2A, when experimentally obtained values of ATPout⁄ ADPout ratios Table 1 and [16] are used in the calculation, inhibition with palmitoyl-CoA would cause an increase in [AMP]out of 17% an
Trang 1nucleotide translocator determines palmitoyl-CoA effects Implications for a mechanism linking obesity and type 2 diabetes Jolita Ciapaite1,5, Stephan J L Bakker2, Michaela Diamant3, Gerco van Eikenhorst1,
Robert J Heine3, Hans V Westerhoff1,4and Klaas Krab1
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: jolita.ciapaite@falw.vu.nl
(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 (QH2⁄ 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 H2O2 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 C¼ 0.13) and phosphorylation (C ¼ 0.14) flux It controlled intramitochondrial (C¼)0.70) and extramitochondrial ATP ⁄ ADP ratios (C¼ 0.23) more strongly, whereas the control exerted over the QH2⁄ Q ratio (C¼)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, P1,P5-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 Xmby module i; C J k
i , flux control coefficient, quantifying control of flux Jkby module i; Dw, membrane potential, i.e electrical potential across the inner mitochondrial membrane; J 1 , 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.
Trang 2In 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 (Jo) by 56 ± 3% and phosphoryla-tion flux (Jp) by 58 ± 7%, and increased proton-leak flux ( J1) 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 (QH2⁄ Q) increased by 42 ± 9%, and Dw increased by 13 ± 1 mV (9%) The QH2⁄ Q
Trang 3ratio 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 H2O2
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 H2O2 production in mitochondria respiring on succinate Figure 1A shows that palmitoyl-CoA induced H2O2 production in state 3 in a concentration-dependent manner The palmitoyl-CoA-induced H2O2 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 H2O2 formation, although to a lower extent (Fig 1B)
To test whether palmitoyl-CoA metabolism via b-oxidation contributes to increased H2O2 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 H2O2 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 H2O2 pro-duction However, partial inhibition of palmitoyl-CoA-induced H2O2 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
+ 5 l M
Palmitoyl-CoA
Jo[nmol O2Æmin)1Æ(mg protein))1] 53 ± 3 23 ± 2**
Jp[nmol ADPÆmin)1Æ(mg protein))1] 375 ± 26 160 ± 15**
J 1 (nmol O 2 Æmin)1Æ(mg protein))1] 3.3 ± 0.3 4.5 ± 0.4**
*P < 0.05 and **P < 0.01 versus no palmitoyl-CoA.
Fig 1 Effect of palmitoyl-CoA on H2O2production in isolated mitochondria respiring on succinate (A) Dependence of H2O2production on palmitoyl-CoA concentration (B) Comparison of the effects of various inhibitors on H2O2production St 3, State 3; p-CoA, palmitoyl-CoA (5 l M ), protonophore S-13 (0.2 l M ); AT, atractyloside (1.5 l M ); CAT, carboxyatractyloside (0.1 l M ); Oligo, oligomycin (0.5 l M ); Ro, rotenone (2 l M ); M-CoA, malonyl-CoA (0.1 m M ); PC, palmitoyl- L -carnitine (5 l M ) 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.
Trang 4Palmitoyl-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 (ATPout⁄ ADPout) (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 P1,P5-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 ATPout⁄ ADPout
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 ATPout⁄ ADPout 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 ATPout⁄ ADPout ratios, a small decrease leads to a large increase in [AMP]out, whereas [AMP]out changes relatively little in the range
of high ATPout⁄ ADPout ratios As indicated in Fig 2A, when experimentally obtained values of ATPout⁄ ADPout 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 ATPout⁄ ADPout 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 ATPtotal⁄ ADPtotal 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 ATPtotal⁄ ADPtotal ratio reflect changes in the ATPout⁄ ADPout ratio Palmitoyl-CoA caused a signi-ficant concentration-dependent decrease in the ATPtotal⁄ ADPtotal ratio and increase in [AMP]total, which corresponded quite well to the correlation of [AMP]out and the ATPout⁄ ADPout 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 (ATPin⁄ ADPin) as an 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 (Jp) 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)
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
2 m M The points show experimentally determined dependence of
[AMP]total on the ATPtotal⁄ 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.
Trang 5Further analysis with the ATPin⁄ ADPin 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 (Jo) and phosphorylation flux (Jp) for both respiratory sub-strates are summarized in Table 2 The distribution pattern of the control over Jpamong the modules was similar to that of Jo 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
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 modmod-ule 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 oxidatitra-tion module with 0–55 n M rotenone when the phosphorylation module was blocked with 0.3 l M oligomycin J p
was calculated as: Jp¼ J o ) J h at the same value of Dw; J 1 was measured as Join 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.
