Hypothyroidism Reduces Tricarboxylate Carrier Activity and Expression in Rat Liver Mitochondria by Reducing Nuclear Transcription Rate and Splicing Efficiency
2006; Elsevier BV; Volume: 281; Issue: 28 Linguagem: Inglês
10.1074/jbc.m507237200
ISSN1083-351X
AutoresLuisa Siculella, Simona Sabetta, Anna Maria Giudetti, Gabriele V. Gnoni,
Tópico(s)Adipose Tissue and Metabolism
ResumoThe tricarboxylate carrier (TCC), also known as citrate carrier, is an integral protein of the mitochondrial inner membrane. It is an essential component of the shuttle system by which mitochondrial acetyl-CoA, primer for both fatty acid and cholesterol synthesis, is transported into the cytosol, where lipogenesis occurs. The effect of hypothyroidism on the activity and expression of the hepatic mitochondrial TCC was investigated in this study. TCC activity was significantly decreased in hypothyroid rats as compared with euthyroid animals. This hormone deficiency effect was due to a reduction in the amount of carrier protein, which resulted from a proportionate decrease of the specific mRNA. Hypothyroidism did not influence TCC mRNA stability. On the other hand, nuclear run-on assay revealed that the transcriptional rate of TCC mRNA decreased by ∼40% in the nuclei from hypothyroid versus euthyroid rats. In addition, the ribonuclease protection assay showed that, in the nuclei of hypothyroid rats, the ratio of mature to precursor RNA decreased, indicating that the splicing of TCC RNA is affected. Furthermore, we found that the ratio of polyadenylated/unpolyadenylated TCC RNA as well as the length of the TCC RNA poly(A) tail were similar in both euthyroid and hypothyroid rats. Thus, the rate of formation of the TCC 3′-end is not altered in hypothyroidism. These results suggest that hypothyroidism affects TCC expression at both the transcriptional and post-transcriptional levels. The tricarboxylate carrier (TCC), also known as citrate carrier, is an integral protein of the mitochondrial inner membrane. It is an essential component of the shuttle system by which mitochondrial acetyl-CoA, primer for both fatty acid and cholesterol synthesis, is transported into the cytosol, where lipogenesis occurs. The effect of hypothyroidism on the activity and expression of the hepatic mitochondrial TCC was investigated in this study. TCC activity was significantly decreased in hypothyroid rats as compared with euthyroid animals. This hormone deficiency effect was due to a reduction in the amount of carrier protein, which resulted from a proportionate decrease of the specific mRNA. Hypothyroidism did not influence TCC mRNA stability. On the other hand, nuclear run-on assay revealed that the transcriptional rate of TCC mRNA decreased by ∼40% in the nuclei from hypothyroid versus euthyroid rats. In addition, the ribonuclease protection assay showed that, in the nuclei of hypothyroid rats, the ratio of mature to precursor RNA decreased, indicating that the splicing of TCC RNA is affected. Furthermore, we found that the ratio of polyadenylated/unpolyadenylated TCC RNA as well as the length of the TCC RNA poly(A) tail were similar in both euthyroid and hypothyroid rats. Thus, the rate of formation of the TCC 3′-end is not altered in hypothyroidism. These results suggest that hypothyroidism affects TCC expression at both the transcriptional and post-transcriptional levels. Thyroid hormones have a profound influence on normal development, differentiation, and metabolism (1Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1552) Google Scholar). Mitochondria are considered possible subcellular loci of thyroid hormone action in view of their crucial role in energy metabolism (2Goglia F. Moreno M. Lanni A. FEBS Lett. 1999; 452: 115-120Crossref PubMed Scopus (148) Google Scholar). Mitochondrial function can be regulated by thyroid hormones both indirectly in a "nuclear mediated" way and directly through interaction with some mitochondrial component (2Goglia F. Moreno M. Lanni A. FEBS Lett. 1999; 452: 115-120Crossref PubMed Scopus (148) Google Scholar, 3Weitzel J.M. Iwen K.A. Seitz H.J. Exp. Physiol. 