From the Centro de Investigaciones Biológicas,
Consejo Superior de Investigaciones Científicas, 28006 Madrid,
Spain, § Centre de Recherche sur l'Endrocrinologie
Moléculaire et le Développement, CNRS, Meudon F-92190,
France, and the ¶ Neurosciences Institute, Ninewells Medical
School, Dundee University, Dundee DD1 9SY, United Kingdom
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ABSTRACT |
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The activity of the brown fat uncoupling protein
(UCP1) is regulated by purine nucleotides and fatty acids. Although the
inhibition by nucleotides is well established, the activation by fatty
acids is still controversial. It has been reported that the ADP/ATP carrier, and possibly other members of the mitochondrial carrier family, mediate fatty acid uncoupling of mitochondria from a variety of
sources by facilitating the transbilayer movement of the fatty acid
anion. Brown fat mitochondria are known to be more sensitive to fatty
acid uncoupling, a property that has been assigned to the presence of
UCP1. We have analyzed the transport properties of UCP1 and conclude
that fatty acids are not essential for UCP1 function, although they
increase its uncoupling activity. In order to establish the difference
between the proposed carrier-mediated uncoupling and that exerted
through UCP1, we have studied the facility with which fatty acids
uncouple respiration in mitochondria from control yeast and strains
expressing UCP1 or the mutant Cys-304 Gly. The concentration of
free palmitate required for half-maximal activation of respiration in
UCP1-expressing mitochondria is 80 or 40 nM for the mutant
protein. These concentrations have virtually no effect on the
respiration of mitochondria from control yeast and are nearly 3 orders
of magnitude lower than those reported for carrier-mediated uncoupling.
We propose that there exist two modes of fatty acid-mediated
uncoupling; nanomolar concentrations activate proton transport through
UCP1, but only if their concentrations rise to the micromolar range do
they become substrates for nonspecific carrier-mediated uncoupling.
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INTRODUCTION |
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The ability of long-chain fatty acids to uncouple mitochondrial respiration from ATP synthesis (see Ref. 1; reviewed in Ref. 2) has been generally assumed to resemble that of other weak acids, which can permeate through the lipid bilayer in both protonated and unprotonated forms. However, recent data have established that fatty acid anion flip-flop across the phospholipid bilayer occurs far too slowly (t > 1 min) (3) to allow the anion to complete a classic protonated/deprotonated cycle as for classical uncouplers. The protonophoric activity observed when extremely high fatty acid concentrations (above 0.1 mM) are employed is probably caused by the disruption of the lipid bilayer in a detergent-like mode (1, 4, 5), inasmuch as, for example, effects are not reversed by albumin. The situation is less clear when lower fatty acid concentrations are employed and detergent effects are negligible. It has been shown recently that, under these conditions, fatty acid uncoupling becomes sensitive to inhibitors such as carboxyatractylate, and consequently it has been proposed that permeation of the fatty acid anion is mediated by the ADP/ATP carrier (6-9) and possibly by other anion carriers of the mitochondrial inner membrane (10, 11).
Brown fat is a specialized tissue, the function of which is to produce heat. The thermogenic mechanism is centered around the brown fat uncoupling protein (UCP1),which allows dissipation of the proton electrochemical potential gradient and therefore uncouples respiration from ATP synthesis (12, 13). Recently, three new uncoupling proteins have been identified with homology to UCP1 and termed UCP2 (14), UCP3 (15), and StUCP (16). Free fatty acids, liberated by the noradrenergic stimulation of lipolysis, are the substrate for brown fat thermogenesis and also act as the cytosolic second messengers by which noradrenaline activates UCP1 (17, 18).
The mechanism by which fatty acids activate UCP1 is a matter of debate. Two main hypothesis are current. The first is an extension of the observation of carboxyatractylate-sensitive mitochondrial fatty acid uncoupling, and proposes that UCP1 and other mitochondrial anion carriers (19, 20) can catalyze the transport of the fatty acid anion (21, 22). The second model proposes that fatty acids act as a prosthetic group in UCP1 delivering protons to a site from which they are translocated to the other side of the membrane (23). The anion transporting activity would have little physiological importance and thus would be a vestige of its evolutionary relationship with the rest of mitochondrial metabolite carriers.
