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Differential effects of coconut oil- and fish oil-enriched diets on tricarboxylate carrier in rat liver mitochondria

2003; Elsevier BV; Volume: 44; Issue: 11 Linguagem: Inglês

10.1194/jlr.m300237-jlr200

1539-7262

Anna Maria Giudetti, Simona Sabetta, Roberta di Summa, Monica Leo, Fabrizio Damiano, Luisa Siculella, Gabriele V. Gnoni,

Peroxisome Proliferator-Activated Receptors

The mitochondrial tricarboxylate carrier (TCC) plays an important role in lipogenesis being TCC-responsible for the efflux from the mitochondria to the cytosol of acetyl-CoA, the primer for fatty acid synthesis. In this study, we investigated the effects of two high-fat diets with different fatty acid composition on the hepatic TCC activity. Rats were fed for 3 weeks on a basal diet supplemented with 15% of either coconut oil (CO), abundant in medium-chain saturated fatty acids, or fish oil (FO), rich in n-3 polyunsaturated fatty acids. Mitochondrial fatty acid composition was differently influenced by the dietary treatments, while no appreciable change in phospholipid composition and cholesterol level was observed. Compared with CO, the TCC activity was markedly decreased in liver mitochondria from FO-fed rats; kinetic analysis of the carrier revealed a decrease of the Vmax, with no change of the Km. No difference in the Arrhenius plot between the two groups was observed. Interestingly, the carrier protein level and the corresponding mRNA abundance decreased following FO treatment.These data indicate that FO administration markedly decreased the TCC activity as compared with CO. This effect is most likely due to a reduced gene expression of the carrier protein. The mitochondrial tricarboxylate carrier (TCC) plays an important role in lipogenesis being TCC-responsible for the efflux from the mitochondria to the cytosol of acetyl-CoA, the primer for fatty acid synthesis. In this study, we investigated the effects of two high-fat diets with different fatty acid composition on the hepatic TCC activity. Rats were fed for 3 weeks on a basal diet supplemented with 15% of either coconut oil (CO), abundant in medium-chain saturated fatty acids, or fish oil (FO), rich in n-3 polyunsaturated fatty acids. Mitochondrial fatty acid composition was differently influenced by the dietary treatments, while no appreciable change in phospholipid composition and cholesterol level was observed. Compared with CO, the TCC activity was markedly decreased in liver mitochondria from FO-fed rats; kinetic analysis of the carrier revealed a decrease of the Vmax, with no change of the Km. No difference in the Arrhenius plot between the two groups was observed. Interestingly, the carrier protein level and the corresponding mRNA abundance decreased following FO treatment. These data indicate that FO administration markedly decreased the TCC activity as compared with CO. This effect is most likely due to a reduced gene expression of the carrier protein. A large body of evidence shows that hepatic lipogenesis is regulated by both nutritional and hormonal factors [for review see Ref. (1Hillgartner F.B. Salati L.M. Goodridge A.G. Physiological and molecular mechanism involved in nutritional regulation of fatty acid synthesis.Physiol. Rev. 1995; 75: 47-76Crossref PubMed Scopus (401) Google Scholar)]. In particular, it is well documented that dietary polyunsaturated fatty acids (PUFAs) are noticeably effective in inhibiting hepatic lipogenesis and in lowering hypertriglyceridemia, n-3 fatty acids being more potent than n-6 lipids in this respect (2Harris W.S. Connor W.E. McMurry M.P. The comparative reduction of the plasma lipids and lipoproteins by dietary polyunsaturated fats: salmon oil versus vegetable oils.Metabolism. 1983; 32: 179-184Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 3Rustan A.C. Christiansen E.N. Drevon C.A. Serum lipids, hepatic glycerolipid metabolism and peroxisomal fatty acid oxidation in rats fed ϖ-3 and ϖ-6 fatty acids.Biochem. J. 1992; 283: 333-339Crossref PubMed Scopus (108) Google Scholar). 