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(The FASEB Journal. 2007;21:1137-1144.)
© 2007 FASEB

Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers

Valeri Beck*, Martin Jaburek{dagger}, Tatiana Demina*,1, Anne Rupprecht*, Richard K. Porter{ddagger}, Petr Jezek{dagger} and Elena E. Pohl*,2

* Institute of Cell Biology and Neurobiology, Centre for Anatomy, Charité Universitätsmedizin, Berlin, Germany;

{dagger} Department of Membrane Transport Biophysics, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; and

{ddagger} School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland

2Correspondence: Institute of Cell Biology and Neurobiology, Charité Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany. E-mail: elena.pohl{at}charite.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uncoupling proteins 1 (UCP1) and 2 (UCP2) belong to the family of mitochondrial anion transporters and share 59% sequence identity with each other. Whereas UCP1 was shown to be responsible for the rapid production of heat in brown adipose tissue, the primary function and transport properties of ubiquitously expressed UCP2 are controversially discussed. Here, for the first time, the activation pattern of the recombinant human UCP2 in comparison to the recombinant human UCP1 are studied using a well-defined system of planar lipid bilayers. It is shown that despite apparently different physiological functions, hUCP2 exhibited its protonophoric function similar to hUCP1—exclusively in the presence of long-chain fatty acids (FA). The calculated hUCP2 transport rate of 4.5 s–1 is the same order of magnitude, as shown previously for UCP1. It leads to the conclusion that the differences in the activity of both proteins in living mitochondria are based exclusively on their different expression level. Both proteins are activated much more effectively by polyunsaturated than by saturated FA. The proton and total membrane conductances increased in the range palmitic < oleic < eicosatrienoic < linoleic < retinoic < arachidonic acids. The higher uncoupling protein (UCP)—dependent conductance in the presence of polyunsaturated FA is explained on the basis of the FA cycling hypothesis.—Beck, V., Jaburek, M., Demina, T., Rupprecht, A., Porter, R. K., Jezek, P., Pohl, E. E. Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers.


Key Words: mitochondria • anion transporter • proton transport • artificial bilayer membranes • reactive oxygen species


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UNCOUPLING PROTEINS 1 (UCP1) and 2 (UCP2) belong to the subfamily of mitochondrial uncoupling proteins and share 59% sequence identity with each other. The most studied UCP1, specifically localized in brown adipose tissue (BAT) (1) and thymus (2) , mediates the fatty acid (FA)-dependent H+ influx resulting in the uncoupling of electron transport from ATP synthesis that leads to the rapid production of heat. According to alternative hypotheses (3 4 5 6 7) , UCP1 is either a proton channel or a FA anion carrier. Unlike UCP1, UCP2 transcript is ubiquitously distributed in all mammalian tissues (1 , 8) . The extremely low-protein concentrations in mitochondria make the involvement of UCP2 in thermogenesis marginal. The variety of proposed physiological roles for UCP2 is surprising. Several research groups (9 10 11 12) have reported that UCP2 down-regulates the production of reactive oxygen species (ROS) by mild uncoupling of the respiratory chain (1 , 13 14 15 16 17) , as suggested by Skulachev (18) . Activated UCP2 may thus play an important antioxidant role in the pathogenesis of multiple sclerosis (19) and atherosclerosis (20) , improving recovery after stroke (21) or prevention of liver disease (22) . UCP2 has been implicated in a wide range of pathological states, including obesity, diabetes, aging, and degenerative, neurological, circulatory, endocrine and immunological diseases (for reviews, see (1 , 14 , 16 , 23 , 24) ).

