Changed Energy State and Increased Mitochondrial -Oxidation Rate in Liver of Rats Associated with Lowered Proton Electrochemical Potential and Stimulated Uncoupling Protein 2 (UCP-2) Expression

Lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver cellular metabolism, which become poised toward peroxisome proliferator-activated receptor (PPAR) α-regulated fatty acid catabolism in mitochondria. After dietary treatment of rats with the hypolipidemic, modified fatty acid, tetradecylthioacetic acid (TTA), the energy state parameters of the liver were altered at the tissue, cell, and mitochondrial levels. Thus, the hepatic phosphate potential, energy charge, and respiratory control coefficients were lowered, whereas rates of oxygen uptake, oxidation of pyridine nucleotide redox pairs, β-oxidation, and ketogenesis were elevated. Moderate uncoupling of mitochondria from TTA-treated rats was confirmed, as the proton electrochemical potential (Δp) was 15% lower than controls. The change affected the ΔΨ component only, leaving the ΔpH component unaltered, suggesting that TTA causes induction of electrogenic ion transport rather than electrophoretic fatty acid activity. TTA treatment induced expression of hepatic uncoupling protein 2 (UCP-2) in rats as well as in wild type and PPARα-deficient mice, accompanied by a decreased double bond index of the mitochondrial membrane lipids. However, changes of mitochondrial fatty acid composition did not seem to be related to the effects on mitochondrial energy conductance. As TTA activates PPARδ, we discuss how this subtype might compensate for deficiency of PPARα. The overall changes recorded were moderate, making it likely that liver metabolism can maintain its function within the confines of its physiological regulatory framework where challenged by a hypolipemic agent such as TTA, as well as others.

oxidizable fatty acid analogues, of which tetradecylthioacetic acid (TTA) 1 is the most studied, have been reviewed (1)(2)(3)(4). A considerable body of evidence points to shifts in the liver cellular metabolism, resulting in channeling of fatty acids to an enhanced mitochondrial ␤-oxidation, at the expense of triacylglyceol synthesis. Simultaneously, there is up-regulation of the inner carnitine palmitoyltransferase II, 2,4-dienoyl-CoA reductase, and mitochondrial 3-hydroxy-3-methyl-CoA synthase. The outer carnitine palmitoyltransferase-I is not affected, suggesting that the rate control of ␤-oxidation and ketogenesis resides in steps beyond acyl group translocation into the matrix (4 -6). Modulation of lipid metabolism with TTA seems at least in part to be related to the role of TTA as a regulator for members of the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors. TTA has been demonstrated to function as a ligand and activator of the PPAR subtypes PPAR␣, PPAR␦, and PPAR␥ (4,(7)(8)(9). PPAR␣ is the predominant subtype in the liver where it controls transcription of genes involved in fatty acid metabolism, such as the genes for peroxisomal acyl-CoA oxidase and fatty acid transport protein, which are up-regulated after TTA treatment (7,9).
Mitochondrial uncoupling by fatty acids has been widely demonstrated during the last decades. Energy coupling is impaired when protons and other ions are allowed to pass through the inner membrane without the production of ATP. Consequently, the stored energy from the mitochondrial proton gradient intended for ATP synthesis is converted to heat. Wojtczak et al. (10) have demonstrated protonophoric behavior in vitro of high concentrations of 3-thia fatty acids toward the mitochondrial inner membrane. The concentration range causing rapid transbilayer movement of acyl chains was on par with that of normal, unipolar long chain fatty acids like palmitic or oleic acids. A similar behavior has been shown to apply to other hypolipidemic fatty acid analogues, such as ␤,␤Ј-methyl-substituted-hexadecane-␣,-dioic acid (11). The molecular basis for fatty acid-mediated uncoupling of respiration remains unclear, but both passive and protein-mediated mechanisms appear to be involved. Skulachev (12) introduced the hypothesis of fatty acid cycling, assuming spontaneous translocation (flip-flop) of the protonated form of the fatty acid in one direction (toward matrix) and a transfer of the anionic form in the other direction, mediated by some inner membrane proteins. Putative candidates for such proteins are the ADP/ATP antiporter and the uncoupling proteins (UCPs) (12,13). UCP homologues form a family of mitochondrial carriers that are capable of depleting the proton gradient. The UCP subtypes, UCP-1, UCP-2, and UCP-3, differ in respect to tissue distribution and probably also function. UCP-1 appears to be solely expressed in brown adipose tissue where it mediates thermogenesis, whereas UCP-2 and UCP-3 are more widely expressed. The functions of UCP-2 and UCP-3 are still unclear, but a mild uncoupling of respiration could prevent the accumulation of oxygen radicals and/or control the NADϩ/NADH ratio and consequently regulate metabolic pathways such as ketogenesis and lipogenesis (14,15). The activities of the UCPs are induced by free fatty acids (16). Furthermore, mono-and polyunsaturated fatty acids, but not saturated fatty acids, were found to increase UCP-2 expression in hepatocytes possibly via a PPAR␣-mediated pathway (17). Others have found that PPAR␣ mediates in vivo regulation of hepatic ucp-2 gene expression and that PPAR␥ has the same property in brown adipose tissue (18).
These observations suggest the possibility that PPAR activation and increased ␤-oxidation rate in liver mitochondria of rats fed TTA might be associated with increased proton conductance across the membrane. We have investigated whether this occurs in vivo after long term feeding of TTA to rats, by measuring energy state parameters at the tissue level, the cellular level, as well as at the level of isolated mitochondria, and if so to assess the extent to which such a mechanism might contribute to increased fatty acid oxidation.

