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First published online August 9, 2007
Journal of Experimental Biology 210, 2866-2872 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003749
Relationship between food availability, glycerol and glycogen levels in low-temperature challenged rainbow smelt Osmerus mordax
Ocean Sciences Centre, Memorial University of Newfoundland, St John's, Newfoundland, A1C 5S7, Canada
* Author for correspondence (e-mail: wdriedzic{at}mun.ca)
Accepted 11 June 2007
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: rainbow smelt, Osmerus mordax, glycerol, low temperature, freeze resistance
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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) and
elevated glycerol levels (Raymond,
1992). These fish remain active and continue to feed in icy
seawater, but face the challenge of continual loss of glycerol across the
epithelia (Raymond, 1993),
resulting in the necessity for an ongoing synthesis of glycerol. Studies
involving injection of radioisotopes and heavy isotopes reveal that glycerol
is produced from glucose and amino acids, which would be obtained from the
diet and/or on-board reserves (Raymond,
1995; Raymond and Driedzic,
1997; Walter et al.,
2006). Liver is a primary site of glycerol production, as
indicated by enzyme complement, gene expression and synthesis by isolated
liver preparations (Driedzic and Ewart,
2004; Driedzic et al.,
2006; Liebscher et al.,
2006). Glycogen in liver is important as a metabolic source of
glycerol. Glycogen in liver decreased by 75 h post capture in fish held
without feeding at –1°C and muscle glycogen content was only a small
percentage of the level in liver (Raymond,
1995). Liver glycogen declined significantly more at
–1°C than +1°C following 14 or 20 days without food
(Raymond et al., 1996) but
even in fed fish, under laboratory conditions, liver glycogen decreased as
winter progressed from December to April
(Treberg et al., 2002). The
picture that emerges is that dietary glucose, amino acids and glycogen
reserves support glycerol synthesis for antifreeze protection. In the present
study, details of the interplay between temperature, food availability,
on-board fuel reserves and tissue glycerol content are addressed.
Glycerol content in plasma of rainbow smelt begins to increase when the
temperature decreases to about 4°C, and may reach levels approaching 500
mmol l–1 (Lewis et al.,
2004; Driedzic et al.,
2006). Glycerol levels in tissues other than plasma are not well
understood. In rainbow smelt sampled at one time point in winter at
–2°C, the glycerol content in a number of different tissues was
similar to that in plasma (Raymond,
1992). In a partial seasonal study, glycerol levels in muscle
followed a similar profile as in plasma; however, at the peak of plasma
glycerol levels the content of glycerol in liver appeared to be lower than in
plasma (Treberg et al., 2002).
A similar relationship was noted in fish that were force-chilled
(Driedzic et al., 2006).
Plasma glycerol content peaks in February and thereafter decreases, even
though water temperature is still below 0°C. Plasma glycerol content
reaches 5 mmol l–1 in May, similar to levels in the fall. As
plasma glycerol decreases, antifreeze protein becomes the dominant freeze
protection mechanism (Lewis et al.,
2004). Although decreases in plasma glycerol content while water
temperatures are still very low are documented, it is not clear if other
tissues follow this pattern and if the decrease in plasma glycerol is
triggered by small upswings in temperature towards 0°C or some other
factor such as photoperiod. Also, it is not known if one of the fates of
plasma glycerol is reincorporation into the glycogen pool.
The current experiment addresses a number of questions relevant to glycogen and glycerol management in freeze-resistant rainbow smelt. Foremost, we tested the hypothesis that continual feeding is a requirement to survive low temperatures. The initial glycogen content in a number of tissues is reported. Thereafter, glycogen levels in liver and glycerol level in numerous tissues were determined in fish maintained at low temperature in winter through to the spring period. The quantitative importance of exchange between these two metabolic pools and the relationship between plasma glycerol and tissue glycerol levels as the former changes was assessed.
