Mitochondrial uncoupling protein may participate in futile
cycling of pyruvate and other monocarboxylates
Petr
Je
ek and
Ji
í
Borecký
Department of Membrane Transport Biophysics, Institute of
Physiology, Academy of Sciences of the Czech Republic, CZ-14220
Prague 4, Czech Republic
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ABSTRACT |
The physiological role of monocarboxylate transport in brown
adipose tissue mitochondria has been reevaluated. We studied pyruvate,
-ketoisovalerate,
-ketoisocaproate, and phenylpyruvate uniport
via the uncoupling protein (UCP1) as a GDP-sensitive swelling in
K+ salts induced by valinomycin or
by monensin and carbonyl
cyanide-p-(trifluoromethoxy)phenylhydrazone in Na+ salts. We have demonstrated
that this uniport is inhibited by fatty acids. GDP inhibition in
K+ salts was not abolished by an
uncoupler, indicating a negligible monocarboxylic acid penetration via
the lipid bilayer. In contrast, the electroneutral pyruvate uptake
(swelling in ammonium pyruvate or potassium pyruvate induced by change
in pH) mediated by the pyruvate carrier was inhibited by its specific
inhibitor
-cyano-4-hydroxycinnamate but not by fatty acids.
Moreover,
-cyano-4-hydroxycinnamate enhanced the energization of
brown adipose tissue mitochondria, which was monitored fluorometrically
by 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide and safranin O. Consequently, we suggest that UCP1 might participate in futile cycling
of unipolar ketocarboxylates under certain physiological conditions
while expelling these anions from the matrix. The cycle is completed on
their return via the pyruvate carrier in an
H+ symport mode.
brown adipose tissue; uncoupling protein 1; pyruvate carrier; uniport of monocarboxylates; anion futile cycling
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INTRODUCTION |
NONSHIVERING THERMOGENESIS in newborn mammals,
including humans, or in adulthood, when induced by cold adaptation or
overnutrition or under specific pathological conditions, takes place in
mitochondria of the brown adipose tissue (BAT) (3, 5, 9, 23, 25). BAT
mitochondria generate heat due to uncoupling protein 1 (UCP1), which is
specific for BAT, unlike the ubiquitous uncoupling protein 2 (6). UCP1
dissipates a protonmotive force by mediating
H+ backflow (18, 23). According to
a recent hypothesis, this is carried out by a unique fatty acid cycling
mechanism (7, 11, 15, 31). UCP1 is considered to conduct anionic fatty acids. Simultaneously, fatty acids are able to return in a protonated form via the lipid bilayer. The overall cycle leads to
H+ translocation (7, 31) and,
hence, to uncoupling. Indeed, UCP1 can be regarded as a pure anion
uniporter, strictly specific for monovalent unipolar anions (14), since
it translocates their wide spectrum, alkylsulfonates, oxohalogenides,
and monovalent phosphate analogs (14), including anionic forms of
physiologically abundant fatty acids (7). The transport of all anions
is allosterically inhibited by GDP (11) and other purine nucleotides.
Thus UCP1 exhibits the widest substrate specificity among the
homologous anion transporters of the mitochondrial gene family (18).
For UCP1, only single charged anions and their
unipolarity1
are limiting (14).
Among mitochondrial metabolite anions (19), pyruvate has been
demonstrated to be a UCP1 substrate (14). It is not trivial to ask,
What is the physiological role of such monocarboxylate transport via
UCP1, since a futile cycling of pyruvate might proceed in a manner
similar to that of the cycling of fatty acids? The anionic
monocarboxylates would be expelled from the matrix because of the
negative membrane potential (
) generated by the respiratory chain, whereas their return in an
H+ symport (electroneutral) mode
could proceed via specific metabolite carriers, the pyruvate or the
-ketoisovalerate carriers. Such a mechanism is plausible under the
following conditions: 1) UCP1 should
provide monocarboxylate uniport, 2)
BAT mitochondria should contain carriers allowing for electroneutral
uptake (H+ symport) of
monocarboxylates, the pyruvate carrier (4, 19), or the
-ketoisovalerate carrier (10), and
3) these carriers should be active
at an intermediate thermogenic state, when the anion uniport pathway of
UCP1 is not completely inhibited by ATP and is not saturated by fatty
acids. In this state a minimum change in pH (
pH) should be
established to drive the
pyruvate-H+ symport.
Condition 1 is fulfilled, because a
GDP-sensitive pyruvate uniport in BAT mitochondria was described
previously (14). However, other unipolar ketocarboxylates were not
studied, and it is not known whether fatty acids can compete with
ketocarboxylates. Moreover, regulation of UCP1 activity is complex and
includes variations in the levels of free fatty acids and cytosolic
purine nucleotides (20). The nucleotide inhibitory ability is
negatively modulated by increasing pH and
Mg2+ (16). It has long been known
that full coupling of BAT mitochondria in vitro is possible only when
the fatty acids are removed (by BSA or combustion via the carnitine
cycle) and, at the same time, purine nucleotides such as GDP are
present (17, 26, 28). Why simple removal of the cycling agent, a fatty
acid, is not sufficient to cause full coupling of BAT mitochondria has
not been explained.
