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Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction
The International Journal of Biochemistry & Cell Biology 37 (2005) 809–821
Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction ˇ ˇ Michal R˚uzˇ iˇckaa , Eva Skobisov´ aa , Andrea Dlaskov´aa , Jitka Santorov´ aa , Katar´ına ˇ ceka , Mark´eta Z´ ˇ acˇ kov´aa , Martin Modriansk´yb , Petr Jeˇzeka,∗ Smolkov´aa , Tom´asˇ Spaˇ a
Department of Membrane Transport Biophysics, No. 75 Institute of Physiology, Academy of Sciences of the Czech Republic, V´ıdeˇnsk´a 1083, 14220 Prague 4, Czech Republic b Department of Medical Chemistry, Faculty of Medicine, Palack´ y University, Olomouc, Czech Republic Received 27 July 2004; received in revised form 18 October 2004; accepted 27 October 2004
Abstract Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3 H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3 H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2 O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response. © 2004 Elsevier Ltd. All rights reserved. Keywords: Lipopolysaccharide; Oxidative stress in liver; Mitochondrial uncoupling protein UCP2; Rat liver mitochondria; Reactive oxygen species
∗
Corresponding author. Tel.: +420 296442760; fax: +420 296442488. ˇ E-mail addresses: [email protected] (M. R˚uzˇ iˇcka), [email protected] (E. Skobisov´ a), [email protected] (A. Dlaskov´a), ˇ ˇ cek), [email protected], [email protected] (J. Santorov´ a), [email protected] (K. Smolkov´a), [email protected] (T. Spaˇ ˇ acˇ kov´a), [email protected] (P. Jeˇzek). [email protected] (M. Z´ URL: www.mitonet.cz (P. Jeˇzek). 1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.10.016
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1. Introduction In the liver, a negligible amount of mitochondrial uncoupling protein UCP2 mRNA detectable by Northern blots (Fleury et al., 1997; Gimeno et al., 1997) was originally ascribed to the resident macrophages (Carretero et al., 1998; Hodn´y et al., 1998; Larrouy et al., 1997). Several studies have shown, however, that UCP2 mRNA is transcribed in hepatocytes under certain stimuli (Cortez-Pinto, Lin, Yang, Da Costa, & Diehl, 1999; Faggioni, Shigenaga, Moser, Feingold, & Grunfeld, 1998; Takahashi et al., 2002) or after prolonged incubation in a cell culture medium (Kimura et al., 1999). TNF␣ or other “pyrogenic” cytokines have been found to upregulate UCP2 transcription in hepatocytes (Cortez-Pinto et al., 1998; Faggioni et al., 1998) and to downregulate UCP2 in macrophages (Diehl & Hoek, 1999). It has been suggested that the up-regulation in liver may serve to compensate the increased ROS production from macrophages (Diehl & Hoek, 1999). The physiological role of UCPs is determined by the extent of uncoupling they induce when actiˇ ˇ acˇ kov´a, R˚uzˇ iˇcka,Skobisov´ vated (Jeˇzek, Z´ a, & Jab˚urek, 2004). The UCPs are sluggish transporters, with a very low molecular activity (Echtay, Winkler, Frischmuth, & Klingenberg, 2001; Jab˚urek & Garlid, 2003; Urb´ankov´a, Voltchenko, Pohl, Jeˇzek, & Pohl, 2003; ˇ ˇ acˇ kov´a, Skobisov´ Z´ a, Urb´ankov´a, & Jeˇzek, 2003); accordingly, the extent of uncoupling in a given cell type is determined largely by their level of expression. Thermogenesis in brown fat cells requires a large extent of uncoupling, and this is achieved by a correspondingly high level of UCP1 expression (Jeˇzek, 1999). The physiological role of UCPs in non-thermogenic tissues is suggested to be reduction of mitochondrial production of reactive oxygen species (ROS) (N`egreSalvayre et al., 1997), which can be achieved by low levels of uncoupling (Korshunov, Skulachev, & Starkov, 1997; Korshunov, Korkina, Ruuge, Skulachev, & Starkov, 1998; Kowaltowski, Costa, & Vercesi, 1998; Liu, Fiskum, & Schubert, 2002; Skulachev, 1998) corresponding to the low levels of UCP expression in such cells (Pecqueur et al., 2001). UCP2 mRNA was found in all kinds of human, rat, and mouse tissues studied (Fleury et al., 1997; Gimeno et al., 1997; Jeˇzek, 2002; Pecqueur et al., 2001); detectable amounts of UCP2 protein were found for example in mouse lung, spleen, or stomach under nor-
mal physiological conditions (Pecqueur et al., 2001; Couplan et al., 2002). Divergence between mRNA and protein quantity has been attributed to translational down-regulation (Pecqueur et al., 2001). A 3 H-GTP ˇ acˇ kov´a et al. (2003) indicated rather binding study by Z´ high amounts of UCP2 protein in rat lung and modest amounts in rat kidney. The upper estimates of UCP2 protein content in rat liver mitochondria were ∼10 times less than in lung. Functional activation of UCP2 leads to a decrease in ROS production (Arsenijevic et al., 2000; Duval et al., 2002; Kizaki et al., 2002; N`egre-Salvayre et al., 1997), since even slight uncoupling causes a substantial decrease of resting-state mitochondrial ROS production (Korshunov et al., 1997, 1998; Skulachev, 1998). Duval et al. (2002) have shown that UCP2-mediated uncoupling in endothelial cells is able to decrease even extracellular ROS in co-incubated low-density lipoproteins (LDL). Mice with deleted LDL receptor exhibited extensive dietinduced atherosclerotic plaques when they received bone marrow transplanted from UCP2 (−/−) mice, and the appearance of these plaques was prevented when they received bone marrow transplants from UCP2 (+/+) mice (Blanc et al., 2003). Over-expressed UCP2 had also a preventive role in brain after stroke in mice (Mattiasson et al., 2003). These findings suggest a hypothesis in which ROS-homeostasis of the whole organism is regulated by a “ROS-sink” in tissues where UCP2 (or UCP3)-mediated suppression of ROS production is involved (Jeˇzek & Hlavat´a, unpublished). In order to document the recruitment of UCP2 in hepatocytes in detail and to show that it is accompanied by the uncoupling resulting in a decrease of mitochondrial ROS production, we attempted to access whether a recruited UCP2 can suppress ROS production in mitochondria of stimulated liver, eventually to judge an extent of such expected weak uncoupling. Hence, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection.
2. Materials and methods 2.1. Real-time RT-PCR Sets of two primers and two fluorescent hybridization probes were designed for rat UCP2 and rat -actin
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Table 1 Set of primers and fluorescent hybridization probes used for RT-PCR quantification of UCP2 mRNA Primers (forward – f and reverse – r) and probes were designed for the approximately first third of UCP2 sequence (349 base pairs, 136–485 bp), against segments which substantially differ among all UCP isoforms (34) Protein Rat UCP2 Primer f Primer r Probe 1 Probe 2 Rat -actin Primer f Primer r Probe 1 Probe 2
Sequence (5 → 3 )
Melting point (◦ C)
gAgAgTCAAgggCTAgCgC gCTTCgACAgTgCTCTggTA TCAgAgCATgCAggCATCgg – X R640 – CCgCCTCCTggCAggTAgC – p
57 56 63 65
ACCCACACTgTgCCCATCTA gCCACAggATTCCATACCCA gCCACgCTCggTCAggATCTTCAT – X R640 – AggTAgTCTgTCAggTCCCggCCA – p
58 59 67 66
X denotes the fluorescein fluorophore (donor); R640 is a Roche fluorophore (acceptor). The listed melting points were calculated by the synthesizing company (TIB MOLBIOL, Berlin, Germany).
as a house-keeping gene (Table 1). Their synthesis was provided by TIB MOLBIOL (Berlin). mRNA was isolated from DNAse-I-treated total RNA (Chomczynski & Sacchi, 1987) or directly from powdered frozen rat liver or hepatocytes, using mRNA isolation kit (Roche Biochemicals, Mannheim, Germany). Total hepatocyte RNA was isolated using Trizol reagent (GIBCO BRL). Primers designed for PCR of the approximately first third of rat UCP2 sequence (349 base pairs, 136–485 bp) and adjacent hybridization probes (a gap of 1 bp) were chosen to hybridize the most variant part of the sequence, where various UCP isoforms (UCP1 to 5) differ from each other (Jeˇzek & Urb´ankov´a, 2000). Total RNA and mRNA were quantified from spectra with subtracted light scattering contribution using a Spectronics 3000 diode-array spectrophotometer (Milton–Roy) in 200 l quartz cuvettes. mRNA (60–100 ng) was used for real-time RT-PCR on a LightCycler (Roche) with 0.5 M primers and 0.2 M hybridization probes. Reverse transcription was performed (30 min), followed by transcriptase inactivation for 30 s at 95 ◦ C, and by 40 cycles of annealing for 25 s at 57 ◦ C, incubation for 25 s at 72 ◦ C, and for 1 s at 95 ◦ C. MgCl2 content was optimized to 4 mM. Calibration (Fig. 1a) was provided by dilution series of PCR-amplified amplicon (rat UCP2 136–485 bp from LightCycler RT-PCR) starting from the initial concentration of 2 g/ml. When the calibration was measured with UCP2 amplicon DNA inserted into AdvanTAge vector (Clontech), a higher slope (−4.36) was obtained (not shown), indicating slower reaction.
