Study done on hibernation in tegus, first year.

[VARNYARD]

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Seasonal metabolic depression, substrate utilisation and changes in scaling
patterns during the first year cycle of tegu lizards (Tupinambis merianae)
Silvia Cristina R. de Souza1,*, JosÃ?Æ?Ã?© Eduardo de Carvalho1, Augusto S. Abe2,
JosÃ?Æ?Ã?© Eduardo P. W. Bicudo1 and Marilene S. C. Bianconcini1
1Departamento de Fisiologia, Instituto de BiociÃ?Æ?Ã?ªncias, Universidade de SÃ?Æ?Ã?£o Paulo, 05508-900 SÃ?Æ?Ã?£o Paulo, SP, Brazil
and 2Departamento de Zoologia, Instituto de BiociÃ?Æ?Ã?ªncias, Universidade Estadual Paulista, Caixa Postal 199,
13506-900 Rio Claro, SP, Brazil
*Author for correspondence (e-mail: <!-- e --><a href="mailto:[email protected]">[email protected]</a><!-- e -->)
Accepted 16 October 2003

The Journal of Experimental Biology 207, 307-318 307
Published by The Company of Biologists 2004
doi:10.1242/jeb.00756
The tegus increase in body mass after hatching until
early autumn, when the energy intake becomes gradually
reduced. Resting rates of oxygen consumption in winter
drop to 20% of the values in the active season
(V
.
OÃ?â??Ã?·=0.0636Ã?â??Ã?·mlÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1Ã?â??Ã?·hÃ?¢ââ??‰â?¬Å?1) and are nearly temperature
insensitive over the range of 17Ã?¢ââ??‰â?¬Å?25Ã?â??Ã?°C (Q10=1.55). During
dormancy, plasma glucose levels are 60% lower than those
in active animals, while total protein, total lipids and b-
hydroxybutyrate are elevated by 24%, 43% and 113%,
respectively. In addition, a significant depletion of liver
carbohydrate (50%) and of fat deposited in the visceral fat
bodies (24%) and in the tail (25%) and a slight loss of
skeletal muscle protein (14%) were measured halfway
through the inactive period. Otherwise, glycogen content
is increased 4-fold in the brain and 2.3-fold in the heart of
dormant lizards, declining by the onset of arousal. During
early arousal, the young tegus are still anorexic, although
V .
OÃ?â??Ã?· is significantly greater than winter rates. The fat
deposits analysed are further reduced (62% and 45%,
respectively) and there is a large decrease in tail muscle
protein (50%) together with a significant increase in
glycogen (2Ã?¢ââ??‰â?¬Å?3-fold) and an increase in plasma glucose
(40%), which suggests a role for gluconeogenesis as a
supplementary energy source in arousing animals. No
change is detectable in citrate synthase activity, but b-
hydroxyacyl CoA dehydrogenase activities are strongly
affected by season, reaching a 3-fold and 5-fold increase
in the liver tissue of winter and arousing animals,
respectively, and becoming reduced by half in skeletal
muscle and heart of winter animals compared with late
fall or spring active individuals. From hatching to late
autumn, the increase of the fat body mass relatively to
body mass is disproportionate (b=1.44), and the mass
exponent changes significantly to close to 1.0 during the
fasting period. The concomitant shift in the V
.
OÃ?â??Ã?· mass
exponent in early autumn (b=0.75) to values significantly
greater than 1.0 in late autumn and during winter
dormancy indicates an allometric effect on the degree of
metabolic depression related to the size of the fat stores
and suggests greater energy conservation in the smaller
young.
Key words: dormancy, fasting, oxygen consumption, metabolic
depression, scaling, lipid, glycogen, HOAD, CS, lizard, tegu,
Tupinambis merianae.
Summary
Introduction
Seasonal metabolic depression, substrate utilisation and changes in scaling
patterns during the first year cycle of tegu lizards (Tupinambis merianae)
Silvia Cristina R. de Souza1,*, JosÃ?Æ?Ã?© Eduardo de Carvalho1, Augusto S. Abe2,
JosÃ?Æ?Ã?© Eduardo P. W. Bicudo1 and Marilene S. C. Bianconcini1
1Departamento de Fisiologia, Instituto de BiociÃ?Æ?Ã?ªncias, Universidade de SÃ?Æ?Ã?£o Paulo, 05508-900 SÃ?Æ?Ã?£o Paulo, SP, Brazil
and 2Departamento de Zoologia, Instituto de BiociÃ?Æ?Ã?ªncias, Universidade Estadual Paulista, Caixa Postal 199,
13506-900 Rio Claro, SP, Brazil
*Author for correspondence (e-mail: <!-- e --><a href="mailto:[email protected]">[email protected]</a><!-- e -->)
Accepted 16 October 2003 Over the past two decades, the mechanisms of torpor have
been investigated most amply in small mammals that hibernate
in cold environments and involve modulation of the control of
appetite and fattening, thermoregulation, blood clotting and
other functions associated with seasonal fluctuations in
temperature and food supply in normoxic environments (Boyer
and Barnes, 1999; Guppy and Withers, 1999). The ability to
transform a euthermic pattern into dormancy and torpor
apparently constitutes a widespread feature in the endotherms,
as suggested by growing evidence on mammals and birds from
tropical and subtropical climates, which exhibit a state of
torpor during the cool-dry season or during the daytime when
food intake is halted (Bicudo, 1996; Ortmann et al., 1996;
Schmid and Speakman, 2000).
In addition to these classical model systems, many lower
vertebrates are known to enter a state of reduced metabolism
over winter or under the combined influence of air temperature
and relative humidity (Pinder et al., 1992; Abe, 1995; Guppy
and Withers, 1999; Storey, 2002). The metabolic correlates of
seasonality in these animals probably share some fundamental
attributes with mammalian species that undergo a fasting
period during the annual cycle. For example, a substantial
proportion of the energy supply during the hypometabolic state
may derive from lipid oxidation, and the carbohydrate stores 308
apparently constitute a limited potential for sustaining energy
expenditure at the whole body level, as opposed to the
adjustments seen under anoxic conditions (Storey, 1996).
Remarkably, unlike the situation in mammal and bird species,
a large reduction in aerobic metabolism can be achieved in
the lower vertebrates without the predominant effect on the
energy requirements associated with endothermy and a
high thermoregulatory capacity. The regulatory mechanisms
involved in metabolic depression appear to be coupled to an
endogenous, circannual rhythm superimposed on the circadian
pattern of thermal behaviour and locomotor activity, as shown
in terrestrial reptiles (Hismiller and Heldmaier, 1988; FoÃ?Æ?Ã?  et
al., 1994). These aspects have been studied in detail in only a
few species, and considerable research remains to be done in
order to identify the general principles of regulation acting on
these events.
Another largely unstudied aspect of seasonal fasting and
hypometabolism concerns how these profound changes
develop in newly born animals of small body mass and limited
capacity to store substrates. Since much evidence suggests that
metabolic depression is endogenous in nature, it is important
to investigate how early this phenomenon manifests in the
life cycle. Furthermore, the ontogenetic adjustment of the
relationship between energy use and storage capacity as an
essential part of the make up of seasonal dormancy also may
be predicted. We have begun to address these questions
by examining the occurrence of metabolic depression and
associated changes in energy metabolism during the first year
cycle of tegu lizards (Tupinambis merianae). The tegu is
widely distributed throughout South America (Avila-Pires,
1995) and occurs in large numbers in southeastern Brazil.
Winter at this latitude is synchronized with the dry season,
when insects and other food sources become scarce. An innate
rhythm is apparent soon after hatching and, in the adult stage,
the reproductive activities are concentrated in the spring
months, with foraging and energy intake becoming gradually
reduced during summer until the lizards enter a state of
continuous inactivity, spending 4Ã?¢ââ??‰â?¬Å?5Ã?â??Ã?·months underground at
temperatures around 17Ã?â??Ã?°C in the autumn and winter months
(Abe, 1995). Abe (1995) verified a marked reduction in oxygen
consumption during winter to 20Ã?¢ââ??‰â?¬Å?30% of the resting values in
lizards with a mean mass of 1270Ã?â??Ã?·kg, consumption rates
becoming nearly temperature insensitive over the range of
17Ã?¢ââ??‰â?¬Å?25Ã?â??Ã?°C. Anorexia is also a marked feature of the annual cycle
in tegu lizards and develops from late summer through early
autumn irrespective of the high temperatures and wide food
availability at this time of the year (H. R. Lopes and A. Abe,
unpublished observation).
The present study is concerned with the questions of how
fasting and the magnitude of metabolic depression in young
tegus compare with these events in their adult counterparts and
whether such changes are influenced by body mass in the
growing lizards. We also focus on the absence of reproduction
at this early stage of development, emphasising correlative
shifts in the levels of metabolites, substrate stores and enzymes
over the first year cycle. The results support the notion of an
endogenous rhythm acting via appetite control and energy
metabolism in the tegu and suggest a shift in the balance
between energy expenditure and body mass during the
hypometabolic state, related to fat storage capacity and
increased energy conservation in the smaller progeny.
Materials and methods
Animal supply and maintenance
Young tegu lizards (Tupinambis merianae DumÃ?Æ?Ã?©ril and
Bribon) were obtained from a population reared outdoors in
large pens in Rio Claro, southeastern Brazil. About two months
after eclosion in the summer, the animals were moved to the
laboratory and used in the experiments during their first annual
cycle. The lizards were kept indoors in 120Ã?â??Ã?·litre cages
equipped with incandescent lights set on an 8Ã?â??Ã?·h:16Ã?â??Ã?·h L
photo- and thermal-period, in addition to the sunlight diffusing
from outside. The lizards could freely alternate between
