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1 Biochemistry and 2 Zoology Departments, University of Western Australia, Crawley, Western Australia 6009, Australia
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Protein synthesis is downregulated during metabolic depression in a number of systems where the metabolic depression is effected by obvious extrinsic cues. The metabolic depression of the estivating land snail Helix apersa occurs in the absence of any obvious physiological stress and has an intrinsic component independent of temperature, pH, O2 status, or osmolality. We show that this metabolic depression is accompanied by a downregulation of protein synthesis in vivo. The rate of protein synthesis decreases in two major tissues during estivation: to 23% and 53% of the awake rate in hepatopancreas and foot muscle, respectively. We show from calculations of the theoretical contribution of protein synthesis to total O2 consumption that the depression of protein synthesis must be a significant, obligate, in vivo component of metabolic depression in H. aspersa.
pulmonate snail; metabolic depression; mollusc
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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MANY ORGANISMS ENTER A STATE of quiescence and survive for extended periods on stored fuels in the face of adverse environmental conditions. Associated with this quiescence is a depression of basal metabolic rate by 60-100% (11). Such a significant change in metabolic rate for an extended period necessitates a coordinated reduction and rebalancing of ATP production and utilization. Protein synthesis is a major contributor to cell energy expenditure in a variety of organisms, tissues, and cell types and typically accounts for 18-26% of the total ATP turnover (13). Protein synthesis, if maintained at a similar rate during metabolic depression, would become an impossibly costly process in terms of its contribution to total energy consumption. We would therefore expect that this major energy-consuming process is substantially downregulated during metabolic depression.
Accordingly, protein synthesis has been shown to be considerably reduced in types of metabolic depression that involve a physiological stress, such as desiccation, hypoxia, or hypothermia. For example, during cryptobiosis, where cell water is removed or restructured and all metabolic activity appears to cease (11), protein synthesis cannot be measured, but reactivation of translation on rehydration has been observed in anhydrobiotic Artemia gastrulae (27). Similarly, protein synthesis is downregulated during metabolic depression caused by lowered (but nonfreezing) temperature. For example, hibernating bats have an undetectable level of protein synthesis in pectoral muscle and liver in vivo (32), and hibernating ground squirrels have a lowered rate of incorporation of protein into liver microsomes (30). In metabolic depression associated with severe hypoxia or anoxia, there is likewise evidence that protein synthesis is decreased. This has been demonstrated in cell-free lysates and mitochondria from Artemia, turtle hepatocytes, turtle heart, and various tissues of carp (2, 16, 19, 20, 24). Intrinsic metabolic depression is another category of metabolic depression where a reduced metabolic rate occurs in anticipation of a physiological stress or as part of a developmental cue. Therefore, the metabolic depression occurs without an obvious physiological stress (11) and, in the case of some diapausing animals, even when conditions for development seem optimal (23). Examples of intrinsic metabolic depression include estivating pulmonate snails and frogs and also some diapausing animals (11). In two studies that addressed the question of metabolic depression and protein synthesis during diapause (aerobic diapause by Artemia and aerobic diapause by killifish embryos), metabolic depression was associated with a large (90%) decrease in the rate of protein synthesis (6, 23). The only study of protein synthesis in a nondiapausing estivator, a desert frog, showed a 67% reduction in the rate of protein synthesis by liver slices (8). Metabolic depression in which the intrinsic factors play a significant role is of fundamental interest to researchers examining the regulation of metabolic depression. Organisms that demonstrate this type of metabolic depression, such as snails, can potentially tell us more about regulatory principles involved in normal cell metabolism than can an organism in which most of the depression is a result of a massive decrease in body temperature, such as a mammalian hibernator or an organism that is anhydrobiotic and lacks functional cell water (11).
The land snail Helix aspersa is an excellent model in which
to study intrinsic metabolic depression. H. aspersa, when
deprived of food and water, readily enters a state of estivation, which is characterized by withdrawal into the shell, construction of one or
more epiphragms covering the shell aperture, and a decrease in standard
metabolic rate. Within 4 wk after the removal of food and water from
H. aspersa, there is an 84% metabolic depression from the
normal standard (awake) state as measured by in vivo O2
consumption (
O2) (22).