EXPERIMENTAL PROCEDURES
Materials-TTA was synthesized as described previously (19). 3  Animals-Male Wistar rats were obtained from Møllegaard Breeding Laboratory, Eiby, Denmark. They were housed in pairs in wire cages and maintained on a 12-h cycle of light and dark at 20 Ϯ 3°C. The rats had free access to pellet food and water, and they were acclimatized to these conditions for at least 1 week prior to the experiment. Each test and control group consisted of at least 4 animals. If not otherwise stated, palmitic acid and TTA were separately dissolved in acetone and sprayed on pellets to an amount of 3 g/kg of pellets, resulting in an approximate daily dose of 300 mg/kg body weight (estimated consumption of pellets per rat, 20 g; body weight near 200 g). At day 7, the rats were subjected to a 12-h fast before termination. The PPAR Ϫ/Ϫ and PPAR ϩ/ϩ mice (20 -25 g) were a generous gift from Frank J. Gonzales (National Cancer Institute, Bethesda, MD) and have been described elsewhere (20). The PPAR Ϫ/Ϫ and PPAR ϩ/ϩ lines were purebred on a sv129 background. The mice were given a diet consisting of 21.8% casein, 10% soy oil, 17% vitamin/mineral mixture (5.83% vitamin mixture, AIN-93VX, Dyets Inc.; 17.4% mineral mixture, AIN-93G, Dyets Inc.; 11.6% cellulose; 63,9% sucrose; 1.2% cholintartrate) and dextrin (49.5-51.2%), supplemented with 0.5% fenofibrate (gift from Alan Edgar, Fournier, France) or 1.7% TTA. At termination the animals were anesthetized with a subcutaneous injection of Hypnorm Dormicum TM (fentanyl/fluanisone midazol-am, 0.2 ml/100 g body weight). Livers were either immediately removed, placed in ice-cold homogenizing medium and weighed, or freeze-clamped in situ and stored in liquid N 2 . Cardiac puncture was performed to obtain blood samples in EDTA vacutainers; one aliquot being frozen in liquid N 2 for later measurement of blood nucleotides. The Norwegian State Board of Biological Experiments with Living Animals approved the protocol.
Extraction and Measurement of Nucleotides-Nucleotides, including NAD ϩ and NADP ϩ , were extracted with perchloric acid, whereas pyridine nucleotides in reduced form were obtained by alkaline extraction according to Williamson and Corkey (21). Extracted nucleotides were separated, identified, and quantified on an ion pair reversed-phase high-performance liquid chromatographic system (22). Inorganic phosphate was determined by an assay based on the production of phosphomolybdate, which can be measured photometrically at 340 nm (Bayer AG, Leverkusen, Germany). The liver contents of each component were corrected for the amounts due to blood contaminating the tissue as described by Hohorst et al. (23), by measurement of the oxyhemoglobin concentration, in the presence of saponin, of blood and liver tissue. Tissue energy parameters were calculated as follows: energy charge ϭ 1 ⁄2{ ( Isolation of Mitochondria and Measurements of Enzyme Activities-The livers of individual animals were homogenized in an ice-cold medium consisting of 0.25 M sucrose, 10 mM HEPES buffer, pH 7.4, 1 mM EGTA. The mitochondrial fraction was obtained by differential centrifugation as described earlier (25) at 0 -4°C. The Bio-Rad protein kit (Bio-Rad) was used for protein determination with bovine serum albumin (Sigma) as standard. Until used the mitochondrial fractions were stored at 0°C at a concentration of 100 mg of protein/ml. Rates of oxygen consumption were measured polarographically as described earlier (26), the system being calibrated with air-saturated distilled water. Measurements were made at 25.0°C, and where not specified otherwise the medium contained the following components at the indicated concentrations: 83 mM KCl, 4 mM KH 2 PO 4 , 20 mM K-HEPES buffer, pH 7.4, 1 mM EGTA, 1 mM MgCl 2 , and 4 mg of mitochondrial protein, in a total volume of 1.0 ml. Substrates were added as follows: 240 M palmitoyl-L-carnitine ϩ 5 mM malate (or 0.5 mM malate as indicated), 210 M pamlitoyl-CoA ϩ 1 mM L-carnitine ϩ 5 mM malate (or 0.5 mM malate as indicated), or 5 mM sodium succinate ϩ 1 g of rotenone. Where indicated 40 M FCCP was added near the end of the experiment. Rates of ␤-oxidation were determined by incubating mitochondria at 30°C with [1-14 C]palmitoyl-L-carnitine or [1-14 C]palmitoyl-CoA ϩ 1 mM L-carnitine, as described in Ref. 6.