The present report will examine the characteristics of fatty
acid-mediated uncoupling of yeast mitochondria expressing
recombinant UCP1. Two questions are addressed. First, does
UCP1 function in the absence of both nucleotides and fatty
acids? Second, what is the difference between UCP1-mediated
uncoupling and that mediated by other carriers? We conclude
that UCP1 retains the capacity to transport
H+(OH) in the absence of fatty acids
and that fatty acids activate UCP1 at much lower
concentrations than are required for other mitochondrial
carriers.
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EXPERIMENTAL PROCEDURES |
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Expression Vector, Site-directed Mutagenesis, and Yeast
Growth--
The coding sequences for wild type UCP1 and the mutant
Cys-304 Gly were introduced in the pYeDP-1/8-10 vector as
described previously (24). Gene expression was under the control of the gal10-cyc1 promoter, induced by galactose and repressed by
glucose. The diploid Saccharomyces cerevisiae strain W303
was transformed by electroporation, and clones containing the
expression vector were plated on SD minimal medium (2% glucose, 0.67%
yeast nitrogen base, 0.1% casamino acids, 20 mg/liter tryptophan, 40 mg/liter adenine, 2% agar) and selected for uracil autotrophy. Control yeasts (UCP
) contained the same vector but with the UCP1
cDNA in the inverse orientation (25). For preparation of
mitochondria, yeast were incubated aerobically at 28 °C in SP medium
(0.1% glucose, 2% lactic acid, 0.67% yeast nitrogen base, 0.1%
KH2PO4, 0.12%
(NH4)2SO4, 0.1% casamino acids, 20 mg/liter tryptophan, 40 mg/liter adenine, pH 4.5), and 14 h before
harvesting were transferred to SG medium (2% galactose, 0.67% yeast
nitrogen base, 0.1% casamino acids, 20 mg/liter tryptophan, 40 mg/liter adenine).
Isolation of Mitochondria and Analysis of the Palmitate Stimulation of Respiration-- Mitochondria were isolated from protoplasts as described previously (25). The final mitochondrial pellet was resuspended in 0.65 M mannitol, 0.5 mM phosphate, 2 mM EGTA, 1 mM EDTA, 32 µM bovine serum albumin (fatty acid content 0.01%, i.e. molar ratio of fatty acid/albumin approximately 0.04), 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml pepstatin A, 10 mM Tes,1 pH 6.8, to a final concentration of 15-20 mg/ml protein. The sensitivity of mitochondrial respiration to fatty acids was evaluated from the effect of varying palmitate:albumin ratios on the NADH oxidation rate. For respiration assays, mitochondria (0.15 mg/ml protein) were incubated at 20 °C in a buffer containing 0.6 M mannitol, 10 mM phosphate, 0.5 mM EGTA, 1 mg/ml bovine serum albumin (fatty acid content 0.01%), 10 mM Tes, pH 6.8. Respiration was initiated by addition of 3 mM NADH. Other additions are described in the text and/or figure legends. Mitochondrial buffers and stock palmitate and nucleotide solutions were prepared fresh every day. Protein was determined by the Biuret method using albumin as standard.
Palmitate binding to yeast mitochondria was determined under conditions identical to those maintained during respiration experiments. Mitochondria (0.15 mg/ml) were added to respiration buffer that also contained 40 nM [9,10-3H]palmitate (1.5 µCi/ml) and 0.4 µCi/ml [14C]sucrose. 90 s after the addition of NADH, two aliquots were withdrawn and spun for 1 min at 14,000 rpm in an Eppendorf centrifuge. Subsequently, palmitate additions were made to achieve the desired molar ratio to albumin and left to incubate for another 90 s before two additional aliquots were withdrawn and centrifuged. This period was sufficient to measure an stable rate of respiration in a parallel experiment in the oxygen electrode. To investigate the removal by albumin of membrane-bound palmitate, 140 µM palmitate was added to an aliquot of stock mitochondria (adjusted to 10.5 mg/ml protein), mixed for 30 s and then added to respiration buffer to give a final concentration of 0.15 mg/ml protein. NADH was then added, and, after another 2 or 4 min, aliquots were withdrawn and spun. Supernatants were removed immediately after centrifugation and pellets dissolved in 25 µl of 5% SDS. Radioactivity was determined both in pellets and the original incubation and from these data bound palmitate determined. Two to three independent experiments were performed with each strain (UCP ![]() |
RESULTS |
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In order to establish how the presence of UCP1 modifies the
bioenergetic properties of mitochondria, it is necessary to
characterize appropriate controls without UCP1. This has been performed
previously with warm- and cold-acclimated animals because the brown fat
mitochondria of the two appear only to differ in their UCP1 content
(18, 27, 28). The recombinant expression of UCP1 in yeasts allows not
only the comparison of mitochondria incorporating UCP1 with appropriate
negative controls but also the use of mutants to further characterize
transport functions. We have recently described that mutations in the
C-terminal end of the protein modify the Km for the
activation of ion transport by fatty acids (24). We will use one of
these mutants, Cys-304 Gly, to re-examine the transport properties
of UCP1 both in the presence and absence of fatty acids.