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 Jp
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.
Trang 6was 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 Jo and Jp 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 Jo and Jp signifi-cantly increased upon inhibition with palmitoyl-CoA The control of Joincreased by 67% and 55% with suc-cinate and glutamate plus malate, respectively, whereas the control of Jp was affected more strongly: it increased by 87% and 83% with succinate and glutam-ate plus malglutam-ate, 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, QH2⁄ 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, h1and 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; QH2ox,
QH2-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
No p-CoA + 5 l M p-CoA No p-CoA + 5 l M p-CoA
Succinate
ATP synth 0.060.00 0.120.04* 0.060.01 0.150.05*
Glutamate plus malate
Leak 0.040.00 0.140.01** ) 0.03 0.00 ) 0.06 0.01 **
*P < 0.05 and **P < 0.01 versus no palmitoyl-CoA.
Trang 7Control of the QH2/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 QH2⁄ Q
ratio was almost solely controlled by the respiratory
chain enzymes, with the coenzyme Q reducing module
exerting a positive control and the coenzyme QH2
oxidizing module exerting a negative control, while the
control of Dw was shared equally between the
gen-erating (positive control) and 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
bc1 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
QH2⁄ Q ratio and Dw (Table 3) This is because activa-tion of the ANT stimulates the phosphorylaactiva-tion branch of oxidative phosphorylation, which consumes
Dw The negative control over the QH2⁄ Q ratio is explained similarly
Palmitoyl-CoA had hardly any effect on the control
of the QH2⁄ Q ratio when glutamate plus malate was used as a substrate With succinate, palmitoyl-CoA mainly affected the control of the QH2⁄ Q ratio by respiratory-chain modules: control by both coen-zyme Q-reducing and coencoen-zyme QH2-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 ATPin⁄ ADPin ratio and ATPout⁄ ADPout ratio is summarized in Table 3 For both sub-strates used, control of the ATPin⁄ ADPin 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
CQH2 =Q
i CiD CATPin =ADP in
i CATPout =ADP out
i
Succinate
Glutamate plus malate
*P < 0.05 and **P < 0.01 versus no palmitoyl-CoA.
Trang 8over the ATPin⁄ ADPin 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
QH2-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 ATPin⁄
ADPinratio by ATP synthesis and the negative control
by the proton leak The negative control of the ATPin⁄
ADPinratio 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 ATPout⁄ ADPout 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
ATPin⁄ ADPin ratio, the distribution of the control of
the ATPout⁄ ADPout ratio within the respiratory chain
depended on the substrate used Comparing the two
substrates, ATP synthesis exerted less control over the
ATPout⁄ ADPout ratio in the case of succinate
oxida-tion Palmitoyl-CoA increased the control of the
ATPout⁄ ADPout 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 QH2-oxidizing module and hexokinase with
succinate The control of the ATPout⁄ ADPout 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 Jo, 68% of the decrease in Jp, 56% of the increase in the QH2⁄ Q ratio, 70% of the increase in Dw, 72% of the increase
in the ATPin⁄ ADPinratio, and 59% of the decrease in the ATPout⁄ ADPout 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 Jo, 76% of the reduction in Jp, 68% of the increase in Dw, 88% of the increase in the ATPin⁄ ADPinratio, and 69% of the reduction in the ATPout⁄ ADPout ratio The exception was the QH2⁄ 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.
pCoA iIRJp
pCoA iIRQH2 =Q
pCoA iIRDpCoA i IRATPin =ADP in
pCoA iIRATPout =ADP out
pCoA Ie i
pCoA
Succinate
Glutamate plus malate
Trang 9ANT contributed only 29% of the overall increase in
the QH2⁄ 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 ATPout⁄
ADPout 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 (EC50@ 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 ATPout⁄ ADPoutratio With respect to the interrelation between ATPout⁄ ADPout ratios and [AMP]out, we have shown here how [AMP]out increases with decreasing ATPout⁄ ADPout 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 ATPtotal⁄ ADPtotal 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 Km 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
Trang 10consumption 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 H2O2 production
in mitochondria respiring on succinate The sensitivity
of the palmitoyl-CoA-induced H2O2 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 QH2⁄ 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 H2O2 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 H2O2
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 Vmax 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 QH2⁄ Q ratio to the least extent, and indeed it was the least affected by palmitoyl-CoA
Jo, Jp, ATPin⁄ ADPin and ATPout⁄ ADPout 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 QH2⁄ 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 QH2⁄ Q ratio, their immediate produc-ers and consumproduc-ers, 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 QH2⁄ 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 Jo 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