The freeze-avoidance response of smelt Osmerus mordax : initiation and subsequent suppression of glycerol, trimethylamine oxide and urea accumulation
1 Ocean Sciences Centre, Memorial University of Newfoundland, St John's
Newfoundland, Canada A1C 5S7
2 NRC Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova
Scotia, Canada B3H 3Z1
* Author for correspondence (e-mail: wdriedzic{at}mun.ca )
Accepted 27 February 2002
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Summary |
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Key words: smelt, Osmerus mordax, freeze-avoidance response, thermal hysteresis, glycerol, trimethylamine oxide, urea, glycerol-3-phosphate dehydrogenase, antifreeze
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Introduction |
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Levels of small (non-protein) organic solutes are higher in wild smelt
caught in winter than in specimens caught in summer and fall (Raymond,
1992,
1994
). Tissue and plasma
levels of glycerol, TMAO and urea can also be significantly changed under
laboratory conditions by acclimation temperature. In fish caught during winter
conditions (less than -1°C) solute concentrations are lower following
acclimation for 14-15 days to temperatures above freezing, whereas in fish
caught during fall-type conditions (5°C) solute levels are increased when
fish are maintained at -1°C for 21 days (Raymond,
1993
,
1994
;
Raymond et al., 1996
).
Organic osmolytes are either synthesized or obtained through feeding. The
carbon sources for glycerol appear to be stored glycogen and amino acids,
which are accessible via the gluconeogenic pathway
(Raymond, 1995;
Raymond and Driedzic, 1997
).
Furthermore, because smelt may lose approximately 10% of their total glycerol
stores per day when winter-adapted
(Raymond, 1993
), it is very
likely that dietary amino acids are a key precursor to glycerol on a continual
basis. Driedzic et al. (1998
)
have shown that levels of glycerol and amino acid metabolizing enzymes are
severalfold higher in the liver of smelt than in other teleosts collected from
the same region during the winter. For amino acids to be utilized for glycerol
synthesis there must be a high capacity for deamination and subsequent
excretion or detoxification of the ammonia that would be produced. The high
liver amino transferase activities
(Driedzic et al., 1998
) suggest
that these may play a critical role in amino acid flux into the glycerol pool.
Since ammonia is toxic, even in ammoniotelic teleosts
(Wright, 1995
), the production
of urea may be a means of detoxifying ammonia produced from the deamination of
amino acids. Thus if glycerol production from amino acids increases, we might
expect to see an increase in the levels of urea. Glycerol-3-phosphate
dehydrogenase (GPDH) activity is very high in winter-caught smelt relative to
other teleosts and appears to be a key enzyme in glycerol synthesis
(Driedzic et al., 1998
). Since
GPDH has such high activity in the liver, which is probably the major site of
glycerol synthesis, it may be an essential component in total glycerol
synthesis and could have a regulatory role. Consistent with this hypothesis,
winter-caught smelt held at elevated temperatures show decreased levels of
liver GPDH mRNA, in parallel with a decrease in plasma glycerol concentration
(Ewart et al., 2001
).
Previous studies have been restricted to comparisons of a limited number of time points and thus only provide a snapshot glimpse into the smelt antifreeze response. Moreover, they have included smelt from different geographic locations and these fish may respond differently to seasonal stimuli. The present study is the first to examine a number of aspects involved in cold acclimation in smelt from a consistently cold region under laboratory conditions over an entire winter season. Unlike previous work which utilized short-term acclimation, we used two groups of smelt, one maintained at approximately 5°C and another that was allowed to track ambient winter water temperatures. We measured the accumulation of glycerol, TMAO, urea and antifreeze protein activity (thermal hysteresis), along with the expression of antifreeze protein (AFP) mRNA, to determine the temporal sequence of the actual antifreeze response in smelt. By measuring liver alanine aminotransferase (AlaAT), aspartate aminotransferase (AspAT) and GPDH activities, together with the expression of GPDH mRNA, we could examine the temporal regulation of the levels of these enzymes with respect to seasonal expression of glycerol synthesis. Finally, we tested the hypothesis that smelt maintained over winter at temperatures well above freezing suppress their antifreeze mechanisms and thus do not accumulate the organic osmolytes or AFP.
