Bioscience Reports, Vol. 25, Nos. 3/4, June/August 2005 ( 2005)
DOI: 10.1007/s10540-005-2884-7
Role of Sarco/Endoplasmic Reticulum Ca2+-ATPase
in Thermogenesis
Leopoldo de Meis,1,3 Ana Paula Arruda,1 and Denise P. Carvalho2
Enzymes are able to handle the energy derived from the hydrolysis of phosphate compounds
in such a way as to determine the parcel that is used for work and the fraction that is
converted into heat. The sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) is a family
of membrane-bound ATPases that are able to transport Ca2+ ion across the membrane
using the chemical energy derived from ATP hydrolysis. The heat released during ATP
hydrolysis by SERCA may vary from 10 up to 30 kcal/mol depending on the SERCA
isoform used and on whether or not a Ca2+ gradient is formed across the membrane. Drugs
such as heparin, dimethyl sulfoxide and the platelet-activating factor (PAF) are able to
modify the fraction of the chemical energy released during ATP hydrolysis that is used for
Ca2+ transport and the fraction that is dissipated in the surrounding medium as heat. The
thyroid hormone 3,5,3¢-triiodo L-thyronine (T3) regulates the expression and function of the
thermogenic SERCA isoforms. Modulation of heat production by SERCA might be one of
the mechanisms involved in the increased thermogenesis found in hyperthyroidism.
KEY WORDS: Ca2+-ATPase; thermogenesis; Ca2+ transport; heat production; ATP
hydrolysis; thyroid hormone.
THERMOGENESIS
Heat generation plays a key role in the regulation of cellular energy balance, and
altered thermogenesis is noted in several diseases, such as obesity and thyroid dysfunction [1–8]. The maintenance of body temperature depends on the balance between heat production and dissipation. Heat production by an organism at
thermoneutrality is called obligatory thermogenesis, which is a consequence of all
metabolic reactions inherent to life. Cold exposure leads to increased heat production the so-called facultative thermogenesis, which is conceptually divided into
shivering and nonshivering thermogenesis [6, 8]. In this review we will focus in the
1
From the Instituto de Bioquı́mica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, 21941-590, RJ, Brasil.
2
Instituto de Biofisica Carlos Chagas F, Universidade Federal do Rio de Janeiro, Cidade Universitária,
21941-590, RJ, Brasil.
3
To whom correspondence should be addressed. E-mail: [email protected]
181
0144-8463/05/0800-0181/0 2005 Springer Science+Business Media, Inc.
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De Meis, Arruda, and Carvalho
heat produced during ATP hydrolysis by muscle Ca2+-ATPase, a probable source of
heat contributing to both obligatory and facultative thermogenesis.
ENERGY INTERCONVERSION BY ENZYMES
Evidence reported during the past five years indicate that enzymes are able to
handle the energy derived from the hydrolysis of phosphate compounds in such a
way as to determine the parcel that is used for work and the fraction that is converted into heat [9–20]. This is particularly noticeable in membrane-bound enzymes
that are involved in the transport of ions. The ability to modulate the conversion of
energy into either heat or work varies depending on both the enzyme and the
experimental conditions used. Examples of enzymes that are able to modulate the
conversion of energy are hexokinase, inorganic pyrophosphatase and the various
SERCA isoforms. Bianconi [21] noticed a small change of enthalpy for the reactions
of yeast hexokinase that depends on the hexokinase isoenzyme used. Recently, it was
shown that the amount of heat produced during PPi hydrolysis varies greatly
depending on whether the reaction is catalyzed by yeast soluble pyrophosphatase
(PPase) or by the membrane-bound pyrophosphatase responsible for proton transport in corn vacuoles [11]. When the soluble inorganic PPase catalyzes the hydrolysis
of pyrophosphate, the amount of heat released is 6.3 kcal/mol PPi cleaved. However,
with the membrane bound PPase, the heat released varies between 7.5 and 23.5 kcal/
mol depending on the KCl concentration in the medium and on whether or not a H+
gradient is formed across the vacuoles membrane (Fig. 1). From the various enzymes
-25
∆ Η cal, kcal/mol
-20
-15
-10
-5
0
0 20 40 60 80 100
KCl, mM
Fig. 1. Effect of K+ on the DHcal of PPi hydrolysis. The
assay medium composition was 50 mM MOPS–Tris buffer
(pH 7.5), 0.3 mM PPi and 0.6 mM MgCl2 and inorganic
PPase from yeast (D) or vacuolar PPase in the absence (O) or
presence (d) of 5 lM FCCP. For details, see Ref. [11].