warming and cooling their bodies by climbing onto a small
platform or hiding in a wooden shelter among the sheets of
paper covering the box floor. The animals were separated into
small groups according to size to minimise fighting and
competition for food and were fed every two days on raw meat,
egg and fruits, enriched with minerals, having continuous
access to drinking water.
A noticeable change in daily activity was seen in the early
autumn, when the time spent on thermoregulation became
progressively shorter and food intake gradually reduced until
the lizards became continuously inactive inside the shelter. The
animals were kept in the shade throughout the winter months,
being routinely inspected and weighed every 15Ã?â??Ã?·days with
minimal disturbance. By early spring, they expelled a dried
pellet of uric acid and began moving outside the shelter,
promptly reacting when mechanically stimulated. These
changes were taken as an indication of arousal, and the animals
were then returned to the previous photo- and thermal-period,
with free access to drinking water. During the first days of
arousal they were still anorexic, and a gradual increase in the
time spent on thermoregulation and food intake took place over
the following weeks.
Minimum and maximum air temperatures were recorded
daily using a thermometer placed in the shelter area. The mean
ranges for each seasonal period were: early autumn, 21Ã?¢ââ??‰â?¬Å?26Ã?â??Ã?°C;
late autumn, 18Ã?¢ââ??‰â?¬Å?23Ã?â??Ã?°C; winter, 15Ã?¢ââ??‰â?¬Å?20Ã?â??Ã?°C; early spring,
20Ã?¢ââ??‰â?¬Å?26Ã?â??Ã?°C; late spring, 23Ã?¢ââ??‰â?¬Å?30Ã?â??Ã?°C.
Oxygen consumption measurements
Resting oxygen consumption rates (V
.
OÃ?â??Ã?·) were measured
during the annual cycle on groups of tegus as follows: Ã?¢ââ??¬Ã?Å?autumn
activityÃ?¢ââ??‰â??¢, subdivided into early and late autumn to include
lizards exhibiting behaviour characteristic of the onset of
dormancy; Ã?¢ââ??¬Ã?Å?winter dormancyÃ?¢ââ??‰â??¢, including fasting and totally
inactive lizards over 50Ã?¢ââ??‰â?¬Å?60 winter days; Ã?¢ââ??¬Ã?Å?arousalÃ?¢ââ??‰â??¢ for lizards
emerging from dormancy (90Ã?¢ââ??‰â?¬Å?100Ã?â??Ã?·days from the first winter
day), subdivided into rehydrated, unfed animals (48Ã?¢ââ??‰â?¬Å?96Ã?â??Ã?·h after
arousal) and fed animals (~1Ã?â??Ã?·week after arousal); Ã?¢ââ??¬Ã?Å?spring 308
apparently constitute a limited potential for sustaining energy
expenditure at the whole body level, as opposed to the
adjustments seen under anoxic conditions (Storey, 1996).
Remarkably, unlike the situation in mammal and bird species,
a large reduction in aerobic metabolism can be achieved in
the lower vertebrates without the predominant effect on the
energy requirements associated with endothermy and a
high thermoregulatory capacity. The regulatory mechanisms
involved in metabolic depression appear to be coupled to an
endogenous, circannual rhythm superimposed on the circadian
pattern of thermal behaviour and locomotor activity, as shown
in terrestrial reptiles (Hismiller and Heldmaier, 1988; FoÃ?Æ?Ã?  et
al., 1994). These aspects have been studied in detail in only a
few species, and considerable research remains to be done in
order to identify the general principles of regulation acting on
these events.
Another largely unstudied aspect of seasonal fasting and
hypometabolism concerns how these profound changes
develop in newly born animals of small body mass and limited
capacity to store substrates. Since much evidence suggests that
metabolic depression is endogenous in nature, it is important
to investigate how early this phenomenon manifests in the
life cycle. Furthermore, the ontogenetic adjustment of the
relationship between energy use and storage capacity as an
essential part of the make up of seasonal dormancy also may
be predicted. We have begun to address these questions
by examining the occurrence of metabolic depression and
associated changes in energy metabolism during the first year
cycle of tegu lizards (Tupinambis merianae). The tegu is
widely distributed throughout South America (Avila-Pires,
1995) and occurs in large numbers in southeastern Brazil.
Winter at this latitude is synchronized with the dry season,
when insects and other food sources become scarce. An innate
rhythm is apparent soon after hatching and, in the adult stage,
the reproductive activities are concentrated in the spring
months, with foraging and energy intake becoming gradually
reduced during summer until the lizards enter a state of
continuous inactivity, spending 4Ã?¢ââ??‰â?¬Å?5Ã?â??Ã?·months underground at
temperatures around 17Ã?â??Ã?°C in the autumn and winter months
(Abe, 1995). Abe (1995) verified a marked reduction in oxygen
consumption during winter to 20Ã?¢ââ??‰â?¬Å?30% of the resting values in
lizards with a mean mass of 1270Ã?â??Ã?·kg, consumption rates
becoming nearly temperature insensitive over the range of
17Ã?¢ââ??‰â?¬Å?25Ã?â??Ã?°C. Anorexia is also a marked feature of the annual cycle
in tegu lizards and develops from late summer through early
autumn irrespective of the high temperatures and wide food
availability at this time of the year (H. R. Lopes and A. Abe,
unpublished observation).
The present study is concerned with the questions of how
fasting and the magnitude of metabolic depression in young
tegus compare with these events in their adult counterparts and
whether such changes are influenced by body mass in the
growing lizards. We also focus on the absence of reproduction
at this early stage of development, emphasising correlative
shifts in the levels of metabolites, substrate stores and enzymes
over the first year cycle. The results support the notion of an
endogenous rhythm acting via appetite control and energy
metabolism in the tegu and suggest a shift in the balance
between energy expenditure and body mass during the
hypometabolic state, related to fat storage capacity and
increased energy conservation in the smaller progeny.
Materials and methods
Animal supply and maintenance
Young tegu lizards (Tupinambis merianae DumÃ?Æ?Ã?©ril and
Bribon) were obtained from a population reared outdoors in
large pens in Rio Claro, southeastern Brazil. About two months
after eclosion in the summer, the animals were moved to the
laboratory and used in the experiments during their first annual
cycle. The lizards were kept indoors in 120Ã?â??Ã?·litre cages
equipped with incandescent lights set on an 8Ã?â??Ã?·h:16Ã?â??Ã?·h L
photo- and thermal-period, in addition to the sunlight diffusing
from outside. The lizards could freely alternate between
warming and cooling their bodies by climbing onto a small
platform or hiding in a wooden shelter among the sheets of
paper covering the box floor. The animals were separated into
small groups according to size to minimise fighting and
competition for food and were fed every two days on raw meat,
egg and fruits, enriched with minerals, having continuous
access to drinking water.
A noticeable change in daily activity was seen in the early
autumn, when the time spent on thermoregulation became
progressively shorter and food intake gradually reduced until
the lizards became continuously inactive inside the shelter. The
animals were kept in the shade throughout the winter months,
being routinely inspected and weighed every 15Ã?â??Ã?·days with
minimal disturbance. By early spring, they expelled a dried
pellet of uric acid and began moving outside the shelter,
promptly reacting when mechanically stimulated. These
changes were taken as an indication of arousal, and the animals
were then returned to the previous photo- and thermal-period,
with free access to drinking water. During the first days of
arousal they were still anorexic, and a gradual increase in the
time spent on thermoregulation and food intake took place over
the following weeks.
Minimum and maximum air temperatures were recorded
daily using a thermometer placed in the shelter area. The mean
ranges for each seasonal period were: early autumn, 21Ã?¢ââ??‰â?¬Å?26Ã?â??Ã?°C;
late autumn, 18Ã?¢ââ??‰â?¬Å?23Ã?â??Ã?°C; winter, 15Ã?¢ââ??‰â?¬Å?20Ã?â??Ã?°C; early spring,
20Ã?¢ââ??‰â?¬Å?26Ã?â??Ã?°C; late spring, 23Ã?¢ââ??‰â?¬Å?30Ã?â??Ã?°C.
Oxygen consumption measurements
Resting oxygen consumption rates (V
.
OÃ?â??Ã?·) were measured
during the annual cycle on groups of tegus as follows: Ã?¢ââ??¬Ã?Å?autumn
activityÃ?¢ââ??‰â??¢, subdivided into early and late autumn to include
lizards exhibiting behaviour characteristic of the onset of
dormancy; Ã?¢ââ??¬Ã?Å?winter dormancyÃ?¢ââ??‰â??¢, including fasting and totally
inactive lizards over 50Ã?¢ââ??‰â?¬Å?60 winter days; Ã?¢ââ??¬Ã?Å?arousalÃ?¢ââ??‰â??¢ for lizards
emerging from dormancy (90Ã?¢ââ??‰â?¬Å?100Ã?â??Ã?·days from the first winter
day), subdivided into rehydrated, unfed animals (48Ã?¢ââ??‰â?¬Å?96Ã?â??Ã?·h after
arousal) and fed animals (~1Ã?â??Ã?·week after arousal); Ã?¢ââ??¬Ã?Å?spring308
apparently constitute a limited potential for sustaining energy
expenditure at the whole body level, as opposed to the
adjustments seen under anoxic conditions (Storey, 1996).
Remarkably, unlike the situation in mammal and bird species,
a large reduction in aerobic metabolism can be achieved in
the lower vertebrates without the predominant effect on the
energy requirements associated with endothermy and a
high thermoregulatory capacity. The regulatory mechanisms
involved in metabolic depression appear to be coupled to an
endogenous, circannual rhythm superimposed on the circadian
pattern of thermal behaviour and locomotor activity, as shown
in terrestrial reptiles (Hismiller and Heldmaier, 1988; FoÃ?Æ?Ã?  et
al., 1994). These aspects have been studied in detail in only a
few species, and considerable research remains to be done in
order to identify the general principles of regulation acting on
these events.
