n-6 PUFAs downregulate expression of the tricarboxylate carrier in rat liver by transcriptional and posttranscriptional mechanisms
2004; Elsevier BV; Volume: 45; Issue: 7 Linguagem: Inglês
10.1194/jlr.m400061-jlr200
ISSN1539-7262
AutoresLuisa Siculella, Fabrizio Damiano, Simona Sabetta, Gabriele V. Gnoni,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe tricarboxylate (citrate) carrier (TCC), a protein of the mitochondrial inner membrane, is an obligatory component of the shuttle system by which mitochondrial acetyl-CoA is transported into the cytosol, where lipogenesis occurs. The aim of this study was to investigate the molecular basis for the regulation of TCC gene expression by a high-fat, n-6 PUFA-enriched diet. Rats received for up to 4 weeks a diet enriched with 15% safflower oil (SO), which is high in linoleic acid (70.4%). We found a gradual decrease of TCC activity and a parallel decline in the abundance of TCC mRNA, the maximum effect occurring after 4 weeks of treatment. At this time, the estimated half-life of TCC mRNA was the same in the hepatocytes from rats on both diets, whereas the transcriptional rate of TCC mRNA, tested by nuclear run-on assay, was reduced by ∼38% in the rats on the SO-enriched diet. The RNase protection assay showed that the ratio of mature to precursor RNA, measured in the nuclei, decreased with the change to the n-6 PUFA diet.These results suggest that administration of n-6 PUFAs to rats leads to changes not only in the transcriptional rate of the TCC gene but also in the processing of the nuclear precursor for TCC RNA. The tricarboxylate (citrate) carrier (TCC), a protein of the mitochondrial inner membrane, is an obligatory component of the shuttle system by which mitochondrial acetyl-CoA is transported into the cytosol, where lipogenesis occurs. The aim of this study was to investigate the molecular basis for the regulation of TCC gene expression by a high-fat, n-6 PUFA-enriched diet. Rats received for up to 4 weeks a diet enriched with 15% safflower oil (SO), which is high in linoleic acid (70.4%). We found a gradual decrease of TCC activity and a parallel decline in the abundance of TCC mRNA, the maximum effect occurring after 4 weeks of treatment. At this time, the estimated half-life of TCC mRNA was the same in the hepatocytes from rats on both diets, whereas the transcriptional rate of TCC mRNA, tested by nuclear run-on assay, was reduced by ∼38% in the rats on the SO-enriched diet. The RNase protection assay showed that the ratio of mature to precursor RNA, measured in the nuclei, decreased with the change to the n-6 PUFA diet. These results suggest that administration of n-6 PUFAs to rats leads to changes not only in the transcriptional rate of the TCC gene but also in the processing of the nuclear precursor for TCC RNA. PUFAs are potent regulators of cellular metabolism. PUFAs, as components of dietary lipids, not only influence hormonal signaling events by modifying membrane lipid composition but also have a direct effect on the molecular events that govern gene expression (1Clarke S.D. Gasperikova D. Nelson C. Lapillonne A. Heird W.C. Fatty acid regulation of gene expression.Ann. N. Y. Acad. Sci. 2002; 967: 283-298Google Scholar). PUFA ingestion leads to a noticeable inhibition of the activity of many proteins/enzymes involved in both carbohydrate metabolism and lipogenesis (2Jump D.B. Clarke S.D. Regulation of gene expression by dietary fat.Annu. Rev. Nutr. 1999; 19: 63-90Google Scholar). In this regard, PUFAs have long been known to decrease the capacity of liver cells to synthesize fatty acids de novo (3Clarke S.D. Romsos D.R. Leveille G.A. Specific inhibition of hepatic fatty acid synthesis exerted by dietary linoleate and linolenate in essential fatty acid adequate rats.Lipids. 