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in PresS. Am J Physiol Cell Physiol (August 30, 2006). doi:10.1152/ajpcell.00208.2006
1
Mechanisms of mitochondrial response to variations in energy demand in
eukaryotic cells
Anne Devin and Michel Rigoulet*
IBGC du CNRS, UMR 5095, Université Victor Segalen Bordeaux 2, 1 rue Camille Saint
Saëns, 33077 Bordeaux cedex, France. Tel/Fax (33) 5-56-99-90-40
Running head: energy demand and mitochondrial adaptations
*To whom correspondence should be addressed: [email protected]
Copyright © 2006 by the American Physiological Society.
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Abstract
This review focuses on the different mechanisms involved in the adjustment of
mitochondrial ATP production to cellular energy demand. The oxidative phosphorylations
steady state at constant mitochondrial enzyme content can vary in response to energy demand.
However, such an adaptation is tightly linked to a modification in both oxidative
phosphorylations yield and phosphate potential and is obviously very limited in eukaryotic
cells. We herein describe the three main mechanisms involved in mitochondrial response to
energy demand. In heart cells, a short-term adjustment can be reached mainly through
metabolic signaling via phosphotransfer networks by the compartmentalized energy transfer
and signal transmission. In such a complex regulatory mechanism, calcium signaling
participates in an activation of matricial dehydrogenases as well as of the mitochondrial ATP
synthase. These processes allow a large increase in ATP production rate without any
important modification in thermodynamic forces. For a long term adaptation two main
mechanisms are involved: the modulation of the mitochondrial enzyme content as a function
of energy demand and/or kinetic regulation by covalent modifications (phosphorylations) of
some respiratory chain complexes subunits. Regardless of the mechanism involved (kinetic
regulation by covalent modification or adjustment of the mitochondrial enzymatic content),
the cAMP signaling pathway plays a major role in the molecular signaling leading to the
mitochondrial response. We will discuss the energetic advantages of these mechanisms.
Keywords: yeast, C6 glioma cells, muscle, kinetic regulation.
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Introduction
Mitochondria are intracellular organelles that play an important role in eukaryotic
cells. They are now known to intervene not only in energy transduction and some
intermediary metabolism pathways but also in calcium homeostasis, cell signaling or
apoptosis. One important feature of these organelles is that they harbor their own circular
genome. In mammalian cells, the mitochondrial DNA contributes 13 mRNA, 22 tRNA, and 2
rRNA molecules that are essential to mitochondrial function. The thirteen mRNA molecules
all encode components of the oxidative phosphorylations apparatus. These thirteen
components are combined with nuclear encoded proteins to form multi-subunit holoenzymes:
cytochrome oxidase, respiratory chain complexes 1 and 3 and ATPsynthase. The function of
these holoenzymes is clearly impaired if contribution from either mitochondrial or nuclear
genome is absent (20). Indeed, most of the proteins belonging to mitochondria are encoded by
the nuclear genome. Thus, mitochondrial biogenesis involves the coordinated expression of
hundreds of genes both at the nuclear and at the mitochondrial level. The understanding of the
interaction between these two genomes, leading to functional mitochondria has gained some
insights over the past few years. Depending on the physiological stimulus, several
transcription factors are now known to be activated and their recognition sequence on various
mitochondrial protein encoding genes have been identified. Moreover, mitochondrial
composition can change. Indeed, mitochondrial content is commonly estimated by (i) the
maximal respiratory capacity, (ii) the activity of a typical marker enzyme e.g. citrate synthase
or (iii) the content of a single mitochondrial protein. This assumption is valid if one considers
the mitochondrial content to be regulated as a whole i.e. every single mitochondrial protein
expression is regulated in the same way. This assumption should be seriously reconsidered
since mitochondrial protein composition has been shown to vary, for example, in response to
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chronic exercise (19, 21, 31, 43). Such a peculiar organelle biogenesis might be linked to the
fact that mitochondrial biogenesis requires the cooperation of both, the nuclear and the
mitochondrial genomes. Thus, depending on the signaling pathway involved and the way it
regulates the expression of genes encoding mitochondrial proteins, different mitochondrial
compositions might be set up.