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Sterol regulation element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats.J. Biol. Chem. 1999; 274: 23577-23583Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). The n-3 PUFAs also play a crucial role as "fuel partitioners," in that they direct fatty acids away from triacylglycerol storage and toward oxidation (11Clarke S.D. Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome.J. Nutr. 2001; 131: 1129-1132Crossref PubMed Scopus (324) Google Scholar). They act by upregulating the expression of genes encoding proteins involved in fatty acid oxidation while downregulating genes encoding proteins of lipid synthesis (11Clarke S.D. Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome.J. Nutr. 2001; 131: 1129-1132Crossref PubMed Scopus (324) Google Scholar). On the other hand, monounsaturated fatty acids like oleate (C18:1, n-9) or saturated fatty acids like palmitate (C16:0) and medium-chain fatty acids [as present in coconut oil (CO)] do not inhibit either the activities or the expression of the lipogenic enzymes (6Jump D.B. Clarke S.D. Thelen A. Liimatta M. Ren B. Badin M. Dietary polyunsaturated fatty acid regulation of gene transcription.Prog. Lipid Res. 1996; 35: 227-241Crossref PubMed Scopus (95) Google Scholar, 8Baltzell J.K. Berdanier C.D. Effect of the interaction of dietary carbohydrate and fat on the responses of rats to starvation-refeeding.J. Nutr. 1985; 115: 104-110Crossref PubMed Scopus (26) Google Scholar, 12Clarke S.D. Abraham S. Gene expression: nutrient control of pre- and posttranscriptional events.FASEB J. 1992; 6: 3146-3152Crossref PubMed Scopus (114) Google Scholar). Lipogenesis requires cooperation between mitochondrial and cytoplasmic enzymes and involves fluxes of metabolites across the mitochondrial membranes (13Watson J.A. Lowenstein J.M. Citrate and the conversion of carbohydrate into fat. Fatty acid synthesis by a combination of cytoplasm and mitochondria.J. Biol. Chem. 1970; 245: 5993-6002Abstract Full Text PDF PubMed Google Scholar). The mitochondrial tricarboxylate carrier (TCC) (also known as citrate carrier), an integral protein of the mitochondrial inner membrane, catalyzes the efflux of citrate from the mitochondrial matrix in exchange for tricarboxylates, dicarboxylate (malate), or phosphoenolpyruvate across the mitochondrial inner membrane (14Krämer R. Palmieri F. Metabolite carriers in mitochondria.in: Ernster L. Molecular Mechanism in Bioenergetics. Elsevier Science Publishers, Amsterdam1992: 359-384Crossref Scopus (116) Google Scholar). This carrier protein plays a pivotal role in intermediary metabolism by connecting carbohydrate with the lipid metabolism. In fact, it transports in the form of citrate acetyl-CoA, mainly derived from the sugar source, from mitochondria to the cytosol. Here, by the action of ATP-citrate lyase, citrate provides the carbon units for fatty acid and cholesterol biosynthesis. In addition, it supplies NAD+ and NADPH, which support cytosolic glycolysis and lipid biosynthesis, respectively (15Kaplan R.S. Mayor J.A. Structure, function and regulation of the tricarboxylate transport protein from rat liver mitochondria.J. Bioenerg. Biomembr. 1993; 25: 503-514Crossref PubMed Scopus (30) Google Scholar). TCC has been extensively characterized in mammalian (16Bisaccia F. De Palma A. Palmieri F. Identification and purification of the tricarboxylate carrier in rat liver mitochondria.Biochim. Biophys. Acta. 1989; 977: 171-176Crossref PubMed Scopus (102) Google Scholar, 17Clayes D. Azzi A. Tricarboxylate carrier of bovine liver mitochondria.J. Biol. Chem. 1989; 264: 14627-14630Abstract Full Text PDF PubMed Google Scholar, 18Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. Purification and characterization of the tricarboxylate carrier from rat liver mitochondria.J. Biol. Chem. 1990; 265: 13379-13385Abstract Full Text PDF PubMed Google Scholar) and fish (19Zara V. Iacobazzi V. Siculella L. Gnoni G.V. Palmieri F. Purification and characterization of the tricarboxylate carrier from eel liver mitochondria.Biochem. Biophys. Res. Commun. 