Mitoproteome Plasticity of Rat Brown Adipocytes in Response to Cold

Mitoproteome Plasticity of Rat Brown Adipocytes in Response to Cold Acclimation Rachel Navet,§,† Gregory Mathy,§,† Pierre Douette,§ Rowan Laura Dobson,# Pierre Leprince,‡ Edwin De Pauw,# Claudine Sluse-Goffart,§ and Francis E. Sluse*,§ Laboratory of Bioenergetics and Laboratory of Mass Spectrometry, Baˆt. B6c, Alleé de la Chimie 3, and Centre de Recherche en Neurobiologie Cellulaire et Molećulaire, Baˆt. B36, Avenue de l’Hoˆpital1, 4000 Lie`ge, Belgium Received February 23, 2006

Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein patterns found in rat control and cold-acclimated BAT was performed. A total of 58 proteins exhibiting significant differences in their abundance was unambiguously identified. Proteins implicated in the major catabolic pathways were up-regulated as were ATP synthase and mitofilin. Moreover, these results support the fact that adipocytes can balance their ATP synthesis and their heat production linked to UCP1-sustained uncoupling. Keywords: Proteomics • 2 D-DIGE • Brown adipose tissue • Uncoupling protein 1 • Cold acclimation • Thermogenesis

Introduction Brown adipose tissue (BAT) is present only in mammals and is more abundant in hibernating, cold-acclimated, and newborn mammals.1,2 It plays an important role in thermoregulation during acute cold exposure and in cold acclimation. In both cases, the increased activity of the tissue, that is, more heat production (due to its ability to greatly increase its respiratory rate) and an increase in adipocyte recruitment processes (an increase in the total number of differentiated brown adipocytes that results in a higher thermogenic capacity) are under the control of norepinephrine released from sympathetic nerves. Then, the extensive vascularization of BAT allows heat to diffuse through the animal’s body. This process is termed “non-shivering thermogenesis” (for very extensive reviews of BAT function, see refs 1 and 2). In the 1970s, research on BAT evidenced that its ability to increase its respiratory rate in response to hormonal stimulation is specifically due to the dramatic up-regulation of an uncoupling protein (UCP1) located in the mitochondrial inner membrane: UCP1 catalyzes a proton conductance that dissipates the proton electrochemical gradient built up by the respiratory chain activity. It was also shown that UCP1 sustains the respiratory burst when cellular FFA content increases3,4 and that purine nucleotides (PN) (di- and triphospate) inhibit the proton conductance induced by the FFA-activated UCP1mediated respiration. As such, UCP1 is able to share the proton electrochemical gradient with ATP synthase (proton partitioning) during the oxidative phosphorylation and therefore un* To whom correspondence should be addressed. E-mail: [email protected]. § Laboratory of Bioenergetics. † These authors contributed equally to the work. # Laboratory of Mass Spectrometry. ‡ Centre de Recherche en Neurobiologie Cellulaire et Molećulaire. 10.1021/pr060064u CCC: $37.00

 2007 American Chemical Society

couples respiration from ATP synthesis. One important particularity of BAT is the low capacity (maximal activity) of ATP synthase compared to that of the respiratory chain14,15 and of UCP1. Thus, in cold-adapted BAT, a hormone-dependent FFA release greatly activates up-regulated UCP1 and increases the respiratory chain activity independently of the ATP synthase activity. Moreover, the FFA β-oxidation provides reduced coenzymes (NADH and FADH2) to feed the respiratory chain. Thus, the thermogenic property of the cold-adapted BAT results from the concomitant increase in the weight of the tissue, the number of mitochondria, and the concentration of UCP1 within mitochondria, as well as the greater availability of FFA.2 It is noteworthy that since 1995, UCP1 homologues have been discovered throughout the eukaryotic world (plants,5 animals,6 and protists7-9), except in fermentative yeast.10 Because these UCP1 homologues were found to be expressed in nonthermogenic tissues and in unicellular organisms, their physiological role is still debated.11-13 Besides these well-known changes in BAT, it is questionable if cold acclimation could also implicate a wider mitoproteome adaptation. Therefore, two-dimensional differential in-gel electrophoresis (2D-DIGE) was performed to gain a comparative proteomic analysis of mitochondrial adaptation occurring in rat BAT following a 20-day cold exposure. Apart from the wellknown increase in UCP1 content, changes in mitochondrial proteins involved in the respiratory chain, FFA uptake, and β-oxidation, as well as in the Krebs cycle, are observed. Moreover, an increase in the ATP synthase content and a decrease in the creatine kinase content are also observed. Our proteomic survey shows that the energy metabolism of the BAT is adapted following cold adaptation in order to produce more heat for adaptative thermogenesis and simultaneously to sustain ATP synthesis for housekeeping of brown adipocytes. Journal of Proteome Research 2007, 6, 25-33

