Vol. 274, Issue 6, R1578-R1587, June 1998
Impact of starvation-refeeding on kinetics and protein
expression of trout liver NADPH-production systems
Juan B.
Barroso1,
Juan
Peragón1,
Constanza
Contreras-Jurado2,
Leticia
García-Salguero2,
Francisco J.
Corpas3,
Francisco J.
Esteban1,
María A.
Peinado1,
Manuel
De La
Higuera4, and
José A.
Lupiáñez2
1 Department of Biochemistry
and Molecular Biology, Faculty of Experimental Sciences, University
of Jaén, E23071 Jaén;
2 Department of Biochemistry and
Molecular Biology and
4 Department of Animal Biology and
Ecology, Centre of Biological Sciences, University of Granada,
E-18001 Granada; and 3 Department
of Biochemistry, Cellular and Molecular Biology of Plants,
Estación Experimental del Zaidín Consejo Superior de
Investigaciones Científicas, E18008 Granada, Spain
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ABSTRACT |
Herein we
report on the kinetic and protein expression of glucose-6-phosphate
dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase, and malic
enzyme (ME) in the liver of the trout (Oncorhynchus mykiss) during a long-term starvation-refeeding
cycle. Starvation significantly depressed the activity of these enzymes
by almost 60%, without changing the Michaelis constant. The time
response to this nutritional stimulus increased with fish weight. The
sharp decline in G6PDH and ME activities was due to a specific
protein-repression phenomenon, as demonstrated by molecular and
immunohistochemical analyses. Also, the dimeric banding pattern of
liver G6PDH shifted from the fully reduced and partially oxidized
forms, predominant in control, to a fully oxidized form, more sensitive
to proteolytic inactivation. Refeeding caused opposite effects in both
protein concentration and enzyme activities of about twice the control values in the first stages, later reaching the normal enzyme activity levels. Additionally, the partially oxidized form of G6PDH increased. The kinetics of these enzymes were examined in relation to the various
metabolic roles of NADPH. These results clearly indicate that trout
liver undergoes protein repression-induction processes under these two
contrasting nutritional conditions.
rainbow trout; low-fat and high-carbohydrate diet; glucose-6-phosphate dehydrogenase; 6-phosphogluconate dehydrogenase; malic enzyme; dimeric banding pattern; immunohistochemistry
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INTRODUCTION |
THE SUPPLY OF REDUCING equivalents in the form of NADPH
is one of the most important factors related to cell growth,
proliferation, and detoxification (3, 23, 24). NADPH, one
of the principal end products of several metabolic pathways, is also an
indispensable substrate of reductive biosynthetic reactions (34).
Hexose monophosphate shunt dehydrogenases, both glucose-6-phosphate
dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH),
together with malic enzyme (ME), are the key cytoplasmic dehydrogenases
generating reducing power in the form of NADPH.
It is also well established that in higher vertebrates the activity of
hexose monophosphate dehydrogenases in various tissues changes under
different nutritional and hormonal conditions (10, 16, 23, 24, 34).
Nevertheless, in fish, the adaptive response of these NADPH-production
enzyme systems to such conditions has not been completely characterized
(2, 4, 18, 27).
As opposed to mammals, fish can survive rather prolonged starvation
(4). This survival capacity is influenced by age, sex, temperature, and
salinity, as well as other environmental and seasonal factors. There is
evidence that this starvation tolerance is owed to processes of
metabolic adaptation, regulated by the nervous and endocrine systems,
in which different pancreatic and thyroid hormones play a major part
(21, 30).
It is clear that the liver is the principal site of lipogenesis in
teleostean fish (4, 12), whereas adipose tissue is dedicated primarily
to the incorporation and storage of fatty acids produced de novo in
hepatic tissue (33). In addition, starvation provokes a generalized
weight loss, which translates directly as reduced cell growth.
Therefore, the relationship between the production of reducing
equivalents, such as NADPH, and protein synthesis (15) implies a
decline in the activity of these enzyme systems during prolonged
starvation.
Although in rainbow trout the behavior and role of the liver and
skeletal muscle in relation to the variations in cell growth differ
according to the physiological situation (2, 3), during prolonged
starvation, both tissues lose significant weight, although each to a
different degree. In analyzing the nature of tissue growth, it is
necessary to consider two types of growth: 1) hyperplasia and
2) hypertrophy. The number of cells
or nuclei (indicator of hyperplasia) can be estimated by determining
the total DNA content, whereas the cell size or cell area controlled by
a single nucleus (indicating hypertrophy) can be estimated by the
protein-to-DNA ratio (39). Depending on total DNA content and the
protein-to-DNA ratio, during a prolonged starvation, the liver weight
loss is caused by two cumulative factors, reducing both cell number and
size. Subsequent refeeding tends to restore the original values of
these two cell indexes (5).
It is well known that the metabolic response of ectotherms depends on
temperature and adaptational periods (4, 37), although under different
conditions these animals need long periods for intracellular enzyme
activity to regain equilibrium (4). Wacke et al. (36) conclude that
teleost fish, and therefore rainbow trout, which are genetically
adapted to enduring prolonged food deficits, maintain relatively high
and stable levels of enzymatic activities. Nevertheless, controversy
surrounds the attainment and maintenance of equilibrium in these
NADPH-production enzyme systems, not only during starvation-induced
weight loss but also during compensatory cell growth stimulated by
refeeding (2, 4, 21, 40).
In general terms, the activity of an enzyme reflects the number of
enzyme molecules per cell or the regulation of the catalytic efficiency
of a constant number of enzyme molecules per cell (11). We have
assessed the effects of long-term starvation and refeeding on the
kinetic adaptive behavior and expression of the hepatic NADPH-production systems in trout of different body sizes, determining the specific protein content by Western blot, kinetic, and
immunohistochemical analysis. Our aim is to provide a detailed
understanding of the main regulatory mechanisms of these enzyme systems
and thereby clarify the metabolic roles of each.
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MATERIALS AND METHODS |
Chemicals.
