Tài liệu Báo cáo khoa học: Metabolic control of mitochondrial properties by adenine nucleotide translocator determines palmitoyl-CoA effects Implications for a mechanism linking obesity and type 2 diabetes pdf

Metabolic control of mitochondrial properties by adenine
nucleotide translocator determines palmitoyl-CoA effects
Implications for a mechanism linking obesity and type 2 diabetes
Jolita Ciapaite
1,5
, Stephan J. L. Bakker
2
, Michaela Diamant
3
, Gerco van Eikenhorst
1
,
Robert J. Heine
3
, Hans V. Westerhoff
1,4
and Klaas Krab
1
1 Department of Molecular Cell Physiology, Institute for Molecular Cell Biology, Faculty of Earth and Life Sciences, VU University,
Amsterdam, the Netherlands
2 Department of Internal Medicine, University of Groningen and University Medical Center Groningen, the Netherlands
3 Department of Endocrinology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, the Netherlands
4 Manchester Centre for Integrative Systems Biology, MIB, University of Manchester, UK
5 Centre of Environmental Research, Faculty of Nature Sciences, Vytautas Magnus University, Kaunas, Lithuania
Keywords
metabolic control analysis; oxidative
phosphorylation; palmitoyl-CoA; reactive
oxygen species; type 2 diabetes
Correspondence
J. Ciapaite, Centre of Environmental
Research, Faculty of Nature Sciences,

Vytautas Magnus University, Kaunas,
Vileikos 8, LT-44404, Lithuania
Fax: +370 37 327904
Tel: +370 37 327905
E-mail:
(Received 21 June 2006, revised 19 August
2006, accepted 4 October 2006)
doi:10.1111/j.1742-4658.2006.05523.x
Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by
long-chain acyl-CoA esters has been proposed to contribute to cellular dys-
function in obesity and type 2 diabetes by increasing formation of reactive
oxygen species and adenosine via effects on the coenzyme Q redox state,
mitochondrial membrane potential (Dw) and cytosolic ATP concentrations.
We here show that 5 lm palmitoyl-CoA increases the ratio of reduced to
oxidized coenzyme Q (QH
2
⁄ Q) by 42 ± 9%, Dw by 13 ± 1 mV (9%), and
the intramitochondrial ATP ⁄ ADP ratio by 352 ± 34%, and decreases the
extramitochondrial ATP ⁄ ADP ratio by 63 ± 4% in actively phosphorylat-
ing mitochondria. The latter reduction is expected to translate into a 24%
higher extramitochondrial AMP concentration. Furthermore, palmitoyl-
CoA induced concentration-dependent H
2
O
2
formation, which can only
partly be explained by its effect on Dw. Although all measured fluxes and
intermediate concentrations were affected by palmitoyl-CoA, modular kin-
etic analysis revealed that this resulted mainly from inhibition of the ANT.
Through Metabolic Control Analysis, we then determined to what extent

the ANT controls the investigated mitochondrial properties. Under steady-
state conditions, the ANT moderately controlled oxygen uptake (control
coefficient C ¼ 0.13) and phosphorylation (C ¼ 0.14) flux. It controlled
intramitochondrial (C ¼ )0.70) and extramitochondrial ATP ⁄ ADP ratios
(C ¼ 0.23) more strongly, whereas the control exerted over the QH
2
⁄ Q
ratio (C ¼ )0.04) and Dw (C ¼ )0.01) was small. Quantitative assessment
of the effects of palmitoyl-CoA showed that the mitochondrial properties
that were most strongly controlled by the ANT were affected the most.
Our observations suggest that long-chain acyl-CoA esters may contribute
to cellular dysfunction in obesity and type 2 diabetes through effects on
cellular energy metabolism and production of reactive oxygen species.
Abbreviations
[AMP]
out
, concentration of extramitochondrial AMP; AMPK, AMP-activated protein kinase; ANT, adenine nucleotide translocator;
Ap5A, P
1
,P
5
-di(adenosine-5¢)-pentaphosphate; ATP
in
⁄ ADP
in
ratio, ATP to ADP ratio in the mitochondrial matrix; ATP
out
⁄ ADP
out
ratio,

extramitochondrial ATP to ADP ratio; C
X
m
i
, concentration control coefficient, quantifying control of intermediate X
m
by module i; C
J
k
i
, flux
control coefficient, quantifying control of flux J
k
by module i; Dw, membrane potential, i.e. electrical potential across the inner mitochondrial
membrane; J
1
h
, proton leak flux; J
o
, oxygen uptake flux; J
p
, phosphorylation flux; LCAC, long chain acyl-CoA ester; QH
2
⁄ Q ratio, ratio of
reduced to oxidized coenzyme Q; ROS, reactive oxygen species; S-13, 5-chloro-3-t-butyl-2¢-chloro-4¢-nitrosalicylanilide.
5288 FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS
In mammals, mitochondria produce the majority
of ATP required to drive energy-dependent cellular
processes. However, mitochondria also play more
indirect roles. Impaired mitochondrial function is

emerging as an important factor in insulin-resistant
states: less efficient mitochondrial oxidative phos-
phorylation has been demonstrated in both the
elderly and insulin-resistant offspring of patients with
type 2 diabetes compared with young, healthy con-
trols [1,2]. Although under physiological conditions
nonesterified fatty acids are an important source of
fuel for many tissues because they can yield relatively
large quantities of ATP, obesity-related persistent
oversupply of nonesterified fatty acids and accumula-
tion of triacylglycerols in nonadipose tissues is
thought to contribute to the molecular mechanisms
underlying both insulin resistance and b-cell dysfunc-
tion in type 2 diabetes [3,4]. Nonesterified and esteri-
fied fatty acids interfere with mitochondrial oxidative
phosphorylation in vitro [5,6]. Furthermore, an imbal-
ance in fatty acid metabolism resulting in activation
of nonoxidative rather than oxidative pathways and
accumulation of biologically active molecules [e.g.
long-chain acyl-CoAs (LCACs), ceramide, diacylglyc-
erol] could adversely affect cellular function by direct
effects on a variety of enzymes and induction of
apoptosis [4].
Tight regulation of intracellular concentrations of
free LCACs by acyl-CoA-binding protein can be
impaired under pathological conditions with excess
lipid supply (e.g. obesity) because of inadequate
expression of the latter [7]. LCACs modulate the
activity of the mitochondrial adenine nucleotide
translocator (ANT) from both the outer and matrix

sides of the inner mitochondrial membrane by com-
petitive displacement of the nucleotide from its bind-
ing site on the protein [8]. It has been hypothesized
that increased concentrations of free LCACs interfere
with mitochondrial function through inhibition of
the ANT, leading to lower cytosolic ATP and matrix
ADP availability, increased mitochondrial membrane
potential (Dw), and reduction level of coenzyme Q
[9]. The two latter events are expected to promote
the formation of reactive oxygen species (ROS)
[10,11], resulting in impaired cellular functions and
cell death. Moreover, increased AMP production by
adenylate kinase caused by the low cytosolic
ATP ⁄ ADP ratio and further breakdown of AMP to
adenosine is expected to result in an increase in
extracellular adenosine concentration [12]. The latter
is a potent vasodilator [13], which can promote
sodium retention in the kidney and stimulate sympa-
thetic nervous system activity [14].
Fatty acid-induced insulin resistance in liver is one
of the main causes of hyperglycemia in type 2 diabetes
[15], and the role of mitochondria in this dysfunction
is not fully elucidated. We have shown that, in isolated
rat liver mitochondria oxidizing succinate, palmitoyl-
CoA inhibited the ANT and induced working-condi-
tion-dependent changes in intramitochondrial and
extramitochondrial ATP concentrations and Dw [16].
The relative contribution of a particular enzyme to the
control of metabolic pathway flux and concentrations
of reaction intermediates determines to what extent

