Download Metabolic rate depression in animals

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Biol. Rev. (2004), 79, pp. 207–233. f Cambridge Philosophical Society
DOI : 10.1017/S1464793103006195 Printed in the United Kingdom
Metabolic rate depression in animals:
transcriptional and translational controls
Kenneth B. Storey* and Janet M. Storey
College of Natural Sciences, Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6
(E-mail : [email protected])
(Received 6 June 2002 ; revised 28 March 2003; accepted 14 April 2003)
ABSTRACT
Metabolic rate depression is an important survival strategy for many animal species and a common element
of hibernation, torpor, aestivation, anaerobiosis, diapause, and anhydrobiosis. Studies of the biochemical
mechanisms that regulate reversible transitions to and from hypometabolic states are identifying principles of
regulatory control that are conserved across phylogenetic lines and that are broadly applied to the control of
multiple cell functions. One such mechanism is reversible protein phosphorylation which is now known to
contribute to the regulation of fuel metabolism, to ion channel arrest, and to the suppression of protein synthesis
during hypometabolism. The present review focuses on two new areas of research in hypometabolism : (1) the role
of differential gene expression in supplying protein products that adjust metabolism or protect cell functions for
long-term survival, and (2) the mechanisms of protein life extension in hypometabolism involving inhibitory
controls of transcription, translation and protein degradation. Control of translation examines reversible phosphorylation regulation of ribosomal initiation and elongation factors, the dissociation of polysomes and storage of
mRNA transcripts during hypometabolism, and control over the translation of different mRNA types by differential sequestering of mRNA into polysome versus monosome fractions. The analysis draws primarily from current research on two animal models, hibernating mammals and anoxia-tolerant molluscs, with selected examples
from multiple other sources.
Key words : hypometabolism, metabolic rate depression, hibernation, anoxia tolerance, aestivation, protein synthesis, reversible phosphorylation, gene expression, polysome profiles.
CONTENTS
I. Introduction .................................................................................................................................................
II. Coordinate regulation of metabolic rate depression ..............................................................................
(1) Reversible phosphorylation regulation of metabolic enzymes ........................................................
(2) Reversible phosphorylation regulation of membrane ion channels and receptors ......................
III. Metabolic arrest and suppression of protein synthesis ...........................................................................
(1) Control of translation by reversible protein phosphorylation .........................................................
(2) Ribosome aggregation state .................................................................................................................
(a) Differential distribution of individual mRNA species ................................................................
(b) Anticipatory up-regulation .............................................................................................................
IV. Metabolic arrest and suppression of protein degradation .....................................................................
V. Metabolic arrest and gene expression ......................................................................................................
(1) RNA synthesis .......................................................................................................................................
(2) Gene discovery ......................................................................................................................................
(3) Hypometabolism-induced gene expression .......................................................................................
(4) Hibernation-induced gene expression ................................................................................................
* Address for correspondence.
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Kenneth B. Storey and Janet M. Storey
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(a) a2-Macroglobulin ............................................................................................................................
(b) PDK4 ................................................................................................................................................
(c) Myosin ..............................................................................................................................................
(d ) Fatty acid binding proteins ............................................................................................................
(e) Uncoupling proteins ........................................................................................................................
(5) Anoxia-induced gene expression .........................................................................................................
(a) Gene expression during anoxia in turtles .....................................................................................
(b) Gene expression during anoxia in marine snails ........................................................................
(c) cGMP mediation of anoxia-induced gene expression ................................................................
VI. Conclusions ..................................................................................................................................................
VII. Acknowledgements ......................................................................................................................................
VIII. References ....................................................................................................................................................
I. INTRODUCTION
The ability to suppress metabolic rate strongly and sink into
a hypometabolic state is a life-saving mechanism for many
organisms. When animals are faced with environmental
stresses that threaten normal life, limit food availability or
impose severe challenges to their physiology, they can
retreat into a hypometabolic or dormant state where they
remain until conditions are again conducive to active life.
Examples of hypometabolism abound as animal responses
to high and low temperature, oxygen deprivation, food
restriction and water limitation (Storey & Storey, 1990). In
mammals and birds, hypometabolism ranges from nightly
torpor in many small species to winter hibernation that can
last as long as nine months for some mammals living in cold
Arctic or alpine habitats (Geiser, 1988 ; Wang & Wolowyk,
1988). Aestivation allows many animals, including lungfish,
frogs, toads and snails, to survive through long dry seasons
(Pinder, Storey & Ultsch, 1992; Abe, 1995; Land & Bernier,
1995) whereas diapause arrests the developmental cycle of
many insects and other animals to permit survival through
harsh conditions and/or coordinate transitions to the next
life stage among the many individuals in a population
(Danks, 1987 ; Hairston & Caceres, 1996; Podrabsky &
Hand, 1999; Denlinger, 2001). Many organisms are excellent facultative anaerobes and one of the keys to survival of
long-term oxygen deprivation is metabolic rate depression
that lowers tissue energy demand to a level that can be
supplied by pathways of fermentative ATP production alone
(Storey, 1993; Grieshaber et al., 1994; Lutz & Storey, 1997;
Hochachka & Lutz, 2001; Jackson, 2001). In most of
the cases of hypometabolism cited above, metabolic rate
is lowered to between 5 and 40 % of the resting rate of
the normal animal and most experience little or no change
to the internal environment of their cells and organs so
that arousal can be rapid when favourable environmental
conditions return (Guppy & Withers, 1999). Hypometabolism can also be taken to extremes by cryptobiotic
organisms (including many seeds, spores, cysts, and eggs).
In these metabolic rate is typically less than 5% of normal
and, in many cases, a virtually ametabolic state exists
(Crowe, Hoekstra & Crowe, 1992 ; Guppy & Withers, 1999;
Clegg, 2001). Such organisms experience a profound change
in their internal environment often involving extreme
dehydration and the addition of high levels of protectants to
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stabilize cellular macromolecules for periods of dormancy
that can extend to many years.
Studies of hypometabolism in nature have been, and still
are, episodic ; both physiological and biochemical studies
have progressed to different stages in different animal models
and the primary focus of research is often quite different
with each animal model. Much of the early work with all
models centred in two areas : (1) quantifying physiological
parameters of metabolic suppression including the percentage reduction in metabolic rate and factors such as
bradycardia, apnoeic breathing patterns, hypercapnia, and
acidosis that are common elements of hypometabolism
across phylogeny, and (2) analyzing changes in the patterns
of fuel use, end product accumulation, and the biosynthesis
of the low molecular weight protectants that are present in
various systems. More recently the focus of much research
has shifted to the biochemistry of metabolic rate depression
and studies with multiple systems (e.g. hibernating mammals, anoxia-tolerant molluscs and turtles, estivating amphibians and snails, diapausing insects, anhydrobiotic brine
shrimp) are uncovering the molecular mechanisms that
support and regulate metabolic suppression (recent reviews :
Storey & Storey, 1990; Guppy, Fuery & Flanigan, 1994 ;
Hand & Hardewig, 1996 ; Hochachka et al., 1996 ; Storey,
1996, 2001, 2002 a ; Brooks & Storey, 1997; Guppy &
Withers, 1999; Bickler, Donohoe & Buck, 2001 ; Denlinger,
2001; Hochachka & Lutz, 2001). Principles of biochemical
regulation are emerging that are widely conserved across
phylogenetic lines and show the unifying elements of metabolic arrest.
The present review focuses on recent advances in understanding the role of differential gene expression in metabolic
depression and the regulatory controls on transcription and
translation that allow these energy-expensive processes to be
suppressed when organisms enter a hypometabolic state.
However, first we will briefly review some of the earlier work
on the biochemical regulation of metabolic rate depression,
highlighting in particular the central role of reversible protein phosphorylation in coordinating the suppression of
many metabolic loci. It should be noted that the research
considered herein derives from laboratory experimentation
using animals induced to enter a hypometabolic state by the
acute imposition of stress (e.g. exposure to a nitrogen gas
atmosphere for studies of anoxia tolerance in marine snails).
The use of controlled experimental conditions allows the
Metabolic rate depression in animals
biochemist to identify definitive and typically maximal responses to an imposed stress. However, in nature, the stress
conditions that initiate hypometabolism may be much more
variable (e.g. less severe, shorter duration, build up slowly
over a long period, or be mixed with other stresses) and
where possible, biochemical responses identified from laboratory studies should also be assessed in animals sampled
from field conditions.
II. COORDINATE REGULATION OF
METABOLIC RATE DEPRESSION
In recent years, a major focus of research in hypometabolism has been the molecular mechanisms that regulate
metabolic rate depression. To maintain homeostasis in
any cell, the rate of ATP utilization by the myriad of
ATP-consuming processes must match the rate of ATP
production by central pathways of fuel catabolism. All
cells have mechanisms for increasing ATP production
when energy demand is high and for scaling back production when energy demand declines. Conversely, situations that limit ATP production will quickly affect the rates
of ATP-utilizing processes. Indeed, studies have shown
that a hierarchy of sensitivity to ATP supply exists, the
pathways of macromolecular biosynthesis (such as protein
synthesis, RNA/DNA synthesis) being more sensitive to
ATP availability than various other activities such as
transmembrane ion pumping (Buttgereit & Brand, 1995).
When ATP supply is compromised in intolerant systems,
the ability to compensate or adjust to this energy stress
is often very limited and cells/organisms quickly succumb
to injury or death. The highly injurious effects of hypoxia
or hypothermia on the core organs of most mammals
offer prime examples of the metabolic damage caused
when energy supplies fail (Hochachka, 1986). In particular,
ATP limitation has a major effect on one of the most energyexpensive functions of cells, the maintenance of membrane
potential difference. ATP limitation causes a rapid imbalance in the opposing rates of ion transport across
membranes by ATP-dependent ion pumps versus ATPindependent ion channels so that membrane potential
difference is quickly dissipated with multiple negative
consequences (Perez-Pinzon et al., 1992 ; Hochachka & Lutz,
2001). It is obvious, then, that the transition from an active
state to a hypometabolic or dormant state where metabolic
rate (a measure of ATP turnover) may be as little as 1–5% of
the former resting metabolic rate of the active organism
must require a rebalancing of the rates of both ATPproducing and ATP-utilizing reactions. Metabolic controls
must be utilized to coordinate a net suppression of all
metabolic functions to achieve a new net rate of ATP turnover that can be sustained over the long-term by the ‘ onboard ’ fuel reserves of the organism. The question is how is
this achieved ?
Energy savings during hypometabolism come from several sources. Hypometabolism typically goes hand-in-hand
with a lack of voluntary muscle movements so the energy
costs of skeletal muscle work are nominal. Heart beat
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and respiration rate are greatly reduced, breathing often
becomes intermittent, and kidney filtration rate is reduced.
Organisms do not eat so the energetic costs of digestion,
nutrient absorption, and peristalsis are eliminated. A substantial part of total energy savings comes from the suppression of these physiological activities. Metabolic rate is
also influenced by factors including the partial pressures
of O2 and CO2, pH and temperature. Changes in these extrinsic parameters during hypometabolism can account for a
portion of the metabolic inhibition (Barnhart & McMahon,
1988 ; Geiser, 1988; Pedler et al., 1996 ; Guppy et al., 2000).
Major energy savings in the hypometabolic state come
from the specific inhibition of the rates of multiple cellular
processes providing an intrinsic component to metabolic
arrest. For example, mitochondria isolated from tissues of
torpid ground squirrels (Spermophilus sp.) show rates of state
3 respiration that are 30–66 % lower than the rates in
euthermic animals (summer active or interbout arousal)
yet this stable inhibition is rapidly reversed upon arousal
(Fedotcheva et al., 1985; Gehnrich & Aprille, 1988 ; Martin
et al., 1999). Whole cells and tissues from hypometabolic
animals can also retain an intrinsic metabolic depression
when assessed in vitro. The metabolic rate of hepatopancreas
cells from estivating land snails (Helix aspersa) was only onethird to one-half that of controls (Bishop & Brand, 2000;
Guppy et al., 2000) and the metabolic rate of liver slices from
estivating frogs (Neobatrachus centralis) was 45% of that for
slices from control frogs (Fuery et al., 1998).