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Materials and methods |
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Some fish were held at 8±0.5°C for the duration of the experiments in both the 2003/2004 and the 2005/2006 studies. Most of the experiments were conducted over the 2003/2004 season with fish subjected to a controlled decrease in water temperature. In these studies, rainbow smelt were transferred to a 500 l tank set at 8°C, 2 weeks prior to a reduction in water temperature. On 15 January 2004, temperature was decreased over a 19-day period to –1°C and subsequently maintained at approximately –1°C until the final sampling point on 11 May 2004. In this experiment, fish either continued to receive food or were starved from the initiation of the water temperature decrease. A population of fish was also allowed to track ambient water temperature during the 2003/2004 season. A group of fish from the October 2005 collection was fed and maintained at 8°C until sampled on 26 January 2006. Thereafter, this population was starved for 26 days and sampled again. All experiments involved both male and female fish.
At sampling times, fish were randomly selected, weighed, measured for length, and blood drawn via a caudal vessel. Fish were then killed with a blow to the head, tissues removed and stored at temperatures below –65°C for later glycerol and/or glycogen analysis. Blood was centrifuged at 9300 g immediately after sampling, plasma was collected and frozen in liquid nitrogen.
Biochemical assays and wet weight determinations
Glycerol level in the plasma was determined directly using a colorimetric
detection kit (F6428, Sigma-Aldrich, St Louis, MO, USA). Samples were read at
540 nm after a 15 min incubation at room temperature. Tissues were homogenized
in 9 vol. 10% perchloric acid, the homogenate centrifuged at 1500
g and the supernatant assayed for glycerol. Glycogen was
measured by the method of Walaas and Walaas (Walaas and Walaas, 1950) as
described by Driedzic et al. (Driedzic et
al., 1998).
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Data analysis
Unless otherwise indicated, values (means ± s.e.m.) were compared
with a one-way analysis of variance (ANOVA) for all measurements followed by
Duncan's post-hoc test. In some situations involving comparisons
between two values, a t-test was applied; P<0.05
considered as statistically significant.
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Results |
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The cumulative mortalities associated with the controlled decrease in temperature are shown in Fig. 1B. The figure is set with day 0 as the start of temperature decrease. Each group started with 130 fish, of which six healthy fish were removed on each of the dates shown in Fig. 1A. There is a clear difference in the spontaneous death rate between fed and starved rainbow smelt. Fish that were fed survived well. By day 30 at –1.2°C, there were nine recorded deaths with 11 more between day 30 and day 115 at temperatures of –0°C or lower. In contrast, in the group of starved fish, all individuals had succumbed by 30 days (14 February 2004), when the temperature had reached –1.2°C. Many of the fish died at temperatures well above the anticipated freezing point of body fluids. For instance, 50 fish died by day 20 (20 February 2004), when the water temperature was still above –0.8°C. A typical teleost should be able to survive these temperatures even in the absence of antifreeze mechanisms.
In the 2003/2004 experiment, there were no inexplicable mortalities in fish that were fed and maintained at either ambient or elevated temperatures over the time window of the above described experiment. In the 2005/2006 study, eight apparently healthy fish that had been maintained on heated water, were food deprived beginning 16 January 2006. One fish died over the next 26 days, when all had been sampled.
Glycogen levels
Tissue-specific glycogen levels were determined in rainbow smelt held at
8°C (Fig. 2A). Fish were
sampled on 15 January 2004. Glycogen content was highest in liver at
172±64 µmol glucosyl unit g–1, followed by heart at
50±12 µmol glucosyl unit g–1. Muscle and gut levels
were approximately 17 µmol glucosyl unit g–1, with lower
levels being noted in the other tissues.
