00
2.50
0.50
6.50
30/16/32
44.09
4.41
6.00
32.00
2.00
7.00
2.50
0.50
1.50
30/12/41
44.09
1.91
4.50
41.00
2.00
7.00
2.50
0.50
30.61
12.15
13.52
17.53
17.46
30.85
8.47
13.37
17.68
17.45
24.78
20.94
II.83
18.08
13.71
25.23
16.17
11.95
17.84
14.14
25.66
12.09
12.44
17.88
14.35
30.59
20.57
13.36
19.30
15.85
30.40
15.99
12.29
18.99
16.01
31.37
11.48
11.37
18.99
16.52
41127132
41/19/40
36142122
33136131
34127139
32142126
38133129
39124137
*All the diets are labelled by their protein/fat/carbohydrate
(%) content.
TAccording to De la Higuera et al. (1989).
ICalculated
on the basis of 23.6, 39.5 and 17.2 kJ/g of protein, fat and cooked
@Rounded values.
starch,
respectively.
Diet composition
in relation
to eel growth
oil and fish oil) and carbohydrate
(supplied by
cooked
starch)
content.
These
diets were
grouped into three categories (dietary groups 1,
2 and 3), according to their gross energy content
(17.5, 18.0 and 19.0 MJ/kg, respectively)
and
protein:energy
ratio (17.5, 14 and 16 g/MJ, respectively).
Within each group of diets, the
protein
level was constant,
with a varying
proportion
of the other two energy-yielding
nutrients (lipids and carbohydrates).
Each diet
was fed to three experimental
lots, twice daily,
in a paste form prepared by mixing SO% of
the dry mixture with 50% distilled water and
distributed
by placing the diet into a plastic
mesh basket near the water surface.
The food intake of each lot was recorded
daily, and the growth rate was measured
by
individually
weighing the eels of each group at
the beginning
and end of the experimental
period.
Composition analysis
Eels from an initial control group, at the
beginning
of the experimental
period, and ten
from each experimental
dietary treatment (randomly selected from the corresponding
three
replicates at the end of this period), were used
to determine body and liver composition,
(according to AOAC, 1980), as follows:
-Water
content:
by desiccation
at 105°C.
--Protein
content:
by the Kjeldahl method
(N x 6.25).
--Fat content: by the Soxhlet method.
Tissue -sample preparation
At the end of the experiment,
ten fish from
each dietary treatment were randomly collected
from the three replicate
tanks, individually
weighed and their livers immediately
dissected
and weighed. A liver portion was immediately
homogenized
for enzyme determinations,
and
the remaining
portion was frozen in liquid NZ
and stored at - 2OC until the liver-composition
analysis. Glycogen
determination
was carried
Table 2. Influence
of diet composition
423
and metabolism
out by the method
(1974).
of Keppler
and
Decker
Enzyme assays
Enzyme activities were determined
in crudeliver extracts. Liver samples were homogenized
in ice-cold buffer (100 mM Tris, 250 mM sucrose, pH 7.6), the extracts were centrifuged at
3O,OOOg, for 30 min at 4C, and the supernatant
was assayed for enzyme activity. When required
(glutamate
dehydrogenase
activity
determination).
mitochondrial
membranes
were ruptured by sonication
before assay and then
dialysed in sucrose buffer. The soluble protein
content of homogenates
was determined by the
method of Bradford (1976).
Measurements
were performed at 25’ C with
the appropiate
enzyme and reagent. Activities
were estimated by the change in ExdOdue to the
oxidation
of NADH
(PK and GDH) or reduction of NADP (FBPase and G6PDH). using
a spectrophotometer.
The results were expressed
as mUnits/mg
protein and Units/g tissue.
The following procedures
were used: glutamate dehydrogenase
(GDH, EC 1.4.1.2) according to Schmidt (1974); pyruvate
kinase (PK,
EC 2.7.1.40)
according
to Carbonell
et ul.
(1973); fructose
1,6_bisphosphatase
(FBPase,
EC 3.1.3.11) according
to Latzko and Gibbs
(1974) and glucose-6-phosphate
dehydrogenase
(G6PDH,
EC 1.l. 1.49) according to Lohr and
Waller ( 1960).
Statistical analysis
A one-way
ANOVA,
followed
by LSD
test, was used to evaluate the significance
of
differences
attributable
to diet composition
(P < 0.05). Analysis of regression was also used
when necessary.