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Materials and methods |
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Fish were maintained in two experimental groups: one tank was maintained at approximately 5°C and the other at ambient seawater temperatures. Fish were initially sampled in mid-December, when the ambient water temperature was approximately 5°C, and then at approximately monthly intervals. Sample dates were December 15, 1999, January 11, 2000, February 29, 2000, March 30, 2000 and May 15, 2000. Ambient water temperatures were 0.8°C, 0°C and -0.8°C by January 11, February 29 and March 30, respectively (Fig. 1). Initially, three fish from each tank were sampled. There was no significant difference in all measured characteristics between these fish and thus the data were pooled into a single initial point. Due to lack of experimental animals, no ambient-temperature fish were sampled in May.
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Over the duration of the experiment, mean body mass (50.9±17.9g) was not significantly different between the initial sample (65.6±30.4g) and all following periods for both groups (P>0.05, Student's t-test).
Sample collection and preparation
Fish were killed by a blow to the head. Blood was drawn by caudal puncture
through a 25-gauge heparinized syringe and centrifuged at approximately
5000g for 3 min at 4°C. Plasma was frozen at -60°C for
later analysis. Liver and muscle samples were quickly removed and either used
immediately for enzyme assays (GPDH) or frozen with liquid nitrogen for later
analysis.
Biochemical assays
Plasma, muscle and liver were analyzed for glycerol, urea and TMAO.
Glycerol was determined directly from plasma, but muscle and liver samples
were first homogenized in 10 volumes of distilled water. For urea and TMAO
analysis, plasma was diluted 1:10 with 5% trichloracetic acid (TCA) (w/v) and
muscle and liver samples were homogenized in 9 volumes of 5 % TCA. Any further
dilutions were also done with 5 % TCA. Glycerol and urea contents were
determined using Sigma diagnostic kits 337-40A and 535, respectively. TMAO was
determined by the method of Wekell and Barnett
(1991), modified for use with
small sample volumes. As in Raymond
(1998
), disposable plastic
cuvettes were used. TMA was determined for a number of tissues and found to be
very low relative to TMAO; as in Raymond
(1994
), all TMAO data were not
corrected for the minor TMA content. Glycogen was measured by the method of
Walaas and Walaas (1950) as described by Driedzic et al.
(1998
).
For enzyme assays, liver samples were weighed and homogenized with 9
volumes of ice-cold buffer (20 mmol l-1 imidazole, 5.0 mmol
l-1 EDTA, 5.0 mmol l-1 EGTA, 10 mmol l-1
mercaptoethanol, 50 mmol l-1 Naf and 0.1 mmol l-1 PMSF,
pH 7.4) for determination of GPDH, AlaAT and AspAT. Samples were homogenized
using a Polytron tissue homogenizer for 10 s and centrifuged at approximately
10,000 g in an Eppendorf centrifuge for 5 min at 4°C to
remove cellular debris. Enzyme assay conditions were modified from Driedzic et
al. (1998). Assay conditions
were as follows. GPDH: 20 mmol l-1 imidazole, pH 7.2, 0.15 mmol
l-1 NADH; the reaction was initiated by 2.0 mmol l-1
dihydroxyacetone phosphate. AlaAT: 50 mmol l-1 imidazole, pH 7.4,
0.05 mmol l-1 pyridoxal phosphate, 0.2 mmol l-1 NADH, 2
U ml-1 lactate dehydrogenase (LDH), 200 mmol l-1
alanine; the reaction was initiated by adding 10 mmol l-1
-ketoglutarate (
-KG). AspAT: 50 mmol l-1 imidazole,
pH 7.4, 0.05 mmol l-1 pyridoxal phosphate, 0.2 mmol l-1
NADH, 1 U ml-1 malate dehydrogenase (MDH), 30 mmol l-1
aspartate; the reaction was initiated by adding 10 mmol l-1
-KG. All assays were done on a Gilford Model 2600 spectrophotometer
with a circulating water-jacketed cell holder maintained at 15°C. GPDH
activity was determined on fresh tissues; AlaAT and AspAT assays were done on
frozen samples.
Antifreeze activity measurement
Plasma thermal hysteresis (antifreeze activity) was measured as the
difference between the melting and freezing points using a Clifton nanolitre
osmometer (Clifton Technical Physics, Hartford, NY, USA) as described by Ewart
et al. (2000).
Gene expression analysis
Total RNA was isolated from smelt livers using the RNeasyTM Mini kit
(Qiagen) according to the manufacturer's instructions and stored at -80°C
in the presence of RNAasinTM RNAase inhibitor (Promega) until analysis.