All biochemicals were obtained from Sigma Chemical (St. Louis, MO) or
Boehringer (Mannheim, Germany). Other chemicals came from Merck and
were of highest purity available.
Fish and maintenance.
Juvenile rainbow trout (Oncorhynchus
mykiss) of different body weights (30, 100, and 180 g) were obtained from a local fish farm (Riofrio, Granada, Spain). Fish
were kept in 350-liter fiberglass tanks with continuous aeration,
dechlorinated water with a flow rate of 1.5 l · min
1 · kg1
of fish at 15.0 ± 0.5°C. The light-dark period was a 12:12-h cycle. During 2 wk of acclimation, fish were fed a standard diet (composition in proteins, lipids, and carbohydrates was 45, 12, and 8 g/100 g, respectively, whereas the amount of gross energy was 14.9 kJ/g
diet). For the enzyme activity experiments, the timing for starvation
differed for each fish weight group, choosing those that reached a
significant reduction in the enzyme activity levels. For this, the fish
groups of 30, 100, and 180 g were starved for 35, 77, and 133 days,
respectively. All fish were refed with a low-fat and high-carbohydrate
diet (LF-HC; composition in proteins, lipids, and carbohydrates was 40, 8, and 23 g/100 g, respectively, whereas the amount of gross energy was
15.0 kJ/g diet) for 40 days. For the SDS-PAGE, immunoblot, and
immunohistochemical analyses for G6PDH and ME, only fish of 30 g were
used, with a starvation period of 63 days and a refeeding time of 20 days with the standard diet as previously defined.
Tissue preparation for analytic procedures.
Fish were killed by a sharp blow to the head. Livers were immediately
removed and homogenized (1:10, wt/vol) in 100 mM
Tris · HCl containing (in mM) 250 sucrose, 1 EDTA,
0.1 NADP, and 0.57 phenylmethylsulfonyl fluoride, pH 7.6. All
procedures were performed at 4°C. Homogenates were centrifuged at
105,000 g for 60 min. The supernatant
fraction was used for biochemical and immunochemical assays.
Enzyme activity assays.
G6PDH
(D-glucose-6-phosphate:NADP+
1-oxido-reductase, EC 1.1.1.49) and 6PGDH
(6-phospho-D-gluconate:NADP+
2-oxidoreductase-decarboxylating, EC 1.1.1.44) were determined as
described by Barroso et al. (3), based on the reduction of
NADP+ at 340 nm in 50 mM HEPES, pH
7.6, containing 2 mM MgCl2, 0.8 mM
NADP+, and a variable
concentration of substrate. For kinetic studies, the range of substrate
concentration for both G6PDH and 6PGDH was 0.005-5 mM [13
concentrations were used: 5, 7.5, 10, 12.5, 15, 20, 50, 100, 250, 500, 1,000, 2,500, and 5,000 µM; of these, 4 concentrations were below the
Michaelis constant
(Km) value, 2 around its value, and 7 above
Km]. The
G6PDH activity was corrected for 6PGDH activity as described Corpas et
al. (8). ME
(L-malate:NADP+
oxidoreductase-oxaloacetate-decarboxylating, EC 1.1.1.40) was assayed
according to the following protocol: the reaction mixture contained, in
a total volume of 1 ml, 50 mM HEPES, pH 7.6, 2 mM MgCl2, 0.4 mM NADP, and a variable
concentration of L-malate. For
kinetic studies the range of
L-malate concentrations was
0.05-10 mM (11 malate concentrations were used: 0.050, 0.075, 0.1, 0.15, 0.175, 0.2, 0.5, 1, 2.5, 5, and 10 mM; of these, 4 concentrations were below the Km
value, 2 around its value, and 5 above
Km).
A milliunit of activity (mU) is defined as the amount of enzyme
required to reduce 1 nmol NADP/min at 25°C. Proteins were determined according to the Bradford method (6) using BSA as a
standard. Enzyme activity is expressed as specific activity in terms of
milliunit of activity per milligram of protein.
Determination of DNA concentration.
The method of Munro and Fleck (22) was used for the DNA separation,
purification, and quantification. The RNA and DNA fractions were
separated by digestion in alkali (0.3 N KOH) at 37°C for 1 h,
followed by acidification in
HClO4, 1.2 N. The DNA
concentration was estimated by the indole test (7).
Nondenaturing gel electrophoresis and detection of G6PDH activity.
Liver samples were separated by PAGE in 5% acrylamide tube gels.
Before electrophoresis, samples were prepared with 20% glycerol and 8 mM NADP+ (final concentration).
Samples were electrophoresed at a constant current of 1.5 mA/gel. The
isoforms of G6PDH were visualized by staining of the enzyme activity
after the incubation of the gel in 50 mM Tris · HCl,
pH 7.6, containing (in mM) 10 G6P, 0.8 NADP+, 5 EDTA, 2 MgCl2, 0.24 nitroblue tetrazolium,
and 65 µM phenazine methosulfate in the dark, until precipitated
formazan appeared (about 15 min). The reaction was stopped
by immersing the gel in 7% acetic acid. Gels were scanned using a gel
scanner and then photographed.
Antibodies.
Polyclonal antibodies against G6PDH and ME from rat liver were used
(35).
SDS-PAGE and immunoblot analyses.
Samples from high-speed liver supernatant fractions were heated to
95°C for 3 min in 62 mM Tris · HCl, pH 6.8 buffer, containing 2% (wt/vol) SDS, 10% (vol/vol) glycerol, and 10 mM
1,4-dithiothreitol. Polypeptides were separated by 7.5% SDS-PAGE using
a Bio-Rad Mini-Protein II apparatus and electroblotted onto 0.2 µm
polyvinylidene difluoride membrane (Immobilon-P, Millipore) using a
semi-dry transfer apparatus (Hoeffer) at 1.5 mA/cm2 membrane for 90 min in 25 mM Tris, 192 mM glycine, and 10% methanol, pH 9.4. The
membranes were blocked with 10 mM Tris · HCl, 100 mM
NaCl, pH 7.5 buffer (TBS) containing 5% nonfat dry milk and 0.05%
Tween 20. The blots were then incubated overnight at 4°C with
either rabbit anti-G6PDH or rabbit anti-ME antisera (diluted 1:1,000
and 1:1,500 in blocking solution, respectively). The blots were washed
with TBS buffer containing 0.1% Tween 20. Immunodetection was
performed using an enhanced chemiluminescence kit (Amersham). The blots
were scanned with a computer-assisted videodensitometer and
photographed.