inhibition of that enzyme would affect pathway flux
and intermediate concentrations. The control can be
quantitatively assessed using Metabolic Control Analy-
sis [17–19]. The control of fluxes and intermediates is a
system property, i.e. it is determined by all enzymes
constituting the pathway. For this reason here we
quantitatively assessed the control of fluxes and inter-
mediates of oxidative phosphorylation not only by the
ANT but also by other components of oxidative phos-
phorylation. Furthermore, we tested parts of the above
hypothesis by determining the effects of palmitoyl-
CoA on actively phosphorylating (state 3) mitochon-
dria oxidizing a more physiological NADH-delivering
substrate, i.e. glutamate plus malate. To investigate
which mitochondrial enzymes are involved in the
multiple effects that we encountered, we implemented
modular kinetic analysis. We found that palmitoyl-
CoA acts directly on the ANT, and then indirectly
induces ROS production and a concomitant reduction
in the extramitochondrial ATP ⁄ ADP ratio. The extent
to which palmitoyl-CoA affected different mitochond-
rial properties can largely be explained by the magni-
tude of the control exerted by the ANT over these
properties.
Results
Palmitoyl-CoA effects on steady-state fluxes
and intermediate concentrations
Table 1 summarizes the effects of 5 lm palmitoyl-CoA
on the steady-state fluxes and intermediate concentra-
tions in isolated rat liver mitochondria respiring on

glutamate plus malate. Palmitoyl-CoA decreased
oxygen uptake flux (J
o
) by 56 ± 3% and phosphoryla-
tion flux (J
p
) by 58 ± 7%, and increased proton-leak
flux ( J
1
h
) by 37 ± 6%. The opposite effect was found
on the extramitochondrial and matrix ATP ⁄ ADP
ratios: the former decreased by 63 ± 4%, and the lat-
ter increased by 352 ± 34%. The reduced to oxidized
coenzyme Q ratio (QH
2
⁄ Q) increased by 42 ± 9%,
and Dw increased by 13 ± 1 mV (9%). The QH
2
⁄ Q
J. Ciapaite et al. Palmitoyl-CoA and control of mitochondrial function
FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS 5289
ratio in mitochondria respiring on succinate was
4.9 ± 0.3 and 5.4 ± 0.2 in the absence and presence
of 5 lm palmitoyl-CoA, respectively. We conclude that
palmitoyl-CoA affects virtually all steady-state proper-
ties of these mitochondria, albeit to various extents.
Palmitoyl-CoA effects on mitochondrial H
2
O

2
production
We have shown that 5 lm palmitoyl-CoA caused a
significant increase in Dw in actively phosphorylating
mitochondria (state 3) respiring on succinate [16] and
NADH-delivering substrate (Table 1). To test the
notion that the palmitoyl-CoA-induced increase in Dw
would stimulate ROS production [10], we determined
the effect of palmitoyl-CoA on H
2
O
2
production in
mitochondria respiring on succinate. Figure 1A shows
that palmitoyl-CoA induced H
2
O
2
production in
state 3 in a concentration-dependent manner. The
palmitoyl-CoA-induced H
2
O
2
production was partially
sensitive to protonophore S-13, suggesting dependence
on Dw (Fig. 1B). In line with this, inhibition of the
ANT with atractyloside or carboxyatractyloside and
ATP synthase with oligomycin also induced H
2

O
2
formation, although to a lower extent (Fig. 1B).
To test whether palmitoyl-CoA metabolism via
b-oxidation contributes to increased H
2
O
2
production,
we determined the effect of palmitoyl-l-carnitine (sub-
strate for b-oxidation) and malonyl-CoA (inhibitor
of palmitoyl-carnitine transferase 1, part of the mito-
chondrial acyl-CoA transport system). Palmitoyl-
l-carnitine (5 lm) alone and in combination with
atractyloside (to test whether the effect of palmitoyl-
CoA requires both its oxidation and its inhibition of
the ANT) stimulated H
2
O
2
production rate less than
5 lm palmitoyl-CoA (Fig. 1B), suggesting that b-oxi-
dation was not involved. Furthermore, rotenone, an
inhibitor of respiratory chain complex I, had no
significant effect on palmitoyl-CoA-induced H
2
O
2
pro-
duction. However, partial inhibition of palmitoyl-CoA-

induced H
2
O
2
production with malonyl-CoA (Fig. 1B)
suggests that palmitoyl-CoA partially exerts its effect
from the matrix side.
Table 1. Steady-state values of fluxes and intermediates, as affec-
ted by palmitoyl CoA. Values are mean ± SEM from four experi-
ments.
No
Palmitoyl-CoA
+5l
M
Palmitoyl-CoA
J
o
[nmol O
2
Æmin
)1
Æ(mg protein)
)1
] 53 ± 3 23 ± 2**
J
p
[nmol ADPÆmin
)1
Æ(mg protein)
)1

] 375 ± 26 160 ± 15**
J
1
h
(nmol O
2
Æmin
)1
Æ(mg protein)
)1
] 3.3 ± 0.3 4.5 ± 0.4**
Dw (mV) 150 ± 2 163 ± 3**
ATP
in
⁄ ADP
in
0.67 ± 0.04 3.03 ± 0.04**
ATP
out
⁄ ADP
out
0.16 ± 0.01 0.06 ± 0.01*
QH
2
⁄ Q 6.7 ± 1.0 9.4 ± 1.3*
AMP
out
(calculated) (lM) 70.02 86.68
*P<0.05 and **P<0.01 versus no palmitoyl-CoA.
A

B
Fig. 1. Effect of palmitoyl-CoA on H
2
O
2
production in isolated mitochondria respiring on succinate. (A) Dependence of H
2
O
2
production on
palmitoyl-CoA concentration. (B) Comparison of the effects of various inhibitors on H
2
O
2
production. St 3, State 3; p-CoA, palmitoyl-CoA
(5 l
M), protonophore S-13 (0.2 lM); AT, atractyloside (1.5 lM); CAT, carboxyatractyloside (0.1 lM); Oligo, oligomycin (0.5 lM); Ro, rotenone
(2 l
M); M-CoA, malonyl-CoA (0.1 mM); PC, palmitoyl-L-carnitine (5 lM). All inhibitors were added in state 3. Values are mean ± SEM from
four experiments. *P<0.001 versus state 3; #P<0.02 and $P<0.002 versus 5 l
M palmitoyl-CoA.
Palmitoyl-CoA and control of mitochondrial function J. Ciapaite et al.
5290 FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS
Palmitoyl-CoA effects on extramitochondrial AMP
concentration
We have shown that 5 lm palmitoyl-CoA caused a sig-
nificant decrease in the extramitochondrial ATP ⁄ ADP
ratio (ATP
out
⁄ ADP

out
) (Table 1). However, in this par-
ticular experiment it was not possible to determine the
effect of decreased extramitochondrial ATP availability
on extramitochondrial AMP formation experimentally,
as we used P
1
,P
5
-di(adenosine-5¢)-pentaphosphate
(Ap5A) as inhibitor of adenylate kinase to prevent
depletion of available ATP and ADP and to maintain
steady-state respiration. Instead, we did a theoretical
calculation of the extramitochondrial AMP concentra-
tion ([AMP]
out
) expected at different ATP
out
⁄ ADP
out
ratios. This calculation assumes that the adenylate kin-
ase reaction is at equilibrium, which is a safe assump-
tion because there is not much net flux expected
through this enzyme under the conditions investigated.
Figure 2A shows [AMP]
out
predicted to be present at
different steady-state ATP
out
⁄ ADP

out
ratios when the
total adenylate concentration is 0.1 mm, with the
assumption that the proportions of adenine nucleotides
are regulated by the adenylate kinase equilibrium. In
the range of relatively low ATP
out
⁄ ADP
out
ratios, a
small decrease leads to a large increase in [AMP]
out
,
whereas [AMP]
out
changes relatively little in the range
of high ATP
out
⁄ ADP
out
ratios. As indicated in
Fig. 2A, when experimentally obtained values of
ATP
out
⁄ ADP
out
ratios (Table 1 and [16]) are used in
the calculation, inhibition with palmitoyl-CoA would
cause an increase in [AMP]
out

of 17% and 24% with
succinate and glutamate plus malate as substrates,
respectively.
However, such a low ATP
out
⁄ ADP
out
ratio (< 0.2)
obtained using excess hexokinase and low concentra-
tion of total adenylates (i.e. 100 lm) is not likely to
be relevant under physiological conditions. For this
reason we performed an experiment without adenylate
kinase inhibitor and with higher and more physiologi-
cally relevant total adenylate concentration (2 mm).
We determined how palmitoyl-CoA (5 and 10 lm)
affects the ATP
total
⁄ ADP
total
ratio and [AMP]
total
in
actively phosphorylating (state 3) mitochondria respir-
ing on succinate and compared the experimental and
calculated values (Fig. 2B). Because the total adeny-
late concentration in the medium was high (2 mm)
and the contribution of the matrix adenylates was
relatively low (% 10 lm), we assumed that changes
in the ATP
total