Condition 2 is also valid: because BAT
mitochondria can respire with pyruvate (21), they exhibit an uptake of
[14C]pyruvate and
swell in ammonium pyruvate (4). However, condition 3 needs to be investigated, and this is the main issue
of this article. We have found that, in addition to pyruvate, UCP1 also translocates other unipolar ketocarboxylates and that their transport is inhibited by fatty acids. The presence of the pyruvate carrier in
BAT mitochondria was confirmed, and inhibition of this carrier by
-cyano-4-hydroxycinnamate (
-CHC) was shown to enhance
mitochondrial energization. Hence, it is suggested that futile cycling
of ketocarboxylates might partially contribute to uncoupling of
mitochondria in BAT.
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MATERIALS AND METHODS |
Most of the chemicals were purchased from Sigma Chemical or Fluka;
2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASMPI) was a kind
gift of Prof. J. Rafael (University of Heidelberg, Heidelberg,
Germany). Syrian hamsters were cold adapted for at least 3 wk at
5°C. BAT mitochondria were isolated in a medium containing 250 mM
sucrose, 10 mM Tris-MOPS, 1 mM Tris-EGTA, pH 7.4, and BSA (5 mg/ml);
the final washing was performed in a BSA-free medium. BSA was omitted
during the isolation of mitochondria for some 
measurements.
Anion uniport indicated by osmotic swelling was measured by following
the decrease in apparent absorbance given by light scattering in the
530- to 550-nm range with use of a diode-array spectrophotometer (Spectronics 3000, Milton Roy). Light-scattering intensity reflects the
inverse volume (2). All media contained 0.1 mM Tris-EGTA, 2-8 µM
rotenone, and 1 µM oligomycin. Passive swelling in the absence of
respiration was assayed routinely in the presence of 0.25 µg/ml
antimycin. Usually, 0.2 mg protein/ml of BAT mitochondria were used per
assay in a medium of 40% isotonic osmolarity (270 mosmol taken as
100%), i.e., in 54 mM salts of monovalent anions buffered to pH 7.2 with 5 mM Tris-MOPS. The transport rates (in min
1) were calculated as
the time changes in a normalized light-scattering parameter
(2),
which was calculated as follows
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(1)
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where
P is the mitochondrial concentration (mg/ml),
PS is the standard mitochondrial
concentration of 1 mg/ml,
A
1 is the inverse
absorbance of the suspension, and
is a machine constant (2) equal
to 0.1163 for the Spectronics 3000 diode-array spectrophotometer.
O2 consumption of mitochondria was
measured at 37°C in a 5-ml thermostatically controlled chamber
equipped with a Clark polarographic electrode in a medium containing
250 mM sucrose, 10 mM Tris-MOPS, 1 mM Tris-EGTA, pH 7.4, and 40 µM
rotenone. Energization (uncalibrated 
) of a mitochondrial
suspension was continuously monitored (17, 22, 28) in the same medium
by a fluorescence probe, DASMPI (3 µM), with excitation at 467 nm and
emission at 561 nm with use of 2-nm slit widths on an Ortec
fluorescence spectrometer (model 9200, EG & G) or a Shimadzu
fluorometer (model RF5301 PC), with excitation vertically polarized
(5-nm slit width) and the emission collected through a 10-nm slit and a
polarizer in cross orientation to eliminate scattering. 
was
estimated according to Mewes and Rafael (22). For calibration, we
assumed that under maximum energization (GDP + BSA) 
of 170 mV is
established and with BSA only 
of 37.5 mV is established (26),
while
pH is 50 mV at pH 7.2 (26) and overall protonmotive force is
220 and 87.5 mV, respectively. A net fluorescence intensity was
obtained when light scattering and a fluorescence increase due to BSA
interaction with the probe were taken into account. Alternatively,

was monitored by 12.5 µM safranin, as described elsewhere
(13), in the same medium in which DASMPI was used.
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RESULTS |
Uncoupling protein mediates uniport of monovalent ketocarboxylates.
In accordance with our previous observations and conclusions (14), we
reevaluated the ability of UCP1 to conduct physiologically relevant
"metabolic" anions. We studied valinomycin-induced uptake of
K+ salts of pyruvate (Fig.
1A)
and phenylpyruvate (Fig. 1B) in
nonrespiring BAT mitochondria pretreated with BSA. An inhibitor of the
pyruvate carrier (27),
-CHC, was routinely present to eliminate the pyruvate carrier-mediated flux. The pyruvate uptake was
highly sensitive to GDP (Figs. 1 and 2),
and the inhibition was not released by carbonyl
cyanide-p-(trifluoromethoxy) phenylhydrazone
(FCCP; Fig. 1A). This excludes the
possibility that monocarboxylates pass through the membrane as neutral
acids and that UCP1 participates only in
H+
reentry.2
Hence, the observed GDP-sensitive fluxes must be ascribed to the
function of UCP1. This is also confirmed by the absence of valinomycin-induced swelling of rat liver mitochondria in potassium pyruvate in the presence of
-CHC (Fig.