Another largely unstudied aspect of seasonal fasting and
hypometabolism concerns how these profound changes
develop in newly born animals of small body mass and limited
capacity to store substrates. Since much evidence suggests that
metabolic depression is endogenous in nature, it is important
to investigate how early this phenomenon manifests in the
life cycle. Furthermore, the ontogenetic adjustment of the
relationship between energy use and storage capacity as an
essential part of the make up of seasonal dormancy also may
be predicted. We have begun to address these questions
by examining the occurrence of metabolic depression and
associated changes in energy metabolism during the first year
cycle of tegu lizards (Tupinambis merianae). The tegu is
widely distributed throughout South America (Avila-Pires,
1995) and occurs in large numbers in southeastern Brazil.
Winter at this latitude is synchronized with the dry season,
when insects and other food sources become scarce. An innate
rhythm is apparent soon after hatching and, in the adult stage,
the reproductive activities are concentrated in the spring
months, with foraging and energy intake becoming gradually
reduced during summer until the lizards enter a state of
continuous inactivity, spending 4Ã?¢ââ??‰â?¬Å?5Ã?â??Ã?·months underground at
temperatures around 17Ã?â??Ã?°C in the autumn and winter months
(Abe, 1995). Abe (1995) verified a marked reduction in oxygen
consumption during winter to 20Ã?¢ââ??‰â?¬Å?30% of the resting values in
lizards with a mean mass of 1270Ã?â??Ã?·kg, consumption rates
becoming nearly temperature insensitive over the range of
17Ã?¢ââ??‰â?¬Å?25Ã?â??Ã?°C. Anorexia is also a marked feature of the annual cycle
in tegu lizards and develops from late summer through early
autumn irrespective of the high temperatures and wide food
availability at this time of the year (H. R. Lopes and A. Abe,
unpublished observation).
The present study is concerned with the questions of how
fasting and the magnitude of metabolic depression in young
tegus compare with these events in their adult counterparts and
whether such changes are influenced by body mass in the
growing lizards. We also focus on the absence of reproduction
at this early stage of development, emphasising correlative
shifts in the levels of metabolites, substrate stores and enzymes
over the first year cycle. The results support the notion of an
endogenous rhythm acting via appetite control and energy
metabolism in the tegu and suggest a shift in the balance
between energy expenditure and body mass during the
hypometabolic state, related to fat storage capacity and
increased energy conservation in the smaller progeny.
Materials and methods
Animal supply and maintenance
Young tegu lizards (Tupinambis merianae DumÃ?Æ?Ã?©ril and
Bribon) were obtained from a population reared outdoors in
large pens in Rio Claro, southeastern Brazil. About two months
after eclosion in the summer, the animals were moved to the
laboratory and used in the experiments during their first annual
cycle. The lizards were kept indoors in 120Ã?â??Ã?·litre cages
equipped with incandescent lights set on an 8Ã?â??Ã?·h:16Ã?â??Ã?·h L
photo- and thermal-period, in addition to the sunlight diffusing
from outside. The lizards could freely alternate between
warming and cooling their bodies by climbing onto a small
platform or hiding in a wooden shelter among the sheets of
paper covering the box floor. The animals were separated into
small groups according to size to minimise fighting and
competition for food and were fed every two days on raw meat,
egg and fruits, enriched with minerals, having continuous
access to drinking water.
A noticeable change in daily activity was seen in the early
autumn, when the time spent on thermoregulation became
progressively shorter and food intake gradually reduced until
the lizards became continuously inactive inside the shelter. The
animals were kept in the shade throughout the winter months,
being routinely inspected and weighed every 15Ã?â??Ã?·days with
minimal disturbance. By early spring, they expelled a dried
pellet of uric acid and began moving outside the shelter,
promptly reacting when mechanically stimulated. These
changes were taken as an indication of arousal, and the animals
were then returned to the previous photo- and thermal-period,
with free access to drinking water. During the first days of
arousal they were still anorexic, and a gradual increase in the
time spent on thermoregulation and food intake took place over
the following weeks.
Minimum and maximum air temperatures were recorded
daily using a thermometer placed in the shelter area. The mean
ranges for each seasonal period were: early autumn, 21Ã?¢ââ??‰â?¬Å?26Ã?â??Ã?°C;
late autumn, 18Ã?¢ââ??‰â?¬Å?23Ã?â??Ã?°C; winter, 15Ã?¢ââ??‰â?¬Å?20Ã?â??Ã?°C; early spring,
20Ã?¢ââ??‰â?¬Å?26Ã?â??Ã?°C; late spring, 23Ã?¢ââ??‰â?¬Å?30Ã?â??Ã?°C.
Oxygen consumption measurements
Resting oxygen consumption rates (V
.
308
apparently constitute a limited potential for sustaining energy
expenditure at the whole body level, as opposed to the
adjustments seen under anoxic conditions (Storey, 1996).
Remarkably, unlike the situation in mammal and bird species,
a large reduction in aerobic metabolism can be achieved in
the lower vertebrates without the predominant effect on the
energy requirements associated with endothermy and a
high thermoregulatory capacity. The regulatory mechanisms
involved in metabolic depression appear to be coupled to an
endogenous, circannual rhythm superimposed on the circadian
pattern of thermal behaviour and locomotor activity, as shown
in terrestrial reptiles (Hismiller and Heldmaier, 1988; FoÃ?Æ?Ã?  et
al., 1994). These aspects have been studied in detail in only a
few species, and considerable research remains to be done in
order to identify the general principles of regulation acting on
these events.
Another largely unstudied aspect of seasonal fasting and
hypometabolism concerns how these profound changes
develop in newly born animals of small body mass and limited
capacity to store substrates. Since much evidence suggests that
metabolic depression is endogenous in nature, it is important
to investigate how early this phenomenon manifests in the
life cycle. Furthermore, the ontogenetic adjustment of the
relationship between energy use and storage capacity as an
essential part of the make up of seasonal dormancy also may
be predicted. We have begun to address these questions
by examining the occurrence of metabolic depression and
associated changes in energy metabolism during the first year
cycle of tegu lizards (Tupinambis merianae). The tegu is
widely distributed throughout South America (Avila-Pires,
1995) and occurs in large numbers in southeastern Brazil.
Winter at this latitude is synchronized with the dry season,
when insects and other food sources become scarce. An innate
rhythm is apparent soon after hatching and, in the adult stage,
the reproductive activities are concentrated in the spring
months, with foraging and energy intake becoming gradually
reduced during summer until the lizards enter a state of
continuous inactivity, spending 4Ã?¢ââ??‰â?¬Å?5Ã?â??Ã?·months underground at
temperatures around 17Ã?â??Ã?°C in the autumn and winter months
(Abe, 1995). Abe (1995) verified a marked reduction in oxygen
consumption during winter to 20Ã?¢ââ??‰â?¬Å?30% of the resting values in
lizards with a mean mass of 1270Ã?â??Ã?·kg, consumption rates
becoming nearly temperature insensitive over the range of
17Ã?¢ââ??‰â?¬Å?25Ã?â??Ã?°C. Anorexia is also a marked feature of the annual cycle
in tegu lizards and develops from late summer through early
autumn irrespective of the high temperatures and wide food
availability at this time of the year (H. R. Lopes and A. Abe,
unpublished observation).
The present study is concerned with the questions of how
fasting and the magnitude of metabolic depression in young
tegus compare with these events in their adult counterparts and
whether such changes are influenced by body mass in the
growing lizards. We also focus on the absence of reproduction
at this early stage of development, emphasising correlative
shifts in the levels of metabolites, substrate stores and enzymes
over the first year cycle. The results support the notion of an
endogenous rhythm acting via appetite control and energy
metabolism in the tegu and suggest a shift in the balance
between energy expenditure and body mass during the
hypometabolic state, related to fat storage capacity and
increased energy conservation in the smaller progeny.
Materials and methods
Animal supply and maintenance
Young tegu lizards (Tupinambis merianae DumÃ?Æ?Ã?©ril and
Bribon) were obtained from a population reared outdoors in
large pens in Rio Claro, southeastern Brazil. About two months
after eclosion in the summer, the animals were moved to the
laboratory and used in the experiments during their first annual
cycle. The lizards were kept indoors in 120Ã?â??Ã?·litre cages
equipped with incandescent lights set on an 8Ã?â??Ã?·h:16Ã?â??Ã?·h L
photo- and thermal-period, in addition to the sunlight diffusing
from outside. The lizards could freely alternate between
warming and cooling their bodies by climbing onto a small
platform or hiding in a wooden shelter among the sheets of
paper covering the box floor. The animals were separated into
small groups according to size to minimise fighting and
competition for food and were fed every two days on raw meat,
egg and fruits, enriched with minerals, having continuous
access to drinking water.
A noticeable change in daily activity was seen in the early
autumn, when the time spent on thermoregulation became
progressively shorter and food intake gradually reduced until
the lizards became continuously inactive inside the shelter. The