⁄ ADP
total
ratio reflect changes in the
ATP
out
⁄ ADP
out
ratio. Palmitoyl-CoA caused a signi-
ficant concentration-dependent decrease in the
ATP
total
⁄ ADP
total
ratio and increase in [AMP]
total
,
which corresponded quite well to the correlation of
[AMP]
out
and the ATP
out
⁄ ADP
out
ratio predicted by
the calculation.
Palmitoyl-CoA specifically affects the ANT
To identify the sites of oxidative phosphorylation
directly affected by palmitoyl-CoA, we applied modu-
lar kinetic analysis in two different ways: with either
Dw or matrix ATP ⁄ ADP ratio (ATP

in
⁄ ADP
in
)asan
intermediate. Modular kinetic analysis with Dw as con-
necting intermediate revealed that palmitoyl-CoA
inhibits the phosphorylating module (Fig. 3A), as the
flux through the module ( J
p
) was significantly lower in
the presence of palmitoyl-CoA than in its absence,
when both conditions were compared for the same lev-
els of Dw. The flux through the substrate-oxidation
module was slightly, although not significantly, higher
in the presence of palmitoyl-CoA (Fig. 3B), indicating
a tendency of palmitoyl-CoA to stimulate the activity
of this module, possibly via its effect on b-oxidation.
The proton-leak module was not affected directly by
palmitoyl-CoA (Fig. 3C).
A

B

Fig. 2. Dependence of AMP concentration on the ATP ⁄ ADP ratio.
(A) Dependence of AMP concentration on the ATP ⁄ ADP ratio when
the total concentration of adenylates is 100 l
M. [AMP] was calcula-
ted as described in Experimental procedures using an equilibrium
constant for adenylate kinase equal to 0.442 [42]. The points on the
curve show [AMP] expected to be present at the experimentally

obtained mean values of ATP
out
⁄ ADP
out
for succinate [16] and
glutamate plus malate (Table 1), respectively, if adenylate kinase
was not inhibited. (B) Dependence of AMP concentration on the
ATP ⁄ ADP ratio when the total concentration of adenylates is
2m
M. The points show experimentally determined dependence of
[AMP]
total
on the ATP
total
⁄ ADP
total
ratio in actively phosphorylating
(state 3) mitochondria respiring on succinate with no adenylate kin-
ase inhibitor added. The points correspond to conditions with 0, 5
or 10 l
M palmitoyl-CoA added, and are mean ± SEM from three
independent experiments. *P<0.05 versus no palmitoyl-CoA.
Succ, Succinate; g + m, glutamate plus malate; p-CoA, palmitoyl-
CoA. Open symbols, no palmitoyl-CoA; closed symbols, + palmi-
toyl-CoA.
J. Ciapaite et al. Palmitoyl-CoA and control of mitochondrial function
FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS 5291
Further analysis with the ATP
in
⁄ ADP

in
ratio as an
intermediate showed that the ATP-consuming module
(comprising the ANT and hexokinase) was inhibited
by palmitoyl-CoA (Fig. 4A), as concluded from lower
flux through the module in the presence of palmitoyl-
CoA, while the ATP-producing module was not
affected (Fig. 4B). We have shown previously that
hexokinase is not inhibited by palmitoyl-CoA [16].
Therefore our current data indicate that, also in
mitochondria respiring on the NADH-delivering sub-
strate, ANT is the only component of oxidative phos-
phorylation affected by palmitoyl-CoA, although a
stimulatory effect on substrate oxidation cannot be
excluded. Thus the multitude of effects on steady-state
fluxes and intermediate concentrations exerted by
palmitoyl-CoA is achieved through inhibition of the
ANT.
Metabolic control of mitochondrial properties
To determine whether palmitoyl-CoA affected the
properties it would be expected to affect for its direct
action on the ANT, and to see if we could account for
the observation that some properties were affected
more than others, we used the systems biology method
of Metabolic Control Analysis. To assess the control
of fluxes and intermediates, we took a modular
approach (Fig. 5).
Metabolic control of fluxes
Control coefficients of the six modules of oxidative
phosphorylation over the oxygen uptake (J

o
) and
phosphorylation flux (J
p
) for both respiratory sub-
strates are summarized in Table 2. The distribution
pattern of the control over J
p
among the modules was
similar to that of J
o
for both substrates used except
for the negative control exerted by the proton leak
(because it dissipates Dw which is needed to drive
ADP phosphorylation and adenine nucleotide trans-
location). In all conditions, the control distribution
A B C
Fig. 3. Effect of palmitoyl-CoA on the kinetics of the oxidative phosphorylation modules around Dw. (A) Kinetics of the phosphorylation mod-
ule as determined by titration of the substrate oxidation module with 0–25 n
M myxothiazol. (B) Kinetics of the substrate oxidation module
determined by titrating the phosphorylation module with 0–0.3 l
M oligomycin. (C) Kinetics of the proton leak module as determined by titra-
tion of the substrate oxidation module with 0–55 n
M rotenone when the phosphorylation module was blocked with 0.3 lM oligomycin. J
p
was calculated as: J
p
¼ J
o
) J

h
at the same value of Dw; J
1
h
was measured as J
o
in the absence of ADP phosphorylation [46]. Values are
mean ± SEM from four experiments. Open symbols, no palmitoyl-CoA; closed symbols, +5 l
M palmitoyl-CoA.
A B
Fig. 4. Effect of palmitoyl-CoA on the kinetics of the modules of
oxidative phosphorylation around the intramitochondrial ATP ⁄ ADP
ratio. (A) Kinetics of the ATP-consuming module as determined by
titration of the ATP-producing module with 0–20 n
M myxothiazol.
(B) Kinetics of the ATP-producing module as determined by titration
of the ATP-consuming module with 0–0.75 l
M atractyloside. J
p
was calculated as: J
p
¼ J
o
) J
h
at the same value of Dw and multi-
plied by the ADP ⁄ O ratio [16] (ADP ⁄ O ¼ 2.7 ± 0.1). Values are
mean ± SEM from four experiments. Open symbols, no palmitoyl-
CoA; closed symbols, +5 l
M palmitoyl-CoA.

Palmitoyl-CoA and control of mitochondrial function J. Ciapaite et al.
5292 FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS
was as expected for state 3: the bulk of flux control
was shared between the respiratory chain and the mod-
ules involved in the production of extramitochondrial
ATP (actually glucose 6-phosphate), with hardly any
control by the proton-leak module. The contribution
of the ANT to the control of J
o
and J
p
was moderate
and similar with both respiratory substrates.
When the two substrates are compared, using glu-
tamate plus malate instead of succinate, control of the
fluxes shifts from the respiratory chain to ATP synthe-
sis. Furthermore, the distribution of the control within
the respiratory chain shifts from the part downstream
of coenzyme Q with succinate to the part upstream of
coenzyme Q with glutamate plus malate.
In agreement with the fact that the ANT is the only
target of palmitoyl-CoA in the system of oxidative
phosphorylation under these experimental conditions,
we found that, with both respiratory substrates, the
control exerted by the ANT over J
o
and J
p
signifi-
cantly increased upon inhibition with palmitoyl-CoA.