Mitochondria are responsible for countless functions, one of them being the generation
of ATP from metabolic fuels through oxidative phosphorylations. Strong reducing agents
such as NADH and FADH2 donate electrons to the respiratory chain resulting in the
establishment of an electrochemical potential difference in protons across the mitochondrial
inner membrane. This proton electrochemical potential difference is in turn used by the
mitochondrial F0F1-ATPsynthase, which couples the proton input to ATP synthesis.
Mitochondria have to make energy conversion meet energy demand, which can vary by
consequential lengths. To do so, one can expect mitochondria to use at least three means,
which are not exclusive: (i) a change in the oxidative phosphorylations rate, as it is now well
established that oxidative phosphorylations in the living cell are not functioning to their fullest
(i.e. the maximal respiratory capacity is usually higher than the spontaneous respiratory rate);
an increase or a decrease in oxidative phosphorylations rate would thus allow an increase or a
decrease in energy conversion flux (i.e. the rate of ATP synthesis); such an adaptation
involves a modification in associated forces and in oxidative phosphorylations yield (15, 32);
(ii) a change in the oxidative phosphorylations steady state by kinetic regulations of one or
more controlling steps such as complex I and cytochrome-c-oxidase; (iii) energy demand
could also be met by a change in the amount of mitochondrial enzymes per cell, with a
constant steady state in the activity of each compound. In this latter case, associated forces
and oxidative phosphorylations yield may not be affected.
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In this short review, we will take stock of what is known on the mechanisms of
mitochondrial adaptation to changes in cell energy demand.
Mitochondrial response through variations in the cellular mitochondrial
enzymatic content.
1-In yeast, there is a strict adjustment between growth and mitochondrial enzyme
content
Microbial growth is a striking adaptation of microorganisms to environmental changes
aiming at reaching a compromise between growth rate, growth yield and thermodynamic
growth efficiency. The growth yield is the part of the energy input (substrate consumption)
converted into biomass during growth. Over the last two decades, the complete
thermodynamic description of growth processes has been obtained by establishing the
balanced chemical reactions for anabolism and catabolism. Thus, by applying nonequilibrium thermodynamics, two parameters have been defined to assess growth efficiency:
thermodynamic Gibbs energy efficiency ( G) and thermodynamic enthalpy efficiency ( H).
The estimation of the former parameter for microbial growth is still difficult due to the lack of
knowledge about the Gibbs energy change of substrates, products and biomass. In contrast,
the quantification of enthalpy efficiency has been successfully achieved for microorganisms
as well as for cultured mammalian cells. This approach is based on the continuous
measurement of heat production and on the exhaustive determination of substrates and byproducts concentrations, thus allowing the construction of enthalpy balances (see 17, 50 and
51 for reviews). In microorganisms, the strategy of optimizing growth rate and growth yield
has mainly been studied under conditions of active exponential growth (17, 26, 42). We
analyzed the variation in enthalpy growth yield under conditions where a long-term
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adaptation of yeast cells occurs in response to carbon source depletion in the culture medium.
The enthalpy growth yield is defined as the enthalpy variation corresponding to the complete
combustion of the formed biomass divided by the enthalpy variation corresponding to the
complete combustion of the consumed substrates. We particularly focused on the role played
by the mitochondrial compartment during the transition from exponential growth to stationary
phase, during aerobic metabolism.
As stated above, growth is the result of the coupling between substrate catabolism and
various anabolic reactions taking place during net biomass synthesis. In this process, ATP
cycling plays a central role (figure 1). When eukaryotic cells are grown aerobically with a
non-fermentable substrate as sole carbon and energy source, ATP synthesis relies mainly on
mitochondrial oxidative phosphorylations, as opposed to substrate level phosphorylations. In
this global scheme, the growth yield represents the amount of carbon substrate assimilated
into biomass as compared to the total amount of substrate used for all the metabolic processes.