Metabolic depression in H. aspersa is induced without any
change in ambient temperature, without any significant water loss, and
with minimal changes in hemolymph PO2
(3); i.e., H. aspersa initiates the behavioral,
and then the physiological, parts of estivation before the
manifestation of any physiological stress. The most major and immediate
threat is water loss, but even after 1-3 mo of estivation there is
no significant increase in the osmolality of hemolymph
(22). In this study, we have measured the in vivo extent
of downregulation in the rate of protein synthesis in two major
tissues, hepatopancreas and foot muscle from H. aspersa,
during estivation.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Chemicals. L-[2,6-3H]phenylalanine (specific activity 54.0 Ci/mmol), 14C-methylated proteins, and NCS II tissue solubilizer were purchased from Amersham Life Science (Buckinghamshire, UK). Phenylalanine, tyrosine, HEPES, trifluoroacetic acid (TFA), and reagents used to determine phenylalanine concentration (procedure no. 60-F, Sigma Diagnostics) were obtained from Sigma Chemical (St. Louis, MO). High-performance liquid chromatography (HPLC)-grade acetonitrile was purchased from EM Science (Merck, Darmstadt, Germany). All other reagents were obtained from BDH Chemicals.
Animal collection and maintenance.
H. aspersa were collected from the grounds of the University
of Western Australia and surrounding suburban gardens. Snails were kept
in glass aquariums, which were kept constantly moist and between 22 and
25°C. They were maintained on an artificial diet (28)
for 
2 wk before being placed into estivation. Snails were induced to
enter estivation by placement in separate containers without food or
water and were kept undisturbed in the dark. Snails used in the
experiments had been estivating for 2 mo. Control (nonestivating)
snails were obtained by arousal of estivating snails by placement in
moist aquariums. Control snails were kept in this environment without
food for 48 h and were seen to be active before use in experiments.
Measurement of rates of protein synthesis in vivo.
The rate of protein synthesis was measured using a modified version of
the method described previously (9). One microliter of
L-[2,6-3H]phenylalanine (120 mM, 100 µCi/ml) was injected per 400 mg of shell-free weight. Shell-free
weight was estimated by using data obtained in a previous pilot study.
The shell-free weight of awake snails (total weight 4-6 g) was
83.7 ± 0.7% (n = 34), and the shell-free weight
of estivating snails (total weight 4-6 g) was 75.7 ± 0.8%
(n = 6). The apparent difference in shell-free weight between awake and estivating snails could at least be partly explained by the fact that epiphragm material associated with the estivating snails was classified as shell material. The error in estimating shell-free weight could result in 4.8% and 2.6% errors in the flooding dose for awake and estivating snails, respectively. The injectate was introduced into the snails between the shell and mantle
edge through a 1-mm hole carefully punctured through the shell just
above the mantle using a dissecting needle. Any of the estivating
snails that showed any signs of arousal from estivation at any stage
during the experiment were discarded. The metabolic rate of a group of
estivating snails was determined before and after an injection by
measuring O2.
O2 was measured using a Servomex
paramagnetic O2 analyzer at an airflow rate of 30 ml/min, controlled by a Sierra mass-flow controller.
O2 was measured for 2 h before the
injection, snails were removed from the chamber and injected (see
above), and O2 was measured for a
further 2 h. O2
(ml · g
1 · h1) was
calculated by adjusting O2 for standard
pressure and temperature and the shell-free weight of the snails. The
average O2 was 0.021 ± 0.003 and
0.022 ± 0.005 ml · g1 · h1
(n = 6) before and after injection, respectively. A
two-tailed paired t-test indicated no difference in
O2 before and after the injection.
80°C.
A 10% homogenate of the tissues was prepared with deionized water
using an Ultra-Turrax T25 homogenizer (Janke and Kunkel IKA
Labortechnik) at 20,500 rpm for 30 s at room temperature. Protein
was precipitated with perchloric acid. The homogenate was kept on ice
for 30 min and then centrifuged at 4,000 g for 20 min at
4°C. The supernatant (S1) was removed and frozen. The protein pellet was redissolved in sodium hydroxide at 37°C. A fraction of the solubilized protein pellet was removed for
determination of protein concentration using a Bio-Rad DC protein assay
kit and BSA as a standard. The solubilized protein was reprecipitated with perchloric acid and collected by centrifugation as described above. The supernatant from this centrifugation was removed, and the
pellet was solubilized with NCS II tissue solubilizer. To determine the
amount of incorporation of radiolabeled phenylalanine into protein, the
NCS-solubilized protein was counted in a liquid scintillation counter
(model LS 3800, Beckman). Protein-free supernatant (S1) was
also counted to determine the amount of radiolabeled phenylalanine in
the free pool.