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Fish that were fed and held under ambient temperature conditions had a liver glycogen level of 73.3±10.5 µmol glucosyl unit g–1 (sampled 6 February 2004; –1°C) (Fig. 2B; square symbol). This value tended to be lower than the levels of 172±64 and 153±23 µmol glucosyl unit g–1 noted in liver of fish held at 8°C and sampled on 15 January 2004 (Fig. 2) or 26 January 2006 (Table 1), respectively but significantly (ANOVA; P=0.001) higher than the 2.0±0.9 µmol glucosyl unit g–1 in fish subjected to the forced decreased in temperature (Fig. 2B; day 22). In the 2006 experiment, the liver glycogen content in rainbow smelt held at 8°C and fed was no different than in fish subjected to 26 days of starvation at this temperature (Table 1).
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Plasma glycerol levels
Plasma glycerol in fish that were force chilled and fed increased from 5
µmol ml–1 to levels in excess of 300 µmol
ml–1 at day 42 (27 February 2004; –1°C)
(Fig. 3A). Thereafter, plasma
glycerol concentration decreased to 37.4±9.7 µmol
ml–1 on the final sample date (11 May 2004;
–1.2°C).
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Rainbow smelt maintained at elevated temperatures had plasma glycerol levels of less than 10 µmol ml–1 (Fig. 3A). Food deprivation for 26 days at 8°C resulted in a significant decrease in plasma glycerol level (Table 1).
Tissue glycerol levels
The content of glycerol in various tissues in fish that were subjected to a
forced decrease in water temperature and fed is presented in
Fig. 4. These values follow the
general pattern of that in plasma with an increase to day 42 (17 February
2004; –1°C), followed by a continual decrease. This pattern occurred
in all tissues sampled. The level of tissue glycerol at the apex of the curves
was highest in kidney at 263±37 µmol g–1 and lowest
in gill at 125±13 µmol g–1. Glycerol levels in fish
that were deprived of food, under the same thermal conditions, followed the
same pattern up to day 22, at which time the study ended (data not shown).
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As plasma glycerol increased to the apex on day 42 the level of glycerol was either close to equilibrium between the two compartments or there was a gradient from the plasma space to the tissue space. Late in the experiment (i.e. days 104 and 116), as plasma glycerol levels decreased, the average ratio for all tissues was in excess of 1.0. Although the value for any specific tissue is not significantly different from 1.0, the overall picture shows them to be consistently positive, suggesting that further study of this may be required in order to determine whether glycerol content in tissue water might exceed that in plasma.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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An imposed temperature decrease leads to increases in glycerol levels in
plasma and other tissues, as has been observed in earlier studies
(Driedzic et al., 2006). Even
though the temperature was held below –1°C, glycerol in plasma and
other tissues began to decrease in samples taken after 27 February 2004 (i.e.
day 42), with tissue levels of glycerol generally following the same pattern
as plasma glycerol. This pattern of glycerol accumulation and subsequent
decrease is strikingly similar to that observed in two other experiments in
which rainbow smelt were maintained under natural thermal and light conditions
(Treberg et al., 2002;
Lewis et al., 2004). A decrease
in temperature alone is sufficient to activate the glycerol accumulation
mechanism and occurs at about 5°C, but the signal for reducing the level
of glycerol resides elsewhere. Potential candiates are photoperiod or an
inherent circannual rhythm, possibly in association with sufficient levels of
antifreeze protein.
The highest level of glycogen occurs in liver, followed by heart, with
lower levels in a number of other tissues. At 8°C, starvation alone for a
26-day period did not result in a significant decrease in liver glycogen,
although the mean value decreased by 22%. This is common for fish, for example
Atlantic cod held at 8°C showed about a 60% decrease in liver glycogen
following 39 days of food deprivation (Hall
et al., 2006). In rainbow smelt, however, a decrease in
temperature from 8° to –1.2°C over only 12 days resulted in
liver glycogen levels close to zero. The decrease in liver glycogen occurs
even in fed fish, but not to as great an extent. As such, either starvation or
low temperature results in glycogen mobilization, with the combination of both
having the most impact.