Preparation and Incubation of Primary Hepatocytes-Hepatocytes were prepared from rats by collagenase perfusion by a modification (27) of the method of Berry and Friend (28). The final wash of cells in Ca 2ϩ -free phosphate-buffered saline, pH 7.4, was performed at 37°C in the presence of 1 mM L-carnitine 20 min prior to incubation, to compensate for carnitine lost from cells during preparative procedures (29,30). Production of acid-soluble products was measured using fatty acids as substrates. The assay mixture contained the following components in a total volume of 1.0 ml: 400 M 1-14 C-labeled fatty acid (0.25 Ci/ml) in Ca 2ϩ -free phosphate-buffered saline, pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , and 1% (w/v) fatty acid-free bovine serum albumin. Incubation was for 1 h at 30°C in the presence of 2 ϫ 10 6 cells. Acid-soluble products were measured essentially as given by Christiansen et al. (31). Rates of oxygen uptake by cells were measured polarographically at 37°C as given above for isolated mitochondria, except that the reaction mixture contained in a total volume of 1.0 ml: 400 M fatty acid in Ca 2ϩ -free phosphatebuffered saline, pH 7.4, containing 1% fatty acid-free bovine serum albumin and 2 ϫ 10 6 cells. The experiment was started with the addition of fatty acid substrate after a 10-min preincubation at 37°C with an open measuring chamber. Where used, 5 M FCCP was added near the end of each 10-min experiment. Rates of ␤-oxidation were measured as for isolated mitochondria (above) except that [1-14 C]palmitic acid was used as substrate. Production of ketone bodies was assessed by measuring the amount of D-␤-hydroxybutyrate (Sigma, kit number 310A) present in a neutralized, perchloric acid extract of the reaction mixture after completion of the polarographic experiment, and corrected for the contents of that component in unincubated cells.
Measurement of Protein Motive Force-The proton electrochemical potential (⌬p) were measured by recording the distribution across the mitochondrial inner membrane of labeled TTP as the permeant cation and labeled 5,5-dimetyloxazolidine-2,4-dione as the permeant weak acid, via centrifugation through an oil layer essentially as given by Dawson et al. (32). Briefly, the electrical potential difference (⌬) was measured by incubating mitochondria in a medium containing the following concentrations of components: 150 mM KCl, 5 mM K-HEPES, pH 7.4, 2.5 mM Tris phosphate, 0.5 mM malate, 10 mM 5,5-dimetyloxazolidine-2,4-dione, 100 M inulin, 10 M TTP, 50 mg of dextran 40 (to facilitate passage of mitochondria though an oil layer (33)), 0.18 Ci/ml [ 14 C]TTP, and 10 mg of mitochondrial protein in a total of 2.0 ml.
Incubation was performed in uncapped tubes at 25°C. For measurement of the transmembrane pH difference (⌬pH), labeled TTP was exchanged for 0.18 Ci of [ 14 C]5,5-dimetyloxazolidine-2,4-dione/ml ϩ 0.6 Ci of [ 3 H]inulin/ml. Experiments were started by addition of substrate, either 40 nmol of palmitoyl-L-carnitine/mg of mitochondrial protein, in the presence of 1 mM malate, pH 7.4, or 2.5 mM Tris succinate, pH 7.4, in the presence of 10 g of rotenone. At 3 and 5 min appropriate samples were withdrawn and added to tubes previously charged with a silicon oil layer ( ϭ 1.5) above a layer of 10% (w/v) perchloric acid ( ϭ 1.8), followed by centrifugation for 1 min at 15,000 rpm (Eppendorf microcentrifuge). For the ⌬ experiments, the disappearance of [ 14 C] from the upper, incubation medium layer was recorded by subjecting aliquots of that layer to scintillation counting, whereas for the ⌬pH experiments, aliquots were withdrawn from both the upper as well from the bottom (perchloric acid) layers, and subjected to dual-channel scintillation counting. Determination of intra-mitochondrial volume was made by recording the transmembrane distributions of [ 14 C]sucrose and 3 H 2 O (32). The sucrose impermeable space of liver mitochondria isolated from animals given dietary palmitate was 1.27 Ϯ 0.11 versus 1.28 Ϯ 0.14 l/mg of protein for the TTA-treated ones (n ϭ 12). No correction was applied for possible overestimation of ⌬ because of passive TTP binding since the measured ⌬ was always higher than the figure where deviation from Nernst behavior has been demonstrated (34). Parallel, polarographic incubations were used to verify that a steady state rate of oxygen uptake existed within the time frame of withdrawal of aliquots.
Mitochondrial Fatty Acid Composition-TTA suspended in 0.5% carboxymethylcellulose was administered to rats by orogastric intubation (150 mg/kg body weight) once daily for 10 days. Control animals received carboxymethylcellulose only. PPAR Ϫ/Ϫ and PPAR ϩ/ϩ mice were given the diet described above. Lipids were extracted from the liver mitochondrial fractions of rats and mice, transesterified with BF 3methanol, and analyzed essentially as described in Ref. 35. The methyl esters of fatty acids were analyzed on a GC 8000 Top gas chromatograph (Carlo Erba Instrument), equipped with a flame ionization detector, programmable temperature of vaporization injector, AS 800 autosampler (Carlo Erba Instrument), and a capillary column (60 m ϫ 0.25 mm) containing a highly polar SP 2340 phase with film thickness 0.20 mm (Supelco). Natural occurring fatty acids were positively identified by comparison to known standards (Larodan Fine Chemicals, Malmö, Sweden) and verified by mass spectrometry. Quantification of the fatty acids was based on heneicosanoic acid (21:0) as an internal standard.