Comparison of UCP2’s transport mechanism and its regulation by ligands with the properties of UCP1 might provide a key to understanding its function. In our previous studies, we have shown that both UCP1 isolated from hamster BAT and human recombinant UCP1 are activated exclusively in the presence of long chain FA (5 , 25) . Although the presence of FA has long been accepted to be an absolute requirement for UCP1-mediated H+ transport (1 , 6 , 26) , it has been recently questioned (27 28 29) . The molecular transport mechanism of UCP2 is in dispute as well, despite numerous experiments involving liposomes reconstituted with UCPs, isolated mitochondria, cell cultures and knockout mice (30) . Because of the sequence similarity between UCP1 and UCP2, the proton conductance enhancement by FA and inhibition by purine nucleotides shown for UCP1 (5) is supposed to be shared by UCP2 (31 , 32) . On the other hand, UCP2 lacks the histidine pair (H145, H147), which has been considered to be decisive for proton translocation in UCP1 (33 , 34) . It has been reported that when UCP2 is expressed in S. cerevisiae under the same conditions as UCP1, mitochondria only show a modest increase in the basal rate of respiration and lack of nucleotide and FA sensitivity (35) . Several investigators have doubts about whether UCP2 and UCP3 are H+ transporters at all, arguing that the response of expressing UCP2 and UCP3 in mammalian cells to many stimuli often seemed not to be compatible with an uncoupling function of UCP2 and UCP3 (36 37 38 39) .

The question of FA specificity in the UCP activation is unclear. With numerous FA tested mostly on their own in different systems (32 , 35 , 40 41 42 43) , no regularity has been revealed (Table 1 ). First attempts to study a range of FA systematically were undertaken on liposomes (31 , 32) . Among those tested, the FA oleic acid exhibited the highest transport rate, but {omega}-6 polyunsaturated fatty acids (PUFAs), such as cis-8,11,14-eicosatrienoic (C20:3, {omega}-6) and cis-6,9,12 octadecatrienoic (C18:3, {omega}-6) acids exhibited both high Vmax and the highest apparent affinity (1/Km); the efficiency of lauric, palmitic, and linoleic acid was reported to be much lower and roughly the same if compared to each other.


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  		Table 1. Activation of UCP1 and UCP2 by different FA

The aim of the study is to test the hypothesis that UCP2 transports protons by the same mechanism as UCP1, despite the apparently different functions assigned to them. Our data answer the question of whether the apparent lower uncoupling capacity of UCP 2 in mitochondria/cells/organisms is due to 1) a lower inherent activity and/or differential activation/inhibition of the UCP 2 protein compared with UCP 1 or/and 2) a lower constitutive level of expression of UCP 2 in tissues. We investigate whether there is a differential level of fatty acid-dependent proton transfer activity, by measuring the single-molecule, protein-mediated proton conductance, in UCP 1 compared to UCP 2, depending on the degree of fatty acid saturation. On the basis of these experiments, the mechanism of UCP-mediated proton transfer is evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
hUCP1 and hUCP2 expression, extraction, purification, and reconstitution into liposomes
Human UCP2-containing plasmids were transformed into the bacterial strain BL21 (Novagen) as described previously (31 , 44 45 46) . Human UCP 1 was expressed in E. coli strain Rosetta pLysS (Novagen) transformed with vectors containing cDNA coding for human UCP1, as described previously (47) . Solubilization of inclusion bodies and purification of hUCP2 and hUCP1 followed a previously described protocol (44) . Here, we used 10 mg of the total phospholipid extract from E. coli (Sigma) per mg of isolated protein. The obtained samples of purified recombinant hUCP2 and hUCP1 were reconstituted into liposomes using previously described procedures based on detergent removal by Bio-Beads SM-2 (Bio-Rad) in 30 mM K-TES, 80 mM K2SO4, 2 mM EDTA, pH 7.2. The obtained proteoliposomes were stored at –80°C and thawed the day of experiment. Liposomes, containing FA or UCP were extruded through the filter of 400-nm pore diameter using a small-volume extrusion apparatus (Avestin, Ottawa, Canada), as described earlier (48) . Proteoliposomes were mixed with FA-containing liposomes in required proportions and added to the buffer solution, where a monolayer containing both FA and protein was built spontaneously.