1E).

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Fig. 1.
Uniport of pyruvate and other unipolar ketocarboxylates via uncoupling
protein in brown adipose tissue (BAT) mitochondria
(A-D) and its absence in liver
mitochondria (E). Uniport of
pyruvate (A), phenylpyruvate
(B), -ketoisovalerate
(C), and -ketoisocaproate
(D) was measured as a passive
swelling monitored by light scattering. Swelling was induced by 1 µM
valinomycin (Val) in 55 mM K+
salts buffered to pH 7.2 with 5 mM Tris-MOPS
(A and
B) or by a mixture of 1 µM
monensin (Mon) and 1 µM carbonyl
cyanide-p-(trifluoromethoxy)phenylhydrazone
(FCCP) in 55 mM Na+ salts
(C and
D) and 5 mM Tris-MOPS, pH 7.2, all
in presence of 1 mM -cyano-4-hydroxycinnamate ( -CHC). +GDP and
+GDP + FCCP, traces measured with 1 mM GDP and with 1 mM GDP and 1 µM
FCCP, respectively; oxamate, trace measured in
K+ salt of oxamate
( -amino- -ketoacetate). All assays contained 0.1 mM Tris-EGTA, 2 µM rotenone, 0.25 µg/ml antimycin, and 0.2 mg protein/ml of BAT or
liver mitochondria.
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Fig. 2.
Dose responses for inhibition of pyruvate and phenylpyruvate uniport
via uncoupling protein 1 (UCP1) by GDP. GDP dose responses for pyruvate
( ) and phenylpyruvate ( ) uniport were constructed from titrations
by GDP. Measurements were performed under conditions described in Fig.
1 legend. Inhibitory ability in percent is expressed as 100%
(v0) minus remaining activity in percent at
given GDP dose (v). Curves were drawn on assumption that
infinite GDP would yield 100% inhibition. Dose responses were fitted
on basis of linearization of Hill plots
(inset), and fits were drawn
according to Hill's equation (solid lines). This procedure yielded
Hill's coefficient
(nH) of 0.95 for pyruvate and phenylpyruvate and inhibition constants
(Ki) of 155 and
2,076 µM, respectively.
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Typical rates of UCP1-mediated transport were 0.2 and 1.0 min
1 in pyruvate and
phenylpyruvate, respectively. For comparison, the rate in a
Cl
medium was 0.3 min
1 (not shown). UCP1 also
translocates
-ketoisovalerate (Fig.
1C) and
-ketoisocaproate (Fig.
1D), as demonstrated by
GDP-sensitive swelling induced by monensin in the
Na+ salts in the presence of FCCP.
The typical rates reached 0.6 and 0.7 min
1, respectively. GDP (1 mM) caused incomplete inhibition in Fig. 1,
B-D. This is due in part to an
apparently lower affinity for GDP in inhibiting these anions (see
below). All monocarboxylate substrates tested always have a second
polar (carbonyl) group near the carboxyl. When this unipolarity
requirement is not fulfilled (14) or when a third polar group is
attached, as in the case of oxamate (
-amino-
-ketoacetate, Fig.
1A), then the anion is not
translocated by UCP1.
GDP inhibition of transport of various UCP1 anionic substrates was
previously found to exhibit different inhibition constants (Ki).
Particularly, Ki
values increased with the increasing hydrophobicity or size of anions
(14). This phenomenon is probably due to interference of hydrophobic
anions with GDP binding, as demonstrated by Nedergaard and Cannon (24)
in the case of benzenesulfonate. We evaluated the
Ki values for GDP
inhibition of uniport of all four carboxylates shown in Fig. 1 at pH
7.2, and two representative dose-response curves are illustrated in
Fig. 2. The curves were drawn on the assumption that infinite GDP would
yield 100% inhibition, and Ki values were
derived from the Hill plots (Fig. 2,
inset). Indeed, Ki increased with
increasing hydrophobicity.
Ki for inhibition of pyruvate uniport was 155 µM, whereas
Ki values for the
more hydrophobic carboxylates were between 1 mM
(
-ketoisovalerate) and 2 mM (phenylpyruvate and
-ketoisocaproate).
Fatty acids and monocarboxylates share the same pathway in UCP1.
The mutual competition of fatty acids and other anionic substrates on
UCP1 has repeatedly been reported (7, 11, 15, 29). Because some
artificial derivatives such as azido fatty acid,
12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA), were previously
shown to inhibit Cl
uniport
via UCP1 more potently than natural fatty acids (15), we investigated
whether the UCP1-mediated pyruvate uniport is also inhibited by AzDA.