Heat Production During ATP Hydrolysis
183
that are able to control the conversion of energy into either work or heat, the best
known are the sarco/endoplasmic reticulum Ca2+-ATPases (SERCA).
THE VARIOUS SERCA ISOFORMS
This is a family of membrane-bound ATPases that are able to translocate Ca2+
ions across the membrane using the chemical energy derived from ATP hydrolysis.
Different genes encode the SERCA isoforms [22, 23]. SERCA 1 is the isoform found
in white muscle and rat brown adipose tissue [9, 15, 22, 23]. Red muscle expresses
both SERCA 1 and SERCA 2a while blood platelets and lymphoid tissues express
SERCA 2b and SERCA 3 [9, 20, 22–24]. From the various isoforms, SERCA 1 is the
only form able to modulate the amount of heat produced during ATP hydrolysis
being able to release from 10 up to 30 kcal/mol ATP depending on whether or not a
Ca2+ gradient is formed across the membrane [9, 14, 18, 20]. The catalytic cycle for
Ca2+ transport of the various SERCA isoforms is the same (reactions 1–6 in Fig. 2).
The cycle of SERCA 1 however, has an additional step (reaction 7 in Fig. 2) that is
only activated when the vesicles accumulate Ca2+ and a gradient is formed across
the membrane. This ramification allows SERCA 1 to catalyze the hydrolysis of ATP
through two different catalytic routes [9, 10, 14, 19, 25]. In one of them hydrolysis is
coupled with the translocation of Ca2+ through the membrane and is the same in all
the isoforms known (reactions 1–6). The second route found in SERCA 1 is not
coupled to the transport of Ca2+ and all the energy derived from ATP hydrolysis is
converted into heat (reaction 7 in Fig. 2). In the presence of a Ca2+ ionophore (leaky
vesicles) Ca2+ is translocated across the membrane during ATP cleavage but there is
no Ca2+ accumulation in the vesicles lumen. With leaky vesicles the heat release by
the various isoforms is the same and varies between 9 and 11 kcal/mol ATP cleaved.
With intact vesicles, Ca2+ is accumulated in the vesicles lumen and for the SERCA 1
this triggers the shortcut of the transport cycle where the cleavage of ATP is completed in a step that precedes the translocation of Ca2+ through the membrane
(reaction 7 in Fig. 2). Therefore, through this second route SERCA 1 operates as a
thermogenic device that enhances the amount of heat produced during ATP cleavage
from ~10 kcal/mol to the range varying from 20 up to 30 kcal/mol [14]. Different
from SERCA1, the heat released during ATP hydrolysis by SERCA 2 and 3 (Fig. 3)
are the same (~10 kcal/mol) in presence or absence of a gradient [18, 20].
During transport, the amount of ATP cleaved through the uncoupled route
varies depending on the source of the vesicles. In rabbit white muscle vesicle, the rate
of ATP cleaved through the uncoupled thermogenic route (reaction 7 in Fig. 2) is
2–8-folds faster than the activity coupled to Ca2+ transport. In BAT the ratio
between the uncoupled and coupled ATPase activities is much higher than that of
white muscle; in these vesicles, after formation of a Ca2+ gradient, only one out of