Of course, under steady state conditions, where ATP synthesis matches ATP consumption, the
growth yield may depend on two variables. First, the fraction of ATP utilized for cell
maintenance versus that used for biomass synthesis per se. Second, the growth yield may be a
function of the amount of ATP synthesized per oxygen consumed during oxidative
phosphorylations (i.e. ATP/O ratio). The actual ATP/O ratio has been shown, in vitro in
isolated mitochondria, to vary according to the proton leak, the degree of redox proton pump
slipping (15, 32) and the functional steady state of mitochondria i.e. ATP/O ratio varies from
zero under non-phosphorylating conditions (state 4) to the maximal value that can be
sustained in the presence of saturating amounts of Pi, ADP and respiratory substrate (state 3)
(figure 2). Thus, by extrapolating these results to the in vivo situation, the growth yield should
be decreased when the ATP turnover decreases, because the consumption of the respiratory
substrate required to compensate for the proton-leak and proton-slipping increases.
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During the growth of yeast in a medium containing a limiting amount of a nonfermentable substrate such as lactate, we estimated the growth yield from the measure of four
parameters: the net substrate (lactate) consumption, the by-product formation, the biomass
formed and the heat production which is the part of the energy input lost for maintenance and
energy transduction processes. Three distinct growth phases can be defined: (i) in the early
exponential phase, the heat production increased in parallel with biomass production; (ii) in
the transition (or late exponential) phase, occurring when about one third of the lactate had
been consumed, the heat dissipation pattern no longer correlated with the biomass increase;
(iii) in the stationary phase, lactate was exhausted and the heat production rate eventually
reached a constant and low value, consistent with the growth arrest (10). Energy balance in
each phase may be calculated with integrated values of net substrate consumption (energy
input) and net product formation, including biomass and heat dissipation (energy output),
transformed into energy units. The part of the energy input converted into biomass was nearly
the same during the early exponential and transition phases indicating that the enthalpy
growth yield remained constant (10). However, during the transition phase, the ATP demand
for growth decreased and the question arose as of the nature of the mechanism(s) responsible
for the maintenance of the enthalpy growth yield.
We observed a parallel decrease in the activities of all the measured mitochondrial
enzymes (i.e. D,L-lactate dehydrogenase and citrate synthase) as well as a decrease in the
amount of cytochromes, indicating that the adaptive process involves a decrease in the
amount of mitochondria per cell but not a change in the oxidative phosphorylations steady
state (10). Taking into account the part of energy used for maintenance, it can be concluded
that mitochondria themselves are the major heat dissipative system in a fully aerobic
metabolism (see also 11), and that the parallel decrease in the ATP demand and in the amount
of mitochondria when growth rate decreases leads to a constant enthalpy growth yield.
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2- The cAMP/protein kinase A signaling pathway is involved in the yeast mitochondrial
adaptive response.
In mammalian cells, several cAMP targets and transcription factors seem to be
involved in the up-modulation of mitochondrial biogenesis when energy demand increases
(see below). In the yeast Saccharomyces cerevisiae, the RAS/cAMP/protein kinase pathway is
involved in many physiological adaptations of cells upon environmental changes. This
includes the diauxic shift, responses to nutrient starvation, oxidative stress and heat shock (6,
23, 40, 44, 46, 53). We analyzed the ability of various mutants of the RAS/cAMP/protein
kinase pathway to develop their mitochondrial compartment when grown on lactate. In the
yeast RAS cascade, represented in Figure 3, CDC25 catalyzes the conversion of GDP-RAS1
and 2 into GTP-RAS1 and 2, which are the activators of CYR1, the adenylate cyclase (47)
catalyzing cAMP synthesis. The cAMP intracellular concentration thus depends on the
respective activities of CYR1 and the phosphodiesterases PDE1 and 2. High cAMP
concentrations promote the dissociation of the regulatory subunit (BCY1 (49)) from the
catalytic subunits (TPK1,2,3 (48)), thus activating the catalytic subunits of the protein kinases
A, which phosphorylate a variety of substrates. The mutants used in our study (12) were the
following: (i) a mutant disrupted for RAS2 which leads to under-activation of the RAS/cAMP
signaling pathway; (ii) three mutants in which the RAS/cAMP/protein kinase signaling
pathway is over-activated: IRA1 and 2 genes disruptant, RAS2val19 point-mutant and a BCY1
inactivation mutant. The mutant disrupted for the RAS2 gene is characterized by a slight
decrease in the content of all the respiratory chain cytochromes and in maximal respiratory
capacity. By contrast, whatever the mutation considered, over-activation of this signaling
pathway induced a twofold increase in the cellular content of all the cytochromes, except for
cytochrome oxidase in the RAS2val19 mutant. These changes in cytochrome content correlated
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with the variation in respiratory capacity.