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Published on Web 12/14/2006

research articles Materials and Methods Materials. Cyanine dyes (CyDyes: Cy2, Cy3, and Cy5) and immobilized IPG strips were purchased from GE Healthcare. Sequencing-grade modified trypsin was from Roche. All other chemicals were of the highest purity grade and were purchased from Sigma. CyDye, DeCyder, and Ettan are trademarks of G.E. Healthcare. Animal Handling. Twenty Wistar male rats (200 g) were shared in two groups: 10 control rats were kept at room temperature (22 °C) and 10 rats were exposed to cold conditions according to a progressive protocol of cooling. After four steps of 5 °C decreases (2 days at each temperature), coldexposed rats were submitted to a 20-day stay at 2 °C. They were kept in groups of 5 rats per cage and fed with standard pellet chow and water ad libitum. Litters (sawdust) were changed every day to avoid humidity and to ensure comfort. After sacrifice, the interscapular BAT lobules were collected and immediately cooled in an ice-cold mitochondrial isolation medium. Careful dissection and removal of adjacent tissues (white adipose tissue, nerves, and conjunctive tissues) were carried out in a cold room before homogenization. All animal experiments were conducted in accordance with the Belgian Ethical Commitee guideline (Nο. 207). Isolation and Purification of BAT Mitochondria. Isolation was performed according to Lin and Klingenberg.16 Briefly, the BAT was homogenized in an ice-cold isolation medium (250 mM sucrose, 1 mM EGTA, and 5 mM K/TES, pH 7.2) at the ratio of 1 g of tissue per 20 mL of isolation medium and filtered through two layers of gauze. The resulting suspension was centrifuged at 8500g for 10 min at 4 °C. The pellet was resuspended in the same volume of isolation medium. Cell debris was removed by centrifugation at 700g for 10 min at 4 °C. Mitochondria were finally sedimented by centrifugation of the supernatant at 8500g for 10 min at 4 °C. The mitochondrial pellet was washed in isolation medium, resuspended (10 mg/ mL), and centrifuged at 150 000g for 1 h at 4 °C in a discontinuous Percoll gradient (30-70%). Purified mitochondria were washed twice to remove the Percoll. Mitochondrial samples from each of the 10 rats were pooled for subsequent analysis. Totals of 3.4 mg and 18.6 mg of mitochondrial proteins were recovered after differential centrifugations from 3.4 g of control and 6.2 g of cold-acclimated BAT, respectively. CyDye Labeling and Two-Dimensional Differential In-Gel Electrophoresis (2D-DIGE). CyDye labeling of mitochondrial proteins was performed as described in Douette et al.17 Briefly, the mitochondrial proteins were extracted into the lysis buffer (7 M urea, 2 M thiourea, and 2% (w/v) ASB-14 (zwitterionic detergent)) at 10 mg of proteins/mL and vortexed for 10 min. After removing the insoluble material by centrifugation, the pH was adjusted to 8.5 with 100 mM NaOH suitable for efficient CyDye labeling. Protein concentration was evaluated with the RC/DC Protein Assay (Bio-Rad Laboratories). Mitochondrial protein samples (25 µg) were set to a final protein concentration of 5 mg/mL with lysis buffer and labeled separately with 0.2 nmol of CyDye (Cy3, Cy5) (Amersham Bioscience), vortexed, and incubated 30 min in the dark. At the same time, a pooled sample composed of equal amounts of mitochondrial proteins from control and cold-acclimated samples was labeled with Cy2, that constituted an internal standard. This pooled sample was introduced in all the gels and was used for matching and normalization.18,19 Indeed, the main problem in comparative gel-based proteomics is the inter-gel variability (i.e., a given spot will never have the same abundance in two different gels). 26