Immunohistochemical studies.
Fish were anesthetized in water containing 0.3 ml ethylene
glycol-mono-phenylether per liter, weighed, and then heparinized through the dorsal aorta (500 IU, Rovi). For hepatic perfusion, abdominal and heart cavities were exposed, and a blunt 20-gauge cannula
(Abbott) was inserted into the major tributary to the hepatic portal
vein and tied securely in place. The liver was cleared of blood by an
in situ perfusion with 3-4 ml of carbogenated 10 mM Na-phosphate,
pH 7.4, containing 0.9% (wt/vol) NaCl (PBS), at room temperature, with
a flow rate of 5.2 ml of
PBS · min1 · kg
body wt1, using a
peristaltic pump (Gilson minipuls). After the flow was initiated, a
small cut was made in the tail kidney to allow the blood and perfusate
to escape from the portal venous system. Livers were fixed in 100 mM
Na-phosphate, pH 7.4, containing 4% paraformaldehyde, at the same flow
rate. Fixed livers were removed, cut into cubes of 8-10
mm3, and incubated for 3 h at room
temperature with the previous fixative solution. Liver blocks were kept
overnight at 4°C in 100 mM Na-phosphate, pH 7.4, containing 30%
(wt/vol) sucrose. Blocks were covered with OCT compound and then frozen
in 2-methylbutane prechilled in liquid nitrogen. Serial sections of 30 µm were prepared using a cryostat (2800 Frigocut E, Reicher-Jung).
Inhibition of endogenous peroxidase was made on free-floating sections
with 0.03%
H2O2
in PBS for 30 min. After several washes in PBS, these free-floating
sections were incubated with antibodies of either rabbit anti-rat G6PDH
or rabbit anti-rat ME, diluted 1:50 in PBS containing 0.2% Triton
X-100 overnight at 4°C, washed in PBS, and then incubated with
biotinylated goat anti-rabbit IgG (Vector Laboratories) followed by
peroxidase-linked avidin-biotin complex. Peroxidase activity was
detected by nickel-enhanced diaminobenzidine procedure (32). Sections
were then mounted on slides using DePeX. Control
procedures were carried out when the primary antibody was either
omitted or replaced with an equivalent concentration of preimmune
serum.
Kinetic parameters.
Kinetic data were analyzed using a nonlinear regression analysis
program (Enzfitter, Elsevier Biosoft) and EZ-FIT (Dupont de Nemours,
Glenolden Laboratory). The activity ratio is the relationship between
enzyme activity at subsaturating substrate concentration and maximum
rate. Catalytic efficiency, defined as the ratio between enzyme
activity and Km,
was determined at a substrate-saturating concentration. This parameter
relates total enzyme concentration to the interaction between enzyme
and the substrate.
Another way to express the different kinetic parameters used in the
present work is by their relationship to the cell unit, given that the
number of cell nuclei is represented by the total quantity of DNA (39).
The total activity corresponds to the total number of units of enzyme
present in the complete organ and is expressed in total units of
tissue. The activity by cell unit represents the enzyme activity per
cell and is expressed in units per milligram DNA. The maximum rate per
cell unit indicates the initial rate of the enzyme at
substrate-saturating concentrations per cell and is determined
consistently under the same experimental conditions, expressed in
milliunits of activity per milligram DNA. The specific activity per
cell unit reflects the specific activity of the enzyme per cell,
expressed in milliunits of activity per milligram protein per milligram
DNA. The catalytic efficiency of activity per cell unit corresponds to
the catalytic efficiency per cell and is expressed in milliunits of
activity per milligram DNA per 106
M.
Statistical analysis.
All values are reported as means ± SE. The normal distribution of
variables was analyzed using a computerized Kolmogorov-Smirnov test.
This statistical test accepted the hypothesis of a normal distribution,
and the results obtained for age groups and different nutritional
situation were compared using the one-way ANOVA followed, in the
appropriate cases, by a Duncan or Newman-Keuls multiple-range test.
Also, statistical significance between means was determined using an
unpaired two-tailed Student's t-test.
The possibility of a tank effect was tested for each parameter, also
using the unpaired Student's t-test,
with no differences being found between tanks of the same experimental
group (data not shown).The difference was considered significant at a
level of P < 0.05.
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RESULTS |
Liver growth, protein, and DNA content.
Loss of mass, a clear sign of long-term starvation, is especially
critical in the liver, skeletal muscle, adipose tissue, and intestine
(2). Our results indicate that, at the end of the starvation period
(133 days) in the trout weighing 180 g, the liver weight diminished by
80%, with a loss of 63% of the total DNA content. At 8 days of
refeeding, the liver had regained 74% of its original weight (a gain
of 259% over starvation weight) without registering significant
changes in the total DNA content. Cell size, represented by the
relationship between the cell protein and hepatic DNA concentrations,
showed opposite changes during the starvation-refeeding cycle, that is,
the protein-to-DNA ratio fell 23% during starvation and rose 76%
during refeeding (Table 1).
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Table 1.
Influence of long-term starvation-refeeding cycle on liver weight,
protein, and nucleic acid contents in rainbow trout
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Hepatic G6PDH and 6PGDH activities.