A direct involvement of cAMP in the regulation of cellular mitochondrial content has
been further underlined by studying the OL556 strain (6), in which the high affinity
phosphodiesterase (PDE2) and CDC25 were inactivated rendering this strain sensitive to
exogenous cAMP. Consequently, in the presence of cAMP in the culture medium, OL556
cells possess all the characteristics of RAS/cAMP pathway overactivated mutant strains (13).
OL556 cells were aerobically grown with either 0.2% or 2% lactate as carbon source. In the
presence of O.2% lactate, addition of 3mM cAMP increased the growth rate from 0.15 to 0.2
per hour, the latter value being close to the one measured in the presence of 2% lactate (Table
1). In this case, the growth rate remained unchanged after cAMP addition. Moreover, for cells
grown with 0.2% lactate, cAMP addition did not significantly change the cell protein content
expressed as dry mass. In contrast, cAMP treatment led to a markedly high protein
accumulation in cells grown with 2% lactate. By multiplying the cellular protein content by
the respective growth rate, we observed that cAMP addition increased the protein synthesis
rate to about the same extent, regardless of the concentration of lactate in the medium (45%
and 55% increase in the presence of 0.2% and 2% lactate, respectively). At the same time,
cAMP addition significantly changed the respiratory activity of cells grown in the presence of
2% lactate and to a lesser extent in the presence of 0.2% lactate. This suggested a global
activation of the metabolism of these cAMP-treated cells. Moreover, the effect of 3mM
cAMP on the enthalpy growth yield was dependent on the carbon source limitation i.e. in
0.2% lactate, the decrease in enthalpy growth yield was 20% whereas it reached 40% in the
presence of 2% lactate (Table 1). This was further confirmed by the assessed enthalpy
balance. The part of the energy input lost as heat or stored as biomass was not affected by
variations in lactate concentration. cAMP treatment in the presence of 0.2% lactate increased
the heat production and decreased the biomass yield. Finally, a more pronounced effect of
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cAMP was observed in the presence of 2% lactate: an increase in both by-product formation
(pyruvate plus acetate) and heat production and a large decrease in biomass formation (13).
The decrease in the enthalpy growth yield is not due to an alteration in mitochondrial
oxidative phosphorylation, leading to a higher respiration rate. Indeed, in permeabilized
spheroplasts (1), the (non-phosphorylating) state 4 and the ADP-induced state 3 respiration
were about two times higher when cells were grown with cAMP than without. Moreover, the
respiratory control ratio was slightly higher for cells grown in the presence of cAMP.
Additional data (electron microscopy, citrate synthase and D+L-lactate dehydrogenase
activities, cytochrome content) confirmed that the number of mitochondria is almost two
times higher in cells grown in the presence of cAMP. However, the isolated mitochondria are
not significantly different when prepared from cAMP treated or untreated cells (13). In
conclusion, under conditions where the growth rate was already optimal (high lactate
concentration), addition of exogenous cAMP led to the proliferation of well-coupled
mitochondria within the cells. This phenomenon led to uncoupling between biomass synthesis
and catabolism and consequently to a decrease in the enthalpy growth yield. This confirms
that mitochondria by themselves are a major heat dissipative system in a fully aerobic
metabolism and that a subtle adaptation of the amount of mitochondria to the growth rate is
necessary to maintain the enthalpy growth yield. Taken together our results show that the
RAS/cAMP/protein kinase cascade plays an important role in this adaptation but do not
exclude the participation of other signaling pathways.