To ensure that free phenylalanine was not being trapped in the protein
pellet, hepatopancreas and foot muscle from control snails were
homogenized as described above. Then 5 µl of a solution containing
L-[2,6-3H]phenylalanine (12 mM, 0.2 µCi/µl) were added per milliliter of homogenate. The samples were
then treated as described above, and radioactivity in the supernatant
from each stage and in the final protein pellet was determined. A
negligible amount of free phenylalanine was trapped in the final
pellet. The radioactivity in the pellet was 0.0036 ± 0.001% and
0.0027 ± 0.004% (n = 3) of the amount in
S1 for hepatopancreas and foot muscle, respectively.
To determine the percent recovery of protein after perchloric acid
precipitation, 14C-methylated proteins were added to
control hepatopancreas homogenate, and the samples were treated as
described above. The amounts of radioactivity in each of the
supernatants and the final protein pellet were determined as described
above, and 99.29 ± 1.94% (n = 5) of the
radioactivity originally added to the homogenate was found to be in the
final protein pellet.
Spectrophotofluorometric determination of phenylalanine. Phenylalanine concentration in the free pool (S1 samples) was measured using an assay modified from a kit from Sigma Diagnostics (procedure no. 60-F) for phenylalanine fluorometric determination in serum and plasma. A peptide control, i.e., omitting L-leucyl-L-alanine, was used to control for nonspecific fluorescence (1). Phenylalanine recovery in the assay was determined using phenylalanine-spiked samples. The recovery for phenylalanine in foot muscle extracts was 97 ± 3.4% (mean ± SE, n = 4), but phenylalanine recovery from hepatopancreas extracts varied between individuals because of the presence of compounds that quenched the fluorescence. Therefore, to assay the concentration of phenylalanine in hepatopancreas, test samples, spiked test samples, peptide control, and spiked peptide control samples were assayed simultaneously for each sample.
HPLC of acid extracts. Breakdown products of phenylalanine (principally tyrosine) can confound the measurement of protein synthesis using labeled phenylalanine (9). Therefore, it was necessary to determine whether any of the label was appearing in other compounds. For this reason, snails were injected with L-[2,6-3H]phenylalanine (120 mM, 100 µCi/ml) as described above. Protein-free supernatants were prepared from awake and estivating hepatopancreas and foot muscle 8 h after the injection as described above.
The extracts were neutralized and concentrated fivefold in a Speed-Vac concentrator (model SCV 100H, Savant). HPLC was performed on the samples using a 250 mm × 4.6 mm C18 macrosphere column. The samples were chromatographed using a linear acetonitrile concentration gradient from 0% acetonitrile-0.06% TFA to 80% acetonitrile-0.06% TFA over 30 min with a flow rate of 0.5 ml/min. The absorbance of the eluant was measured at 270 nm. Elution times of tyrosine and phenylalanine were determined using samples spiked with cold phenylalanine and tyrosine. One-milliliter (2 min) fractions were collected, and the amount of radioactivity contained in each fraction was determined by scintillation counting. A sample of the injectate solution and an extract from awake hepatopancreas spiked with the injectate solution were also chromatographed.Calculations and statistics.
To determine whether specific activity was changing over time, a
one-way ANOVA was used to compare the different time points. Rates of
protein synthesis (nmol phenylalanine · mg
protein1 · h1) were calculated
using 1) the average specific activity (dpm/nmol phenylalanine) observed between 60 and 240 min and 2) the
slope of the linear regression equation describing the disintegrations per minute incorporated per milligram of protein at 60, 120, and 240 min. The rates of protein synthesis were compared between states for
each tissue by comparing the slopes of the cumulative incorporation
curves using t-tests (33). All other
comparisons were made using unpaired t-tests. Values are
means ± SE. Data were considered significantly different at
P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Stability of radiolabeled free phenylalanine.