The suppression of multiple individual metabolic processes must be coordinated to achieve a net suppression that
balances the rates of ATP-producing and ATP-consuming
processes at a new lower net rate of ATP turnover. Rates of
substrate utilization are always strongly suppressed in hypometabolic states in order to extend greatly the time that
the internal fuel reserves can sustain survival. ATP-consuming processes must similarly be suppressed. The concept
of ‘channel arrest’ was one of the first ideas to be explored
(Perez-Pinzon et al., 1992). If the rate of ATP production is
reduced in the hypometabolic state, then the rate of ATP
expenditure by ATP-dependent ion pumps must also be
reduced and, to maintain homeostasis, the opposing
facilitated diffusion of ions through ion channels must also
be suppressed. Channel arrest predicts a coordinated suppression of membrane channels and pumps in both plasma
and organelle membranes and considerable evidence for
the mechanism has accumulated (Bickler et al., 2001;
Hochachka & Lutz, 2001). In neurons of anoxia-tolerant
turtles (Pseudemys scripta), the main energy-saving mechanism
might be the down regulation of firing rates or synaptic
transmission involving localized channel suppression and
termed ‘ spike arrest ’ by Sick et al. (1993). Other ‘opposing ’
functions should also be coordinately suppressed during
entry into the hypometabolic state such as the rates of synthesis versus degradation of macromolecules including DNA,
mRNA, proteins and membrane phospholipids. All rate
processes should be slowed so that all macromolecules
experience significant life extension (perhaps 10-fold or
more) and cells remain fully competent to return rapidly to
the active state with all metabolic functions intact even after
very long periods of dormancy.
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Priorities for energy expenditure are also re-arranged
during hypometabolism to sustain critical functions at the
expense of others (Hochachka et al., 1996). This principle
was illustrated in studies using isolated hepatocytes from
anoxia-tolerant turtles (Chrysemys picta bellii ). Incubation
under anoxic conditions resulted in a rapid 94 % decrease in
ATP turnover and a reorganization of the proportion of ATP
turnover devoted to five main ATP-consuming processes in
liver cells : Na+,K+-ATPase activity, protein synthesis,
protein degradation, gluconeogenesis, and urea synthesis.
Respectively, these consumed 28, 36, 17, 17 and 3 % of
ATP turnover in normoxic cells (Hochachka et al., 1996).
However, even though the activity of Na+,K+-ATPase
decreased by 75 % in anoxic hepatocytes (supporting
the theory of channel arrest) (Buck & Hochachka, 1993), the
other energy-consuming processes were even more strongly
suppressed (e.g. gluconeogenesis was undetectable). As a
result the proportion of cellular ATP turnover supporting
Na+K+-ATPase activity actually rose to nearly 75% in
anoxic cells.
However, although multiple metabolic processes are virtually shut down in the hypometabolic state, they must
be retained in readiness so that normal metabolic function
can be rapidly restored during arousal. Thus, hypometabolism is not generally associated with a major loss of
metabolic capacity but instead reversible controls are applied
coordinately to suppress the rates of all metabolic processes
while leaving intact the potential to return rapidly to the
normal state.
(1 ) Reversible phosphorylation regulation of
metabolic enzymes
Much recent work has been aimed at understanding the
molecular mechanisms that provide reversible metabolic
suppression and coordinate the transitions to and from the
hypometabolic state. The regulation of fuel catabolism in
hypometabolism was the first area to receive attention, and
studies by our laboratory and others showed the repeated
occurrence across wide phylogenetic lines of one mechanism
that had powerful regulatory control over key enzymes of
fuel catabolism. This is reversible protein phosphorylation
controlled by the actions of protein kinases and protein
phosphatases. The importance of this mechanism was first
demonstrated in the regulation of pyruvate kinase (PK) in
anoxia-tolerant marine molluscs (reviewed in Storey &
Storey, 1990 ; Storey, 1993). Anoxia-induced phosphorylation of PK caused a strong decrease in enzyme activity and
major changes in kinetic properties that virtually shut off the
enzyme during anaerobiosis. This facilitates a rerouting of
carbohydrate flux that directs phosphoenolpyruvate away
from PK and instead into the phosphoenolpyruvate carboxykinase reaction and onwards into the reactions of succinate synthesis. Subsequently, the suppression of multiple
enzymes of anaerobic glycolysis has been shown to be
coordinated by this same reversible phosphorylation mechanism in various species of marine molluscs and other invertebrates and, furthermore, the same mechanisms regulate the
suppression of carbohydrate catabolism during aestivation
in land snails (Storey, 1993; Brooks & Storey, 1997).
Kenneth B. Storey and Janet M. Storey
Reversible phosphorylation control over enzymes of fuel
metabolism is now recognized as one of the major principles
of metabolic regulation in hypometabolism and is known to
contribute to metabolic suppression and the reorganization
of fuel metabolism in anoxia-tolerant animals, estivating
anurans and molluscs, and hibernating mammals (for
reviews see Storey, 1996, 1997, 2002 a).
For example, inhibition of carbohydrate catabolism during mammalian hibernation (facilitating a switch to lipid
oxidation) is aided by a strong phosphorylation-mediated
inhibition of pyruvate dehydrogenase (PDH), the enzyme
that gates carbohydrate entry into the tricarboxylic acid
cycle. During hibernation, the amount of PDH present in
the dephosphorylated active a form drops precipitously
(Fig. 1 A) so that the percentage of the enzyme that is
active falls from 60–80 % in euthermia to less than 5% in
hibernation (Brooks & Storey, 1992 a ; Storey, 1997). PDH
activity is similarly suppressed in estivating snails (Brooks
& Storey, 1992b). The application of metabolic control
analysis to isolated hepatopancreas cells from control versus
estivating Helix aspersa showed that at least 75 % of the reduction in mitochondrial respiration in aestivation was due
to primary changes in the kinetics of substrate oxidation,
most probably the result of reduced activities of substratesupplying enzymes (e.g. PDH and others) or enzymes of
mitochondrial oxidative metabolism (e.g. citrate synthase
and cytochrome c oxidase both decreased in estivating
H. aspersa and other land snails) (Bishop, St.-Pierre & Brand,
2000). Reversible phosphorylation control over selected
enzymes is a key element in this regulation.
( 2) Reversible phosphorylation regulation of
membrane ion channels and receptors
Given the role of reversible phosphorylation in the control
of fuel catabolism in hypometabolism, and therefore in the
rate of ATP generation, it seemed likely that this mechanism
should also be involved in regulating the myriad of ATPutilizing processes in cells. Recent studies have addressed the
control of energy-expensive metabolic processes beginning
with the control of membrane ion pumps. These utilize a
huge proportion of total cellular energy to pump ions against
their concentration gradients ; for example, the Na+,K+ATPase alone is responsible for 5–40 % of total ATP turnover depending on cell type (Claussen, 1986). Recent
experimental studies are showing that reversible phosphorylation control of ion pumps is an integral mechanism of
natural hypometabolism. For example, in hibernating
ground squirrels, Spermophilus lateralis, Na+,K+-ATPase
activities were uniformly reduced in most organs to just
40–60 % of the euthermic values (assayed at 25 xC) (Fig. 1 B).
Notably, activity was not suppressed in heart, an organ that
must continue to perform muscle work through torpor.
In vitro studies confirmed that the mechanism of this inhibition was protein phosphorylation. The activity of skeletal
muscle Na+,K+-ATPase from euthermic animals was
strongly reduced by incubations that stimulated protein
kinases whereas activity was restored after alkaline phosphatase treatment (Fig. 1 C) (MacDonald & Storey, 1999).
Activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase is
Metabolic rate depression in animals
1.4
A
Pyruvate
dehydrogenase a
1.2
1.0
0.8
0.6
0.4
0.2
0
*
*
Heart
Kidney
Activity (units g –1 wet mass)
Activity (units g –1 wet mass)
1.6
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300
250
200
150
B
Na+, K+– ATPase
*
Euthermic
Hibernating
100
80
40
*
20
0
12
Activity (units g –1 wet mass)
*
60
Muscle
Heart
C
b
Kidney
Liver
Reversible phosphorylation
of Na+, K+– ATPase
10
Control
ATP + cAMP
Alk. P’tase
8
6
a,b
4
2
a
0
Euthermic
Hibernating
Fig. 1. Effect of hibernation on the activities of (A) the active form of pyruvate dehydrogenase (PDHa) and (B) Na+,K+-ATPase in
tissues of ground squirrels (Spermophilus lateralis). (C) Effects of stimulating endogenous protein kinase A (by incubation with
10 mmol lx1 ATP, 10 mmol lx1 MgCl2, 0.3 mmol lx1 cAMP) and subsequent alkaline phosphatase (Alk. P’tase) treatment (10 units)
on Na+,K+-ATPase activity in skeletal muscle extracts from euthermic and hibernating ground squirrels. Data are means¡SEM,
N=4. *, Significantly different from the corresponding euthermic value, P<0.05. a – Significantly different from the untreated
control sample ; b – significantly different from the protein kinase treated sample, P<0.05. From Brooks & Storey (1992a) and
MacDonald & Storey (1999).
similarly reduced during hibernation ; both a decrease in
enzyme amount (per milligram of SR protein) and in
enzyme specific activity (per milligram of Ca2+-ATPase
protein) occur in hibernator muscle (Malysheva et al., 2001).
The reduction in specific activity is probably due to protein
phosphorylation. Other proteins involved in SR calcium
signaling are also suppressed during hibernation ; the SR
calcium-release channel (ryanodine receptor) decreased by
50 % in hibernation and levels of most SR calcium binding
proteins (e.g. sarcalumenin, calsequestrin) were 3–4-fold
lower in hibernating, compared with summer-active, animals (Malysheva et al., 2001).
The control of neuronal activity, including the regulation
of ion pumps, ion channels, and membrane receptors, has
been extensively studied in brain of anoxia-tolerant freshwater turtles as a model system for exploring the effects of
ischemia on the vertebrate brain (recent reviews Bickler
et al., 2001 ; Hochachka & Lutz, 2001). Multiple mechanisms
of ion channel arrest have been identified with reversible
phosphorylation of channels or receptor subunits playing a
substantive role (Bickler et al., 2001). Indeed, all voltagegated channels (Na+, Ca2+, K+) are sensitive to phosphorylation by multiple protein kinases and roles for reversible
protein phosphorylation in hypoxic suppression are being
identified. One of the most complete pictures of reversible
phosphorylation control of neuronal function during hypoxia/anoxia is the regulation of the N-methyl-D-aspartatetype glutamate receptor (NMDAR) (Bickler et al., 2001).
Several kinases including protein kinase A, protein kinase C
and tyrosine kinase phosphorylate NMDARs and increase
activity whereas the opposing phosphatases decrease activity.
Suppression of NMDAR activity is critical for the survival
of turtle neurons in anoxia and inhibition of phosphatase
action (via specific phosphatase inhibitors or by flooding with
cAMP to stimulate kinases) prevents the normal silencing of
NMDARs under anoxia (Bickler et al., 2001).
Most recently, the focus of research on metabolic arrest
by our laboratory and others has turned to the regulation of
Kenneth B. Storey and Janet M. Storey
212
Table 1. Protein synthesis inhibition during hypometabolism : selected examples from hibernation, anoxia tolerance, aestivation
and diapause. The rate of protein synthesis in hypometabolism is shown as a percentage of the corresponding control rate
Hypometabolic rate
of protein synthesis
( % of control)
Species
Condition
Tissue
Spermophilus tridecemlineatus
Hibernation
Littorina littorea
Trachemys scripta
Anoxia
Anoxia
Anoxia
Carassius carassius
Anoxia
Helix aspersa
Neobatrachus centralis
Austrofundulus limnaeus
Artemia franciscana
Aestivation
Aestivation
Diapause
Anaerobic quiescence
Brain
Brain
Kidney
Brown adipose
Hepatopancreas
Hepatocytes
Heart, liver, brain,
muscle, others
Liver
Heart, muscle
Hepatopancreas
Liver
Whole embryo
Whole embryo
0.04*
34
15
104
50
8
y0
5
y50
30
33
7
8
Reference
Frerichs et al. (1998)
Frerichs et al. (1998)
Hittel & Storey (2002 b)
Hittel & Storey (2002 b)
Larade & Storey (2002 c)
Land et al. (1993)
Fraser et al. (2001)
Smith et al. (1996 b)
Smith et al. (1996 b)
Guppy et al. (2000)
Fuery et al. (1998)
Podrabsky & Hand (1999, 2000)
Hofmann & Hand (1994)
* Compares in vivo protein synthesis rates at euthermic (37 xC) versus hibernating (7.5 xC) body temperatures. Other values for hibernators
are for in vitro assays that compare tissue extracts from euthermic and hibernating animals at a constant temperature.
other energy-expensive cellular processes (e.g. transcription
and translation) and to the role that gene expression plays in
producing proteins with specific functions in hypometabolism. The remainder of this review focuses primarily on
new studies in these two areas and on our studies with two
animal systems in particular – hibernating mammals (ground
squirrels of the genus Spermophilus) and anoxia-tolerant
marine snails (the periwinkle, Littorina littorea). The new data
demonstrate striking parallels in principles and mechanisms
of metabolic control that are involved in hypometabolism in
both systems.