animals were kept in the shade throughout the winter months,
being routinely inspected and weighed every 15Ã?â??Ã?·days with
minimal disturbance. By early spring, they expelled a dried
pellet of uric acid and began moving outside the shelter,
promptly reacting when mechanically stimulated. These
changes were taken as an indication of arousal, and the animals
were then returned to the previous photo- and thermal-period,
with free access to drinking water. During the first days of
arousal they were still anorexic, and a gradual increase in the
time spent on thermoregulation and food intake took place over
the following weeks.
Minimum and maximum air temperatures were recorded
daily using a thermometer placed in the shelter area. The mean
ranges for each seasonal period were: early autumn, 21Ã?¢ââ??‰â?¬Å?26Ã?â??Ã?°C;
late autumn, 18Ã?¢ââ??‰â?¬Å?23Ã?â??Ã?°C; winter, 15Ã?¢ââ??‰â?¬Å?20Ã?â??Ã?°C; early spring,
20Ã?¢ââ??‰â?¬Å?26Ã?â??Ã?°C; late spring, 23Ã?¢ââ??‰â?¬Å?30Ã?â??Ã?°C.
Oxygen consumption measurements
Resting oxygen consumption rates (V
.
OÃ?â??Ã?·) were measured
during the annual cycle on groups of tegus as follows: Ã?¢ââ??¬Ã?Å?autumn
activityÃ?¢ââ??‰â??¢, subdivided into early and late autumn to include
lizards exhibiting behaviour characteristic of the onset of
dormancy; Ã?¢ââ??¬Ã?Å?winter dormancyÃ?¢ââ??‰â??¢, including fasting and totally
inactive lizards over 50Ã?¢ââ??‰â?¬Å?60 winter days; Ã?¢ââ??¬Ã?Å?arousalÃ?¢ââ??‰â??¢ for lizards
emerging from dormancy (90Ã?¢ââ??‰â?¬Å?100Ã?â??Ã?·days from the first winter
day), subdivided into rehydrated, unfed animals (48Ã?¢ââ??‰â?¬Å?96Ã?â??Ã?·h after
arousal) and fed animals (~1Ã?â??Ã?·week after arousal); Ã?¢ââ??¬Ã?Å?spring Energy metabolism and scaling patterns in tegu lizards 309
activityÃ?¢ââ??‰â??¢, including fully active lizards 30Ã?¢ââ??‰â?¬Å?40Ã?â??Ã?·days after
arousal.
The metabolic chamber consisted of a well-sealed acrylic
box, having an inlet and an outlet fixed to the top of the lid.
The lizards were transferred into individual metabolic
chambers by mid-afternoon and kept in the shade for at least
two hours prior to experiments, during which time room air
could diffuse freely through an aperture. The chamber lids
were then screwed shut, providing an effective gas-tight seal,
and V
.
OÃ?â??Ã?· was measured. The initial and final fractional O2
concentrations were obtained by taking two samples, each of
10Ã?â??Ã?·ml, with a gas-tight syringe, the first after closing the
chamber and the second after 60Ã?â??Ã?·min, in two consecutive series
of measurements. Prior to sampling, the air inside the chamber
was gently mixed. The sample was carefully injected into an
oxygen analyzer (S-3; Applied Eletrochemistry, Pittsburgh,
PA, USA), drawn through silica gel and ascarite and
subsequently into the oxygen sensor via a pump at low speed.
Reproducibility of the procedure was verified by the repeated
injection of samples of gas mixtures containing known O2
concentrations: the error was always less than 1%. Oxygen
concentrations below 19% were avoided during the
experiments. V
.
OÃ?â??Ã?· was calculated according to the equation
developed by Hill (1972) and expressed as mlÃ?â??Ã?·O2Ã?â??Ã?·hÃ?¢ââ??‰â?¬Å?1Ã?â??Ã?·gÃ?¢ââ??‰â?¬Å?1 at
standard temperature and pressure.
Measurements were performed from 19.30Ã?â??Ã?·h to 23.30Ã?â??Ã?·h.
Previous experiments revealed two V
.
OÃ?â??Ã?· peaks during the daily
cycle of the tegu, one in the late morning and the other in the
early afternoon, followed by a progressive drop in V
.
OÃ?â??Ã?· to the
minimum values at 02.00Ã?¢ââ??‰â?¬Å?04.00Ã?â??Ã?·h. In one experimental series,
resting rates were measured in six different groups of animals
at 25Ã?â??Ã?±1Ã?â??Ã?°C, except during dormancy when 17Ã?â??Ã?±1Ã?â??Ã?°C was used.
Both temperatures are representative of the mean variation
encountered by the tegu within its underground shelter (Abe,
1995). The lizards were then killed to obtain blood and tissue
samples for each specific seasonal period, as detailed below.
In the other experimental series, resting V
.
OÃ?â??Ã?· was measured on
the same group of animals at 25Ã?â??Ã?°C and 17Ã?â??Ã?°C throughout the
year to verify seasonal changes in the temperature effect, as
calculated from the Q10 ratio. In all cases, body temperature
was assumed to be in equilibrium with the air inside the
chamber after the acclimation period.
Blood and tissue sampling
For blood and tissue analysis, one group of animals was
killed in the morning after V
.
OÃ?â??Ã?· was measured, and named
according to the seasonal period as above. In these
experiments, Ã?¢ââ??¬Ã?Å?autumn activityÃ?¢ââ??‰â??¢ refers to late autumn, and
Ã?¢ââ??¬Ã?Å?arousalÃ?¢ââ??‰â??¢ to rehydrated, unfed animals. The animals were
decapitated and blood samples were taken directly into preheparinised
tubes (0.2Ã?â??Ã?·mg heparin per ml blood). A 0.1Ã?â??Ã?·ml
sample was vigorously mixed with 0.2Ã?â??Ã?·ml of 0.6Ã?â??Ã?·molÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1
perchloric acid (v/v), centrifuged at 6000Ã?â??Ã?·g and 4Ã?â??Ã?°C for 10Ã?â??Ã?·min,
and stored at 10Ã?â??Ã?°C for lactate assay. The remaining volume
was centrifuged to obtain plasma samples, then frozen in liquid
nitrogen and stored at Ã?¢ââ??‰â?¬Å?80Ã?â??Ã?°C until analysis. The whole brain,
liver, heart ventricle, white portion of the iliofibularis muscle,
and a sample of the longitudinal tail muscle were quickly
dissected and immediately frozen in liquid nitrogen and stored
at Ã?¢ââ??‰â?¬Å?80Ã?â??Ã?°C for metabolite and enzyme assays. Finally, the two
abdominal fat bodies were removed and weighed, and a sample
of the whole tail was removed from the proximal third to assess
cyclic changes in fat content. All tissue samples were stored at
Ã?¢ââ??‰â?¬Å?80Ã?â??Ã?°C until analysis.
Analysis of blood and tissue metabolites
Blood osmolality was measured in 10Ã?â??Ã?·ml plasma samples
using a vapour pressure Osmometer (5500; Wescor, Logan, UT,
USA). Total protein was assayed in blood samples according to
Lowry et al. (1951), using bovine serum albumin as a standard.
Total lipids and b-hydroxybutyrate were measured in plasma
samples using diagnostic kits purchased from LabTest (Belo
Horizonte, MG, Brazil) and Sigma (St Louis, MO, USA),
respectively. The first method is based on the sulpho-phosphovaniline
colorimetric reaction, and the second follows the
enzymatic oxidation of b-hydroxybutyrate to acetoacetate. Dglucose
and L-lactate concentrations were assayed in
deproteinised samples according to standard enzymatic
procedures (Bergmeyer, 1984); NAD+ and NADP+ reactions
were monitored at 340Ã?â??Ã?·nm using a spectrophotometer (DU-70;
Beckman, Fullerton, CA, USA) at 25Ã?â??Ã?°C. Values are expressed
as mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1.
Total tissue water and protein were measured in duplicate
samples of skeletal (tail) muscle. Water content was estimated
by the accompanying mass loss in each tissue sample at 80Ã?â??Ã?°C
until constant mass. For total protein analysis, tissue samples
were homogenised in four volumes (v/w) of 0.6Ã?â??Ã?·molÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 PCA.
The homogenate was centrifuged for 5Ã?â??Ã?·min at 10Ã?â??Ã?·000Ã?â??Ã?·g and the
pellet redissolved in 0.6Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 PCA, the procedure being
repeated twice. The precipitate was solubilised in 2.5% KOH
and protein content was measured (Lowry et al., 1951).
Glycogen content was assayed in liver, skeletal (tail) muscle,
heart and brain. Frozen samples were homogenized in ice-cold
0.6Ã?â??Ã?·molÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 PCA, and two aliquot samples were taken. One was
incubated with amyloglucosidase at 40Ã?â??Ã?°C for glycogen
hydrolysis and D-glucose analysis, and the other was used to
estimate background glucose. The assays were conducted
following standard enzymatic procedures (Bergmeyer, 1984),
and glycogen standards were used to control hydrolysis efficacy.
Total lipid content in liver, skeletal (tail) muscle and whole
tail samples was measured in freshly thawed samples as
described by Folk et al. (1957). Fresh masses of the abdominal
fat bodies were taken as an estimate of the amount of fat in
this deposit at different times during the annual cycle.
Enzyme assays
The activities of citrate synthase (CS), an indicator of
tissue total aerobic capacity, and of b-hydroxyacyl CoA
dehydrogenase (HOAD), an indicator of the capacity for fatty
acid utilisation, were measured in skeletal muscle (iliofibularis,
white portion), heart ventricle, liver and brain tissue. Freshly
thawed samples were homogenised at approximately 4Ã?â??Ã?°C in 310
nine volumes of buffer (w/v) with a teflonÃ?¢ââ??‰â?¬Å?glass homogenizer,
using the following composition: 20Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 imidazol-HCl,
pH 7.4, 2Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 EDTA, 0.1% Triton X-100. Homogenates
assigned to the CS assay were consecutively frozen at Ã?¢ââ??‰â?¬Å?80Ã?â??Ã?°C
and thawed at 4Ã?â??Ã?°C three times for complete membrane
disruption before centrifugation. All homogenates were
centrifuged at 17Ã?â??Ã?·000Ã?â??Ã?·g and 4Ã?â??Ã?°C for 10Ã?â??Ã?·min, and the
supernatant fractions kept ice-cold until assay.
Enzyme activities were measured spectrophotometrically
(Beckman DU-70) at 25Ã?â??Ã?°C, by following the nicotinamide
adenosine dinucleotide (NADH) and 5,5Ã?â??Ã?¢-dithiobis 2-
nitrobenzoic acid (DTNB) reactions at 340Ã?â??Ã?·nm and 412Ã?â??Ã?·nm,
respectively, under saturating, non-inhibitory substrate
conditions. Buffers and enzyme assays followed the standard
approaches given in Bergmeyer (1984), and preliminary
experiments were performed to check for control reaction rates
(reactions omitting substrate) and to establish the optimal