The control of J
o
increased by 67% and 55% with suc-
cinate and glutamate plus malate, respectively, whereas
the control of J
p
was affected more strongly: it
increased by 87% and 83% with succinate and glutam-
ate plus malate, respectively. Owing to the summation
property of flux control coefficients [17,18], an increase
in the control strength of one component of the system
automatically leads to a decrease in the control
strength of other component(s). In our case, an
increase in the control of fluxes by the ANT was
mainly compensated for by decreased control by the
respiratory chain modules (Table 2). Furthermore, the
control by the proton-leak module slightly but signifi-
cantly increased because palmitoyl-CoA increases Dw,
moving the system to a new steady state that is closer
to state 4, where control by proton leak is high.
Fig. 5. Division of the oxidative phosphorylation into modules. The modules: 1, Q-reducing module, comprising dicarboxylate carrier and sub-
strate dehydrogenases (malate and NADH dehydrogenases in the case of glutamate plus malate oxidation, or succinate dehydrogenase in
the case of succinate oxidation); 2, QH
2
-oxidizing module, comprising cytochrome bc
1
and cytochrome c oxidase; 3, proton leak module,
comprising passive membrane permeability to protons and cation cycling; 4, ATP synthesis, comprising ATP synthase and phosphate carrier;
5, adenine nucleotide translocator; 6, hexokinase. The intermediates: a,QH
2

⁄ Q ratio; b , membrane potential (Dw); d, matrix ATP ⁄ ADP ratio
(ATP
in
⁄ ADP
in
); c, extramitochondrial ATP ⁄ ADP ratio (ATP
out
⁄ ADP
out
). Arrows marked e, h
1
and p indicate electron flux, transmembrane pro-
ton flux, and ATP flux, respectively. The dashed arrow h
1
going from Q-reducing module to Dw is valid only when glutamate + malate is
used as a substrate.
Table 2. Metabolic control of fluxes. The control coefficients were
calculated from elasticity coefficients (Supplementary material,
Table S2) and steady-state fluxes (Table 1 and [16] for glutamate
plus malate and succinate, respectively). Values are mean ± SEM
from three (succinate) or four (glutamate plus malate) experi-
ments (indicated as subscript). Q red, Q-reducing module; QH
2
ox,
QH
2
-oxidizing module; Leak, proton-leak module; ATP synth, ATP-
synthesis module; ANT, adenine nucleotide translocator; Hk, hexo-
kinase; p-CoA, palmitoyl-CoA.
Module, i

C
J
o
i
C
J
p
i
No p-CoA + 5 lM p-CoA No p-CoA + 5 lM p-CoA
Succinate
Q red 0.13
0.02
0.09
0.02
** 0.14
0.02
0.09
0.02
**
QH
2
ox 0.45
0.01
0.31
0.04
* 0.46
0.01
0.30
0.03
*

Leak 0.02
0.00
0.06
0.01
** ) 0.03
0.01
) 0.04
0.01
ATP synth 0.06
0.00
0.12
0.04
* 0.06
0.01
0.15
0.05
*
ANT 0.12
0.03
0.20
0.02
* 0.13
0.03
0.24
0.02
**
Hk 0.22
0.01
0.21
0.01

0.24
0.01
0.26
0.02
Glutamate plus malate
Q red 0.26
0.04
0.14
0.02
* 0.27
0.04
0.16
0.02
*
QH
2
ox 0.14
0.02
0.14
0.01
0.15
0.02
0.16
0.02
Leak 0.04
0.00
0.14
0.01
** ) 0.03
0.00

) 0.06
0.01
**
ATP synth 0.28
0.04
0.22
0.02
0.31
0.04
0.28
0.03
ANT 0.13
0.01
0.20
0.01
* 0.14
0.01
0.26
0.01
**
Hk 0.15
0.03
0.15
0.01
0.16
0.04
0.20
0.01
*P<0.05 and **P<0.01 versus no palmitoyl-CoA.
J. Ciapaite et al. Palmitoyl-CoA and control of mitochondrial function

FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS 5293
Control of the QH
2
/Q ratio and Dw
Coenzyme Q reduction level and Dw are among the
factors that determine ROS production by the mitoch-
ondrial respiratory chain [11,12]. The control of these
intermediates by the six modules of oxidative phos-
phorylation is summarized in Table 3. The sum of all
concentration (also Dw) control coefficients in a path-
way is zero, by definition [17,18]. Accordingly, the val-
ues of the coefficients can be positive or negative
depending on whether an enzyme is involved in the
production or the consumption of an intermediate,
respectively. For both substrates used, the QH
2
⁄ Q
ratio was almost solely controlled by the respiratory
chain enzymes, with the coenzyme Q reducing module
exerting a positive control and the coenzyme QH
2
oxidizing module exerting a negative control, while the
control of Dw was shared equally between the Dw-gen-
erating (positive control) and Dw-consuming or Dw-
consumption-stimulating processes (negative control)
(Table 3). In the case of succinate oxidation, most of
the control of Dw within the respiratory chain resided
in the part downstream of coenzyme Q (cytochrome
bc
1

complex and cytochrome c oxidase), whereas, in
the case of glutamate plus malate, the part upstream
of coenzyme Q (dicarboxylate carrier and substrate
dehydrogenases) had slightly more control of Dw, poss-
ibly because NADH dehydrogenase, a proton-pumping
enzyme, becomes active. With both respiratory
substrates, the ANT exerted negative control over the
QH
2
⁄ Q ratio and Dw (Table 3). This is because activa-
tion of the ANT stimulates the phosphorylation
branch of oxidative phosphorylation, which consumes
Dw. The negative control over the QH
2
⁄ Q ratio is
explained similarly.
Palmitoyl-CoA had hardly any effect on the control
of the QH
2
⁄ Q ratio when glutamate plus malate was
used as a substrate. With succinate, palmitoyl-CoA
mainly affected the control of the QH
2
⁄ Q ratio by
respiratory-chain modules: control by both coen-
zyme Q-reducing and coenzyme QH
2
-oxidizing mod-
ules has decreased. Furthermore, palmitoyl-CoA had
little effect on the control of Dw except that the con-

trol by the coenzyme Q-reducing and ATP-synthesis
module significantly decreased with glutamate plus
malate as substrate, and for both substrates the negat-
ive control exerted by the proton leak slightly increased
because of the effect of palmitoyl-CoA on Dw.
Control of matrix and extramitochondrial ATP/ADP
ratios
The control of the ATP
in
⁄ ADP
in
ratio and ATP
out
⁄
ADP
out
ratio is summarized in Table 3. For both sub-
strates used, control of the ATP
in
⁄ ADP
in
ratio was
shared among all modules of oxidative phosphoryla-
tion, with a slight negative control exerted by the
proton leak. The ANT exerted a large negative control
Table 3. Metabolic control of intermediates. The control coefficients were calculated from elasticity coefficients (Supplementary material,
Table S2) and steady-state fluxes (Table 1 and [16] for glutamate plus malate and succinate, respectively). Values are mean ± SEM from
three (succinate) or four (glutamate plus malate) experiments (indicated as subscript). Q red, Q-reducing module; QH
2
ox, QH

2
-oxidizing
module; Leak, proton-leak module; ATP synth, ATP-synthesis module; ANT, adenine nucleotide translocator; Hk, hexokinase; p-CoA, palmi-
toyl-CoA.
Module, i
C
QH
2
=Q
i
C
Dw
i
C
ATP
in
=ADP
in
i
C
ATP
out
=ADP
out
i
No p-CoA + 5 lM p-CoA No p-CoA + 5 lM p-CoA No p-CoA + 5 lM p-CoA No p-CoA + 5 lM p-CoA
Succinate
Q red 0.57
0.11
0.38

0.06
0.02
0.00
0.01
0.00
0.20
0.05
0.18
0.10
0.25
0.05
0.14
0.04
*
QH
2
ox ) 0.30
0.06
) 0.14
0.04
0.06
0.00
0.06
0.01
0.78
0.12
0.79
0.31
0.85
0.03

0.46
0.06
*
Leak ) 0.01
0.00
) 0.02
0.00
0.00
0.00
) 0.01
0.00
) 0.04
0.01
) 0.11
0.05
) 0.05
0.01
) 0.06
0.01
ATP synth ) 0.04
0.01
) 0.05
0.01
) 0.01
0.00
) 0.01
0.00
0.26
0.02
0.79