III. METABOLIC ARREST AND SUPPRESSION
OF PROTEIN SYNTHESIS
Protein synthesis is an energy-expensive process, requiring
about five ATP equivalents per peptide bond formed and
consuming a substantial portion of the total ATP turnover
of all cells and organs (e.g. 36 % in normoxic turtle hepatocytes ; Hochachka et al., 1996). Protein synthesis is well
known to be sensitive to the availability of energy and amino
acids and the rate of protein synthesis is suppressed during
starvation and during severe hypoxia in oxygen-sensitive
systems (Casey et al., 2002; DeGracia et al., 2002 ; Mordier
et al., 2002). Thus, it is reasonable to predict that protein
synthesis would be strongly suppressed in order to save
energy during hypometabolism. Indeed, recent studies with
several systems confirm this ; strong reductions in the rate
of protein synthesis occur as part of hibernation, anoxia
tolerance, aestivation and diapause (Table 1) ( Joplin &
Denlinger, 1989 ; Hofmann & Hand, 1992b ; Land, Buck &
Hochachka, 1993 ; Fuery et al., 1998 ; Hand, 1998;
Podrabsky & Hand, 2000; Fraser et al., 2001 ; Larade &
Storey, 2002c). For example, in hibernating thirteen-lined
ground squirrels (S. tridecemlineatus), the rate of 14C-leucine
incorporation into protein in vivo in brain of torpid animals
was only 0.04% of the mean value in active squirrels (Frerichs et al., 1998). Part of this rate suppression is due to the
body temperature differences between the two states (37 xC
for euthermia versus approximately 7.5 xC for torpor) but
when protein synthesis by brain extracts was assessed in vitro
at 37 xC (eliminating the extrinsic effect of temperature), the
intrinsic rate of synthesis in extracts from hibernator brain
was still just 34 % of the euthermic value (Frerichs et al.,
1998). Similarly, the rate of 3H-leucine incorporation into
protein in S. tridecemlineatus kidney extracts in vitro was just
15% of the euthermic value but, interestingly, brown adipose tissue showed no reduction in protein biosynthesis
during torpor (Hittel & Storey, 2002b).
In addition to an overall suppression of protein synthesis,
the proportion of cellular ATP turnover devoted to protein
synthesis drops in hypometabolism. In anoxic turtle hepatocytes, for example, ATP use by protein synthesis fell from
24.4% of total ATP turnover under normoxia to just 1.6 %
in anoxia (Land et al., 1993). In killifish embryos, the proportion dropped from 36% in normal developing embryos
to negligible when embryos entered diapause (Podrabsky &
Hand, 2000). Furthermore, suppression of protein synthesis
can be a rapid event ; the rate of 3H-leucine incorporation
into protein in hepatopancreas extracts from Littorina littorea
was reduced by 50 % within just 30 min when snails were
transferred from aerobic to anoxic conditions and this value
was sustained over 48 h of anoxia exposure (Larade &
Storey, 2002 c). This rapid suppression suggests that protein
synthesis inhibition is a pro-active response by cells that is
an integral part of metabolic arrest rather than a reactive
response to ATP limitation. Indeed, ATP content and
energy charge did not change significantly over the course
of 72 h of anoxia exposure in L. littorea tissues (Churchill
& Storey, 1996).
Metabolic rate depression in animals
Two factors may contribute to the inhibition of protein
synthesis during hypometabolism : (1) mRNA substrate availability, and (2) specific inhibition of the ribosomal translational machinery. Substrate availability is always a factor
in any metabolic process but there appears to be little, if any,
change in global mRNA levels in most hypometabolic systems. In hibernator tissues, for example, neither total RNA
nor mRNA levels changed and transcript levels of four
constitutive genes did not fall during hibernation, implicating no net loss of the mRNA for constitutively active
genes during torpor (Frerichs et al., 1998 ; O’Hara et al.,
1999 ; Knight et al., 2000). Evidence from cDNA array
screening of hundreds of genes in multiple tissues of ground
squirrels and bats also indicates that transcript levels of most
genes are unaffected during hibernation (Eddy & Storey,
2001 ; Hittel & Storey, 2001). In anoxia-tolerant turtles, total
RNA levels were unaffected in all organs and poly(A)+
RNA levels remained constant in four organs over 16 h of
submergence anoxia at 15 xC but rose by 30% in white
skeletal muscle (Douglas et al., 1994). Studies with crucian
carp (Carassius carassius) suggested that total RNA synthesis
(of which the largest component is rRNA) is reduced in
brain but increased in heart and liver during anoxia (Smith
et al., 1999 a) ; the implications for mRNA content are not
clear. Translatable RNA levels did not change in Artemia
franciscana during anoxia exposure (Hand, 1998). Furthermore, mRNA appears to remain intact during hypometabolism ; analysis of various specific gene transcripts via
Northern blotting failed to detect reductions in transcript
sizes during hypometabolism (Cai & Storey, 1996 ; Fahlman, Storey & Storey, 2000 ; Knight et al., 2000 ; Hittel &
Storey, 2001, 2002 a, b ; Larade, Nimigan & Storey, 2001;
Larade & Storey, 2002 b) and the distribution of poly(A)
tail lengths showed no general shortening of tails in torpid
ground squirrels as would be expected if transcripts were
degrading during hibernation (Knight et al., 2000). Overall,
then, global mRNA substrate availability does not appear to
be a factor in protein synthesis inhibition in hypometabolism. Control of protein synthesis is vested instead in
strong regulation of translation by the ribosomes. Two
mechanisms that contribute to the arrest of protein synthesis
have received experimental attention : (1) reversible phosphorylation control of the translational machinery, and
(2) the state of ribosome assembly.
(1 ) Control of translation by reversible
protein phosphorylation
In brain of hibernating ground squirrels both translation
initiation and polypeptide elongation are inhibited (Frerichs
et al., 1998). Inhibition of both was traced to the regulation
of specific functional proteins in ribosomes. The inhibition
of translation initiation was linked with the eukaryotic
initiation factor 2 (eIF2) which introduces initiator methionyl-tRNA into the 40S ribosomal subunit (Fig. 2). The
mechanism involved is phosphorylation of the alpha-subunit
of eIF2 (eIF2a). This mechanism is widely known as a
means of inhibiting eukaryotic protein synthesis and even
small amounts of phospho-eIF2a significantly inhibit
global protein synthesis by blocking the initiation of nascent
213
polypeptides (Rhoads, 1993 ; Mikulits et al., 2000). For example, in rat brain homogenates obtained after 90 min reperfusion following 10 min cardiac arrest, protein synthesis
was reduced by 85 % and this was associated with approximately 23 % of eIF2a in the phosphorylated state compared
with approximately 1 % under control conditions (DeGracia
et al., 2002). Phosphorylated eIF2a acts as a dominant
inhibitor of the guanine nucleotide exchange factor eIF2B
and prevents the recycling of eIF2a between successive
rounds of peptide synthesis (Clemens, 2001 ; DeGracia et al.,
2002). Conditions known to increase eIF2a phosphorylation
include virus infection, heat shock, iron deficiency, nutrient
deprivation, changes in intracellular [Ca2+], induction of
apoptosis, the unfolded protein response, hypoxia/anoxia
and postischemic reperfusion (Munoz et al., 2000 ; Althausen
et al., 2001; Clemens, 2001; Martin de la Vega et al., 2001;
Casey et al., 2002 ; DeGracia et al., 2002).
Analysis of eIF2a regulation uses two kinds of antibodies – one that detects eIF2a protein in general and one
that is specific for the peptide containing the phosphorylated
residue. This analysis has consistently shown a strong increase in the amount of phospho-eIF2a in hypometabolic
states. For example, phospho-eIF2a content increased
markedly in kidney of hibernating squirrels but with no
change in total eIF2a protein (Hittel & Storey, 2002 b). In
brain of euthermic ground squirrels phospho-eIF2a content
was less than 2% of the total but rose to approximately 13%
during hibernation (Frerichs et al., 1998). The same mechanism occurs during anoxia in the marine snail, L. littorea.
Total eIF-2a content in hepatopancreas was unaffected over
a cycle of anoxia and aerobic recovery but the amount
of phospho-eIF2a rose approximately 15-fold in anoxic
animals, compared with aerobic controls (Fig. 3) (Larade &
Storey, 2002c). However, when oxygen was reintroduced,
phospho-eIF2a fell to control levels or below within 1 h.
Although the focus to date on the suppression of translation initiation in hypometabolism has been on eIF2a, other
regulatory factors may be involved and these await study
in hypometabolic systems. For example, the eukaryotic initiation factor 5 (eIF5), which acts as a GTPase-activating
protein to promote GTP hydrolysis within the 40S initiation
complex (consisting of 40S*eIF3*AUG*Met-tRNA(f)*eIF2*
GTP) (Fig. 2), is also regulated by reversible protein phosphorylation (Majumdar et al., 2002). Eukaryotic initiation
factor 4E binding protein (4E-BP1) is also regulated in this
manner (Fig. 2) and was significantly dephosphorylated
during ischemia in rat brain (at the same time as eIF2a was
phosphorylated) (Martin de la Vega et al., 2001). Dephosphorylation allows the 4E-BP1 to bind to and inhibit eIF4.
Another subunit of this initiation factor, eIF4G, shows
proteolytic fragmentation after ischemia/reperfusion in rat
brain. Fragmentation changes the types of mRNAs that
can be translated because intact eIF4G is needed to allow
eIF4E-bound m7G-capped mRNAs (the vast majority of
cellular mRNAs) to bind to the small ribosomal subunit
(DeGracia et al., 2002). Without intact eIF4G, message
selection changes dramatically to favour only those messages that contain an internal ribosome entry site (IRES)
(Gingras, Raught & Sonenbert, 1999). In mammals, many
of the messages that contain an IRES code for proteins
214
Kenneth B. Storey and Janet M. Storey
Fig. 2. Mechanism of translation initiation focusing on the control of eukaryotic initiation factors 2 (eIF2) and 4 (eIF4) via reversible
protein phosphorylation. Hexagonal figures show initiation factors, indicated with numbers ; ovals show ribosomal subunits and eIF
Metabolic rate depression in animals
involved in apoptosis and recent work suggests that the
protein synthesis inhibition response, characterized by eIF2a
phosphorylation and eIFG fragmentation, is typical of the
initiation of apoptosis in response to various stresses (summarized by DeGracia et al., 2002). Amino acid starvation
also inhibits protein synthesis through these same mechanisms and leads to up-regulation of selected gene transcripts
containing an IRES despite the general inhibition of the
cap-dependent translational apparatus during starvation
(Fernandez et al., 2001 ; Mordier et al., 2002). Hypometabolism is, of course, a form of starvation ; organisms stop
eating and switch to a dependence on internal fuel reserves.
It is possible, then, that the protein synthesis inhibition
response and/or other selected adjustments for hypometabolism grew out of the pre-existing mechanisms that regulate suppression of non-essential processes either (a) during
starvation or (b) in response to ischemic stress. Modified
controls or an additional layer of control could be added to
avoid or reverse signals that would in other organisms lead
to apoptosis. Furthermore, it is seems probable that message
selection (via an IRES or other mechanism) represents the
key to the specific up-regulation of selected genes during
natural hypometabolism.
Of note in this regard is a key new study that demonstrates the presence of an IRES in the mRNA transcript
of hypoxia-inducible factor-1a (HIF-1a) (Lang, Kappel
& Goodall, 2002). HIF-1 mediates a variety of gene upregulation responses to hypoxia in oxygen-sensitive organisms to produce proteins that either increase anaerobic ATP
production such as glycolytic enzymes and glucose transporters or enhance oxygen delivery such as erythropoietin
and vascular endothelial growth factor (VEGF) (Bunn &
Poyton, 1996). HIF-1 activity is primarily regulated via
availability of the HIF-1a subunit which is expressed constitutively but rapidly degraded ; by contrast, HIF-1b subunit levels are always substantial. When oxygen is low,
degradation is inhibited and HIF-1a content increases, dimerizes with HIF-1b and translocates to the nucleus to
activate gene expression. However, to be effective in hypoxia
signaling, the translation of HIF-1a itself must continue
under hypoxia. Using polysome profile analysis, Lang et al.
(2002) demonstrated that HIF-1a mRNA remained in the
polysome fraction during hypoxia (despite an overall 50%
decrease in protein synthesis in hypoxic cells). The long and
GC-rich 5k-untranslated region (UTR) of HIF-1a suggested
the presence of an IRES and this was confirmed with a series
of transfection studies using luciferase downstream reporters
(Lang et al., 2002). The presence of this IRES allows HIF-1a
translation to be maintained under hypoxic conditions that
are inhibitory to cap-dependent translation. Interestingly,
VEGF also contains an IRES (Miller et al., 1998). As will
be discussed in a later section, new research with both mammalian hibernators and anoxia-tolerant snails shows that
215
a number of genes that are up-regulated during hypometabolism are distributed very differently across a polysome/
monosome gradient than are constitutively expressed genes
and it can be proposed that the mRNA transcripts of these
genes may contain an IRES in the 5kUTR that allow their
translation during hypometabolism.