1996; 233: 508-513Crossref Scopus (25) Google Scholar, 20Zara V. Palmieri L. Franco M.R. Perrone M. Gnoni G.V. Palmieri F. Kinetics of the reconstituted tricarboxylate carrier from eel liver mitochondria.J. Bioenerg. Biomembr. 1998; 30: 555-563Crossref PubMed Scopus (16) Google Scholar) liver mitochondria. The rat liver cDNA was cloned (21Kaplan R.S. Mayor J.A. Wood D.O. The mitochondrial tricarboxylate transport protein. cDNA cloning, primary structure and comparison with other mitochondrial transport proteins.J. Biol. Chem. 1993; 268: 13682-13690Abstract Full Text PDF PubMed Google Scholar) and overexpressed in bacteria (22Xu Y. Mayor J.A. Gremse D. Wood D.O. Kaplan R.S. High yield bacterial expression, purification, and functional reconstitution of the tricarboxylate transport protein from rat liver mitochondria.Biochem. Biophys. Res. Commun. 1995; 207: 783-789Crossref PubMed Scopus (45) Google Scholar). The cDNA sequence of yeast (23Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. High level expression and characterization of the mitochondrial citrate transport protein from the yeast Saccharomyces cerevisiae.J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), cow (24Iacobazzi V. De Palma A. Palmieri F. Cloning and sequencing of the bovine cDNA encoding the mitochondrial tricarboxylate carrier protein.Biochim. Biophys. Acta. 1996; 1284: 9-12Crossref PubMed Scopus (16) Google Scholar), and human (25Heisterkamp N. Mulder M.P. Langeveld A. Ten Haeva Z. Rose B.A. Graffen J. Localization of the human mitochondrial citrate transporter protein gene to chromosome 22Q11 in the DiGeorge Syndrome critical region.Genomics. 1995; 29: 451-456Crossref PubMed Scopus (44) Google Scholar) are also known. The nucleotide sequence of the human TCC gene has been determined (26Iacobazzi V. Lauria G. Palmieri F. Organization and sequence of the mitochondrial citrate transport protein.DNA Seq. 1997; 7: 127-139Crossref PubMed Scopus (29) Google Scholar). However, despite its important metabolic role and unlike the lipogenic enzymes, little is known about the regulation of TCC activity. A coordinate regulation of lipogenic enzyme activities in the cytosol and citrate transport activity across the mitochondrial inner membrane by nutritional factors was found (27Zara V. Gnoni G.V. Effect of starvation on the activity of the mitochondrial tricarboxylate carrier.Biochim. Biophys. Acta. 1995; 1239: 33-38Crossref PubMed Scopus (39) Google Scholar, 28Zara V. Giudetti A.M. Siculella L. Palmieri F. Gnoni G.V. Covariance of tricarboxylate carrier activity and lipogenesis in liver of polyunsaturated fatty acid (n-6) fed rats.Eur. J. Biochem. 2001; 268: 5734-5739Crossref PubMed Scopus (31) Google Scholar). In particular, a decrease of TCC activity and lipogenesis in the liver cytosol of PUFA (n-6)-fed rats was observed (28Zara V. Giudetti A.M. Siculella L. Palmieri F. Gnoni G.V. Covariance of tricarboxylate carrier activity and lipogenesis in liver of polyunsaturated fatty acid (n-6) fed rats.Eur. J. Biochem. 2001; 268: 5734-5739Crossref PubMed Scopus (31) Google Scholar). Moreover, a recent study from our laboratory showed that the starvation-induced decrease of TCC activity in rat liver is parallel with a reduction of TCC mRNA accumulation. This latter effect was due to a posttranscriptional control of the carrier gene expression (29Siculella L. Sabetta S. di Summa R. Leo M. Giudetti A.M. Palmieri F. Gnoni G.V. Starvation-induced posttranscriptional control of rat liver mitochondrial citrate carrier expression.Biochem. Biophys. Res. Commun. 