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The internal standard introduction overcomes the inter-gel variability problem. The normalization process is the calculation, in a given gel, of the spot abundance ratio of the Cy3 and Cy5 differentially labeled proteins against the Cy2 labeled internal standard (Cy3/Cy2 and Cy5/Cy2). As all the gels contain the same internal standard, the ratios from the different gels enable the calculation of protein changes regardless of the spot abundance and thus of inter-gel variability. After 30 min in the dark, the reaction was stopped with 10 mM lysine. To control the labeling efficiency, labeled proteins (0.5 µg) were subjected to SDS-PAGE separation, and the gels were scanned with the Typhoon 9400 scanner (G.E Healthcare) at the wavelengths corresponding to each CyDye, namely, 480 nm (Cy2), 532 nm (Cy3) and 633 nm (Cy5). Differentially labeled mitochondrial samples (25 µg of each Cy2-, Cy3-, and Cy5-labeled sample) were pooled and resolved isoelectrically on 24-cm IPG strips, pH 3-10, NL on an IPGphor isoelectric focusing unit (G.E. Healthcare). Isoelectric focusing (IEF) was successively conducted at 200 V for 200 Vh, 500 V for 500 Vh, 1 kV for 1 kVh, and 8 kV for 60 kVh at 20 °C with a maximum current setting of 50 µA per strip. IPG strips were then equilibrated according to Go¨rg and co-workers20 and sealed on top of 12% w/v acrylamide gels. The seconddimension electrophoresis was performed overnight at 20 °C in an Ettan Dalt II system (G.E. Healthcare) at 1 W per gel. Each gel was finally scanned with the Typhoon 9400 scanner (G.E. Healthcare) at the wavelengths corresponding to each CyDye. To account for experimental bias due to in-gel protein aggregation, 2D-gels of mitochondrial samples were run in triplicate. Image Analysis. Images were analyzed with the DeCyder software 6.5 (G.E. Healthcare) according to the manufacturer. In brief, co-detection of the three CyDye-labeled forms of each spot was performed using the DIA (Differential In-gel Analysis) module. The DIA software allowed the calculation of ratios between samples and internal standard abundances for each spot. Inter-gel variability was corrected by normalization of the Cy2 internal standard spot maps present in each gel by the BVA (Biological Variance Analysis) module. Protein spots that showed a statistically significant Student’s t-test (p < 0.05) for an increased or decreased in intensity were accepted as being differentially expressed in control and cold-acclimated samples. Protein Identification. Spots that showed a significant variation in their abundance were automatically excised from the gel with the Ettan Spot Picker and submitted to tryptic digestion following protein reduction (135 mM DTT) and alkylation (55 mM iodoacetamide). The resulting digested peptides were extracted and rehydrated in 4 µL of formic acid (1%). One microliter of rehydrated sample was mix with 1 µL of HCCA matrix and subsequently drop on a MTP 384 target plate matt steel according to Karas and Hillenkamp.21 Samples were analyzed with an UltraFlex II MALDI-TOF-TOF (Bruker Daltonics) by MS fingerprint (spectra acquisition mass range: 70-4240 m/z). Peaks with the highest intensities obtained in TOFMS mode were subsequently analyzed by LIFT MS/MS (mass range 40-1100). Proteins identifications were carried out with the biotools software (Bruker) using the Mascot search engine.

Results and Discussion Two-Dimensional Differential In-Gel Electrophoresis (2DDIGE) and Comparative Analysis of BAT Mitochondria Proteomes from Control and Cold-Acclimated Rats. Since we

Mitoproteome Plasticity in Cold-Acclimated BAT

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Figure 1. Mitochondrial protein pattern by 2D-DIGE (partial image of analytical gel). Rat BAT mitochondria were isolated, and mitochondrial proteins were extracted by using lysis buffer (7 M urea, 2 M thiourea, and 2% ASB-14). Control and cold-adapted mitochondria were differentially labeled with Cy3 and Cy5. An internal standard composed of equal amount of each mitochondrial sample and labeled with Cy2 was added to improve comparative analysis. Labeled samples (25 µg of each Cy2, Cy3, and Cy5) were loaded on 24-cm, 3-10 NL IPG-strips and subjected to isoelectric focusing. Second-dimension was performed in 12% acrylamide gels. Gels were then scanned in a wavelength-selective way, and subsequent image analyses were performed with Decyder (DIA and BVA softwares, Amersham Biosciences). Seven identified proteins were used as apparent mass and pH (theoretical pI) labels indicated by arrows on both axes (uncoupling protein 1 and electron-transfer flavoprotein have almost the same apparent mass, NADH-ubiquinone oxidoreductase 75 kDa and ubiquinol-cytochrome c reductase complex core protein have almost the same theoretical pI).

found a specific adaptation of the mitoproteome of recombinant yeast cells expressing UCP1 that was accompanied by an increase in total mitochondrial mass,17 we questionned whether rat cold acclimation, which results in overexpression of UCP1 and increased mitochondrial mass, could also trigger a specific adaptative response of the mitoproteome. Two-dimensional differential in-gel electrophoresis has been successfully used for quantitative comparative proteomic analysis of wild-type (WT) versus UCP1-carrying yeast mitochondria,17 of WT versus alternative oxidase (AOX)-carrying yeast mitochondria,22 and of genetically obese ob/ob mice versus lean mice liver mitochondria.23 Consequently, the 2D-DIGE technology was used to compare the mitoproteomes (mainly soluble) of control and cold-acclimated rat BAT mitochondria. As already discussed elsewhere,13 2D-DIGE enables visualization of multiple protein samples on one 2D-gel by means of a differential preelectrophoretic labeling of the protein samples with spectrally resolvable fluorescent dyes (Cy2, Cy3, and Cy5). This technology allows the detection of subtle changes in protein expression profiles as low as 10% with high statistical confidence (up to 95%).19 This low technical threshold enables us to observe low biological modification in the expression of a protein of high metabolic importance, including minute modification in the expression of proteins that have a very high substrate flux control within metabolic pathways. Scanning of the gel using the three wavelengths specific for the Cydyes led to the detection of 1706 spots. According to our