Results
Table 2 shows the results of food
weight-increase
and
feed efficiency
on weight
intake,
indices
gain and food utilization
Diets
Group
Weight gain
(O/o)
Food intake
(gl I00 g fish/day)
Feed efficiency
(weight gain/
food intake)
30/16/23
31.30”
+ I .02
3.28”
AO.29
0.14
+0.01
30/12/32
43.70kd
* 1.21
3.07b
kO.30
0.20ah
+0.01
I
Group
30/8/41
45.90hd
kO.69
2.80b
kO.24
0.23k
+0.01
25;20/23
39.47’h
f 2.09
3.99’
kO.06
0.14”
kO.01
25/16:32
42.53”
F5.51
2.57”h
+0.03
0.22”
kO.02
2
Group
25/12/41
53.17d’
F4.85
2.60dh
kO.05
0.27’
kO.02
Values are mean + SEM of the three experimental
groups fed on each diet.
“““‘Values within each row with different superscripts
are significantly
different
30/20/23
52.17’d’
+ I .82
2.76”h
kO.55
0.27’
+0.05
(P -C 0.05)
3
30,‘16/32
46.40h“
+6.24
2.58””
) 0.03
0.24”
+0.02
3Oil2/41
59.50’
,2.71
2.03”
kO.08
0.38d
+o.oo
424
M. D. Suarez
Table 3. Influence
ef al.
of diet composition
on body composition
Diets*
Group 1
Moisture
Initial
valuest
69.10
& 1.30
66.40
(%)
Protein
(% d.m.)
Fat
(% d.m.)
+2.70
32.80
& 3.20
30/16/23
6?.67”b
+0.43
59.84$d
+0.69
33.87”
50.66
30/12/32
68.90b
kO.87
59.90d
Group
30/8/41
68.50ab
+0.40
61.23’
+ 1.96
kO.03
33.93”
k 1.76
33.03”
kO.09
25/20/23
67.83”b
kO.29
59.17td
25/16/32
67.35”
+0.26
55.30pb
kO.66
37.20b
+ 0.69
Table 4. Influence
of diet composition
Group
25112141
68.47”b
kO.15
56.93$”
+ 1.15
40.4or
kO.87
*r~ = 10 animals/diet.
tn = 40 animals:
$Final values are significantly
different from initial ones ($P < 0.05).
aMceValues within each row with different superscripts
are significantly
obtained with the experimental diets. Group 1
of the eels fed on diets with slightly lower energy
contents showed the highest intake, although lot
1 (fed on diet 25/20/23)
from dietary group 2
showed the highest values. Furthermore, within
each group of diets, a tendency of decreasing
food intake was observed when dietary carbohydrates content increased, associated with
lower dietary lipid levels.
The best indices of growth were obtained for
diets of a higher energy density, and a positive
correlation between body weight increase and
total dietary energy was found 0, = 7.44 x
-89.37, r = 0.62,
P < 0.05).
Within each group
of diets, high carbohydrate levels associated
with low lipids concentrations yielded the best
growth results. Similary, higher feed-efficiency
indices were reached for diets with higher levels
of carbohydrates and total energy.
Results for body composition are shown in
Table 3. Eels from group 3, with the best growth
and feed-efficiency indices (except for diet
2
kO.15
37.13b
kO.38
different
30/20/23
67.40”
+0.92
53.87$”
* 0.49
41.20$’
kO.55
3
30/16/32
70.43’
f0.03
59.40fd
kO.23
34.53”
kO.38
30/12/41
67.20”
kO.06
54.40yb
kO.69
39.33fk
kO.15
(P < 0.05).
30/16/32), significantly (P < 0.05)increased
their body fat stores when compared with initial
values. Eels from group 1, fed on diets with the
lowest energy content, showed a significantly
lower body-fat content. Body-nitrogen content
was inversely related with body-lipid levels.
Dietary influences on the liver composition
and hepatosomatic ratio are shown in Table 4.
For any given group, fish fed on diets of a lower
dietary carbohydrate content had livers with a
significantly lower glycogen and higher protein
content. The results for enzyme activities, expressed as mUnits/mg liver protein, are presented in Table 5. Glutamate dehydrogenase
(GDH) activities showed significantly higher
values for diets with a higher protein:energy
ratio (group 1, except lots fed on lowest fat and
highest carbohydrate content; diet 30/g/41), regardless of whether the dietary-protein content
from the other two groups was lower (25%
protein, group 2) or the same (30% protein,
group 3). Within each group, the GDH activity
on hepatic
somatic
index (HSI) and liver composition
Diets*
._____-.