2.2. Animal treatment Wistar or Long Evans rats were used for all experiments. 1 mg LPS in sterile physiological saline solution was intraperitoneally (i.p.) injected to rats, which were, while starving, decapitated after 18 or 24 h (or at 6 h intervals when estimating a time course). Livers were removed and put into liquid nitrogen. Liver mitochondria were isolated by a standard procedure with albumin, usually from rats sacrificed 18 h post injection, while their protein content was evaluated using a Bio-Rad kit or the Lowry standard assay. For hepatocyte isolation, male Wistar rats (6–8week-old) were injected (i.p.) with 0.5 mg LPS/kg in sterile phosphate buffered saline (PBS) or sterile PBS alone. 2.3. Immunodetection of UCP2 Although UCPs belong to a large family of mitochondrial anion carriers with a common architecture and homologous common hydrophilic sequences extending to the matrix and cytosol (Jeˇzek & Jeˇzek 2003), and hence there is a high chance of cross-reactivity of anti-UCP2 antibodies with respect to other family members, we also attempted immunodetection by Western blotting. Goat anti-UCP2 antibodies (raised against the UCP2 carboxy terminus) were purchased from Santa Cruz Biotechnology (those of Calbiochem were less specific), while the secondary rabbit anti-goat IgG horse-radish-peroxidase-conjugated
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30 min in 200 ml of 100 mM sucrose, 20 mM HEPESTris, 1 mM EDTA, 2 M rotenone, 5 M CAT, pH 7.0 ˇ acˇ kov´a et al., 2003). Mitochondria were then filtered (Z´ through nitrocellulose filters (0.45 mm pores, Millipore) which were subsequently washed twice by sucrose medium and placed onto scintillation solution. The counted radioactivity of the samples containing excess of non-radioactive GTP was always subtracted. Scatchard plots were constructed for each measurement, combined into one graph and the data were fitted by linear regressions. Errors of x-axis intercepts were taken from linear regression fits. 2.5. Mitochondrial H2 O2 generation
Fig. 1. Real-time RT-PCR quantification of UCP2 mRNA in rat liver and hepatocytes: (a) calibration curve – a relation of the initial amount of rat UCP2 amplicon cDNA (first third of rat UCP2 sequence, 349 base pairs, 136–485 bp) to the time of crossing point TCP . Linear regression yielded: TCP = −3.399 log c − 19.19, which is in agreement with the predicted theoretical slope of 3.3, meaning that 3.3 cycles of difference are obtained when the two samples differ in mRNA by one order of magnitude. This is equivalent to doubling the amplicon amount in each cycle. (b) Increase of UCP2 mRNA after LPS injection in comparison with untreated rats—the obtained times of crossing points TCP were converted to the relative amounts according to the respective calibrations. (a) One milligram was intraperitoneally injected to Wistar rats. Animals were decapitated after 18 h (24 h for hepatocyte isolation, see experimental procedures). mRNA was isolated and quantified on a LightCycler.
antibodies (Santa Cruz Biotechnology) were used together with ECL Plus kit for chemiluminiscence detection (Amersham Biosciences). 2.4. 3 H-GTP binding assay for liver mitochondria BSA-washed liver mitochondria (0.2 mg protein) isolated from control or LPS-treated rats (18 h) were mixed with 3 H-GTP aliquots (of 8–320 pmol and with 0.6 mM cold GTP in parallel series) and incubated for
Mitochondrial H2 O2 generation was measured by fluorescent monitoring of oxidation of scopoletin by horseradish peroxidase (Loschen, Flohe, & Chance, 1971) in rat and mice liver mitochondria respiring with 10 mM succinate in the medium containing 125 mM sucrose, 65 mM KCl, 10 mM Tris–HEPES, 1 mM Tris–EGTA, pH 7.2, 1 M cyclosporine A, 10 M rotenone. For H2 O2 detection, 1 M scopoletin and 0.5 M horseradish peroxidase were added and maximum rates were taken from the 10 to 15 min run. Fluorescence was monitored on a RF5301 PC fluorometer (Shimadzu, Japan), with polarization filters (Polaroid) in cross-orientation in order to decrease light scattering. Aliquots of H2 O2 were added for calibration. 2.6. Isolation of rat hepatocytes Hepatocytes were isolated by two-step collagenase perfusion (Moldeus, Hogberg, & Orrenius, 1978). Samples were centrifuged (60 × g, 2 min) to separate non-parenchymal cells from hepatocytes, which were then washed in Williams’ medium E (WME) supplemented with 2 mM glutamine, 10 M streptomycin, 100 U/ml penicillin, 350 nM insulin, and 1 M dexamethasone. Cells were first counted (viability was assessed by Trypan Blue exclusion) and plated on collagen-coated cultivation plates at a density of 2 × 105 cells/cm2 in WME, supplemented with 5% calf serum. Culture was maintained in a moisture incubator at 37 ◦ C with 5% CO2 (air:CO2 was 95:5). Following a 3-h stabilization period, cells were washed and stained as described below.
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2.7. In situ mitochondrial membrane potential (Ψ m ) of rat hepatocytes Hepatocytes were washed twice in ice-cold PBS and then incubated for 30 min at 37 ◦ C in PBS containing 0.5 M fluorescent probe JC-1 (Molecular Probes, Eugene, OR), a voltage sensitive dye (Cossarizza, Ceccarelli, & Masini, 1996) that selectively enters mitochondria. When excited at 490 nm, the dye will emit green fluorescence at low Ψ and red at high Ψ . Following incubation, the cells were washed once with PBS and covered with a thin layer of PBS. This treatment allows better photography while preventing cell death due to drying. Olympus IX 70 fluorescence microscope equipped with digital camera Camedia C3030 (Olympus C&S, Prague) was used for all in situ fluorescence measurements. 2.8. In situ detection of apoptotic markers in primary rat hepatocytes
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The mRNA levels of -actin remained the same. The time-course of activation (not shown) evolved so that at 10 h post injection an average increase in UCP2 mRNA was 1.7 fold and maximum, up to 4-fold occurred after 18 and 24 h. The increase was sex-independent and Long Evans rats, tested if they have different sensitivity to LPS, responded similarly (2.8-fold increase, n = 3). 3.2. Possible UCP2 protein increase in liver mitochondria upon LPS-stimulation of rats The possible increase in UCP2 protein was indicated by Western blotting (Fig. 2). The specifi-city of immunodetection was not 100% as demonstrated using mitochondria from yeast expressing UCP2 (see ˇ acˇ kov´a et al., 2003) vs. those lacking expression Z´ vector. A non-specific antibody interaction was still indicated with the latter, directed most probably to the other mitochondrial carriers. Such a nonspecific interaction represents a background. Nevertheless,
Hepatocytes seeded on 24-well plates were washed once in ice-cold PBS and incubated for 15 min at room temperature in Annexin-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 , pH 7.4) containing 10 l of Annexin V Alexa Fluor 594 conjugate (Molecular Probes, Eugene, OR). The incubation volume was 150 l containing 105 cells per well. Following incubation the cells were washed once with the binding buffer and covered with a thin layer of the same buffer. Fluorescence microscopy and imaging was similar as described above.
3. Results 3.1. LPS stimulation increases UCP2 mRNA in liver and hepatocytes The amount of UCP2 mRNA in liver of untreated Wistar rats accounted for ∼8.10−7 fraction of mRNA (calculated from calibrations, Fig. 1a), confirming the low transcript levels in liver as compared e.g. to 5.10−6 ˇ fraction in kidney (Santorov´ a et al., unpublished). Fig. 1b illustrates the average 3.3 ± 0.7-fold increase in UCP2 mRNA (n = 7) found in LPS-stimulated rats and 3.0 ± 0.2-fold increase in hepatocytes isolated from LPS-treated rats sacrificed 24 h post injection (Fig. 1b).
Fig. 2. Immunodetection of UCP2 in yeast and liver mitochondria. Western blots are shown for mitochondrial samples of yeast expressing UCP2 (“YUCP2”) vs. those lacking expression vector (“YE”) and for liver mitochondria from LPS-treated (“LPS”) and untreated rats (“CTRL”).
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Fig. 3. 3 H-GTP binding to isolated liver mitochondria from control rats (䊉) or rats injected with 1 mg LPS for 24 h ( , ), In controls, an average value (± S.D., five isolations of mitochondria) of the number of binding corresponded to 21 ± 4 pmol/mg protein, whereas for the two representative Scatchard plots measured after LPS-treatment it was 72 (data fit displayed) and 64, respectively. The derived binding constants Kd s were 0.23 ± 0.03 M and 0.26 or 0.3 M in mitochondria from controls (average) and two isolations of LPS-treated rats, respectively.