The specific activity of the free phenylalanine in the tissues was
determined from the radioactivity in the protein-free supernatant (which was assumed to be phenylalanine) and the concentration of free
phenylalanine. Therefore, it was necessary to determine whether a
significant amount of phenylalanine was being converted to any other
compounds, the primary concern being the enzymatic conversion of
phenylalanine to tyrosine. During HPLC of protein-free supernatant
(spiked with phenylalanine and tyrosine) prepared from H. aspersa hepatopancreas, tyrosine eluted at ~17.7 min and phenylalanine at ~21 min (Fig.
1A). In foot
muscle, 8 h after injection, 91% of the radioactivity eluted at
the peak corresponding to phenylalanine (Fig. 1B). In
hepatopancreas, 8 h after injection, 90% of the radioactivity
eluted at the time corresponding to the phenylalanine peak (Fig.
1C). There was no visible peak of radioactivity eluting at
the time corresponding to a tyrosine peak in either tissue. Any
radioactivity due to tyrosine would have eluted in the fraction
collected between 16.25 and 18.75 min, which represented ~11% of the
peak corresponding to phenylalanine. There was ~10% of the
radioactivity in earlier fractions from both tissues and from the
injectate (Fig. 1D) in a broad peak that eluted at ~8 min.
Mixing the radiolabeled phenylalanine solution with protein-free supernatant from awake or estivating hepatopancreas had no effect on
the radioactive elution profile. Therefore, the radioactivity eluting
in the broad peak at 8 min is due to impurities in the radiolabeled
phenylalanine stock solution. For both tissues, there was no
significant difference between awake and estivating samples in the
amount of radioactivity in the peak corresponding to the elution time
of phenylalanine.
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Time course of free phenylalanine specific activity.
To calculate the rate of protein synthesis, it is necessary to
1) know the specific activity of the free phenylalanine at the start of the measurement period and 2) assume that it
remained constant over the measurement period. The data (Fig.
2) indicate that the specific activity
reaches a maximum by 60 min and that there are no differences in
specific activity between 60 and 240 min (P < 0.05)
for either tissue. The average specific activity over this period was
905 ± 140 and 860 ± 70 dpm/nmol phenylalanine (n = 27) for awake and estivating snail hepatopancreas,
respectively. The average specific activity over this period was
716 ± 67 and 870 ± 79 dpm/nmol phenylalanine
(n = 27) for awake and estivating snail foot muscle,
respectively. There was also no difference (P < 0.05)
in the specific activity at 60 and 240 min between the two tissues. In
all four cases, the average specific activity of the free pool between
60 and 240 min was not different from the specific activity of the
injectate solution (909 ± 32.3 dpm/nmol phenylalanine). However,
as determined by one-way ANOVA, there was a significant difference
(P < 0.05, by Fisher's protected least significant
difference test statistic) in specific activity between the 15-min time
point and the 60-, 120-, and 240-min time points for all four
combinations of tissues and states. The 30-min time point is an
intermediate value in all cases and is not significantly different from
the 60- to 240-min time points in any of the tissues. The 30-min time
point is significantly different from the 15-min time point only in
awake foot and estivating hepatopancreas.
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Free phenylalanine concentration. Because the injectate had been delivered at 120 mM phenylalanine at a dose of 1 µl/400 mg shell-free wt, the final concentration of phenylalanine would be expected to increase by 0.3 nmol/mg shell-free wt. Mean phenylalanine concentrations at 240 min were 0.36 ± 0.08, 0.30 ± 0.01, 0.33 ± 0.05, and 0.28 ± 0.04 nmol/mg tissue (n = 9) for awake snail hepatopancreas, estivating snail hepatopancreas, awake snail foot muscle, and estivating snail foot muscle, respectively. Free phenylalanine concentrations in uninjected snails were below the sensitivity of the assay (0.05 nmol/mg tissue).
Incorporation of radiolabel into protein.
Incorporation of radiolabel was linear between 60 and 240 min for all
the tissues and states investigated (Fig.
3, A and B).
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1 · h1 (n = 27) for awake and estivating hepatopancreas, respectively, and 231 ± 48 and 146.4 ± 30 dpm · mg
protein1 · h1 (n = 27) for awake and estivating foot muscle, respectively. Comparison of
the slopes of the incorporation curves between states for each tissue
using a two-tailed t-test (33) indicates a
significant difference between the awake and estivating state for each
tissue (P < 0.05).
Rates of protein synthesis.