Isolation of mRNA and Quantitation by Real-time PCR Analysis-Total RNA was extracted from freeze-clamped liver using Trizol TM reagent (Invitrogen). Quantitative real-time PCR was carried out using ABI PRISM TM 7900 HT sequence detection system (Applied Biosystems, Foster City, CA) with conditions and reagents as recommended by the manufacturer. Each sample was analyzed in triplicate. Sequencespecific PCR primers and TaqMan probes for UCP-2 and the GAPDH cDNAs were designed using Primer Express software (Applied Biosystems). The following primers and probes were used: GAPDH: primers, 5Ј-TGCACCACCAACTGCTTAGC-3Ј and 5Ј-CAGTCTTCTGAGTGGC-AGTGATG-3Ј, and probe, 5Ј-TGGAAGGGCTCATGACCACAGTCCA-3Ј; UPC-2: primers, 5Ј-TGGCCTCTACGACTCTGTAAAGC-3Ј and 5Ј-CAG-GGCACCTGTGGTGCTA-3Ј, and probe, 5-CAAGGGCTCAGAGCATG-CAGGCA-3Ј. The GAPDH was used as endogenous control for normalization of cDNA amounts. This analysis was also performed on isolated hepatocytes (performed as described above) that were purified by centrifugation on a 45% Percoll cushion to minimize the influence from Kuppfer cells (17).
Western Analysis-Protein from extracts were separated by SDS-PAGE and transferred to nitrocellulose membrane (Hybond ECL, Amersham) according to standard techniques. Blots were probed with a polyclonal goat antibody to UCP-2 (sc-6525, Santa Cruz Biotechnology Inc.) and goat horseradish peroxidase-conjugated anti-rabbit antibody (Bio-Rad).
Statistics and Presentation of Results-The data are presented as mean Ϯ S.D., and differences were evaluated by a two-sample Student's t test (two-tailed distribution) where relevant. p Ͻ 0.05 was regarded as statistical significant.

Energy Parameters in Rat
Liver-To investigate whether the hypolipidemia caused by supplementing rat diets with 3-thia fatty acids is associated with perturbation of the liver energy state, we established a 7-day regime of feeding animals TTA compared with a set of control animals receiving palmitic acid (which does not cause hypolipidemia). TTA treatment resulted in a lowering of energy state parameters such as phosphate potential by 30% and energy charge by 13%, compared with control livers (Table I). Simultaneously, the liver NAD ϩ /NADH and NADP ϩ /NADPH redox pairs became more oxidized. Whereas the total amounts of adenine nucleotides or inorganic phosphate remained unaffected by the dietary treatment, the amounts of nicotinamide adenine dinucleotide almost doubled and that of nicotinamide adenine dinucleotide phosphate increased by 30%, strongly suggesting that TTA stimulates the biosynthetic pathways for pyridine nucleotides in liver or, alternatively, inhibits glycohydrolases, which converts NAD to nicotinamide.
Oxidative Capacities of Primary Hepatocytes from TTAtreated Rats-Hepatocytes prepared from animals given TTA versus palmitic acid in their diets would be expected to expose facets of a mechanism for lowering the liver energy state. Accordingly, a study was undertaken of cellular production of acid-soluble products from labeled fatty acid substrates (as a measure of ␤-oxidation) as well as of fatty acid-stimulated ketogenesis and respiratory rates. As shown in Table II, TTA feeding caused increases in rates of cellular ␤-oxidation by 1.4 -1.9-fold, eicosapentaenoic acid (EPA) being the better substrate among the fatty acids supplied. Stimulation by TTA feeding on ketogenesis in cells was more pronounced for palmitic acid (2-fold) and EPA (1.9-fold) than for docosahexaenoic acid (DHA; 1.5-fold) as sources of carbon. The fatty acid-stimulated rates of oxygen uptake responded similarly (1.3-fold increase) to TTA feeding of source animals. The oxygen uptake rates measured in the uncoupled state (preincubation with FCCP) were almost identical with any fatty acid substrate, regardless of feeding regime, indicating that the capacity of the mitochondrial respiratory chain had not been altered by the diet supplement. However, as a consequence there was some indication of a lowering of energy transducing activity in cells from TTA-treated rats compared with controls. Thus, respiratory control ratios for all substrates were significantly lower: from 2.8 to 1.9 for palmitic acid, from 2.8 to 2.0 for EPA, and from 2.9 to 2.2 for DHA. These changes, mitochondrial in origin, may plausibly be interpreted as increased proton conductance, leading to lower rates of ATP production.
To test for possible lack of citric acid cycle intermediates in mitochondria, hepatocytes were preincubated with malate. This addition affected neither ␤-oxidation nor oxygen uptake rates with palmitic acid as substrate but, surprisingly, did cause a stimulation of ketogenesis 7-fold in cells from palmitic acid-treated rats against 4.4-fold for the TTA-fed rat cells. As neither O 2 uptake rates, nor rates of ␤-oxidation were correspondingly elevated in the presence of added malate, carbon from malate would appear to be channeled to ketone bodies, possibly from pyruvate via malic enzyme (the precise source of carbon for ketogenesis under these conditions is under investigation).
Effect of TTA as Diet Supplement on the Energy Metabolism of Isolated Rat Liver Mitochondria-One probable cause of the low energy state of the liver in rats subjected to dietary treatment with TTA would be deficient ATP production. Consequently, liver mitochondria isolated from the two groups of animals were examined for energy transducing properties. TTA treatment stimulated the oxygen uptake rates of mitochondria oxidizing palmitoyl-CoA or palmitoyl-L-carnitine by 25 and 20%, respectively (Table III). The measured rates of O 2 uptake were not different from those recorded where the mitochondria had been preincubated with 0.3 nmol of oligomycin/mg of mitochondrial protein, and were thus regarded as representing state 4. However, the chosen concentration of added malate (5 mM) may have been excessive as replenishment of lost citric acid cycle intermediates and competed with the products of ␤-oxidation as source of reducing equivalents for the respiratory chain. Thus, in the presence of 0.5 mM malate O 2 uptake rates with fatty acyl substrates were lower, but stimulation of state 4 in mitochondria from TTA-treated rats became even more pronounced (50% increase). Oxidation rates with succinate (ϩ rotenone) were approximately equal with mitochondria from the two groups of rats. Yet, the overall parameters of energy transduction, respiratory control, and ADP/O ratios, decreased significantly by 10% with mitochondria from TTAtreated rats versus controls regardless of the substrate used, suggesting that an increase in proton conductance had taken place. A similar effect has been detected in liver mitochondria of mice subjected to long term dietary treatment with TTA (36).