365 ATP cleaved is used to pump Ca2+ across the membrane [15].
FUTILE CYCLE OF ATP HYDROLYSIS
Activation of BAT thermogenic activity is associated with an increase of the
mitochondrial respiration rate. This has been attributed to the leakage of protons
across the inner mitochondrial membrane promoted by activation of UCP 1. In
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De Meis, Arruda, and Carvalho
Fig. 2. The catalytic cycle of the Ca2+-ATPase. The sequence includes
two distinct enzymes conformations, El and E2. The Ca2+ binding sites
in the E1 form face the external surface of the vesicle and have a high
affinity for Ca2+ (Ka = 10)6 M at pH 7). In the E2 form the Ca2+
binding sites face the vesicle lumen and have a low affinity for Ca2+
(Ka=10)3 M at pH 7). The enzyme form El is phosphorylated by ATP
but not by Pi, forming the high-energy phosphoenzyme 2Ca:E1 ~ P (Keq
hydrolysis ~ 106, DG ~ )8.4 kcal/mol). The enzyme form E2 is phosphorylated by Pi but not by ATP, forming the low energy phosphoenzyme 2Ca:E2-P (Keq hydrolysis ~1, DG ~ 0 kcal/mol). When the Ca2+
concentration on the two sites of the membrane is inferior to 50 lM
(leaky vesicles), reaction 4 is irreversible and this forces the sequence to
flow forward from reaction 1 to 6. Reaction 7 (dashed lines) has only
been described for SERCA 1 and is only detected when during transport
the Ca2+ concentration in the vesicle lumen raises to a value higher than
1 mM. This permits the reversal of reactions 4 and 3, condition that
triggers accumulation of the phosphoenzyme form 2Ca:E1 ~ P and
activation of the uncoupled ATPase activity mediated by reaction 7. For
details, see Refs. [10, 15, 17, 19, 25–34].
order to compensate for this leak, the cell would then increase the rate of oxygen
consumption to maintain the proton gradient at a competent level for ATP synthesis
[3, 6, 35–41]. Heat is produced during the uncoupled ATPase activity of SERCA 1
from either white muscle or BAT vesicles and, in the living cell the ADP produced by
the uncoupled ATPase activity should also lead to an increase of the mitochondrial
oxygen consumption in order to maintain the cytosolic ATP concentration. Therefore, the SERCA 1 uncoupled ATPase activity of BAT and white muscle may represent one of the routes of heat production that contributes for the thermogenic
function of cells, both cleaving ATP for the sole purpose of producing heat and by
Heat Production During ATP Hydrolysis
Heat released, mcal / mg
1000
185
120
(a)
(b)
100
800
80
600
60
400
40
200
20
0
0
0
20 40 60 80
µmol Pi /mg
0
2
4
6
8
10
µmol Pi /mg
Fig. 3. White and Red muscle: Correlation between heat release and ATP
hydrolysis in the presence and absence of a Ca2+ gradient. The assay
medium composition was 50 mM MOPS–Tris buffer (pH 7.0), 4 mM
MgCl2, 100 mM KCl, 5 mM NaN3, 10 mM Pi,1 mM ATP, 0.10 mM
EGTA, 0.112 mM CaCl2. The free Ca2+ concentration calculated was
10 lM. The reaction was performed at 35C and it was started by the
addition of vesicles derived from the sarcoplasmic reticulum of white (a)
or red muscle (b), in the presence (O) or absence (d) of a Ca2+ gradient.
The same result was observed using either blood platelets or red muscle
vesicles. For details see Refs. [18, 20].
activating mitochondria oxygen consumption, a condition that increases the rate of
all reactions involved in the respiration and that generate heat.
REGULATION OF THE THERMOGENIC ACTIVITY OF SERCA
With intact vesicles (gradient) derived from white muscle sarcoplasmic reticulum it was found that the amount of heat produced during ATP hydrolysis varies
depending on the Ca2+ concentration in the medium. In presence of a Ca2+ concentration similar to that found in the cytosol of a relaxed muscle fiber (1 lM) the
heat produced was 32 kcal/mol ATP cleaved, and decreased to 23 kcal/mol ATP as
the Ca2+ concentration was raised to a level similar to that found in the cytosol
during muscle contraction (~10 lM). This was not observed in leaky vesicles, where
the heat produced varied between 10 and 12 kcal/mol ATP regardless of the Ca2+
concentration in the medium [18].
The thermogenic activity of rabbit SERCA 1 is only active in the physiological
temperature range ()35C). Both the uncoupled ATPase activity and the amount of
heat produced during ATP hydrolysis decrease as the temperature of the medium is
lowered and at 20C it is abolished, the amount of heat produced both in presence
and absence of gradient being the same (~10 kcal/mol). Inhibition of the uncoupled
ATPase activity is also observed at 35C when the water activity of the medium is
decreased by the addition of dimethyl sulfoxide [10, 12].