The rates of phenylalanine incorporation were 0.51 ± 0.12, 0.12 ± 0.09, 0.32 ± 0.07, and 0.17 ± 0.03 nmol
phenylalanine · mg
protein1 · h1 for awake snail
hepatopancreas, estivating snail hepatopancreas, awake snail foot
muscle, and estivating snail foot muscle, respectively (Fig.
4). In both tissues, the rate of protein
synthesis decreased during estivation (P < 0.05). This
represents a decrease in protein synthesis rate to 23% of the awake
rate during estivation in hepatopancreas and to 53% of the awake rate
in foot muscle.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Methods validation. The study presented here satisfies all the generally accepted criteria that define a valid measurement of protein synthesis: rapid flooding of the phenylalanine free pool, stabilization of the free pool1 specific activity over the measurement period, no difference in specific activity between the free pool and the injectate, and linear incorporation of radioactivity into protein over the measurement period.
In addition, we have addressed a concern, i.e., that in some systems phenylalanine has been shown to be readily converted to tyrosine in vivo (9). One way to overcome this problem is to enzymatically convert free phenylalanine (and phenylalanine as a product of the hydrolysis of protein) to
-phenylethylamine and then
extract the -phenylethylamine and measure its specific activity
(25). In this study, in which all measurements were complete within 4 h of adding the injectate, we have shown using HPLC that there is no significant conversion of phenylalanine to
tyrosine after 8 h. The final possible concern about the
methodology is the assumption that there is no difference between the
two states in the proportion of phenylalanine being incorporated into newly synthesized protein. The few data on this issue are at least consistent with the assumption. Methionine labeling of newly
synthesized proteins in awake and estivating snails (4)
has shown the synthesis of only a few estivation-specific proteins, and
in quiescent Artemia embryos, there are no qualitative or
quantitative changes in the mRNA pool with quiescence
(15).
The sham injection had the effect of increasing the rate of
incorporation between 180 and 240 min in the hepatopancreas. The extent
of the increase is similar to the cumulative incorporation in awake and
estivating snails between 0 and 60 min, and therefore the experimental
manipulation associated with the injection itself is responsible for
the initially high rate of incorporation seen in estivating
hepatopancreas. The O2 data demonstrate
that this increase in rate occurs without an increase in measurable
O2 of the whole animal.
Rates of protein synthesis.
This is the first study to measure downregulation in the rate of
protein synthesis in vivo for an estivating animal. The absolute rates
reported here vary between 0.12 and 0.51 nmol
phenylalanine · mg
protein1 · h1. Other studies
involving ectotherms, where the absolute rates of protein synthesis are
expressed in a way similar to the data presented here, report similar
rates. For example, the rate of protein synthesis in flight muscle of
Manduca sexta is 1-2 nmol phenylalanine · mg
protein1 · h1 (26),
and the rates in Neobatrachus centralis liver
(8), Bufo marinus liver (7), and
turtle ventricle (2) (with the assumption that protein
content of the tissue is ~10% by weight) are 0.6, 0.6, and
0.15-0.5 nmol phenylalanine · mg
protein1 · h1, respectively.
Contribution of protein synthesis to metabolic rate.
Cycloheximide, an inhibitor of protein synthesis, is often used to
directly measure the contribution of protein synthesis to
O2. Although this measurement is not
possible for individual tissues in an in vivo study, we have estimated
the contribution by making a number of assumptions. We have assumed
that 5 ATP equivalents are required per peptide bond, giving the value
of 46 mmol ATP/g protein synthesized. We have assumed that the rate of
O2 is the same per gram in all
tissues2 and that the P/O
ratio is 3, which is the value used in a number of other studies
(13, 17, 18). The proportion of phenylalanine, compared
with other amino acids, in protein from hepatopancreas and foot muscle
was taken from data from a related species, O. lactea
(5). Using these assumptions, we estimate that protein synthesis accounts for 12 ± 3% and 18.4 ± 14% of
O2 by hepatopancreas in awake and
estivating snails, respectively. For the foot muscle, protein synthesis
accounts for 9.8 ± 2% and 31.6 ± 6% of
O2 of the foot muscle in the awake and
estivating snail, respectively. The values we estimate here are lower
than in other ectotherms, where the minimal cost of protein synthesis
has been estimated using fractional rates of protein synthesis. The
estimated cost of whole body protein synthesis was calculated to be
19-37% of O2 for shore crabs
Carcinus maenas (18), 35-51% for O. vulgaris (17) (depending on whether the estimation
used 46 or 68 mmol ATP/g protein synthesized), and 20% for the mussel
M. edulis (14). Few studies have measured the
contribution of protein synthesis to metabolic rate by direct means,
and the variation in values is large. In liver of B. marinus, as determined by inhibition with cycloheximide, protein
synthesis accounted for ~12% of O2 (7). However, in N. centralis, protein
synthesis accounted for ~49% of O2 of
the liver (8). Other estimates for ectotherms based on
direct measurements include 36% for nondiapausing killifish embryos
(23), 80% for trout hepatocytes (21), 36%
for normoxic turtle hepatocytes (20), 66% (whole body
protein synthesis) for the isopod Glyptonotus antarcticus,
and 22% (whole body protein synthesis) for the isopod Idotea
rescata (29). Very probably one source of this
variation is the concentration of cycloheximide used in these studies,
inasmuch as it has recently been reported for fish hepatocytes that
O2 in the presence of cycloheximide is
dependent on the concentration of the inhibitor (31). The most reliable studies are probably those where cycloheximide
concentration is carefully titrated against protein synthesis to
determine the absolute minimum required for a measurable decrease in
rate of protein synthesis and O2.