Northern blotting was performed on 10µg samples of total RNA and
hybridization was carried out with -32P-labelled DNA probes.
For smelt GPDH, the probe consisted of a 1012 base pair (bp) fragment of the
GPDH cDNA; for smelt AFP, the probe was a full-length smelt AFP cDNA (Ewart et
al., 1992
,
2001
). Probes were labelled by
random priming using a commercially supplied kit (Roche) and hybridized with
the blot membrane. Following each probing, the membrane was washed and exposed
to film (Biomax MR, Kodak). The autoradiographic images were analysed by
densitometry as previously described (Ewart
et al., 2001
).
Statistical analysis
Means were compared with a one-way analysis of variance (ANOVA) for all
measurements and P<0.05 was taken as significant.
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Results |
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Glycerol
Mean initial plasma glycerol concentration was 80 mmol l-1
(Fig. 3A). Plasma glycerol
increased by approximately threefold, peaking at 234 mmol l-1 by
January 11 in the smelt held at ambient water temperatures, which was
significantly greater than the 68 mmol l-1 found in the fish
maintained in warm water. Glycerol levels in the ambient group remained
significantly higher than those in the warm temperature group for the duration
of the experiment; however, the concentration decreased from February 29 to
March 30 and was then not significantly different from the levels in the
initial sample. Plasma glycerol levels in the warm temperature group steadily
decreased to significantly less than the initial values on March 30 and to an
almost negligible amount by May 15.
|
Muscle glycerol levels followed a very similar trend to plasma levels (Fig. 3B), but were consistently lower. In the ambient temperature group, the mean initial glycerol level in muscle was 49 µmol g-1 and increased to a maximum of 116 µmol g-1 on February 29. Glycerol levels tended to decrease from February 29 to March 30. As with plasma, muscle glycerol levels in the warm temperature group began decreasing in January, were significantly less than the initial value by February and continued to decrease throughout the experiment. Similar to plasma, muscle glycerol levels in warm-and ambient-temperature groups were significantly different at all sampling periods.
Liver glycerol levels also followed a trend of increasing significantly in the ambient temperature group from the initial level of 56 µmol g-1 to a maximum of 153 µmol g-1 in January (Fig. 3C). Glycerol levels were maintained in this group throughout the experiment and were significantly greater than the initial values and those found in fish held at warm temperatures. Interestingly, in the warm-acclimated group, liver glycerol levels did not show any significant decrease from initial values throughout the experiment.
Trimethylamine oxide
The mean plasma TMAO concentration was initially 14 mmol 1-1 and
increased in both groups by January 11
(Fig. 4A). On February 29, when
the ambient temperature fell below 0°C, the ambient group reached its peak
TMAO concentration at 19 mmoll-1. Plasma TMAO had significantly
decreased to 9 mmoll-1 by February 29 in the warm-acclimated group
and, like glycerol, continued to decrease throughout the experiment. Unlike
glycerol, plasma TMAO concentrations in the ambient-temperature smelt group
remained elevated in March.
|
Muscle TMAO levels did not change significantly from the initial sample values with either treatment at any time (Fig. 4B). There was a significant difference between warm and ambient groups on February 29; however, neither value was significantly different compared to the initial values.
Liver TMAO levels mirrored the trend seen in plasma, where levels significantly increased from the initial 13 µmol g-1 to 17 µmol g-1 in the ambient group, whereas TMAO levels continually decreased from the initial concentration in the warm-acclimated fish (Fig. 4C).
Urea
Initially, concentrations of plasma urea followed a very similar pattern to
glycerol, with an approximately threefold increase from 3 mmoll-1
on December 15 to 9 mmoll-1 by February 29 in the
ambient-temperature fish (Fig.
5A). As with glycerol, plasma urea concentrations began decreasing
within the first month in warm-temperature fish and remained low throughout
the experiment. However, in cold acclimated smelt plasma urea remained
elevated, like the TMAO concentration.
|
Muscle urea followed a very similar trend to plasma urea, with levels in the ambient group increasing from 3 µmol g-1 initially to a maximum of 9 µmol g-1 on February 29 (Fig. 5B). The urea concentration in the warm-temperature group was significantly different from the initial value on March 30, but not in any other month.
Urea levels in liver followed the same trend as in plasma and muscle (Fig. 5C). Liver urea levels in the warm group on February 29 and March 30 were significantly less than the initial values.