statistical threshold (p < 0.05, Student’s t-test), 163 protein spots exhibited differences in normalized spot volume ratios exceeding 1.2. Among them, 90 spots were increased, while 73 spots were decreased in the “cold-adapted” mitoproteome. Protein spots that varied between control and “cold-adapted” mitoproteomes were collected and subjected to in-gel digestion and peptide mass fingerprint plus peptides sequencing by MS/ MS mass spectrometry (Figure 1). In total, 58 proteins (29 unique and 29 redundant) were identified with significant scores (Table 1). Notably, all MS-identified proteins were of mitochondrial origin. Adaptation of Enzymes Related to Increase in Heat Production. It is well-known that the thermogenic properties of BAT are due to its ability to increase its total respiratory rate, which is mainly sustained by FFA-activated UCP1.3,4 In our comparative 2D-DIGE analysis, we found that UCP1 is strongly up-regulated by cold adaptation (3.7-fold for the same amount of mitochondrial proteins, Figure 2A). This represents only a part of the overall UCP1 up-regulation in response to cold acclimation. Indeed, the mass of mitochondria per gram of BAT was also increased at least 3-fold (10 mg/g in control and 30 mg/g in cold-adapted samples), and moreover, the brown adipocyte recruitment led to almost a doubling (1.8-fold) of the BAT mass per animal (0.34 g/control animal and 0.62 g/cold-adapted animal). Therefore, the average total amount of UCP1 per animal was increased at least 20-fold following 20-day cold acclimation. This means that the total capacity per Journal of Proteome Research • Vol. 6, No. 1, 2007 27

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Table 1. List of Proteins Exhibiting Different Abundance in Cold-Adapted versus Control BAT Mitochondriaa DIGE analysis SP

Q91VD9 Q91VD9 Q91VD9 Q91VD9 Q91VD9 Q9DCT2 Q9CZ13 Q9CZ13 P10719 P10720 P04633 P15650 P15650 P15650 P15650 P45953 P45953 P45953 P45953 P45953 P08503 Q60587 P23965 P14604 Q99LC5 Q99LC6 Q921G7 P52873 P52874 P52873 P49432 P08460 P08461 P08461 P08461 Q9ER34 Q9ER34 Q9ER34 Q9ER34 Q9ER34 Q9ER34 O08749 Q9D6R2 P04636 P14408 Q704S8 P18886 Q6P8J7 Q9Z0X1 Q8CAQ8 Q9D0K2 Q920F5 Q02253 Q02253 Q02253 P35571 P35571 P35571

name

number

Complex I NADH-ubiquinone oxidoreductase 75 kDa subunit 478 NADH-ubiquinone oxidoreductase 75 kDa subunit 471 NADH-ubiquinone oxidoreductase 75 kDa subunit 472 NADH-ubiquinone oxidoreductase 75 kDa subunit 466 NADH-ubiquinone oxidoreductase 75 kDa subunit 462 NADH-ubiquinone oxidoreductase 30 kDa subunit 1619 Complex III Ubiquinol-cytochrome-c reductase complex core protein 924 Ubiquinol-cytochrome-c reductase complex core protein 980 ATP Synthase ATP synthase beta chain 822 ATP synthase beta chain 825 UCP-1 Mitochondrial brown fat uncoupling protein 1 1319 Beta Oxidation Acyl-CoA dehydrogenase, long-chain specific 937 Acyl-CoA dehydrogenase, long-chain specific 1054 Acyl-CoA dehydrogenase, long-chain specific 1053 Acyl-CoA dehydrogenase, long-chain specific 1051 Acyl-CoA dehydrogenase, very-long-chain specific 629 Acyl-CoA dehydrogenase, very-long-chain specific 538 Acyl-CoA dehydrogenase, very-long-chain specific 611 Acyl-CoA dehydrogenase, very-long-chain specific 595 Acyl-CoA dehydrogenase, very-long-chain specific 791 Acyl-CoA dehydrogenase, medium-chain specific 1037 Trifunctional enzyme beta subunit 1590 3,2-trans-enoyl-CoA isomerase 1529 Enoyl-CoA hydratase 1580 ETF Electron-transfer flavoprotein alpha-subunit 1294 Electron-transfer flavoprotein alpha-subunit 1308 Electron-transfer flavoprotein alpha-subunit 1306 Pyruvate Carboxylase Pyruvate carboxylase 191 Pyruvate carboxylase 197 Pyruvate carboxylase 195 Pyruvate Dehydrogenase Pyruvate dehydrogenase E1 component beta subunit 2400 Dihydrolipoyllysine-residue acetyltransferase 633 Dihydrolipoyllysine-residue acetyltransferase 644 Dihydrolipoyllysine-residue acetyltransferase 649 Dihydrolipoyllysine-residue acetyltransferase 640 Krebs Cycle Aconitate hydratase 547 Aconitate hydratase 491 Aconitate hydratase 554 Aconitate hydratase 596 Aconitate hydratase 593 Aconitate hydratase 549 Dihydrolipoyl dehydrogenase 802 Isocitrate dehydrogenase [NAD] subunit alpha 1207 Malate dehydrogenase 1256 Fumarate hydratase 923 Fatty Acid Import Carnitine O-acetyltransferase 595 Carnitine O-palmitoyltransferase II 608 Others Creatine kinase 2408 Programmed cell death protein 8 679 Mitochondrial inner membrane protein (Mitofilin) 409 Succinyl-CoA:3-ketoacid-coenzyme A transferase 1 768 Malonyl-CoA decarboxylase 873 Methylmalonate-semialdehyde dehydrogenase 750 Methylmalonate-semialdehyde dehydrogenase 798 Methylmalonate-semialdehyde dehydrogenase 2406 Glycerol-3-phosphate dehydrogenase 504 Glycerol-3-phosphate dehydrogenase 508 Glycerol-3-phosphate dehydrogenase 586