Contribution of the downregulation of protein synthesis to metabolic depression. Downregulation in the rate of protein synthesis accounts for 60% of the metabolic depression in hepatopancreas but only 2.5% of the metabolic depression of the whole animal, given that the hepatopancreas makes up about 4.1 ± 1.1% (n = 18) of the wet shell-free weight of the animal. The depression of protein synthesis in the foot muscle accounts for 28% of the metabolic depression in the foot muscle but only 1.4% of the metabolic depression of the whole animal, given that the foot muscle represents 5.2 ± 1.3% (n = 18) of the wet shell-free weight of the animal.
The rate of protein synthesis reported here accounts for a significant proportion of the metabolic rate of the tissues in which it is measured. As a result, the change in the rate of protein synthesis that occurs between the awake and estivating state accounts for a significant proportion of the metabolic depression of the tissue involved. In other metabolically depressed systems, the downregulation of protein synthesis makes a similar contribution to the total metabolic depression. The change in the rate of protein synthesis in isolated liver from the estivating frog N. centralis accounted for 52% of the metabolic depression in the tissue and 4% of the metabolic depression of the whole frog (8). In anoxic hepatocytes from the turtle, the decrease in the rate of protein synthesis accounted for 38% (20) of the metabolic depression, and in diapausing killifish embryos, the decrease in the rate of protein synthesis accounted for 36% of the metabolic depression (23). It is reasonable to assume that a similar downregulation in protein synthesis would be seen throughout the various tissues of the snail. Therefore, protein synthesis would be a major contributor to metabolic depression, and without depression of protein synthesis the metabolic depression would be limited, and a large percentage of the estivating metabolic rate would be dedicated to support protein synthesis. From our estimates, if the rate of protein synthesis was not downregulated, during metabolic depression it would account for 78% and 60% of theO2 in hepatopancreas and foot muscle, respectively.
Conclusions. We have reported for two tissues, hepatopancreas and foot muscle, that protein synthesis is downregulated during estivation. This downregulation is quantitatively an essential component of metabolic depression in the estivating snail, a theme that is becoming accepted dogma in the field of metabolic depression, but the characterization of this phenomenon in an estivator has wider implications. During estivation, there is a metabolic depression that occurs without any significant physiological stress and without changes in water, solute, or O2 status (11), and downregulation is likely to be coordinated by an intrinsic cue. Previous in vitro studies of H. aspersa have shown a stable intrinsic component to metabolic depression that can be isolated from the changes in pH and PO2 seen in vivo (10, 22). It is the mechanism by which this intrinsic component is regulated that is of interest. By identifying an energy-consuming process such as protein synthesis, which is downregulated during metabolic depression, it may be possible to delineate the pathway by which the process is regulated and, therefore, find the intrinsic cue that leads to metabolic depression. There is accumulating evidence that regulation of protein synthesis during metabolic depression occurs at the translational level (for review see Ref. 12). Further studies on protein synthesis during metabolic depression will need to focus on regulatory mechanisms, i.e., perhaps investigating changes in the specific activity of the components of the translational machinery.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Pakay, Biochemistry Dept., Univ. of Western Australia, 35 Stirling Hwy., Crawley, WA 6009, Australia (E-mail: jlpakay{at}cyllene.uwa.edu.au).