0.13
* 0.11
0.01
0.23
0.07
*
ANT ) 0.07
0.01
) 0.08
0.01
) 0.02
0.00
) 0.02
0.00
) 0.39
0.02
) 0.78
0.11
* 0.24
0.05
0.37
0.03
*
Hk ) 0.15
0.03
) 0.09
0.02
) 0.04
0.01
) 0.03

0.01
) 0.81
0.19
) 0.88
0.20
) 1.41
0.04
) 1.13
0.01
*
Glutamate plus malate
Q red 0.56
0.14
0.60
0.11
0.03
0.00
0.02
0.00
* 0.55
0.14
0.46
0.18
0.41
0.06
0.28
0.03
*
QH
2

ox ) 0.40
0.13
) 0.42
0.08
0.02
0.00
0.02
0.00
0.23
0.08
0.59
0.21
0.23
0.04
0.30
0.04
Leak ) 0.01
0.00
) 0.03
0.00
* 0.00
0.00
) 0.01
0.00
* ) 0.05
0.01
) 0.20
0.08
) 0.04
0.01

) 0.11
0.02
**
ATP synth ) 0.08
0.02
) 0.05
0.01
) 0.02
0.00
) 0.01
0.00
* 0.70
0.11
1.30
0.40
0.48
0.06
0.53
0.07
ANT ) 0.04
0.00
) 0.06
0.02
) 0.01
0.00
) 0.01
0.00
) 0.70
0.14
) 1.28

0.36
* 0.23
0.02
0.48
0.03
**
Hk ) 0.04
0.01
) 0.04
0.01
) 0.01
0.00
) 0.01
0.00
) 0.73
0.11
) 0.88
0.25
) 1.31
0.03
) 1.47
0.09
*P<0.05 and **P<0.01 versus no palmitoyl-CoA.
Palmitoyl-CoA and control of mitochondrial function J. Ciapaite et al.
5294 FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS
over the ATP
in
⁄ ADP
in
ratio, because it functions as a

‘consumer’ of matrix ATP by transporting it from
mitochondria to the intermembrane space. The
distribution of control within the respiratory chain
depended on the substrate used: for succinate, the
QH
2
-oxidizing module exerted more control than
Q-reducing module, whereas, with glutamate plus ma-
late as substrate, it was the opposite. Palmitoyl-CoA
tended to increase the positive control of the ATP
in
⁄
ADP
in
ratio by ATP synthesis and the negative control
by the proton leak. The negative control of the ATP
in
⁄
ADP
in
ratio by the ANT increased by 100% and 82%
with succinate and glutamate plus malate as substrate,
respectively.
For both substrates, hexokinase exerted the highest
negative control on the ATP
out
⁄ ADP
out
ratio
(Table 3). The remainder of the control was distributed

among the respiratory-chain modules, ATP synthesis,
and the ANT, with negligible negative control exerted
by the proton leak. Similarly to the control of the
ATP
in
⁄ ADP
in
ratio, the distribution of the control of
the ATP
out
⁄ ADP
out
ratio within the respiratory chain
depended on the substrate used. Comparing the two
substrates, ATP synthesis exerted less control over the
ATP
out
⁄ ADP
out
ratio in the case of succinate oxida-
tion. Palmitoyl-CoA increased the control of the
ATP
out
⁄ ADP
out
ratio by ATP synthesis and proton
leak, and decreased the control by the Q-reducing
module with both respiratory substrates, and the con-
trol by the QH
2

-oxidizing module and hexokinase with
succinate. The control of the ATP
out
⁄ ADP
out
ratio by
the ANT increased by 56% and 113% with succinate
and glutamate plus malate as substrate, respectively.
Partial integrated responses to palmitoyl-CoA
Table 4 summarizes integrated elasticities to palmitoyl-
CoA and partial integrated responses of system fluxes
and intermediates to palmitoyl-CoA mediated through
each module of oxidative phosphorylation. With both
respiratory substrates, the ANT had the largest elasti-
city to palmitoyl-CoA, in agreement with the finding
that, under our experimental conditions, the ANT is
the main target of palmitoyl-CoA in oxidative phos-
phorylation. As a consequence, the response mediated
through the ANT contributed most to the overall
response of the system fluxes and intermediates to
palmitoyl-CoA, i.e. the response through the ANT was
responsible for 68% of the decrease in J
o
, 68% of the
decrease in J
p
, 56% of the increase in the QH
2
⁄ Q
ratio, 70% of the increase in Dw, 72% of the increase

in the ATP
in
⁄ ADP
in
ratio, and 59% of the decrease in
the ATP
out
⁄ ADP
out
ratio with succinate as substrate.
Similar results were obtained when glutamate plus
malate was used as substrate: the response through the
ANT contributed 75% of the reduction in J
o
, 76% of
the reduction in J
p
, 68% of the increase in Dw, 88% of
the increase in the ATP
in
⁄ ADP
in
ratio, and 69% of the
reduction in the ATP
out
⁄ ADP
out
ratio. The exception
was the QH
2

⁄ Q ratio, where the response through the
Table 4. Contribution of individual modules of oxidative phosphorylation to the overall response of system variables to palmitoyl-CoA. The
partial integrated responses (IR) of each module to 5 l
M palmitoyl-CoA were calculated using control coefficients (Tables 2 and 3) and integ-
rated elasticity coefficients (Ie) of modules to palmitoyl-CoA as described in [21]. Values are mean ± SEM from three (succinate) or four (glu-
tamate plus malate) experiments (indicated as subscript). Modules: 1, Q reducing; 2, QH
2
oxidizing; 3, proton leak; 4, ATP synthesis;
5, ANT; 6, hexokinase. p-CoA, Palmitoyl-CoA; OR, overall response.
i
i
IR
J
o
pÀCoA
i
IR
J
p
pÀCoA
i
IR
QH
2
=Q
pÀCoA
i
IR
Dw
pÀCoA

i
IR
ATP
in
=ADP
in
pÀCoA
i
IR
ATP
out
=ADP
out
pÀCoA
Ie
i
pÀCoA
Succinate
1 ) 0.02
0.00
) 0.02
0.00
) 0.08
0.03
0.00
0.00
) 0.02
0.00
) 0.03
0.01

) 0.14
0.04
2 0.07
0.01
0.07
0.01
) 0.05
0.02
0.01
0.00
0.11
0.01
0.13
0.03
0.15
0.03
3 0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.01
0.01
0.01
) 0.07
0.10

4 ) 0.05
0.02
) 0.05
0.02
0.04
0.02
0.01
0.00
) 0.22
0.08
) 0.10
0.04
) 0.83
0.29
5 ) 0.37
0.04
) 0.40
0.04
0.24
0.04
0.07
0.00
1.27
0.10
) 0.75
0.08
) 3.32
0.43
6 ) 0.04
0.00

) 0.04
0.00
0.03
0.00
0.01
0.00
0.14
0.01
0.26
0.04
) 0.18
0.03
OR ) 0.41
0.02
) 0.44
0.01
0.18
0.04
0.09
0.00
1.28
0.03
) 0.49
0.04
Glutamate plus malate
1 0.16
0.04
0.16
0.05
0.33

0.08
0.02
0.01
0.33
0.11
0.25
0.07
0.62
0.17
2 0.01
0.05
0.01
0.05
) 0.10
0.13
0.00
0.00
) 0.01
0.03
0.02
0.08
0.25
0.44
3 0.01
0.00
0.00
0.00
0.00
0.00
0.00

0.00
) 0.01
0.00
) 0.01
0.00
0.17
0.08
4 0.03
0.10
0.03
0.11
) 0.01
0.03
0.00
0.01
0.09
0.27
0.05
0.18
0.02
0.38
5 ) 0.66
0.12
) 0.71
0.13
0.18
0.06
0.06
0.01
3.79

1.44
) 1.10
0.18
) 5.00
0.89
6 ) 0.02
0.00
) 0.02
0.00
0.00
0.00
0.00
0.00
0.09
0.01
0.16
0.01
) 0.12
0.01
OR ) 0.47
0.08
) 0.53
0.08
0.40
0.10
0.08
0.01
4.28
1.68
) 0.62

