Interplay among energy metabolism, organ mass and digestive enzyme activity in the mouse-opossum Thylamys elegans: the role of thermal acclimation
1 Centro de Estudios Avanzados en Ecología y Biodiversidad,
Departamento de Ecología, Pontificia Universidad Católica de
Chile, PO Box 6513677, Santiago, Chile
2 Departmento de Ciencias Ecológicas, Facultad de Ciencias,
Universidad de Chile, Casilla 653, Santiago, Chile
* Author for correspondence (e-mail: rnespolo{at}genes.bio.puc.cl)
Accepted 6 June 2002
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Summary |
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Key words: basal metabolic rate, maximum metabolic rate, marsupial, thermal acclimation, organ mass, Thylamys elegans, aminopeptidase-N
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Introduction |
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Small eutherian mammals and birds are classic examples of animals that use
the processes listed above (for a review, see
McNab, 2002). However, it is
not well known what happens in other endotherms such as marsupials. The
physiology of marsupials is similar to eutherians in several respects
(Hallam and Dawson, 1993
;
Chappell and Dawson, 1994
;
Gibson and Hume, 2000
;
Holloway and Geiser, 2001
),
but with respect to BMR and thermal acclimation, there are some differences.
The BMR in marsupials is comparatively low, which makes their factorial
aerobic scope (FAS=MMR/BMR) unusually high (for marsupials, FAS is near 8; for
birds and mammals, FAS=5) (see Hinds and
MacMillen, 1984
; Smith and
Dawson, 1985
; Hinds et al.,
1995
). In addition, the body temperature of marsupials is
considerably lower than in eutherians
(Hume, 1999
). Interestingly,
in some marsupial species, thermal acclimation does not induce MMR changes
(Smith and Dawson, 1985
;
Dawson and Olson, 1988
), and
this was attributed to the absence of brown adipose tissue (BAT)
(Dawson and Olson, 1988
;
Rose et al., 1999
). However,
cold acclimation can increase the speed and reduce energy expenditure of
re-warming after torpor (Opazo et al.,
1999
), which suggests that acclimation could induce some changes
in the thermogenic capacity of marsupials, even in the absence of BAT. Birds
do not have BAT either, but they do exhibit changes in MMR in response to
thermal acclimation (Swanson,
2001
), which demonstrates that BAT is not the only way of changing
thermogenic capacity during thermal acclimation.
To evaluate the underlying causes of differences in organismal performance,
the phenotype needs to be investigated simultaneously at several levels. This
integrative approach correlates organismal performance at one level with
changes at the lower level in the same individual subjected to a variety of
experimental treatments (e.g. Garland,
1984; Garland and Else,
1987
; Hammond and Janes,
1998
; Chappell et al.,
1999
; Konarzewski et al.,
2000
; Hammond et al.,
2001
). We used this approach to assess the effects of thermal
acclimation in a small didelphid marsupial, the mouse-opossum (Thylamys
elegans), which inhabits Mediterranean environments of central Chile.
This species shows conspicuous phenotypic flexibility in the activity of its
intestinal disacharidases, both on a seasonal basis
(Sabat and Bozinovic, 1994
)
and in response to diet acclimation (Sabat
et al., 1995
).
Our studies are useful not only for determining the underlying causes of
the observed differences in organismal performance in a wild endothermic
species, but also because little is known about the flexibility of MMR in
marsupials. For example, in an exhaustive review on marsupial nutrition and
energetics there is no mention of interspecific variability or intraspecific
plasticity in MMR (Hume,
1999), whereas ecophysiological variables such as field metabolic
rate, water turnover and BMR received a thorough analysis
(Hume, 1999
).
The aim of this work was to determine the effect of laboratory thermal
acclimation of T. elegans on (1) aerobic metabolism (BMR and MMR),
(2) mass of metabolically active organs and (3) digestive enzymatic activity.
Since T. elegans is mainly an insectivorous species
(Sabat et al., 1993), and the
intestinal aminopeptidase-N shows clear phenotypic flexibility in birds and
mammals (Sabat et al., 1998
,
1999
), we chose this enzyme to
determine if physiological flexibility exists at this level.