liver mitochondria isolated from LPS-treated rats indicated a higher amount of UCP2 antigen over the amount (or a background) seen in controls (Fig. 2). UCP2 protein content increase was more credibly evaluated from the increase in 3 H-GTP binding sites in mitochondria of LPS-treated rats vs. control mitochondria (Fig. 3). The resulting Scatchard plots indicate that the number of 3 H-GTP binding sites increased up to three times after the LPS treatment. The relative increase of 3 H-GTP binding sites well matches the increase in UCP2 mRNA amount detected after the LPStreatment. Such correlation with the mRNA increase for UCP2 suggests that at least part of the increase in 3 H-GTP binding sites could be due to enhanced UCP2 expression. Since liver tissue contains 85% of hepatocytes and the rest is represented by non-parenchymal liver cells in which LPS reduces UCP2 mRNA (CortezPinto et al., 1998), one must ascribe the possible UCP2 increase to mitochondria originating from hepatocytes. The binding was evaluated in the presence of CAT to exclude the possible ADP/ATP carrier contribution. 3.3. H2 O2 production in liver mitochondria of LPS-stimulated rats is enhanced by GDP Considerable suppression of mitochondrial ROS production is expected when Ψ is decreased even by
several mV (Korshunov et al., 1997). Hence, the subtle Ψ drop predicted for the fully functional UCP2 at the levels existing in LPS-stimulated liver (Jeˇzek et al., 2004) can be amplified into a much larger change in the ROS production. As Fig. 4a illustrates, H2 O2 generation in liver mitochondria from control rats respiring with succinate under the state-4 conditions was not sensitive to GDP, whereas the rate of H2 O2 production after LPS treatment for 18 h substantially increased upon GDP addition (Fig. 4b). The rates amounted to 0.40 ± 0.13 and 0.45 ± 0.11 nmol H2 O2 min−1 mg protein−1 in controls (n = 5, n is number of mitochondrial isolations) and after LPS treatment (n = 7), respectively. GDP raised them up to 0.6 ± 0.17 nmol H2 O2 min−1 mg protein−1 after LPS treatment. Note, that liver mitochondria were isolated in the presence of albumin, hence the observed rates do not reflect the rates in vivo which should be lower for mitochondria with activated UCP2 (under LPS treatment) or at least if they were equal they had to be much higher if the UCP2 “antioxidant” function would not be recruited (such high rates as observed in vitro with GDP). The observed similar rates under in vitro conditions reflect the same and intact state of the respiratory chain, and lack of its damage upon LPS treatment. GDP dose–response for H2 O2 generation in mitochondria of LPS-treated liver in the presence of linoleic acid exhibited activation constant AC50 of about 90 M, when half-maximum rate is considered between the limits of maximum activation by 2.5 mM GDP and no activation in the absence of GDP (Fig. 4c). Linoleic acid (5 or 10 M) without GDP decreased H2 O2 generation in control mitochondria and after LPS-treatment. The accelerating effect of GDP on H2 O2 production in liver mitochondria from LPSstimulated rats was not abolished by CAT (Fig. 4b), indicating no participation of the ADP/ATP carrier. The GDP effect after LPS stimulation was observed also in the presence of linoleic acid, which is consistent with the fact that fatty acid is a required cofactor (cycling substrate) for UCP-mediated uncoupling (Jeˇzek, ˇ acˇ kov´a et al., 2003). We 2002; Urb´ankov´a et al., 2003; Z´ have also demonstrated that any accelerated H+ backflow was reducing H2 O2 generation in liver mitochondria of both control and LPS-treated rats, since in both cases uncoupler addition (1 M FCCP) diminished the H2 O2 generation significantly (0.12 ± 0.04 nmol H2 O2 min−1 mg protein−1 found in controls versus
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Fig. 4. GDP enhances H2 O2 production in respiring liver mitochondria of LPS-treated but not control rats: (a) liver mitochondria from control rats, (b) LPS-treated rats (1 mg LPS, i.p.) – H2 O2 production was measured as described in Section 2. Control rats (a) exhibited nearly identical rates of H2 O2 production in the absence (thin solid line) and presence of 1 mM GDP; whereas after LPS treatment GDP accelerated H2 O2 production (b, lower solid dotted trace) when compared to rates in the absence of GDP (b, upper solid dotted trace). 10 M linoleic acid did not enhance H2 O2 production as shown for (b) (thin solid trace “+Lin”) and 1 M carboxyatractyloside (CAT) did not prevent the GDP effect in mitochondria of LPS-stimulated rats as illustrated by the thin solid trace measured also in the presence of 10 M linoleic acid. If the linoleic acid-induced uncoupling due to the ADP/ATP carrier was involved CAT had to prevent it. But, this is not the case. Consequently, the effect can be ascribed to the recruited UCP2. (c) GDP dose–response for activation of H2 O2 production in liver mitochondria from LPS-treated rats – in the presence of 10 M linoleic acid. A half-maximum was considered when at 50% between the limits of maximum activation by 2.5 mM GDP and no activation without GDP. The activation constant AC50 of 90 M was yielded.
0.2 ± 0.06 nmol H2 O2 min−1 mg protein−1 after LPS treatment). Also oxidative phosphorylation started by ADP addition in the presence of Mg2+ and phosphate reduced H2 O2 generation transiently until ADP was exhausted during the period of ATP-synthesis (not shown). Without phosphate or Mg2+ , ADP did not reduce the rates of H2 O2 generation. Fig. 5 shows the relative rates of H2 O2 generation corrected for FCCP “background” levels (rates obtained with1 M FCCP were subtracted from each rate) as percent of the corrected rates observed with GDP. It is clearly recognized that GDP had a very subtle effect, if any, in the untreated liver mitochondria. GDP (or GTP, 84% of GDP response) was doubling or tripling
the corrected rates in liver mitochondria of LPS-treated rats. Most likely, the UCP2-mediated uncoupling is responsible for suppression of H2 O2 generation to the basal level, which increased when UCP2 was blocked by GDP. The added or endogenous fatty acids (unremoved during isolations) enabled the uncoupling function of UCP2. 3.4. Mitochondrial membrane potential (Ψ m ) in primary hepatocytes from LPS-treated rats Overcoming difficulties with exact in vitro simulation of an in vivo stimulation by LPS while accessing consequences of the increased UCP2 expression, we
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Fig. 5. Association of GDP-induced enhancement of H2 O2 production in liver mitochondria with LPS-treatment. Graph summarizes the data of five control isolations and seven isolations of mitochondria from LPS-treated rats. Background rates given by the rates in the presence of 1 M FCCP were subtracted from the measured rates of H2 O2 generation and the resulting corrected rates were related as percentage of the maximum rates obtained in the presence of 1 mM GDP. When indicated, 10 M linoleic acid or 1 M CAT were present. Student t-test results are represented as follows: **** p < 0.002, *** p < 0.01, * p < 0.05.
Fig. 6. In situ mitochondrial membrane potential of hepatocytes isolated from LPS-treated or untreated rats, panel A; PBS (control cells), panel B; PBS + 1 M CCCP, panel C; LPS, panel D; LPS + 1 M CCCP. Magnification 350×. The red staining represents formation of JC1aggregates and higher membrane potential. 1 M CCCP was added to cells to verify the presence of green fluorescence only in the uncoupled state.
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studied in situ mitochondrial membrane potential (Ψ m ) using the voltage-sensitive fluorescent probe JC-1. As shown in Fig. 6, the basal Ψ m in control hepatocytes is higher than in hepatocytes isolated from rats 20 h post injection (i.p.) of 0.5 mg LPS/kg weight. Control hepatocytes maintain the signature floor tile
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pattern with distinct cell contacts and nuclei (Fig. 6, panels A and B), whereas cells isolated from LPStreated rats are rounded with minimum cell contacts perhaps as a result of higher sensitivity to collagenase perfusion (Fig. 6, panels C and D). Indeed, the viability of cells isolated from LPS-treated rats was typically 15–20% lower than that of hepatocytes from untreated rats. Hepatocytes from LPS-treated rats display lower Ψ m , as indicated by decreased occurrence of red JC-1 aggregates (Fig. 6, panel A versus C). Uncoupler (1 M CCCP) caused Ψ m collapse, reflected by a complete disappearance of red JC1-aggregates and very dim green fluorescence. The latter indicates that Ψ m was lower than basal Ψ m (Fig. 6, panels B, D). The observed lower Ψ m in hepatocytes isolated from LPS-treated rats could indicate ensuing apoptosis due to a loss of cytochrome c. A loss of cytochrome c has been linked to oxidation and appearance of phosphatidylserine (PS) in the outer leaflet of cell membrane (Jiang et al., 2003). Using fluorescence of Annexin V conjugates for PS detection, we observed very few cells marginally stained by Annexin V in the control primary rat hepatocytes (Fig. 7, panel A). The number of stained cells slightly increased when isolated from LPS-treated rats (Fig. 7, panel B). A positive control for PS-dependent apoptosis, however, was obtained by treating the primary hepatocytes for 1 h with 1 M camptothecin (Fig. 7, panel C), triggering apoptosis. Since the camptothecin effect was identical when used in primary hepatocytes from untreated or LPS-treated rats and since only a minor part of hepatocytes from LPS-treated rats displayed this marker, we can assume that the observed decrease in Ψ m was not associated with massive apoptosis.
4. Discussion
Fig. 7. In situ detection of early apoptosis in primary rat hepatocytes, panel A; PBS (control cells), panel B; LPS, panel C; PBS + 1 M camptothecin. Magnification 350×. Annexin V Alexa Fluor 594 reveals the presence of phosphatidylserine in the outer leaflet of cell membrane.