1977; 11: 485-490Google Scholar, 4Wilson M.D. Blake W.L. Salati L.M. Clarke S.D. Potency of polyunsaturated and saturated fats as short-term inhibitors of hepatic lipogenesis in rats.J. Nutr. 1990; 120: 544-552Google Scholar). This pathway is most active in liver and adipose tissue and involves a set of enzymes referred to as lipogenic enzymes (5Hillgartner F.B. Salati L.M. Goodridge A.G. Physiological and molecular mechanism involved in nutritional regulation of fatty acid synthesis.Physiol. Rev. 1995; 75: 47-76Google Scholar). These include FAS, acetyl-CoA carboxylase (ACC), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD), and 6-phosphogluconate dehydrogenase. Consistent with their role in energy metabolism, the activities of these enzymes are induced when animals are fed a high-carbohydrate diet and decreased during starvation or by a high-fat, PUFA-enriched diet [for review, see ref. (5Hillgartner F.B. Salati L.M. Goodridge A.G. Physiological and molecular mechanism involved in nutritional regulation of fatty acid synthesis.Physiol. Rev. 1995; 75: 47-76Google Scholar)]. Similarly, the activities of key enzymes involved in regulating the flux of glucose to fatty acids, such as glucokinase, pyruvate kinase (L-PK), and pyruvate dehydrogenase, are influenced by a high-PUFA diet (6Jump D.B. Clarke S.D. Thelen A. Liimatta M. Coordinate regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids.J. Lipid Res. 1994; 35: 1076-1084Google Scholar). It has been shown that PUFAs inhibit rodent hepatic lipogenesis by suppressing the mRNA encoding for several proteins, including ACC, FAS, L-PK, G6PD, ME, and the S14 protein (2Jump D.B. Clarke S.D. Regulation of gene expression by dietary fat.Annu. Rev. Nutr. 1999; 19: 63-90Google Scholar). Different mechanisms are responsible for the regulation of the lipogenic enzymes at the molecular level. The PUFA-mediated inhibition of ACC, FAS, L-PK, stearoyl-CoA desaturase (SCD-1), and, in part, S14 protein occurs at the transcriptional level (6Jump D.B. Clarke S.D. Thelen A. Liimatta M. Coordinate regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids.J. Lipid Res. 1994; 35: 1076-1084Google Scholar, 7Toussant M.J. Wilson M.D. Clarke S.D. Coordinate suppression of liver acetyl-CoA carboxylase and fatty acid synthetase by polyunsaturated fat.J. Nutr. 1981; 111: 146-153Google Scholar, 8Blake W.L. Clarke S.D. Suppression of hepatic fatty acid synthase and S14 gene transcription by dietary polyunsaturated fatty acid.J. Nutr. 1990; 120: 1727-1729Google Scholar, 9Ntambi J.M. Dietary regulation of stearoyl-CoA desaturase 1 gene expression in mouse liver.J. Biol. Chem. 1992; 267: 10925-10930Google Scholar, 10Landschultz K.T. Jump D.B. MacDougald O.A. Lane M.D. Transcriptional control of the stearoyl-CoA desaturase-1 gene by polyunsaturated fatty acids.Biochem. Biophys. Res. Commun. 1994; 200: 763-768Google Scholar, 11Jump 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: 227-241Google Scholar), whereas PUFA suppression of G6PD and ME is thought to occur through a posttranscriptional mechanism (1Clarke S.D. Gasperikova D. Nelson C. Lapillonne A. Heird W.C. Fatty acid regulation of gene expression.Ann. N. Y. Acad. Sci. 2002; 967: 283-298Google Scholar, 5Hillgartner F.B. Salati L.M. Goodridge A.G. Physiological and molecular mechanism involved in nutritional regulation of fatty acid synthesis.Physiol. Rev. 1995; 75: 47-76Google Scholar, 12Hodge D.L. Salati L.A. Nutritional regulation of glucose-6-phosphate dehydrogenase is mediated by a nuclear post-transcriptional mechanism.Arch. Biochem. Biophys. 