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Materials and methods |
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Physiological measurements (i.e. BMR and MMR, see below) were made before and after acclimation. At the end of each measurement we recorded body mass, using an electronic balance (sensitivity ±0.1 g), and rectal body temperature (Tb) using a Cole-Parmer copper-constant thermocouple.
Basal metabolic rate (BMR)
Prior to BMR measurements animals were fasted for 6 h. BMR was determined
according to the following protocol. Oxygen consumption
(VO2) was measured in a computerized (Datacan
V) open-flow respirometry system (Sable Systems, Henderson, Nevada, USA).
Measurements of animals were made in steel metabolic chambers of 1000 ml, at
ambient temperature (Ta) 30.0±0.5 °C, which is
within the thermoneutral zone for this species (M. Rosenmann, personal
communication). The metabolic chamber received dried air at a rate of 505 ml
min-1 from mass flow controllers (Sierra Instruments, Monterey,
California, USA), which was enough to ensure adequate mixing in the chamber.
Air passed through CO2-absorbent granules of Baralyme and Drierite
before and after passing through the chamber and was monitored every 5 s by an
Applied Electrochemistry O2-analyzer, model S-3A/I (Ametek,
Pittsburgh, Pennsylvania, USA). Oxygen consumption values were calculated
using equation 4a of Withers
(1977). Since T.
elegans is a nocturnal species, all metabolic trials were completed
during the rest phase of activity (between 08.00 and 16.00 h).
The complete VO2 trial lasted 2.5 h and BMR
was taken as the lowest sample registered during the last hour of
VO2 recording, which was comparable with
previous results obtained using the same species (e.g.
Sabat et al., 1995;
Opazo et al., 1999
), and
yielded the same values as the lowest obtained during a 5 min period over the
complete VO2 record. Previous BMR measurements
to determine the optimal time to reach minimum metabolism indicated that this
species reaches a steady state after 15-20 min, with no changes of
VO2 >15% in the following 3 h.
Maximum metabolic rate (MMR)
We measured MMR in a He-O2 atmosphere following the procedure of
Rosenmann and Morrison (1974),
using an open circuit respirometer, as described by Chappel and Bachman
(1995). In brief, a mixture of He (80%) and O2 (20%) was passed
through a volumetric flowmeter before entering the chamber (i.e. a positive
pressure system), and was maintained at a rate of 1000±3 ml
min-1. Such a flow rate prevented the partial oxygen pressure from
falling below 150 Torr, a value far above hypoxia
(Rosenmann and Morrison,
1975
). The mixture passed through CO2-absorbent
granules of Baralyme and Drierite before and after passing through the
chamber, which was tightly sealed with teflon and vaseline. The chamber
temperature (0.0±0.5 °C) was continuously recorded. To be sure that
individuals attained MMR, we (1) finished each record when the decline in
VO2 was evident (usually after 8-10 min of
measurement), and (2) measured Tb after each trial to
ensure animals reached hypothermia (Tb<30 °C in all
cases; normothermic Tb=34 °C, see Results).
Organ masses and enzyme assays
After the second set of metabolic measurements all animals were killed by
decapitation and dissected abdominally. We extracted first the colon and small
intestine, and then heart, lungs, liver and kidneys, and the remaining carcass
was weighed. Organs were washed with 0.9% NaCl solution and immediately
weighed (fresh mass). Stomach, caecum and total intestine were weighed with
and without content. Except for the small intestine, all organs were dried at
60 °C to constant mass (48 h), and weighed again (dry mass). No
significant trends (i.e. acclimation effect, dependence with
Mb, metabolism or enzyme activity) on total content and
water content of fresh organs were observed (data not shown).
We weighed the entire small intestine before cutting it into four sections of equal length (defined operationally as duodenum, jejunum A, jejunum B and ileum) for statistical comparisons. The sections were washed with 0.9% NaCl and stored in criovials with liquid nitrogen. To measure intestinal aminopeptidase-N activity, tissues were thawed and homogenized (30 s in an Ultra Turrax T25 homogenizer at maximum setting) in 20 volumes of 0.9% NaCl solution. We measured enzyme activity in the whole-tissue homogenate to avoid any underestimation of activity.