1 The flooding dose was much larger than the endogenous free pool. The normal free phenylalanine concentration in hepatopancreas and foot muscle was below the lower limit of the phenylalanine assay used here. However, if the concentration for free phenylalanine in whole body for H. aspersa (0.02 mM) is used (5), then in this study the flooding dose of phenylalanine raises the concentration of the tissue free pool ~15-fold, to 0.3 mM.
2
O2, from Ref.
22, was 134 ± 7 and 22 ± 3 µl
O2 · g shell-free wet tissue
wt1 · h1 for awake and estivating
snails, respectively.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 7, 2002;10.1152/ajpregu.00636.2001
Received 26 October 2001; accepted in final form 20 February 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Ambrose, JA.
A shortened method for the fluorometric determination of phenylalanine.
Clin Chem
15:
15-23,
1969[Abstract].
2.
Bailey, JR,
and
Driedzic WR.
Decreased total ventricular and mitochondrial protein synthesis during extended anoxia in the turtle heart.
Am J Physiol Regulatory Integrative Comp Physiol
271:
R1660-R1667,
1996
3.
Barnhart, MC.
Respiratory acidosis and metabolic depression in dormant invertebrates.
In: Living in the Cold II, edited by Malan A,
and Canguilhem B.. Paris: Colloque INSERM/John Libbey Eurotext, 1989, p. 321-331.
4.
Brooks, SPJ,
and
Storey KB.
Evidence for aestivation specific proteins in Otala lactea.
Mol Cell Biochem
143:
15-20,
1995[Web of Science][Medline].
5.
Campbell, JW,
and
Speeg KV.
Arginine biosynthesis and metabolism in terrestrial snails.
Comp Biochem Physiol
25:
3-32,
1968[Medline].
6.
Clegg, JS,
Drinkwater LE,
and
Sorgeloos P.
The metabolic status of diapause embryos of Artemia franciscana.
Physiol Zool
69:
49-66,
1996.
7.
Fuery, CJ,
Withers PC,
and
Guppy M.
Protein synthesis in the liver of Bufo marinus
cost and contribution to oxygen consumption.
Comp Biochem Physiol A Physiol
119:
459-467,
1998.
8.
Fuery, CJ,
Withers PC,
Hobbs AA,
and
Guppy M.
The role of protein synthesis during metabolic depression in the Australian desert frog Neobatrachus centralis.
Comp Biochem Physiol A Physiol
119:
469-476,
1998.
9.
Garlick, PJ,
McNurlan MA,
and
Preedy VR.
A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine.
Biochem J
192:
719-723,
1980[Web of Science][Medline].
10.
Guppy, M,
Reeves DC,
Bishop T,
Withers P,
Buckingham JA,
and
Brand MD.
Intrinsic metabolic depression in cells isolated from the hepatopancreas of estivating snails.
FASEB J
14:
999-1004,
2000
11.
Guppy, M,
and
Withers P.
Metabolic depression in animals: physiological perspectives and biochemical generalizations.
Biol Rev Camb Philos Soc
74:
1-40,
1999[Medline].
12.
Hand, SC.
Downregulation of cellular metabolism during environmental stress: mechanisms and implications.
Annu Rev Physiol
58:
539-563,
1996[Web of Science][Medline].
13.
Hawkins, A.
Protein turnover: a functional appraisal.
Funct Ecol
5:
222-233,
1991.
14.
Hawkins, AJS,
Widdows J,
and
Bayne BL.
The relevance of whole-body protein metabolism to measured costs of maintenance and growth in Mytilus edulis.
Physiol Zool
62:
745-763,
1989.
15.
Hofmann, GE,
and
Hand SC.
Comparison of messenger RNA pools in active and dormant Artemia franciscana embryos: evidence for translational control.
J Exp Biol
164:
103-116,
1992
16.
Hofmann, GE,
and
Hand SC.
Global arrest of translation during invertebrate quiescence.
Proc Natl Acad Sci USA
91:
8492-8496,
1994
17.
Houlihan, DF,
McMillan DN,
Agnisola C,
Genoino IT,
and
Foti L.
Protein synthesis and growth in Octopus vulgaris.