t-test

ratio

MW

pI

0.022 0.0014 0.0026 0.0002 0.013 0.0008

1.41 1.47 1.73 2.13 1.48 1.51

80724.00 80724.00 80724.00 80724.00 80724.00 30360.00

5.51 5.51 5.51 5.51 5.51 6.4

0.0044 0.035

1.5 1.25

53420.00 53420.00

5.75 5.75

0.0068 0.0043

1.32 1.41

56318.00 56318.00

5.18 5.18

0.011

3.65

33080

9.21

0.045 0.036 0.0074 0.0067 0.013 0.034 0.004 0.0027 0.0039 0.013 0.015 0.0009 0.002

1.33 1.35 1.48 1.51 1.31 1.41 1.34 1.67 1.47 1.29 -1.96 3 -1.46

80724.00 48242.00 48242.00 48242.00 71047.00 71047.00 71047.00 71047.00 71047.00 46925.00 51667.00 32348.01 31516

7.63 7.63 7.63 7.63 9.01 9.01 9.01 9.01 9.01 8.63 9.5 9.55 8.4

0.017 0.036 0.0015

-1.56 -1.54 -1.7

35360.01 35360.00 35360.00

7.62 7.62 7.62

0.0061 0.029 0.025

1.51 1.48 1.5

130349.00 130344.00 130349.00

6.25 6.25 6.25

0.0016 0.023 0.03 0.018 0.0037

1.42 1.36 1.36 1.34 1.37

38848 59126.01 59126.00 59126.00 59126.00

5.94 5.7 5.7 5.7 5.7

0.0031 0.016 0.0006 0.0061 0.011 0.015 0.0004 0.029 0.004 0.0067

1.39 1.41 1.62 1.37 1.48 1.29 1.78 -1.23 1.34 1.76

86121.00 86121.00 86121.00 86121.00 86121.00 86121.00 54212 40069.00 36089.00 54714.00

7.87 7.87 7.87 7.87 7.87 7.87 8 6.27 8.92 9.06

0.0027 0.017

1.67 1.41

71211.00 74634.00

8.73 6.89

0.0036 0.027 0.018 0.0007 0.0042 0.0019 0.0003 0.01 0.034 0.04 0.0004

-1.58 1.4 1.34 -2.87 1.29 -1.99 -1.94 -1.36 1.54 1.6 1.78

47899.00 66952.00 84247.00 56352.00 55298.00 58227.00 58227.00 58227.00 81549 81549 81549

8.64 9.23 6.18 8.73 8.96 8.47 8.47 8.47 6.2 6.2 6.2

a Proteins that significantly varied in cold-acclimated BAT mitochondria versus control BAT mitochondria (p < 0.05, Student’s t-test) are organized according to their general function. Legend abbreviations: SP, access number on Swiss-Prot; number, spot number allocated by the DeCyder software; t-test, value of the Student’s t-test; ratio, amplitude of variation where a positive value means that the amount of protein is increased in cold-acclimated BAT mitochondria; MW, molecular weight; pI, isoelectric point. Score, coverage (percentage of the protein sequence identified), and queries (number of peptides hits for each proteins) for MS fingerprint and score and coverage for MS/MS are also displayed in the table in Supporting Information.

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Figure 2. Comparative analysis of eight protein spot intensities using the BVA module of the DeCyder software. The selected spots are displayed as partial view of the 2D-gel (top panels) and as three-dimensional images (bottom panels). Spot boundary of selected proteins is displayed in pink. The left and right sides of each panel correspond to the control and cold-acclimated gels, respectively. MMSA is methylmalonate-semialdehyde dehydrogenase.

animal (maximal activity of fully FFA-activated UCP1) of UCP1 is increased by such a factor. These results are in perfect agreement with those reported by Zaninovich and co-workers after 4-month cold exposure of rats, although they showed a 6-fold increase in UCP1 by Western blot analysis24 (for the same amount of mitochondrial proteins) with a 2-fold increase in