Aminopeptidase-N assays were done using L-alanine-p-nitroanilide
as a substrate. Briefly, 100 µl of homogenate diluted with 0.9% NaCl
solution were mixed with 1 ml of assay mix (2.04 mmol l-1
L-alanine-p-nitroanilide in 0.2 mol µl-1
NaH2PO4/Na2HPO4, pH 7). The
reaction was incubated at 37 °C and arrested after 10 min using 3 ml of
ice-cold acetic acid (2 mol l-1), and absorbance was measured at
384 nm. Standardized intestinal enzymatic activities were calculated on the
basis of absorbance. The protein content of intestinal homogenates was
determined using Coomassie-Plus Protein Assay (Pierce, Rockford, Il, USA).
Enzyme activity was standardized to rate per g wet tissue of intestine and per
mg protein (Sabat et al.,
1998), and the activities of all enzymes are presented as
standardized hydrolytic activity (UI g-1 wet tissue, and UI
mg-1 protein, where UI=µmol hydrolyzed min-1).
Statistical analyses
We performed all analyses both with and without sex as a factor. Since the
results were similar we report only the latter values here. For analyses of
VO2 (i.e. BMR and MMR) and factorial aerobic
scope (FAS=MMR/BMR), there were two factors: thermal acclimation and
laboratory effect (i.e. each individual was measured twice, before and after
thermal acclimation). Since VO2 is correlated
with Mb, we included it as a changing covariate in
repeated-measures analysis of covariance (ANCOVA) (StatSoft, 1996). The effect
of acclimation on organ masses was tested by ANCOVA with carcass mass as the
covariate, to avoid confounding effects due to the part-whole correlation (see
Christians, 1999). The
correlation among organ masses was assessed using residuals from the linear
regression between organ mass and Mb of pooled data (i.e.
cold- and warm-acclimated individuals). No transformations were necessary to
meet analysis of variance (ANOVA) assumptions (see below).
Aminopeptidase-N activity (i.e. UI mg-1 protein; no dependence
on Mb) was measured in the four sections of the small
intestine, which were considered as repeated-measures
(Meynard et al., 1999). The
effect of thermal acclimation and position on this variable was evaluated
using a repeated-measures ANOVA. This analysis was repeated for total enzyme
activity (UI per total intestine) using ANCOVA and Mb as
covariates.
To assess the association between organ masses and VO2, we performed stepwise multiple regressions for BMR and MMR separately, using residuals of organ masses as independent variables, and residuals of VO2 as the dependent variable.
All data were tested for homogeneity of variance using the Levene test and for normality using the KolmogorovSmirnov test. For ANCOVA analyses we tested for parallelism between both levels of the factor (i.e. acclimation temperature) using a test of interaction with covariate (StatSoft, 1996).
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Results |
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Organ masses
Analyses using fresh organ mass gave similar results as for dry mass, thus,
for organs other than small intestine (for which we have only fresh mass,
since it was used for the enzyme assay), we report only the results of the dry
mass analyses. Carcass dry mass showed no significant difference between cold-
and warm-acclimated individuals (cold=14.1±1.02 g; warm=16.1±1.5
g, ANOVA, F1,17=1.16, P=0.296). We observed
significant differences in the dry mass of some organs between acclimation
groups, however (Fig. 1).
Masses were higher in cold acclimated individuals for kidneys (ANCOVA,
F1,15=10.73, P=0.005), caecum (ANCOVA,
F1,11=7.80, P=0.017), and liver (ANCOVA,
F1,16=6.38, P=0.022).
|
We did not observe significant effects of thermal acclimation on the wet mass of small intestine (cold=26.8±2.8 g; warm=19.8±2.2 g; ANCOVA, F1,16=0.984, P=0.098, Table 2) or colon (ANCOVA, F1,13=3.29, P=0.093), although masses tended to be greater for cold-acclimated individuals than for warm-acclimated individuals. We did not observe significant differences in the dry mass of heart, lungs or stomach between acclimation groups (Fig. 1).