Group
Hepatosomatic
index
Moisture (%)
Initial
values?
1.12
io.01
73.80
* 0.80
14.00
kO.20
11.20
kO.40
17.41
k1.90
Protein
(% d.m.)
Fat
(% d m.)
Glycogen
(% d.m.)
*n = 10 animals/diet.
tn = 40 animals.
SFinal values are significantly
“kd’Values
within
30116123
1.12”
10.01
65.681”
4 1.99
21.31Sd
kO.39
3.821”
kO.63
21.96”
+2.62
different
30/12/32
1.14”
+0.01
77.80’
kO.83
14.67”
kO.45
13.16Cd
* 1.17
40.96$c
f 1.36
Group
1
30/8/41
l.28*b
F 0.05
76.09cd
+2.09
16.48&
&0.50
5.58jab
* 0.05
32.03$&
+2.71
25120123
1.14”
io.01
73.25k
+ 0.44
17.41’
* 0.07
1 I .27Cd
f 1.07
30.77Sbc
+ 1.78
2
25116132
I .26fb
kO.01
75.69cd
+0.44
16.45bc
k 0.23
13.68d
+0.98
35.55$
& 1.70
25/12/41
1.22$b
kO.03
76.99cd
+0.34
15.71”b
kO.28
10.35’
+ 1.16
38.73fde
k 1.29
from initial ones ($P < 0.05).
each row with different
superscripts
are significantly
different
_.Group
(P < 0.05).
30/20/23
30/16/32
I.384
I .37:
50.01
kO.01
68.54tab
76.89cd
+0.91
& 2.22
20.751d
15.23”
L-o.01
+0.48
4.79:“s
8.04k
+ 1.35
+ 1.16
27.83”b
34.671
* 1.94
k2.78
3
.._~.
30/12/41
1.12”
i-o.01
73.94”
+0.53
17.12’
+0.39
7.98k
+ 1.01
34.741
to.93
Diet composition
Table
5. Influence
of diet composition
in relation
on hepatic
to eel growth
enzymatic
425
and metabolism
activity
(mUnits/mg
soluble
protein)
Diets*
Group
GDH
FBPase
G6PDH
PK
30116123
28.99’
k2.43
3.71’
kO.19
2.41db
+0.31
I .75”
kO.16
1
30/12/32
28.44’
+I.06
2.23”b
kO.33
3.59”s
+0.67
I .93dh
kO.31
Group
30/8/41
2O.W
k2.25
2.33”b
kO.22
6.49*
+ 6.49
1.63”
+0.12
*n = 10 animals/diet.
“kd’d’Values within each row with different
25120123
22.60”’
+2.50
2.69b
+0.38
1.16”
+0.17
1.8Sab
kO.09
superscripts
25/16/32
12.67”
k2.08
2.60b
+0.20
4.36k
+ 1.10
1.99&b
kO.18
are significantly
decreased (group 1) or did not change (groups
2 and 3) when increasing dietary carbohydrates
or decreasing the total lipid content in the diet.
The GDH activity showed a positive correlation
with protein intake, expressed either as mU/mg
protein ( y = 0.37 x - 90.08, r = 0.7 1, P < 0.05)
or as Units/g liver (y = 0.06 x - 15.84, r = 0.85,
P < 0.005).
As an index of gluconeogenesis,
the enzyme
fructose
1,6_bisphosphatase (FBPase) showed
an increased activity in the livers of fish fed on
diets with the lowest (23%) carbohydrate
content and highest total lipid concentrations.
A
lower gluconeogenic
activity was also observed
in fish fed on diets with a lower protein content
(group 2) although these differences were not
significant. Furthermore,
an inverse relationship
appeared
between enzyme activity and total
carbohydrate
intake
(y = -0.35
x +67.65,
r = -0.60, P < 0.05 when expressed as mUnits/
mg protein and _t’= -0.04 .\: + 5.99, r = -0.75,
P < 0.05 when expressed as Units/g liver), but
not between enzyme activity and protein intake.