Figure 3 shows strong inhibition by 40 µM
AzDA in the dark (i.e., not
photoactivated),3
independently of the presence of
-CHC. A similar result was also
obtained with lauric acid, a natural fatty acid, but at a much higher
concentration of 500 µM (Fig. 3B,
+laurate). As a gauge for nonspecific changes that could be caused by
AzDA and lauric acid, we tested swelling in potassium acetate with
nigericin, which acts independently of a protein carrier (not shown).
This swelling was not affected by concentrations of up to 50 µM with AzDA or up to 500 µM with lauric acid. Figure
4 illustrates the dose responses for
inhibition of pyruvate uniport by AzDA and lauric acid. Apparent
Ki was ~10
µM, irrespective of whether
-CHC was present. The inhibitory
effect of lauric acid was lower
(Ki = 306 µM).
Similar data were found for other monocarboxylates (not shown).

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Fig. 3.
Inhibition of pyruvate uniport via UCP1 by fatty acids.
Valinomycin-induced passive swelling of BAT mitochondria in potassium
pyruvate was measured in absence (A)
and presence (B) of 1 mM
-CHC. Effect of 500 µM lauric acid (+laurate) and 40 µM
12-(4-azido-2-nitrophenylamino)dodecanoic acid (+AzDA) was studied.
Rates were 0.25 and 0.045 min 1 in control and with
AzDA (82% inhibition) in A and 0.23, 0.053, and 0.048 min 1 in
control and with lauric acid (77% inhibition) and AzDA (79%
inhibition), respectively, in B with
-CHC. See Fig. 1 legend for other details.
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Fig. 4.
Dose responses for fatty acid inhibition of pyruvate uniport via UCP1
and lack of fatty acid inhibition of pyruvate carrier. Dose responses
for AzDA inhibition were measured in presence ( ) and absence ( )
of 1 mM -CHC and for lauric acid inhibition ( ) of pyruvate
uniport via UCP1. Lack of inhibition by AzDA of pyruvate
carrier-mediated electroneutral
pyruvate-H+ symport is also
illustrated when assay was assessed as swelling in ammonium pyruvate
( , see Fig. 5 legend) or as pH-induced swelling ( , see Fig. 6
legend). Inhibitory ability is expressed as 100% minus remaining
activity in percent at a given AzDA dose. Theoretical curves (solid
lines) were fitted using linearization of Hill plots (not shown) and
drawn on basis of Hill's equation with assumption that infinite AzDA
(lauric acid) would yield 100% inhibition. For AzDA,
nH was 1.2 with 1 mM -CHC and without -CHC and
Ki was 11.3 and
9.3 µM, respectively. For lauric acid,
nH was 0.98 and
Ki was 306 µM.
See Fig. 1 legend for other details.
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Pyruvate carrier in BAT mitochondria is inhibited by
-CHC but not by fatty acids.
A symport of H+ and
monocarboxylates can be provided in mitochondria by the pyruvate and
the
-ketoisovalerate carrier (10, 19). The former has been assumed
to allow for respiration of BAT mitochondria with pyruvate (21).
Therefore, our further goal was to reevaluate a non-UCP1-mediated
electroneutral pyruvate uptake in BAT mitochondria that can be ascribed
to the pyruvate carrier. For this purpose, we first studied the
swelling of BAT mitochondria in ammonium pyruvate (Fig.
5). The pyruvate uptake representing such
swelling (4) must be electroneutral, proceeding as a symport with
H+, since only the neutral
NH3 is able to pass through the
membrane. The transport was inhibited by
-CHC with a
Ki of 5.5 mM
(Fig. 5C). On the contrary, it was
not inhibited by AzDA (Fig. 4) or by lauric acid. The electroneutral
nigericin-induced uptake of pyruvate in the presence of GDP and the
absence of
-CHC was quite slow in nonrespiring BAT mitochondria
(0.05 min
1) as well as
its
-CHC-sensitive part (0.03 min
1, Fig.
5A). This shows that the activity of
the pyruvate carrier is low under these conditions. However, another
electroneutral transport induced by nigericin, a
phosphate-H+ symport via the
phosphate carrier, was not obstructed, as shown in a parallel
experiment (Fig. 5A).

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Fig. 5.
A and
B: pyruvate carrier function as
assessed by passive swelling. Two assays for pyruvate carrier based on
passive swelling were tested. 1)
Swelling was assessed in ammonium pyruvate
(B,
NH4-pyr), with no nigericin (Nig)
added. This assay was conducted by mixing BAT mitochondria with a
medium of 54 mM ammonium pyruvate, 5 mM Tris-MOPS, and 0.1 mM
Tris-EGTA, pH 7.2, containing 0.5 µg/ml antimycin, 4 µM rotenone,
and 1 µM oligomycin. 2) Passive
swelling of BAT mitochondria was assessed in potassium pyruvate
containing 1 mM GDP (A, K-pyr) induced
by 0.13 µM nigericin in absence or presence of 1 mM -CHC
(+ -CHC), which was quite slow (rates reached 0.05 and 0.02 min 1, respectively). To
verify that nonrespiring BAT mitochondria possess ability to conduct an
electroneutral transport in presence of 0.13 µM nigericin, a
Pi-H+
symport mediated by the Pi carrier
was tested (A,
KPi) by passive swelling in
KPi (44 mM, pH 7.2). Other
conditions were as for potassium pyruvate.