substrate and cofactor concentrations for the final procedure.
Each tissue sample was assayed in duplicate, and enzyme
activities were described as units per mg of tissue wet mass.
Soluble protein concentration was measured in all tissue
fractions using bovine serum albumin standards (Lowry et al.,
1951), and enzyme activities were calculated per soluble
protein mass to verify any biased tendency due to a change in
tissue water content and/or to unspecific effects on the soluble
protein content. Assay conditions were as follows. CS:
50Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 Tris-HCl (pH 8.0), 0.3Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 acetyl CoA,
0.1Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 DTNB, 0.5Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 oxaloacetate; HOAD:
50Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 Tris-HCl (pH 7.0), 0.15Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 NADH,
0.1Ã?â??Ã?·mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1 acetoacetyl CoA.
Statistical analysis
A one-way analysis of variance (ANOVA) or the
KruskalÃ?¢ââ??‰â?¬Å?Wallis ANOVA on ranks procedure was used to test
for differences between groups over the annual activity cycle.
Means were then compared by the StudentÃ?¢ââ??‰â?¬Å?NewmanÃ?¢ââ??‰â?¬Å?Keuls or
DunnÃ?¢ââ??‰â??¢s tests for multiple comparisons, where appropriate. The
correlations between V
.
OÃ?â??Ã?·, fat body mass or Q10 values
and body mass were assessed using a least-squares linear
regression method on log-transformed data, and the
contribution of body mass in predicting the dependent variable
was evaluated by the F-test. The analyses were based on Zar
(1999) and performed using SigmaStat statistical software
(Jandel Co.). The probability of error in the test results was
generally assumed to be significant at PÃ?â??Ã?²0.05.
Results
Changes in oxygen consumption rates (VÃ?â?¹Ã¢â??¢ OÃ?â??Ã?·)
The resting, mass-specific V
.
OÃ?â??Ã?· decreased by a mean of 50%
from early to late autumn in newly hatched lizards. In winter,
V .
OÃ?â??Ã?· stabilised at values 75Ã?¢ââ??‰â?¬Å?85% lower than those recorded
in early autumn, with a concomitant decrease in body
(experimental) temperature from 25Ã?â??Ã?°C to 17Ã?â??Ã?°C (TableÃ?â??Ã?·1). The
neonates became continuously inactive and did not eat or drink
over at least 90Ã?â??Ã?·days during winter. At the onset of arousal,
they drank water abundantly but remained anorexic, while V
.
OÃ?â??Ã?·
and the overall activity increased to the levels seen in late
autumn. Food intake began within ~1Ã?â??Ã?·week, leading to a further
increase in V
.
OÃ?â??Ã?· to values only 25% lower than those seen in
early autumn, a non-significant difference. Thereafter, the
lizards resumed intense activity, and V
.
OÃ?â??Ã?· values measured
30Ã?¢ââ??‰â?¬Å?40Ã?â??Ã?·days after arousal were similar to those of early autumn,
a state that continued during the first half of summer.
Body mass did not differ significantly among the
experimental groups above (P=0.333). The scaling effect on
seasonal fluctuations in V
.
OÃ?â??Ã?· and on the degree of metabolic
depression was then analysed in lizards whose body mass
ranged from 37Ã?â??Ã?·g to 257Ã?â??Ã?·g. The relationship between body
mass (Mb) and V
.
OÃ?â??Ã?· changed with season during the first year
cycle of the tegu (TableÃ?â??Ã?·1). In early autumn, V
.
OÃ?â??Ã?· correlates with
Mb0.75, small animals having higher mass-specific metabolic
rates than their larger counterparts. Mass exponents for the
reduced metabolic rates were >1.0 during late autumn and
winter dormancy and close to 1.0 during unfed arousal,
implying the lack of increase in mass-specific metabolism with
S. C. R. de Souza and others
Table 1. Resting rates of mass-specific oxygen consumption (VÃ?â?¹Ã¢â??¢ OÃ?â??Ã?·) and the scaling relationship with body mass in tegu lizards
during the first annual cycle
Seasonal activity state VÃ?â?¹Ã¢â??¢O2 (mlÃ?â??Ã?·O2Ã?â??Ã?·hÃ?¢ââ??‰â?¬Å?1Ã?â??Ã?·gÃ?¢ââ??‰â?¬Å?1) Body mass (g) a b r2
Autumn activity
Early 0.0627Ã?â??Ã?±0.0057bÃ?¢ââ??‰â?¬Å?e 92.6Ã?â??Ã?±14.9 Ã?¢ââ??‰â?¬Å?0.75Ã?â??Ã?±0.37 0.75Ã?â??Ã?±0.19 0.60
Late 0.0309Ã?â??Ã?±0.0033a,cÃ?¢ââ??‰â?¬Å?e,f 111.1Ã?â??Ã?±18.7 Ã?¢ââ??‰â?¬Å?2.09Ã?â??Ã?±0.47 1.27Ã?â??Ã?±0.24 0.74
Winter dormancy 0.0119Ã?â??Ã?±0.0013a,b,dÃ?¢ââ??‰â?¬Å?f 104.4Ã?â??Ã?±12.1 Ã?¢ââ??‰â?¬Å?2.18Ã?â??Ã?±0.68 1.12Ã?â??Ã?±0.34 0.52
Arousal
Unfed 0.0273Ã?â??Ã?±0.0023aÃ?¢ââ??‰â?¬Å?c,e,f 94.1Ã?â??Ã?±9.1 Ã?¢ââ??‰â?¬Å?1.50Ã?â??Ã?±0.72 0.94Ã?â??Ã?±0.37 0.40
Fed 0.0477Ã?â??Ã?±0.0042aÃ?¢ââ??‰â?¬Å?d,f 113.7Ã?â??Ã?±8.4 Ã?¢ââ??‰â?¬Å?2.81Ã?â??Ã?±0.60 1.72Ã?â??Ã?±0.29 0.77
Spring activity 0.0636Ã?â??Ã?±0.0045bÃ?¢ââ??‰â?¬Å?e 129.6Ã?â??Ã?±14.8 Ã?¢ââ??‰â?¬Å?1.13Ã?â??Ã?±0.429 0.96Ã?â??Ã?±0.20 0.70
Values are the mean Ã?â??Ã?± 1 S.E.M. from 10Ã?¢ââ??‰â?¬Å?12 different animals measured at 25Ã?â??Ã?°C, except in winter dormancy when 17Ã?â??Ã?°C was used. Linear
regressions were performed on log-transformed VÃ?â?¹Ã¢â??¢O2 (mlÃ?â??Ã?·O2Ã?â??Ã?·hÃ?¢ââ??‰â?¬Å?1) and body mass (g) as described by log10 VÃ?â?¹Ã¢â??¢O2=a+b log10 Mb, where a is the
intercept Ã?â??Ã?± S.E.M., b is the slope Ã?â??Ã?± S.E.M. and Mb is body mass; a indicates significant differences from early autumn, b from late autumn, c from
winter dormancy, d from unfed arousal, e from fed arousal, f from spring activity (P<0.05). decreasing mass during the hypometabolic state. According to
the equations, a 3-fold increase in body mass leads to a 4-fold
increase in V
.
OÃ?â??Ã?· in late autumn and a 3.4-fold increase during
winter dormancy. In both cases, the slopes are significantly
different from that obtained for early autumn individuals
(P<0.001), and the degree of metabolic depression in late
autumn would be 30% versus 61% for lizards weighing 180Ã?â??Ã?·g
and 60Ã?â??Ã?·g, respectively; later in winter, metabolic depression
would reach 73% and 83% for the same body mass values.
Thus, size-related differences may influence either the
magnitude of V
.
OÃ?â??Ã?· decrease and/or the time of entry into
dormancy. At the onset of arousal, the V
.
OÃ?â??Ã?· is somewhat
enhanced in aphagic animals although the percentage of
depression is still high, irrespective of body mass. The slope
at this step is b=0.94 and is significantly different from that for
winter dormancy (P<0.01). When feeding reinitiates, the
exponent shifts remarkably to b=1.72 and, assuming a 3-fold
increase in body mass, V
.
OÃ?â??Ã?· would increase almost 7-fold. The
calculated percentage of V
.
OÃ?â??Ã?· depression would still be 53% in
the smaller individuals as opposed to <10% in larger lizards,
indicating that the larger the individual, apparently less time is
necessary to accomplish the transition from dormancy to full
activity. Later in spring, the slope for resting V
.
OÃ?â??Ã?· is b=0.96 and
within one month of arousal metabolic rates are similar,
irrespective of body mass. Body mass accounted for more than
50% of V
.
OÃ?â??Ã?· variability in most groups, with inter-individual
variation being predominant in unfed, arousing animals. After
correcting V
.
OÃ?â??Ã?· for body mass, the slope of the relationship
between V
.
OÃ?â??Ã?· and body mass was significantly different from 0
only in fed individuals during arousal (b=0.722; P=0.035). In
the other groups, variability was large, perhaps preventing a
statistically significant correlation.
The V
.
OÃ?â??Ã?· rates measured at 25Ã?â??Ã?°C and 17Ã?â??Ã?°C in the single group
used for Q10 analysis showed a pattern similar to the above.
Entry into dormancy occurred earlier that year and, although
typical early autumn data were not available, aerobic
metabolism at 25Ã?â??Ã?°C stabilised at values 77% lower during
dormancy compared with rest in spring. A significant decrease
was seen at 17Ã?â??Ã?°C, the magnitude being less along this lower
temperature line (55%). There was no statistical difference
between V
.
OÃ?â??Ã?· values at these two temperatures during late
autumn or winter dormancy, and the calculated Q10 is ~1.5 for
these two groups (TableÃ?â??Ã?·2). During early arousal, there was a
significant difference between the V
.
OÃ?â??Ã?· values measured at 25Ã?â??Ã?°C
and 17Ã?â??Ã?°C, causing an increase in Q10 values prior to food
intake, which then further increased to almost 3.0 after feeding
was reinitiated, increasing above this value in active spring
individuals. Statistical analysis failed to show any significant
correlation between body size and the Q10 effect in most
groups in this experimental series, possibly due to the reduced
sample size (N=6). An exception, however, was the spring
activity group, in which a strong correlation was found
(r2=0.90; P<0.004). The extremely high mass exponent
(b=9.35) suggests a much greater temperature sensitivity of V
.
OÃ?â??Ã?·
between 17Ã?â??Ã?°C and 25Ã?â??Ã?°C in larger individuals at this period of
the first annual cycle.
Changes in body mass and composition
The tegus weighed ~15Ã?â??Ã?·g upon hatching and, in the
laboratory, their mass increased by 5Ã?¢ââ??‰â?¬Å?7-fold during the 4Ã?¢ââ??‰â?¬Å?5
months up to mid-autumn. Thereafter, feeding and other
activities became gradually reduced, and mass loss during
dormancy reached 15% of the maximum mass in late autumn,
as calculated on an individual basis for six animals (TableÃ?â??Ã?·2).
More than half of this loss (62%) was quickly offset after water
intake during the first days of arousal, suggesting that mass
change is partially due to evaporative water loss, besides the
use of other body stores. Body mass increased at progressively
higher rates after feeding was reinitiated and increased more
than 10-fold in the young lizards by the end of the first year
cycle.
During dormancy, plasma protein concentration increased
concomitantly with a peak in osmolality, which also increased