The upstream regulators of the initiation factors also need
study. Mammalian cells have four eIF2 kinases, each activated by a distinct stress. Heme-regulated inhibitor (HRI)
is activated by hemin deprivation, double-stranded RNAdependent kinase (PKR) is activated by double-stranded
RNA, PKR-like endoplasmic reticulum kinase (PERK) is
activated by unfolded proteins and ischemia/reperfusion,
and the general amino acid control eIF2 protein kinase
(GCN2) is activated by serum or amino acid starvation
(Kumar et al., 2001). Identification of the protein kinase(s)
that mediates the suppression of eIF2a during hypometabolism will be a major advance since this kinase will likely
also regulate various other downstream targets. Furthermore, Harding et al. (2000) showed that whereas the eIF2a
kinases, PERK and GCN2, repress translation of most
mRNAs by targeting eIF2a, they also selectively increase
translation of the transcription factor ATF4 (activating
transcription factor) which in turn increases transcription of
selected genes. This suggests a method by which a single
signal, in this case a signal acting via PERK, can have both
generalized (inhibition of protein synthesis) and specific
(up-regulation of selected genes) actions and may provide
a model for understanding similar situations of selective gene
up-regulation during hypometabolism.
Regulation of eIF2a could also come from control over
the dephosphorylation of the phosphoprotein. Both protein
phosphatase (PP) 1 and 2A can dephosphorylate phosphoeIF2a and PP-1 appears to have this role in vivo (Redpath &
Proud, 1990). However, studies to date have given mixed
results as to whether inhibitory control of phosphatases is
a factor in phospho-eIF2a accumulation during ischemia/
reperfusion (Munoz et al., 2000 ; DeGracia et al., 2002).
Interestingly, analysis of the effects of natural hypometabolism (in estivating or anoxia-tolerant organisms) on protein phosphatase activities have shown that the general
response is a reduction in PP-1 and PP-2A activities in tissues (Mehrani & Storey, 1995 ; Cowan et al., 2000 ; Cowan &
Storey, 2001) consistent with the general metabolic rate
depression and with the dominant role of protein kinases
in suppressing metabolic functions during entry into hypometabolism.
Protein translation is also regulated at the level of polypeptide elongation and reversible phosphorylation control
over specific elongation factors is again the mechanism. For
example, in both mouse and rat models of brain ischemia,
phosphorylation of the eukaryotic elongation factor-2
(eEF-2) increased during ischemia and decreased again
inhibitor proteins. The figure shows the order of assembly of various eIFs onto the 40S ribosomal subunit including eIF2 which
carries the initiator methionyl-tRNA and the eIF4 family that assemble on the m7G-capped mRNA, with its start codon (AUG), and
introduce it to the 40S subunit. Shaded boxes show (a) the recycling of eIF2 and inhibitory control of this factor via phosphorylation
of its a-subunit which stabilizes the interaction of eIF2-GDP with its binding protein, eIF2B, and (b) inhibitory control of eIF4E via
interaction with its binding protein (4E-BP) which is released when 4E-BP is phosphorylated.
Kenneth B. Storey and Janet M. Storey
216
Protein
?
Ribosomal Protein L26
Gene
60S
Normoxia
Anoxia
Recovery
P
elF-2a
A
Protein
40S
Met-tRi
elF-2a-P
elF-2
GTP
Fig. 3. Analysis of ribosomal components during anoxia exposure in Littorina littorea. The top left panel shows the increased
expression of ribosomal protein L26 mRNA in hepatopancreas over the course of anoxia exposure (12, 24, 96 and 120 h anoxia) and
aerobic recovery (1 h recovery after 120 h anoxia). The bottom left panel shows changes in the phosphorylation of the a subunit of
eukaryotic initiation factor 2 (eIF-2a) after 24 h anoxia or 1 h recovery after anoxia. Both L26 and eIF-2 play roles in efficient
translation, and the right panel shows the location of both components on the intact ribosome. L26 is located at the subunit interface
and functions during the peptide transfer from the A (aminoacyl) site to the P (peptidyl) site, whereas eIF-2, which is involved in
initiation, is associated with the A site. Modified from Larade et al. (2001) and Larade & Storey (2002 c).
during reperfusion (Althausen et al., 2001; Martin de la
Vega et al., 2001). To date, control of elongation factors in
natural hypometabolism has only been studied in hibernating ground squirrels. Frerichs et al. (1998) demonstrated that
mean transit times for polypeptide elongation by ribosomes
were three-fold longer in extracts from brain of hibernating
ground squirrels, compared with euthermic animals. Subsequently, elevated amounts of phospho-eEF-2 were found
in brain and liver of hibernating ground squirrels, compared
with euthermic controls (Chen et al., 2001). Regulation was
due to both an approximately 50 % higher activity of eEF-2
kinase in hibernator tissues and a 20–30 % decrease in
PP-2A activity (which opposes eEF-2 kinase). This latter was
the result of a 50–60% increase in levels of the specific
inhibitor of PP2A, I2PP2A (Chen et al., 2001). Interestingly,
recent work by Ryazanov (2002) has cloned and sequenced
eEF-2 kinase and found that it represents a new family of
kinases. Sequence comparison has identified two other
family members, so-called channel-kinases, that represent a
novel type of signaling molecule – a protein kinase fused to
an ion channel. Thus, another huge field of protein kinase
mediated signal transduction is now open for exploration.
For the field of hypometabolism, this new family of kinases
offers an intriguing potential way of coordinating both
channel arrest and protein synthesis arrest.
The involvement of protein phosphorylation mechanisms
in the control of both translation initiation and peptide
elongation further emphasizes the key role that this regulatory mechanism plays in multiple aspects of metabolic
suppression and links the suppression of protein synthesis
with the control of multiple other metabolic functions
(e.g. enzymes of fuel metabolism, ion-motive ATPases) discussed previously. The important principle here is that
multiple cell functions are controlled via a single regulatory
mechanism (reversible protein phosphorylation) to achieve a
coordinated suppression of metabolic rate.
( 2) Ribosome aggregation state
The activity state of the protein-synthesizing machinery
in a cell/tissue can generally be inferred from the state of
ribosomal assembly. Active translation occurs on polysomes (aggregates of ribosomes moving along a strand
of mRNA) whereas monosomes are translationally silent.
Metabolic rate depression in animals
0.06
217
A
0.05
B
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M
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A 254
0.04
M
0.03
0.02
0.01
0
Relative band
intensity
1
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10
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3
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7
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3000
2000
1000
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Relative band
intensity
1
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6
7
8
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10
1
15%
30%
2
3
4
5
6
7
8
9 10
3000
2000
1000
30%
Linear sucrose gradient
Linear sucrose gradient
15%
Fig. 4. Polysome profiles of Littorina littorea hepatopancreas extracts. Tissue samples were gently homogenized in buffer containing
300 mmol lx1 sucrose and centrifuged at 13 000 g to pellet mitochondria. Supernatants were removed and then centrifuged at
40 000 g on 15–30 % continuous sucrose density gradients and then fractions were collected. The absorbance at 254 nm (A254) (upper
panel of each triplet) shows the relative amount of total RNA in each fraction. Pairs of fractions were then pooled, separated by
agarose gel electrophoresis and stained with ethidium bromide (middle panel) or blotted onto nitrocellulose for Northern blotting
with 32P-labeled a-tubulin probe ; the scanned intensity of tubulin bands is shown in the lower panel. (A) Aerobic, (B) 24 h anoxia, (C)
72 h anoxia, (D) 72 h anoxia followed by 3 h aerobic recovery. Polysome region [P] ; monosome peak [M]. Plots are representative
of N=3 trials per condition. Modified from Larade & Storey (2002 c).
Hence, an effective way to gauge the effects of a stress on
cellular translational activity is to analyze changes in the
proportions of polysomes versus monosomes. A polysome
profile is generated by separating ribosomes on a sucrose
gradient (Surks & Berkowitz, 1971). Polysomes appear in the
denser fractions (e.g. see fractions 3–6 in Fig. 4) with the
lighter weight monosomes and messenger ribonuclear proteins (mRNP) in the less dense fractions (fractions 7 and
higher). Ribosome presence throughout the gradient is
quantified by detecting ribosomal RNA in one of several
ways : measurement of absorbance at 254 nm, ethidium
bromide staining, or Northern blotting with a 32P-labelled
probe specific for 18S rRNA.
Stresses that compromise cellular energy or amino acid
availability lead to dissociation of polysomes and an increase
in the number of free translationally silent monosomes. For
218
example, hypoxia/anoxia stress, starvation and diabetes
have this effect (Surks & Berkowitz, 1971 ; Metter & Yanagihara, 1979 ; Harmon, Proud & Pain, 1984). Studies with
several systems have now shown that polysome disaggregation is a component of natural hypometabolism. For example, in response to anoxia exposure A. franciscana embryos
show a decrease in polysome content as one part of a 92 %
suppression of overall metabolic rate (Hofmann & Hand,
1992). Polysome disaggregation also occurs in mammalian
hibernation and during anaerobiosis in marine molluscs
(Frerichs et al., 1998; Knight et al., 2000 ; Hittel & Storey,
2002b ; Larade & Storey, 2002 c). Fig. 4 illustrates this principle showing the effects of anoxia exposure on ribosome
distribution patterns in extracts of L. littorea hepatopancreas.
Under normoxic conditions, a high proportion of ribosomes
was present in the high-density polysome fractions as
confirmed by both absorbance measurements at 254 nm
and ethidium bromide staining for RNA. The mRNA for
a constitutively expressed gene, tubulin, was also mainly in
the polysome fraction during normoxia (Fig. 4 A). This is
consistent with a state of active translation under aerobic
conditions. After 24 h of anoxia exposure, however, the
peak of ribosomal RNA began to shift towards a lower
density indicating disagreggation of polysomes into monosomes (Fig. 4B) and after 72 h of anoxia there was little
evidence of polysomes remaining in hepatopancreas extracts
(Fig. 4 C). Tubulin mRNA was also sequestered into the
monosome peak during anoxia which is consistent with
the idea that the mRNA pool is largely maintained in
the hypometabolic state but is not translated. Within 3 h of
the shift back to an oxygenated atmosphere, however, all
indicators showed a major reassembly of polysomes and
a return of mRNA for constitutively active genes into the
polysome pool (Fig. 4D).
Comparable studies with several tissues (brain, liver, kidney) of ground squirrels showed the same principle of polysome disaggregation during hibernation and a shift of mRNA
for constitutively active genes into the monosome fraction
(Frerichs et al., 1998; Knight et al., 2000 ; Hittel & Storey,
2002b). For example, in euthermic kidney extracts there
was a distinct distribution of the mRNA for the constitutively
expressed gene, cytochrome c oxidase subunit 4 (Cox4 ), between the heavy polyribosome and the monosome/mRNP
fractions. However, in hibernator extracts, Cox4 mRNA
transcripts were strongly shifted into the monosome fraction
(Hittel & Storey, 2002 b). Knight et al. (2000) found a similar
shift of the mRNA for another constitutive enzyme (glyceraldehyde-3-phosphate dehydrogenase) into the monosome
fraction during hibernation with mRNA returning to polysome fractions during arousal. Furthermore, by sampling
ground squirrels at different body temperatures during entry
into and arousal from hibernation, van Breukelen & Martin
(2001) showed a temperature dependence of polysome disaggregation in liver. The distribution of the mRNA for
actin, a constitutively expressed gene, was monitored during
cooling, and 18 xC was found to be the critical temperature
where polysome disaggregation began, as judged by a large
increase in actin mRNA and rRNA in the monosome fraction. Similarly, reaggregation of polysomes also occurred at
this temperature during arousal from hibernation. Whether
Kenneth B. Storey and Janet M. Storey
this effect of temperature on ribosome aggregation state is
a passive influence of temperature or results from regulation
of one of the initiation factors remains to be seen. Several
well-known examples of temperature-dependent metabolic
switches are in fact mediated by the differential effects of
temperature on the activities of selected protein kinases and
phosphatases. The activation of cryoprotectant synthesis at
5 xC in cold-hardy insects is a prime example of this, the
effect deriving from a specific low temperature inhibition
of glycogen phosphorylase phosphatase (PP-1) which then
allows phosphorylase kinase activity to dominate and lead
to an activation of glycogenolysis (Storey & Storey, 1991).