C: dose response for -CHC
inhibition of pyruvate carrier. Pyruvate carrier was assayed as
swelling in ammonium pyruvate ( , cf.
B) or pH-induced swelling ( ,
see Fig. 6). Percent inhibitory ability was calculated as described in
Fig. 4 legend. Solid line, corresponding fit of data for passive
swelling by use of Hill's equation;
nH was 1.2 and
Ki was 5.5 mM.
For pH-induced swelling (fit not shown)
nH was 1 and
Ki was 9 mM.
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We have also developed a new assay for electroneutral pyruvate
transport in which we employ the valinomycin-induced swelling in
potassium pyruvate under conditions when fully coupled BAT mitochondria
(by GDP and BSA) are respiring with
-glycerophosphate (Fig.
6). Propranolol was also present to exclude
most of the inner membrane anion channel-mediated flux (1). Under these conditions, respiratory H+ pumping
compensates the pyruvate-H+
symport and the simultaneous K+
uniport (influx). Moreover, addition of valinomycin to coupled mitochondria is known to create an initial
pH jump (1). Thus
pH
drives the pyruvate-H+ symport. As
demonstrated in Fig. 6, such electroneutral
pyruvate-H+ symport was sensitive
to
-CHC with a
Ki of 9 mM (Fig.
5C), indicating the participation of
the pyruvate carrier. Similar results were obtained with the other
known substrates of the pyruvate carrier such as phenylpyruvate (Fig.
6B), lactate (Fig.
6C), and chloroacetate (Fig.
6D). The residual
inhibitor-insensitive portion could be attributed to the nonionic
diffusion of pyruvic and other ketocarboxylic acids across the lipid
bilayer or to the uninhibited part of the inner membrane anion
channel-mediated flux. A contribution of the former process should be
minor, as documented by the very slow passive swelling of BAT
mitochondria in potassium pyruvate in the presence of nigericin and
-CHC (Fig. 5A).

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Fig. 6.
pH-induced swelling of monocarboxylates mediated by pyruvate carrier
in BAT mitochondria. Transport of pyruvate
(A), phenylpyruvate
(B), lactate
(C), and chloroacetate
(D) was measured in respiring
coupled BAT mitochondria in presence of 1 mM GDP and BSA (1.5 mg/ml)
and 5 mM -glycerophosphate in media containing 5 mM Tris-MOPS, pH
7.2, and 1 mM Tris-EGTA in absence of antimycin as a pH-induced
swelling initiated by 1 µM valinomycin. Valinomycin is known (cf.
Ref. 1) to create a pH jump in respiring coupled mitochondria. pH
then drives a monocarboxylate-H+
symport. Inhibition by 1 mM -CHC (+ -CHC) indicates participation
of pyruvate carrier. Other conditions were similar to those described
in Fig. 1 legend.
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Effect of
-CHC on coupling of BAT mitochondria.
It is well known (17, 26, 28) that addition of GDP to fatty
acid-depleted BAT mitochondria leads to their maximum coupling (
of 170 mV) (26). Here we demonstrate that addition of
-CHC to fatty
acid-depleted BAT mitochondria leads to some coupling (Fig.
7). Monitoring 
fluorometrically by
DASMPI (22), we found that a nearly uncoupled state of fatty
acid-depleted BAT mitochondria in the absence of GDP can be further
coupled by
-CHC in the presence of endogenous (not shown) or
externally added pyruvate (Fig. 7). This intermediate coupling with
-CHC and BSA was estimated to reach 112-135 mV (the maximum

and the sole BSA-induced energization served as the 2 calibration points). We have explained this as a result of the
elimination of pyruvate cycling.
-CHC enhanced the energization,
independently of whether it was added before (not shown) or after BSA
(Fig. 7A) or before (Fig.
7B) or after (Fig.
7A) the respiratory substrate
-glycerophosphate. Pyruvate served only as a cofactor of the
putative pyruvate cycling, since rotenone was always present. Figure
7B shows that the energization in the
presence of pyruvate and rotenone was higher with than without
-CHC.
A sole pyruvate addition rather led to a slight uncoupling (Fig.
7B, only pyr). We have also verified
that
-CHC added after FCCP did not cause any effect, nor did it
interfere with the fluorescence of DASMPI (not shown). We have also
confirmed the well-known fact (17, 26, 28) that the sole addition of
GDP (not shown) or sole fatty acid removal (Fig. 7) did not lead to
complete coupling, but only to small 
. Also, GDP added after
-CHC was still able to induce maximum energization (Fig. 7A, dashed trace). Note that the scale
in millivolts is nonlinear with regard to the fluorescence;
consequently, this energization appears to be exaggerated.

