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,

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,

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

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

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

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

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
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

⁄ 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

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

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

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.

⁄ 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

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

) 0.06
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

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-

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

0.46
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

0.01
) 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

) 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

‘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

-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

⁄ 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

) 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

) 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

0.00
) 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

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

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

. 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-

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

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,

. 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

)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

)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

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

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

⁄ 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-

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

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

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.

in the insulin-resistant offspring of patients with type
2 diabetes. N Engl J Med 350, 664–671.
3 Felber JP & Golay A (2002) Pathway from obesity to
diabetes. Int J Obes Relat Metab Disord 26, S39–S45.
4 Unger RH (2002) Lipotoxic diseases. Annu Rev Med 53,
319–336.
5 Schonfeld P, Wieckowski MR & Wojtczak L (2000)
Long-chain fatty acid-promoted swelling of mitochon-
dria: further evidence for the protonophoric effect of
fatty acids in the inner mitochondrial membrane. FEBS
Lett 471, 108–112.
6 Chua BH & Shrago E (1977) Reversible inhibition of
adenine nucleotide translocation by long chain acyl-
CoA esters in bovine heart mitochondria and inverted
submitochondrial particles. J Biol Chem 252, 6711–
6714.
7 Franch J, Knudsen J, Ellis BA, Pedersen PK, Cooney GJ
& Jensen J (2002) Acyl-CoA binding protein expression
is fiber type- specific and elevated in muscles from the
obese insulin-resistant Zucker rat. Diabetes 51, 449–454.
8 Woldegiorgis G, Yousufzai SY & Shrago E (1982) Stu-
dies on the interaction of palmitoyl coenzyme A with
the adenine nucleotide translocase. J Biol Chem 257,
14783–14787.
9 Bakker SJL, IJzerman RG, Teerlink T, Westerhoff HV,
Gans ROB & Heine RJ (2000) Cytosolic triglycerides
and oxidative stress in central obesity: the missing link
between excessive atherosclerosis, endothelial dysfunc-
tion, and b-cell failure? Atherosclerosis 148, 17–21.
10 Korshunov SS, Skulachev VP & Starkov AA (1997)

18 Heinrich R & Rapoport TA (1974) A linear steady-state
treatment of enzymatic chains. General properties, con-
trol and effector strength. Eur J Biochem 42, 89–95.
19 Groen AK, Wanders RJ, Westerhoff HV, van der Meer
R & Tager JM (1982) Quantification of the contribution
of various steps to the control of mitochondrial respira-
tion. J Biol Chem 257, 2754–2757.
20 Kholodenko BN (1988) How do external parameters
control fluxes and concentrations of metabolites? An
additional relationship in the theory of metabolic con-
trol. FEBS Lett 232, 383–386.
21 Ainscow EK & Brand MD (1999) Quantifying elasticity
analysis: how external effectors cause changes to meta-
bolic systems. Biosystems 49, 151–159.
22 Schwenke WD, Soboll S, Seitz HJ & Sies H (1981)
Mitochondrial and cytosolic ATP ⁄ ADP ratios in rat
liver in vivo. Biochem J 200, 405–408.
23 Soboll S, Seitz HJ, Sies H, Ziegler B & Scholz R (1984)
Effect of long-chain fatty acyl-CoA on mitochondrial
and cytosolic ATP ⁄ ADP ratios in the intact liver cell.
Biochem J 220, 371–376.
24 Akerboom TP, Bookelman H & Tager JM (1977)
Control of ATP transport across the mitochondrial
membrane in isolated rat-liver cells. FEBS Lett 74,
50–54.
25 Hardie DG (2004) The AMP-activated protein kinase
pathway – new players upstream and downstream.
J Cell Sci 117, 5479–5487.
26 van den Berghe G, Bontemps F & Vincent MF
(1988) Cytosolic purine 5¢-nucleotidases of rat liver

33 Tager JM, Groen AK, Wanders RJ, Duszynski J, West-
erhoff HV & Vervoorn RC (1983) Control of mitochon-
drial respiration. Biochem Soc Trans 11, 40–43.
34 Duszynski J, Groen AK, Wanders RAJ, Vervoorn RC
& Tager JM (1982) Quantification of the role of the
adenine nucleotide translocator in the control of mito-
chondrial respiration in isolated rat-liver cells. FEBS
Lett 146, 262–266.
35 Bohnensack R, Kuster U & Letko G (1982) Rate-con-
trolling steps of oxidative phosphorylation in rat liver
mitochondria. A synoptic approach of model and
experiment. Biochim Biophys Acta 680, 271–280.
36 Westerhoff HV, Plomp PJ, Groen AK, Wanders RJ,
Bode JA & van Dam K (1987) On the origin of the lim-
ited control of mitochondrial respiration by the adenine
nucleotide translocator. Arch Biochem Biophys 257,
154–169.
37 Gellerich FN, Bohnensack R & Kunz W (1983) Control
of mitochondrial respiration. The contribution of the
adenine nucleotide translocator depends on the ATP-
and ADP-consuming enzymes. Biochim Biophys Acta
722, 381–391.
38 Wanders RJA, Groen AK, van Roermund CWT &
Tager JM (1984) Factors determining the relative contri-
bution of the adenine nucleotide translocator and the
ADP-regenerating system to the control of oxidative
phosphorylation in isolated rat-liver mitochondria.
Eur J Biochem 142, 417–424.
39 Mildaziene V, Nauciene Z, Baniene R & Grigiene J
(2002) Multiple effects of 2,2,5,5-tetrachlorbiphenyl on

of metabolic control theory. Eur J Biochem 188,
313–319.
47 Westerhoff HV & Kell DB (1987) Matrix method for
determining the steps most rate- limiting to metabolic
fluxes in biotechnological processes. Biotechnol Bioeng
30, 101–107.
48 Echtay KS, Winkler E & Klingenberg M (2000) Coen-
zyme Q is an obligatory cofactor for uncoupling protein
function. Nature 408, 609–613.
49 Giersch C (1994) Determining elasticities from multiple
measurements of steady-state flux rates and metabolite
concentrations: theory. J Theor Biol 169, 89–99.
50 Hofmeyr JH, Cornish-Bowden A & Rohwer JM (1993)
Taking enzyme kinetics out of control; putting control
into regulation. Eur J Biochem 212, 833–837.
51 Westerhoff HV & Chen YD (1984) How do enzyme
activities control metabolite concentrations? An addi-
tional theorem in the theory of metabolic control.
Eur J Biochem 142, 425–430.
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