1997; 348: 303-312Google Scholar). Lipogenesis requires cooperation between mitochondrial and cytoplasmic enzymes and involves fluxes of metabolites across the mitochondrial membranes (13Goodridge A.G. Regulation of fatty acid synthesis in isolated hepatocytes. Evidence for a physiological role for long chain fatty acyl-CoA and citrate.J. Biol. Chem. 1973; 248: 4318-4326Google Scholar). The tricarboxylate (citrate) carrier (TCC), an integral protein of the mitochondrial inner membrane, plays a fundamental role in intermediary metabolism because it represents a link between carbohydrate catabolism and fatty acid synthesis. Indeed, definitive evidence was obtained indicating that citrate is an obligatory component of the shuttle system by which mitochondrial acetyl-CoA, mainly derived from a sugar source, is transported to the cytosolic compartment, where it becomes a substrate for ACC (14Allred J.B. Reilly E. Short-term regulation of acetyl-CoA carboxylase in tissues of higher animals.Prog. Lipid Res. 1997; 35: 371-385Google Scholar). In addition, this shuttle supplies NAD+ and NADPH to support 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: 503-514Google Scholar). TCC has been extensively characterized in liver mitochondria from mammals (16Bisaccia F. De Palma A. Palmieri F. Identification and purification of the tricarboxylate carrier in rat liver mitochondria.Biochim. Biophys. Acta. 1989; 977: 171-176Google Scholar, 17Clayes D. Azzi A. Tricarboxylate carrier of bovine liver mitochondria.J. Biol. Chem. 1989; 264: 14627-14630Google 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: 13379-13385Google 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: 508-513Google 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: 555-563Google Scholar). The TCC cDNA sequences of rat (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: 13682-13690Google Scholar), yeast (22Kaplan 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: 4108-4114Google Scholar), cow (23Iacobazzi V. De Palma A. Palmieri F. Cloning and sequencing of the bovine cDNA encoding the mitochondrial tricarboxylate carrier protein.Biochim. Biophys. Acta. 1996; 1284: 9-12Google Scholar), and human (24Heisterkamp 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: 451-456Google Scholar) are known. The nucleotide sequence of the human TCC gene has been determined (25Iacobazzi V. Lauria G. Palmieri F. Organization and sequence of the mitochondrial citrate transport protein.DNA Sequence. 1997; 7: 127-139Google Scholar). A coordinated reduction of lipogenic enzyme and of TCC activities has been described in starved rats (26Zara V. Gnoni G.V. Effect of starvation on the activity of the mitochondrial tricarboxylate carrier.Biochim. Biophys. Acta. 1995; 1239: 33-38Google Scholar). The simultaneous decrease of TCC mRNA level, observed in the mitochondria of these animals, was ascribed to a posttranscriptional mechanism (27Siculella 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: 418-423Google Scholar). Moreover, a recent study from our laboratory showed that lipogenic enzyme and TCC activities were reduced in parallel by a diet supplemented with 15% safflower oil (SO), which is rich in n-6 PUFAs (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: 5734-5739Google Scholar). A lower content in the liver of both immunoreactive TCC protein and its mRNA was demonstrated to be responsible for the observed reduction (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: 5734-5739Google Scholar). The aim of this study was to investigate the molecular basis for the regulation of TCC gene expression as mediated by a high-fat, n-6 PUFA-enriched diet. We showed that in hepatic cells, the effect of a SO-supplemented diet on TCC gene expression is modulated by both transcriptional and posttranscriptional mechanisms. [α-32P]dATP (3,000 Ci/mmol) and [α-32P]UTP (3,000 Ci/mmol) were purchased from Perkin-Elmer Life Sciences (Milano, Italy). [14C]citrate (specific activity, 100 mCi/mmol) and Hybond N+ nylon filters were purchased from Amersham Biosciences (Milano, Italy). Restriction enzymes were obtained from Promega (Milano, Italy). RNase-free DNase I, α-amanitin, actinomycin D, and 1,2,3-benzenetricarboxylic acid were purchased from Sigma-Aldrich