Glucose&phosphate-dehydrogenase
(G6PDH)
showed a trend to increase when dietary carbohydrate
concentration
rose or when dietary
lipids concentrations
fell. Those differences
were statistically significant for diets included in
groups 1 and 3, but not when group 2 was
considered. Nevertheless, a negative correlation
was observed between total lipid intake and
enzyme activity, expressed either as mUnits/mg
protein
(y = -0.17
I + 111.73, r = -0.80,
P < 0.01) or as mUnits/g
liver (y = -0.009 x
+6.63,
r = -0.81,
P <O.OOl). Similarly,
a
positive correlation
was demonstrated
between
dietary carbohydrate
levels and G6PDH activity. either expressed
as mUnits/mg
protein
(y = 2.94 .Y -38.88,
r =0.79,
P <O.Ol) or
as mUnits/g
liver (_r = 0.15 .Y- 1.48, r = 0.76,
P < 0.01).
Pyruvate
kinase activity,
as an index of
glucose
utilization
by the eel, showed
its
highest values for intermediate
or high carbo-
Group
2
2511214 1
16.3Vb
+2.01
I .65”
kO.13
2.79”b
+0.52
7 04”b
A.
kO.27
different
30/20/23
17.60ab
f 1.23
4.17d
kO.27
2.38”b
kO.29
2.10”b
+0.21
3
30/16/32
13.26”
+I.89
2.21dh
+0.20
2.81dh
+0.46
3.50’
+0.24
30/12/41
13.81”
k 1.26
3 [3”h
A.
kO.17
5.24
i_ 1.54
2.40h
kO.18
(P < 0.05).
hydrate contents within each dietary group. The
overall highest values were obtained
in many
eels fed on the highest energy diets (dietary
group 3).
Discussion
An evaluation of diet acceptance, on the basis
of intake, indicated that the diets with the lowest
energy level were, in general terms, the best
accepted. Within each dietary group, a trend of
increasing food intake appeared, corresponding
to an increase in lipid levels and a decrease in
carbohydrate
levels. It is well known that one of
the main factors involved in the amount of food
intake is the energy content of the diet (Ringler,
1979; Weiswerg and Lotrich, 1982). Other possibilities that cannot be ruled out to explain the
results obtained in food intake, include a positive effect of lipid addition on diet palatability
and/or some type of imbalace in macronutrients
which causes an increase in the intake of the
diets with a lower carbohydrate
content.
The higher feed efficiency obtained for diets
of a higher carbohydrate
content, irrespective of
the diet groups assayed, appears to indicate a
great capacity of carbohydrate
to replace protein with success, especially
for high-energy
diets (group 3). In fact, previous work on nutritive utilization of diets of the same composition
as those assayed in this work has demonstrated
that protein replacement by carbohydrates
produced higher indices of dietary protein efficiency
and retention. In addition, for a given group of
diets, with the same total energy content and
P: E ratio, the efficiency of carbohydrates
in
replacing
protein
appears to exceed that of
lipids in promoting
growth and higher conversion rates, as observed in previous studies (Garcia Gallego et al., 1993; Hidalgo et al., 1993:
Sanz et al., 1993). As will be discussed later, a
reduced
gluconeogenesis,
together
with an
increased
NADPH
production
for lipid synthesis would indicate a metabolic adaptation
of
426
M. D. Suirez
the European
eel to utilize dietary
carbohydrates.
Eels fed on diets of a higher energy content
(group 3) had the highest growth, food efficiency
and body-fat depots. The capacity of eel to store
lipids has been shown repeatedly (Love, 1980;
Gallagher
et al., 1984; Degani et al., 1986).
Body-fat depots constitute the principal way of
storing energy in fish (Shul’man,
1974), especially in migrating species, in which the fat is
utilized during migration
or during starvation.
In the present study, no differences
were
found in the capacity of carbohydrates
and fat
to induce body storage of lipids, the total dietary energy content of which is probably responsible mainly for the observed differences.
In
other studies, it has been shown that the increase in the levels of dietary fat (Dosoretz and
Degani,
1987) and carbohydrates
(Nose and
Arai, 1973; Degani and Viola, 1987; Hidalgo
et al., 1993) stimulates the body fat store. Similarly, liver glycogen content was directly related
to dietary carbohydrate
concentrations
(Degani.
1987).