The effects of long-term starvation and refeeding on the behavior of
the hepatic pentose-phosphate cycle dehydrogenases, G6PDH and
6-phosphogluconate dehydrogenase (6PGDH) in trout of different body
weights are shown in Tables 2 and
3 and Figs. 1,
2, and 3. The results were
qualitatively similar in the three experimental groups. Starvation
significantly inhibited the rates of hepatic G6PDH and 6PGDH, although
the larger the trout, the longer the response time to the nutritional
regimen. In all cases, the enzymes followed a Michaelian curve for G6P
and 6PG, respectively (results not shown). In the trout
of 180 g, starvation did not significantly alter any of the kinetic
parameters of hepatic G6PDH or 6PGDH during the first 35 days. At 98 days, activity of both dehydrogenases was significantly inhibited,
reaching the highest reduction level (65%) at 133 days in specific
activity as well as in maximum rate and catalytic efficiency (Tables 2
and 3, and Fig. 1), without significant changes in
Km, compared with
control values. This reduction in the enzyme activities, recorded at
all substrate concentrations, was proportionally constant over the
saturating curves, indicating a reduction in enzyme concentration. This
idea is supported by the constancy of the activity ratio values (Tables 2 and 3).
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Table 2.
Kinetic behavior of hepatic glucose-6-phosphate dehydrogenase during
long-term starvation-refeeding cycle in rainbow trout of 180 g
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Table 3.
Kinetic behavior of hepatic 6-phosphogluconate dehydrogenase during
long-term starvation-refeeding cycle in rainbow trout of 180 g
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Fig. 1.
Time course of hepatic glucose-6-phosphate dehydrogenase (G6PDH,
A), 6-phosphogluconate dehydrogenase
(B), and malic enzyme (ME,
C) activities during the
starvation-refeeding cycle. Starved rainbow trout of 30 g ( ), 100 g
( ), and 180 g ( ) were refed (solid symbols) a low-fat and
high-carbohydrate diet at different times (arrows) when activity levels
were significantly decreased. Activity results are the means of 5 experimental observations and are expressed as milliunits of activity
per milligram protein. SE was always <10% and was omitted to clarify
the figure. * Degree of significance
(P < 0.05) between control and
starved and control and refed fish.
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Fig. 2.
Western blot analysis of G6PDH protein content from rainbow trout liver
during starvation-refeeding cycle. Protein from cytosolic supernatants
of trout weighing 30 g were electrophoresed and then blotted to
polyvinylidene difluoride (PVDF). The 97-kDa G6PDH protein,
corresponding to a dimeric form of the enzyme, was detected with
specific polyclonal anti-G6PDH rabbit serum.
Bottom shows immunoblot (80 µg/lane), indicating time-dependent effects of starvation-refeeding
cycle on G6PDH protein levels. This is representative of 5 separate
experiments. Top shows quantification
by scanning densitometry of G6PDH levels; results are expressed as
arbitrary densitometric units and are means of 5 observations. SE was
always <5% and was omitted to clarify the figure.
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Fig. 3.
Activity staining of rainbow trout liver G6PDH on a 5% polyacrylamide
tube gels. Lanes a, b, and
c contain cytosolic samples (80 µg
total protein/lane) from controls, starved, and refed fish,
respectively. This is representative of 5 separate experiments. A
variability <5% was found.
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On the contrary, during refeeding with the LF-HC experimental diet, the
response of both dehydrogenases was much quicker and abrupt, both the
specific activity and the catalytic efficiency rising significantly
without changes in the
Km or activity
rate. At 8 days of refeeding, the specific activity of both G6PDH and of 6PGDH increased by 75% with respect to the final starvation value;
however, after 20 days, values rose roughly 5.5-fold, and at 40 days
returned to control values (Tables 2 and 3, and Fig. 1).
In relation to the changes in tissue weight and in the concentrations
of cell protein and DNA, the total activity of both dehydrogenases
diminished 91% during starvation as a consequence of the decrease of
75% of the activity per cell unit (Tables 2 and 3), without
significant changes in the other kinetic parameters per cell unit. At 8 days of refeeding (on the LF-HC diet), the hepatic tissue weight
increased 3.6-fold with respect to starvation values and recovered 74%
with respect to the initial weight (results not shown). Therefore, the
total activity, both of G6PDH and 6PGDH, increased fivefold over
starvation values. This sharp increase reflects a rise of twofold in
the specific activity per cell unit and fourfold in the activity per
cell unit.
The kinetic parameters of the hepatic enzymes in the lower-weight trout
during the starvation-refeeding cycle followed a similar pattern as
that described for the 180-g fish but with less response time in the
former. In 100-g trout, the specific activity of both dehydrogenases
fell significantly from day 55 of
starvation and from day 35 in 30-g
individuals (Fig. 1). Refeeding the LF-HC diet raised these activities
significantly, even exceeding control values at 20 days, although to a
lesser degree than in the 180-g weight category (Fig. 1). In all cases,
at the end of refeeding (40 days), the enzyme activities values
returned to the control levels.
When 30-g trout were refed on a standard diet (45/12/8) instead of the
LF-HC (40/8/23) one, only a gradual rise to control values was
recorded, without the overcompensation responses noted previously. The
analysis of G6PDH by Western blot showed a single band with an apparent
molecular size of 97 kDa, corresponding to the dimeric form of the
protein. Figure 2 presents the evolution of the G6PDH protein content,
indicating a progressive decline in the quantity of G6PDH protein from
day 35 of starvation, being 92% at 63 days and a gradual recovery of the protein levels to control values at
20 days of refeeding.
Figure 3 shows G6PDH activity on nondenaturing gel electrophoresis of
high-speed supernatants of control (lane
a), long-term starvation (lane
b), and refeeding (lane
c) 180-g trout. This is the first time in fish that
hepatic G6PDH has been shown to exist in three dimeric forms with
different electrophoretic mobility [as occurs in mammals (13,
19)], designated from 1 to 3, depending on its distance to the
anode (lane a). Densitometric scan
analysis, as well as the relative ratios expressed as percentages of
total activity (results not shown) of the bands
1, 2, and
3, revealed a significant shift (about
45%) toward band 1 in the starved
fish (lane b) compared with control,
without any significant changes in bands
2 and 3. During
refeeding, the dimeric pattern of bands 1 and 3 shifted
selectively toward band 2 (lane c).