Yeast harbors three cAMP protein kinase (PKA) catalytic subunits, which have greater
than 75% identity and are encoded by the TPK (TPK1, TPK2 and TPK3) genes (48). Although
they are redundant for viability and functions such as glycogen storage regulation, the three
kinases are not redundant for other functions such as pseudohyphal growth, regulation of
genes involved in trehalose degradation and water homeostasis as well as iron uptake, which
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are all regulated by Tpk2p (33, 34, 38). Tpk1p is required for the derepression of branched
chain amino acid biosynthesis genes that seem to have another role in the maintenance of iron
levels and DNA stability within mitochondria (39). These data provide evidence for a
specificity of signaling through the three PKA catalytic subunits. In order to elucidate a
potential role of one or more of these subunits in the regulation of mitochondrial biogenesis in
response to energy demand during growth, we investigated the role of each one of the TPKs
for this process. We showed that the yeast protein kinase Tpk3p is specifically involved in the
regulation of mitochondrial enzymatic content at the time of the transition phase when cells
are grown in 2% lactate (8). Indeed, compared to wild type, the +tpk3, but not +tpk1 or +tpk2,
strain showed a decrease in the spontaneous respiration rate as well as in the growth rate
during the transition phase. Thus, this PKA is involved in the cellular response leading to a
decrease in the growth rate when reaching the stationary phase (i.e., transition phase). In order
to further characterize the mitochondrial compartment in these cells, we isolated mitochondria
from both the wild type and the mutant strains. Phosphorylating and uncoupled respiratory
rates and enzymatic activities (i.e., citrate synthase, cytochrome-c-oxidase and oligomycinsensitive ATPase) were decreased (around 40%) in the mutant compared to the wild type.
However, when cytochrome content was measured in both strains, the respiratory chain
composition was clearly modified. Indeed, whereas the cytochromes a+a3, b and c1 were not
significantly affected in the mutant strain, the c content was largely decreased (40%). The
decrease in phosphorylating and uncoupled respiratory rates, as well as cytochrome-c-oxidase
activity, originated in this decrease in cytochrome c content. Under non-phosphorylating
conditions, the mitochondria isolated from the mutant exhibited a lower respiratory rate than
the wild type mitochondria for a comparable protonmotive force, indicating that the energy
waste was decreased in these mitochondria. This is due to a decrease in the slipping process at
the level of cytochrome-c-oxidase (8) originating in an enhancement of the kinetic constraints
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on the electron flux at the level of cytochrome c leading to an increase in reactive oxygen
species (ROS) production (unpublished results). This raises the question of an involvement of
these species in the adjustment of mitochondrial enzymatic content in response to energy
demand.
In conclusion, from studies of various strains in different growth phases, it is clear that
in yeast, there is a homeostasis of growth yield due to a tight adjustment of mitochondrial
enzyme content to the growth rate. The signaling pathway responsible for such an adjustment
is the Ras/cAMP pathway. However, upstream of the ras proteins and downstream of the
PKA, the molecular mechanisms remain to be elucidated even though the reactive oxygen
species seem to act as signaling molecules in this process, downstream of the PKA.
3-In muscle, the mitochondrial enzymatic content is increased in response to energy
demand upon exercise
Muscle mitochondrial content can be increased by 50 to 100 % within 6 weeks of
endurance training. Chronic contractile activity produced by electrical stimulation of the
motor nerve can mimic this mitochondrial biogenesis. Williams et al. (52) were the first to
show that chronic contractile activity led to increases in mRNA levels encoding both nuclear
and mitochondrial gene products.
Numerous rapid events occur at the onset of contractile activity. Two of them have
been shown to be involved in mitochondrial biogenesis: calcium signaling and ATP turnover.
When released from the sarcoplasmic reticulum and in addition to its role in actin/myosin
interaction, calcium can also activate a number of kinases (Ca2+calmodulin kinase II, protein
kinase C) and phosphatases, which translocate their signals to the nucleus and alter the rate of
gene transcription. Moreover, increases in cytosolic calcium levels are known to be matched
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within the mitochondria and directly influence the rate of mitochondrial respiration (25). This
occurs via the activation of dehydrogenases, which require calcium for full activity (30). It
has also been shown that the mitochondrial ATP synthase was activated by Ca2+ (18). Thus,
calcium itself is a signaling molecule allowing an integrated activation of the overall oxidative
phosphorylations process. This could lead to an increase in mitochondrial ATP synthesis
without any change in both mitochondrial and cytosolic phosphate potentials (24). However,
quantitative estimates of calcium effects in mitochondria are in conflict with the magnitude of
changes in the respiratory rate in vivo upon contractile activity (9, 45). More likely, the
regulation of oxidative phosphorylations in these cells involves a high level of organization of
the metabolic signaling network. In this regard, a new role of spatially arranged intracellular
enzymatic networks, catalyzed by creatine kinase, adenylate kinase, carbonic anhydrase and
glycolytic enzymes in supporting high-energy phosphoryl transfer and signal communication
between ATP-generating and ATP-consuming/ATP-sensing processes had emerged. This
dynamic concept points out that metabolic signaling through near-equilibrium enzymatic
networks, along with other homeostatic mechanisms (3) contributes to efficient intracellular
energetic communication in maintaining the balance between cellular ATP consumption and
production. As this research area is currently very productive and in progress and as the
present review is mainly focused on the adaptation of oxidative phosphorylation to long-term
variations in cell energy demand, the readers are referred to recent more specialized reviews
(4, 5, 14 and 41).