Studies with mammalian systems have linked the suppression of protein synthesis and the phosphorylation of
eIF2a during nonlethal disruptions of cellular homeostasis
with the assembly of stress granules, ribonuclear protein
structures that reversibly bind and protect mRNAs (Kedersha et al., 1999). Storage of mRNAs in such stress granules
during hypometabolism would be a useful mechanism that
could (a) preserve the valuable pool of untranslated mRNAs
until normal conditions were re-established, and (b) provide
for a very rapid re-initiation of the translation of key
transcripts to provide protein products that are needed
immediately during arousal from the hypometabolic state.
Prominent protein constituents of stress granules are
poly(A)-binding protein 1 (PABP-1) and T-cell intracellular
antigen-1 (TIA-1), a self-aggregating RNA-binding protein
(Kedersha et al., 1999). To determine whether these proteins
played a role in hypometabolism during hibernation,
Western blotting was used to assess their presence in the
polysome profiles of kidney extracts from euthermic versus
hibernating ground squirrels (Hittel & Storey, 2002b). TIA1 was restricted to the monosome/RNP fractions in both
situations but a significant redistribution of PABP-1 was
seen during hibernation (Fig. 5). In extracts from euthermic
animals, PABP-1 was found only in monosome fractions
(fractions 8–10) whereas in extracts from hibernating individuals PABP-1 was also detected in fractions 4, 5, and 7,
fractions that also contained substantial amounts of Cox4
and Oct2 mRNA. This suggests that a significant amount of
the mRNA in these fractions is sequestered into untranslatable pools. Furthermore, the presence of ultrastructural changes in the nuclei of hibernating animals and
indirect evidence for the binding of PABP to hibernator
mRNAs (Wang & Lee, 1996 ; Knight et al., 2000) strengthens the argument for the presence of stress granules during
torpor.
If most polysomes break down during hypometabolism
and if most mRNA transcripts are sequestered into untranslatable pools, then how are genes that are up-regulated
during torpor handled ? Studies with hibernating S. tridecemlineatus are highlighting some interesting variations on the
general principle.
( a ) Differential distribution of individual mRNA species
Brown adipose tissue (BAT) is a thermogenic organ that is
critical to survival of the hibernator because it is a major
producer of the heat that is needed to rewarm the animal
during arousal. Unlike the situation in other tissues of the
Metabolic rate depression in animals
219
A
B
Relative transcript levels
E
2.5
H
OCT2
COX4
C
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2.0
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4 5 6 7
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Fig. 5. Effect of hibernation on the organic cation transporter 2 (OCT2) and mRNA binding proteins in Spermophilus tridecemlineatus
kidney. (A) Western blots showing levels of OCT2 protein compared with a constitutive protein, cytochrome c oxidase subunit 4
(COX4), in euthermic (E) versus hibernating (H) animals. (B) Oct2 mRNA transcript levels. Values are means¡SEM, N=3. (C)
Kidney extracts were separated on 0.5–1.5 mol lx1 sucrose gradients drained into 10 fractions (fraction numbers increase with
decreasing density), and total RNA in each fraction was run on an agarose gel, blotted onto nylon membranes and Northern blots
were hybridized with 32P-dCTP-labeled Oct2 cDNA probe. (D) Blots in C were scanned and mRNA band intensity in each fraction
was plotted as a percentage of the total mRNA signal detected. Data points are means for N=3 trials with triangles showing
euthermic samples and squares showing hibernating samples. (E) Western blot analysis of poly(A)-binding protein 1 (PABP-1 ;
81 kDa) and T-cell intracellular antigen-1 (TIA-1 ; 43 kDa) protein levels in the polysome profile fractions. Modified from Hittel &
Storey (2002b).
hibernator, the rate of protein synthesis in BAT was not
reduced in hibernation (Table 1) and there was actually an
increased abundance of 18S rRNA in the higher density
polysome fractions of BAT from hibernating S. tridecemlineatus compared with euthermic animals (Hittel & Storey,
2002 b). The distribution of two mRNA transcripts on the
polysome profile was followed, one that is up-regulated
during hibernation (the H isoform of fatty acid binding
protein ; H-FABP) and one that is constitutively expressed
(Cox4 ) (Fig. 6). H-FABP has a critical function in BAT
of hibernators in the intracellular transport of fatty acids,
the fuel for thermogenesis. Transcript levels of H-FABP
increased approximately threefold in BAT during hibernation (Hittel & Storey, 2001) and a high proportion of this
message was found associated with the heaviest polysomes (Hittel & Storey, 2002 b). Compared with euthermic
controls, this represented a substantial enrichment of the
H-FABP transcript in the heavy polysome fraction and
correlated with the observed threefold increase in H-FABP
protein in BAT from hibernating animals. By contrast, when
the distribution of mRNA for the constitutively expressed
gene was assessed, most Cox4 mRNA was sequestered into
the monosome and mRNP fractions during hibernation
and COX4 protein levels remained constant. This indicates
that individual transcript species can be treated differently during hibernation and suggests that another level of
metabolic control in hibernation is the differential distribution of individual mRNA species between translationally
active and inactive ribosomes. The heavy polyribosomes
in hibernator BAT appear to contain those mRNAs (such
as H-FABP) that are crucial to the hibernation phenotype
while mRNA species that are not needed during hibernation
are relegated into the translationally silent monosome
fractions.
Kenneth B. Storey and Janet M. Storey
220
D
A
4 5 6 7 8 9 10
% mRNA
1 2 3
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Fig. 6. Effect of hibernation on heart-type fatty acid binding protein (H-FABP) compared with a constitutive protein, cytochrome c
oxidase subunit 4 (COX4), in brown adipose tissue of Spermophilus tridecemlineatus. (A–C) Northern blots showing the distribution of
18S rRNA transcripts (which tracks the position of the 40S ribosomal subunit), H-FABP mRNA transcripts, and COX4 transcripts
in polysome profiles of brown adipose tissue from euthermic (E) versus hibernating (H) squirrels. (D–F) Band intensities plotted as a
percentage of the total signal detected. Other information as in Fig. 5. (G) Western blot analysis of H-FABP and COX4 protein
levels. Modified from Hittel & Storey (2002b).
(b ) Anticipatory up-regulation
Another variation on translational control is the concept of
anticipatory up-regulation of genes. Transcript levels of
some genes are elevated in the hypometabolic state but no
increase in the level of the corresponding protein product
occurs. Analysis of polysome profiles indicates that this is
because the up-regulated mRNA transcripts are sequestered
into the monosome fraction in the hypometabolic state. The
gene for the organic cation transporter type 2 (Oct2 ) in
hibernator kidney represents a case in point (Fig. 5). Organic
cation transporters are transmembrane protein pumps that
actively absorb and/or excrete endogenous and exogenous
organic ions against their concentration gradients (Burckhardt & Wolff, 2000 ; Urakami et al., 2000). OCT2 protein
is found primarily in kidney where it is localized in the
basolateral membranes of cells lining the proximal tube
(outer medulla) of the nephron. During hibernation transcript levels of Oct2 were 2–3-fold higher in kidney compared with controls (assessed by both Northern blots and
cDNA arrays) (Hittel & Storey, 2002b). Despite this, Western blots showed that OCT2 protein levels dropped by 66%
in hibernator kidney. This dichotomy was explained by the
distribution of Oct2 mRNA on the polysome profile. In
euthermic extracts, Oct2 mRNA was partitioned between
the heaviest polysomes (fractions 1 and 2) and the unbound
mRNA or mRNPs (fraction 9) (Fig. 5 D). During hibernation, however, the profile showed both a strong increase
in the total amount of Oct2 message (by 2.1-fold) and surprisingly, that the vast majority of this mRNA was localized
in fractions 6–9. Thus, although total Oct2 transcript levels
clearly increased during hibernation in kidney, most of the
transcripts were stored in the translationally silent monosome and mRNP fractions.
Hence, hibernation stimulated transcription of Oct2 but
not its translation into protein. Why would this be ? One
reason may be that OCT2 protein could be particularly
sensitive to some form of damage that accrues during
hibernation ; for example, the protein may be damaged
by oxygen free radicals or by low temperature, leading to
its degradation. Another possibility is that part of the process
of shutting down kidney function during hibernation may
be the suppression of membrane transport functions that are
major energy consumers in kidney. Some transporters may
be controlled with reversible mechanisms such as protein
phosphorylation whereas deactivation of others may be only
possible via protein degradation. In either case it could make
sense that the gene is up-regulated as animals enter hibernation and its transcripts are stored in the monosome/
mRNP fractions during torpor. That way the transcripts are
Metabolic rate depression in animals
already present and ready to be translated as soon as arousal
begins in order to allow the fastest resumption of kidney
functions during the brief hours of the interbout.
IV. METABOLIC ARREST AND SUPPRESSION
OF PROTEIN DEGRADATION
Net protein turnover is governed by the rates of both protein
synthesis and protein degradation. Given that protein synthesis is strongly suppressed in hypometabolic states but
that, upon arousal, animals show no significant deficit of
cellular proteins, it is obvious that the rates of protein
degradation must also be strongly suppressed during hypometabolism. Indeed, these two opposing processes are undoubtedly coordinately regulated to achieve a lower net rate
of protein turnover. In addition to the ATP savings associated with suppressed protein turnover during hypometabolism, the protein ‘life extension ’ that results would also
minimize the amounts of nitrogenous end products that accumulate as by-products of proteolysis and reduce the costs
of processing and storing these during torpor. For example,
the rate of urea synthesis by anoxic turtle hepatocytes was
reduced by 70% compared with controls (Hochachka et al.,
1996).
Studies aimed at assessing proteolysis in hypometabolic
states are considerably fewer and more variable in their
methodology than those that have addressed protein synthesis but estimates of protein longevity in hypometabolic
states indicate that proteolysis must be suppressed. For example, the half-life of cytochrome oxidase was increased by
as much as 77-fold in encysted gastrulae of the brine shrimp,
A. franciscana, indicating strong suppression of proteolysis
(Anchordoguy & Hand, 1995). Land & Hochachka (1994)
assessed proteolysis in both labile and stable protein pools
in isolated hepatocytes of anoxia-tolerant painted turtles
(C. p. bellii ). Compared with controls, half-lives of the labile
protein pool increased by 40 % in anoxic hepatocytes, halflives of stable proteins doubled, and the mean suppression of
proteolysis for both pools combined was 36 %. Furthermore,
proteolysis in aerobic hepatocytes accounted for 22% of
total energy use by hepatocytes but this was suppressed to
just 0.7 % under anoxia. Recall that the proportion of ATP
turnover devoted to protein synthesis in aerobic versus anoxic
turtle hepatocytes was comparable (Land et al., 1993),
indicating that rates of protein synthesis and degradation are
coordinately regulated.
The mechanisms of proteolytic suppression in hypometabolism are just beginning to be identified. Ubiquitindependent proteolysis is one of the major modes of cellular
proteolysis; conjugation with a polymer of ubiquitin tags
proteins for degradation by the 26S proteasome whereas
monoubiquitylated proteins are targeted for endocytosis and
degradation in lysosomes (Pickart, 2001). A recent study
with ground squirrels showed that the level of ubiquitinconjugated protein rose by 2–3-fold during hibernation, an
occurrence that is best explained by an inhibition of proteolysis during torpor, a process that in mammalian cells is
highly sensitive to temperature reduction (van Breukelen &
221
Carey, 2002). Other studies with A. franciscana, suggest that
the block on proteolysis during hypometabolism is placed
at the level of ubiquitin conjugation because the ubiquitin
conjugate levels in anoxic embryos fell to just 7% of
normoxic values (Anchordoguy & Hand, 1994). The resumption of ubiquitination in embryos during recovery
from anoxia was dependent on a rise in ATP levels (and/or
reduced AMP concentration) and a re-alkalization of the
cytoplasm (Anchordoguy & Hand, 1995). Data from anoxiatolerant turtles might also support control of proteolysis
at the level of protein ubiquitination because the activity
of the multicatalytic proteinase complex (a key part of the
26S proteosome) did not change in liver of anoxic versus
control turtles (Willmore & Storey, 1996). However, there is
not yet sufficient information from different animal systems
to suggest whether either, both or none of these represent
the common mode of proteolysis control in hypometabolic
animals nor are the regulatory mechanism(s) of proteolysis
suppression known.