The results of the G6PDH immunohistochemical analysis on trout
hepatocytes are presented in Fig. 4,
A and
B. Figure
4A illustrates a strong cytoplasmic
immunoreaction (p-diaminobenzidine-nickel deposits) in
control samples, although the reaction intensity showed pronounced
individual cell variations, yielding a mottled appearance. The immunolabeling of the hepatocytes appears
to be slightly related to venous patterns, with an increase in the
perivenous areas; the bile duct epithelia are also stained. Figure
4B indicates a significant
immunolabeling decrease in the starved fish, although some hepatocytes
located in perivenous areas are still marked. The bile duct epithelia
remain immunostained.
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Fig. 4.
G6PDH immunohistochemistry. Light micrographs of G6PDH immunoreactivity
in sections 30 µm thick from control
(A) or starved
(B) trout liver. bd, bile duct; pv,
portal vein; cv, central vein; note the immunoreaction product inside
hepatocytes (arrowheads).
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Hepatic ME activity.
The results obtained followed a pattern similar to that described for
the dehydrogenases of the pentose-phosphate cycle. The specific
activity, maximal velocity
(Vmax), and
catalytic efficiency of hepatic enzyme in the 180-g trout diminished
significantly from day 77, registering
60% after 133 days of starvation (Table 4
and Fig. 1). These values persisted until day
8 of refeeding (on the LF-HC diet), when values
practically reached control; a fivefold increase in 20 days raised
values to control levels (Table 4 and Fig. 1). Neither situation caused
significant changes in the
Km or in the
activity rate.
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Table 4.
Kinetic behavior of hepatic malic enzyme during a long-term
starvation-refeeding cycle in rainbow trout of 180 g
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According to analysis of changes per cell unit, the total activity of
ME fell 90% during starvation, due to a 74% reduction in the activity
per cell unit; no significant changes were recorded in the maximum rate
per cell unit, in the specific activity per cell unit, or in the
catalytic efficiency per cell unit. After 8 days of refeeding (on the
LF-HC diet), the total enzyme activity in the liver rose 434% over
starvation values. In this sense, the catalytic activity, specific
activity, and efficiency per cell unit, registered significant
increases, although of less magnitude (Table 4).
In the 100-g trout, the kinetic parameters followed the same pattern
described for heavier fish, although the reduction was significant from
day 70 of starvation. This period was
reduced for 30-g trout, which showed significant changes in specific
activity at 35 days of starvation. During LF-HC refeeding of both sizes of trout, the ME activity followed a similar pattern to that of 180-g
trout (Fig. 1).
As with G6PDH and 6PGDH, refeeding 30-g trout on the standard diet
instead of the LF-HC one gradually raised activity values to control,
without registering the overcompensation noted previously. Western blot
analysis of ME showed a single band with an apparent molecular size of
62 kDa, corresponding to the monomeric form of the protein. Figure
5 shows the evolution of the ME protein content, reflecting a progressive decline from day
35 of starvation, reaching 77% at 63 days, as well as
a gradual recovery of protein levels to control values at 20 days of
refeeding.
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Fig. 5.
Western blot analysis of ME protein content from rainbow trout liver
during starvation-refeeding cycle. Protein from cytosolic supernatants
of trout weighing 30 g were electrophoresed and then blotted to PVDF.
The 62-kDa ME protein was detected with specific polyclonal anti-ME
rabbit serum. Bottom shows immunoblot
(40 µg/lane), indicating time-dependent effects of
starvation-refeeding cycle on ME protein levels. This is representative
of 5 separate experiments. Top shows
quantification by scanning densitometry of ME levels; results are
expressed as arbitrary densitometric units and are means of 5 observations. SE was always <5% and was omitted to clarify the
figure.
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Finally, Fig. 6 reflects ME
immunohistochemical analysis of trout hepatocytes. Figure
6A illustrates, in the control trout, an immunoreactive distribution similar to that of G6PDH, although with
a less pronounced differential venous profile and weakly reacting bile
ducts. In starved fish, the cell immunoreaction significantly declined
but without disappearing completely and remaining in perivenous areas
and bile ducts (Fig. 6B).
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Fig. 6.
ME immunohistochemistry. Light micrographs of ME immunoreactivity in
sections 30 µm thick from control
(A) or starved
(B) trout liver. bd, bile duct; pv,
portal vein; cv, central vein; arrowheads, note immunoreaction product
inside hepatocytes.
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DISCUSSION |
One of the primary aspects of relationships between major metabolic
pathways is metabolic change during the starvation-refeeding cycle,
enabling variable fuel consumption to meet fluctuating metabolic
demand. A clearly lipolytic situation such as prolonged starvation
implies the mobilization of fat deposits, thereby depressing lipid
biosynthesis. The supply of reducing equivalents, necessary for the
synthesis of fatty acids and such processes as detoxification systems,
as well as the processes of cell growth and proliferation, must also be
inhibited. Higher vertebrates reportedly have an enormous adaptive
capacity for dehydrogenases of the phosphogluconate cycle, altering
their activity in response to different nutritional situations, for
example, decreasing their activity during starvation (11, 25).
In addition, given that rainbow trout liver is the principal organ of
de novo fatty-acid synthesis, a swift and abrupt reduction might be
expected in the activity of the enzymes involved in lipogenesis after
the beginning of starvation, for example, in the eel the levels of
lipid synthesis fall some sixfold during the first week of starvation
(1). Nevertheless, to clarify some of the differences found in the
literature in relation to the adaptive capacity of the hepatic G6PDH,
6PGDH, and ME systems of fish during starvation, we have studied the
effects on the kinetic behavior and protein concentration of these
systems in trout of different sizes, noting the significant variations
in the adaptive capacity of these systems in relation to the size of
the fish.