In parallel experiments, rates of ␤-oxidation from palmitoyl-CoA had increased by 40% (not shown) over controls, in agreement with previous studies (6,37).
Energy transduction in rat liver mitochondria from the two groups of rats was investigated by measuring proton electrochemical potentials (⌬p) under conditions where O 2 uptake rates were in a steady state. From Fig. 1A it appears that mitochondria from TTA-treated rats, respiring on succinate (ϩ rotenone) or palmitoyl-L-carnitine, maintained a ⌬p 15% lower than that of control mitochondria. Furthermore, this change affected the ⌬ component only (Fig. 1B), leaving the ⌬pH component unaltered. Thus, although the O 2 uptake rates with fatty acyl substrates had increased (Table III), this increase was clearly insufficient to compensate for the loss of ⌬. It follows that TTA as a dietary supplement for rats results in an increase in proton conductance across liver mitochondrial membranes.
Also addressed was the question of whether the concentration of nonesterified TTA, which obtains in the liver under the given feeding conditions, would be sufficiently high to acutely cause the observed loss of ⌬. According to Ref. 38 rats receiving 300 mg of TTA/kg of body weight as a dietary supplement yield a total liver concentration of 20 nmol of nonesterified TTA/g wet tissue. Calculations to express this figure in terms of nanomoles/mg of mitochondrial protein were as outlined by Fleischer et al. (39), using values of 0.12 g of total protein/g wet liver and 0.25 g of mitochondrial protein/g of total protein that yields the figure of 30 mg of mitochondrial protein/g wet liver.
The amount of nonesterified TTA in the liver of treated animals would therefore be of the order of 20/30 ϭ 0.67 nmol/mg of mitochondrial protein. In accordance with this, experiments were made in which mitochondria from control animals were preincubated with 0.6 nmol of TTA/mg of protein for 10 min prior to measurements of energy transduction parameters. As shown in Table III and Fig. 1 this amount of TTA did not measurably affect any parameter, findings that practically exclude the possibility that the observed lowering of ⌬ was directly effected by the endogenous, nonesterified TTA present.
Mitochondrial Fatty Acid Composition-TTA can be converted to CoA thioester-like natural fatty acids (40) and is incorporated into different lipid classes including phospholip-TABLE II Oxidation of fatty acids by primary hepatocytes as a function of dietary treatment by palmitic acid or TTA Experiments were performed as described under "Experimental Procedures." Production of acid-soluble radioactivity from [1-14 C] fatty acid is given as nanomoles of fatty acid consumed/h/2 ϫ 10 6 cells at 30°C, ketogenesis is given as production of ␤-hydroxybutyrate as micromoles/h/2 ϫ 10 6 cells at 37°C, and rates of oxygen consumption are given as nanogram atoms of oxygen/h/2 ϫ 10 6 cells at 37°C. The concentrations of FCCP and malate were 5 M and 10 mM, respectively. PA, palmitic acid. ids (38,41). To test the hypothesis that TTA action on ⌬ could be mediated by alterations of fatty acid composition, mitochondria were isolated from TTA-treated rats and controls. The treatment markedly changed the total fatty acid composition in liver mitochondria (Table IV). TTA accumulated in the mitochondria while the levels of the saturated fatty acids 14:0, 15:0, and 17:0 decreased. The total amount of monounsaturated fatty acids was increased, especially the ⌬ 9 -monounsaturated fatty acids. The ⌬ 9 -desaturated product of TTA was also detected. TTA treatment altered the levels of polyunsaturated fatty acids of the n-3 and n-6 families. The amounts of 18:2 n-6, 18:3 n-3, and 20:5 n-3 were significantly lowered, whereas 18:3 n-6 and 20:3 n-6 were increased. Altogether, these changes resulted in lowering of the double bond index. It is worth noting that the levels of mitochondrial arachidonic acid (20:4 n-6) as well as 22:6 n-3 were unchanged after administration of TTA. Expression of UCP-2-Members or the UCP family of proteins are able to deplete the mitochondrial proton gradient by allowing transmembrane proton transfer without the production of ATP. The biochemical activities and biological functions of the recently identified UCP-2 and UCP-3 proteins are not well known, but studies have suggested that they play roles in energy expenditure for adaptation of cellular metabolism to an excessive supply of substrates to regulate the ATP level, NAD ϩ / NADH ratio, and various metabolic pathways. UCP-2 may also exert a protective role against formation of free radicals (15). In light of the hypothesized function of UCP-2 in energy regulation, and the central role of the liver in overall energy metabolism, we proposed that hepatocyte UCP-2 would be regulated by TTA. Indeed, TTA treatment of rats stimulated UCP-2 mRNA expression in the liver ( Fig. 2A), whereas expression of UCP-1 was not detected (data not shown). Previously, it has been shown that Kupffer cells are responsible for a major portion of the UCP-2 expression in rat liver (42). We therefore measured the UCP-2 mRNA expression in isolated and purified primary hepatocytes from control rats and TTA-treated rats. According to Armstrong and Towle (17) such a procedure allows us to measure UCP-2 in the hepatocyte. Our results clearly showed that the mRNA level of UCP-2 was elevated in hepatocytes from TTA-treated animals ( Fig. 2A). This demonstrates that the enhanced UCP-2 mRNA level observed in liver tissue from animals treated with TTA at least in part can be explained by an increased UCP-2 expression in the hepatocytes.