Formation of bilayer membranes from protein-containing monolayer
Bilayer membranes containing UCP were formed on the tip of conventional dispensable plastic pipettes (Eppendorf epT.I.P.S. Geloader, Fisher Scientific, Hamburg, Germany) with a diameter of 240 ± 45 µm, as described previously (25) . At the beginning of the experiment, the pipette was filled with the buffer solution and attached to the pipette holder manually. A plastic dispensable container with the volume of 0.75 ml and the diameter of 12 mm was filled with the proteoliposome suspension, which was continuously stirred and maintained at 37°C. On top of the suspension, a protein/lipid monolayer was formed spontaneously. The automatic raising above the monolayer and lowering of the pipette into suspension was stopped after the bilayer was formed, as indicated by capacitance measurements. The solutions of UCP-containing liposomes (proteoliposomes) or FA-containing liposomes were used in a final concentration of 1–1.5 mg lipid/ml buffer. Polar or total lipid extracts from E. coli used for the formation of planar bilayer membrane were obtained from Avanti Polar Lipids (Alabaster, AL); hexane, hexadecane, K2SO4, TES, and EGTA were obtained from Sigma Aldrich (Steinham, Germany). Long-chain fatty acids (palmitic, oleic, eicosatrienoic, linoleic, retinoic, arachidonic) were purchased as chloroform solutions from Sigma Aldrich and added to the lipid phase prior to final extrusion through the LiposoFast. The required concentration of FA in the lipid membrane was achieved by the mixing of UCP- und FA-containing liposomes at definite ratios.

Measurements of the total and proton membrane conductance
Current-voltage (I-V) characteristics were measured by a patch-clamp amplifier (EPC 10; from Dr. Schulze, HEKA Elektronik, Hannover, Germany). For conductance measurements, a ramp voltage signal was applied. Membrane conductance G was determined at zero voltage from a fit of voltages in the linear interval between –50 and 50 mV. For noise reduction the Power-Line Conditioner (Warner Instruments, Hamden, CT) was used. Data acquisition and processing were performed by Software Pulse (v. 8.65; Dr. Schulze, HEKA Elektronik).

In the same setup the proton conductance, GH/OH, was measured, as described in details elsewhere (5) . Well-buffered solutions on out (cis) and in (trans) sides of the membrane had similar osmolarities, ionic strength, and concentrations of all ions except H+ and OH–. pHin and pHout were adjusted to 7.0 and 7.4, respectively, using HEPES or MES (49 , 50) . The experimental proton Nernst potential (49) at a pH gradient is equal to the shift of the reversal potential, V0, because there are no cis/trans gradients of buffer cations and anions (49 , 50) . V0 was obtained from current-voltage characteristics, which were measured in the presence of a transmembrane gradient of 0.4 pH units. The final proton conductance, GH/OH, in S cm–2 was calculated as:

Formula 1(1)
where {Psi}N is the theoretical value of Nernst equilibrium potential (23.8 mV for a pH gradient of 0.4). The protein turnover number, k, in s–1, can be estimated as

Formula 2(2)
where j is a flux through the protein channel and can be defined as

Formula 3(3)
where z and F are the ion charge and the Faraday constant, respectively. Current through one channel, i, is calculated as:

Formula 4(4)
where P is the amount of the protein channels, reconstituted in the membrane with surface A and I, the total current through the membrane. Taking into account that I = GU, lipid-protein molecular ratio, r = P/L, the current is

Formula 5(5)
where b is the surface of the lipid molecule; rm, lipid/protein mass ratio; ML and MP, lipid and protein molecular weights, respectively. On the basis of the equations 3 , 4 , and 5 we have the final equation for the calculation of protein turnover number, k, as:

Formula 6(6)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of UCP2 by FA and purine nucleotides
The total conductance of membranes, reconstituted either with UCP2 (G0=(18±6) nS/cm2) or with different FA at a concentration of 15 mol%, was comparable to the conductance of the pure lipid membrane made from Escherichia coli polar lipid extract ((15±5) nS/cm2). Conductance at 0 mV was determined from current-voltage (I-V) characteristics, which were linear in the range from –50 to + 50 mV.