Liver glycogen reserves can only account for a small proportion of the
glycerol that accumulates. For instance, liver glycogen with an initial level
of 175 µmol glucosyl units g–1 could produce 350 µmol
glycerol g–1. Of this about 150 µmol g–1
would be retained in the liver with the remainder (i.e. 350–150=200
µmol g–1) available for export to other tissues. A 50 g
fish with a liver mass of 0.75 g could release 150 µmol of glycerol, which
if distributed evenly across all tissues, would result in a glycerol increment
of only 3 µmol g–1. With the possible exception of heart,
none of the other tissues have adequate glycogen reserves to account for the
increase in glycerol content. There is a clear need for dietary intake of
fuels or additional on-board reserves such as amino acids from protein to
support glycerol production. In this context it is important to note that
glycerol from triglycerides is not considered to be a contributor to glycerol
production (Raymond,
1995).
The decrease in glycerol content in all tissues observed late in the study is associated with an increase in glycogen content in liver, and this occurs while the temperature is below –1°C. It is likely that one fate of glycerol is incorporation into the glycogen pools. Between days 89 and 116 the content of glycogen in liver increased on average by about 125 µmol glucosyl units g–1, or the equivalent of 250 µmol glycerol g–1. This could account for the decrease in liver glycerol level of about 50 µmol g–1 and still provide room to accommodate some of the decrease of glycerol in the plasma and other tissues. Glycogen levels may cycle in other tissues as well. The important point is that glycogen synthesis can occur at low temperatures and could serve as a sink for glycerol, suggesting that glycerol need not be lost to the environment nor metabolized to CO2 during this period.
Glycerol level in tissues follows the same general profile as that in
plasma, with an increase as temperature is reduced followed by a decrease in
level. As plasma glycerol increased there was either a gradient from the
plasma space to the tissue space or the level of glycerol was close to
equilibrium between the two compartments. In liver, on day 15, there is no
significant difference between glycerol in tissue water and in plasma, but for
all other tissues tested glycerol is lower in the tissue water than in plasma.
This would be consistent with a movement of glycerol from liver into plasma
and subsequently down a concentration gradient from plasma into other tissues.
This contention is supported by the release of glycerol by isolated
preparations (Driedzic et al.,
1998; Driedzic and Ewart,
2004). However, on day 42, when plasma glycerol is at its highest
level, glycerol in liver tissue water is less than in plasma. This situation,
of lower than expected concentration of glycerol in liver at times of high
plasma glycerol, has been observed in two previous experiments
(Treberg et al., 2002;
Driedzic et al., 2006). There
are a number of possible explanations to account for this. As previously
suggested, based on findings with yeast, there may be active pumping of
glycerol out of liver into plasma (Driedzic
et al., 2006). An alternative explanation is that the process of
glycerol sequestering into glycogen may be starting with the intracellular
metabolic steps proceeding faster than diffusion of glycerol into the liver. A
third possibility is that other tissues, such as kidney (a known gluconeogenic
tissue), may become a site of glycerol release as the season progresses.
Finally, when plasma glycerol returns to minimal levels there appear to be
higher levels of glycerol in tissues than would be expected if glycerol was in
equilibrium between the intra- and extracellular space.
General conclusions
Rainbow smelt must feed to live at low temperatures; in food-deprived fish
death ensues at about 5°C. Glycerol accumulation is activated by low
temperature alone but the decrease in glycerol that occurs in late
winter/early spring is temperature-independent. The early phase of glycerol
production is associated with mobilization of liver glycogen; however,
glycogen from liver and in all other tissues can account for only a small
fraction of the total glycerol that accumulates. This is consistent with the
need to continue feeding at low temperature with a constant supply of ingested
carbohydrate and amino acids for glycerol synthesis. During late winter/early
spring, even while the temperature is still subzero, glycerol levels decrease
with a concomitant increase in glycogen.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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