Co.; the Bio-Rad protein assay kit was purchased from Bio-Rad Laboratories (Milano, Italy). T3 RNA polymerase, RPAIII kit, RNase inhibitor, and β-actin antisense control template were obtained from Celbio (Milano, Italy). All other reagents were of analytical grade. Male Wistar rats (200–250 g), purchased from Harlan, were housed individually at 22 ± 1°C with a 12 h light/12 h dark cycle. Animal treatment was the same as reported by Zara et al. (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: 5734-5739Google Scholar). Briefly, control rats were fed ad libitum with a standard rodent diet (25% protein, 4.3% lipid, 59.7% carbohydrate, of which 7.1% was cellulose, and a salt and vitamin mixture), whereas a second group of rats was fed a high-fat diet [the standard diet supplemented with 15% (w/w) SO/kg diet] for the indicated periods of time. The SO diet was freshly prepared each week and stored at −20°C until required. The fatty acid composition of dietary lipids, determined by gas-liquid chromatographic analysis of their respective fatty acid methyl esters, is reported in Table 1. The animals had free access to food and water. Body weight and food intake of the animals were recorded throughout the experiment, and no significant differences were found between the two dietary groups. All of the experiments were performed in accordance with local and national guidelines regarding animal experiments.TABLE 1Fatty acid composition of control and SO-supplemented dietsFatty AcidControl DietSO DietC14:00.97 ± 0.130.32 ± 0.09C16:016.56 ± 0.508.66 ± 0.13C16:1 (n-7)1.16 ± 0.090.38 ± 0.06C18:02.18 ± 0.191.54 ± 0.13C18:1 (n-9)28.78 ± 1.5116.84 ± 0.56C18:2 (n-6)49.46 ± 1.2870.40 ± 1.98C20:5 (n-3)1.40 ± 0.150.19 ± 0.06C22:6 (n-3)1.14 ± 0.120.30 ± 0.05Σ saturated19.71 ± 0.4510.52 ± 0.38Σ unsaturated81.94 ± 0.8788.11 ± 0.92Σ saturated/Σ unsaturated0.24 ± 0.020.12 ± 0.02Values are percentages of total fatty acids. Results are expressed as means ± SE of five determinations. Interbatch variation in composition did not exceed 0.5% in each case. SO diet indicates a control diet supplemented with 15% (w/w) safflower oil. Fatty acids were extracted from the two different diets and analyzed by gas-liquid chromatography as fatty acid methyl esters. Σ saturated, sum of saturated fatty acids; Σ unsaturated, sum of unsaturated fatty acids. Open table in a new tab Values are percentages of total fatty acids. Results are expressed as means ± SE of five determinations. Interbatch variation in composition did not exceed 0.5% in each case. SO diet indicates a control diet supplemented with 15% (w/w) safflower oil. Fatty acids were extracted from the two different diets and analyzed by gas-liquid chromatography as fatty acid methyl esters. Σ saturated, sum of saturated fatty acids; Σ unsaturated, sum of unsaturated fatty acids. The citrate transport assay was carried out in freshly isolated rat liver mitochondria essentially as described by Palmieri et al. (29Palmieri F. Stipani I. Quagliariello E. Klingenberg M. Kinetic study of the tricarboxylate carrier in rat liver mitochondria.Eur. J. Biochem. 1972; 26: 587-594Google Scholar). Briefly, mitochondria (∼50 mg of protein) were resuspended in 100 mM KCl, 20 mM Hepes, 1 mM EGTA, and 2 μg/ml rotenone (pH 7.0) in a final volume of 10 ml and loaded with 0.75 mM malate as described by Zara and Gnoni (26Zara V. Gnoni G.V. Effect of starvation on the activity of the mitochondrial tricarboxylate carrier.Biochim. Biophys. Acta. 1995; 1239: 33-38Google Scholar). Transport was initiated by the addition of 0.5 mM [14C]citrate (specific activity, 100 mCi/mmol) to malate-loaded mitochondria and terminated by adding 12.5 mM 1,2,3-benzenetricarboxylic acid. Total RNA from liver was isolated as described by Chomczynski and Sacchi (30Chomczynski P. Sacchi N. Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 1987; 162: 156-159Google Scholar). Total RNA (15 and 30 μg) was separated, blotted, and hybridized as reported by Siculella et al. (27Siculella 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: 418-423Google Scholar). The RNA blots were hybridized with [α-32P]cDNA probe, labeled by random primer technique (31Feinberg A.P. Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Anal. Biochem. 1983; 132: 6-13Google Scholar), corresponding to nucleotides 459–1,421 of the rat 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: 13682-13690Google Scholar). For the normalization of the hybridization signals, a probe encoding part of the human β-actin was used. After autoradiography, the intensity of the bands was determined by densitometry. Nuclei were isolated from hepatocytes obtained by liver perfusion and collagenase digestion as reported by Gnoni et al. (32Gnoni G.V. Geelen M.J.H. Bijleveld C. Quagliariello E. Van den Bergh S.G. Short-term stimulation of lipogenesis by triiodothyronine in maintenance cultures of rat hepatocytes.Biochem. Biophys. Res. Commun. 1985; 128: 525-530Google Scholar). After collecting and washing twice with cold PBS, hepatocytes were lysed in 1 ml of buffer 1 [0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 15 mM Hepes (pH 7.4), 2 mM EDTA, 0.5 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 14 mM 2-mercaptoethanol, and 0.5% (v/v) Nonidet P40] by homogenization with a Dounce homogenizer. Nuclei were purified from hepatocytes by centrifugation through a 2.0 M sucrose cushion as described by Siculella et al. (27Siculella 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: 418-423Google Scholar). The nuclei were stored at −80°C in 210 μl of storage buffer [50% (v/v) glycerol, 50 mM Hepes (pH 7.4), 150 mM NaCl, 0.1 mM EDTA, 10 mM DTT, and 0.25 mM phenylmethylsulfonyl fluoride] before use in a nuclear run-on assay, which was carried out as described by Liu, Sun, and Jost (33Liu Y. Sun L. Jost J.P. In differentiating mouse myoblasts DNA methyltransferase is posttranscriptionally and posttranslationally regulated.Nucleic Acids Res. 1996; 24: 2718-2722Google Scholar). Total RNA was extracted as indicated above. Denatured DNA was applied to Hybond N+ nylon membranes by a dot-blot apparatus. The filter-bound DNA was hybridized as reported by Siculella et al. (27Siculella 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: 418-423Google Scholar). Hybridization signals were quantified as described above. Hepatocytes from control and SO-fed rats were maintained on plastic Petri dishes (60 mm) until monolayer formation (32Gnoni G.V. Geelen M.J.H. Bijleveld C. Quagliariello E. Van den Bergh S.G. Short-term stimulation of lipogenesis by triiodothyronine in maintenance cultures of rat hepatocytes.Biochem. Biophys. Res. Commun. 1985; 128: 525-530Google Scholar) and were further incubated in Ham's F12 medium in the presence of 4 μg/ml actinomycin D. At different times, 10 plates (∼4 × 106 cells) from each group were washed with cold PBS and total RNA was extracted as above described. For each time point, 10 μg of RNA was separated on 1% agarose gel with formaldehyde and Northern blot hybridization was carried out as indicated above, using TCC cDNA as a probe. The same filter was stripped by washing twice in a boiling solution of 0.1% SDS. The membrane was rehybridized with β-actin cDNA. The autoradiogram was quantified by densitometric scanning. The isolated nuclei were lysed by adding 4 ml of denaturing solution [4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 100 mM β-mercaptoethanol, and 0.5% N-lauroylsarcosine]. All remaining RNA isolation steps were as described by Chomczynski and Sacchi (30Chomczynski P. Sacchi N. Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 1987; 162: 156-159Google Scholar). Final RNA pellets were resuspended in 100 μl of diethyl pyrocarbonate-treated water and stored at −80°C. Two probes were designed for use in the RNase protection assay and were obtained by PCR amplification using genomic clone p5B8 containing the TCC gene as a template (data not shown). The first probe, designated intron2-exon3 (I2-E3), was obtained by PCR amplification using the following primers: rp1, 5′-GAATTCTGCTGCAGGAACGACCAGGA-3′, and rp2, 5′-AAGCTTCACGGTCTCCATGGG-3′. The second probe, designated exon7-intron7 (E7-I7), was obtained by PCR amplification using the following primers: rp3, 5′-GAATTCGGCCTGGAGGCACACAAATAC-3′, and rp4, 5′-AAGCTTCTGGGTAGAGCAGAGAGCC-3′. For subcloning purposes, an EcoRI site was added at the 5′-end of primers rp1 and rp3, whereas a HindIII site was added at the 5′-end of primers rp2 and rp4 (underlined). After amplification by PCR, the amplified fragments were subcloned into the EcoRI and HindIII sites of pBluescript II vector. The subclones were linearized with EcoRI (Promega) and used in the in vitro transcription reactions. The transcribed RNA was larger than the protected fragments, so that incompletely digested probe could be differentiated from the target signal in the RNase protection assay. RNA probes complementary to TCC RNA were synthesized by an in vitro transcription reaction using 10 units of T3 RNA polymerase in a buffer containing 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2, 2 mM spermidine, 5 mM DTT, 500 μM NTPs (A, G, C), 50 μCi of [α-32P]UTP (specific activity, 800 Ci/mmol), and 5 units of RNase inhibitor. For each reaction, 0.3 μg of plasmid DNA was used as a template. The reaction mixture was incubated for 60 min at 37°C and stopped by adding 1 unit of RNase-free DNase I for 15 min at 37°C. The integrity of the synthesized RNA probes was checked by 6% denaturing polyacrylamide gel electrophoresis. The RNase protection assay was performed using the RPAIII kit, following the instructions given by the supplier. Nuclear RNA (10 μg) was hybridized with 2 × 105 cpm of 32P-labeled RNA probe in 20 μl of hybridization solution at 50°C for 16 h. In each hybridization reaction, a β-actin antisense 32P-labeled RNA probe was added for normalization purposes. As a control for testing the RNase activity, probes were also hybridized with 10 μg of yeast RNA. The hybrids protected from digestion with RNase were resolved on a 6% denaturing polyacrylamide gel. Gels were dried and exposed for autoradiography, and the intensity of the bands was evaluated by densitometry with Molecular Analyst Software. All data are presented as means ± SE for the number of experiments indicated in each case. Statistical analysis was performed by Student's t-test. Differences were considered statistically significant at P < 0.05. Previous data from our laboratory (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: 5734-5739Google Scholar) have shown that feeding rats a high-fat diet (15% SO) for 4 weeks resulted in a noticeable decrease in TCC activity. To search for an earlier effect, experiments were carried out for up to 4 weeks using the same SO treatment. Equal amounts of liver mitochondrial proteins from the two groups of animals were used. Figure 1gives the citrate exchange by malate-loaded liver mitochondria, isolated from control animals and from rats fed the SO-enriched diet for the indicated periods of time. Compared with control animals, a gradual decrease of the carrier activity was observed, reaching ∼50% inhibition after 4 weeks of SO diet administration (15.6 ± 1.1 vs. 30.3 ± 1.3 nmol/min × mg protein for the control). The reduction at 4 weeks is in agreement with our previous finding (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: 5734-5739Google Scholar). No significant changes in the carrier activity were detected with longer treatment times (data not shown). To investigate the molecular basis of the regulation of TCC activity by dietary n-6 fat, Northern blot analysis