2003; 88.1: 121-128Crossref Scopus (170) Google Scholar). Hypothyroidism is associated with a considerable decrease in basic metabolic rate, oxygen consumption, and rates of oxidation of glucose, fatty acids, and amino acids (4Tata J.R. Ernster L. Lindberg O. Arrhenius E. Pedersen S. Hedman R. Biochem. J. 1963; 86: 408-428Crossref PubMed Scopus (316) Google Scholar, 5Moreno M. Lanni A. Lombardi A. Goglia F. J. Physiol. (Lond.). 1997; 505: 529-538Crossref Scopus (115) Google Scholar). Moreover, thyroid hormones dramatically affect the extent to which liver contributes to total lipogenesis in rat. This contribution ranges from 5% in the hypothyroid animals to 34% in the hyperthyroid animals (6Freake H.C. Schwartz H.L. Oppenheimer J.H. Endocrinology. 1989; 125: 2868-2874Crossref PubMed Scopus (59) Google Scholar). Fatty acid synthesis in rat liver is extremely responsive to the thyroid status of the organism. Hypothyroidism in developing or adult rats depresses hepatic fatty acid synthase (FAS) 2The abbreviations used are: FAS, fatty acid synthase; TCC, tricarboxylate carrier; ACC, acetyl-CoA carboxylase; T3, tri-iodothyronine; pre-mRNA, precursor mRNA; UTR, untranslated region; nt, nucleotide(s). activity by 50% (7Hoch F.L. Prog. Lipid Res. 1988; 27: 199-270Crossref PubMed Scopus (138) Google Scholar). Cultures of hepatocytes obtained from hypothyroid rats synthesize fatty acids from acetate half as fast as euthyroid hepatocytes (8Gnoni G.V. Geelen M.J.H. Bijleveld C. Quagliariello E. Van den Bergh S.G. Biochem. Biophys. Res. Commun. 1985; 128: 525-530Crossref PubMed Scopus (28) Google Scholar). The enzymatic activities of de novo fatty acid synthesis, i.e. acetyl-CoA carboxylase (ACC) and FAS, are greatly increased in hyperthyroid rats (9Roncari D.A. Murthy V.K. J. Biol. Chem. 1975; 250: 4134-4138Abstract Full Text PDF PubMed Google Scholar, 10Landriscina C. Gnoni G.V. Quagliariello E. 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Sci. 1986; 478: 46-62Crossref PubMed Scopus (41) Google Scholar) and in chick embryo hepatocytes (14Stapleton S.R. Mitchell D.A. Salati L.M. Goodridge A.G. J. Biol. Chem. 1990; 265: 18442-18446Abstract Full Text PDF PubMed Google Scholar), as well as the putative lipogenic enzyme S14 protein in primary cultures of rat hepatocytes (15Mariash C.N. Jump D.B. Oppenheimer J.H. Biochem. Biophys. Res. Commun. 1984; 123: 1122-1129Crossref PubMed Scopus (26) Google Scholar) and in rat liver (16Jump D.B. J. Biol. Chem. 1989; 264: 4698-4703Abstract Full Text PDF PubMed Google Scholar), are transcriptionally regulated by T3, whereas it has been proposed that, in rats, T3 is involved not only in ACC transcription (17Huang C. Freake H.C. Biochem. Biophys. Res. Commun. 1998; 249: 704-709Crossref PubMed Scopus (20) Google Scholar) but also in ACC mRNA stability (18Katsurada A. Iritani N. Fukuda H. Matsumura Y. Noguchi T. Tanaka T. Eur. J. Biochem. 1990; 190: 435-441Crossref PubMed Scopus (136) Google Scholar). Transcriptional and post-transcriptional mechanisms have been suggested for T3 regulation of the malic enzyme gene (19Song M.