2002; 299: 418-423Crossref PubMed Scopus (23) Google Scholar). In the present study, we showed that the fatty acid composition of a high-fat diet specifically affects TCC activity in rat liver mitochondria. Compared with CO-fed rats, a fish oil (FO)-supplemented diet markedly reduced the TCC activity in these organelles. This reduction can most likely be attributed to a lower content of both an immunoreactive carrier protein and an mRNA abundance in rat liver cells. Bio-Rad protein assay was purchased from Bio-Rad Laboratories (Milano, Italy). Nylon filters, Hybond N+, and nitrocellulose paper (0.45 μm) were purchased from Amersham Biosciences (Milano, Italy). [1,5-14C]citrate (specific activity, 100 mCi/mmol) was obtained from Amersham Pharmacia Biotech (Milano, Italy). [α-32P]dATP (specific activity 3,000 Ci/mmol) was purchased from Perkin Elmer Life Sciences (Milano, Italy). The 1,2,3-benzenetricarboxylic acid (1,2,3-BTA) and horseradish peroxidase-conjugated anti-rabbit immunoglobulin secondary antibodies were obtained from Sigma-Aldrich Co. (Milano, Italy). CO and FO were from Mucedola (Milano, Italy). All other reagents were of analytical grade. Male Wistar rats (150–200 g) were used throughout this study. They were housed in cages in a temperature (22 ± 1°C)- and light (light on 8:00–20:00)-controlled room and randomly assigned to one of two different groups. The first group received a basal pellet diet (Morini, S. Polo D'Enza, Reggio Emilia, Italy) enriched with 15% (wt/wt) CO for three weeks, while the second group was fed on a 15% (wt/wt) FO-enriched basal diet for the same treatment period. The basal diet consisted of: 18.8% crude protein, 3.5% crude fat with adequate amounts of essential fatty acids, 4% crude fiber, 6% ash, and a salt and vitamin mixture. The diets were prepared weekly and stored at −20°C. Fatty acid composition of the dietary lipids, determined by gas-liquid chromatographic analysis of the fatty acid methyl ester (FAME) derivatives, is reported in Table 1. In the CO-enriched diet, lauric acid (C12:0) was the most representative fatty acid, which was absent in the FO diet. Together with myristic acid (C14:0), it represented almost 60% of total CO fatty acids; the Σ saturated/Σ unsaturated fatty acid ratio was much higher in the CO diet than in the FO diet. In the latter, the n-3 series fatty acids were particularly abundant and represented about 30% of the total fatty acids. The animals had free access to food and water. Food consumption was monitored daily, and no significant difference was recorded between the two groups. The experimental design was in accordance with local and national guidelines covering animal experiments.TABLE 1Fatty acid composition (mol%) of experimental dietsFatty AcidCOFOC10:04.95 ± 0.33NDC12:040.78 ± 0.39NDC14:015.65 ± 0.306.51 ± 0.34aP < 0.001, statistically significant diet effect.C16:011.43 ± 0.1017.62 ± 0.82aP < 0.001, statistically significant diet effect.C16:1 (n-7)0.28 ± 0.059.12 ± 0.38aP < 0.001, statistically significant diet effect.C18:09.08 ± 0.093.39 ± 0.18aP < 0.001, statistically significant diet effect.C18:1 (n-9)5.18 ± 0.4817.84 ± 1.2aP < 0.001, statistically significant diet effect.C18:2 (n-6)12.90 ± 0.7213.99 ± 1.1C18:3 (n-3)ND2.44 ± 0.15C20:5 (n-3)0.42 ± 0.1313.55 ± 0.90aP < 0.001, statistically significant diet effect.C22:5 (n-3)ND2.31 ± 0.15C22:6 (n-3)0.43 ± 0.1011.76 ± 0.92aP < 0.001, statistically significant diet effect.Σ Saturated81.89 ± 0.4527.52 ± 0.40aP < 0.001, statistically significant diet effect.Σ Unsaturated18.31 ± 0.5671.01 ± 0.77aP < 0.001, statistically significant diet effect.Σ Saturated/Σ unsaturated4.48 ± 0.170.39 ± 0.02aP < 0.001, statistically significant diet effect.CO, coconut oil; FO, fish oil; ND, not detected; Σ saturated, sum of saturated fatty acids; Σ unsaturated, sum of unsaturated fatty acids. Results are expressed as means ± SD of six determinations. Basal diet was supplemented with 15% CO or 15% FO, respectively. Fatty acids were extracted from the two different diets and analyzed by gas-liquid chromatography.a P < 0.001, statistically significant diet effect. Open table in a new tab CO, coconut oil; FO, fish oil; ND, not detected; Σ saturated, sum of saturated fatty acids; Σ unsaturated, sum of unsaturated fatty acids. Results are expressed as means ± SD of six determinations. Basal diet was supplemented with 15% CO or 15% FO, respectively. Fatty acids were extracted from the two different diets and analyzed by gas-liquid chromatography. Rat liver mitochondria were prepared following standard procedures. TCC activity was assayed in freshly isolated rat liver mitochondria as reported in Palmieri et al. (30Palmieri F. Stipani I. Quagliariello E. Klingenberg M. Kinetic study of the tricarboxylate carrier in rat-liver mitochondria.Eur. J. Biochem. 