V. METABOLIC ARREST AND GENE
EXPRESSION
(1 ) RNA synthesis
Protein synthesis has two major energetic costs, the cost of
transcription and the cost of translation. As discussed above,
the suppression of protein translation in hypometabolic
states is a major contributor to energy savings. Is transcription similarly suppressed ? It is estimated that transcription
consumes 1–10 % of the energy budget of cells (Rolfe &
Brown, 1997). Presumably global transcription rates would
also be suppressed in hypometabolism although there has
not been much study of this. Work with A. franciscana showed
that during quiescence induced by anoxia the global arrest
of protein synthesis was accompanied by mRNA levels that
remained constant (Hand & Hardewig, 1996). This indicated that (a) protein synthesis inhibition is not caused
by message limitation, and (b) the rate of transcription must
also be arrested. The latter point was further confirmed
using nuclear run-on assays. These showed that the rate of
transcript elongation was reduced by 79 and 88% after
4 and 24 h of anoxia exposure, respectively (increasing
mRNA half-lives by at least 8.5-fold), yet was fully restored
within 1 h when embryos were reoxygenated (van Breukelen, Maier & Hand, 2000). Artificial acidification of aerobic
embryos indicated that approximately half of the anoxiainduced suppression of transcription could be accounted for
by pH effects on the process. A similar analysis of transcription during hibernation showed an approximately
twofold decrease in transcript initiation in liver of hibernating S. lateralis versus interbout aroused animals. This, coupled
with Q 10 values of 2–3 for the rate of mRNA transcript
elongation in vitro indicated that transcript elongation would
be virtually halted in vivo at the low body temperatures
typical of hibernation (van Breukelen & Martin, 2002).
Application of the nuclear run-on technique to L. littorea
hepatopancreas showed a similar suppression of the rate
of mRNA transcription during anaerobiosis ; the rate of
222
mRNA elongation under anoxic conditions, measured as
32
P-UTP incorporation into nascent mRNAs by isolated
nuclei, dropped to less than one-third of the normoxic rate
(Larade & Storey, 2002 a).
(2 ) Gene discovery
The transition to a hypometabolic state is certainly not the
time for a major restructuring of cellular metabolism and,
indeed, evidence from a variety of sources indicates that
the vast majority of cellular proteins are unaltered when
organisms make this transition. Instead, reversible regulatory controls are applied to multiple key loci to provide both
a coordinated suppression of net ATP turnover and a readjustment of selected functions to serve the needs of the
hypometabolic state. The prominence of reversible controls
in hypometabolism means that relatively few changes to the
protein composition of cells may be required to support the
transitions to and from the hypometabolic state. Indeed,
based on the results of various survey studies of stressinduced gene expression (described below), this is proving to
be the case. All studies seem to show that changes in gene
and protein expression associated with metabolic depression
are highly selective and tend to serve specific needs of the
hypometabolic state.
Analysis of the gene expression changes that support
hypometabolism is in its infancy and, to date, the results have
been quite spotty and derived from multiple species/tissues
and diverse techniques. Common principles that extend
across phylogenetic lines have yet to be identified but major
advances in the technology for gene screening, as well as
emerging technologies for protein screening, are certain to
allow important discoveries over the next few years. Various
methods of gene discovery have been used to date including
cDNA library screening, differential display polymerase
chain reaction (PCR) and cDNA array screening. Differential screening of cDNA libraries is especially useful for the
detection of novel genes that are specific to the animal or
stress under consideration. For example, the anoxia-induced
gene, kvn, was discovered this way in L. littorea hepatopancreas, its sequence showing no similarities to any other
gene/protein sequences present in international databases
(Larade & Storey, 2002 b). Novel genes specific to freezing
survival have also been found in freeze-tolerant frogs
(Storey, 1999).
One new advance in molecular biology will revolutionise
gene screening over the next few years: the use of cDNA
arrays. These will allow broad-based and comprehensive
screening for stress-induced gene expression patterns. Stateof-the-art glass slide microarrays can have up to 31 500 nonredundant cDNAs bound to them (Boer et al., 2001) and
offer one-step screening of the responses of broad families of
genes (e.g. signal transduction systems, glycolytic enzymes,
molecular transporters, transcription factors) to an imposed
stress. Array screening is currently having a major impact in
medical research allowing, for example, broad screening of
gene expression changes during aging, disease, and carcinogenesis (Hal et al., 2000; Boer et al., 2001 ; Helmberg,
2001). We have recently used both nylon macroarrays
(Clontech Rat ATLASTM containing cDNAs for 588 genes)
Kenneth B. Storey and Janet M. Storey
and human 19 000 cDNA microarrays (Ontario Cancer
Institute) to screen for hibernation-specific gene expression
in ground squirrels and bats (Eddy & Storey, 2001, 2002 ;
Hittel & Storey, 2001) and anoxia-induced gene expression
in L. littorea (Larade & Storey, 2002 a). Nylon macroarrays
were used to examine gene expression in brown adipose
tissue of hibernators. These showed that transcript levels
of most genes did not change during hibernation but
expression of a few (<1 %) was elevated. Prominent among
these were isoforms of FABP (Hittel & Storey, 2001). Similar
screening of S. tridecemlineatus skeletal muscle showed that
several genes that encode components of the small and large
ribosomal subunits were consistently down-regulated during
hibernation including L19, L21, L36a, S17, S12 and S29
(Eddy & Storey, 2002). This further implicates control of the
ribosomes as critical to the inhibition of protein synthesis in
hibernation.
One relevant issue to all uses of cDNA arrays in comparative animal systems is that of heterologous probing –
the use of arrays containing immobilized cDNAs from one
species to screen the mRNA populations of another species.
Clearly, there are gene sequence differences between species
that could prevent the cDNA probes synthesized from the
mRNA of the test species from hybridizing with the bound
cDNA fragments on the arrays so that a certain percentage
of the genes on the array will not cross-react. This is a
problem if the goal of the screening is to evaluate the responses of specific genes to the stress but if the goal is instead
to screen broadly to find new stress-responsive genes, then
the positive ‘hits ’ from cDNA array screening can easily
provide a researcher with months or years of follow-up work
to investigate the roles of each of these genes in hypometabolism.
In our experience, heterologous probing works very well
for mammalian hibernators. In our first studies with rat
macroarrays we found that the percentage of cDNAs that
cross-reacted was 93% for S. tridecemlineatus and 73% for the
little brown bat, Myotis lucifugus (Eddy & Storey, 2002). Using
the human 19K microarrays, the initial results were poor
with a cross-reaction rate of only 15–20 % but we were
able raise this to 85–90% using a simple reduction in the
post-hybridization washing temperature from 50 xC, as
suggested by the manufacturer, to room temperature (21 xC)
(Eddy & Storey, 2002). We have also used human 19K
cDNA arrays to screen for anoxia-induced changes in gene
expression in hepatopancreas of L. littorea. Not unexpectedly
considering the phylogenetic distance between gastropods
and humans, cross-reactivity was low at only 18.35 %. Of
those genes that did show significant cross-reaction due to
high sequence identity, most showed no change in transcript
levels in anoxia (88.8 %) and only 0.6 % showed reduced
transcripts ; these data are in agreement with the idea that
mRNA content is largely preserved during hypometabolism.
However, 10.6 % of genes on the array were putatively
up-regulated by twofold or more during anoxia. This
represented over 300 genes and indicates scope for a widespread response to anoxia by many cellular functions in this
facultative anaerobe. The genes identified included protein
phosphatases and kinases, mitogen-activated protein kinase
interacting factors, translation factors, antioxidant enzymes,
Metabolic rate depression in animals
and nuclear receptors (Larade & Storey, 2002 a). Not all
of these candidate genes will be confirmed as anoxia upregulated when studied in detail using homologous probes
and techniques such as Northern blotting or quantitative
RT-PCR, but nonetheless, the results are sure to provide
several new leads to follow.
The use of cDNA array screening will certainly lead to
novel discoveries about any system to which it is applied
but a few caveats need mentioning. Apart from the sometimes low cross-reactivity with heterologous probing, array
screening may still be limited in its ability to detect transcripts that are in low copy number or to confirm significant
differences for changes of less than twofold. Furthermore,
changes in mRNA transcript levels do not always imply
changes of equal magnitude in protein levels or in the impact on metabolism. Protein levels are influenced by several
additional layers of control including post-transcriptional
processing of mRNA, differential mRNA stability, selective
translation of transcripts by ribosomes, etc. The impact of
altered gene expression on metabolism will also vary from
protein to protein. For example, small changes in transcript
and protein levels of a regulatory enzyme may have a much
greater impact on metabolism than a much greater upregulation of a high activity equilibrium enzyme.
(3 ) Hypometabolism-induced gene expression
Despite the strong overall suppression of protein synthesis in
hypometabolic states, examples of the specific up-regulation
of selected genes and synthesis of specific proteins can
be found in virtually every hypometabolic system that has
been examined. For example, the gene for riboflavin binding protein was up-regulated during aestivation in liver of
spadefoot toads Scaphiopus couchii and may contribute to vitamin storage during the months of dormancy (Storey, Dent
& Storey, 1999). Various genes are up-regulated during
insect or nematode diapause (e.g. Flannagan et al., 1998;
Cherkasova et al., 2000; Denlinger, 2001; Moribe et al.,
2001). For example, heat shock protein 70 was strongly upregulated at the initiation of diapause and persisted throughout 55 days of diapause, but disappeared within 12 h when
diapause was terminated in pupae of the flesh fly Sarcophaga
crassipalpis (Rinehart, Yocum & Denlinger, 2000). Gene upregulation during hypometabolism in hibernating mammals
and anoxia-tolerant animals is summarized in the following
sections.
(4 ) Hibernation-induced gene expression
Several groups are studying hibernation-responsive gene
expression using multiple techniques and several animal and
organ models. To date, one consistent finding from these
studies is that entry into (and arousal from) hibernation apparently occurs with very few changes in gene expression.
Thus, screening of a cDNA library made from heart of hibernating S. lateralis retrieved only two clones that were
confirmed as up-regulated (Fahlman et al., 2000) whereas
screening of a bat (M. lucifugus) skeletal muscle library found
approximately 60 candidate clones but none that could
be confirmed as up-regulated (Eddy & Storey, 2001). Use
223
of differential display PCR similarly failed to find hibernation-responsive genes in S. lateralis muscle (Eddy & Storey,
2001) and cDNA array screening showed low numbers of
putatively up-regulated genes in ground squirrel and bat
tissues (Eddy & Storey, 2002). O’Hara et al. (1999) found
29 candidate cDNAs that appeared to differ between
euthermic and hibernating states in S. lateralis brain out of
4500 PCR products compared by differential display but
none showed significant changes when examined with
Northern blots. The apparent lack of major changes in gene
expression during hibernation that is illustrated by these
studies is not unexpected because (a) an energy-limited torpid state is not a time for major metabolic reorganization,
and (b) cells and organs must remain competent to resume
normal body functions rapidly during interbout arousals.
However, these results also mean that the control of hibernation may be vested in only a handful of critical gene/
protein changes.
To date, the genes that are known to be hibernation-responsive in ground squirrels include a2-macroglobulin in
liver (Srere et al., 1995), moesin in intestine (Gorham, Bretscher & Carey, 1998), isozyme 4 of pyruvate dehydrogenase
kinase (PDK) and pancreatic lipase in heart (Andrews et al.,
1998 ; Buck, Squire & Andrews, 2002), isoforms of uncoupling protein and FABP in multiple tissues (Boyer et al.,
1998 ; Hittel & Storey, 2001), the ventricular isoform of
myosin light chain 1 (MLC1v) in heart and skeletal muscle
(Fahlman et al., 2000), organic cation transporter 2 in kidney
(Hittel & Storey, 2002b), the melatonin receptor (Mel1A)
(McCarron et al., 2001) and four genes on the mitochondrial
genome : NADH ubiquinone-oxidoreductase subunit 2
(ND2), cytochrome c oxidase subunit 1 (COX1) and ATPase
subunits 6 and 8 (Fahlman et al., 2000; Hittel & Storey,
2002 a). Given that the mitochondrial genome is transcribed
as a unit, the identification of the mitochondria-encoded
genes suggests that a general up-regulation of the mitochondrial genome occurs during hibernation. This is
particularly significant because transcript levels of related
nuclear-encoded genes (e.g. subunit 4 of COX and ATPa,
a nuclear encoded subunit of the mitochondrial ATP
synthase) did not change during hibernation (Fig. 7) (Hittel
& Storey, 2002 a). Interestingly, up-regulation of mitochondrial genes also occurs in freeze-tolerant and anoxia-tolerant
animals (Storey, 1999; see Section V.5 below) so this may
be a generalized response by stress-tolerant animals that
requires further investigation. Other gene expression changes
have been reported in brain of hibernating ground squirrels ;
a 98 kDa protein with a phosphotyrosine moiety was found
in membrane fractions during a hibernation bout but
disappeared within 1 h of arousal (Ohtsuki et al., 1998)
and increased expression of ‘ intermediate-early’ genes that
code for selected transcription factors (c-fos, junB, c-Jun)
occurred during late torpor and peaked during arousal
(O’Hara et al., 1999). Notably, a substantial number of
hibernation-responsive genes have been found in heart
(Fig. 7). This may be related to the fact that the heart must
continue to work during torpor and adjustments in gene
expression are undoubtedly necessary to optimize cardiac
muscle function with respect to the changes in temperature,
work load and fuel availability that occur in torpor.