Figure 1 shows that a significant increase in the current of the UCP2-containing membrane occurs only after the addition of FA (arachidonic acid, AA, Fig. 1 ). In contrast to the experiments carried out on mitochondria and reconstituted liposomes, all FA in the maximal molar concentration 15 mol% were added to the lipid phase containing UCP prior to membrane formation. This allowed minimization of artifacts related to different FA solubility in buffer solution. The increase in FA-mediated UCP2 conductance was inhibited in the presence of ATP (Fig. 1) , ADP, and GTP (data not shown). One millimolar of purine nucleotide (PN) inhibited UCP2-mediated proton conductance to various extents, depending on FA/PN and the FA/lipid ratio. This activation/inhibition pattern leads to the definitive conclusion that UCP2 exhibits its protonophoric function analogously to UCP1, despite their possibly different physiological functions.


Figure 1
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  		Figure 1. Nucleotide-sensitive increase of UCP2-mediated membrane conductance in the presence of arachidonic acid, AA. Current-voltage relationships of UCP2-containing membranes were measured in the absence (open circles) or in the presence (solid circles) of AA, and in the presence of AA and ATP (shaded circles). The concentrations of Escherichia coli polar lipid, UCP2 and ATP were 1 mg/ml, 12.5 µg/mg of lipid and 1 mM, respectively; the concentration of AA was 15 mol%. The buffer solution contained 20 mM KCl, 25 mM TES, 0.6 mM EGTA, pH 7.0, T = 37°C.

The measured increase in proton conductance significantly depended on the UCP content in the membrane (Fig. 2 ) and/or the concentration of added FA (Fig. 2 , Fig. 3 ). Although the dependence on protein content seems to be trivial, there are some examples where physiological changes in UCP2 or UCP3 mRNA or protein abundance were found to correlate with mitochondrial proton conductance (liver mitochondria from ob/ob mice (51) ), and others, where they do not correlate (muscle mitochondria (52 , 53) ). The lack of consistent correlation between UCP2 or UCP3 content and proton conductance can be explained by the interfering of two processes in living systems: the alteration of protein amount due to its overexpression and the alteration of protein activity due to the involvement or suppression of additional regulating mechanisms. In our experiments, protein activity in the presence of oleic acid remains the same, although the protein amount is changed, indicating that in vivo the simple overexpression of protein should lead to the uncoupling enhancement. Because the different batches of prepared protein contained different amounts of protein, we compared the relative values, G/G0, where G and G0 are, respectively, the membrane conductances in the presence of both protein and FA, and of protein alone (Fig. 2) .


Figure 2
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  		Figure 2. The relative total conductance of planar lipid membrane containing 28 µg/ml UCP2 (squares), 19 µg/ml uncoupling proteins 1 (UCP2) (circles) and no protein (triangles) in the presence of different concentrations of oleic acid. The relative conductance is the ratio of G/G0, where G0 is the conductance of membrane reconstituted with protein in the absence of FA and G is the conductance of the 1) pure lipid bilayer or 2) the bilayer reconstituted with protein in the presence of FA. Lipid content was 1.5 mg/ml. The buffer contained 50 mM K2SO4, 25 mM TES, 0.6 mM EGTA, pH 7.35, T = 37°C.


Figure 3
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  		Figure 3. Total membrane conductance of the UCP2-reconstituted membranes in the presence of fatty acids with various saturation degrees added in a concentration of 10 mol% (gray bars) and 15 mol% (solid bars). Open bars indicate the conductances of the corresponding fatty acid (FA) in the absence of protein. Conductances were calculated at 0 mV from current-voltage characteristics measured in the range from –50 to + 50 mV. Lipid (polar lipid extract from E. coli) content was 1.5 mg/ml, concentration of UCP2, 7 µg/mg of lipid. The buffer contained 50 mM K2SO4, 25 mM TES, 0.6 mM EGTA, pH 7.35, T = 37°C.