was carried out. Total RNA from livers of control and SO-fed rats was separated onto a denaturing agarose gel, transferred to Hybond N+ membranes, and hybridized with the TCC [α-32P]cDNA probe and normalized with the β-actin [α-32P]cDNA probe. The latter was used for the normalization, as described previously (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: 5734-5739Google Scholar), in agreement with other findings (11Jump 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: 227-241Google Scholar, 34Jump D.B. Clarke S.D. MacDougald O. Thelen A. Polyunsaturated fatty acids inhibit S14 gene transcription in rat liver and cultured hepatocytes.Proc. Natl. Acad. Sci. USA. 1993; 90: 8454-8458Google Scholar) that the hepatic abundance of its mRNA is not affected by PUFA feeding. Densitometric analysis showed a time-dependent reduction of mRNA abundance, reaching an inhibition of ∼35% at 4 weeks of SO treatment with respect to control animals (Fig. 2). Like the TCC activity, a prolonged treatment of rats with SO-added diet did not further decrease the TCC mRNA level (data not shown). Dietary treatment of 4 weeks was then used in all subsequent experiments. Previous studies have shown that mRNAs encoding for several different proteins change in stability after dietary treatment (35Goldman M.J. Back D.W. Goodridge A.G. Nutritional regulation of the synthesis and degradation of malic enzyme messenger RNA in duck liver.J. Biol. Chem. 1985; 260: 4404-4408Google Scholar, 36Tebbey P.W. McGowan K.M. Stevens J.M. Buttke T.M. Pekala P.H. Arachidonic acid down-regulates the insulin-dependent glucose transporter gene (GLUT4) in 3T3-L1 adipocytes by inhibiting transcription and enhancing mRNA turnover.J. Biol. Chem. 1994; 269: 639-644Google Scholar, 37Sessler A.M. Kaur N. Palta J.P. Ntambi J.M. Regulation of stearoyl-CoA desaturase I mRNA stability by polyunsaturated fatty acids in 3T3-L1 adipocytes.J. Biol. Chem. 1996; 271: 29854-29858Google Scholar). To investigate the half-life of TCC mRNA, isolated hepatocytes from control and SO-fed rats were cultured in the presence of actinomycin D (4 μg/ml). At the indicated times (Fig. 3), total RNA was extracted from hepatocytes and analyzed as reported in Experimental Procedures. For each time point, 10 μg of RNA was separated onto a 1% agarose gel containing formaldehyde and Northern blot analysis was performed. Autoradiograms were quantified by densitometric scanning. The log of TCC and β-actin mRNA abundance was reported as a function of time. The semilog plot represents the decay curve of TCC mRNA (Fig. 3, top) and β-actin mRNA (Fig. 3, bottom) from both control and SO-fed rats. Results from five independent experiments showed that the apparent half-life of the TCC mRNA in cultured hepatocytes from both groups of rats was very similar (10.9 ± 0.8 h in SO-fed rats vs. 10.7 ± 0.7 h in control rats). Moreover, in the same RNA preparation, the relative rate of degradation of β-actin mRNA also remained constant. To determine whether the reduction in the steady-state level of the mRNA observed after SO treatment (Fig. 2) was attributable to a decrease in gene transcription, nuclear run-on assays were performed. To this end, nuclei isolated from control and SO-treated rat hepatocyte suspensions were allowed to incorporate [α-32P]UTP. Then, [α-32P]RNA was extracted and hybridized to dots (5 μg) of TCC cDNA, β-actin cDNA, pUC19, and FAS cDNA applied to the filters. Nonrecombinant plasmid pUC19 was used as a negative control, whereas β-actin cDNA represented a control for the normalization and selectivity of the response. FAS cDNA was used as a positive control, as it has been reported that n-6 PUFA-supplemented diet modulates this gene at the transcriptional level (8Blake W.L. Clarke S.D. Suppression of hepatic fatty acid synthase and S14 gene
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