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Fig. 7.
Coupling effect of -CHC in BAT mitochondria.
A and
B: energization ( ) of
mitochondria was monitored by increasing fluorescence of 6 µM
2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASMPI), added at
beginning of each trace. BAT mitochondria were resuspended to 1 mg
protein/ml in an O2-saturated
medium containing 250 mM sucrose, 10 mM Tris-MOPS, 1 mM Tris-EGTA, pH
7.4, and 40 µM rotenone. A: BAT
mitochondria containing natural endogenous fatty acids. Solid trace,
effect of -CHC added after -glycerophosphate ( -GP) and
pyruvate. First, BSA (5 mg/ml) was added to eliminate fatty acids;
corresponding fluorescence increase is mostly due to BSA interaction
with DASMPI. Subsequently, a substrate, 10 mM -glycerophosphate,
Na+ salt, and a cofactor, 5 mM
Tris-pyruvate, were added (arrow, -GP + pyr), causing a slight
signal decrease. Addition of 1 mM -CHC caused a biphasic
fluorescence ( ) increase, which was terminated by anoxia (no
O2). Dashed trace, coupling
effect of 1 mM GDP added after 1 mM -CHC. Order of additions before
GDP was as described above. A complete uncoupling induced by 2 µM
FCCP is also demonstrated; fluorescence ( ) dropped to original
low value. B: BAT mitochondria were
isolated with BSA, which was also present in assay medium (5 mg
BSA/ml). Top trace, -CHC effect
when 1 mM -CHC was added before -glycerophosphate and pyruvate
( -GP + pyr). Because 40 µM rotenone was present, only former
represents a respiratory substrate. This is compared with same
measurement without -CHC (dashed trace) and without both -CHC and
-glycerophosphate (bottom trace,
only pyr). C: -CHC-induced coupling
of BAT mitochondria monitored by
O2 consumption. BAT mitochondria
(0.4 mg protein/ml) were injected into a 5-ml chamber with a Clark
polarographic O2 probe containing
an O2-saturated medium of same
composition used for  monitoring, supplemented by 7.5 mg BSA/ml.
As indicated by arrows, 10 mM Tris-pyruvate (pyr), 4 mM
-glycerophosphate, Na+ salt
( -GP), 8 mM -CHC, and 4 µM FCCP were added. Numbers under trace
are respiratory rates in nmol
O · min 1 · mg
protein 1.
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Parallel measurements of respiration by a Clark
O2 probe confirmed that the
-CHC-induced coupling decreases
O2 consumption of BAT
mitochondria, whereas the addition of an uncoupler accelerates their
respiration (Fig. 7C). Rat liver
mitochondria respiring with endogenous substrates (i.e., without
rotenone, Fig.
8A) or nonenergized (with rotenone), in the presence (Fig.
8A, bottom trace) or absence of
pyruvate (Fig. 8B), exhibited no
increase in 
after addition of
-CHC. Instead, a slight
decrease of 
was noted. Succinate, when added after
-CHC, was
still able to energize rat liver mitochondria to a maximum coupling
(Fig. 8B).

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Fig. 8.
Lack of -CHC effect in rat liver mitochondria. Energization ( )
of rat liver mitochondria (1 mg protein/ml) was monitored by increasing
fluorescence of DASMPI as described in Fig. 7 legend. In
A, 1 mM -CHC was added to
mitochondria energized almost completely by endogenous substrates
[no rotenone, top trace; note
that 10 mM Tris-succinate (succ) did not further increase  ]
or to nonenergized mitochondria (bottom
trace), where effect of 5 mM Tris-pyruvate was also
evaluated (pyr). In B, 10 mM succinate
(succ) added after -CHC was still able to induce maximum coupling;
uncoupling induced by 2 µM FCCP is illustrated as well.
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The alternative monitoring of 
by safranin also confirmed the
coupling effect of
-CHC in BAT mitochondria (Fig.
9). In contrast to DASMPI, safranin
fluorescence is quenched with increasing 
, so one could expect
almost "mirror" changes in the fluorescence records, and this was
indeed the case.
-CHC enhanced the energization independently
of whether added after BSA (Fig.
9A) or before BSA (Fig. 9.
B and
C). The latter effect of
-CHC was
higher in the presence of externally added pyruvate (Fig. 9,
B vs.
C). As expected, ATP added after BSA
induced the maximum energization (Fig. 9, B and
C). These results again suggest that
the inhibition of the pyruvate carrier may eliminate pyruvate
cycling, in which this carrier participates with UCP1.

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Fig. 9.
Monitoring -CHC effect in BAT mitochondria by safranin. Energization
( ) of BAT mitochondria (1 mg/ml) partially depleted of fatty
acids was monitored by decreasing fluorescence of 12.5 µM safranin in
an O2-saturated medium containing
250 mM sucrose, 10 mM Tris-MOPS, 1 mM Tris-EGTA, pH 7.4, 10 mM
-glycerophosphate, Na+ salt,
and 40 µM rotenone. Traces A and
B were measured with 5 mM
Tris-pyruvate and trace C without
pyruvate. -CHC (1 mM), added after
(A) or before
(B and
C) BSA enhanced  . Increase in
 was higher in presence of 5 mM Tris-pyruvate
(B vs.