Evaluation of FA specificity
To evaluate the dependence of the proton influx on FA saturation degree, we compared the activities of UCP2 in the presence of saturated palmitic acid (PA), monounsaturated oleic acid (OA), and polyunsaturated acids such as {omega}-6-eicosatrienoic (EA), linoleic acid (LA), and arachidonic acid (AA), the latter containing four double bonds. Fatty acids were added directly to the lipid phase containing UCP2 before bilayer membranes were formed, assuring thereby the protein-lipid interaction. Figure 3 demonstrates that increased membrane conductance was measured in the presence of all FA and depended on the FA/lipid ratio (10 mol% FA—gray bars, 15 mol% FA—black bars). White bars demonstrate similar low conductivities for all FA (15 mol%) measured at zero voltage in the absence of UCP, which supports earlier observations (5 , 54) but contradicts results obtained in decane-containing membranes (55) . UCP2-mediated membrane conductance, G, differed in the presence of various FA: it was very small in the presence of palmitic acid in comparison with FA-free membranes, increased significantly in the presence of FAs having one, two, and three double bonds (oleic, linoleic, and eicosatrienoic acids, respectively), and reached maximum values in the presence of polyunsaturated arachidonic FA (Fig. 3) . The addition of all-trans retinoic acid (RA), which has been claimed to be the most potent activator of UCP1 (43) , demonstrates that the increase in the amplitude of the RA-mediated membrane conductance fits well with the observed conductance dependence on FA saturation (Fig. 3) .

Measurements of proton conductance and estimation of substrate turnover number for hUCP2
Proton conductance for hUCP2, GH/OH, was estimated as described in the section Materials and Methods. The example of current–voltage curves in the absence and in the presence of pH gradient is shown in Fig. 4 . The calculated values for the activation by arachidonic acid were 142.5 nS/cm2 and 105 nS/cm2 for 15 and 10 mol% of AA in lipid phase, respectively, which exceeded the total conductances in the presence of all other studied acids.


Figure 4
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  		Figure 4. Current-voltage characteristics of membranes containing UCP2 and arachidonic acid (15 mol%) in the presence (solid circles) and in the absence (gray circles) of transmembrane proton gradient. The voltage from –50 to + 50 mV was applied. From the interception of the I-V curve with y axis, the reversal potential V0 was estimated.

The substrate turnover numbers per protein molecule, k, were calculated according to the equation 6 . For maximal concentration of arachidonic acid in lipid 15 mol% a turnover number of 4.8 s–1 was calculated, which is similar to the k of UCP1 estimated earlier at similar conditions (5) .

Evaluation of protein isoform specificity
To reveal a possible protein specificity, we have measured G in the presence of hUCP1 (Fig. 5 , inset) and compared its relative conductance increase in the presence of different fatty acids with the relative conductance for hUCP2 (Fig. 3) . The results show that the activities of both hUCP1 and hUCP2 are significantly more pronounced in the presence of arachidonic acid, a polyunsaturated fatty acid.


Figure 5
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  		Figure 5. Comparison of relative total membrane conductances, GFA/GLA, mediated by UCP1 (solid squares) and UCP2 (open squares) in the presence of FA with various saturation degrees. GFA and GLA are conductances in the presence of the studied fatty acid and linoleic acid, respectively. Inset: Conductance of membranes reconstituted with hUCP1 in the presence of fatty acids with various saturation degrees. Current-voltage characteristics of membranes containing UCP1 (0.7 µg/mg lipid) and FA (15%) were measured in the range from –50 to + 50


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using planar lipid membranes reconstituted with purified recombinant human uncoupling proteins, which allowed us to measure and to compare the single-molecule protein-mediated proton conductances of hUCP1 and hUCP2, we have shown for the first time that they are similar for both proteins. These results solve the long-standing controversy concerning the UCP2-mediated proton transport. The lower UCP2 "activity" seen in the experiments with isolated mitochondria can be attributed exclusively to the low-protein expression level in the corresponding tissues.