|
Correlations of residuals (from linear regressions with Mb) between the dry mass of various organs (Table 3) demonstrated the following significant positive associations: kidneys with liver, colon, caecum and stomach; liver with colon and stomach; heart with lungs; and colon with caecum and stomach (see Table 3 for statistics). However, after a Bonferroni correction, just colon and liver remained significantly correlated. Stepwise multiple regressions between residuals of BMR (pooled data from both acclimation groups) and residuals of organ mass (kidneys, liver, heart, lungs, colon, caecum, stomach, carcass, and fresh small intestine) revealed that 77% of the variance in BMR is explained by the mass of colon, small intestine and heart (multiple r2=0.77, P=0.011). A similar analysis for MMR did not give significant results (P=0.15).
|
We repeated the stepwise regression analysis for BMR using only residuals of digestive organ mass as independent variables (stomach, liver, kidneys, small intestine, colon and caecum) and residuals of BMR as the dependent variable. We found that 71 % of the variance in BMR was explained by colon, small intestine and kidneys (r2=0.71, P=0.028). The stepwise multiple regression between residuals of MMR and residuals of thermogenic organ mass (heart, lungs and carcass) were marginally significant (r2=0.42, P=0.06).
Digestive aminopeptidase-N
There were no significant differences in aminopeptidase-N activity between
acclimation groups when expressed as UI mg-1 protein (ANOVA,
F1,17=0.9, P=0.78,
Table 2), but there was a
significant effect of position along the small intestine on enzyme activity
(ANOVA, F3,51=3.78, P=0.016,
Table 2). The a
posteriori Scheffe test revealed that jejunum B had the largest enzyme
activity compared with the other three small intestine sections
(P=0.017, Table 2). Cold-acclimated individuals presented significantly higher total
aminopeptidase-N activity (using Mb as a covariate) than
warm-acclimated animals (ANCOVA, F1,16=16.88,
P<0.001).
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Discussion |
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It is a recognized fact that most small eutherian mammals change their
thermogenic capacity after cold acclimation (e.g.
Rosenmann et al., 1975;
Hammond et al., 2001
;
Merritt et al., 2001
, and
references therein). This phenotypic flexibility (sensu
Hammond et al., 2001
) is
observed at different levels of the physiological phenotype. For example,
during intense thermogenesis and/or exercise, total thermogenic capacity (in
eutherians) is driven by non-shivering thermogenesis (NST) and shivering
thermogenesis (ST) (Jansky,
1973
), and each of these components present changes after thermal
acclimation (e.g. Jansky,
1973
; Rosenmann et al.,
1975
; Bockler and Heldmaier,
1983
). Nevertheless, NST is considered the most plastic component
since it depends on the metabolism of BAT, which is specialized to hypertrophy
during cold acclimation (Kronfeld-Schor et
al., 2000
). Marsupials do not demonstrate NST, at least not in the
same way as small eutherians (i.e. associated with BAT)
(Rose et al., 1999
), which may
explain why thermal acclimation did not have an effect on MMR in T.
elegans. It could also be argued that the thermal gradient we selected
for this study (10-30 °C) was not steep enough to induce a change in MMR.
However, previous studies have shown that this same thermal gradient is enough
to elicit large and significant differences in MMR in several species of
rodents (Nespolo et al.,
2001
). Moreover, our results are in agreement with previous
reports on marsupials (Smith and Dawson,
1985
; Dawson and Olson,
1988
).
Recently, Holloway and Geiser
(2001) documented significant
seasonal differences in MMR for sugar gliders Petraurus breviceps.
However, MMR and BMR values in their study are reported (and statistically
compared) in terms of mass-specific units, in spite of the fact that these
authors reported Mb changes between seasons. As many
authors have claimed, statistical comparisons made on mass-specific metabolism
are unreliable (Packard and Boardman,
1999
; Christians,
1999
; Hayes,
2001
), and there are several robust statistical procedures that
control for Mb (e.g. residual analysis, ANCOVA, multiple
regression) (see Christians,
1999
). Moreover, since VO2 is
measured on whole animals, in the absence of other options (e.g. when sample
size is small), units of VO2 per animal should
be used (Hayes, 2001
). For
this reason, unfortunately, it is not possible to decide whether there were
significant differences in MMR between seasons for the data presented by
Holloway and Geiser
(2001
).