Glutamate
dehydrogenase,
a representative
enzyme of amino acid catabolism,
is the main
agent for ammonia
production
in fish (Van
Waarde,
1983). Glutamate
dehydrogenase
activity correlates with ammonia excretion, and its
K, values, although
with species dependent
differences (Walton and Cowey, 1989) are generally similar or above the mean tissue amino
acid concentrations
which would determine
a
first order kinetics, increasing catabolism
with
increasing
substrate
concentrations
(Beamish
and Thomas, 1984; Degani et al., 1985) following the intake of diets with a high dietary
protein content (Ogata et al., 1985). Eels fed on
diets with the highest
protein:energy
ratio
(17.5 g/MJ, group
1) showed a significantly
higher liver GDH activity with respect to diets
of both group 2 and group 3 in which the lower
protein:energy
ratio (14 and 16, respectively)
was obtained by increasing fat or carbohydrate
levels in the diet. Consequently,
protein-energy
substitution
by non-protein
energy nutrients
results in a lower catabolism of the amino acids,
as shown by lower GDH activity values. Furthermore,
the positive
correlation
between
protein intake (in g/lOOg fish/day) and GDH
activity
showed
the influence
of substrate
availability
on enzyme adaptation,
as has also
been demonstrated
in the rainbow
trout by
different authors (Walton, 1986; Lupiaiiez et al.,
1989).
In relation to the capacity of trout liver to
adapt the rate of gluconeogenesis
to variations
in diet composition,
a decreased FBPase activity
was observed when decreasing dietary protein
or increasing the dietary carbohydrate
content.
et al.
A decrease of in totum gluconeogenesis
from
14C-alanine or 14C-glutamate has been observed
in the trout (Cowey et al., 1977a; de la Higuera
and Cardenas,
1985) when decreasing dietary
protein
associated
with increasing
dietary
carbohydrate
concentrations.
A high-protein
diet has also been shown to produce an increase
of liver FBPase activity in the trout (Cowey et
al., 1977b. 1981). In dietary carbohydrate
levels,
a negative correlation
was observed between
FBPase activity and carbohydrate
intake. In
fact, for any group of diets, increasing dietary
carbohydrate
levels (diets containing
32 or 41%
digestible carbohydrates)
decreased the FBPase
activity, suggesting
that dietary carbohydrate
influence
was at least as important
as that
observed for low protein concentrations.
Carbohydrate substitution
by fat in diets from any
dietary group also significantly
decreased this
enzyme activity.
The relative importance
of
lipids regulating
the gluconeogenesis
rate remains to be demonstrated.
Nevertheless,
when
eels were fed on diets with increasing fat levels,
at the same concentrations
of protein
and
cooked starch, FBPase activity did not vary
(Suarez et al., 1993) showing the most important
role of carbohydrate
and protein regulating the
activity of this enzyme.
An alternative pathway for glucose utilization
is the pentose-phosphate
cycle, which is associated with NADPH production
to be used for
different biosynthetic
purposes, i.e. lipogenesis.
Eels have a great capacity to increase their body
fat stores (in this study reaching concentrations
of about 40% in dry matter), with glucose being
an alternative
precursor.
Hence, G6PDH
activity could reflect metabolic
adaptations
to
different
nutritional
and physiological
situations. The results obtained
support the idea
that nutritional
lipogenic circumstances,
such as
a high carbohydrate
intake, can stimulate the
activity of liver G6PDH and other NADPHproducing
systems in flsh (Hilton and Atkinson, 1982; Fynn-Aikins
et al., 1992; Barroso,
1993). Conversely,
the substitution
of dietary
carbohydrates
by fat leads to enzyme repression. Furthermore,
when dietary carbohydrate
and protein are maintained
constant, increasing
dietary lipids levels induced a progressive decrease of G6PDH activity (Suarez et al., 1993).
Similar results were obtained in the trout (Jiirss
et al., 1985). Adaptive changes of G6PDH from
a lipogenic to a lipolytic situation, or vice versa,
are presumably
similar to those observed in
the rat where the activity of this enzyme is
related to induction
or repression phenomena.
through
the corresponding
changes in intracellular-messenger-RNA
concentrations
proportional
to G6PDH activity (Prostko et al..
1989).
Diet composition
in relation
As shown in two experimental
groups, higher
levels of carbohydrates
increased PK activity
coinciding
with a decrease of FBPase activity.