In muscle cells, since ATP production is able to match ATP consumption in a wide
range and without any variation in phosphate potential, the hypothesis that disturbance in
energy metabolism, leading to either ATP depletion or a change in the phosphorylation
potential, could initiate a compensatory response, ultimately leading to an increase in
mitochondrial content (37), seems unlikely. However, an increase in ATP turnover without
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any variation in cellular ATP levels can also lead to mitochondrial biogenesis. Calcium
induces an increase in cytochrome c mRNA levels mediated by the activation of PKC
isoforms (16). However, it also appears that an increase in calcium cannot, by itself, lead to an
increase in overall mitochondrial biogenesis. Indeed, subsequent studies have shown that
whereas a number of nuclear genes encoding mitochondrial subunits expression are increased
along with cytochrome c, a number of others that would be critical for mitochondrial
biogenesis, such as COX subunits IV, Vb and VIc, are not. Two interpretations arise: either
calcium only forms part of a broader series of signals that mediate modifications in the
synthesis of mitochondrial components or the overall stoichiometry of the respiratory chain is
modified.
Under conditions of partial mitochondrial uncoupling which mimics the intense
exercise, ATP production matches ATP consumption at lower phosphate potential, and an
induction of the nuclear respiratory factor 1 (NRF-1) is observed. Subsequent to this
induction, an increase in the expression of its target genes has been observed. It appears that
the increase in the mitochondrial respiration or the imbalance between ATP demand and ATP
supply provides a stimulus for the sequential induction of a variety of genes involved in the
biogenesis of the organelle.
A number of transcription factors have been implicated in mitochondrial biogenesis.
They include NRF-1 and NRF-2, peroxisome proliferator-activated receptors
and (PPAR-
and PPAR-K ), and Sp1 (27). PGC-1 is a transcriptional co-activator that binds PPAR and
thus regulates its activity. It has recently been shown that PGC-1 mRNA increases between
1.5 and 7 to 10 fold following a single bout of exercise (2, 36). Contractile activity has been
shown to induce an increase in the mRNA and/or protein levels of several of these
transcription factors, consistent with their roles in mediating phenotypic changes as a result of
exercise (22). The upstream regulatory regions of genes encoding mitochondrial proteins are
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highly variable in their composition (27, 29). This variability suggests that a coordinated upregulation of genes transcription in response to contractile activity would be difficult to
achieve, unless the multiple transcription factors mentioned above were effective in uniformly
up-regulating the transcriptional activity of numerous genes. A complete coordination among
the responses is not achieved at the protein level (19) and does not seem to be required for an
increase in mitochondrial content and activity i.e. physiological function.
These results tend to show that, similarly to what was shown in yeast, energy
production meets energy demand mainly through an adjustment in mitochondrial enzyme
content. It is obvious that in muscle cells, this adjustment in mitochondrial enzyme content
cannot be continuously correlated to energy demand. Indeed, in these peculiar cells, energy
demand varies rapidly (with a very different time span than mitochondrial turnover) and by
great lengths. Thus, the fact that upon exercise a muscle harbors more mitochondria implies
that at rest there is an increase in energy waste (futile cycles, mitochondrial activity).
In muscle, mitochondrial response to energy demand involves at least two distinct
mechanisms: (i) a short-term event can be reached mainly through metabolic signaling via
phosphotransfer networks by the compartmentalized energy transfer and signal transmission.
In such a complex regulatory mechanism, calcium signaling participates in an activation of
matricial dehydrogenases as well as of the mitochondrial ATP synthase; (ii) a long-term
adaptation involving an enhancement of mitochondrial biogenesis.