Compared with warm-acclimated mouse-opossums, cold-acclimated individuals
presented higher BMR. This observation is not new, as many authors have
demonstrated for both eutherians and metatherians
(Dawson and Olson, 1988;
Rose et al., 1999
). An
increase in BMR is interpreted as an increment in maintenance costs due to the
enlargement of digestive organs (Konarzewsky and Diamond, 1995). This
functional dependence is supported by our results because 71 % of the variance
in BMR was significantly explained by the intestines and kidneys. Indeed,
cold-acclimated individuals had significantly larger caecum, kidneys and liver
than warm-acclimated animals. It is known that the kidneys and liver of
vertebrates respond to cold acclimation with an increase in mass
(Hammond and Wunder, 1995
;
Hammond et al., 2001
).
Similarly, the comparatively large caecum suggests that T. elegans
may present some hindgut fermenter activity
(Hume, 1999
), and that under
cold conditions (i.e. high energy demands) this activity is probably
increased. This is not surprising since most mouse-opossums are described as
hindgut fermenters (Hume,
1999
).
An acclimation response of intestinal enzymes has been reported elsewhere
in several vertebrate species, including marsupials (see
Martínez del Río et al.,
1995; Sabat et al.,
1995
,
1998
). These changes,
according to the adaptive modulation hypothesis (see
Hume and Stevens, 1996
), are
responses to the intake of specific dietary substrates. In addition,
conditions favoring hyperphagia, such as cold exposure, lead to increases in
intestinal mass (Toloza et al.,
1991
; Karasov,
1996
). These responses by the small intestine lead to an increase
in total hydrolytic capacity, matching biochemical features with changes in
the intake of dietary substrates. The null response of aminopeptidase-N
specific activity (per mg protein), and the marked differences observed in
total activity of aminopeptidase-N (after controlling for
Mb), suggest that tissue growth (a quantitative response),
rather than differential expression (a qualitative response), of enzymes was
the acclimation response at the digestive level. Phenotypic plasticity in
enzyme activity has been reported for T. elegans in particular
(Sabat et al., 1995
) as well
as for mammals in general (Buddington,
1994
; Sabat et al.,
1999
, and references therein). Similarly, the effect of
acclimation on the size of the small intestine in vertebrates has received a
great amount of interest, in particular diet acclimation
(Piersma and Lindstrom, 1997
)
and thermal acclimation (Hammond and
Wunder, 1995
; Hammond et al.,
2001
). However, the effects of thermal acclimation on digestive
enzyme activities are poorly known (e.g.
Harada and Kano, 1976
;
Das and Das, 1982
), and to the
best of our knowledge no studies have been published dealing with this issue
in marsupials.
So, how does T. elegans cope with cold periods without increasing
thermogenic capacity? A partial answer to this question may be the use of
torpor (Silva-Durán and Bozinovic,
1999); torpor could be used as an evasive strategy to avoid low
Ta (e.g. Geiser,
1994
). Still, interestingly, there are several species of
eutherian mammals that hibernate or use torpor as an energy saving strategy,
but at the same time are able to express a high phenotypic flexibility in
thermogenic capacity (Heldmaier et al.,
1982
; Merritt et al.,
2001
). Moreover, the fact that the thermogenic capacity of T.
elegans did not increase after cold acclimation does not imply that they
cannot survive this Ta in euthermia, rather that T.
elegans cannot increase its maximum capacity for heat production in
excess of requirements.
A very different response was observed at the digestive level since acclimation induced changes in the mass of caecum, kidneys and small intestine. As expected, these changes were accompanied by significant differences in BMR. These changes suggest that cold-acclimated individuals can adjust for high-energy demands. However, the adjustment is mostly in tissue mass rather than specific enzyme activity, at least for aminopeptidase-N activity.
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Acknowledgments |
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