In this work we documented a correlation between increased UCP2 mRNA levels in liver and hepatocytes, increased UCP2 protein content in liver mitochondria estimated as 3 H-GTP binding sites, the ability to slightly uncouple hepatocyte mitochondria, and the ability to decrease liver mitochondrial H2 O2 production in the absence of UCP2 inhibitors GDP or GTP which was manifested only upon LPS-stimulation of rats. In spite of a theoretical possibility of translational down-regulation explaining low amounts of detectable
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UCP2 protein at modest levels of mRNA in many tisˇ acˇ kov´a et al., 2003), the sues (Pecqueur et al., 2001; Z´ described situation in rat liver shows a nearly direct correlation between the increase in mRNA and in 3 HGDP-binding site which may reflect the increase in UCP2 protein amount. We can conclude that elevated UCP2 expression upon LPS-stimulation of liver is now well documented and we provide a clear confirmation (Fig. 1b) that it indeed takes place in hepatocytes. Possible functional activation of UCP2 at the corresponding capacity was suggested by GDP-induced restoration of higher H2 O2 production associated with the LPS induction. Mitochondria in situ could produce equivalently much more ROS, if UCP2 was not recruited and activated. These results seem to obey similar principles as the previously obtained data on H2 O2 production in rat heart mitochondria. The latter was found to decrease by 57% when Ψ diminished by 10% (Korshunov et al., 1997). Hence, detection of H2 O2 production points to an amplified effect of a presumably subtle (Jeˇzek et al., 2004) uncoupling mediated by UCP2. Possible functional activation of UCP2 was also demonstrated by indications of lower membrane potential of hepatocyte mitochondria in situ. The remaining questions are: why Nature has developed such a stimulating mechanism and what is the physiological role of elevated UCP2 expression in liver? Two possibilities exist. The recruitment of UCP2 molecules has been suggested as pre-determined thermogenic (febrile) response (Faggioni et al., 1998) and it remains to be proven that under such conditions FAinduced uncoupling is initiated in liver or skeletal muscle with concomitant heat release in a sufficiently high amount which is able to increase the body temperature. For the liver, even if we did not measure temperature increase, we may doubt a significant UCP2 participation in the eventual LPS-induced thermogenesis due to the inability of UCP2 to cause an extensive uncoupling (see below). On the other hand, compatible with a low extent of UCP2-mediated uncoupling, the purpose of UCP2 recruitment in liver, as part of the systemic response to bacterial infection (simulated by LPS in our case), can be the resetting of ROS homeostasis. During infection the whole body ROS content is elevated due to macrophage attack (Parola & Robino, 2001). Thus UCP2-induced uncoupling should decrease ROS production in liver mitochondria themselves (Korshunov
et al., 1997, 1998; Skulachev, 1998; Figs. 4 and 5). Upon LPS stimulation, the released TNF␣ activates NF-B pathway which leads to a decrease of reduced glutathione and increase of intracellular ROS (Parola & Robino, 2001). Such an elevated H2 O2 production is seen in mitochondria of LPS-treated rats with inhibited UCP2 (by GDP). The recruited UCP2 can at least partly counteract these changes, since activation of its function can suppress ROS production in mitochondria back to a low state. An instant drop in the ROS production then immediately releases a free capacity of the ROS-detoxification mechanisms which reduce the ROS content. This consideration is supported by the findings showing that UCP2-related uncoupling in mitochondria of endothelial cells can decrease ROS externally to these cells and even in co-incubated LDL lipoproteins (Duval et al., 2002); and by demonstrations of increased atherosclerotic plaques after bone marrow transplantation from UCP2 (−/−) mice to LDL receptor (−/−) mice (Blanc et al., 2003) and by the observed UCP2 preventive role after the stroke (Mattiasson et al., 2003). Accordingly, the purpose of LPS/cytokineinduction of UCP2 may be to decrease ROS content in the tissues. Any possible ROS increase during signaling or pathophysiological events in cells, or in body fluids like blood, which induces UCP2 expression, can be considered as a compensation mechanism, in which the increased UCP2-mediated uncoupling of mitochondria sets ROS levels back to the (original) steady state. An instant component of such compensation mechanism may also exist, activating merely the function of existing UCP2 levels, as based upon the direct interaction of UCP2 with lipoperoxidation products – reactive aldehydes, according to Brand group (Echtay et al., 2003; Murphy et al., 2003), or PUFAhydroperoxides (Jab˚urek, Miyamoto, Di Mascio, Garlid, & Jeˇzek, 2004). Note, that always the conditions must be established which permit the UCP2 uncoupling function (Jab˚urek & Garlid, 2003; Jeˇzek, 2002; Jeˇzek et al., ˇ acˇ kov´a et al., 2003): re2004; Urb´ankov´a et al., 2003; Z´ lease of bound inhibitory ligands (such as nucleotides) must take place and activating fatty acids (probably cycling substrates of UCP2), must be present. However, not many details about UCP2 influence on mitochondrial energetics have yet been described. One can derive some estimations of UCP2 uncoupling
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Fig. 8. Contribution of UCP1 to uncoupling in BAT mitochondria and UCP2 to uncoupling in liver mitochondria of LPS-stimulated rats, A scheme based on the experimental data for BAT mitochondria (Nicholls, 1974), where UCP1 contribution to uncoupling causes increase in respiration from the coupled rate of 43 nmol O min−1 mg protein−1 to a maximum of 140 nmol O min−1 mg protein−1 . For 11times less abundant UCP2 in liver mitochondria of LPS-stimulated rats (taking the number of 3 H-GTP binding sites as a maximum estimate, Fig. 2 the maximum uncoupled rate is only a fraction of the extent in BAT mitochondria and the related protonmotive force decrease would correspond to ∼5 mV. These calculations are based on the relationship valid for BAT mitochondria above 166 mV: R = 415–1.7 p, where R is respiration in nmol O min−1 mg protein−1 (Nicholls, 1974). Hence p = 244.1 − R/1.7. Using this for UCP2, the maximum UCP2 contribution (in case of its 11 times lower amounts) to decrease in p would be 4.3–5.1 mV.
strength from classic experiments with brown adipose tissue (BAT) mitochondria, where uncoupling is given by UCP1 (Nicholls, 1974). Using a “battery curve” i.e. protonmotive force p relationship to respiration for BAT mitochondria respiring with sn-glycerolphosphate (Nicholls, 1974), one obtains maximum respiration of 140 nmol O/min mg as limited by the dehydrogenase (Fig. 8). Since it has been achieved either in the presence of FCCP and GDP blocking UCP1 or with an uninhibited UCP1, one can relate the whole respiratory increase starting from 43 nmol O/min mg (Nicholls, 1974) to be under control of UCP1-mediated H+ backflow. Relating this to the amount of dimeric UCP1 present in BAT (800 pmol/mg protein) one obtains 0.121 nmol O min−1 pmol−1 of UCP1 dimer. Simply assuming that UCP2 would provide the same straightforward uncoupling (all UCP2 molecules active and uninhibited), and assuming its 11–13.3 times less abundancy (∼60–72 pmol/mg protein, see Fig. 3) in liver mitochondria of LPS-stimulated rats, full uncoupling due to UCP2 could provide respi-
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ration increase of 7.3–8.7 nmol O min−1 mg protein−1 . These considerations rely on the assumption of similar Vmax exerted by UCP1 and UCP2, which is reasonable as proven by measurements of reconstituted proteins (Jab˚urek & Garlid, 2003; Jeˇzek, 2002; Jeˇzek et al., ˇ acˇ kov´a et al., 2003). 2004; Urb´ankov´a et al., 2003; Z´ In this work, we focused on UCP2 function in liver mitochondria. However, similar principles and hypotheses stated above can be applied also to the other tissues. Indeed, LPS also induced UCP2 mRNA in brain (Busquets et al., 2001), lung, and stomach (Pecqueur et al., 2001). Nevertheless, most relevant for possible involvement in fever or defense against excessive ROS would be the elevated UCP2 expression in skeletal muscle. UCP3 has also been suggested to be molecular determinant of thyroide-stimulated adaptive thermogenesis (Lanni, Moreno, Lombardi, & Goglia, 2003). The intravenous injection or administration of TNF␣ to rats resulted in enhanced UCP2 and UCP3 mRNA in skeletal muscle (Busquets et al., 1998; Masaki et al., 1999). The question, whether also protein levels are elevated as well as uncoupling awaits further research. Surprisingly, our estimations using 3 HGTP binding gave a quite low amount of sum of both UCP2 plus UCP3 ∼30 pmol of dimers per mg protein ˇ acˇ kov´a et al., 2003). High muscle mass could com(Z´ pensate for this low relative amount. In conclusion, in the liver due to a small extent of potential decrease (justified also by the above theoretical calculations) mediated by the activated UCP2 upon its recruitment by LPS (or cytokines), documenting that UCP2 causes only a weak uncoupling, the recruited UCP2 is able to decrease mitochondrial ROS production but is unable to produce enough of heat for thermogenesis and participation in a febrile response.
Acknowledgement The project was supported by the grants of the Grant Agency of the Czech Republic, No. 301/02/1215 to P.J. & M.M. and 301/01/P084 to M.R.; by the Internal Grant Agency of the Academy of Sciences of the Czech Republic (No. A5011106); the research project AVOZ5011922 and the Fogarty international NIH grant (FIRCA) No. TW01487. An excellent technical assistance of Jana Koˇsaˇrov´a, Jana Brucknerov´a and Jitka Smikov´a is gratefully acknowledged as well as fruitful
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discussion of Prof. K.D. Garlid, Portland State University, Portland, OR, USA.