Mar Biol (Berl)
106:
251-259,
1990.
18.
Houlihan, DF,
Waring CP,
Mathers E,
and
Gray C.
Protein synthesis and oxygen consumption of the shore crab Carcinus maenus after a meal.
Physiol Zool
63:
735-756,
1990.
19.
Kwast, KE,
and
Hand SC.
Oxygen and pH regulation of protein synthesis in mitochondria from Artemia franciscana embryos.
Biochem J
313:
207-213,
1996.
20.
Land, SC,
Buck LT,
and
Hochachka PW.
Response of protein synthesis to anoxia and recovery in anoxia tolerant hepatocytes.
Am J Physiol Regulatory Integrative Comp Physiol
265:
R41-R48,
1993
21.
Pannevis, MC,
and
Houlihan DF.
The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout Oncorhynchus mykiss.
J Comp Physiol [B]
162:
393-400,
1992[Medline].
22.
Pedler, S,
Fuery CJ,
Withers PC,
Flanigan J,
and
Guppy M.
Effectors of metabolic depression in an estivating pulmonate snail (Helix aspersa): whole animal and in vitro tissue studies.
J Comp Physiol [B]
166:
375-381,
1996[Medline].
23.
Podrabsky, JE,
and
Hand SC.
Depression of protein synthesis during diapause in embryos of the annual killifish Austrofundulus limnaeus.
Physiol Biochem Zool
73:
799-808,
2000[Web of Science][Medline].
24.
Smith, RW,
Houlihan DF,
Nilsson GE,
and
Brechin JG.
Tissue-specific changes in protein synthesis in vivo during anoxia in crucian carp.
Am J Physiol Regulatory Integrative Comp Physiol
271:
R897-R904,
1996
25.
Suzuki, O,
and
Yagi K.
A fluorometric assay for -phenylethylamine in rat brain.
Anal Biochem
75:
192-200,
1976[Web of Science][Medline].
26.
Tischler, ME,
Wu M,
Cook P,
and
Hodsden S.
Ecdysteroids affect in vivo protein metabolism of the flight muscle of the tobacco hornworm (Manduca sexta).
Insect Physiol
36:
699-708,
1990.
27.
Wahba, AJ,
and
Woodley CL.
Molecular aspects of development in the brine shrimp Artemia.
Prog Nucleic Acid Res Mol Biol
31:
221-265,
1984[Web of Science][Medline].
28.
Watkins, B,
and
Simkiss K.
Interactions between soil bacteria and the molluscan alimentary tract.
J Molluscan Stud
56:
267-274,
1990
29.
Whiteley, NM,
Taylor EW,
and
El Haj AJ.
A comparison of the metabolic cost of protein synthesis in stenothermal and eurythermal isopod crustaceans.
Am J Physiol Regulatory Integrative Comp Physiol
271:
R1295-R1303,
1996
30.
Whitten, BK,
and
Klain GJ.
Protein metabolism in hepatic tissue of hibernating and arousing ground squirrels.
Am J Physiol
214:
1360-1362,
1968
31.
Wieser, W,
and
Krumschnabel G.
Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects.
Biochem J
355:
389-395,
2001[Web of Science][Medline].
32.
Yacoe, ME.
Protein metabolism in the pectoralis muscle and liver of hibernating bats, Eptesicus fuscus.
J Comp Physiol
152:
137-144,
1983.
33.
Zar, JH.
Biostatistical Analysis. Upper Saddle River, NJ: Prentice Hall, 1996.
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) and estivating
(
) snails. Values are means ± SE
(n = 3). C: elution profile of radiolabel (8 h after injection) from protein-free supernatant (spiked with cold
phenylalanine and tyrosine) of hepatopancreas from control
(
) snails. Values are
means ± SE (n = 3). D: elution profile
of radiolabel in injectate solution (
) and elution
profiles of protein-free supernatant from awake (
) hepatopancreas spiked with injectate
solution. Values are from an isolated experiment.
, cumulative incorporation at 240 min for estivating
foot muscle after sham injection (n = 3). Linear
regression analysis was performed on all incorporation curves: for
awake foot muscle, r = 0.99 (n = 25);
for estivating foot muscle, r = 1.00 (n = 25); for awake hepatopancreas, r = 1.00 (n = 27); for estivating hepatopancreas,
r = 0.92 (n = 26).