Mitochondrial response through changes in the kinetic regulation of
mitochondrial enzymes
C6 glioma cells are cancer cells with a high proliferation rate. Mitochondrial response
to energy demand has been studied in C6 glioma cells during growth. When grown on a 3D
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support, these cells, similarly to yeast cells (see above), exhibit different growth phases.
Unlike some kind of cancer cells, these cells are highly dependent on their respiratory
metabolism.
We
estimated
the
relative
contribution
of
mitochondrial
oxidative
phosphorylations to ATP synthesis to be between 70 and 85% (28). All throughout growth,
the ATP/ADP ratio remained constant while when the growth rate decreased, both glycolysis
and oxidative phosphorylations activities decreased in a similar way. Between the beginning
of the exponential phase and the confluence, the cellular respiratory rate and the maximal
respiratory capacity were decreased by a factor 5. This is not due to a decrease in
mitochondrial enzyme content since, in the meantime, the activities of citrate synthase, and
complexes III and IV were maintained almost constant (28, 35). In contrast, the specific
activity of complex I decreased continuously as the cell density increased. Moreover, there
was a good positive correlation between the respiratory rate and the activity of complex I,
indicating that the cellular respiratory rate is mainly controlled by the activity of this complex
(35). Western blot analysis showed that the amount of complex I was maintained constant all
throughout growth. Immunodetection of the phosphoserine-containing proteins of
immunoprecipitated complex I revealed, among several bands, a major protein band with an
apparent molecular mass of about 16-18 kDa. This phosphorylated protein is likely the
subunit ESSS which has been identified in bovine heart mitochondria as one of two complex I
phosphorylation sites, the other one being MWFE a 10kD protein whose expression is
supposed to be linked to cAMP signaling (7). The phosphorylation level of this 18kDa
subunit decreased during growth of C6 glioma cells, indicating that complex I activity and
thus respiratory rate are kinetically regulated by phosphorylation during growth. Further
experiments demonstrated that the 18 kDa subunit phosphorylation is at least under the
control of the cAMP signaling pathway (35).
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Even though we were not able to decipher the exact molecular mechanism modulating
the phosphorylation state of the 18kDa subunit, it is clear that in C6 glioma cells, the
adjustment of oxidative phosphorylations to energy demand through the growth rate is mainly
due to the regulation of the phosphorylation level of a subunit of complex I.
Concluding remarks:
From the studies reported here, it seems that the mitochondrial adaptation to variation
in cellular energy demand through modifications of oxidative phosphorylations steady state
without kinetic regulation or modification in mitochondrial enzyme content is only observed
as a short-term adjustment. For instance, when yeast cells reach the stationary growth phase
or when C6 glioma cells are grown in a medium deprived of glutamine, one can observe a
growth arrest associated to a decrease in respiratory rate which gets closer to a nonphosphorylating respiratory rate (10, 28). It is noteworthy that in heart cells the short-term
adjustment can be reached mainly through metabolic signaling via phosphotransfer networks
by the compartmentalized energy transfer and signal transmission. In such a complex
regulatory mechanism, calcium signaling participates in an activation of matricial
dehydrogenases as well as of the mitochondrial ATP synthase. This concerted response leads
to an increase in mitochondrial ATP synthesis at nearly constant forces.
For a long-term adaptation two main mechanisms are involved: the modulation of
mitochondrial enzyme content as a function of energy demand and/or the kinetic regulation by
covalent modifications of some respiratory chain complexes subunits. In yeast, the main
process is a tight adjustment of mitochondrial enzyme content to the growth rate in such a
way that the oxidative phosphorylations steady state and consequently the growth yield
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remain constant. In contrast, in C6 glioma cells, the main process is a modulation of the
phosphorylation status of at least the ESSS subunit of complex I, leading to a change in the
activity of this controlling step at constant mitochondrial enzymatic content. It is noteworthy
that whatever the mechanism involved (kinetic regulation by covalent modification or
adjustment of mitochondrial enzyme content), the cAMP pathway plays a major role in the
molecular signaling leading to the mitochondrial response.