Kenneth B. Storey and Janet M. Storey
224
torpor. For example, up-regulation of a2-macroglobulin
during hibernation drew attention to the clotting cascade in
hibernators. a2-Macroglobulin is a protease inhibitor that
binds and inhibits several of the proteases that catalyze steps
in the clotting cascade, including thrombin which cleaves
fibrinogen to release fibrin monomers that polymerize into a
fibrin clot. Increased a2-macroglobulin synthesis and export
by liver (Srere et al., 1995), along with reduced platelet
numbers and other adjustments to components of the clotting cascade, contribute to the observed decrease in clotting
capacity of blood during torpor, an adaptation that minimizes spontaneous clot formation in the microvasculature
under the low blood flow (ischemic) conditions of the
hibernating state (Drew et al., 2001).
7
Ratio hibernating: euthermic
Heart genes
6
5
4
3
2
1
AT
P–
a
AT
P6
/8
Co
x4
Co
x1
FA
BP
–A
N
ad
2
FA
BP
–H
M
LC
1v
0
Fig. 7. Histograms showing the effect of hibernation on gene
up-regulation in ground squirrel heart. Relative transcript levels
(determined from Northern blots) in hibernating versus euthermic
heart are shown for eight genes : MlC1v, myosin light chain 1
ventricular isoform ; Nad2, subunit 2 of NADH-ubiquinone
oxidoreductase ; FABP-H and FABP-A, heart and adipose isoforms of fatty acid binding protein, respectively ; Cox1 and
Cox4, subunits 1 and 4 of cytochrome c oxidase ; ATP6/8,
ATPase 6/8 bicistronic mRNA ; and ATPa, alpha subunit
of the mitochondrial ATP synthase. Nad2, Cox1 and ATP6/8
are mitochondria-encoded subunits and Cox4 and ATPa are
nuclear-encoded subunits of mitochondrial proteins. Data are
means¡SEM, N=3. Data on MLC1v and Nad2 transcripts
are from Spermophilus lateralis (Fahlman et al., 2000) ; all others
are from S. tridecemlineatus (Hittel & Storey, 2001, 2002a).
Some proteins are also down-regulated during hibernation (Kondo & Kondo, 1992 ; Soukri et al., 1996; Schmidt
& Kelley, 2001). Particularly interesting is the suppression of
insulin-like growth factor and of its plasma binding protein
(IGFBP-3) during hibernation (Schmidt & Kelley, 2001).
The growth-regulatory IGF axis controls energy-expensive
somatic growth in skeleto-muscular and other tissues and its
suppression during hibernation could be a key contributor
to the hypometabolic state. Prostaglandin D2 synthase activity was reduced in late torpor in hypothalamus of S. lateralis but rebounded during arousal (O’Hara et al., 1999).
Four proteins in the blood of euthermic chipmunks (Tamius
asiaticus) also disappeared when the animals were hibernating (Kondo & Kondo, 1992) ; one of these showed high
homology with a1-antitrypsin. Interestingly, a1-antitrypsin
and three other acute-phase blood proteins were unaffected
during hibernation in S. richardsonii but levels of a fifth
member of the family, a2-macroglobulin, increased significantly (Srere et al., 1995). Results from both species suggest
modification of the acute phase response to injury and
infection during hibernation.
(a ) a2-Macroglobulin
The known hibernation-specific gene responses suggest some
of the metabolic adjustments that are needed to support
( b ) PDK4
PDK4 expression during hibernation has a role in the control of fuel selection for the torpid animal (Andrews et al.,
1998; Buck et al., 2002). Pyruvate dehydrogenase (PDH) is
the entry point for carbohydrate into the tricarboxylic acid
cycle. During hibernation, most organs switch to an almost
exclusive dependence on lipid oxidation for energy generation in order to spare carbohydrate for selected tissues such
as brain. A key enzyme in the control of carbohydrate
catabolism is PDH and the amount of active PDH is
strongly reduced during hibernation or daily torpor (Storey,
1997; Heldmaier et al., 1999), the mechanism being PDKmediated phosphorylation. Isozyme 4 of PDK is strongly
expressed in heart and skeletal muscle of mammals and
is responsive to physiological stresses including starvation
and diabetes (Buck et al., 2002). PDK4 transcripts and protein were strongly induced in heart, skeletal muscle and
white adipose tissue of ground squirrels during the winter
with high levels maintained during both hibernation and
interbout arousals (Buck et al., 2002). High PDK4 levels
during the winter would keep PDH in an inactive state,
thereby minimizing carbohydrate catabolism by muscles or
carbohydrate use as a substrate for lipogenesis by adipose
tissue.
( c ) Myosin
Gene expression studies also give evidence of myosin restructuring in support of low-temperature cardiac function
in hibernators. Myosin is made up of two heavy chains
(MHC) and four light chains (MLC) that are classified as
either alkali (MLC1, MLC3) or regulatory (MLC2) light
chains ; the latter are subject to reversible phosphorylation.
Transcript levels of the ventricular isoform of MLC1 rose 2–
3-fold during hibernation in S. tridecemlineatus heart and
skeletal muscle (Fig. 7) implicating an increased content
of this subunit in the myosin motor at low temperature
(Fahlman et al., 2000). Other changes occurred in hamster
(Cricetus cricetus) heart; the phosphorylation state of MLC2
decreased from 45% in summer to 23% in torpor and the
proportions of MHC isoforms changed from a dominance
of the b isoform (79% of total) in summer hamsters and
winter-active animals at 22 xC to near-equal amounts of the
Metabolic rate depression in animals
a (53%) and b (47%) forms in hibernators (Morano et al.,
1992). Changes in the expression of myosin genes and in
the mix of myosin isoforms represented in a muscle is a
well-known response to various stimuli (stretch, electrical
stimulation, work load) in mammals and to temperature
change in fish and is key to adapting the myosin motor to
optimize contractile properties for different demands placed
on the muscle (Goldspink & Yang, 2001). It is not surprising,
then, that myosin restructuring occurs during hibernation in
heart. During hibernation, the heart continues to beat but at
a much lower body temperature and with a much lower rate
than normal. However, peripheral resistance increases substantially and to compensate for this the force of contraction
actually increases (Wang, 1989). Changes in sarcoplasmic
reticulum Ca2+ storage and release have been tested as a
possible reason for enhanced contractility in hibernation
(Wang & Lee, 1996) but a change in the composition of
muscle proteins would also provide an optimal mix of isoforms to adapt the contractile apparatus to the new work
load and thermal conditions of the torpid state.
225
for triglyceride delivery via the blood. Hence, expression
of A-FABP in hibernator heart provides the organ with
access to both intracellular and extracellular lipid reserves
for fuel.
Analysis of the sequence of H-FABP from ground squirrels also showed three unique amino acid substitutions that
place polar amino acids in positions previously filled by nonpolar or hydrophobic amino acids (Hittel & Storey, 2001).
The substitutions occur in positions that would alter the
flexibility of the protein and may contribute to the effective
function of H-FABP at low temperatures. Studies with the
liver isoform of FABP show that fatty acid binding by
ground squirrel L-FABP is temperature insensitive over the
range 5–37 xC whereas kinetic properties of rat L-FABP are
compromised at low temperature (Stewart, English & Storey, 1998). Amino acid substitutions to H-FABP may similarly create temperature insensitive properties that allow the
protein to function optimally in fatty acid transport over a
wide range of body temperatures.
( e) Uncoupling proteins
( d ) Fatty acid binding proteins
An up-regulation of the genes for heart (H) and adipose (A)
isoforms of FABP during hibernation in BAT and heart of
ground squirrels and the confirmation of enhanced synthesis
of FABP protein in BAT during hibernation (FABP mRNA
is in the heavy polysome fraction, Western blots show increased protein content) emphasizes the importance of lipid
oxidation in these organs during hibernation (Figs 6 and 7)
(Hittel & Storey, 2001, 2002 b). Heart switches to an almost
total reliance on lipid oxidation during torpor and lipidfueled thermogenesis by uncoupled mitochondria in BAT is
critical for rewarming the hibernator during arousal. The
occurrence of both isoforms in both tissues is unusual but
can be explained by their different functions in fatty acid
transport. The A isoform of FABP is believed to carry fatty
acids to and from intracellular lipid droplets and because it
can form a complex with hormone-sensitive lipase, a prime
function seems to be to carry fatty acids away from intracellular lipid droplets after triglyceride hydrolysis and to the
mitochondria for oxidation (Vogel-Hertzel & Bernlohr,
2000). By contrast, the role of H-FABP is to pick up incoming fatty acids at the plasma membrane and transport
them to the mitochondria. The presence of both isoforms
in BAT reflects the fact that the tissue uses both its own internal lipid reserves and fatty acids imported from white
adipose tissue to fuel nonshivering thermogenesis. Vertebrate
heart typically displays only the H-isoform because it imports its fatty acids and induction of A-FABP in heart of
hibernating ground squirrels (transcripts were not found
in euthermic heart) is a first in the mammalian literature.
Its presence could serve two functions : (1) because A-FABP
also occurs in the hearts of Antarctic teleost fishes (Vayada
et al., 1998), the presence of A-FABP may be important
for low-temperature function, or (2) hearts of hibernating
ground squirrels maintain substantial intracellular triglyceride lipid droplets (Burlington et al., 1972) that are
probably needed to meet the demand for high rates of fatty
acid oxidation during arousal that could exceed the capacity
Hibernation-associated changes in the expression of uncoupling protein (UCP) isoforms also occur (Boyer et al.,
1998). High rates of fatty acid oxidation fuel thermogenesis
in hibernator organs both to support a minimum body
temperature during torpor and to reheat the body rapidly
during arousal. Heat production comes from nonshivering
thermogenesis in BAT and also from shivering in skeletal
muscles. Nonshivering thermogenesis in BAT involves the
uncoupling protein UCP1 that dissipates the proton motive
force across the inner mitochondrial membrane to release
energy as heat rather than trapping it in the synthesis of
ATP. The amount and activity of UCP1 in rodent BAT is
strongly stimulated by cold exposure (Nizielski, Billington &
Levine, 1995). Recent studies have explored the expression
of UCP1 and its two homologues in hibernators (Boyer et al.,
1998 ; Praun et al., 2001). UCP2 is expressed in multiple
tissues and UCP3 occurs mainly in skeletal muscles. Increased mRNA levels for all three isoforms were found in
BAT during cold exposure in hamsters but UCP2 and
UCP3 mRNA levels in muscle did not change (Praun et al.,
2001). By contrast, during hibernation in ground squirrels,
UCP2 mRNA levels increased in white adipose tissue by
1.6-fold and UCP3 mRNA rose by threefold in skeletal
muscle whereas UCP1 mRNA was not affected in BAT
when hibernation was at ambient temperatures above 0 xC
(Boyer et al., 1998). However, when thermogenic demands
were increased by lowering the ambient temperature to
subzero values, UCP1 mRNA increased approximately
twofold in BAT compared with transcript levels in animals
hibernating above 0 xC ; UCP2 mRNA in white adipose
and UCP3 mRNA in skeletal muscle also rose. Although
the thermogenic capacity of BAT was believed to be fully
developed before hibernation begins, these results show that
the torpid animal still has a capacity to respond with further
UCP1 expression if thermal stress during hibernation is
severe. Furthermore, the results for UCP2 and UCP3
suggest that tissues other than BAT may contribute to nonshivering thermogenesis in the hibernator.
226
(5 ) Anoxia-induced gene expression
The anoxic state is an energy-limited one in which energyexpensive processes such as protein synthesis are restricted
by mechanisms discussed earlier. Nonetheless, selected
gene expression continues and various examples of anoxiastimulated up-regulation or induction of genes are now
known, presumably representing genes whose protein
products play key roles in anaerobic metabolism or in
protecting cells from damage.