Induction of UCP-2 expression was also found at the protein level in liver from TTA-treated rats (Fig. 2B).

Effect of TTA on UCP-2 Expression and Mitochondrial Fatty Acid Composition in Wild Type and PPAR␣-deficient Mice-
To evaluate more conclusively that the effects of TTA are at the level of PPAR␣ activation, as opposed to effects because of incorporation into mitochondrial membrane lipids, a study was performed in wild type and PPAR␣-deficient mice. A non-fatty acid PPAR activator, fenofibrate, was chosen as a reference control to differentiate effects because of TTA versus simple PPAR activation. Fig. 2C shows that UCP-2 mRNA was increased more than 6-fold in wild type mice given fenofibrate. This effect was completely abolished in PPAR␣-deficient mice, demonstrating that fenofibrate induce UCP-2 mRNA expression via PPAR␣, confirming the results reported by Kelly et al. (18). It is noteworthy that TTA induced UCP-2 expression both in wild type and PPAR␣-deficient mice, suggesting that UCP-2 induction also may be mediated by alternative pathways.
The effect of TTA on mitochondrial fatty acid composition in wild type and PPAR␣-deficient mice was assessed (Table V). The treatment markedly changed the total fatty acid in mitochondria of both mice strains. TTA and ⌬9-desaturated TTA accumulated in the mitochondria to a much higher level in PPAR␣-deficient mice (Table V). Thus, the changes in mitochondrial fatty acid composition in the liver of PPAR␣-deficient mice reflected the changes in the rat, i.e. the total amount of monounsaturated fatty acids was increased, especially the ⌬9 monounsaturated fatty acids; the amounts of 18:2 n-6 and 20:5 n-3 were decreased; the amounts of 22:6 n-6 and 20:4 n-6 were unchanged, resulting in lowering of the double bound index. Differences in fatty acid composition in mitochondria from rats versus PPAR␣-deficient mice were observed in the level of saturated fatty acids and especially on 20:3 n-6.

DISCUSSION
The main finding in this investigation was the lowered energy state of the liver in rats receiving a diet containing the hypolipidemic fatty acid TTA as compared with control rats receiving palmitic acid. This conclusion is evident from data on the phosphorylation state and energy charge pertaining in the liver in vivo.
The measured phosphorylation state (Table I) may not be a proper reflection of the effective "phosphate potential" as it TABLE III Energy transducing parameters and rates of oxygen uptake of isolated mitochondria from palmitate-and TTA-treated rats Oxygen uptake was measured at 25°C with a Clark electrode system under the conditions given in "Experimental Procedures." Uptake rates are presented as nanogram atoms of oxygen/min/mg of mitochondrial protein. Final concentrations of substrates were 50 nmols/mg of mitochondrial protein for palmitoyl-CoA (ϩ1 mM L-carnitine) or palmitoyl-L-carnitine, and 5 mM succinate (ϩ1 g of rotenone/mg protein). Preincubation was for 10 min with TTA at a concentration of 0. 6  . In this context, the findings of a lowered phosphorylation state and a more oxidized state of both pyridine nucleotide redox pairs in livers of rats receiving TTA would be readily explainable. Present experiments, performed with 12-h fasted animals, did not examine rates of gluconeogenesis and it is therefore not known whether the TTA treatment elevates rates above those of controls although lowered figures for the calculated energy charge might be indicative of an increase in the rate of ATP utilization or decreased ATP generation. The marked elevation of the total tissue con-tents of pyridine nucleotides could indicate increased biosynthesis of these components from nicotinamide, and it is tempting to speculate that this resulted from the TTA-stimulated peroxisomal and mitochondrial proliferation (44) causing upregulated expression of enzymes and other proteins that bind pyridine nucleotides, which might reflect adaptation for increased ␤-oxidation flux. The regulation of pyridine nucleotide synthesis in the liver is not completely understood, but is said to function under hormonal control (see Ref. 45).