From an energetic standpoint, with these long-term adaptation mechanisms, the cell
favors being able to change ATP turnover to a large extent without significant modifications
in the forces associated to oxidative phosphorylations and ATP consumption (mitochondrial
electrochemical potential difference in protons, intramitochondrial and cytosolic phosphate
potentials, mitochondrial and cytosolic redox potentials). This implies that the maintenance of
thermodynamic force homeostasis is vital.
Acknowledgments
The authors wish to thank Dr C. Schwimmer for her contribution to the editing of the
manuscript. The work from our laboratory reported in this review was supported by grants
from the ‘‘Association pour la Recherche contre le Cancer’’, from the ‘‘Conseil Régional
d’Aquitaine’’, from the ‘‘Programme de Recherche CNRS: Génie des procédés chimiques,
physiques et biotechnologiques’’ and from the French National Research Agency (ANR).
Legend to figures
Figure 1: Microbial growth is the result of the coupling between substrate catabolism and all
anabolic reactions taking place during net biomass synthesis. Under respiratory metabolism,
energy transduction is mainly achieved through mitochondrial oxidative phosphorylations. In
this process, a central role is played by ATP cycling, which depends on the phosphate
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potential (ATP/ADP.Pi). The energy delivered by substrate catabolism (energy input) is partly
used for biomass synthesis and dissipated as heat. The part of energy output necessary for cell
homeostasis and life is referred to as maintenance.
Figure 2: Mitochondrial oxidative phosphorylations. Oxidative phosphorylations result in a
chemiosmotic coupling between F0F1-ATP synthase and the proton pumps of the respiratory
chain. At any steady state (e.g. constant proton electrochemical potential difference across the
inner membrane, constant ATP synthesis and respiratory rates), the proton efflux by the
respiratory chain compensates the proton leak and the proton influx through the ATP
synthase. The insert represents the ATP/O ratio as a function of the respiratory rate during Pi
titration of oxidative phosphorylations in isolated yeast mitochondria, and in the presence of
saturating amounts of ADP and NADH as respiratory substrate. State 4 is the nonphosphorylating respiration which compensates the proton leak and proton pump slipping.
State 3 is the maximal phosphorylating steady state (i.e. at saturated concentrations of Pi and
ADP) that gives the maximal yield (i.e. ATP synthesis-oxygen consumption flux ratio).
Figure 3: The Ras/cAMP cascade in yeast.
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Table 1: Effect of cAMP on growth characteristics of OL556 cells on lactate
D,L lactate medium
SC 0.2%
SC 2%
cAMP
-
+
-
+
Growth rate
(P) (h-1)
0.15 ± 0.02
0.20 ± 0.01
0.20 ± 0.01
0.20 ± 0.01
Protein content
(mg/mg dry weight)
0.61 ± 0.02
0.65 ± 0.03
0.58 ± 0.03
0.88 ± 0.05
Protein synthesis rate
(Pg/min/mg dry weight)
1.5 ± 0.2
2.2 ± 0.2
1.9 ± 0.2
2.9 ± 0.3
JO growth
(nat. O/min/mg dry weight)
210 ± 20
300 ± 20
195 ± 20
350 ± 15
Enthalpy growth yield
(YH) (%)
55 ± 3
44 ± 5
55 ± 4
34 ± 3
OL556 cells were grown aerobically in a minimal medium supplemented with either
0.2% or 2% D,L-lactate as carbone source. When added in the culture medium, cAMP was 3
mM. Heat production rate, respiratory rate, biomass and metabolites in the culture medium
were measured throughout the entire exponential phase. Data were from (13).
Page 29 of 31
Energy input
Catabolism
Oxidative
Phosphorylation
ATP/ADP.Pi
Figure 1
Energy output
Anabolism (BIOMASS)
Heat
Maintenance
Page 30 of 31
1.4
ATP/O ratio (nmol ATP/natO)
1.2
1.0
State 3
0.8
0.6
0.4
State 4
0.2
0.0
0
100
200
300
400
500
Respiratory rate (natO/min/mg prot)
Figure 2
600
700
Page 31 of 31
GDP
GTP
cdc25
Ras1,2
Ras1,2
Ira1,2
ATP
Cyr1
Cap
Adenylate
cyclase
Target proteins
Figure 3
cAMP
Pde1;2
Tpk
1,2,3
Tpk
1,2,3
cAMP
Bcy1
cAMP
Bcy1
AMP