1972; 26: 587-594Crossref PubMed Scopus (173) Google Scholar). Briefly, the mitochondria (40–50 mg protein) were incubated for 2 min at 20°C in 10 ml 100 mM KCl, 20 mM HEPES, 1 mM EGTA (pH 7.0), and 2 μg/ml rotenone and loaded with l-malate (0.75 mM) essentially as described (27Zara V. Gnoni G.V. Effect of starvation on the activity of the mitochondrial tricarboxylate carrier.Biochim. Biophys. Acta. 1995; 1239: 33-38Crossref PubMed Scopus (39) Google Scholar). The exchange reaction was started by the addition to malate-loaded mitochondria (1–1.5 mg protein) of 0.5 mM [14C]citrate, unless otherwise specified, and terminated by adding 12.5 mM of the inhibitor 1,2,3-BTA (30Palmieri F. Stipani I. Quagliariello E. Klingenberg M. Kinetic study of the tricarboxylate carrier in rat-liver mitochondria.Eur. J. Biochem. 1972; 26: 587-594Crossref PubMed Scopus (173) Google Scholar). Mitochondria were then isolated by centrifugation at 18,000 g for 5 min at 2°C, washed, and extracted with 20% HClO4. The mixture was centrifuged, and the radioactivity in the supernatant was counted. Total lipids were extracted from mitochondria (10 mg protein) by the Bligh and Dyer procedure (31Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Med. Sci. 1959; 37: 911-917Google Scholar). The extracts were dried under N2 flow and resuspended in a proper volume of CHCl3. Phospholipids were separated by HPLC, as previously described (32Ruggiero F.M. Landriscina C. Gnoni G.V. Quagliariello E. Lipid composition of liver mitochondria and microsomes in hyperthyroid rats.Lipids. 1984; 19: 171-178Crossref PubMed Scopus (107) Google Scholar), by using a Beckman System Gold chromatograph equipped with an ultrasil-Si column (4.6 × 250 mm) (Chemtek Analytica, Bologna, Italy). The chromatographic system was programmed for gradient elution by using two mobile phases: solvent A, hexane-2-propanol (6:8; v/v), and solvent B, hexane-2-propanol-water (6:8:1.4; v/v/v). The percentage of solvent B in solvent A was increased in 15 min from 0% to 100%. Flow rate was 2 ml/min, and detection was at 206 nm. Single phospholipids were identified by using known standards and quantitatively assayed by determining inorganic phosphate by the procedure reported in Nakamura (33Nakamura G.R. Microdetermination of phosphorus.Anal. Chem. 1952; 241372Crossref Scopus (74) Google Scholar). To analyze fatty acids, liver mitochondria were saponified with ethanolic KOH for 2 h at 90°C. Fatty acids were extracted as in Muci et al. (34Muci M.R. Cappello A.R. Vonghia G. Bellitti E. Zezza L. Gnoni G.V. Change in cholesterol levels and in lipid fatty acid composition in safflower oil fed lambs.Int. J. Vitam. Nutr. Res. 1992; 62: 330-333PubMed Google Scholar), and their corresponding methyl esters were prepared by trans-esterification with methanolic boron trifluoride (17% BF3) at 65°C for 30 min. FAMEs were then analyzed by gas-liquid chromatography. The helium carrier gas was used at a flow rate of 1 ml · min−1. FAMEs were separated on a 30 m × 0.32 m HP5 (Hewlett Packard) capillary column. The injector and detector temperatures were maintained at 250°C. The column was operated isothermally at 150°C for 4 min and then programmed to 250°C at 4°C/min. Peak identification was performed by using known standards, and relative quantitation was automatically carried out by peak integration. Cholesterol was assayed by HPLC as described (34Muci M.R. Cappello