C). Addition of BSA and ATP led to
further coupling, whereas 2 µM FCCP led to uncoupling, despite a
signal shift due to addition of BSA.
|
|
 |
DISCUSSION |
Je
ek and Garlid (14) reported for the first time that UCP1 can
translocate pyruvate and acetate. We have now extended these findings
to the entire class of unipolar ketocarboxylates. Our data show that
UCP1 allows for the uniport of phenylpyruvate,
-ketoisovalerate, and
-ketoisocaproate, which have not been identified as the UCP1
substrates. We can also suggest that fatty acids and unipolar
monocarboxylates compete within a single pathway of the UCP1. A model
fatty acid, AzDA (15, 29, 30), and the natural lauric acid inhibit the
uniport of pyruvate via UCP1, but not electroneutral pyruvate
transport, mediated by the pyruvate carrier. The latter was clearly
identified in BAT mitochondria, and its inhibition by
-CHC resulted
in an increased coupling of BAT mitochondria (which was incomplete).
Our data suggest that, even in the absence of fatty acids, futile
cycling of pyruvate and other monocarboxylate anions might mimic the
fatty acid cycling uncoupling mechanism (7, 31). All monovalent anions,
which may enter into the matrix by a symport with
H+, could be involved. This could
be ensured by the pyruvate carrier or by the
-ketoisovalerate
carrier. Hence, the existence of the futile cycling of monocarboxylates
could contribute, at least partially, to the uncoupling and enable a
fine regulation of coupling in BAT mitochondria (Fig.
10).

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Fig. 10.
Proposed futile cycling of monocarboxylates involved in BAT
thermogenesis. Decrease in ATP level results in opening of uncoupling
protein (UCP) for pyruvate efflux. Pyruvate might return together with
an H+ on pyruvate carrier, thus
completing uncoupling cycle. Uncoupling decreases
electrochemical proton gradient
( ).
|
|
Features of the uncoupling protein.
The UCP1 is now a well-characterized uniporter of monovalent anions.
Striking analogy between the character and size of fatty acids and UCP1
amphiphilic substrates such as alkylsulfonates [translocation of
which was directly proven (7, 14)] and the mutual competition of
fatty acids and alkylsulfonates (7, 11, 15, 29) supports the fatty acid
cycling mechanism (7). We now suggest that not only can long-chain
fatty acids undergo such cycling but also the compounds with a short
chain, namely, the monocarboxylic
-ketoacids (Fig. 10). We have
unambiguously characterized pyruvate and other unipolar
ketocarboxylates as the transport substrates of UCP1. We may assume
that maximal reaction velocity
(Vmax) for
pyruvate uniport via UCP1 will be close to the value found for
Cl
uniport (7) and that the
Michaelis-Menten constant is on the order of 10 mM. Even if
Vmax is lower and
the assumed affinity (the inverse Michaelis-Menten constant) is much
lower than the corresponding parameters for the fatty acid uniport,
pyruvate uniport in the absence of fatty acids would still be able to
contribute to the uncoupling at a pyruvate physiological concentration
of 0.1 mM (10). Under these conditions, 1% of
Vmax amounts to
100 nmol
H+ · min
1 · mg
UCP1
1, which is equal to
the turnover of 7 s
1.
Because it is known that free Mg2+
also prevents nucleotide inhibition of anion uniport via UCP1, we can
expect that at least 35% of in vitro measured pyruvate uniport
activity will remain (16) at the physiological concentrations of
Mg2+ and ATP, 0.5 mM in a
thermogenic state (20).
The ketocarboxylate uniport via UCP1 is presumably competitively
inhibited by fatty acids (Figs. 3 and 4) and allosterically (11) by GDP
(Figs. 1 and 2). With unipolar ketocarboxylates, we confirmed both
trends, which were previously revealed for the UCP1-substrate pattern,
namely, that the transport rates are enhanced with increasing
hydrophobicity of the anion (14), e.g., phenylpyruvate vs. pyruvate. A
second feature, that GDP inhibition of anion uniport is decreasing with
the hydrophobicity of the anion (with increasing Ki), was also
confirmed for ketocarboxylates. Moreover, residual GDP-insensitive
transport in Na+ salts of
-ketoisovalerate and
-ketoisocaproate might indicate that BAT
mitochondria contain a branched-chain
-ketoacid carrier (10).
Regulation of thermogenesis in BAT.
Regulation of thermogenesis in BAT has not yet been satisfactorily
explained. A central mechanism should involve a single third messenger
(or several such messengers) between norepinephrine-stimulated cAMP
levels and UCP1 (23). The most plausible candidate according to LaNoue
et al. (20) is ATP, which drops to 0.5 mM in thermogenic BAT
cells. At physiological free Mg2+
concentrations and pH, this ATP level is not inhibitory and allows the UCP1 transport pathway to open (16). At the same time, fatty acid levels are elevated, and this leads to the thermogenic state.