Under our experimental conditions, prolonged starvation significantly
diminished, at all body weights studied, the maximum rate and specific
activity both of ME and of the dehydrogenases of the pentose-phosphate
cycle in hepatic tissue, without changing the
Km values and
activity rate. This kinetic behavior corresponded to a clear enzyme
repression, indicated by a strong decrease in both the protein G6PDH
content and ME obtained by Western-blot analysis as the
immunohistochemical marker for both hepatic enzymes.
In this sense, the qualitative immunohistochemical results complement
the quantitative data, demonstrating that starvation decreases the
accumulation of both enzymes in the trout liver. As in other salmonids,
the liver of rainbow trout differs structurally from mammalian liver
(14, 26), but despite these differences, the fish liver parenchyma can
also function as a "metabolitostat" (28). Until now, the location
of the two NADPH-generating enzymes G6PDH and ME in the trout liver
has, to our knowledge, been demonstrated only by histochemical
procedures (28); hence, the present study provides the first
immunohistochemical detection, as well as their expression of both
enzymes. In the control trout livers, the distribution patterns of
G6PDH and ME reflected a slight increase in the liver parenchymal cells
located in the perivenous area. Starved trout revealed
that the immunohistochemical reaction decreased for both enzymes and
that the remaining proteins were located mainly in perivenous areas.
These results concur with the previous general metabolic zonation of
the trout liver in relation to the mammalian liver (20, 28), indicating
that lipogenesis reaches its activity maxima in perivenous areas.
Activity and Western blot analyses reflect a substantial decrease in
the activity and total amount of G6PDH and ME, respectively, in the
liver of the starved trout. The immunohistochemical results not only
support these findings but also show that the low amount of these
enzymes remaining in the liver are located in the perivenous areas,
although we should take into account that their marked decrease
corresponds to these areas also. Thus the hepatocytes located in the
liver perivenous areas are mainly responsible for lipogenic changes
during starvation.
In addition, this phenomenon of enzyme depression is reinforced when we
analyze the evolution of total enzyme activity and the kinetic
parameters as a group, expressed by cell unit. In our case both the
total activity of G6PDH, 6PGDH, and ME, as well as the activity per
cell unit, plunged drastically without significant changes in the
specific activity per cell unit and catalytic efficiency per cell unit,
reflecting a reduction both in the number and size of cells as a
consequence of generalized rather than specific repression of the
enzymatic proteins studied.
In this sense, on analyzing the G6PDH activity in gel, we observed that
the banding pattern in the livers of 180-g trout agrees with the
results of other studies concerning this enzyme (13, 19), which
reported that bands 1 and
3 represent fully oxidized and fully
reduced forms of the enzyme, respectively. We also found that
band 2 (predominant in our banding
pattern) represents a partially oxidized form (Fig. 3). During
long-term starvation the dimeric pattern of bands moved toward
band 1 (fully oxidized), given that
this dimeric form for the protein is more susceptible to proteolytic
inactivation (13, 17, 19) than are bands 2 and 3. This shift in
the dimeric banding pattern toward band 1 reflects an early event in the degradation of G6PDH
during long-term starvation, because the turnover of this enzyme
involves first oxidation followed by inactivation, possibly also by a
microsomal system (19).
Finally, the smallest trout showed comparable behavior in these
NADPH-production systems, although the time needed for a significant fall in these activities increased markedly with the size of the trout.
In addition, it is well established that in starving mammals these
NADPH-production systems decline in activity, clearly as a consequence
of enzyme depression. Thus Zelewski and Swierczynski (41) observed that
starvation significantly depresses dehydrogenase activities of the
pentose-phosphate cycle and of the ME in the liver and brown adipose
tissue in the rat, whereas refeeding with a diet rich in carbohydrates
significantly raises these activity levels to above control values.
Similar trends were reported for fish (31), although for these
NADPH-production systems the overcompensation described for mammals was
not evident (4, 40).
With regard to refeeding, our results agree basically with some
described above, in the significant increase of these enzymatic activities from day 8 onward.
Nevertheless, we have demonstrated the influence of the nutritional
variable in the modulation of these enzyme systems during refeeding,
because the low-carbohydrate diet did not lead to the overcompensation
registered for the hepatic G6PDH, 6PGDH, and ME at 20 days of refeeding
with the LF-HC diet. Finally, after 40 days of refeeding, the
parameters studied returned to control values. In addition, our
analyses of the activity values per organ and per cell of the three
hepatic enzyme systems (G6PDH, 6PGDH, and ME) indicate that refeeding
raised values significantly due to greater cell growth, characterized
both by greater cell number (total quantity of DNA) and size
(protein-to-DNA ratio). In addition, the specific activity per cell
unit increased significantly during refeeding, indicating that,
together with enzyme stimulation in general, there was a clear specific
stimulation of these enzymes. This explains the overcompensation in the
enzymatic activity found in this nutritional situation.
In this sense, when we analyzed the results of enzymatic activity in
gel during the final stage of refeeding, we found a movement in the
dimeric pattern of bands toward band 2 (intermediate oxidation stage of the protein), indicating that de novo
synthesis of the protein takes place in this form, before the
intracellular levels of enzymatic activity reach a new equilibrium in
protein turnover and consequently equilibrium between the different
dimeric forms.
Therefore, we conclude that nutritional situations such as long-term
starvation and refeeding significantly alter activities of the three
enzymatic NADPH-production systems in the liver of rainbow trout. These
kinetic alterations are intimately related to changes in protein
expression (enzyme repression or induction), the adaptive response
depending on the size of the fish.