An intriguing finding was that SERCA 2b and SERCA 3 found in blood
platelets become thermogenic, i.e., behave as SERCA 1 in the presence of platelet
activating factor-PAF [18]. PAF is produced when cells involved in inflammatory
process are activated and the effect of this phospholipid is specific for blood platelet
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De Meis, Arruda, and Carvalho
vesicles. This finding indicates that some substances, like cytokines, hormones and
neurotransmitters might modulate the uncoupled ATPase route and thus heat production by Ca2+-ATPases.
REGULATION BY THYROID HORMONES
The thyroid hormone 3,5,3¢-triiodo L-thyronine (T3) modulates oxygen consumption in different organs through the regulation of different metabolic routes. T3
regulates both obligatory and facultative thermogenesis since hypothyroid animals
cannot survive during acute cold exposure [42, 43], and mice lacking T3 receptor al
have decreased body temperature at thermoneutrality [44, 45]. In contrast, hyperthyroidism leads to increased energy expenditure, which may promote body weight
loss. In hypothyroid humans, the ingestion of thyroid hormones induces significant
modifications in the resting energy expenditure thus demonstrating the physiological
importance of T3 in the control of basal metabolic rate [46]. In small mammals, the
activation of BAT thermogenic activity requires the concerted action of T3 and
norepinephrine. [35, 36, 39, 42, 43]. T3 regulates the expression of different proteins
that seem to be involved in thermogenesis. This includes the mitochondrial uncoupling proteins 1 and 3 (UCP1 and UCP3) and the various SERCA isoforms. The
direct involvement of uncoupling proteins in thermogenesis is not clearly defined [35,
47, 48]. In fact, in spite of its pronounced effects on thermogenesis, the mechanism by
which T3 acts is still unclear.
In animals devoid of BAT, the main thermogenic tissue seems to be the skeletal
muscles, which corresponds to approximately 40% of human body. Thus, a small
change in the rate of heat production in skeletal muscle represents a large contribution to the heat produced by the whole body [2, 3]. The phenotype of white and
red muscles and the transcription of SERCA1 and SERCA 2a genes are regulated by
T3 [9, 49–57]. In red muscle hyperthyroidism induces an increase in SERCA 1
expression accompanied by a decrease in SERCA 2, whereas hypothyroidism causes
a decrease in both SERCA 1 and SERCA 2 content. In white muscles the effect of T3
is less pronounced. In hyperthyroidism a small or no increase of SERCA 1 are
detected [9, 54].
Apart from the effects of T3 on SERCA expression, it was recently shown that
hyperthyroidism promotes a modification of the kinetic activity of SERCA 1, with a
45% increase in both the rate of ATP hydrolysis and the rate of heat production [9].
In this case, the increased heat production is not related to a modification of the
amount of heat released during the cleavage of each ATP molecule (DHCal). In red
muscles, however, the increase in SERCA 1 expression, accompanied by a decrease
in SERCA 2, leads to a twofold increase in the rate of ATP hydrolysis and a fourfold
increase in the rate of heat production [9]. The discrepancy between the rates of ATP
hydrolysis and heat production is related to the different DHCal of ATP hydrolysis of
SERCA 1 and SERCA 2 (Fig. 4).
Finally, T3 also regulates the expression of the Na+/K+-ATPase. This enzyme
is also involved in the process of heat production during thermogenesis, however at
present there are no data available indicating whether or not the Na+/K+-ATPase
can handle the energy derived from ATP hydrolysis in such a way as to determine the
parcel that is used for work and the fraction that is converted into heat.
Heat Production During ATP Hydrolysis
187
Fig. 4. White and Red muscle: ATP hydrolysis (a) and (d), heat release (b) and (e) and
DHcal of ATP hydrolysis (c) and (f). Upper panel – White muscle, Lower panel – Red
muscle. Values of DHCal were calculated by dividing the amount of heat released by the
amount of ATP hydrolyzed. Negative values indicate that the reaction is exothermic. (O)
control and (d)hyperthyroid rabbits. The figure shows a representative experiment. For
details see Ref. [9].
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