We may hypothesize that the sole removal (combustion) of fatty acids
can arrest the uncoupling cycle of fatty acids but not of
monocarboxylates. The latter would be blocked only when high levels of
ATP or other purine nucleotides are also present. Thus, in the absence
of fatty acids as cycling agents, pyruvate cycling may play an
important regulatory role for thermogenesis, when fatty acids are
rapidly depleted from the triglyceride droplets. Our current data
support this point of view. Despite the fact that
-CHC in fatty
acid-depleted BAT mitochondria does not induce the highest coupled
state, the observed partial 
increase is sufficient for delicate
regulation of coupling. This partial 
increase can be interpreted
as a result of blockage of pyruvate cycling by inhibition of pyruvate
uptake via the pyruvate carrier.
Features of the pyruvate carrier in BAT mitochondria.
The presence of the pyruvate carrier in BAT mitochondria has previously
been indicated by the high capacity of BAT mitochondria (21) and BAT
cells (25) to oxidize pyruvate as a respiratory substrate by the
existence of swelling of BAT mitochondria in ammonium pyruvate and by
the respiratory-driven uptake of
[14C]pyruvate into BAT
mitochondria (4). We have confirmed the existence of swelling in
ammonium pyruvate, and we have demonstrated the respiratory-driven
pH-induced electroneutral transport of pyruvate and other
monocarboxylates in BAT mitochondria. Both processes are sensitive to
-CHC, a specific inhibitor of the pyruvate carrier (27), but are
insensitive to fatty acids (Fig. 4). The former is driven by
NH3 permeation into the membrane, and subsequent matrix alkalinization results from the formation of
NH+4, whereas the latter process is driven by
pH of the same orientation, i.e., by the increased
pH under conditions when K+ uptake by
valinomycin discharges the 
component of the proton electrochemical gradient
(
). Swelling in ammonium pyruvate is fast (4), because a much
greater NH+4 gradient is established and
concomitant matrix alkalinization creates
pH comparable in magnitude
to that during respiration.
Slow electroneutral pyruvate transport was detected only when we
attempted to induce passive swelling of BAT mitochondria in potassium
pyruvate by nigericin in the presence of GDP. First, it shows that
pyruvate, unlike acetate, is poorly permeant through the mitochondrial
membrane itself. Otherwise, swelling in potassium pyruvate and
nigericin should be as rapid as any swelling independent of a protein
carrier. Because during a passive swelling nigericin collapses
pH,
we might conclude that electroneutral pyruvate transport is slow at low
pH. This has been suggested also for the dicarboxylate carrier (12).
Such a
pH regulation would represent a rate-limiting step in
pyruvate cycling. Consequently, pyruvate cycling is expected to be a
fine regulatory mechanism rather than a major thermogenic mechanism.
In conclusion, pyruvate (monocarboxylate) cycling might contribute only
partially to the overall thermogenesis but should play an important
role in the fine control of coupling. This could be exerted even by
sequential fluxes via the pyruvate carrier and UCP1. Because of the
limited penetration of pyruvic acid via the lipid bilayer, pyruvate
cycling cannot proceed as freely as fatty acid cycling but is regulated
on both proteins involved. With a partially inhibited pyruvate carrier
(by low
pH), such cycling will play only a minor role. However, on
combustion of fatty acids and concomitantly enhanced 
and
pH,
the activated pyruvate carrier will enable the pyruvate cycling in
concert with the open UCP1 pathway (unsaturated with nucleotides and
fatty acids).
 |
ACKNOWLEDGEMENTS |
The use of a fluorometer provided by Drs. Martin Nikl and Karel
Polák (Institute of Physics, Prague, Czech Republic) is
gratefully acknowledged.
 |
FOOTNOTES |
This work was partly supported by Academy of Sciences of the Czech
Republic Grant 51151 and later by Grant Agency of the Czech Republic
Grants 301/95/0620 and 301/98/0568 and by US-Czechoslovak Science and
Technology Program Grant 94043, which aided the purchase of a new
fluorometer.
1
Unipolarity refers to a condition stating that,
if there is a polar group besides the carboxyl (or other charged
group), it should be located close to the carboxyl group.
2
H+
transport mediated by UCP1 is most probably a result of fatty acid
cycling (6). Residual endogenous fatty acids that were not removed by
BSA treatment could therefore provide such an
H+-conducting pathway.
3
Photoactivated AzDA inhibited UCP1 more
strongly (cf. Ref. 15).
Address for reprint requests: P. Je
ek, Dept. of Membrane
Transport Biophysics (No. 375), Institute of Physiology, Academy of
Sciences of the Czech Republic, Víde
ská 1083, CZ-14220 Prague 4, Czech Republic.
Received 26 February 1996; accepted in final form 15 April 1998.
 |
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