0.11
J. Ciapaite et al. Palmitoyl-CoA and control of mitochondrial function
FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS 5295
ANT contributed only 29% of the overall increase in
the QH
2
⁄ Q ratio, most of the rest of the increase stem-
ming from stimulation of the Q-reducing module
(52%).
The response of a system variable to an external
effector mediated through a specific module is deter-
mined by the control exerted by that module over a
system variable and the elasticity of that module to the
effector [20,21]. Table 4 shows that the overall effects
of palmitoyl-CoA on system fluxes and intermediates
were mainly mediated through the ANT and that the
properties that were controlled most strongly by the
ANT were affected the most.
Discussion
We have shown that palmitoyl-CoA induces ROS pro-
duction in actively phosphorylating isolated rat liver
mitochondria. Furthermore, it influences the ATP
out
⁄
ADP
out
ratio in such a way that these changes result
in increased [AMP]
out
. This is in line with a mechanism

we proposed to underlie the association between obes-
ity and type 2 diabetes [9,12]. However, owing to mul-
tiple interactions in living systems, it can be difficult to
differentiate whether all the effects relate to one or
many primary effects. To acknowledge this, using
modular kinetic analysis, we established that the pri-
mary cause of the effects of palmitoyl-CoA was inhibi-
tion of the ANT. Assessment of the metabolic control
of fluxes and intermediate concentrations by the ANT
then revealed that this enzyme partially controls many
fluxes, concentrations and potentials. This then con-
firmed that an increase in AMP concentration and, at
least partly, stimulation of ROS production are effects
of ANT inhibition. This study thereby shows how sys-
tems biology methodologies might help in dissecting
the convoluted cause–effect chains in multifactorial
diseases such as obesity and type 2 diabetes.
Both starvation and incubation with fatty acids have
been shown to cause a concomitant decrease in cyto-
solic ATP ⁄ ADP ratios and an increase in total LCAC
concentrations in perfused rat liver and isolated
hepatocytes, indicating that inhibition of the ANT by
LCACs may be relevant in vivo [22–24]. It has been
suggested that modulation of ANT activity by LCACs
might be physiologically significant in the regulation of
gluconeogenesis by fatty acids through effects on the
intramitochondrial ATP ⁄ ADP ratio [23].
A decrease in the extramitochondrial concentration
of ATP may result in increased formation of AMP by
adenylate kinase. This may subsequently stimulate a

cellular response to stress through activation of AMP-
dependent processes [25] or lead to the breakdown of
AMP to adenosine and extracellular release of the lat-
ter [12]. The primary mechanism of intracellular
adenosine production is hydrolysis of AMP by a cyto-
solic 5¢-nucleotidase [26]. Increased concentrations of
free ADP and AMP in the cytosol are major determi-
nants of adenosine production, with extracellular
adenosine release correlating linearly with free cyto-
solic AMP concentration [27]. Exogenous adenosine is
a potent vasodilator (EC
50
@ 0.1 lm), and, under phy-
siological conditions, it facilitates tissue recovery after
intensive workload by increasing blood flow and sup-
ply of oxygen and metabolic substrates. Under patho-
logical conditions characterized by inappropriate
intracellular triacylglycerol accumulation, a low cyto-
solic ATP ⁄ ADP ratio may persist because of constant
inhibition of the ANT leading to a sustained increase
in extracellular adenosine concentrations, resulting in
hyperperfusion, hypertension, increased urate produc-
tion, and other abnormalities common to insulin-resist-
ant states [12].
We have shown that inhibition of the ANT with
palmitoyl-CoA results in a significantly lower ATP
out
⁄
ADP
out

ratio. With respect to the interrelation between
ATP
out
⁄ ADP
out
ratios and [AMP]
out
, we have shown
here how [AMP]
out
increases with decreasing ATP
out
⁄
ADP
out
ratio, with larger increases observed at low
ratios and smaller changes at high ratios. This indi-
cates that the effect of LCACs on AMP production
will vary depending on the energy state of the cell. The
theoretical assessment of the correlation was supported
by experimental findings showing that inhibition of the
ANT with palmitoyl-CoA leads to a significant palmi-
toyl-CoA concentration-dependent decrease in the
ATP
total
⁄ ADP
total
ratio and a concomitant increase in
[AMP]
total

. On the basis of our findings, we expect
that, in intact cells, the absolute cytosolic AMP con-
centration will increase moderately in response to a
decrease in the cytosolic ATP ⁄ ADP ratio in the phy-
siologically relevant range. However, even at low
concentrations of AMP, the relative increase in con-
centration would still be substantial and so would the
relative effect on the rate of production of adenosine;
5¢-nucleotidase operates in vivo at substrate concentra-
tions three orders of magnitude below its K
m
of
1.2 mm [28].
Inhibition ⁄ deinhibition of the ANT depending on
LCAC concentration may be relevant in the regulation
of cellular metabolism in vivo via effects on AMP-acti-
vated protein kinase (AMPK). Activation of AMPK
acts as a switch from anabolic to catabolic metabolism
which generates ATP (e.g. stimulation of b-oxidation)
[25]. Thus activation of AMPK would seem to be a
desirable effect in obesity, as it would promote the
Palmitoyl-CoA and control of mitochondrial function J. Ciapaite et al.
5296 FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS
consumption of excess fat. However, the combination
of persistent ANT inhibition by LCACs with constant
activation of AMPK may have some adverse effects,
because stimulation of b-oxidation in response to acti-
vation of AMPK cannot lead to production of ATP
because of lack of mitochondrial ADP. As AMPK sti-
mulates cellular fatty acid uptake [29] and the availab-

ility of circulating fatty acids is increased in obesity,
this may lead to accumulation of intracellular triacyl-
glycerols. Furthermore, owing to the decreased flux
through the tricarboxylic acid cycle in the case of
ANT inhibition, b-oxidation-derived acetyl-CoA may
stimulate pyruvate carboxylase, contributing to
increased rates of gluconeogenesis, or may be used for
ketone body synthesis in the liver. Indeed, short-term
overexpression of AMPK in mouse liver has been
shown to induce fatty liver and increase ketogenesis
[30].
Stimulation of ROS production is thought to con-
tribute to dysfunction of many different cell types,
but, in particular, to b-cell dysfunction in insulin-
resistant states through low expression of antioxidant
enzymes in these cells [31]. Mitochondrial Dw and the
redox state of coenzyme Q are known to affect ROS
formation [10,11]. We have shown that 5 lm palmi-
toyl-CoA caused a substantial increase in Dw (13 mV
with glutamate plus malate and 15 mV with succinate
[16] as substrate, compared with a total state 3–state 4
difference of % 25 mV) and induced H
2
O
2
production
in mitochondria respiring on succinate. The sensitivity
of the palmitoyl-CoA-induced H
2
O

2
production to
protonophore shows that the process is partly
Dw-dependent. This substantiates the part of our
hypothesis suggesting that LCACs bring about ROS
production through an increase in Dw [9]. The effect
of palmitoyl-CoA on the QH
2
⁄ Q ratio with both res-
piratory substrates was less pronounced, casting doubt
on the alternative route by which palmitoyl-CoA may
affect ROS production by the respiratory chain.
Effects through the more elusive local ubiquinone rad-
ical remain an option. Our results indicate that the
palmitoyl-CoA effect on H
2
O
2
production might be
partly exerted from the matrix side, but the effect is
b-oxidation-independent as the substrate of b-oxida-
tion, palmitoyl-l-carnitine, stimulated H
2
O
2
produc-
tion less than did equal amounts of palmitoyl-CoA.
Moreover, palmitoyl-CoA did not enhance respiration
directly, as measured by modular kinetic analysis. A
possibility remains that palmitoyl-CoA increased the

redox level of intramitochondrial NADH and flavo-
proteins, but it was not able to further stimulate res-
piration because it was already operating at V
max
.In
such a case, the most reduced redox potential at the
top of the electron-transfer chain might cause extra
ROS production. Our observation that atractyloside,
a direct inhibitor of the ANT, caused ROS production
that could only partly account for palmitoyl-CoA-
induced ROS production indicates that the ANT is
only partly involved in this process. It is possible that
palmitoyl-CoA decreased mitochondrial antioxidant
capacity by inhibiting nicotinamide nucleotide trans-
hydrogenase [32], an enzyme that provides NADPH
for regeneration of two important antioxidant com-
pounds, glutathione and thioredoxin, in the mitochon-
dria, and in this way contributed to increased ROS
production.
Our data show that the ANT controls many steady-
state concentrations, potentials and fluxes. In agree-
ment with this, the specific effect of palmitoyl-CoA on
the ANT appears to be consistent with its ability to
affect many fluxes, concentrations and potentials.
Table 1 shows that palmitoyl-CoA affects different
mitochondrial properties to different extents. In the
light of these observations, we asked whether Meta-
bolic Control Analysis could have served to predict the
palmitoyl-CoA effects. We showed that the ANT con-
trolled Dw and the QH