A.R. Vonghia G. Bellitti E. Zezza L. Gnoni G.V. Change in cholesterol levels and in lipid fatty acid composition in safflower oil fed lambs.Int. J. Vitam. Nutr. Res. 1992; 62: 330-333PubMed Google Scholar). Approximately 15 and 30 μg of total RNA, extracted from livers of CO- and FO-treated rats according to Chomczynski and Sacchi (35Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63231) Google Scholar), were electrophoretically separated onto 1% formaldehyde-agarose gel under denaturing conditions and transferred to Hybond N+ nylon membrane. The RNA blots were hybridized with an α-32P-labeled probe corresponding to nucleotides 459–1421 of the rat liver TCC cDNA (21Kaplan R.S. Mayor J.A. Wood D.O. The mitochondrial tricarboxylate transport protein. cDNA cloning, primary structure and comparison with other mitochondrial transport proteins.J. Biol. Chem. 1993; 268: 13682-13690Abstract Full Text PDF PubMed Google Scholar). After hybridization, the membranes were washed twice in 2× SSC, 1% SDS at room temperature for 15 min and in 0.1× SSC, 0.1% SDS at 65°C for 30 min. For the normalization of the hybridization signals, the same membranes were stripped by washing them twice in a boiling solution of 0.1% SDS and rehybridized using a probe encoding part of the human β-actin. The membranes were stripped again and rehybridized with a randomly primed, α-32P-labeled, 800 bp FAS cDNA as a positive control. After autoradiography, the intensity of the bands was evaluated by densitometry with Molecular Analyst Software. Equal amounts of mitochondrial proteins from CO- and FO-fed animals were loaded into the wells of 15% polyacrylamide gel (0.75 mm thick). After the electrophoretic run (25 mA/gel), proteins were electroblotted on nitrocellulose membrane and stained with Ponceau. After destaining with water, membranes were subjected to the reaction with antibody directed against a C-terminal peptide of the rat-liver TCC (36Capobianco L. Bisaccia F. Michel A. Sluse F.E. Palmieri F. The N- and C-termini of the tricarboxylate carrier are exposed to the cytoplasmic side of the inner mitochondrial membrane.FEBS Lett. 1995; 357: 297-300Crossref PubMed Scopus (45) Google Scholar) and antibody directed against bovine porin. Porin, the mitochondrial outer membrane channel, was used as a control, because it has been reported that its expression is not affected by dietary PUFA (28Zara V. Giudetti A.M. Siculella L. Palmieri F. Gnoni G.V. Covariance of tricarboxylate carrier activity and lipogenesis in liver of polyunsaturated fatty acid (n-6) fed rats.Eur. J. Biochem. 2001; 268: 5734-5739Crossref PubMed Scopus (31) Google Scholar). The bound antibody was revealed by peroxidase-conjugated anti-rabbit IgG antibody by using 3,3′-diaminobenzidine and hydrogen peroxide as substrates. Blots were evaluated by densitometric analysis with Molecular Analyst Software. Protein was determined by using the method of Lowry et al. (37Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin-Phenol reagents.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with BSA as a standard. Data are the means ± SD of the indicated number of experiments. The Student's t-test was performed to detect significant differences between the two treated groups of animals. Figure 1shows the time course of citrate uptake by malate-loaded mitochondria from liver of the two groups of animals. In all the experiments, the incubation mixture contained equal amounts of mitochondrial proteins and an external citrate concentration of 0.5 mM. When the TCC inhibitor 1,2,3-BTA was added to the incubation mixture before starting the assay with [14C]citrate, the amount of citrate bound to the mitochondria was always the same. This indicates that dietary treatment was without effect on nonspecific citrate binding. As shown in Fig. 1, citrate uptake was strongly reduced in the liver mitochondria of animals fed the FO diet versus the CO diet. In particular, in the first 15 s from starting the reaction (i.e., during the linear