If lowered phosphorylation state of the tissue was caused by impaired mitochondrial energy transduction, then parameters illustrating this effect should respond accordingly. Thus, fatty acid-stimulated rates of oxygen uptake in hepatocytes as well as isolated mitochondria had increased by 30% as a result of TTA treatment. Simultaneously, mitochondria exhibited lowered respiratory control-and ADP/O ratios. That the change in state 4 respiration was a true reflection of altered energy transduction received strong support from lowered mitochondrial proton electrochemical potentials measured under steady-state substrate oxidation (Fig. 1), indicative of higher proton conductance in liver mitochondria of TTA-treated rats. The data presented does not allow a precise prediction of the mechanism involved. However, liver mitochondria have previously been shown to exhibit native proton leakage (46, 47), whereby protons pumped out of the matrix during substrate oxidation are able to re-enter the matrix space through conductance paths that evade the ATP synthase. The nature and properties of the proton leakage, be it uncoupling or electrophoretic ion exchange, has been intensely investigated in recent years (as reviewed Ref. 32), but discrete mechanisms remain incompletely understood. One aspect of this leak has been termed non-ohmic (48) because it is characterized by non-proportionality between the respiratory rate in state 4 and the corresponding ⌬p under conditions where mitochondria maintain a high ⌬. This condition does clearly not apply in the present experiments, in which the range of measured membrane potentials (90 -120 mV) was below the threshold value for the non-ohmic leak (49). Therefore, we do not consider this a valid FIG. 1. Proton electrochemical potentials of isolated liver mitochondria from rats given palmitic acid or TTA in the diet: steady state 4 oxidation. Measurements were performed at 25°C as given under "Experimental Procedures." Numbers on the abscissa refer to the following substrates and treatments: 1, 10 mM succinate (ϩ rotenone), palmitic acid feeding; 2, 10 mM succinated (ϩ rotenone) ϩ 0.6 nmol of TTA/mg of protein, palmitic acid feeding; 3, 10 mM succinate (ϩ rotenone), TTA feeding; 4, 50 nmol of palmitoyl-L-carnitine/mg of protein, palmitic acid feeding; 5, 50 nmol of palmitoyl-L-carnitine/mg of protein ϩ 0.6 nmol of TTA/mg of protein, palmitic acid feeding; 6, 50 nmol of palmitoyl-L-carnitine/mg of protein, TTA feeding. A, ⌬p ϭ ⌬ Ϫ 59 ⌬pH, where numbers refer to mean ⌬p for each column. B, components of ⌬p. Columns are given as mV Ϯ S.D. (n ϭ 5). *, p Ͻ 0.01. explanation for the observed leak rate in liver mitochondria of TTA-treated animals. At this low level of ⌬p any loss of ⌬ should be compensated by a corresponding increase in ⌬pH unless an electrophoretic mechanism for proton re-entry was brought into action. We would therefore suggest that the TTA treatment of rats results in expression of a respiration-dependent electrogenic ion transport system in the mitochondrial membrane, allowing a moderate degree of uncoupling. The question of the cause of the increased proton leak in liver mitochondria of the TTA-treated rats remains. The well known protonophoric effect of fatty acids on the inner membrane (14,50,51) does not apply to the uncoupling that is induced by TTA, because only the ⌬ component, and not ⌬pH, is affected. This is in contrast to what was observed when TTA was provided at a concentration of 30 nmol/mg of protein to liver mitochondria in vitro, which affected both components (10). Calculations (see above) have shown that the content of nonesterified TTA in the liver after TTA dietary treatment is ϳ0.7 nmol/mg of mitochondrial protein, probably even lower because of marked mitochondrial proliferation (44). Addition of this low amount of TTA to mitochondria from control animals in vitro failed to effect any measurable change in energy transduction parameters (Table  III and Fig. 1).
If dietary treatment with TTA directly inflicts aberrations to mitochondrial membranes, likely targets would be changes in phospholipid components. Indeed, TTA and its ⌬ 9 -desaturated product are incorporated into mitochondrial lipids in rat liver (Table IV), even into phospholipids such as phosphatidylethanolamine, cardiolipin, and phosphatidylcholine. 2 According to Brookes et al. (52) variations in the fatty acid composition of mitochondrial membrane phospholipids reconstituted in liposomes did not appear to accommodate large differences in proton conductance. In isolated mitochondria, however, the same authors found that proton leak correlated with several phospholipid fatty acid compositional parameters, including unsaturation index, 14:0, 18:1 n-9, 18:3 n-3, 18:3 n-6, and percent of monounsaturated fatty acids (53). Other reports support the understanding that certain aspects of phospholipid composition appear to be strongly correlated to proton conductance (54). Fatty acid composition in rat liver mitochondria was changed after TTA administration (Table IV). The mitochondrial content of 14:0, 17:0, and 20:5 n-3, but not 22:6 n-3, was decreased, probably because of the increased rate of mitochondrial ␤-oxidation (Table II) (55)(56)(57) and replacement of saturated and unsaturated fatty acids with TTA and ⌬ 9 -desaturated TTA. This is in agreement with data from Table II and our earlier findings that 20:5 n-3 is a better substrate for fatty acid oxidation than 22:6 n-3 (56,58). Free fatty acids may uncouple respiration, e.g. via a flip-flop mechanism. The insertion of sulfur in the hydrocarbon chain of TTA makes the fatty acid more hydrophilic than a normal fatty acid, which probably restrains flip-flop across the hydrophobic phase. In support, addition of physiological amounts of TTA (38) to isolated mitochondria did not increase state 4 oxygen consumption, indicating that the mitochondrial membrane proton conductance remained unchanged.