BAT mass per animal and a 3.8-fold increase of mitochondrial mass per gram of tissue (thus, the total amount of UCP1 per animal was increased 45-fold). If we compare Zaninovich’s results with ours, it seems that there was a kinetics in the adaptative response: adipocyte recruitment process occurred concomitantly with the increase in mitochondrial mass per cell, Journal of Proteome Research • Vol. 6, No. 1, 2007 29

research articles a process that was almost completed after 20 days while upregulation of UCP1 continued afterward. However, the tremendous up-regulation of UCP1 has to be accompanied with up-regulation of upstream enzymes, that is, enzymes from pathways that feed the respiratory chain with electrons and the respiratory chain itself that builds the H+ electrochemical gradient, the driving force of UCP1 activity. We found up-regulation of β-oxidation enzymes. Medium, long, and very long chain Acyl-CoA dehydrogenases that have by far the lowest activity in the FFA catabolic steps of the mitochondrial β-oxidation25 were increased up to 1.4-fold, while trifunctional enzyme was 2.3-fold more abundant. Moreover, 3,2-transenoyl-CoA isomerase, which allows the β-oxidation of unsaturated FFA with cis configuration double bonds, whereas β-oxidation produces trans intermediates, was up-regulated 3-fold (Figure 2B). These results suggest a wide mobilization of all types of FFA. The up-regulation of β-oxidation would require an increase of FFA uptake into the mitochondrial matrix if their import has a significant flux control. Carnitine palmitoyl transferase I (CPTI) is assumed to be rate-limiting for β-oxidation.25 CPTI was not detected by 2D-DIGE as previously observed with steatosis-affected livers mitochondria where fatty acid oxidative enzymes were nevertheless strongly up-regulated.23 This lack of detection by 2D-DIGE is more likely due to the integral-membrane character of CPTI. However, malonylCoA decarboxylase was increased 1.3-fold. On the matrix side, this latter enzyme could scavenge malonyl-CoA formed by the action of propionyl CoA carboxylase on acetyl-CoA, thus, preventing it from inhibiting pyruvate carboxylase,25,26 and on the cytosolic side, malonyl-CoA decarboxylase would prevent malonyl-CoA from inhibiting CPTI.25 Moreover, carnitine Oacetyltransferase was up-regulated 1.7-fold (Figure 2C), indicating an increase in the entry of short chain acyl CoA. Thus, the import of all FFA (from short to long chain) into the matrix seems to be improved. Electron-transfer flavoprotein (ETF) is the primary acceptor for reducing equivalents from the acyl-CoA dehydrogenases, that are then passed to the respiratory chain at the level of Coenzyme-Q by electron-transfer flavoprotein oxidoreductase (ETF:QO). ETF was down-regulated 1.6-fold in cold-adapted mitochondria, and this seems to be in contradiction with a general up-regulation of the β-oxidation flux. However, ETFs are at substrate level concentrations within mitochondria, greatly in excess of the Km values of the acyl-CoA dehydrogenases for ETF.27 Nevertheless, there is no clear explanation for this down-regulation. We also found down-regulation (1.8-fold, Figure 2G) of methylmalonate-semialdehyde dehydrogenase, which is involved in lipogenesis from valine.28 A strong downregulation (2.9-fold) was observed for succinyl-CoA: 3 ketoacidcoenzyme A transferase 1. This would emphasize a strong decrease in ketone-body oxidation29 probably linked to a decrease in their formation from acetyl CoA that is preferentially used by the Krebs cycle (see below). Pyruvate carboxylase was up-regulated 1.5-fold (Figure 2D), while malate dehydrogenase was up-regulated 1.4-fold, allowing oxaloacetate to be over-produced at least 1.5-fold from pyruvate and 1.4-fold from malate. Moreover, as already mentioned, the pyruvate carboxylase inhibitor malonyl-CoA can be more efficiently scavenged by malonyl-CoA decarboxylase. Acetyl-CoA production could also be increased by pyruvate dehydrogenase (1.4-fold) and by β-oxidation (around 1.4-fold). Up-regulation of the two enzymatic competitors of pyruvate strongly suggests that pyruvate availability is increased through 30