Similar
results were obtained
in the trout
(Cowey et al., 1977b; Walton,
1986) where an
opposite response of both routes (glycolysis and
gluconeogenesis,
respectively)
was observed
when comparing the influence of diets only with
extreme opposite levels of protein and carbohydrates. On the contrary, Hilton and Atkinson
(1982) did not find changes in trout liver PK
activity after feeding the fish on isocaloric diets
of different fat and carbohydrate
content. From
the results obtained,
a certain activation
of
glucose
utilization
when increasing
dietary
carbohydrates
can be deduced. Nevertheless, an
inhibition
of PK in eels fed on diets with
increasing
dietary fat content
and the same
levels of carbohydrate
and protein has been
observed (Suarez et al.,1993). In fact, an inhibition of non-reversible
glycolytic enzymes by
lipid catabolism
products such as acetyl-CoA,
ketone-bodies
and ATP, supposed
to be enhanced under high dietary fat conditions,
has
been demonstrated
(Guderley
and Cardenas,
1980; Mommsen
and Suarez, 1984). The increase in PK activity as dietary energy increased
could be explained on the basis of its role in the
production
of pyruvate, a precursor in the synthesis of fatty acids. This fact is in good agreement with the higher body-lipid stores of these
animals.
The relative
influence
of dietary
macronutrient
proportions
on PK and other
glycolytic
enzymes in fish needs further research.
Acknowledgements-This
study was supported
by a grant
from the Comision
Asesora de Investigaci&t
Cientifica y
Tecnica (CAYCIT, no. 012-84) of the Spanish Government.
References
Anderson J., Jackson J. A., Matty A. J. and Capper B. S.
(1984) Effect of dietary carbohydrate
and tibre on the
tilapia
(Oreochromis
niloticus L). Aquaculfure 37,
3033314.
AOAC (1980). Oficial Methods of Analvsis. Association
of
Official Analytical Chemists, Washington,
DC, 1018 pp.
Barroso J. B. (1993) Influencias nutricionales
v de la edad
sobre el comportamiento
cinetico de 10s sistemas productores de NADPH
en diferentes
tejidos de la trucha
arco-iris (Oncorhvnchus mvkiss). Doctoral Thesis. Univ.
of Granada,
262 pp.
Beamish F. W. H. and Thomas E. (1984) Effect of dietary
protein and lipid on nitrogen
loss in rainbow
trout,
Aquaculrure 41, 359-371.
Bergot F. and Brecque J. (1983) Digestibility
of starch by
rainbow trout: effects of the physical state of starch and
of the intake level. Aquaculture 34, 203-212.
Bradford
M. (1976) A rapid sensitive method
for the
quantitation
of microgram
quantities of protein utilizing
the pinciple of protein-dye
binding. Analyr. Biochem. 72,
2488254.
to eel growth
and metabolism
421
Bulnheim
U. (1974) Untersuchungen
zur amylase
und
proteaseaktivitat
im darminhalt
sowie des blutzukker und
leberglykogengehaltes
geim aal (Anguilla anguilla L.) in
abhangigkeit
von der futterqualitlt.
Diss. Univ. Restock
l-97.
Carbonell
J., Feliu J. E., Marco R. and Sols A. (1973)
Pyruvate
kinase. Classes of regulatory
isoenzymes
in
mammalian
tissues. Eur. J. Biochem. 37. 148-156.
Cowey C. B., Cooke J. D., Matty A. J. and Adron J. W.
(1981) Effects of quantity and quality of dietary protein
on certain enzymes activities in rainbow trout. J. Nutr
111, 336-345.
Cowey C. B.. De la Higuera M. and Adron J. W. (1977a)
The effect of dietary composition
and of insulin on
gluconeogenesis
in rainbow trout (Salvo gairdneri). Br. J.
Nun. 38, 3855395.
Cowey C. B., Knox D., Walton
M. J. and Adron
J. W. (1977b) The regulation
of gluconeogenesis
by
diet and insulin in rainbow
trout. Br. J. Nutr. 38,
463-470.
Degani G. (1987) The influence of the relative proportions
of dietary protein and carbohydrate
on body weight
gain, nitrogen retention and feed conversion of european
eels, Anguilla anguilla L. Aquacult. Fish. Man. 18,
151-158.
Degani G.. Horowitz A. and Levanon D. (1985) Effect of
protein level in purified diet and density, ammonia and O?
level on growth of juvenile European eels. Aquaculture 46,
193-200.
Degani G. and Viola S. (1987) The protein sparing effect of
carbohydrates
in the diet of eels (Anguilla anguilla).
Aquaculture 64, 283-291.
Degani G., Viola S. and Levanon
D. (1986) Effects of
dietary carbohydrate
source on growth and body composition of the European eel (Anguilfa anguifla). .4quarulture
52, 97-104.