UCP1 Transports H+(OH) in the Absence of
Fatty Acids--
We have reported previously (24) that, when
mitochondria are isolated from yeasts expressing wild type UCP1
(UCP+ strain), they demonstrate diminished coupling
relative to those from control yeast containing antisense UCP1 cDNA
(UCP
strain). Thus, the respiratory control ratio was
2.67 ± 0.07 (n = 12) for UCP
and
1.75 ± 0.03 (n = 28) for UCP+. GDP
addition lowers the state 4 rate by 33 ± 1.7% (n = 17) in UCP+ mitochondria, consistent with an inhibition
of the uncoupling protein, while having no effect in UCP
mitochondria. The -fold stimulation by FCCP in UCP
is
5.1 ± 0.6 (n = 12), a value close to that
observed in UCP+ if GDP is present (4.6 ± 0.2, n = 5). We can conclude that differences in
bioenergetic properties between the two yeast strains are consistent with the presence of active UCP1.
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UCP1-mediated Versus Carrier-mediated Uncoupling--
Recent
publications have established that the ADP/ATP carrier can also
contribute to the uncoupling of mitochondrial respiration induced by
fatty acids (6-9), and this proposal has been extended to other
metabolite carriers of the mitochondrial inner membrane (10, 11).
Because we now have the protein incorporated into recombinant yeast
mitochondria, we can test the UCP1-independent uncoupling and compare
it to that in its presence. It is to be expected that the other
transporters of the mitochondrial inner membrane are present in equal
amounts in both cases. The yeast are grown under aerobic conditions,
and thus mitochondrial oxidative phosphorylation is fully operative.
Fig. 2 shows the effect on respiration of the same range of
palmitate:albumin ratios, and it becomes apparent that
UCP mitochondria are far less sensitive to palmitate. It
should be noted that, in UCP
mitochondria, the
palmitate-stimulated respiration is not sensitive to atractylate over
the range 10-40 µM, concentrations that do prevent ADP
phosphorylation (data not shown). Recently, it has been demonstrated
that oleate uncouples S. cerevisiae mitochondria at
concentrations ranging from 10 to 50 µM and this
uncoupling could be partially inhibited by bongkrekate (9).
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DISCUSSION |
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The acute control of energy dissipation in brown fat mitochondria has been a subject of debate for more than two decades, preceding the discovery of UCP1 and yet to be resolved. It was recognized in the late 1960s that brown fat mitochondria prepared by conventional methods were uncoupled and that special conditions were needed to observe efficient oxidative phosphorylation, namely removal of endogenous fatty acids and addition of purine nucleotides (29, 30). The mode of action of purine nucleotides has been less controversial than that of fatty acids. Nucleotides bind to UCP1 from the cytosolic side of the mitochondrial inner membrane and inhibit its transport activity (31). The role of fatty acids in the regulation of energy dissipation and their mechanism of action have been the subjects of continuous controversy.
The first question that is addressed in the present paper relates to
the basal activity of UCP1. The earliest studies on the bioenergetic
properties of brown adipose tissue mitochondria established that brown
adipose tissue mitochondria were uncoupled in the absence of both
nucleotides and fatty acids (29, 30). However, recent reports have
questioned these findings by suggesting that simple removal of fatty
acids is sufficient to abolish H+ transport and
consequently that fatty acids are obligatory for UCP1 activity (23). A
separate report (32) questioned the interpretation of nonrespiring
swelling experiments performed in potassium acetate plus valinomycin,
which requires H+(OH) movement for charge and
pH balance, by claiming that the rate observed in the absence of fatty
acids was due to acetate anion transport through the UCP1, although
this explanation fails to account for the high proton conductance of
respiring brown adipose tissue mitochondria in the presence of albumin
calculated from the proton current and proton electrochemical potential
(33). Results presented in this paper demonstrate that there is a
GDP-sensitive conductance that cannot be attributed to endogenous fatty
acids, and that an increased affinity for palmitate in a UCP1 mutant does not result in an increased basal activity. The conclusion is that
fatty acids are not essential for UCP1 function, although they increase
its uncoupling activity. It should be stressed at this point that, if
experiments are performed with low and/or transient diffusion
potentials, rather than during respiration, UCP1 activity in the
absence of fatty acids may not be detected, because in the absence of
nucleotide the H+(OH
) transport rate depends
linearly on the driving force (33).