slightly in the late autumn and arousal groups compared with
active spring animals (TableÃ?â??Ã?·3). There was a significant drop
in the water content of skeletal (tail) muscle during dormancy,
suggesting some loss of fluid from tissue compartments. Other
changes during dormancy included a pronounced drop in
circulating glucose to 40% and an increase in the levels of total
protein (24%), lipids (43%) and b-hydroxybutyrate (113%) in
relation to spring activity values (TableÃ?â??Ã?·3). Particularly
interesting is the almost completely restored glucose levels in
arousing animals before food intake. Blood lactate did not
change significantly, an indication that there is no substantial
alteration in the rates of anaerobic glycolysis associated with
metabolic depression in the tegu.Table 2. Q10 values (mean Ã?â??Ã?± S.E.M.) for the seasonal change
in resting rates of oxygen consumption at 25Ã?â??Ã?°C and 17Ã?â??Ã?°C in
young tegu lizards
Seasonal activity state Q10 Body mass (g)
Autumn activity 1.50Ã?â??Ã?±0.066 99.27Ã?â??Ã?±16.6
Winter dormancy 1.55Ã?â??Ã?±0.053 90.86Ã?â??Ã?±18.1
Arousal
Unfed 1.77Ã?â??Ã?±0.073 86.49Ã?â??Ã?±16.9
Fed 2.94Ã?â??Ã?±0.012 97.26Ã?â??Ã?±23.7
Spring activity 3.54Ã?â??Ã?±0.178 178.03Ã?â??Ã?±43.1
Measurements were made on six animals individually followed
during their first annual cycle. Autumn activity corresponds to late
autumn in TableÃ?â??Ã?·1.
At the tissue level, there was no significant change in the
soluble protein content of tissues examined. Total protein in
tail muscle, however, was 14% less by mid-winter and the
cumulative loss was almost 50% in the tail muscle of arousing
animals compared with the values found in late autumn
(TableÃ?â??Ã?·4). The uric acid pellet expelled on arousal is another
strong indication of protein mobilisation during the prolonged
fast, possibly intensified by the onset of arousal. Tail muscle
glycogen content was reduced to half by mid-winter and
increased almost 3-fold in unfed aroused individuals,
suggesting the use of amino acids resulting from protein 312
catabolism as the carbon source for carbohydrate synthesis
before feeding is reinitiated. Liver glycogen is higher in late
autumn, reaching 474Ã?â??Ã?·mmoles (77Ã?â??Ã?·mg) of glycosyl units in a
100Ã?â??Ã?·g lizard, and reduces by 63% in mid-winter, while glucose
levels remain constant (TableÃ?â??Ã?·4). The liver also regained its
potential to accumulate glycogen in arousing animals,
concomitant with a significant drop in its glucose content. A
distinct trend was seen in the brain tissue, where glycogen is
consistently higher in late fall, winter dormancy and arousal
groups compared with spring activity, reaching a difference of
almost 5-fold in the hypometabolic state. Similarly, heart
glycogen is increased by 2-fold during winter dormancy
compared with levels in late fall and spring activity, suggesting
that these tissues may have the ability to preserve an
endogenous source of glucose during the prolonged fasting.
Together, these results support the idea that carbohydrate
metabolism may be enhanced before food intake at the end of
the prolonged fasting.
Before entry into dormancy, the young lizards deposited an
average of 2.7% of their body mass as fat in the abdominal fat
bodies. The size of this deposit gradually decreased during
dormancy, by 24% after 50Ã?¢ââ??‰â?¬Å?60 days of winter and by 62% on
arousal in early spring; the fat deposits were virtually depleted
in active spring animals 30Ã?¢ââ??‰â?¬Å?40Ã?â??Ã?·days after arousal. The liver
lipid content was fairly constant at 35Ã?â??Ã?·mgÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1 throughout the
year, corresponding to 53Ã?¢ââ??‰â?¬Å?70Ã?â??Ã?·mg in a 100Ã?â??Ã?·g lizard during the
different seasons. In skeletal (tail) muscle, lipids averaged
24Ã?â??Ã?·mgÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1 muscle in late autumn animals, showing a slight
tendency to drop in dormant and arousing animals. The fat
content of the whole tail sample was 2Ã?¢ââ??‰â?¬Å?4-fold higher
(79.2Ã?â??Ã?·mgÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1 tail) than in the skeletal muscle alone. This fat
was reduced 25% by mid-winter and 45% in unfed arousing
animals, compared with active spring individuals, suggesting
that fat from a subcutaneous deposit may constitute another
important energy source in fasting animals.
The correlation between body mass and abdominal fat body
size was examined to test for a scaling effect on the deposition
and mobilization pattern of this energy store. In late autumn
animals, the fat body mass correlates with body mass with
b=1.44 (P<0.000; Fig.Ã?â??Ã?·1), implying that a 3-fold increase in
body mass leads to a 5-fold increase in fat deposits. This
correlation was also significant after transforming fat content
into an index of body mass (b=0.44; P=0.020), confirming that,
on entry into dormancy, larger animals possess substantially
more fat per unit mass available in this deposit. Halfway
through dormancy, the relationship becomes less
disproportionate, although larger animals still have a fat
surplus of 18% in the fat bodies, as calculated for the body size
S. C. R. de Souza and others
Table 3. Seasonal changes in blood plasma parameters during the first annual cycle of tegu lizards
Glucose Lactate Total lipids b-Hydroxybutyrate Protein Urea Osmolality
Seasonal activity state (mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1) (mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1) (mgÃ?â??Ã?·mlÃ?¢ââ??‰â?¬Å?1) (mmolÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1) (mgÃ?â??Ã?·mlÃ?¢ââ??‰â?¬Å?1) (mgÃ?â??Ã?·lÃ?¢ââ??‰â?¬Å?1) (OsmÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1Ã?â??Ã?·H2O)
Autumn activity Ã?¢ââ??‰â?¬Å? Ã?¢ââ??‰â?¬Å? 5.22Ã?â??Ã?±0.63 Ã?¢ââ??‰â?¬Å? 47.4Ã?â??Ã?±2.0 20.4Ã?â??Ã?±4.2 307.1Ã?â??Ã?±5.3b
Winter dormancy 3.08Ã?â??Ã?±0.15b 1.13Ã?â??Ã?±0.16 5.67Ã?â??Ã?±0.36b 2.85Ã?â??Ã?±0.33b 53.3Ã?â??Ã?±2.2b 20.4Ã?â??Ã?±5.2 323.1Ã?â??Ã?±1.5a,b
Arousal 5.87Ã?â??Ã?±0.65 0.67Ã?â??Ã?±0.11 3.02Ã?â??Ã?±0.26a Ã?¢ââ??‰â?¬Å? 40.2Ã?â??Ã?±2.0 21.1Ã?â??Ã?±4.8 315.4Ã?â??Ã?±6.2a,b
Spring activity 7.61Ã?â??Ã?±0.97 0.90Ã?â??Ã?±0.14 3.97Ã?â??Ã?±0.40 1.34Ã?â??Ã?±0.21 43.1Ã?â??Ã?±1.5 35.5Ã?â??Ã?±6.1 293.7Ã?â??Ã?±2.3
Ã?¢ââ??¬Ã?Å?Autumn activityÃ?¢ââ??‰â??¢ corresponds to late autumn, and Ã?¢ââ??¬Ã?Å?arousalÃ?¢ââ??‰â??¢ corresponds to rehydrated, unfed animals in TableÃ?â??Ã?·1. Values are the mean Ã?â??Ã?±
S.E.M. from six animals; a indicates significant differences from autumn activity, b from spring activity (P<0.05).Table 4. Seasonal changes in tissue composition during the first annual cycle of tegu lizards
Seasonal activity state Tail muscle Liver Heart Brain
Protein (mgÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1) Autumn activity 70.5Ã?â??Ã?±2.57 (212.5Ã?â??Ã?±18.6) 121.6Ã?â??Ã?±4.79 64.9Ã?â??Ã?±2.20 62.6Ã?â??Ã?±2.24
Winter dormancy 65.7Ã?â??Ã?±1.85 (183.3Ã?â??Ã?±10.5) 129.6Ã?â??Ã?±6.01 62.1Ã?â??Ã?±2.24 60.1Ã?â??Ã?±2.41
Arousal 63.9Ã?â??Ã?±4.10 (119.8Ã?â??Ã?±21.4)a 120.8Ã?â??Ã?±6.34 59.9Ã?â??Ã?±2.00 66.1Ã?â??Ã?±0.76
Spring activity 60.2Ã?â??Ã?±1.87 (146.1Ã?â??Ã?±14.3)a 132.6Ã?â??Ã?±4.75 61.6Ã?â??Ã?±2.22 70.3Ã?â??Ã?±3.00
Glycogen (mgÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1) Autumn activity 0.83Ã?â??Ã?±0.24 36.2Ã?â??Ã?±6.51bÃ?¢ââ??‰â?¬Å?d 1.04Ã?â??Ã?±0.18 1.22Ã?â??Ã?±0.28d
Winter dormancy 0.36Ã?â??Ã?±0.07c 13.2Ã?â??Ã?±7.33 2.56Ã?â??Ã?±0.48a,c,d 2.59Ã?â??Ã?±0.19a,d
Arousal 1.29Ã?â??Ã?±0.28 21.5Ã?â??Ã?±3.76 1.64Ã?â??Ã?±0.24 1.50Ã?â??Ã?±0.13d
Spring activity 0.78Ã?â??Ã?±0.18 13.4Ã?â??Ã?±2.83 1.10Ã?â??Ã?±0.17 0.49Ã?â??Ã?±0.03
Lipid (mgÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1) Autumn activity 23.6Ã?â??Ã?±6.07b,c 34.5Ã?â??Ã?±0.79 Ã?¢ââ??‰â?¬Å? Ã?¢ââ??‰â?¬Å?
Winter dormancy 11.3Ã?â??Ã?±0.90c 33.0Ã?â??Ã?±4.06 Ã?¢ââ??‰â?¬Å? Ã?¢ââ??‰â?¬Å?
Arousal 15.0Ã?â??Ã?±1.08 38.5Ã?â??Ã?±1.96 Ã?¢ââ??‰â?¬Å? Ã?¢ââ??‰â?¬Å?
Spring activity 20.3Ã?â??Ã?±3.46 29.9Ã?â??Ã?±1.14 Ã?¢ââ??‰â?¬Å? Ã?¢ââ??‰â?¬Å?
Protein values correspond to the soluble fraction after centrifugation; total protein content in tail muscle is indicated in parentheses. Ã?¢ââ??¬Ã?Å?Autumn
activityÃ?¢ââ??‰â??¢ corresponds to late autumn, and Ã?¢ââ??¬Ã?Å?arousalÃ?¢ââ??‰â??¢ to rehydrated, unfed animals in TableÃ?â??Ã?·1. Values are the mean Ã?â??Ã?± S.E.M. from six animals; a
indicates significant differences from autumn; b from winter dormancy; c from arousal; d from spring activity (P<0.05).Energy metabolism and scaling patterns in tegu lizards 313
range considered. The amount of fat mobilised during
dormancy up to mid-winter would be approximately 16% for
a small animal versus 35% for a large animal, and, therefore,
small lizards apparently utilize fat from this deposit at lower
rates than do larger ones. At the onset of arousal, however, the
slope is remarkably high (b=3.53) and the correlation is
statistically significant despite a smaller sample size in this
group (P=0.014, N=6). This shift in scaling pattern implies
ample differences in fat body size according to body mass in
early arousal, and the correlation predicts that this deposit is
virtually exhausted in the smallest individuals. The variability
was very high in this group and the correlation was not
significant after values were corrected for body mass (P>0.2).
In late spring, fat bodies are largely reduced although still vary
with body mass according to b>1, suggesting that the sizerelated
differences observed in late autumn would continue in
the active, growing animal.
Enzyme activities
Maximum CS and HOAD activities in the tissues of young
tegus are given in TableÃ?â??Ã?·5. CS activity was higher in the heart
and brain, both highly oxidative tissues, and typically lower in
skeletal (iliofibularis) muscle. The maximum activity was
constant in brain, liver and skeletal muscle sampl