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Mattiasson, G., Shamloo, M., Gido, G., Mathi, K., Tomasevic, G., Yi, S., et al. (2003). Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma. Nature Medicine, 9, 1062–1068. Moldeus, P., Hogberg, J., & Orrenius, S. (1978). Isolation and use of liver cells. Methods in Enzymology, 52, 60–71. Murphy, M. P., Echtay, K. S., Blaikie, F. H., Asin-Cayuela, J., Cochem´e, H. M., Green, K., et al. (2003). Superoxide activates uncoupling protein by generating carbon-centered radicals and initiating lipid peroxidation. Journal of Biological Chemistry, 278, 48534–48545. N`egre-Salvayre, A., Hirtz, C., Carrera, G., Cazenave, R., Troly, M., Salvayere, R., et al. (1997). A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB Journal, 11, 809–815. Nicholls, D. G. (1974). Hamster brown adipose tissue mitochondria The control of respiration and the proton electrochemical potential gradient by possible physiological effectors of the proton conductance of the inner membrane. European Journal of Biochemistry, 49, 573–583. Parola, M., & Robino, G. (2001). Oxidative stress-related molecules and liver fibrosis. Journal of Hepatology, 35, 297–306. Pecqueur, C., Alves-Guerra, M. -C., Gelly, C., L´evi-Meyrueis, C., Couplan, E., Collins, S., et al. (2001). Uncoupling protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation. Journal of Biological Chemistry, 276, 8705–8712. Skulachev, V. P. (1998). Uncoupling: new approaches to an old problem of bioenergetics. Biochimica et Biophysica Acta, 1363, 100–124. Takahashi, M., Tsuboyama-Kasaoka, N., Nakatani, T., Ishii, M., Tsutsumi, S., Aburatani, H., et al. (2002). Fish oil feeding alters liver gene expression to defend against PPAR␣ activation and ROS production. American Journal of Physiology, 282, G338–G348. Urb´ankov´a, E., Voltchenko, A., Pohl, P., Jeˇzek, P., & Pohl, E. E. (2003). Transport kinetics of uncoupling proteins: analysis of UCP1 reconstituted in planar lipid bilayers. Journal of Biological Chemistry, 278, 32497–32500. ˇ ˇ acˇ kov´a, M., Skobisov´ Z´ a, E., Urb´ankov´a, E., & Jeˇzek, P. (2003). Activating w-6 polyunsaturated fatty acids and inhibitory purine nucleotides are high affinity ligands for novel mitochondrial uncoupling proteins UCP2 and UCP3. Journal of Biological Chemistry, 278, 20761–20769.
Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction ˇ ˇ Michal R˚uzˇ iˇckaa , Eva Skobisov´ aa , Andrea Dlaskov´aa , Jitka Santorov´ aa , Katar´ına ˇ ceka , Mark´eta Z´ ˇ acˇ kov´aa , Martin Modriansk´yb , Petr Jeˇzeka,∗ Smolkov´aa , Tom´asˇ Spaˇ a
Department of Membrane Transport Biophysics, No. 75 Institute of Physiology, Academy of Sciences of the Czech Republic, V´ıdeˇnsk´a 1083, 14220 Prague 4, Czech Republic b Department of Medical Chemistry, Faculty of Medicine, Palack´ y University, Olomouc, Czech Republic Received 27 July 2004; received in revised form 18 October 2004; accepted 27 October 2004
Abstract Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3 H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3 H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2 O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response. © 2004 Elsevier Ltd. All rights reserved. Keywords: Lipopolysaccharide; Oxidative stress in liver; Mitochondrial uncoupling protein UCP2; Rat liver mitochondria; Reactive oxygen species
∗
Corresponding author. Tel.: +420 296442760; fax: +420 296442488. ˇ E-mail addresses: [email protected] (M. R˚uzˇ iˇcka), [email protected] (E. Skobisov´ a), [email protected] (A. Dlaskov´a), ˇ ˇ cek), [email protected], [email protected] (J. Santorov´ a), [email protected] (K. Smolkov´a), [email protected] (T. Spaˇ ˇ acˇ kov´a), [email protected] (P. Jeˇzek). [email protected] (M. Z´ URL: www.mitonet.cz (P. Jeˇzek). 1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.10.016
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1. Introduction In the liver, a negligible amount of mitochondrial uncoupling protein UCP2 mRNA detectable by Northern blots (Fleury et al., 1997; Gimeno et al., 1997) was originally ascribed to the resident macrophages (Carretero et al., 1998; Hodn´y et al., 1998; Larrouy et al., 1997). Several studies have shown, however, that UCP2 mRNA is transcribed in hepatocytes under certain stimuli (Cortez-Pinto, Lin, Yang, Da Costa, & Diehl, 1999; Faggioni, Shigenaga, Moser, Feingold, & Grunfeld, 1998; Takahashi et al., 2002) or after prolonged incubation in a cell culture medium (Kimura et al., 1999). TNF␣ or other “pyrogenic” cytokines have been found to upregulate UCP2 transcription in hepatocytes (Cortez-Pinto et al., 1998; Faggioni et al., 1998) and to downregulate UCP2 in macrophages (Diehl & Hoek, 1999). It has been suggested that the up-regulation in liver may serve to compensate the increased ROS production from macrophages (Diehl & Hoek, 1999). The physiological role of UCPs is determined by the extent of uncoupling they induce when actiˇ ˇ acˇ kov´a, R˚uzˇ iˇcka,Skobisov´ vated (Jeˇzek, Z´ a, & Jab˚urek, 2004). The UCPs are sluggish transporters, with a very low molecular activity (Echtay, Winkler, Frischmuth, & Klingenberg, 2001; Jab˚urek & Garlid, 2003; Urb´ankov´a, Voltchenko, Pohl, Jeˇzek, & Pohl, 2003; ˇ ˇ acˇ kov´a, Skobisov´ Z´ a, Urb´ankov´a, & Jeˇzek, 2003); accordingly, the extent of uncoupling in a given cell type is determined largely by their level of expression. Thermogenesis in brown fat cells requires a large extent of uncoupling, and this is achieved by a correspondingly high level of UCP1 expression (Jeˇzek, 1999). The physiological role of UCPs in non-thermogenic tissues is suggested to be reduction of mitochondrial production of reactive oxygen species (ROS) (N`egreSalvayre et al., 1997), which can be achieved by low levels of uncoupling (Korshunov, Skulachev, & Starkov, 1997; Korshunov, Korkina, Ruuge, Skulachev, & Starkov, 1998; Kowaltowski, Costa, & Vercesi, 1998; Liu, Fiskum, & Schubert, 2002; Skulachev, 1998) corresponding to the low levels of UCP expression in such cells (Pecqueur et al., 2001). UCP2 mRNA was found in all kinds of human, rat, and mouse tissues studied (Fleury et al., 1997; Gimeno et al., 1997; Jeˇzek, 2002; Pecqueur et al., 2001); detectable amounts of UCP2 protein were found for example in mouse lung, spleen, or stomach under nor-
mal physiological conditions (Pecqueur et al., 2001; Couplan et al., 2002). Divergence between mRNA and protein quantity has been attributed to translational down-regulation (Pecqueur et al., 2001). A 3 H-GTP ˇ acˇ kov´a et al. (2003) indicated rather binding study by Z´ high amounts of UCP2 protein in rat lung and modest amounts in rat kidney. The upper estimates of UCP2 protein content in rat liver mitochondria were ∼10 times less than in lung. Functional activation of UCP2 leads to a decrease in ROS production (Arsenijevic et al., 2000; Duval et al., 2002; Kizaki et al., 2002; N`egre-Salvayre et al., 1997), since even slight uncoupling causes a substantial decrease of resting-state mitochondrial ROS production (Korshunov et al., 1997, 1998; Skulachev, 1998). Duval et al. (2002) have shown that UCP2-mediated uncoupling in endothelial cells is able to decrease even extracellular ROS in co-incubated low-density lipoproteins (LDL). Mice with deleted LDL receptor exhibited extensive dietinduced atherosclerotic plaques when they received bone marrow transplanted from UCP2 (−/−) mice, and the appearance of these plaques was prevented when they received bone marrow transplants from UCP2 (+/+) mice (Blanc et al., 2003). Over-expressed UCP2 had also a preventive role in brain after stroke in mice (Mattiasson et al., 2003). These findings suggest a hypothesis in which ROS-homeostasis of the whole organism is regulated by a “ROS-sink” in tissues where UCP2 (or UCP3)-mediated suppression of ROS production is involved (Jeˇzek & Hlavat´a, unpublished). In order to document the recruitment of UCP2 in hepatocytes in detail and to show that it is accompanied by the uncoupling resulting in a decrease of mitochondrial ROS production, we attempted to access whether a recruited UCP2 can suppress ROS production in mitochondria of stimulated liver, eventually to judge an extent of such expected weak uncoupling. Hence, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection.
2. Materials and methods 2.1. Real-time RT-PCR Sets of two primers and two fluorescent hybridization probes were designed for rat UCP2 and rat -actin
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Table 1 Set of primers and fluorescent hybridization probes used for RT-PCR quantification of UCP2 mRNA Primers (forward – f and reverse – r) and probes were designed for the approximately first third of UCP2 sequence (349 base pairs, 136–485 bp), against segments which substantially differ among all UCP isoforms (34) Protein Rat UCP2 Primer f Primer r Probe 1 Probe 2 Rat -actin Primer f Primer r Probe 1 Probe 2
Sequence (5 → 3 )
Melting point (◦ C)
gAgAgTCAAgggCTAgCgC gCTTCgACAgTgCTCTggTA TCAgAgCATgCAggCATCgg – X R640 – CCgCCTCCTggCAggTAgC – p
57 56 63 65
ACCCACACTgTgCCCATCTA gCCACAggATTCCATACCCA gCCACgCTCggTCAggATCTTCAT – X R640 – AggTAgTCTgTCAggTCCCggCCA – p
58 59 67 66
X denotes the fluorescein fluorophore (donor); R640 is a Roche fluorophore (acceptor). The listed melting points were calculated by the synthesizing company (TIB MOLBIOL, Berlin, Germany).
as a house-keeping gene (Table 1). Their synthesis was provided by TIB MOLBIOL (Berlin). mRNA was isolated from DNAse-I-treated total RNA (Chomczynski & Sacchi, 1987) or directly from powdered frozen rat liver or hepatocytes, using mRNA isolation kit (Roche Biochemicals, Mannheim, Germany). Total hepatocyte RNA was isolated using Trizol reagent (GIBCO BRL). Primers designed for PCR of the approximately first third of rat UCP2 sequence (349 base pairs, 136–485 bp) and adjacent hybridization probes (a gap of 1 bp) were chosen to hybridize the most variant part of the sequence, where various UCP isoforms (UCP1 to 5) differ from each other (Jeˇzek & Urb´ankov´a, 2000). Total RNA and mRNA were quantified from spectra with subtracted light scattering contribution using a Spectronics 3000 diode-array spectrophotometer (Milton–Roy) in 200 l quartz cuvettes. mRNA (60–100 ng) was used for real-time RT-PCR on a LightCycler (Roche) with 0.5 M primers and 0.2 M hybridization probes. Reverse transcription was performed (30 min), followed by transcriptase inactivation for 30 s at 95 ◦ C, and by 40 cycles of annealing for 25 s at 57 ◦ C, incubation for 25 s at 72 ◦ C, and for 1 s at 95 ◦ C. MgCl2 content was optimized to 4 mM. Calibration (Fig. 1a) was provided by dilution series of PCR-amplified amplicon (rat UCP2 136–485 bp from LightCycler RT-PCR) starting from the initial concentration of 2 g/ml. When the calibration was measured with UCP2 amplicon DNA inserted into AdvanTAge vector (Clontech), a higher slope (−4.36) was obtained (not shown), indicating slower reaction.