In recent years, protein components, including the novel family of UCPs (UCP-1, UCP-2, and UCP-3) have become implicated as membrane vehicles for proton leakage (15,59). It was of considerable interest to observe that UCP-2 mRNA and protein levels were increased in the liver of rats and mice under dietary treatment with TTA (Fig. 2). UCP-2 expression has been detected both in Kupffer cells (42) and in hepatocytes (17). It was confirmed that TTA-mediated UCP-2 induction takes place in hepatocytes by assessing the expression in isolated and purified hepatocytes (Fig. 2). For EPA (20:5 n-3), oleic acid (18:1 n-9), and arachidonic acid (20:4 n-6), such behavior involves regulation through a prostaglandin/PPAR␣-mediated pathway (17). However, in contrast to the PPAR␣ selective drug fenofibrate, TTA induced UCP-2 expression to an equal level in wild type and PPAR␣-deficient mice (Fig. 2) although the mitochondrial fatty acid composition was differently changed (Table V), especially concerning the level of TTA itself, which accumulated to a much higher degree in mitochondria of PPAR␣-deficient than wild type animals. These data indicate that UCP-2 is up-regulated by TTA through a PPAR␣-independent pathway that is unlikely to involve incorporation of TTA into mitochondrial lipids and accompanied changes of fatty acid composition. TTA is a ligand for PPAR␦ (8, 60) and FIG. 2. UCP-2 expression is increased in liver of TTA-treated rats. A, UCP-2 mRNA expression was measured in liver from male Wistar Rats that were given soy oil (control) or soy oil with TTA (150 or 300 mg/kg body weight per day) by orogastric intubation once daily for 10 days. Determinations were carried out three times for each sample, and the level of UCP-2 was normalized to the GAPDH value. The data is presented as -fold induction compared with control Ϯ S.D. (n ϭ 4). Primary hepatocytes were prepared and purified from animals receiving control diet or diet with TTA (approximate dose of 300 mg/kg/day) for 7 days as described under "Experimental Procedures." The columns represent means of two experiments. B, UCP-2 protein was determined by Western analysis in liver extracts from rats given control, fish oil, or TTA treatment. C, UCP-2 mRNA level was analyzed in livers from wild type (WT) and PPAR␣-deficient (KO) mice receiving control diet or a diet with fish oil, TTA, or fenofibrate. Results are expressed as mean Ϯ S.D. (n ϭ 6). *, p Ͻ 0.01. recently it has been published that this PPAR subtype might play a role in the regulation of muscle lipid homeostasis (61). Thus, it is likely that in liver of PPAR␣-deficient mice, the level of PPAR␦ can compensate for the deficiency of PPAR␣. As mitochondrial fatty acid metabolizing proteins are encoded by PPAR␣, and possibly PPAR␦, target genes, we propose that PPAR␣ and PPAR␦ may play a role in determining the metabolic shift, i.e. increased ␤-oxidation flux and lowered energy state. Elevated expression of the hepatic UCP-2 mRNA is of special interest in this context, in light of the proposal that the UCP-2 protein participates in proton leak mechanisms. The possibility that UCP-2 is involved in electrogenic ion transport in the mitochondrial membrane, allowing a moderate degree of uncoupling, should be considered.
TTA improves insulin sensitivity and reduces adiposity (4,9). Drainage of fatty acids by the liver, relieving the fatty acid pressure on adipose tissue and muscles when fatty acids inhibit glucose uptake and oxidation, is an important effect of TTA. Uncoupling of hepatocyte mitochondria by long chain fatty acids under ketogenic conditions may allow production of ketone bodies at rates that are not limited by liver ATP consumption and requirements. TTA increased liver fatty acid oxidation and ketogenesis (Table II), accompanied by stimulated UCP-2 expression (Fig. 2). Thus, these results indicate a possible role of liver UCP-2 in the control of energy status. Accordingly, the UCP-2 protein may be involved in adaptation of lipid metabolism to an excessive supply of fatty acids to regulate the ATP level, the NAD ϩ /NADH ratio, and various metabolic pathways such as ketogenesis (15).
It has also been suggested that UCP-2 is involved in regulation of free radical formation within the cell. Induction of UCP-2 might decrease the redox pressure by preventing the escape of reactive oxygen species from the electron transport chain (62). Indeed, TTA is reported to have antioxidant effects in vitro and in vivo, including decrease in plasma lipid peroxides and malondialdehyde (63)(64)(65). In agreement with other suggestions (63), these observations provide indirect evidence for UCP-2 playing a role in management of redox homeostasis.
In summary, long term dietary treatment of rats with 3-thia fatty acid results in lowering of the liver energy state while lipid metabolism become poised toward increased mitochondrial fatty acid oxidation accompanied by increased rates of ketogenesis and oxygen uptake, over and above that which is characteristic of the fasted state in control animals. Simultaneously, the liver mitochondria acquire a definitive proton leakage, characterized by a partial loss of the proton electrochemical potential maintained during substrate oxidation in vitro. Whatever the vehicle or mechanism responsible for membrane leakiness, be it induced ion electrogenic transport, induced UCP-2 expression, fatty acid-dependent uncoupling, or compositional change in membrane phospholipids, increased rates of proton re-entry would support higher rates of ␤-oxidation. Elevated ␤-oxidation flux, aided by up-regulated carnitine palmitoyltransferase-II (4), would preferentially channel reducing equivalents to the respiratory chain at the expense of the citric acid cycle (66,67). Simultaneously, there is up-regulation of the mitochondrial hydroxymethylglutaryl-CoA synthase, resulting in a 60% increase in plasma ␤-hydroxybutyrate (6), suggesting that the liver ␤-hydroxybutyrate dehydrogenase should possess an additional capacity for re-oxidation of NADH during fatty acid oxidation. The changes in fatty acid catabolism because of long term dietary treatment with TTA is judged to be sufficiently moderate to warrant the conclusion that liver lipid metabolism remains within the confines of its normal regulatory network.

TABLE V
Mitochondrial fatty acid composition in livers from wild type and PPAR␣-deficient mice after treatment with TTA Fatty acid composition of liver mitochondria was analyzed in control and TTA-treated mice after 5 days. The data are given as mol % Ϯ S.D. (n ϭ 6), (SFA, saturated fatty acids; MUFA, monounsaturated fatty acids).