 

[VARNYARD]

Part 2)

The mechanisms that trigger dormancy are still poorly
100
0.1
1
10
100
1
10
100
0.1
1
Arousal
Autumn activity
Fat body mass (g)
Body mass (g)
Winter dormancy
Spring activity
100
1
10 b=1.44
r2=0.88
b=1.15
r2=0.66
b=1.57
r2=0.61
b=3.53
r2=0.81
Fig.Ã?â??Ã?·1. The allometric relationship between fat body mass (Mfb) and
body mass (Mb) of young tegu lizards (Tupinambis merianae) at
distinct activity states during the first annual cycle. Autumn activity
corresponds to late autumn, and arousal to unfed, rehydrated animals
in TableÃ?â??Ã?·1. Linear regressions were performed on log-transformed
Mfb (g) and Mb (g) as described by log10 Mfb=a+b log10Mb.
Table 5. Enzyme activities in selected tissues of young tegu
lizards during the first annual cycle
Seasonal activity state CS HOAD
IL muscle Autumn activity 1.51Ã?â??Ã?±0.13 1.36Ã?â??Ã?±0.10
Winter dormancy 1.29Ã?â??Ã?±0.12 0.67Ã?â??Ã?±0.04a,d
Arousal 1.54Ã?â??Ã?±0.17 1.01Ã?â??Ã?±0.12a,d
Spring activity 1.57Ã?â??Ã?±0.13 1.31Ã?â??Ã?±0.09
Heart Autumn activity 29.65Ã?â??Ã?±4.60 12.11Ã?â??Ã?±0.26
Winter dormancy 23.04Ã?â??Ã?±1.11 7.19Ã?â??Ã?±0.43a,d
Arousal 28.90Ã?â??Ã?±1.17 8.73Ã?â??Ã?±0.52a,d
Spring activity 22.80Ã?â??Ã?±4.76 12.57Ã?â??Ã?±0.63
Liver Autumn activity 6.53Ã?â??Ã?±0.67 6.67Ã?â??Ã?±0.47
Winter dormancy 6.87Ã?â??Ã?±1.07 18.95Ã?â??Ã?±3.10a,d
Arousal 5.36Ã?â??Ã?±0.36 17.27Ã?â??Ã?±1.58a,d
Spring activity 7.82Ã?â??Ã?±0.78 31.17Ã?â??Ã?±1.80aÃ?¢ââ??‰â?¬Å?c
Brain Autumn activity 16.44Ã?â??Ã?±1.24 2.38Ã?â??Ã?±0.11
Winter dormancy 17.40Ã?â??Ã?±1.37 2.80Ã?â??Ã?±0.19
Arousal 17.80Ã?â??Ã?±0.58 2.44Ã?â??Ã?±0.07
Spring activity 16.93Ã?â??Ã?±1.16 2.71Ã?â??Ã?±0.19
Ã?¢ââ??¬Ã?Å?Autumn activityÃ?¢ââ??‰â??¢ corresponds to late autumn, and Ã?¢ââ??¬Ã?Å?arousalÃ?¢ââ??‰â??¢ to
unfed, rehydrated animals in TableÃ?â??Ã?·1. Values are the mean Ã?â??Ã?± 1 S.E.M.
from six animals in UÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1Ã?â??Ã?·wet mass; a indicates significant
differences from autumn activity; b from winter dormancy; c from
arousal; d from spring activity (P<0.05). IL, iliofibularis, white
portion.314
understood (Guppy et al., 1994; Storey, 2002). In young tegus,
they result in a stepwise depression of aerobic metabolism in
the whole animal, initially causing resting V
.
OÃ?â??Ã?· to drop by 50%
from early to late autumn at 25Ã?â??Ã?°C, associated with anorexia
and a clear departure from the normal routine. This
intermediate condition extends for a few weeks until the
animals remain in their artificial refuges in the mornings,
spending 3Ã?¢ââ??‰â?¬Å?4Ã?â??Ã?·months inactive during the first annual cycle.
During this time, metabolic rates are stable at ~20% of the
resting value without detectable variation within the daily
cycle (S. C. R. de Souza and J. E. de Carvalho, unpublished
observations). The reverse change on arousal is also gradual,
with a partial increase in aerobic metabolism (22Ã?¢ââ??‰â?¬Å?25%)
occurring in aphagic animals measured 48Ã?â??Ã?·h after water intake,
increasing further after food intake recommences a few days
later.
In mammals and birds, the interpretation of changes in V
.
OÃ?â??Ã?·
with torpor is complex mainly because of their inherent
capability to generate heat and thermoregulate in
normothermic conditions. The underlying mechanisms may be
switched off and an ensuing large Q10 effect provides the
animal with an alternative route for substantial reduction in
energy expenditure that may be supplemented by lesser savings
derived from metabolic depression (Guppy and Withers,
1999). In the tegu, the situation is clearly different, with the
hypometabolic condition relying heavily on a temperatureindependent
mechanism for energy conservation on a longterm
basis. During late autumn and winter dormancy, V
.
OÃ?â??Ã?·
decreases markedly in newly hatched lizards compared with
resting V
.
OÃ?â??Ã?· in early autumn, the rates becoming nearly constant
over the temperature range of 17Ã?¢ââ??‰â?¬Å?25Ã?â??Ã?°C. Accordingly, the
calculated Q10 is low, ~1.5 during the hypometabolic
condition, in marked contrast to the large Q10 effect (3.5)
during spring activity, suggesting that the temperature
sensitivity of the metabolic reactions is somehow reduced
during dormancy. Thus, a torpid tegu in its refuge at 17Ã?â??Ã?°C
would acquire total metabolic savings equivalent to a much
larger drop in body temperature, to approximately 8Ã?â??Ã?°C. The
percent depression in winter compared with resting conditions
in spring decreases when calculated along the lower
temperature line, reaching 55% at 17Ã?â??Ã?°C, in contrast to 77% at
25Ã?â??Ã?°C. Overall, the reduced rates of oxidative metabolism
during winter dormancy apparently meet the limit for longterm
survival at a very low energy cost.
The young tegus undergo a period of intense growth from
hatching in early summer until autumn, body mass increasing
by 5Ã?¢ââ??‰â?¬Å?7-fold under laboratory conditions. Thereafter, they
become anorexic and their weight losses result in a
progressively negative balance during winter dormancy,
leading to a net weight loss of 15% until arousal in spring.
Although the size range among siblings is narrow, analysis of
the individual data revealed an allometric effect on the
magnitude of metabolic depression during the first year cycle,
with a clear trend towards higher energy conservation in the
smaller lizards. Initially, the exponent relating V
.
OÃ?â??Ã?· and body
mass shifts from b=0.75 in early autumn individuals to b>1.0
during the hypometabolic condition, an indication that the
extent of depression is largely unproportional. The absolute
rates of metabolism increase by 35%, with a 3-fold increment
in body size in the early stages of depression, a difference of
18% remaining during winter dormancy when metabolic costs
are reduced to their lowest and small differences become
meaningful in terms of substrate savings for the duration of the
hypometabolic condition. During early arousal, V
.
OÃ?â??Ã?· rates are
somewhat enhanced in the aphagic individuals, and at this step
the energy expenditure per mass unity would be similar
irrespective of body mass (b=0.94). However, a few days after
feeding is reinitiated, the exponent rises remarkably to b=1.72,
suggesting that the larger the individual the less time is
necessary to accomplish the full transition from dormancy to
activity. In late spring, the slope for resting V
.
OÃ?â??Ã?· returns to
b=0.96, and this close proportion between body mass and
energy expenditure may be associated with the intense growth
period that follows arousal in young lizards.
Interspecific comparisons of V
.
OÃ?â??Ã?· data from adult,
heterothermic birds and mammals suggest a body mass
influence on the extent of metabolic depression in smaller
species, which undergo deep hibernation (body temperature
<10Ã?â??Ã?°C), the negative slope for the mass-specific rates not being
observed during torpor (Geiser, 1988). This shift in the scaling
pattern has been ascribed to the limited capacity of smaller
species to store energy as fat. A similar effect is not seen when
the different categories of heterotherms are analysed as a single
group, the mass exponents being indistinguishable and <1.0
(Guppy and Withers, 1999). By contrast, the V
.
OÃ?â??Ã?· changes in
young tegus are the product of a complex interaction with a
seasonal rhythm superimposed on the developmental process.
Within the ontogenetic context of mammals and other
vertebrates, the allometric exponent for whole animal
metabolism varies with the stage of development, and different
phases are recognised within this relationship over the life
cycle (Wieser, 1984). Briefly, the exponent is close to 1.0
during the early developmental stages, the period of most rapid
growth, and from then on the basal metabolic rates tend to
follow the surface rule, a pattern that prevails during most of
the life cycle; a crossover point of these two lines is reached
after a given degree of adult body mass is attained. This
suggests that the slope of b=0.75 for autumn activity in the first
year cycle of the tegu may not constitute a definite pattern for
the relationship, being restricted to the transition from a period
of intense growth during entry into dormancy when food intake
declines and growth processes are halted to ensure that a
suitable amount of energy is deposited mostly as fat in the body
stores. In this case, larger animals with their larger fat deposits
may reduce energy expenditure earlier on entry into dormancy.
Roughly, starting from a mean body mass of 15Ã?â??Ã?·g on hatching
(N=24), the young lizards grow at the fastest rate of 1.0Ã?â??Ã?·gÃ?â??Ã?·dayÃ?¢ââ??‰â?¬Å?1
during summer, growth decreasing subsequently to 0.4Ã?â??Ã?·gÃ?â??Ã?·dayÃ?¢ââ??‰â?¬Å?1
and to 0.2Ã?â??Ã?·gÃ?â??Ã?·dayÃ?¢ââ??‰â?¬Å?1 during the early and late autumn months,
until reaching a negative balance. After a prolonged pause in
the anabolic processes during winter dormancy, the young
enter another period of positive energy intake, their growth
S. C. R. de Souza and othersEnergy metabolism and scaling patterns in tegu lizards 315
rates becoming increasingly higher in spring when metabolic
rates show a closer relationship with body mass. Supportive
data suggesting the downregulation of endocrine mechanisms
that promote somatic growth during metabolic depression
were recently obtained with ground squirrels (Spermophilus
lateralis), an effect presumably associated with the change in
nutritional status during the hibernating period (Schmidt and
Kelley, 2001). Thus, while the exact significance of the mass
exponent for aerobic metabolism in early autumn activity
remains unresolved, the significant shifts in scaling pattern,
both on entry and on arousal from the hypometabolic
condition, strongly suggest a body mass influence on the
mechanisms of metabolic depression in young tegus.
A seasonal pattern of lipid cycling coexists with the shifts
in aerobic metabolism during the first annual cycle of tegu
lizards, as revealed by the consistent changes in the mass of
the visceral fat bodies and in the amount of fat in the
subcutaneous tail deposits. Seasonal variation was also found
in the potential for fatty acid oxidation in various tissues, as
shown by the remarkable changes in HOAD activity, which
increases several fold from late autumn to spring activity in
liver tissue, becoming reduced in skeletal and heart muscle in
dormant lizards. Apparently, these effects were independent of
changes in tissue water content and strongly suggest the tissuespecific
regulation of HOAD expression during the annual
cycle, related to the processes of fat deposition and
mobilization as well as to energy spare and overall metabolic
depression during dormancy. The progressive increase of liver
capacity for fatty acid oxidation would limit the use of modest
glycogen reserves, and this pattern, together with the constancy
of CS in most tissues examined, emphasises the aerobic nature
of seasonal dormancy in the tegu.
Seasonal cycles of fat deposition and mobilisation correlate
with food availability in many reptiles, most lipids being stored
subcutaneously and/or in visceral fat bodies (Derickson, 1976).
At most, fat body lipids make up 50% of the total storage
in the species examined, a variable fraction being allocated
for gametogenesis and other processes in preparation for
reproduction during winter and in early spring. The tegus reach
reproductive maturity by their third year cycle; prior to this
point, changes in lipid stores would be closely related to energy
expenditure for whole body maintenance during the fasting
period and for the metabolic increase seen upon arousal. The
allometric patterns for the changes in V
.
OÃ?â??Ã?· and fat body mass are
coherent in this context. In late autumn, the slope for the
correlation between fat body mass and body mass is b=1.44,
revealing that for a body size difference of 3Ã?¢ââ??‰â?¬Å?4-fold, a
disproportionately larger amount of fat will accumulate in larger
young individuals. By mid-winter, the shift in allometric pattern
to a close correlation with body mass suggests that smaller
animals drain fat from the fat body at lower rates than do larger
lizards. This is in good agreement with the greater depression of
aerobic metabolism seen in smaller young individuals. At the
time of arousal, these fat body deposits are virtually exhausted
in the smaller individuals, while substantial fat is still available
in the larger tegus, causing the mass exponent to shift to b>1
again. Given the importance of this fat deposit, this may imply
a limited capability of the smaller animals to sustain the higher
rates of metabolism required to actively hunt for food and to
avoid death by inanition upon return to activity. The events
preparatory to dormancy in the subsequent annual cycles are
probably anticipated, given that the arrest of routine activities
generally occurs earlier, and usually extends further, in
individuals at later stages of development (H. R. Lopes and A.
S. Abe, unpublished observations).
The physiological events relating the size of the fat stores to
energy intake and expenditure are becoming clearer as a
consequence of studies on obesity in humans and laboratory
mammals (for a review, see Ahima and Flier, 2000) and its
correlates in hibernating mammals (Boyer and Barnes, 1999).
The adipocyte is now established as the source of numerous
peptides secreted in the plasma, such as leptin, whose levels
correlate positively with total adipose mass. Leptin secretion
may act as a self-regulating system to sustain both a given
degree of lipid reserve and animal body mass. The action of
leptin on energy expenditure is probably exerted via the
hypothalamus through an effect on the production of thyroid
hormones and by a direct effect on cellular respiration in
peripheral tissues (Reidy and Weber, 2000). In hibernating