range of the citrate uptake curve), the decrease of the transport activity in FO-treated rats was reproducibly found to be ∼60% of that found in the mitochondria from CO-treated rats (2.0 ± 0.45 nmol [14C]citrate-transported/mg protein vs. 4.6 ± 0.75 nmol [14C]citrate-transported/mg protein; P < 0.001). To identify possible changes in the kinetic parameters of the carrier, the rate of [14C]citrate/malate exchange was studied at different external citrate concentrations. On the basis of experimental data (see Fig. 2for a typical experiment), the concentration dependence of citrate uptake by all the mitochondrial preparations is represented by straight lines, thus revealing hyperbolic saturation characteristics. The FO diet induced a remarkable decrease in the Vmax value as compared with the CO diet (8.88 ± 1.49 nmol/min × mg protein vs. 19.52 ± 2.98 nmol/min × mg protein; P < 0.001), while no difference in the affinity of the carrier for citrate, as evidenced by the same Km value (0.11 mM), was observed in liver mitochondria from the two groups of rats. The TCC activity in CO- and FO-treated rats was also studied at different temperatures, and the corresponding Arrhenius plots of a representative experiment are reported in Fig. 3. The results demonstrated that citrate transport was reduced in mitochondria from FO-treated rats at all the tested temperatures compared with CO-fed animals. However, the biphasic profile of the two Arrhenius plots was identical: two linear portions with different slopes intersecting at the same temperature (∼17°C). It has been shown that the transport activities of several mitochondrial carriers are influenced by the lipid composition of the mitochondrial membranes (38Krämer R. Palmieri F. Molecular aspects of isolated and reconstituted carrier proteins from animal mitochondria.Biochim. Biophys. Acta. 1989; 974: 1-23Crossref PubMed Scopus (113) Google Scholar, 39Palmieri F. Indiveri C. Bisaccia F. Krämer R. Functional properties of purified and reconstituted mitochondrial metabolite carriers.J. Bioenerg. Biomembr. 1993; 25: 525-535Crossref PubMed Scopus (78) Google Scholar). The phospholipid and fatty acid composition, as well as the cholesterol content of mitochondrial membranes from treated animals, were, therefore, investigated. No significant variation in the percentage of the main phospholipids of rat liver mitochondria as well as in the levels of total phospholipids and cholesterol (both calculated relative to mitochondrial protein) between the two groups of rats was observed (Table 2). However, the mitochondrial fatty acid composition was noticeably different in the two groups of animals (Table 3). A lack of incorporation of C12:0, the most abundant fatty acid in the CO diet, into mitochondrial phospholipids of CO-fed rats was observed. This confirms earlier observations, which suggest that fatty acids shorter than C14:0 are not incorporated into hepatic phospholipids (40Mayorek N. Bar-Tana J. Medium chain fatty acids as specific substrates for diglyceride acyltransferase in cultured hepatocytes.J. Biol. Chem. 1983; 258: 6789-6792Abstract Full Text PDF PubMed Google Scholar, 41Hargreaves M.K. Pehowich D.J. Clandinin M.T. Effect of dietary lipid composition on rat liver microsomal phosphatidylcholine synthesis.J. Nutr. 1989; 119: 344-348Crossref PubMed Scopus (11) Google Scholar). Moreover, in the CO-enriched mitochondria, palmitic acid (C16:0) and stearic acid (C18:0) were the most prominent saturated fatty acids. In the latter mitochondria, linoleic acid (C18:2, n-6) and arachidonic acid (C20:4, n-6) were more present than in mitochondria from FO-treated rats. Mitochondria from the latter animals showed a content of n-3 fatty acids more than twice as high as those from CO-fed animals. In fact, the FO diet significantly increased the mitochondrial level of eicosapentaenoic acid (C20:5, n-3), docosapentaenoic acid (C22:5, n-3), and docosahexaenoic acid (C22:6, n-3) and, consequently, decreased the proportion of n-