Perspectives
Factors contributing to cell growth include the synthesis and
maintenance of the different elements making up the cell membranes, mainly structural lipids and proteins. In this sense, it is well established that NADPH plays a central role in the reductive
biosynthesis of cholesterol and fatty acids, in the elongation and
desaturation of the latter, as well as in the maintenance of cell
integrity and detoxification processes. It has also been demonstrated
that these reducing equivalents have a key part in the synthesis of protein, the other membrane element. As a result, NADPH is intricately involved in the growth process (29, 38). One of the most outstanding features of antagonistic nutritional situations, such as starvation and
refeeding, concerns sharp changes in cell growth. In this light, the
goal of our research is twofold. First, we are pursuing an in-depth
understanding of the molecular mechanism behind the kinetic changes of
the NADPH-generating systems in situations with significant variations
in cell growth. Second, we seek to link this mechanism with the
intimate relationships between the behavior of these enzyme systems and
the nature of cell growth (29, 38). Only by investigating these factors
in combination, can we achieve a comprehensive understanding of the
molecular nature of this physiological process. Our work, recent (9) and ongoing, reflects this goal.
| |
ACKNOWLEDGEMENTS |
The authors thank Drs. M. Benito and A. M. Valverde for their
generous gifts of antibodies to liver G6PDH and ME and J. Domenzain for
supplying fish. We are grateful to D. Nesbitt for reviewing the English
text.
| |
FOOTNOTES |
This study has been supported by grants from the Comisión
Interministerial de Ciencia y Technología, project No. MAR
92-0412 from Ministerio de Educación y Ciencia (Madrid,
Spain) and the Plán Andaluz de Investigación, project No.
95-3115 (Consolidación de Grupos de Investigación,
Junta de Andalucía, Spain).
Address for reprint requests: J. A. Lupiáñez, Departamento
de Bioquímica y Biología Molecular, Centro de Ciencias
Biológicas, Universidad de Granada, Avenida Fuentenueva s/n,
E-18001 Granada, Spain.
Received 28 May 1997; accepted in final form 10 February 1998.
| |
REFERENCES |
1.
Abraham, S.,
H. J. M. Hansen,
and
F. N. Hansen.
The effect of prolonged fasting on total lipid synthesis and enzyme activities in the liver of the European eel (Anguilla anguilla).
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
79:
285-289,
1984.
2.
Barroso, J. B.
Influencias Nutricionales y de la edad Sobre el Comportamiento Cinético de los Sistemas Productores de NADPH en Diferentes Tejidos de la Trucha Arco Iris (Oncorhynchus mykiss) (MD Thesis). Granada, Spain: University of Granada, 1993.
3.
Barroso, J. B.,
L. García-Salguero,
J. Peragón,
M. de la Higuera,
and
J. A. Lupiáñez.
The influence of dietary protein on the kinetics of NADPH production systems in various tissues of rainbow trout (Oncorhynchus mykiss).
Aquaculture
124:
47-59,
1994.
4.
Bastrop, R.,
R. Spangenberg,
and
K. Jürss.
Biochemical adaptation of juvenile carp (Cyprinus carpio L.) to food deprivation.
Comp. Biochem. Physiol. A Physiol.
98:
143-149,
1991.
5.
Black, D.,
and
R. M. Love.
The sequential mobilization and restoration of energy reserves in tissues of Atlantic cod during starvation and refeeding.
J. Comp. Physiol. [B]
156:
469-479,
1986.
6.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
7.
Ceriotti, G.
A microchemical determination of deoxyribonucleic acid.
J. Biol. Chem.
198:
297-301,
1952[Free Full Text].
8.
Corpas, F. J.,
L. García-Salguero,
J. Peragón,
and
J. A. Lupiáñez.
Kinetic properties of hexose-monophosphate dehydrogenases. I. Isolation and partial purification of glucose 6-phosphate dehydrogenase from rat liver and kidney cortex.
Life Sci.
56:
179-189,
1995[Medline].
9.
De la Higuera, M.,
A. Garzón,
M. C. Hidalgo,
J. Peragón,
G. Cardenete,
and
J. A. Lupiáñez.
Influence of temperature and dietary supplementation either with free or coated lysine on the fractional protein turnover rates in the white muscle of carp.
Fish Physiol. Biochem.
18:
85-95,
1998.
10.
García-Jiménez, C.,
A. Hernández,
M. J. Obregón,
and
P. Santisteban.
Malic enzyme gene expression in differentiating brown adipocytes: regulation by insulin and triiodothyronine.
Endocrinology
132:
1537-1543,
1993[Abstract/Free Full Text].
11.
Goodridge, A. G.
Fatty acid synthesis in eucaryotes.
In: Biochemistry of Lipids, Lipoproteins and Membranes, edited by D. E. Vance,
and J. Vance. Amsterdam: Elsevier, 1991, p. 111-139.
12.
Greene, D. H.,
and
D. P. Selivonchik.
Lipid metabolism in fish.
Prog. Lipid Res.
26:
53-85,
1987[Medline].
13.
Grigor, M. R.
Multiple molecular forms of rat mammary glucose 6-phosphate dehydrogenase: proposed role in turnover of the enzyme.
Arch. Biochem. Biophys.
229:
612-622,
1984[Medline].
14.
Hampton, J. A.,
R. A. McCuskey,
R. S. McCuskey,
and
D. E. Hinton.
Functional units in rainbow trout (Salmo gairdneri, Richardson) liver. I. Arrangement and histochemical properties of hepatocytes.
Anat. Rec.
213:
166-175,
1985[Medline].
15.
Kan, B.,
I. M. London,
and
D. H. Levin.
Role of reversing factor in the inhibition of protein synthesis initiation by oxidized glutathione.
J. Biol. Chem.
263:
15652-15656,
1988[Abstract/Free Full Text].
16.
Kletzien, R. F.,
P. K. W. Harris,
and
L. A. Foellmi.
Glucose 6-phosphate dehydrogenase. A housekeeping enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidant stress.
FASEB J.
8:
174-181,
1994[Abstract].
17.
Levine, R. L.,
J. A. Williams,
E. R. Stadtman,
and
E. Shacter.
Carbonyl assays for determination of oxidatively modified proteins.
Methods Enzymol.
233:
346-357,
1994[Medline].
18.
Lupiáñez, J. A.,
M. J. Sánchez-Lozano,
L. García-Rejón,
and
M. de la Higuera.
Long-term effect of a high-protein/non carbohydrate diet on the primary liver and kidney metabolism in rainbow trout (Salmo gairdneri).
Aquaculture
79:
91-101,
1989.