De la Higuera M. and Cardenas
P. (1985) Influence of
dietary composition
of gluconeogenesis
from L-(U-“‘C)glutamate
in rainbow
trout (Salmo gairdneri). Camp.
Biochem. Physiol. 81, 391-395.
De la Higuera M., Garcia Gallego M., Sanz A., Hidalgo
M. C. and Suarez M. C. (1989) Utilization
of dietary
protein by the eel (Anguilla anguilla): Optimum dietary
protein level. Aquacullure 79, 53-61.
De la Higuera M.. Murillo A., Varela G. and Zamora S.
(1977) The influence of high dietarv fat levels on orotein
utilization by the trout (Sasmo gairdneri). Comp. Br’achem.
Physiol. 56, 37 -4 I
Dosoretz C. and Degani G. (1987) Effect of fat rich diet and
temperature
on growth and body composition
of European eels (Anguilla anguilia). Comp. Biochem. Phy,siol. 87,
733-736.
Fritz R. S. and Kletzien R. F. (1987) Regulation of glucose6 phosphate dehydrogenase
by diet and thyroid hormone.
Molec. Cell. Endocr. 51, 13-17.
Fynn-Aikins
K.. Silas S. O., Hung W. L. and Hongbin L.
(1992) Growth,
lipogenesis
and liver composition
of
juvenile white sturgeon fed different levels of o-glucose.
Aquaculrure 105, 61-72.
Gallagher
M. L., Kane E. and Beringer
R. (1984)
Effect of size on composition
of the American
eel
(Anguilla roslrara).
Comp.
Biochem.
Physiol. 78,
533-536.
Garcia Gallego M., Hidalgo M. C., Suirez M. D., Sanz A.
and De la Higuera M. (1993) Feeding of the European eel
Anguilla anguilla. II. Influence of dietary lipid level.
Comp. Biochem. Physiol. 105, 171-175.
Guderley
H. and Cardenas
J. M. (1980) A study of the
catalitic
properties
of piruvate
kinase isozymes from
salmon and a examination
of their functional
relationships. J. exp. Zool. 212, 269-277.
Hidalgo M. C., Sanz A., Gacia Gallego M., Suarez M. D.
and De la Higuera M. (1993) Feeding of the European eel
428
M. D. SuPrez et al
Anguilla anguilla. I. Influence of dietary carbohydrate
level. Comp. Biochem. Physiol. 105, 165-169.
Hilton J. W. and Atkinson
J. L. (1982) Responses
of
rainbow
trout to increased
levels of carbohydrates
in
practical trout diet. Br. J. Nun. 47, 5977607. _
Jiirss K.. Bittorf T. H. and Viikler T. H. (1985)
Influence of
\
salinity and ratio of lipid to protein in diets on certain
enzyme activities
in rainbow
trout (Salmo gairdneri
Rich.). Comp. Biochem. Physiol. 81, 73-79.
Kaushik S. J., Medale F., Fauconneau
B. and Blank, D.
(1989)
Effect of digestible
carbohydrates
on protein/energy
utilization
and on glucose metabolism
in
rainbow
trout (Salmo gairdneri R.). Aquaculture 79,
63-74.
Kaushik S. J. and Oliva-Teles A. (1985) Effect of digestible
energy on nitrogen and energy balance in rainbow trout.
Aquaculture SO, 899101.
Keppler D. and Decker K. (1974) Glycogen: Determination
with amyloglucosidase.
In Methods of Enzymatic Analysis
(Edited by Bergmeyer
H. U.), Vol. 3, pp. 1127-1131.
Academic Press, London.
Larsson P., Hahrim S. and Okla L. (1990) Fat content as a
factor inducing migratory
behavior in the eel (Anguilla
anguilla L.) to the Sargasso Sea. Naturwissenchaften 71,
488-490.
Latzko E. and Gibbs M. (1974) Alkaline C-fructose-l,6
diphosphatase.
In Methods of Enzymaiic Analysis (Edited
by Bergmeyer H. U.), Vol. 2, pp. 181-184. Academic
Press, London.
Lohr G. W. and Wailer H. D. (1960) Glucose 6-phosphate
dehydrogenase.
In Methods of Enzymatic Analysis. (Edited by Bergmeyer H. U.), Vol 2, pp. 6366641. Academic
Press, London.
Love R. M. (1980) The Chemical Biology of Fishes. Academic Press, London.