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an increase of glycolytic flux. This is more likely connected to the up-regulation of glucose uptake by brown adipocytes during cold exposure.31 Thus, the Krebs cycle substrate availability could be strongly increased. Four enzymes of the Krebs cycle were up-regulated (aconitate hydratase, 1.4-fold; malate dehydrogenase, 1.4-fold; fumarate hydratase, 1.8-fold; and a subunit of ketoglutarate decarboxylase: dihydrolipoyl dehydrogenase, 1.8-fold), sustaining the idea of a greater substrate flux on the Krebs cycle. However, isocitrate dehydrogenase (NAD+-dependent form) was 1.2-fold less abundant. This seems to be in contradiction with an overall increase in the Krebs cycle flux, as the step between isocitrate and ketoglutarate can tightly control the cycle flux. However, two forms of the enzyme catalyze this step, that is, the NAD+-dependent and the NADP+dependent enzymes. The NAD+-dependent isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate into ketoglutatate and NADH, while the NADP+-dependent enzyme sustains the carboxylation of ketoglutarate into isocitrate at the expense of NADPH. The latter reaction is believed to occur when the NADPH level is kept high by the action of the transhydrogenase at the expense of NADH and driven by the H+ electrochemical gradient (∆p).30 Overall, this represents a futile cycle (Figure 3) that costs 1 H+ per isocitrate dehydrogenase cycle, allowing an increased sensitivity to allosteric modifiers of NAD+-isocitrate dehydrogenase (like ADP, citrate). This situation would potentially give rise to a large change in the net flux from isocitrate to R-ketoglutarate. In cold-adapted BAT, the higher activity of UCP1 could decrease NADPH production and in turn the activity of NADPH-dependent carboxylation of R-ketoglutarate. Then, even if NAD-dependent isocitrate dehydrogenase is slightly down-regulated, the flux through the isocitrate f R-ketoglutarate step could be nevertheless increased. Up-regulations of the Krebs cycle and β-oxidation lead to an increase in coenzyme NADH, which is the reducing substrate of respiratory chain complex I. Indeed, we identified its 75 kDa subunit that was up-regulated 1.6-fold (Figure 2H) and its 30 kDa subunit up-regulated 1.5-fold. The increased FADH2 and NADH production through the Krebs cycle and β-oxidation in conjunction with up-regulation of the complex I would lead to an increase in the electron flux within the respiratory chain. Moreover, complex III would also participate to this increased electron flux to the final acceptor, oxygen, as illustrated by its 1.5-fold up-regulation. We also observed up-regulation of glycerol-3-phosphate dehydrogenase, which oxidizes glycerol-3-phosphate formed either from glycerol released by triacylglycerol breakdown by hormone-sensitive lipase or by the reduction of dihydroxyacetone phosphate from glycolysis. This dehydrogenase is a flavoprotein located on the outer face of the inner mitochondrial membrane that has an important role in shuttling reducing equivalents from cytosolic NADH into the mitochondrial matrix by channeling electrons in the respiratory chain at the level of ubiquinone. Reduced ubiquinone is further oxidized by complex III. As previously observed with yeast mitochondria and liver mitochondria,17,22,23 the complex IV does not seem to be affected, suggesting that this enzyme has only very little flux control on the respiratory chain. As a consequence of the up-regulation of complexes I and III, the capacity of the respiratory chain electron transfer increases together with a raise of the proton pumping capacity. Therefore, ∆p, the driving force of UCP1, is more likely better sustained. Moreover, the increased electron flux within the respiratory chain due to up-regulated respiratory enzymes and sustained by UCP1 explains the respiratory burst

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Figure 3. Isocitrate dehydrogenase(ICDH)-transhydrogenase(TH) substrate futile cycle. Activation of UCP1 decreases the activity of the transhydrogenase, and as a consequence, the NADPH-ICDH activity is decreased. Increase in the Krebs cycle flux leads to an increase in citrate, which is an allosteric activator of NAD-ICDH. For further details, see text.

with thermogenic outcome, occurring during adaptative nonshivering thermogenesis. Adaptation of Enzymes Related to ATP Availability. In our comparative 2D-DIGE analysis, we found that, together with a strong up-regulation of UCP1, ATP synthase (b chain) was upregulated (1.4-fold, Figure 2E), while creatine kinase was downregulated (1.6-fold). This could mean that, in order to maintain an appropriate housekeeping, brown adipocytes increased their ability to produce ATP through oxidative phosphorylation and to keep it available by decreasing synthesis of phosphocreatine. Interestingly, ATP synthase up-regulation has been observed in UCP1-recombinant yeast.17 Cell growth was maintained in yeast expressing low amount of UCP1 (1 µg of UCP1/mg of mitochondrial protein) by means of a 1.8-fold cellular upregulation of ATP synthase, but not in the UCP1 highexpression strain (10 µg of UCP1/mg of mitochondrial protein), where growth was decreased by 30% despite a 3.6-fold increase of ATP synthase at the cellular level. Consequently, yeast expressing low amount of UCP1 was efficiently able to counteract the introduction of UCP1 through an up-regulation of its main competitor, ATP synthase, in order to sustain cell growth rate. In cold-adapted BAT, the large increase of UCP1 concentration in the inner mitochondrial membrane is accompanied with the up-regulation of ATP synthase, more likely in order to manage an appropriate H+ partitioning between these two H+ consumers. As in yeast expressing UCP1, the energy metabolic balance could be restored through adequate changes in the mitoproteome in order to ensure a new steadystate between H+-electrochemical gradient-generating (respiratory chain) and -consuming systems (UCP1 and ATP synthase).

Other Adaptations. The mitochondrial inner membrane protein mitofilin controls cristae morphology and is essential for normal mitochondrial function.32 As BAT mitochondria have to sustain a very high activity during cold adaptation, the cristae organization has to be appropriately adapted, since it has an impact on the respiratory activity of the mitochondria through shaping of the mitochondrial inner membrane.33 Here, we showed that mitofilin, a critical organizer of the inner membrane morphology, was 1.3-fold more abundant following cold exposure (Figure 2F). We also observed up-regulation of the apoptosis protein 8 that seems to accumulate in mitochondria. This could correspond to a decrease in its release, and then to an inhibition of apoptosis.34 This observation corroborates the up-regulation of mitofilin, as its inactivation in mouse also leads to an increase of apoptosis frequency.32

Conclusions In this work, we performed an in-depth analysis on the basis of a comparative mitoproteomic approach. This study allowed us to reveal metabolic adaptations that occur during rat cold acclimation (Figure 4). While it was already known that change in UCP1 concentration occurring at the level of mitochondria are amplified through increases in mitochondrial (3-fold) and BAT (1.8-fold) masses, we reported here mitoproteomic adaptations that are part of the response to cold exposure. In such a context, up- and down-regulations of mitochondrial proteins give only a partial insight into the tremendous metabolic adaptation of BAT, although the mitochondrial plasticity represents the molecular basis of the BAT adaptative thermoJournal of Proteome Research • Vol. 6, No. 1, 2007 31

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Navet et al.