2.2. Animal treatment Wistar or Long Evans rats were used for all experiments. 1 mg LPS in sterile physiological saline solution was intraperitoneally (i.p.) injected to rats, which were, while starving, decapitated after 18 or 24 h (or at 6 h intervals when estimating a time course). Livers were removed and put into liquid nitrogen. Liver mitochondria were isolated by a standard procedure with albumin, usually from rats sacrificed 18 h post injection, while their protein content was evaluated using a Bio-Rad kit or the Lowry standard assay. For hepatocyte isolation, male Wistar rats (6–8week-old) were injected (i.p.) with 0.5 mg LPS/kg in sterile phosphate buffered saline (PBS) or sterile PBS alone. 2.3. Immunodetection of UCP2 Although UCPs belong to a large family of mitochondrial anion carriers with a common architecture and homologous common hydrophilic sequences extending to the matrix and cytosol (Jeˇzek & Jeˇzek 2003), and hence there is a high chance of cross-reactivity of anti-UCP2 antibodies with respect to other family members, we also attempted immunodetection by Western blotting. Goat anti-UCP2 antibodies (raised against the UCP2 carboxy terminus) were purchased from Santa Cruz Biotechnology (those of Calbiochem were less specific), while the secondary rabbit anti-goat IgG horse-radish-peroxidase-conjugated
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30 min in 200 ml of 100 mM sucrose, 20 mM HEPESTris, 1 mM EDTA, 2 M rotenone, 5 M CAT, pH 7.0 ˇ acˇ kov´a et al., 2003). Mitochondria were then filtered (Z´ through nitrocellulose filters (0.45 mm pores, Millipore) which were subsequently washed twice by sucrose medium and placed onto scintillation solution. The counted radioactivity of the samples containing excess of non-radioactive GTP was always subtracted. Scatchard plots were constructed for each measurement, combined into one graph and the data were fitted by linear regressions. Errors of x-axis intercepts were taken from linear regression fits. 2.5. Mitochondrial H2 O2 generation
Fig. 1. Real-time RT-PCR quantification of UCP2 mRNA in rat liver and hepatocytes: (a) calibration curve – a relation of the initial amount of rat UCP2 amplicon cDNA (first third of rat UCP2 sequence, 349 base pairs, 136–485 bp) to the time of crossing point TCP . Linear regression yielded: TCP = −3.399 log c − 19.19, which is in agreement with the predicted theoretical slope of 3.3, meaning that 3.3 cycles of difference are obtained when the two samples differ in mRNA by one order of magnitude. This is equivalent to doubling the amplicon amount in each cycle. (b) Increase of UCP2 mRNA after LPS injection in comparison with untreated rats—the obtained times of crossing points TCP were converted to the relative amounts according to the respective calibrations. (a) One milligram was intraperitoneally injected to Wistar rats. Animals were decapitated after 18 h (24 h for hepatocyte isolation, see experimental procedures). mRNA was isolated and quantified on a LightCycler.
antibodies (Santa Cruz Biotechnology) were used together with ECL Plus kit for chemiluminiscence detection (Amersham Biosciences). 2.4. 3 H-GTP binding assay for liver mitochondria BSA-washed liver mitochondria (0.2 mg protein) isolated from control or LPS-treated rats (18 h) were mixed with 3 H-GTP aliquots (of 8–320 pmol and with 0.6 mM cold GTP in parallel series) and incubated for
Mitochondrial H2 O2 generation was measured by fluorescent monitoring of oxidation of scopoletin by horseradish peroxidase (Loschen, Flohe, & Chance, 1971) in rat and mice liver mitochondria respiring with 10 mM succinate in the medium containing 125 mM sucrose, 65 mM KCl, 10 mM Tris–HEPES, 1 mM Tris–EGTA, pH 7.2, 1 M cyclosporine A, 10 M rotenone. For H2 O2 detection, 1 M scopoletin and 0.5 M horseradish peroxidase were added and maximum rates were taken from the 10 to 15 min run. Fluorescence was monitored on a RF5301 PC fluorometer (Shimadzu, Japan), with polarization filters (Polaroid) in cross-orientation in order to decrease light scattering. Aliquots of H2 O2 were added for calibration. 2.6. Isolation of rat hepatocytes Hepatocytes were isolated by two-step collagenase perfusion (Moldeus, Hogberg, & Orrenius, 1978). Samples were centrifuged (60 × g, 2 min) to separate non-parenchymal cells from hepatocytes, which were then washed in Williams’ medium E (WME) supplemented with 2 mM glutamine, 10 M streptomycin, 100 U/ml penicillin, 350 nM insulin, and 1 M dexamethasone. Cells were first counted (viability was assessed by Trypan Blue exclusion) and plated on collagen-coated cultivation plates at a density of 2 × 105 cells/cm2 in WME, supplemented with 5% calf serum. Culture was maintained in a moisture incubator at 37 ◦ C with 5% CO2 (air:CO2 was 95:5). Following a 3-h stabilization period, cells were washed and stained as described below.
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2.7. In situ mitochondrial membrane potential (Ψ m ) of rat hepatocytes Hepatocytes were washed twice in ice-cold PBS and then incubated for 30 min at 37 ◦ C in PBS containing 0.5 M fluorescent probe JC-1 (Molecular Probes, Eugene, OR), a voltage sensitive dye (Cossarizza, Ceccarelli, & Masini, 1996) that selectively enters mitochondria. When excited at 490 nm, the dye will emit green fluorescence at low Ψ and red at high Ψ . Following incubation, the cells were washed once with PBS and covered with a thin layer of PBS. This treatment allows better photography while preventing cell death due to drying. Olympus IX 70 fluorescence microscope equipped with digital camera Camedia C3030 (Olympus C&S, Prague) was used for all in situ fluorescence measurements. 2.8. In situ detection of apoptotic markers in primary rat hepatocytes
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The mRNA levels of -actin remained the same. The time-course of activation (not shown) evolved so that at 10 h post injection an average increase in UCP2 mRNA was 1.7 fold and maximum, up to 4-fold occurred after 18 and 24 h. The increase was sex-independent and Long Evans rats, tested if they have different sensitivity to LPS, responded similarly (2.8-fold increase, n = 3). 3.2. Possible UCP2 protein increase in liver mitochondria upon LPS-stimulation of rats The possible increase in UCP2 protein was indicated by Western blotting (Fig. 2). The specifi-city of immunodetection was not 100% as demonstrated using mitochondria from yeast expressing UCP2 (see ˇ acˇ kov´a et al., 2003) vs. those lacking expression Z´ vector. A non-specific antibody interaction was still indicated with the latter, directed most probably to the other mitochondrial carriers. Such a nonspecific interaction represents a background. Nevertheless,
Hepatocytes seeded on 24-well plates were washed once in ice-cold PBS and incubated for 15 min at room temperature in Annexin-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 , pH 7.4) containing 10 l of Annexin V Alexa Fluor 594 conjugate (Molecular Probes, Eugene, OR). The incubation volume was 150 l containing 105 cells per well. Following incubation the cells were washed once with the binding buffer and covered with a thin layer of the same buffer. Fluorescence microscopy and imaging was similar as described above.
3. Results 3.1. LPS stimulation increases UCP2 mRNA in liver and hepatocytes The amount of UCP2 mRNA in liver of untreated Wistar rats accounted for ∼8.10−7 fraction of mRNA (calculated from calibrations, Fig. 1a), confirming the low transcript levels in liver as compared e.g. to 5.10−6 ˇ fraction in kidney (Santorov´ a et al., unpublished). Fig. 1b illustrates the average 3.3 ± 0.7-fold increase in UCP2 mRNA (n = 7) found in LPS-stimulated rats and 3.0 ± 0.2-fold increase in hepatocytes isolated from LPS-treated rats sacrificed 24 h post injection (Fig. 1b).
Fig. 2. Immunodetection of UCP2 in yeast and liver mitochondria. Western blots are shown for mitochondrial samples of yeast expressing UCP2 (“YUCP2”) vs. those lacking expression vector (“YE”) and for liver mitochondria from LPS-treated (“LPS”) and untreated rats (“CTRL”).