The ability of fatty acids to uncouple mitochondrial respiration has been known for decades (see Ref. 1; reviewed in Ref. 2) but was generally considered "nonspecific," relying on the translocation across the bilayer of the fatty acid in both protonated and in anionic forms and additionally, at high concentrations, on detergent-like effects. In 1988, Skulachev and co-workers (6) demonstrated that carboxyatractylate inhibited the uncoupling effect of palmitate in liver mitochondria and thus proposed that the protonophoric effect of fatty acids was mediated by the ADP/ATP carrier facilitating the translocation of the fatty acid anion. This finding has been supported subsequently by other laboratories, both with intact mitochondria (9, 34) and in reconstituted systems (8). More recently, it has been shown that other members of the family of mitochondrial transporters also appear to participate in this protonophoric action of fatty acids (10, 11). A general model is now emerging, wherein all these carriers, including UCP1, facilitate the transbilayer movement of the fatty acid anion. The protonophoric cycle would be completed with the flip-flop across the bilayer of the protonated acid (21, 22, 35). This H+ cycling mechanism would be analogous to the uncoupling by weak acid protonophores.
These data would appear to question the specificity of the fatty acid uncoupling of brown fat mitochondria. However, in all the reports involving the mitochondrial carriers, the unbound fatty acid concentrations required to observe uncoupling are in the micromolar range (6, 9-11). Thus, for example, in the ADP/ATP carrier of S. cerevisiae mitochondria, the system closest to the one presented in this paper, this form of fatty acid uncoupling requires concentrations in excess of 10 µM (9). The data presented in this paper show that the concentration of free palmitate required for half-maximal stimulation of respiration of UCP+ mitochondria is around 80 nM, a value that is in perfect agreement with that reported for brown fat mitochondria from cold-adapted guinea pigs (27). The extension of the cycling model to the UCP1 has been based mainly on direct measurements of the transport of the fatty acid anion and/or the subsequent H+ delivery across the membrane. Under those conditions, the Km for the anion is around 20 µM (21) and is thus similar to values obtained with other members of the carrier family.
A second model, termed "local H+-buffering," proposes that the fatty acid acts as a prosthetic group for H+ transport and thus the carboxyl group delivers H+ to a site inside UCP1 from which translocation occurs (23). This model would resemble the H+ translocating mechanism in bacteriorhodopsin (23, 36). We consider that the physiological relevance of the two models can be discriminated by the concentrations (Km) required to observe the effects and may represent two separate functions of the UCP1. Nanomolar concentrations of free fatty acids such as can be generated by noradrenaline-evoked lipolysis within brown adipocytes (27) would activate proton transport by UCP1 by the "local H+-buffering" mechanism. Only if unbound fatty acid concentrations were to rise to several micromolar would they themselves become transportable substrates for the UCP1, and presumably for the other mitochondrial carriers. It is important to recall the physiological context of the brown fat UCP1, which was shown several years ago to confer high sensitivity of intact brown adipocytes to fatty acid uncoupling and to be reversibly activated within adipocytes by endogenously generated fatty acids after noradrenaline stimulation of the cells (27). Until similar criteria can be demonstrated for the much less sensitive carrier-mediated uncoupling, the physiological role of the latter, which may provide a mechanistic explanation for what has been known as nonspecific fatty acid uncoupling, remains contentious.
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FOOTNOTES |
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* This work was supported by Spanish Ministry of Education and Culture Grant PB95-0118 and by grants from the French Direction des Recherches, Etudes et Techniques and the Association de Recherches sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Centro de
Investigaciones Biológicas, Consejo Superior de Investigaciones
Científicas, Velázquez 144, 28006 Madrid, Spain. Tel.:
341-5611800 (ext. 4236); Fax: 341-5627518; E-mail:
rial{at}fresno.csic.es.
1 The abbreviation used is: Tes, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.
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REFERENCES |
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