19.
Martins, R. N.,
G. B. Stokes,
and
C. L. Masters.
Regulation of the multiple molecular forms of rat liver glucose 6-phosphate dehydrogenase by insulin and dietary restriction.
Biochem. Biophys. Res. Commun.
127:
136-142,
1985[Medline].
20.
Mommsen, T. P.,
E. Danulat,
M. E. Gavioli,
G. D. Foster,
and
T. W. Moon.
Separation of enzymatically distinct populations of trout hepatocytes.
Can. J. Zool.
69:
420-426,
1991.
21.
Moon, T. W.,
G. D. Foster,
and
E. M. Plisetskaya.
Changes in peptide hormones and liver enzymes in the rainbow trout deprived of food for 6 weeks.
Can. J. Zool.
67:
2189-2193,
1989.
22.
Munro, H. N.,
and
A. Fleck.
The determination of nucleic acids.
In: Methods of Biochemical Analysis, edited by D. Glick. New York: Wiley, 1966, vol. XIV, p. 113-176.
23.
Peragón, J.,
F. Aranda,
L. García-Salguero,
and
J. A. Lupiáñez.
Influence of experimental diabetes on the kinetic behaviour of renal cortex hexose monophosphate dehydrogenases.
Int. J. Biochem.
21:
689-694,
1989[Medline].
24.
Peragón, J.,
F. Aranda,
L. García-Salguero,
A. Vargas,
and
J. A. Lupiáñez.
Long-term adaptive response to dietary protein of hexose monophosphate shunt dehydrogenases in rat kidney tubules.
Cell Biochem. Funct.
8:
11-17,
1990[Medline].
25.
Protsko, C. R.,
R. S. Fritz,
and
R. Kletzien.
Nutritional regulation of hepatic glucose-6-phosphate dehydrogenase.
Biochem. J.
258:
295-299,
1989[Medline].
26.
Rocha, E.,
R. A. F. Monteiro,
and
C. A. Pereira.
Microanatomical organization of hepatic stroma of the brown trout, Salmo trutta fario (teleostei, salmonidae): a qualitative and quantitative approach.
J. Morphol.
223:
1-11,
1995.
27.
Sánchez-Muros, M. J.,
L. García-Rejón,
J. A. Lupiáñez,
and
M. de la Higuera.
Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout (Oncorhynchus mykiss). II. Adaptive response of glucose 6-phosphate dehydrogenase activity to high-carbohydrate/low protein and high-fat/non-carbohydrate diets.
Aquacult. Nutr.
2:
193-200,
1996.
28.
Schär, M.,
I. P. Maly,
and
D. Sasse.
Histochemical studies on metabolic zonation of the liver in the trout (Salmo gairdneri).
Histochemistry
83:
147-151,
1985[Medline].
29.
Segner, H.,
and
R. Böhm.
Enzymes of lipogenesis.
In: Biochemistry and Molecular Biology of Fishes, Analytical Techniques, edited by P. W. Hochachka,
and T. P. Mommsen. Amsterdam: Elsevier, 1994, vol. 3, p. 313-325.
30.
Sheridan, M. A.,
and
T. P. Mommsen.
Effects of nutritional state on in vivo lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch.
Gen. Comp. Endocrinol.
81:
473-483,
1991[Medline].
31.
Shimeno, S.
Properties and distribution of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.
In: Studies on Carbohydrate Metabolism in Fish, edited by V. S. Kothekar. Rotterdam: Balkema, 1982, p. 44-62.
32.
Shu, S.,
and
L. Fan.
The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system.
Neurosci. Lett.
85:
169-171,
1985.
33.
Tocher, D. R.,
J. Carr,
and
J. R. Sargent.
Polyunsaturated fatty acid metabolism in fish cells: differential metabolism of (n-3) and (n-6) series acids by cultured cells originating from a freshwater teleost fish and from a marine teleost fish.
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
94:
367-374,
1989.
34.
Tomlinson, J. E.,
R. Nakayama,
and
D. Holten.
Repression of pentose phosphate pathway dehydrogenase synthesis and mRNA by dietary fat in rats.
J. Nutr.
118:
408-415,
1988.
35.
Valverde, A. M.,
M. Benito,
and
M. Lorenzo.
Hormonal regulation of malic enzyme and glucose-6-phosphate-dehydrogenase expression in fetal brown-adipocyte primary cultures under non-proliferative conditions.
Eur. J. Biochem.
203:
313-319,
1992[Medline].
36.
Wacke, R.,
K. Jürss,
R. Bastrop,
and
T. Vökler.
Time course of enzyme adaptation to dietary change in the rainbow trout (Salmo gairdneri Richardson).
Zool. Jb. Physiol.
93:
11-22,
1989.
37.
Walsh, P. J.,
T. W. Moon,
and
T. P. Mommsen.
Interactive effects of acute changes in temperature and pH on metabolism in hepatocytes from the sea raven Hemitripterus americanus.
Physiol. Zool.
58:
727-735,
1985.
38.
Walzem, R. L.,
T. Storebakken,
S. S. O. Hung,
and
R. J. Hansen.
Relationship between growth and selected liver enzyme activities of individual rainbow trout.
J. Nutr.
121:
1090-1098,
1991.
39.
Waterlow, J. C.,
P. J. Garlick,
and
D. J. Millward.
Protein Turnover in Mammalian Tissues and the Whole Body. North-Holland: Elsevier Biochemical, 1978, p. 804.
40.
Yamauchi, T.,
J. J. Stegeman,
and
E. Goldberg.
The effects of starvation and temperature acclimation on pentose phosphate pathway dehydrogenases in brook trout liver.
Arch. Biochem. Biophys.
167:
13-20,
1975[Medline].
41.
Zelewski, M.,
and
J. Swierczynski.
Organ specific regulation of malic enzyme and hexosemonophosphate shunt dehydrogenases activity by high carbohydrate diet.
Biochem. Int.
19:
1057-1065,
1989[Medline].
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Abstract
Introduction