Liver enzymes
Activities of AspAT, AlaAT and GPDH in liver did not significantly differ
at any time between experimental groups
(Fig. 6). All enzymes had
significantly decreased in activity from the initial values by May 15 in
warm-acclimated fish. Activity of AspAT significantly increased in both groups
by January 11 but was not different from the initial value on February 29 and
March 30. GPDH activity was highest initially and significantly decreased in
both groups by February 29. Complementing the GPDH activity data
(Fig. 6C), levels of GPDH mRNA
isolated from smelt liver showed no significant difference between warm and
ambient groups on January 11 (Fig.
7), although levels were variable within groups and there was a
trend towards lower levels in the warm-temperature fish.
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Liver glycogen
Liver glycogen levels increased dramatically in the warm acclimated fish
but remained low in the ambient group (Fig.
8). However, by March 30, levels of liver glycogen in the warm
group had already decreased from the peak on January 11 to a value not
significantly different from the initial value. Note that the warm group had
significantly higher liver glycogen than the ambient group at all sample
periods.
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Plasma antifreeze protein
Thermal hysteresis, a measure of AFP activity, increased significantly in
both groups by the first month (Fig.
9). However, hysteresis was not significantly different between
experimental groups. In the ambient fish, AFP activity continued to increase
throughout the experiment, while activity in the warm group leveled off after
the initial increase. AFP mRNA expression was significantly higher in January
in the ambient fish than in the warm fish
(Fig. 10), even though there
was no significant difference in AFP activity.
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Discussion |
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The smelt antifreeze response appears to have already been upregulated by the beginning of our experiment. We found that even at water temperatures around 5 °C, in mid-December, the glycerol accumulation response had already begun, with glycerol levels peaking by January or February 29 in fish tracking ambient temperatures. The TMAO accumulation appeared to be well beyond half-complete by this initial sampling period. Urea accumulation did not seem to begin until January, when ambient temperatures consistently dropped below 2 °C. Although ambient temperatures remained below 0 °C from February 29 to March 30, the plasma and muscle glycerol levels decreased over this time in the ambient-temperature fish.
The group of warm fish began decreasing all measured organic osmolytes
either initially (glycerol and urea) or by January (TMAO). This strongly
suggests that the mechanisms involved in accumulation of these solutes are
turning off or downregulating if temperatures remain warm. Thus, although the
accumulation of organic osmolytes may be triggered by decreasing temperature,
as shown by previous acclimation studies (Raymond,
1993,
1994
;
Raymond et al., 1996
),
maintaining these high levels appears to require continuing low
temperatures.
The initial sample had an osmolality of 349 mOsmol kg-1, which
is well within the range given by Holmes and Donaldson
(1969) for Salmoniformes, an
order that is closely related to the Osmeriformes, to which the smelt belong.
This normal value was obtained despite the fact that we found values of 80
mmol l-1, 14 mmol l-1 and 3 mmol l-1 for
glycerol, TMAO and urea, respectively. Therefore these organic osmolytes made
up approximately one third of the total solutes at a water temperature of
5°C. By February 29, plasma osmolality in both groups was well above
normal for marine teleosts, with glycerol, TMAO and urea accounting for
slightly less than a third of the total solutes in fish at 0°C.
There was appreciable plasma AFP activity, based on thermal hysteresis, by the time of our initial sample, again suggesting that the antifreeze response had already begun. The AFP activity, after the initial sample, was in the same range as that found in wild smelt caught in February (Duman and DeVries, 1974). Like the accumulation of the measured organic osmolytes, AFP activity tended to be higher in ambient fish than in warm-temperature fish, with the single exception of the January sample, which appeared to be a result of increased synthesis, as indicated by the increased AFP mRNA expression in the ambient group.
Total plasma organic osmolytes and AFP activity in the ambient group would result in a total freezing-point depression more than adequate for the fish to survive near-freezing marine waters in the ambient group by February 29. These levels were maintained in the ambient group until our final sampling date. It seems that while the colligative antifreeze effect had decreased, the non-colligative AFP activity increased. The data suggest a shift in antifreeze strategy, from an easily regulated but metabolically expensive enzyme activation/osmolyte system to a less expensive, gene expression AFP system as winter progresses.
Liver glycogen
Liver glycogen levels in ambient fish continually decreased and were
significantly less on March 30 than the initial levels in December. However,
glycogen reserves were not entirely depleted over the duration of the
experiment. In wild-caught osmerid fishes, Raymond et al.