mammals, the mechanisms of body mass control may involve
more complex interactions of leptin with other molecules that
effect a seasonal modulation of leptin sensitivity and an
apparent dissociation of its anorectic and metabolic effects
(Boyer et al., 1997; Klingenspor et al., 2000). A leptin-like
molecule has been detected in the plasma and tissues of fish
and reptiles (Johnson et al., 2000; Niewiarowski et al., 2000);
while its physiological function in lower vertebrates has yet to
be elucidated, these findings provide a promising scenario for
the investigation of potential mechanisms linking the control
of body mass and degree of adiposity to the energy expenditure
during the annual cycle of young tegus.
Other sources of energy for the dormant tegu are
carbohydrates in the liver and skeletal muscle. Liver glycogen
in the late autumn averaged 223Ã?â??Ã?·mmolÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1, accounting for 3.6%
of the mass of the liver and corresponding to less than half the
liver glycogen reserve in overwintering amphibians and other
lower vertebrates that display varying capacities of hypoxia
tolerance (Boutilier et al., 1997; Scapin and Giuseppe, 1994;
Lutz and Nilsson, 1997). A rough calculation predicts that
during dormancy a 100Ã?â??Ã?·g lizard could survive for 2Ã?¢ââ??‰â?¬Å?3Ã?â??Ã?·days
exclusively on liver carbohydrates for oxidative processes
(assuming that 0.84Ã?â??Ã?·litres of O2 are required per 1Ã?â??Ã?·g of
carbohydrate oxidized); the same calculation for fat deposited
in the fat bodies provides 170Ã?â??Ã?·days (assuming that 2.0Ã?â??Ã?·litres of
O2 are required per 1Ã?â??Ã?·g of fat oxidized). This carbohydrate store
was reduced to a limit of ~60% in animals sampled halfway
through the inactive period. Plasma lactate remained nearly
constant in relation to active animals, further suggesting that
glycolytic ATP production does not play an important role in
the long-term maintenance of dormant tegus, unlike the case
in vertebrates in which metabolic depression is associated with
oxygen deprivation. However, the small carbohydrate store in316
the liver may be essential during the initial period of fasting,
providing energy at reduced rates for glucose-dependent
tissues such as the brain, renal medulla and retina (Guppy et
al., 1987) until the availability of alternative substrates like
ketone bodies increases in the blood. The high ratio of HOAD
to CS enzyme activities in the liver tissue of dormant tegus and
arousing animals suggests that the liver may be a site of
ketogenesis from the incomplete oxidation of fatty acids
(Stuart and Ballantyne, 1997), accounting for the increase in
b-hydroxybutyrate in the circulating plasma in dormant tegus.
Given the reduced glucose supply, this metabolite may become
a supplementary energy source for specific tissues in the tegu;
in agreement, our recent findings have revealed increased
enzyme activity related to the oxidation of ketone bodies in the
brain of dormant tegus (J. E. de Carvalho, M. S. C. Bianconcini
and S. C. R. de Souza, unpublished results).
The energy supply for such tissues, which typically show a
preference for carbohydrate as an energy substrate, may be
even more challenged in young tegus during emergence from
the hypometabolic condition, when they must rely on reduced
body stores to increase metabolic rate and succeed in the search
for food. Despite the low levels of glucose in the circulating
plasma, glycogen content is increased several fold in the brain
and in the heart of dormant lizards compared with spring active
individuals and may constitute a readily available source of
glucose for these high-priority tissues at the onset of arousal.
Notably, brain glycogen levels reached 2.6Ã?â??Ã?·mgÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1
(16Ã?â??Ã?·mmolÃ?â??Ã?·glycosylÃ?â??Ã?·unitsÃ?â??Ã?·gÃ?¢ââ??‰â?¬Å?1) in dormant lizards, a high content
typically found in the brain of anoxia-tolerant species as
crucian carp, goldfish and freshwater turtles (Lutz and Nilsson,
1994). The delivery rate of glucose to the brain is presumably
lower during metabolic depression, and glycogen deposition in
this condition may be facilitated by a sustained potential of
synthesis and reduced rates of carbohydrate usage. At the onset
of arousal (2Ã?¢ââ??‰â?¬Å?4Ã?â??Ã?·days), brain glycogen decreased by 42%
concomitant with a significant increase in blood glucose and
skeletal muscle glycogen, suggesting that another source of
glucose is made available for the tissues before feeding is
reinitiated.
The substantial reduction in tail muscle total protein in
arousing tegus (50%) is consistent with the general idea that
amino acids from protein breakdown play an important role as
precursors for glucose synthesis at the end of a prolonged
fasting period, in addition to glycerol from fatty acid oxidation
(Moon, 1988). Protein is also an important source of energy
for both large and small hibernating mammals (Cherel et al.,
1995; Tinker et al., 1998), and an adequate balance of fat and
protein use is apparently part of a common repertory in several
spontaneous fasters, the control of which is poorly known
(Robin et al., 1998; Mellish and Iverson, 2001). The degree of
protein catabolism correlates with the size of the initial fat
reserves in the species examined; the young tegus, with their
small size and limited capacity to store fat, may derive more
energy from protein during the fasting period, and particularly
on arousal, than at later stages of development. The high
cumulative protein loss from skeletal white muscle in the
young may nevertheless compromise locomotion and hunting
capabilities upon return to activity. In hibernating bears, net
protein loss varies from 4% to 10% in two muscle types heavily
used for locomotion, with no significant muscle atrophy and
only modest changes in fibre type composition (Tinker et al.,
1998); similarly, the seasonal fast in hedgehogs entails a loss
in total body protein of ~10%, irrespective of the duration of
hypothermia (Cherel et al., 1995). Thus, the comparatively
high loss of protein from the tail muscle of arousing tegus
strengthens the importance of this substrate as an energy source
during the fasting period of the first year cycle and implies a
differential rate of proteolysis in distinct muscle types,
preventing the impairment of functions like locomotion and
lung ventilation.
Our results suggest a downregulatory mechanism that acts
on the energy metabolism of tegu lizards, in which body size
apparently sets a limit to substrate storage capacity in the small
young and thus to their ability to survive during the prolonged
fast soon after hatching. In mammals, the standard rates of
metabolism are due mostly to mitochondrial respiration, of
which ~80% is coupled to ATP synthesis and ~20% is used to
compensate a proton leak across the inner membrane that
bypasses ATP production (Rolfe and Brown, 1997). A similar
composition apparently occurs in ectotherms (Hulbert and
Else, 1981; Brand et al., 1991). A regulatory effect on the rate
of ATP synthesis has been shown during the hypometabolic
condition by altering substrate supply to metabolic pathways
(Storey, 1997) and by influencing other molecular mechanisms
that define the rates of ATP production by the mitochondrial
inner membrane (Martin et al., 1999; St-Pierre et al., 2000).
The ATP demand for protein synthesis, ion pumping and other
energy-consuming processes is reduced in several hibernators
and facultative anaerobes, as discussed in the reviews by
Guppy et al. (1994) and by Boyer and Barnes (1999).
Attempts have been made to quantify the degree of
metabolic change in individual tissues of a few species,
although no general trend is apparent (Flanigan et al., 1991;
Land et al., 1993; Fuery et al., 1998). In the tegu, the
ventilatory pattern becomes episodic during dormancy, with
intervals lasting up to 26Ã?â??Ã?·min at 17Ã?â??Ã?°C; even so, the relative cost
of the work of breathing would account for an estimated 50%
of total metabolic rate (Andrade and Abe, 2000). This high
energy cost implies that the downregulation of cellular
mechanisms is largely unproportional in different tissues and
even among different muscle types in the tegu, involving
tissue-specific modulation of substrate flux through the
metabolic pathways. Given that the skeletal muscle mass
constitutes a high percentage of the vertebrate body, a
substantial reduction of the total energy cost may result from
the pronounced decrease of enzyme activities related to
substrate flux in the glycolytic pathway in this tissue, as
recently observed in dormant tegus (J. E. de Carvalho, M. S.
C. Bianconcini and S. C. R. de Souza, unpublished results). In
hypoxic hibernating frogs, there is a reduction of 50% in proton
leak in skeletal muscle as a consequence of a reduced electron
flux through the mitochondrial membrane (St-Pierre et al.,
S. C. R. de Souza and othersEnergy metabolism and scaling patterns in tegu lizards 317
2000); no change, however, is detectable in normoxic frogs.
While a role played by uncoupling proteins on the regulatory
change of proton leak rate remains controversial, much interest
has emerged regarding the paradoxical findings of an increased
expression of these proteins during induced starvation and its
association with fat metabolism (for a review, see Duloo and
Samec, 2000; Boss et al., 2000). The proteins may be involved
in the regulation of lipid use as an energy fuel and in the
control of body mass, with a more pronounced effect in the
predominantly fast glycolytic white muscles, associated with
their greater capacity to alternate between glucose and lipids
as substrates. Thus, the size of the adipose tissue mass may
affect both the degree of energy expenditure and substrate
preference of the large skeletal muscle mass in animals that
spontaneously undergo fasting periods during their annual
cycle.
To our knowledge, a scaling influence on the magnitude
of metabolic depression has not yet been the subject of
investigation at the cellular level, and therefore the regulatory
mechanisms involved are even less clear. In a phylogenetic
context, many cellular processes are known to be
allometrically related to body mass in the same way as is
standard metabolism (Else and Hulbert, 1985; Porter et al.,
1996). Hulbert and Else (2000) propose that a possible
unifying factor in this arrangement may be the amount and
lipid composition of the cell membranes. Besides the body
mass effect, these authors consider that such factors are
also under the influence of ontogenetic changes, dietary
manipulation and stress conditions, with the ensuing effects
being exerted on the activities of membrane-bound proteins,
such as sodium pumps and mitochondrial uncoupling proteins.
Thus, the potential for a regulatory effect on a variety of
energy-demanding processes would endow the cell membranes
with the capacity to influence energy expenditure during
metabolic depression as well, as shown in a study with
aestivating snails (Stuart et al., 1998). According to this idea,
a change in cell membrane structure and function would
modify the cost of living while maintaining the same factorial
proportion to body size. Given the results obtained with
the tegu, however, it is tempting to speculate that the
disproportionate mass of adipose tissue in young individuals
may transmit a signal of distinct amplitude to the tissues in
which energy is consumed, thus promoting a deviation in
allometric pattern concomitant with the depressing effect.
In conclusion, the present study contributes to our
understanding of the mechanisms underlying metabolic
depression associated with spontaneous fasting in terrestrial
reptiles and provides perspective on the means by which
seasonal events are conciliated with growth and
developmental changes during the early stages of the tegu
lizardÃ?¢ââ??‰â??¢s life cycle. The results obtained should aid in
elucidating potential mechanisms by which the balance
between body mass and energy expenditure may be
modulated, favouring a close relationship with the size of
available substrate stores in a state that allows no energy
intake and demands reduced expenditure.
This study was financed by a research grant from the State
Foundation for Research Support of SÃ?Æ?Ã?£o Paulo (FAPESP)
to S. C. R. de Souza. J. E. de Carvalho received scholarships
from the Federal Council of Research Support (PIBIC-CNPq)
and FAPESP.
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S. C. R. de Souza and others

 

[Beasty]

:shock: Umm... that makes my eyes hurt.
I'll have to read it all later. Thanks for digging it up tho!

 

[HorseCaak]

Too much reading for me... So what was the gist? When I get my Tegu in July from you, should I let it hibernate? I'm going to read the other forums to read more into that but I know you said you don't have to let them hibernate. I'm going to read how now...

 

[Toby_H]

I read the entire thing and I feel dumbier now than when I started....

For those seriously interested in reading it... if you click the link at the top and open the PDF it is MUCH easier to read...

I found this staement early in the article interesting:

The lizards were kept indoors in 120Ã?â??Ã?·litre (30 gallon) cages
equipped with incandescent lights set on an 8Ã?â??Ã?·h:16Ã?â??Ã?·h L
photo- and thermal-period, in addition to the sunlight diffusing
from outside.

So none of the animals used in the experiment had access to UVB light... I wonder what impression that made on their growth pattern... or did I read that wrong?

 

[GOT_TEGUS]

Good Read Bobby... I think I read it before lol

 

[Dom3rd]

Wow thats alot to read...i just printed it off i will read it later

 

[TanMan57]

Omg so many words!

 

[kpelzer]

i wanna know what the article says, i just dont want to read it, lol