2
⁄ Q ratio to the least extent,
and indeed it was the least affected by palmitoyl-CoA.
J
o
, J
p
, ATP
in
⁄ ADP
in
and ATP
out
⁄ ADP
out
ratios were
more strongly controlled by the ANT, and again this
corresponded to a stronger effect of palmitoyl-CoA.
We conclude that the specific effect of palmitoyl-CoA
on the ANT and the varying extent to which the ANT
controls various mitochondrial properties at steady-
state can largely explain the observed palmitoyl-CoA
effects.
The relatively weak control of the QH
2
⁄ Q ratio and
Dw by the ANT is in agreement with the finding that
ANT inhibition by palmitoyl-CoA and the resulting
increase in Dw can only partly account for the
observed increase in ROS production. We found that,

for Dw and the QH
2
⁄ Q ratio, their immediate produc-
ers and consumers, i.e. the respiratory-chain compo-
nents, exerted the strongest control. This indicates
that, if an increase in ROS production is brought
about by alterations in Dw and the QH
2
⁄ Q ratio, inter-
ference with respiratory-chain function will contribute
more than interference with any other component of
oxidative phosphorylation.
It has been shown that the control of J
o
by the
ANT is comparable in isolated liver mitochondria [33]
and isolated hepatocytes [34], indicating that, at least
to a certain extent, results obtained in isolated mito-
chondria can be extrapolated to the intact cell. Our
results reconfirmed the observation that, in isolated rat
liver mitochondria, the ANT has limited control over
J. Ciapaite et al. Palmitoyl-CoA and control of mitochondrial function
FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS 5297
J
o
[19,33,35,36] and that it controls J
p
more strongly
than J
o

. However, it has been demonstrated that ANT
control of J
o
changes depending on intramitochond-
rial and extramitochondrial ATP utilization [37,38].
Accordingly, the effect of LCACs on ANT control of
J
o
must depend on the ATP elasticity of the ATP-util-
izing processes active at the moment of inhibition, e.g.
inhibition of the ANT in rat liver cells with the specific
inhibitor, atractyloside, decreased glucose synthesis to
a greater extent than urea synthesis, even though both
processes require ATP [24].
In conclusion, we have shown that the ANT con-
trolled all investigated properties of the mitochondrial
oxidative phosphorylation to different extents, with the
largest control exerted over the ATP
in
⁄ ADP
in
and
ATP
out
⁄ ADP
out
ratios. The effects of palmitoyl-CoA
largely corresponded to this. Our results suggest that
inhibition of the ANT by LCACs may be important in
the control of cellular energy metabolism, but it

accounts only partly for stimulation of ROS produc-
tion.
Experimental procedures
Materials
Yeast hexokinase was from Roche (Mannheim, Germany).
Horseradish peroxidase, superoxide dismutase, oligomycin,
myxothiazol, atractyloside, carboxyatractyloside, rotenone,
palmitoyl-CoA, malonyl-CoA, Ap5A, p-hydroxyphenyl-
acetic acid and coenzyme Q
1
were from Sigma-Aldrich
(Zwijndrecht, the Netherlands).
Isolation of mitochondria
Liver mitochondria were isolated from male Wistar rats
(250–300 g) as in [39]. Protein content was determined by
the method of Bradford [40], with BSA as a standard.
Measurement of oxygen uptake and Dw
Mitochondria were incubated at 25 °C in a closed, stirred
and thermostatically controlled glass vessel equipped with
Clark-type oxygen electrode and tetraphenylphosphonium
ion (TPP
+
)-sensitive electrode as described [16]. The assay
medium contained 25 mm creatine, 25 mm creatine phos-
phate, 75 mm KCl, 20 mm Tris, 2.3 mm MgCl
2
,5mm
glutamate plus 5 mm malate, 50 lm Ap5A, pH 7.3. An
ADP-regenerating system consisting of excess hexokinase
(5.78 UÆmL

)1
), glucose (12.5 mm) and KH
2
PO
4
(5 mm)
was used to maintain steady-state respiration rates. ATP
at a concentration of 100 lm was added to initiate state 3
respiration.
Determination of adenine nucleotide
concentrations
Adenine nucleotides were extracted with phenol as des-
cribed [41]. Concentrations were measured using a lucifer-
in–luciferase ATP-monitoring kit (BioOrbit, Turku,
Finland). ATP concentrations in the medium and the
mitochondrial matrix were determined from yeast hexo-
kinase kinetics as described [16]. As hexokinase kinetics
were determined in medium containing creatine and creat-
ine phosphate, this medium was used in all experiments
with mitochondria. AMP concentration was determined
spectrophotometrically using a standard enzymatic assay
[42].
Measurement of coenzyme Q reduction
Coenzyme Q reduction levels were determined in a thermo-
statically controlled (25 °C) vessel equipped with platinum
and oxygen electrodes, by polarographically measuring the
redox state of exogenous coenzyme Q
1
(2 lm) [43]. To cal-
ibrate the platinum electrode traces, samples were taken

from incubations of mitochondria in standard assay med-
ium without further additions (state 1) and mitochondria
incubated with substrate (10 mm succinate plus 2 lm rote-
none, or 5 mm glutamate plus 5 mm malate, state 2). Then
1 mL of sample was quenched with 3 mL 0.2 m HClO
4
in
methanol (0 °C), coenzyme Q was extracted with 3 mL pet-
roleum ether (40–60 °C), and reduced and oxidized coen-
zyme Q in the samples was determined by HPLC as
described [44].
Measurement of H
2
O
2
production
The rate of H
2
O
2
production was estimated from the rate
of p-hydroxyphenylacetic acid oxidation (excitation and
emission wavelengths 317 nm and 414 nm, respectively) as
described [45]. Briefly, mitochondria were incubated at
25 °C in 2 mL standard assay medium containing 1 mm
diethylenetriaminepenta-acetic acid, 0.2 mm p-hydroxyphe-
nylacetic acid, 10 UÆmL
)1
horseradish peroxidase and
30 UÆmL

)1
superoxide dismutase under the following condi-
tions: state 3 and state 3 plus inhibitors [palmitoyl-CoA (1,
2.5 and 5 lm), 5-chloro-3-t-butyl-2¢-chloro-4¢-nitrosalicylan-
ilide (S-13, 0.2 lm), atractyloside (1.5 lm), carboxyatrac-
tyloside (0.1 lm), oligomycin (0.5 lm), rotenone (2 lm),
malonyl-CoA (0.1 mm)] or palmitoyl-l-carnitine (5 lm).
Fluorescence signal was quantified using H
2
O
2
as standard.
Calculation of extramitochondrial AMP
concentrations
AMP concentration was calculated as (for derivation see
supplementary data, Appendix S1):
Palmitoyl-CoA and control of mitochondrial function J. Ciapaite et al.
5298 FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS
½AMP¼
aK
eq
r
2
þ r þ K
eq
ð1Þ
where r is the ATP ⁄ ADP ratio (equal to our experimental
extramitochondrial ATP ⁄ ADP ratio only when formation
of AMP is blocked by Ap5A), a is the total amount of
adenylate (100 lm), and K

eq
is the equilibrium constant of
adenylate kinase (K
eq
¼ 0.442 [42]).
Modular kinetic analysis
To localize the sites of action of palmitoyl-CoA, the system
of oxidative phosphorylation was conceptually subdivided
into a small number of functional modules interacting via a
limited number of intermediates. The kinetic response of
flux through the modules to changes in the concentration
of the connecting intermediate was determined in the pres-
ence and absence of palmitoyl-CoA by titrating with speci-
fic inhibitors as described [16,46]. In the first application,
the system was divided into a substrate-oxidation module, a
phosphorylation module, and a proton-leak module, with
Dw as the connecting intermediate [46]. In the second appli-
cation, it was divided into an ATP-producing module and
an ATP-consuming module, with the matrix ATP ⁄ ADP
ratio as the connecting intermediate [16].
Metabolic Control Analysis
Definitions
The flux control coefficient is defined as the fractional
change in the system flux (J
k
) at steady-state in response to
an infinitesimal change in the rate of an enzyme (module)
i (v
i
) [18]:

C
J
i
k ¼
@J
k
J
k
.
@v
i
v
i

ss
¼
@lnJ
k
@lnv
i

ss
ð2Þ
The subscript ss refers to the steady-state condition and is
hereafter omitted, as are the parentheses.
The concentration control coefficient is defined as the
fractional change in the steady-state concentration of inter-
mediate (or ratio of concentrations, Dw)(X
m
) in response

to an infinitesimal direct perturbation of the enzyme (mod-
ule) i rate (v
i
) [17,18]:
C
X
m
i
¼
@X
m
X
m
.
@v
i
v
i
¼
@lnX
m
@lnv
i
ð3Þ
Effectively, the value of the control coefficient of an enzyme
indicates the percentage reduction in a system flux (for flux
control coefficients) or in an intermediate concentration
(for concentration control coefficients) in response to 1%
inhibition of the reaction rate of that enzyme.
The elasticity coefficient is defined as the fractional

change in rate v through enzyme (module) i, caused by the
fractional change in the concentration of intermediate X
m
,
when concentrations of other intermediates are held con-
stant [17,18]:
e
i
X
m
¼
@v
i
v
i
.
@X
m
X
m

intermediates constant
¼
@lnv
i
@lnX
m

intermediates constant
ð4Þ

Calculation of control coefficients
For analysis of metabolic control, we conceptually subdivi-
ded the system of oxidative phosphorylation into six
modules (coenzyme Q-reducing module, coenzyme QH
2
-
oxidizing module, proton-leak module, ATP-synthesis
module, ANT, and hexokinase) connected by four interme-
diates: QH
2
⁄ Q ratio; Dw; ATP
in
⁄ ADP
in
; ATP
out
⁄ ADP
out
(Fig. 5). The control coefficients of the modules for the
oxygen-uptake and phosphorylation fluxes and concentra-
tions of four intermediates were calculated from the system
fluxes and elasticity coefficients (i.e. coefficients quantifying
sensitivity of flux through the module to changes in concen-
tration of an intermediate [17]) using the matrix method
[47]. In the calculation, we assumed that the coen-
zyme Q-reducing and coenzyme QH
2
-oxidizing modules are
insensitive to changes in the ATP
in

⁄ ADP
in
and ATP
out
⁄
ADP
out
ratios (i.e. elasticity coefficients are zero) [46]; in
the case of succinate oxidation, the coenzyme Q-reducing
module is insensitive to Dw, ATP synthesis is sensitive only
to Dw and the ATP
in
⁄ ADP
in
ratio, the hexokinase rate is
sensitive only to the ATP
out
⁄ ADP
out
ratio, whereas proton
leak is sensitive only to Dw [46]; ANT is sensitive to all four
intermediates including the QH
2
⁄ Q ratio [48].
To obtain the elasticity coefficients that were assumed
to have a nonzero value, we used a multiple modulation
method [49], i.e. each module was titrated with a specific
inhibitor (Table 5) and the co-response of the flux and
intermediate concentration was measured. The co-response
coefficients quantifying the ratio of responses of intermedi-

ate X
m
and flux J
k
after perturbation of module i [50] were
determined from the slopes of inhibitor titration curves at
steady-state as:
i
O
X
m
J
k
C
X
m
i
C
J
k
i
¼
@lnX
m
@lnv
i
.
@lnJ
k
@lnv

i
¼
@lnX
m
@lnJ
k
ð5Þ
Table 5. Modulations used to determine the co-response coeffi-
cients. Mal, Malonate (0–0.625 m
M); Oligo, oligomycin (0–0.3 lM);
Atr, atractyloside (0–1.5 l
M); Rot, rotenone (0–30 nM); Myx, myxo-
thiazol (0–25 n
M); Hk, hexokinase (0–5.78 UÆmL
)1
).
Module Succinate Glutamate + malate
Q reducing Myx, Oligo, Atr Myx, Oligo, Atr
QH
2
oxidizing Mal, Oligo, Atr Rot, Oligo, Atr
Proton leak Mal, Myx Rot, Myx
ATP synthesis Mal, Myx, Atr Rot, Myx, Atr
ANT Mal, Myx, Oligo, Hk Rot, Myx, Oligo, Hk
Hk Mal, Myx, Oligo, Atr Rot, Myx, Oligo, Atr
J. Ciapaite et al. Palmitoyl-CoA and control of mitochondrial function
FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS 5299
The response of the flux J
k
to the change in enzyme

(module) i (i „ k) is transmitted via changes in the inter-
mediates X
m
. The response can be approximated by the
expression [18,51]:
C
J
k
i
¼
X
intermediates
C
X
m
i
Á e
k
X
m
ð6Þ
Dividing by the flux control coefficient yields:
1 ¼
X
intermediates
i
O
X
m
J

k
Á e
k
X
m
ð7Þ
Thus when the co-response coefficients are known, the elas-
ticity coefficients of each module to each intermediate can
be calculated from sets of equations such as Eqn 7. For
example, the elasticity coefficients of the coenzyme Q-redu-
cing module for the QH
2
⁄ Q ratio and Dw in the case of
glutamate plus malate oxidation was calculated from the
following set:
1 ¼ e
Qred
QH
2
=Q
Á
Myx
O
QH
2
Q
J
o
þ e
Qred

Dw
Á
Myx
O
Dw
J
o
ð8Þ
1 ¼ e
Qred
QH
2
=Q
Á
Oligo
O
QH
2
Q
J
o
þ e
Qred
Dw
Á
Oligo
O
Dw
J
o

ð9Þ
1 ¼ e
Qred
QH
2
=Q
Á
Atr
O
QH
2
Q
J
o
þ e
Qred
Dw
Á
Atr
O
Dw
J
o
ð10Þ
The calculations for succinate as a respiratory substrate
were performed using data obtained previously [16]. The
inhibitor titration curves (Figs S1 and S2), the co-response
(Table S1) and elasticity coefficients (Table S2), and
detailed calculation of control coefficients (Eqn S6) are
given in Supplementary material, Appendix S2.

Calculation of partial integrated response
Partial integrated responses quantify the response of a sys-
tem variable a (flux J or intermediate X), mediated through
a pathway enzyme (module) i, to a singe-step change in
concentration of external effector q, and were calculated
using control coefficients and integrated elasticity coeffi-
cients as described [21]. The overall response of a system
variable is the sum of all partial responses, i.e. the partial
response indicates how much of the effector-induced change
in a system variable is caused by changes in activity of each
pathway enzyme (module).
Data presentation
Data are expressed as mean ± SEM from n independent
mitochondrial preparations. The statistical significance of
palmitoyl-CoA effects was determined using paired Stu-
dent’s t test. A P < 0.05 that the difference arose by
chance was considered to make the difference statistically
significant.
Acknowledgements
This research was funded by the Dutch Diabetes
Foundation (grant no. 1999.007), and supported by
various other grants [e.g. BioSim and NucSys (FP6
EU)]. We thank G. Wardeh for providing rat livers.
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Supplementary material
The following supplementary material is available
online:
Appendix S1. Equations S1 to S5, used for calculation
of extramitochondrial AMP concentration.
Appendix S2. Calculation of control coefficients.
This material is available as part of the online article
from
Palmitoyl-CoA and control of mitochondrial function J. Ciapaite et al.
5302 FEBS Journal 273 (2006) 5288–5302 ª 2006 The Authors Journal compilation ª 2006 FEBS