(1996) found that levels of
storage compounds such as triacylglycerides and glycogen decreased as water
temperatures approached and fell below 0°C. These decreases were
attributed to the carbon requirement for the synthesis of glycerol. In the
present study, the decline in liver glycogen levels could contribute to an
increase in glycerol in plasma, muscle and liver but could not account for all
of the glycerol production, given a constant loss to the water
(Raymond, 1993
).
Liver enzymes
Tissue levels of glycerol were already elevated by our initial sample and
there was no correlation between glycerol levels and GPDH activity. More
specifically, GPDH activity was highest initially and decreased significantly
in both groups by February 29, even though glycerol levels were still very
high at that time in fish at ambient temperature. Moreover, although the GPDH
mRNA levels were approximately twice as high in January in the ambient fish
than in the warm fish, the difference was not significant. In contrast,
glycerol levels were approximately threefold higher in the ambient fish and
the difference was significant. It appears that if there is a regulatory
interval, it occurs prior to December, when water temperatures are above
5°C. It is possible that GPDH activity increased early in the fall,
facilitating rapid glycerol synthesis initially, followed by a subsequent
decrease to maintenance levels of activity for the balance of the winter. The
observed decrease in GPDH activity by February 29 is followed by relatively
constant levels and precedes the observed decrease in glycerol levels, as
would be expected if, by February 29, the activity of this enzyme decreases to
levels appropriate for maintenance of glycerol metabolism rather than
accumulation. These results contrast with those of previous work on smelt from
the Atlantic coast of Nova Scotia that did show a significant difference in
GPDH mRNA levels between warm- and ambient-acclimated fish in February
(Ewart et al., 2001). It would
be interesting to determine whether this is a population difference or due to
experimental conditions (length of time held at temperature, time of year,
etc.).
Amino acids are a major source of carbon for glycerol production in smelt
via transamination or deamination to pyruvate or oxaloacetate (OAA)
and the phosphorylation of OAA to PEP by PEPCK
(Raymond and Driedzic, 1997).
In accordance with data from radiolabelled precursors, smelt have very high
levels of amino acid-metabolizing enzymes in their liver compared to other
teleosts from the same environment
(Driedzic et al., 1998
). Though
we found much lower AspAT activity and higher AlaAT activity than the previous
study, our data are consistent with the trend shown by Driedzic et al.
(1998
) in that our initial
aminotransferase activities are still several-fold higher than in other
teleosts sampled in the earlier study. Both AspAT and AlaAT did not
significantly differ between the warm- and ambient-maintained fish. Though
AspAT did show a transient increase in January, which was significantly higher
than the initial values in the warm group, both groups returned to activities
roughly equivalent to their initial levels in February and March.
Interestingly, Raymond and Hassel (2000) found that there was a significant
increase in AspAT activity in wild-caught smelt sampled in February compared
to fish caught November. Levels of both aminotransferases significantly
decreased from initial levels in the final sample period and were very close
to what could be thought of as the `normal' teleost range. This suggests that
levels of these enzymes increase prior to winter, which would allow for
increased glycerol production from amino acids.
It is important to note that all respective enzyme activities did not significantly differ between groups. Thus, in warm-acclimated fish, the decrease in glycerol does not appear to be a result of decreased synthetic capability or downregulation at these loci. In fact, because of an expected increased rate of reaction with increased temperature, the warm-acclimated group presumably could convert precursors to glycerol at a greater rate than those held at ambient temperatures.
By early December, the smelt antifreeze mechanisms already appear to be
activated. This may be due to changes in photoperiod, changes in water
temperature or perhaps a combination of factors. There appears to be a
separation of the non-colligative and colligative mechanisms, since in fish
maintained above ambient temperatures levels of the organic osmolytes
glycerol, TMAO and urea all decreased, whereas AFP activity and mRNA
expression remained significantly elevated from initial levels. In addition,
the antifreeze mechanisms appear to activate asynchronously, the initial major
response being the accumulation of glycerol (and other organic osmolytes),
followed by the accumulation of other plasma solutes (presumably inorganic
ions) and, finally, the non-colligative AFP. The decreases in the levels of
GPDH, AlaAT and AspAT would suggest that they play roles in the winter
acclimation of smelt. Amino acids are a major carbon source for glycerol
(Raymond and Driedzic, 1997),
and the urea accumulation, in parallel with glycerol, may be a result of the
deamination of amino acids, as outlined in
Fig. 11.
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Acknowledgments |
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