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Fig. 3. 3 H-GTP binding to isolated liver mitochondria from control rats (䊉) or rats injected with 1 mg LPS for 24 h ( , ), In controls, an average value (± S.D., five isolations of mitochondria) of the number of binding corresponded to 21 ± 4 pmol/mg protein, whereas for the two representative Scatchard plots measured after LPS-treatment it was 72 (data fit displayed) and 64, respectively. The derived binding constants Kd s were 0.23 ± 0.03 M and 0.26 or 0.3 M in mitochondria from controls (average) and two isolations of LPS-treated rats, respectively.
liver mitochondria isolated from LPS-treated rats indicated a higher amount of UCP2 antigen over the amount (or a background) seen in controls (Fig. 2). UCP2 protein content increase was more credibly evaluated from the increase in 3 H-GTP binding sites in mitochondria of LPS-treated rats vs. control mitochondria (Fig. 3). The resulting Scatchard plots indicate that the number of 3 H-GTP binding sites increased up to three times after the LPS treatment. The relative increase of 3 H-GTP binding sites well matches the increase in UCP2 mRNA amount detected after the LPStreatment. Such correlation with the mRNA increase for UCP2 suggests that at least part of the increase in 3 H-GTP binding sites could be due to enhanced UCP2 expression. Since liver tissue contains 85% of hepatocytes and the rest is represented by non-parenchymal liver cells in which LPS reduces UCP2 mRNA (CortezPinto et al., 1998), one must ascribe the possible UCP2 increase to mitochondria originating from hepatocytes. The binding was evaluated in the presence of CAT to exclude the possible ADP/ATP carrier contribution. 3.3. H2 O2 production in liver mitochondria of LPS-stimulated rats is enhanced by GDP Considerable suppression of mitochondrial ROS production is expected when Ψ is decreased even by
several mV (Korshunov et al., 1997). Hence, the subtle Ψ drop predicted for the fully functional UCP2 at the levels existing in LPS-stimulated liver (Jeˇzek et al., 2004) can be amplified into a much larger change in the ROS production. As Fig. 4a illustrates, H2 O2 generation in liver mitochondria from control rats respiring with succinate under the state-4 conditions was not sensitive to GDP, whereas the rate of H2 O2 production after LPS treatment for 18 h substantially increased upon GDP addition (Fig. 4b). The rates amounted to 0.40 ± 0.13 and 0.45 ± 0.11 nmol H2 O2 min−1 mg protein−1 in controls (n = 5, n is number of mitochondrial isolations) and after LPS treatment (n = 7), respectively. GDP raised them up to 0.6 ± 0.17 nmol H2 O2 min−1 mg protein−1 after LPS treatment. Note, that liver mitochondria were isolated in the presence of albumin, hence the observed rates do not reflect the rates in vivo which should be lower for mitochondria with activated UCP2 (under LPS treatment) or at least if they were equal they had to be much higher if the UCP2 “antioxidant” function would not be recruited (such high rates as observed in vitro with GDP). The observed similar rates under in vitro conditions reflect the same and intact state of the respiratory chain, and lack of its damage upon LPS treatment. GDP dose–response for H2 O2 generation in mitochondria of LPS-treated liver in the presence of linoleic acid exhibited activation constant AC50 of about 90 M, when half-maximum rate is considered between the limits of maximum activation by 2.5 mM GDP and no activation in the absence of GDP (Fig. 4c). Linoleic acid (5 or 10 M) without GDP decreased H2 O2 generation in control mitochondria and after LPS-treatment. The accelerating effect of GDP on H2 O2 production in liver mitochondria from LPSstimulated rats was not abolished by CAT (Fig. 4b), indicating no participation of the ADP/ATP carrier. The GDP effect after LPS stimulation was observed also in the presence of linoleic acid, which is consistent with the fact that fatty acid is a required cofactor (cycling substrate) for UCP-mediated uncoupling (Jeˇzek, ˇ acˇ kov´a et al., 2003). We 2002; Urb´ankov´a et al., 2003; Z´ have also demonstrated that any accelerated H+ backflow was reducing H2 O2 generation in liver mitochondria of both control and LPS-treated rats, since in both cases uncoupler addition (1 M FCCP) diminished the H2 O2 generation significantly (0.12 ± 0.04 nmol H2 O2 min−1 mg protein−1 found in controls versus
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Fig. 4. GDP enhances H2 O2 production in respiring liver mitochondria of LPS-treated but not control rats: (a) liver mitochondria from control rats, (b) LPS-treated rats (1 mg LPS, i.p.) – H2 O2 production was measured as described in Section 2. Control rats (a) exhibited nearly identical rates of H2 O2 production in the absence (thin solid line) and presence of 1 mM GDP; whereas after LPS treatment GDP accelerated H2 O2 production (b, lower solid dotted trace) when compared to rates in the absence of GDP (b, upper solid dotted trace). 10 M linoleic acid did not enhance H2 O2 production as shown for (b) (thin solid trace “+Lin”) and 1 M carboxyatractyloside (CAT) did not prevent the GDP effect in mitochondria of LPS-stimulated rats as illustrated by the thin solid trace measured also in the presence of 10 M linoleic acid. If the linoleic acid-induced uncoupling due to the ADP/ATP carrier was involved CAT had to prevent it. But, this is not the case. Consequently, the effect can be ascribed to the recruited UCP2. (c) GDP dose–response for activation of H2 O2 production in liver mitochondria from LPS-treated rats – in the presence of 10 M linoleic acid. A half-maximum was considered when at 50% between the limits of maximum activation by 2.5 mM GDP and no activation without GDP. The activation constant AC50 of 90 M was yielded.
0.2 ± 0.06 nmol H2 O2 min−1 mg protein−1 after LPS treatment). Also oxidative phosphorylation started by ADP addition in the presence of Mg2+ and phosphate reduced H2 O2 generation transiently until ADP was exhausted during the period of ATP-synthesis (not shown). Without phosphate or Mg2+ , ADP did not reduce the rates of H2 O2 generation. Fig. 5 shows the relative rates of H2 O2 generation corrected for FCCP “background” levels (rates obtained with1 M FCCP were subtracted from each rate) as percent of the corrected rates observed with GDP. It is clearly recognized that GDP had a very subtle effect, if any, in the untreated liver mitochondria. GDP (or GTP, 84% of GDP response) was doubling or tripling
the corrected rates in liver mitochondria of LPS-treated rats. Most likely, the UCP2-mediated uncoupling is responsible for suppression of H2 O2 generation to the basal level, which increased when UCP2 was blocked by GDP. The added or endogenous fatty acids (unremoved during isolations) enabled the uncoupling function of UCP2. 3.4. Mitochondrial membrane potential (Ψ m ) in primary hepatocytes from LPS-treated rats Overcoming difficulties with exact in vitro simulation of an in vivo stimulation by LPS while accessing consequences of the increased UCP2 expression, we
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Fig. 5. Association of GDP-induced enhancement of H2 O2 production in liver mitochondria with LPS-treatment. Graph summarizes the data of five control isolations and seven isolations of mitochondria from LPS-treated rats. Background rates given by the rates in the presence of 1 M FCCP were subtracted from the measured rates of H2 O2 generation and the resulting corrected rates were related as percentage of the maximum rates obtained in the presence of 1 mM GDP. When indicated, 10 M linoleic acid or 1 M CAT were present. Student t-test results are represented as follows: **** p < 0.002, *** p < 0.01, * p < 0.05.
Fig. 6. In situ mitochondrial membrane potential of hepatocytes isolated from LPS-treated or untreated rats, panel A; PBS (control cells), panel B; PBS + 1 M CCCP, panel C; LPS, panel D; LPS + 1 M CCCP. Magnification 350×. The red staining represents formation of JC1aggregates and higher membrane potential. 1 M CCCP was added to cells to verify the presence of green fluorescence only in the uncoupled state.
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studied in situ mitochondrial membrane potential (Ψ m ) using the voltage-sensitive fluorescent probe JC-1. As shown in Fig. 6, the basal Ψ m in control hepatocytes is higher than in hepatocytes isolated from rats 20 h post injection (i.p.) of 0.5 mg LPS/kg weight. Control hepatocytes maintain the signature floor tile
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pattern with distinct cell contacts and nuclei (Fig. 6, panels A and B), whereas cells isolated from LPStreated rats are rounded with minimum cell contacts perhaps as a result of higher sensitivity to collagenase perfusion (Fig. 6, panels C and D). Indeed, the viability of cells isolated from LPS-treated rats was typically 15–20% lower than that of hepatocytes from untreated rats. Hepatocytes from LPS-treated rats display lower Ψ m , as indicated by decreased occurrence of red JC-1 aggregates (Fig. 6, panel A versus C). Uncoupler (1 M CCCP) caused Ψ m collapse, reflected by a complete disappearance of red JC1-aggregates and very dim green fluorescence. The latter indicates that Ψ m was lower than basal Ψ m (Fig. 6, panels B, D). The observed lower Ψ m in hepatocytes isolated from LPS-treated rats could indicate ensuing apoptosis due to a loss of cytochrome c. A loss of cytochrome c has been linked to oxidation and appearance of phosphatidylserine (PS) in the outer leaflet of cell membrane (Jiang et al., 2003). Using fluorescence of Annexin V conjugates for PS detection, we observed very few cells marginally stained by Annexin V in the control primary rat hepatocytes (Fig. 7, panel A). The number of stained cells slightly increased when isolated from LPS-treated rats (Fig. 7, panel B). A positive control for PS-dependent apoptosis, however, was obtained by treating the primary hepatocytes for 1 h with 1 M camptothecin (Fig. 7, panel C), triggering apoptosis. Since the camptothecin effect was identical when used in primary hepatocytes from untreated or LPS-treated rats and since only a minor part of hepatocytes from LPS-treated rats displayed this marker, we can assume that the observed decrease in Ψ m was not associated with massive apoptosis.
4. Discussion
Fig. 7. In situ detection of early apoptosis in primary rat hepatocytes, panel A; PBS (control cells), panel B; LPS, panel C; PBS + 1 M camptothecin. Magnification 350×. Annexin V Alexa Fluor 594 reveals the presence of phosphatidylserine in the outer leaflet of cell membrane.