(a ) Gene expression during anoxia in turtles
We first explored anoxia-induced gene expression in the
turtle, Trachemys scripta elegans, a widely studied model species
that can endure total oxygen deprivation for as long as 3–4
months when submerged in cold water with a metabolic rate
only approximately 10 % of the corresponding aerobic rate
( Jackson, 2001). Differential screening of a cDNA library
made from heart of anoxia-exposed adult turtles (20 h submerged in N2-bubbled water at 7 xC) identified the anoxic
up-regulation of three genes coded on the mitochondrial
genome : Cox1 that encodes cytochrome C oxidase subunit 1
(COX1), Nad5 that encodes subunit 5 of NADH-ubiquinone
oxidoreductase (ND5), and the mitochondrial WANCY
(tryptophan, alanine, asparagine, cysteine and tyrosine)
tRNA gene cluster (Cai & Storey, 1996, 1997). All three
transcripts showed a similar pattern of response ; within 1 h
of anoxia exposure transcript levels of Cox1, Nad5 and
WANCY in heart had risen by 4.5-, 3- and 3.5-fold,
respectively (Cai & Storey, 1996, 1997). Levels remained
high over a 20 h anoxia excursion and then declined again
during aerobic recovery. Cox1 and Nad5 were also upregulated in red muscle, brain and kidney during anoxia. A
subsequent study revealed two other mitochondrially encoded genes that were up-regulated in liver of anoxic turtles :
Cytb, encoding the protein cytochrome b and Nad4 which
codes for subunit 4 of ND (Willmore, English & Storey,
2001). Cytb transcript levels were 5.2–5.7-fold higher than
control values over the entire anoxia exposure (1–20 h), decreased by about half within 1 h of aerobic recovery and fell
to near control values by 5 h recovery. Cytb transcripts also
increased by threefold in anoxic kidney. Nad4 transcripts
rose by 13-fold in liver within 1 h of anoxia exposure and
then declined to 5–6-fold higher than controls during longer
term anoxia.
Mitochondrial genes are also up-regulated in several
other instances of low oxygen stress. Freezing is an ischemic
stress that cuts off oxygen delivery to tissues when plasma
freezes. In response to freezing Cox1 and Nad5 were upregulated in freeze-tolerant turtles Chrysemys picta (Cai &
Storey, 1996). Nad4 transcripts rose in liver of freeze-tolerant
wood frogs Rana sylvatica and transcripts for subunits 6 and 8
of the F0F1ATPase complex increased in frog brain during
freezing (S. Wu & K. B. Storey, unpublished observations).
Transcripts of Cox2, another mitochondrially encoded protein subunit, rose 6–7-fold during anoxia in L. littorea
(K. Larade, unpublished data) and, as mentioned previously,
four genes on the mitochondrial genome (Nad2, Cox1 and
ATPase subunits 6 and 8) were also up-regulated during
Kenneth B. Storey and Janet M. Storey
mammalian hibernation (Fahlman et al., 2000; Hittel &
Storey, 2002 a). Hibernation is a situation of greatly reduced
blood flow that is being used as a model for ischemia resistance in mammals (Frerichs et al., 1994 ; Bell et al., 2002).
All of these proteins are large complexes containing multiple
subunits coded on both the nuclear and mitochondrial
genomes (e.g. three of the 13 subunits of COX1 are on the
mitochondrial genome and six of the 41 subunits of ND)
(Azzi, Muller & Labonia, 1989 ; Ohnishi, 1993) and, significantly, we have encountered no case to date where a nuclearencoded subunit of one of these proteins was identified
as upregulated in the hypometabolic state (anoxia, frozen,
hibernation).
The question remains as to why mitochondrial transcript
levels increase under hypometabolic conditions associated
with anoxic or ischemic stress. Mitochondrial DNA contains
only one promoter on each of the L and H strands and all
genes are transcribed as one RNA precursor from the same
initiation site (except for rRNA genes) (Gilham, 1994). The
long polycistronic messages are then cleaved to give the
individual RNA species. This mode of transcription can
explain the parallel increases in Cox1, Nad5 and WANCY
transcripts in anoxic turtle heart but more difficult to explain
are the tissue-specific differences in gene up-regulation. For
example, Cox1 and Nad5 transcripts rise in heart but not
in liver during anoxia whereas Cytb transcripts rise in liver
but not in heart (Cai & Storey, 1996; Willmore et al., 2001).
An extensive analysis of the response to anoxia by all genes
on the mitochondrial genome in multiple tissues of a single
anoxia-tolerant species may resolve the question.
( b ) Gene expression during anoxia in marine snails
Recent studies of anoxia-induced gene expression in the
hepatopancreas of the marine gastropod, L. littorina, have
documented the up-regulation of several genes including
ribosomal protein L26, ferritin heavy chain, cytochrome c
oxidase subunit II (COII), granulin/epithelin, a novel gene
that we have named kvn and various unidentified genes
(Larade et al., 2001; Larade, 2002; Larade & Storey,
2002a, b). Based on their known roles in other organisms,
it is not difficult to infer functions for the protein products
of some of these genes during anoxia.
The gene encoding ribosomal protein L26 was upregulated during anoxia in L. littorea. Northern blots showed
that levels of L26 transcripts rose to a maximum 4.7-fold
increase in hepatopancreas after 96 h of anoxia exposure
whereas a 3.4-fold increase was seen after 48 h in foot,
compared with levels in aerobic snails (Fig. 3) (Larade et al.,
2001). However, transcript levels decreased significantly
within 1 h when snails were returned to aerobic conditions
for recovery. Nuclear run-off assays confirmed that the
elevated transcript levels were the result of an increased rate
of L26 mRNA transcription ; 32P-dUTP incorporation
was approximately twofold higher in nuclei isolated from
hepatopancreas of 48 h anoxic snails than in nuclei from
aerobic animals. The L26 protein resides at the interface
of the large and small ribosomal subunit, apparently at
or near the ribosomal A site where it is likely involved in
subunit interactions (Lee & Horowitz, 1992). L26 can be
Metabolic rate depression in animals
227
Relative transcript level
0
1
2
3
Control
A
B
Anoxia
Freezing
db – cGMP
a
a
a
db – cAMP
PMA
Ca lonophore
Fig. 8. Expression of mRNA transcripts encoding L26 ribosomal protein during in vitro incubation of Littorina littorea hepatopancreas
samples under multiple conditions : anoxia (N2 bubbling at 4 xC for 12 h), freezing (at x7 xC for 12 h ), or incubation (at 4 xC for
2 h) with second messengers dibutyryl cyclic GMP, dibutyryl cyclic AMP, phorbol 12-myristate 13-acetate (PMA) or calcium
ionophore (A23187). After incubation, total RNA was isolated, resolved on a formaldehyde gel, blotted onto nitrocellulose and
hybridized with 32P-labeled L26 probe. The left column shows sample Northern blots for paired incubations of hepatopancreas
samples under (A) normoxic and (B) experimental conditions. The bar graphs on the right show the scanned intensity of experimental samples relative to their paired normoxic controls ; data are means¡SEM, N=3. a, significantly different from control,
P<0.05.
cross-linked to elongation factor 2 (eEF-2), implicating it as a
protein involved in forming the region that binds eEF-2
to the 60S ribosomal subunit preceding translocation of
peptidyl-tRNA from the A to the P site during peptide bond
formation (Nygard, Nilsson & Westermann, 1987). The link
with eEF-2 is interesting because, as discussed earlier, inhibitory control of eEF-2 is one of the mechanisms that
suppresses the rate of protein synthesis during hypometabolism. The function of anoxia-induced up-regulation of
L26 mRNA levels remains elusive but it is possible that
enhanced L26 protein levels during anoxia could contribute
in some way to eEF-2 control. Alternatively, elevated L26
mRNA during anoxia might support a rapid synthesis of this
protein once aerobic conditions were re-established, the
production of L26 protein perhaps being key for the reestablishment of ribosomal function. Ribosome production
is typically strictly coordinated with respect to the synthesis
of component proteins (Mager, 1988) so future studies need
to address the transcriptional and translational responses of
other ribosomal components to anoxia.
Anoxia exposure of L. littorea also stimulated a twofold
increase in mRNA transcripts encoding the ferritin heavy
chain in hepatopancreas; elevated levels were sustained
throughout anoxia but fell within 1 h of oxygenated recovery (Larade, 2002). Western blotting confirmed an increase
in protein content. Ferritin sequesters iron and, by doing so,
plays an important role in antioxidant defence because free
iron is a major catalyst in the production of oxygen free
radicals (Orino et al., 2001). Anoxia-tolerant animals appear
to avoid such oxidative damage by maintaining constitutively high levels of antioxidants and/or improving enzymatic antioxidant defences when stimulated by anoxic/
hypoxic conditions (Hermes-Lima, Storey & Storey, 1998;
Pannunzio & Storey, 1998). Ferritin synthesis, providing
enhanced iron storage, adds another layer of antioxidant
defence.
228
A novel gene, kvn, was also up-regulated during anoxia in
L. littorea hepatopancreas. The gene codes for a protein of 99
amino acids with a predicted molecular weight of 12 kDa
(Larade & Storey, 2002b). Protein function is unknown
but analysis of the putative amino acid sequence showed
domains including casein kinase II phosphorylation sites
and an N-myristoylation site as well as a 15 residue hydrophobic signal sequence at the N terminal. Such signal
sequences generally direct proteins to the endoplasmic reticulum where they are processed and secreted to a final destination. Hence, KVN, which does not contain any known
retention signals or localization motifs, is predicted to be an
extracellular protein (Horton & Nakai, 1997). Levels of kvn
transcripts increased gradually in hepatopancreas during
anoxia, reaching a peak at 5.8-fold higher than control levels
after 48 h anoxia but falling sharply within 1 h of oxygenated recovery. Nuclear run-off assays confirmed that the
rise in kvn transcripts during anoxia was due to increased
transcription, and analysis of kvn distribution on ribosomes
showed a high proportion of kvn in the heavy polysome
fraction (Larade & Storey, 2002b). Hence, it appears that kvn
is actively translated during anoxia. Ongoing studies are
seeking its function.
(c ) cGMP mediation of anoxia-induced gene expression
The regulatory controls on anoxia-induced gene expression
in L. littorea are being explored using in vitro incubations of
hepatopancreas explants (Larade et al., 2001 ; Larade, 2002;
Larade & Storey, 2002 b). Transcript levels of L26, kvn and
ferritin all increased when tissues were incubated in a
medium bubbled with nitrogen gas as compared with aerobic control samples, confirming that anoxia stimulates gene
up-regulation both in vivo and in vitro. Effects of incubation
with stimulators of protein kinases (dibutyryl cAMP, dibutyryl cGMP, calcium ionophore A23187, and phorbol 12myristate 13-acetate) were then tested in aerobic incubations
(Fig. 8). Transcript levels of all three genes were elevated
when tissues were incubated with dibutyryl cGMP whereas
only ferritin message responded to any of the other treatments. This implicates a cGMP-mediated signaling cascade
in the gene expression response to anoxia in L. littorea and
agrees with previous studies that have implicated cGMPdependent protein kinase (PKG) in the regulation of various
metabolic responses to anoxia in marine molluscs. For example, PKG mediates the anoxia-induced phosphorylation
of selected enzymes (e.g. pyruvate kinase) as part of glycolytic rate depression (Storey, 1993). Thus, it appears likely
that the low oxygen signal that stimulates anaerobic adaptations and metabolic rate depression is transmitted
via a cGMP and PKG signal transduction pathway in
anoxia-tolerant molluscs. A well-known activator of guanylyl
cyclases is the diffusible signal molecule, nitric oxide (NO)
(Stamler, Singel & Loscalzo, 1992). In molluscs, intracellular
cGMP levels have been shown to increase in the nervous
system of various species due to activation of soluble
guanylyl cyclase by NO and nitric oxide synthase activity
has been confirmed in a large number of gastropod species
(Moroz, 2000). Recent studies have shown that NO is involved in low oxygen signaling in Drosophila melanogaster
Kenneth B. Storey and Janet M. Storey
(Wingrove & O’Farrell, 1999) and this, coupled with the
evidence cited above of cGMP mediation of anoxia-induced
events in molluscs suggests that the NO/cGMP signaling
pathway may be central in the response to oxygen deprivation in anoxia-tolerant species.
VI. CONCLUSIONS
1. Metabolic arrest is critical for the survival of many
species on Earth ; by suppressing metabolic rate to low
levels, organisms can enter hypometabolic states that allow
them to endure long-term exposure to stressful environmental conditions.
2. The biochemical regulatory mechanisms that control
metabolic rate depression are broadly conserved across
phylogenetic lines and include (a) the use of reversible protein phosphorylation to suppress the rates of major energy
producing and energy consuming pathways to reach a
new lower net rate of ATP turnover, (b) the selected upregulation of specific genes despite an overall suppression of
transcription and translation, and (c) protection and stabilization of macromolecules to provide for a rapid return to
normal life when environmental conditions allow.
3. Many avenues of research on hypometabolism remain
to be explored, particularly the identification and role of
various novel genes that are stress-induced and the mechanisms of signal transduction (hormones, receptors, intracellular messengers, transcription factors) that mediate the
transition to the hypometabolic state.
VII. ACKNOWLEDGEMENTS
Thanks to the many members of the Storey laboratory whose
work is summarized here, especially to K. Larade, S. Eddy, and
D. Hittel for their new work on the regulation of gene expression
and protein synthesis in hibernating mammals and anoxia-tolerant
snails. K. B. Storey holds the Canada Research Chair in Molecular
Physiology.
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