Figure 4. Scheme summarizing the effect of cold adaptation on the BAT mitoproteome. In red (+) and in blue (-) are pathways and proteins that were found to be up-regulated or down-regulated, respectively, in cold-adapted BAT mitochondria. Important pathways and proteins such as FFA uptake and oxidation, Krebs cycle, respiratory chain, ATP synthase, uncoupling protein 1, AIF, and mitofilin are up-regulated by cold adaptation. Creatine kinase, ketone body oxidation, and lipogenesis are down-regulated. Abbreviations: AIF, apoptosis inducing factor; CK, creatine kinase; IMMT, mitofilin; CI, NADH-ubiquinone oxidoreductase; CIII, ubiquinol-cytochrome c oxidoreductase; CIV, cytochrome c oxidase; UCP1, uncoupling protein 1; F0F1, ATP synthase; FFA, free fatty acid; PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; MDH, malate dehydrogenase; CIT, citrate; OAA, oxaloacetate; MAL, malate.

genesis. According to our proteomics data and regarding changes at a supramolecular level, that is, the mitochondrial mass per gram of tissue and BAT mass per animal, we can calculate that the overall respiratory capacity of BAT per animal is increased 10-fold, β-oxidation more than 8-fold, and the ATP synthase capacity 8-fold, while the UCP1 capacity is increased at least 20-fold. These tremendous metabolic adaptations rely mainly on the up-regulation of TCA cycle, β-oxidation, respiratory chain, and ATP synthase and are the results of BAT activation. Interestingly, activation of white adipose tissue (WAT) in ob/ ob mice by rosiglitazone, an agent that enhance insulin sensitivity, results in an increase in adipogenesis, in mitochondrial mass, fatty acid oxidation, and electron transport and more strikingly results in a 20-fold increase in UCP1 gene expression35 indicating also a mitochondrial remodeling similar to the case of BAT activation by cold. A surprising event in BAT is the up-regulation of ATP synthase together with the down-regulation of creatine kinase. An increase in ATP synthase was previously observed in UCP1 recombinant yeast.17 This would be explained as the response from the yeast to the introduction of a deleterious energydissipating system (UCP1) in order to fulfill its purpose of life: ensuring its growth. In UCP1-expressing yeast, the ATP synthase up-regulation illustrated the competition that exists between two H+ electrochemical gradient-consuming systems, including one that is energy-conserving (ATP synthase), while the other one dissipates energy (UCP1). In cold adaptation, the up-regulation of ATP synthase could be a response of the cell in order to sustain the ATP availability needed for the housekeeping of the cold-adapted brown adipocytes. Large increases 32


in mitochondrial mass and activity seem to require two other adaptations. The first is an up-regulation of mitofilin, which could ensure a stable organization of the cristae of the inner mitochondrial membrane, while cristae organization is critical for very active mitochondria. The second consists in the accumulation of the AIF protein 8 that seems to be sequestered in mitochondria in order to prevent apoptosis. In this study, we also provide evidence of the existence of an in vivo mechanism that can balance H + partitioning between UCP1 and ATP synthase through proteomic modifications in front of the tremendous increase of UCP1-sustained uncoupling. Whereas uncontrolled energy dissipation related to an energetic imbalance can potentially lead to adipocyte cell death, a new metabolic steady-state is reached in cold-adapted BAT mitochondria. This new energy balance involves, among others, ATP synthase up-regulation in order to concomitantly increase heat production for adaptative thermogenesis and ATP synthesis for adipocyte housekeeping. Abbreviations: 2D-DIGE, two-dimensional differential ingel electrophoresis; BAT, brown adipose tissue; FFA, free fatty acids; UCP, uncoupling protein.

Acknowledgment. This work was supported by grants from the Fonds National de la Recherche Scientifique (FRFC 2.4532.03, FRSM 9.4573.04) and from the Fonds Spećiaux de Recherche dans les universite´s. It was also co-financed by the Centre d’Analyse des Re´sidus en Traces (CART), the Re´gion Wallone (i-Maldi 114713), Fonds Social Europeén (FSE), and the Centre of Biomedical Integrative Genoproteomics (CBIG).

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P.D. was recipient of a Fonds pour la Recherche Industrielle et Agronomique fellowship. P.L. is a Research Associate of the FNRS.

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Journal of Proteome Research • Vol. 6, No. 1, 2007 33

Mitoproteome Plasticity of Rat Brown Adipocytes in Response to Cold

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