Lupiliiez J. A.. Sanchez Lozano M. J., Garcia Rejon L.
and De la Higuera
M. (1989) Long-term
effect of
a high-protein/non-carbohydrate
diet on the primary
liver and kidney metabolism
in rainbow trout (Salmo
gairdneri) Aquacuhure 79, 9 I-10 1.
Mommsem
T. P. and Suarez R. K. (1984) Control
of
gluconeogenesis
in rainbow
trout hepatocytes:
role of
pyruvate
branchpoint
and phosphoenolpyruvate-pyruvate cycle. Molec. Physiol. 6, 9918.
Nose T. and Arai S. (1973) Optimum
level of protein in
purified diet of eel (Anguilla japonica). Bull. Fresh. Fish.
Res. Lab. 22, 145-155.
Ogata H., Arai S. and Mas Alvarez B. (1985) Effect of
dietary protein levels on free aminoacid content of juvenile European eel. Bull. Japan. Sot. Sci. Fish. 51, 537-578.
Prostko C. R., Fritz R. S. and Kletzien R. (1989) Nutritional regulation of hepatic glucose-6-phosphate
dehydrogenase. Bioehem. J. 258, 295-299.
I
Ringler N. H. (1979) Selective predation
by drift feeding
brown trout, Salmo trutta. J. Fish. Res. Bd Can. 26,
392-403.
Sanz A., Suarez M. D., Hidalgo M. C., Garcia Gallego M.
and De la Higuera M. (1993) Feeding of the European eel
Anguilla anguilla. III. Influence of the relative proportions
of the energy yielding nutrients. Comp. Biochem. Physiol.
105, 1777182.
Schmidt E. (1974) Glutamate
dehydrogenase
UV assay.
In Mefhods of Enzymatic Analysis (Edited by Bergmeyer
H. U.), Vol. 2. pp. 650-656.
Academic
Press, New
York.
Shimeno S., Hosokawa H., Takayama
S., Fukui A., Sasaki
H. and Kajiyama H. (1981) Adaptation
of hepatopancreatic enzymes to dietary carbohydrate
in carp. Adaptation
of hepatopancreatic
enzymes to dietary carbohydrate
in
carp. Bull. Japan. Sot. Sri. Fish. 47, 71-77.
Shul’man G. E. (1974) Life Cycles of Fish. Wiley, New
York. 327 pp.
Suarez M. D., Hidalgo M. C., Sanz A., Garcia Gallego M.
and De la Higuera M. (1993) Intermediary
metabolism
response of the European eel to dietary protein to lipids
ratio. In Fish Nutrition in Practice (Edited by Kaushik S.
J. and Luquet P.), pp. 327-33 1. Les Colloques no 6 I. Ed.
INRA, Paris.
Takeuchi T., Watanabe
T. and Ogino C. (1979) Optimum
ratio of dietary energy to protein for carp. Buh. Japan.
Sot. Sri. Fish. 45, 983-987.
Van Waarde A, (1983) Aerobic and anaerobic
ammonia
production
by fish. Comp. Biochem. Physiol. 74,
675-684.
Viola S. and Rappaport
U. (1979) The “extra caloric
effect” of oil in the nutrition
of carp. Bamidgeh 31,
51-68.
Walton M. J. (1986) Metabolic effects of feeding a high
protein/low
carbohydrate
diet as compared
to a low
protein/high
carbohydrate
diet to rainbow trout Salmo
gairdneri. Fish. Physiol. Biochem. 1, 7-15.
Walton
M. J. and- Cowey C. B. (1989) Intermediary
metabolism.
In Fish Nutrition (Edited bv Halver J. E.).
pp. 2599329. Academic Press, San Diego.
Watanabe
T. (1980) Lipids. In Nutrition in Fish and Die!
(Edited by Ogino C.), pp. 1499186. Koseisha-Koseikaku.
Tokyo.
Watanabe T., Takeuchi T., Satoh S., Ida T. and Yaguchi M.
(1987) Development
of low protein-high
energy diets for
practical carp culture with special reference to reduction
of total nitrogen excretion. Nippon Suisan Gakkaishi 53,
1413-1423.
Weisberg S. B. and Lotrich V. A. (1982) Ingestion, egestion.
excretion, growth and conversion efficiency for the mummichog, Fundulus heteroclirus. J. exp. Mar. biol. Ecol. 62,
2377250.