In this work we documented a correlation between increased UCP2 mRNA levels in liver and hepatocytes, increased UCP2 protein content in liver mitochondria estimated as 3 H-GTP binding sites, the ability to slightly uncouple hepatocyte mitochondria, and the ability to decrease liver mitochondrial H2 O2 production in the absence of UCP2 inhibitors GDP or GTP which was manifested only upon LPS-stimulation of rats. In spite of a theoretical possibility of translational down-regulation explaining low amounts of detectable
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UCP2 protein at modest levels of mRNA in many tisˇ acˇ kov´a et al., 2003), the sues (Pecqueur et al., 2001; Z´ described situation in rat liver shows a nearly direct correlation between the increase in mRNA and in 3 HGDP-binding site which may reflect the increase in UCP2 protein amount. We can conclude that elevated UCP2 expression upon LPS-stimulation of liver is now well documented and we provide a clear confirmation (Fig. 1b) that it indeed takes place in hepatocytes. Possible functional activation of UCP2 at the corresponding capacity was suggested by GDP-induced restoration of higher H2 O2 production associated with the LPS induction. Mitochondria in situ could produce equivalently much more ROS, if UCP2 was not recruited and activated. These results seem to obey similar principles as the previously obtained data on H2 O2 production in rat heart mitochondria. The latter was found to decrease by 57% when Ψ diminished by 10% (Korshunov et al., 1997). Hence, detection of H2 O2 production points to an amplified effect of a presumably subtle (Jeˇzek et al., 2004) uncoupling mediated by UCP2. Possible functional activation of UCP2 was also demonstrated by indications of lower membrane potential of hepatocyte mitochondria in situ. The remaining questions are: why Nature has developed such a stimulating mechanism and what is the physiological role of elevated UCP2 expression in liver? Two possibilities exist. The recruitment of UCP2 molecules has been suggested as pre-determined thermogenic (febrile) response (Faggioni et al., 1998) and it remains to be proven that under such conditions FAinduced uncoupling is initiated in liver or skeletal muscle with concomitant heat release in a sufficiently high amount which is able to increase the body temperature. For the liver, even if we did not measure temperature increase, we may doubt a significant UCP2 participation in the eventual LPS-induced thermogenesis due to the inability of UCP2 to cause an extensive uncoupling (see below). On the other hand, compatible with a low extent of UCP2-mediated uncoupling, the purpose of UCP2 recruitment in liver, as part of the systemic response to bacterial infection (simulated by LPS in our case), can be the resetting of ROS homeostasis. During infection the whole body ROS content is elevated due to macrophage attack (Parola & Robino, 2001). Thus UCP2-induced uncoupling should decrease ROS production in liver mitochondria themselves (Korshunov
et al., 1997, 1998; Skulachev, 1998; Figs. 4 and 5). Upon LPS stimulation, the released TNF␣ activates NF-B pathway which leads to a decrease of reduced glutathione and increase of intracellular ROS (Parola & Robino, 2001). Such an elevated H2 O2 production is seen in mitochondria of LPS-treated rats with inhibited UCP2 (by GDP). The recruited UCP2 can at least partly counteract these changes, since activation of its function can suppress ROS production in mitochondria back to a low state. An instant drop in the ROS production then immediately releases a free capacity of the ROS-detoxification mechanisms which reduce the ROS content. This consideration is supported by the findings showing that UCP2-related uncoupling in mitochondria of endothelial cells can decrease ROS externally to these cells and even in co-incubated LDL lipoproteins (Duval et al., 2002); and by demonstrations of increased atherosclerotic plaques after bone marrow transplantation from UCP2 (−/−) mice to LDL receptor (−/−) mice (Blanc et al., 2003) and by the observed UCP2 preventive role after the stroke (Mattiasson et al., 2003). Accordingly, the purpose of LPS/cytokineinduction of UCP2 may be to decrease ROS content in the tissues. Any possible ROS increase during signaling or pathophysiological events in cells, or in body fluids like blood, which induces UCP2 expression, can be considered as a compensation mechanism, in which the increased UCP2-mediated uncoupling of mitochondria sets ROS levels back to the (original) steady state. An instant component of such compensation mechanism may also exist, activating merely the function of existing UCP2 levels, as based upon the direct interaction of UCP2 with lipoperoxidation products – reactive aldehydes, according to Brand group (Echtay et al., 2003; Murphy et al., 2003), or PUFAhydroperoxides (Jab˚urek, Miyamoto, Di Mascio, Garlid, & Jeˇzek, 2004). Note, that always the conditions must be established which permit the UCP2 uncoupling function (Jab˚urek & Garlid, 2003; Jeˇzek, 2002; Jeˇzek et al., ˇ acˇ kov´a et al., 2003): re2004; Urb´ankov´a et al., 2003; Z´ lease of bound inhibitory ligands (such as nucleotides) must take place and activating fatty acids (probably cycling substrates of UCP2), must be present. However, not many details about UCP2 influence on mitochondrial energetics have yet been described. One can derive some estimations of UCP2 uncoupling
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Fig. 8. Contribution of UCP1 to uncoupling in BAT mitochondria and UCP2 to uncoupling in liver mitochondria of LPS-stimulated rats, A scheme based on the experimental data for BAT mitochondria (Nicholls, 1974), where UCP1 contribution to uncoupling causes increase in respiration from the coupled rate of 43 nmol O min−1 mg protein−1 to a maximum of 140 nmol O min−1 mg protein−1 . For 11times less abundant UCP2 in liver mitochondria of LPS-stimulated rats (taking the number of 3 H-GTP binding sites as a maximum estimate, Fig. 2 the maximum uncoupled rate is only a fraction of the extent in BAT mitochondria and the related protonmotive force decrease would correspond to ∼5 mV. These calculations are based on the relationship valid for BAT mitochondria above 166 mV: R = 415–1.7 p, where R is respiration in nmol O min−1 mg protein−1 (Nicholls, 1974). Hence p = 244.1 − R/1.7. Using this for UCP2, the maximum UCP2 contribution (in case of its 11 times lower amounts) to decrease in p would be 4.3–5.1 mV.
strength from classic experiments with brown adipose tissue (BAT) mitochondria, where uncoupling is given by UCP1 (Nicholls, 1974). Using a “battery curve” i.e. protonmotive force p relationship to respiration for BAT mitochondria respiring with sn-glycerolphosphate (Nicholls, 1974), one obtains maximum respiration of 140 nmol O/min mg as limited by the dehydrogenase (Fig. 8). Since it has been achieved either in the presence of FCCP and GDP blocking UCP1 or with an uninhibited UCP1, one can relate the whole respiratory increase starting from 43 nmol O/min mg (Nicholls, 1974) to be under control of UCP1-mediated H+ backflow. Relating this to the amount of dimeric UCP1 present in BAT (800 pmol/mg protein) one obtains 0.121 nmol O min−1 pmol−1 of UCP1 dimer. Simply assuming that UCP2 would provide the same straightforward uncoupling (all UCP2 molecules active and uninhibited), and assuming its 11–13.3 times less abundancy (∼60–72 pmol/mg protein, see Fig. 3) in liver mitochondria of LPS-stimulated rats, full uncoupling due to UCP2 could provide respi-
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ration increase of 7.3–8.7 nmol O min−1 mg protein−1 . These considerations rely on the assumption of similar Vmax exerted by UCP1 and UCP2, which is reasonable as proven by measurements of reconstituted proteins (Jab˚urek & Garlid, 2003; Jeˇzek, 2002; Jeˇzek et al., ˇ acˇ kov´a et al., 2003). 2004; Urb´ankov´a et al., 2003; Z´ In this work, we focused on UCP2 function in liver mitochondria. However, similar principles and hypotheses stated above can be applied also to the other tissues. Indeed, LPS also induced UCP2 mRNA in brain (Busquets et al., 2001), lung, and stomach (Pecqueur et al., 2001). Nevertheless, most relevant for possible involvement in fever or defense against excessive ROS would be the elevated UCP2 expression in skeletal muscle. UCP3 has also been suggested to be molecular determinant of thyroide-stimulated adaptive thermogenesis (Lanni, Moreno, Lombardi, & Goglia, 2003). The intravenous injection or administration of TNF␣ to rats resulted in enhanced UCP2 and UCP3 mRNA in skeletal muscle (Busquets et al., 1998; Masaki et al., 1999). The question, whether also protein levels are elevated as well as uncoupling awaits further research. Surprisingly, our estimations using 3 HGTP binding gave a quite low amount of sum of both UCP2 plus UCP3 ∼30 pmol of dimers per mg protein ˇ acˇ kov´a et al., 2003). High muscle mass could com(Z´ pensate for this low relative amount. In conclusion, in the liver due to a small extent of potential decrease (justified also by the above theoretical calculations) mediated by the activated UCP2 upon its recruitment by LPS (or cytokines), documenting that UCP2 causes only a weak uncoupling, the recruited UCP2 is able to decrease mitochondrial ROS production but is unable to produce enough of heat for thermogenesis and participation in a febrile response.
Acknowledgement The project was supported by the grants of the Grant Agency of the Czech Republic, No. 301/02/1215 to P.J. & M.M. and 301/01/P084 to M.R.; by the Internal Grant Agency of the Academy of Sciences of the Czech Republic (No. A5011106); the research project AVOZ5011922 and the Fogarty international NIH grant (FIRCA) No. TW01487. An excellent technical assistance of Jana Koˇsaˇrov´a, Jana Brucknerov´a and Jitka Smikov´a is gratefully acknowledged as well as fruitful
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discussion of Prof. K.D. Garlid, Portland State University, Portland, OR, USA.
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