The obtained UCP1- and UCP2-dependent conductances in the presence of polyunsaturated arachidonic acid are severalfold higher than compared to saturated palmitic and monounsaturated oleic acid, which are often used for the experiments on mitochondria. Retinoic acid, which was claimed to be the most potent UCP "activator," is indeed very effective, fitting, however, in the presented G dependence on FA saturation degree. The data lead to the definite conclusion that polyunsaturated acids are the most potent UCP activators (32) .

The higher UCP-dependent conductance in the presence of polyunsaturated FA can be explained on the basis of the FA cycling hypothesis. According to this hypothesis, 1) protonated fatty acids flip-flop along their interleaflet concentration gradient, and 2) the subsequent backward transport of the deprotonated (anionic, FA–) fatty acid is ensured by UCP (3 , 4) . We hypothesize that FA– are transported on the protein-lipid interface. Whereby by lateral diffusion, fatty acid reaches a weak binding site on UCP into which most probably only anionic form can bind. Both steps of the transport are accelerated in the presence of unsaturated FA (Fig. 6 ). Introduction of double bonds leads to decreased order within a membrane and faster molecular reorientation (56 , 57) . High disorder and fluidity promotes flip-flop of unsaturated FAs (58) and lipids with unsaturated fatty acid chains (59) . By decreasing the Born energy, protein efficiently catalyzes this transversal movement of fatty acid anion (60) as shown in Fig. 6 .


Figure 6
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  		Figure 6. Mechanism of faster fatty acid cycling enabled by UCPs in the presence of polyunsaturated FA, in contrast to saturated FA. Membrane contains polyunsaturated arachidonic (A) and stearic (B) acid (red). Both FA can flip-flop through lipid bilayer spontaneously after binding of proton (blue). Subsequently, by lateral diffusion fatty acids reach a weak binding site on UCP into which most probably only anionic forms can bind. The transport of anionic form is facilitated by UCP after initial binding to this FA-binding site.

Interestingly, the basal proton leak in mitochondria from different tissues correlates significantly with the fatty acid composition of inner membrane phospholipids ((13) and references therein). The content of {omega}-3 polyunsaturates, particularly docosahexaenoate [22:6 ({omega}-3)], correlates with high proton conductance, and the content of monounsaturates, particularly oleate [18:1({omega}-9)], correlates with low proton conductance. However, the H+ leak of liposomes made from mitochondrial phospholipids of multiple different organisms is essentially identical, indicating that phospholipid fatty acid composition may control the proton conductance of mitochondria only through phospholipid-protein interactions with nonspecific or specific membrane proteins such as UCPs (13 , 34) .

The preferential activation of UCP by polyunsaturated FA found here may be of great physiological relevance in vivo. UCP-mediated proton flux may decrease the potential across the inner mitochondrial membrane that, in turn, may lead to the attenuation of mitochondrial ROS production (1 , 9 , 12 , 61) . This FA-dependent ROS regulation renders the protein an important player in oxidative stress-related pathologies, including atherosclerosis, ischemia-reperfusion damage, inflammation, type-2 diabetes, Parkinson’s disease, Alzheimer’s disease, and other neurodegenerative diseases (15 , 21 , 62 63 64 65) .


   ACKNOWLEDGMENTS
 
The financial support of the Deutsche Forschungsgemeinschaft (Po-524/2–2, 436 TSE 113/44/0–1) and of the Charité-Universitätsklinikum Berlin to EEP; of the Grant Agency of the Academy of Sciences of the Czech Republic (No. A5011106, AV0Z50110509) to PJ and the Grant Agency of the Czech Republic (No. 301/05/0221 to PJ and No. 204/04/0495 to MJ) is gratefully acknowledged. Tatiana Demina was awarded of the Humbold University International Research Grant (2005). We thank Kimberly Mason for editorial assistance.


   FOOTNOTES
 
1 Present address: Chemistry Department, Moscow State University, Moscow, Russia

Received for publication October 10, 2006. Accepted for publication November 9, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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