-H.K. Dozin B. Grieco J. Rall E. Nikodem V.M. J. Biol. Chem. 1988; 263: 17970-17974Abstract Full Text PDF PubMed Google Scholar, 20Salati L.M. Ma X.J. McCornick C.C. Stapleton S.R. Goodridge A.G. J. Biol. Chem. 1991; 266: 4010-4016Abstract Full Text PDF PubMed Google Scholar). T3 increases the ATP-citrate lyase content by changes in the rate of enzyme synthesis, which in turn correlate with the abundance of the corresponding mRNA (11Hillgartner F.B. Salati L.M. Goodridge A.G. Physiol. Rev. 1995; 75: 47-76Crossref PubMed Scopus (401) Google Scholar). Lipogenesis requires cooperation between mitochondrial and cytoplasmic enzymes and involves fluxes of metabolites across the mitochondrial membranes (21Watson J.A. Lowenstein J.M. J. Biol. Chem. 1970; 245: 5993-6002Abstract Full Text PDF PubMed Google Scholar). The inner membrane of rat liver mitochondria contains a specific carrier, i.e. tricarboxylate carrier, for the outward transport of citrate. The activity of TCC is strictly correlated with the rate of lipogenesis and represents a link between carbohydrate catabolism and fatty acid synthesis. In fact, it exports from mitochondria to the cytosol (in the form of citrate) acetyl-CoA mainly derived from sugar sources, thus providing the carbon units for fatty acid and cholesterol biosyntheses. In addition, it supplies NAD+ and NADPH that support cytosolic glycolysis and lipid biosynthesis, respectively (22Kaplan R.S. Mayor J.A. J. Bioenerg. Biomembr. 1993; 25: 503-514Crossref PubMed Scopus (30) Google Scholar). TCC may have important physiological functions in gluconeogenesis as well (23Kaplan R.S. Oliveira D.L. Wilson G.L. Arch. Biochem. Biophys. 1990; 280: 181-191Crossref PubMed Scopus (52) Google Scholar). The characteristics of this transporting system have been extensively investigated (for review, see Ref. 24Palmieri F. Eur. J. Physiol. 2004; 447: 689-709Crossref PubMed Scopus (631) Google Scholar), but little is known concerning its regulation. It has been reported that TCC activity is altered in type I diabetes (23Kaplan R.S. Oliveira D.L. Wilson G.L. Arch. Biochem. Biophys. 1990; 280: 181-191Crossref PubMed Scopus (52) Google Scholar), can be regulated by exogenous insulin (25Kaplan R.S. Mayor J.A. Blackwell R. Maughan R.H. Wilson G.L. Arch. Biochem. Biophys. 1991; 287: 305-311Crossref PubMed Scopus (27) Google Scholar), and is enhanced during hyperthyroidism (26Paradies G. Ruggiero F.M. Arch. Biochem. Biophys. 1990; 278: 425-430Crossref PubMed Scopus (48) Google Scholar). Recent reports from our laboratory showed that, in parallel with lipogenic enzyme activities, TCC activity is controlled by various nutritional states (27Zara V. Giudetti A.M. Siculella L. Palmieri F. Gnoni G.V. Eur. J. Biochem. 2001; 268: 5734-5739Crossref PubMed Scopus (31) Google Scholar, 28Siculella L. Sabetta S. di Summa R. Leo M. Giudetti A.M. Palmieri F. Gnoni G.V. Biochem. Biophys. Res. Commun. 2002; 299: 418-423Crossref PubMed Scopus (23) Google Scholar, 29Giudetti A.M. Sabetta S. di Summa R. Leo M. Damiano F. Siculella L. Gnoni G.V. J. Lipid Res. 2003; 44: 2135-2141Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). To our knowledge, there has been no report assessing mitochondrial TCC activity relative to the hypothyroid state, and no study so far has gone deep into the hormonal regulation at the molecular level of any gene for a mitochondrial carrier protein. Therefore, the aim of this study was to investigate whether hypothyroidism affects TCC activity and expression and, if so, to characterize the molecular step(s) for this hormonal TCC gene modulation. We showed that the reduced TCC activity observed in the hypothyroid state can be ascribed to either a transcriptional and a post-transcriptional TCC gene regulation. Chemicals—[α-32P]dATP (3000 Ci/mmol) and [α-32P]UTP (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences (Milan, Italy). [14C]Citrate (specific activity, 100 mCi/mmol) and nylon filters Hybond N+ were purchased from Amersham Biosciences. Restriction enzymes were obtained from Promega (Milan, Italy). RNase-free DNase I, α-amanitin, actinomycin D, and 1,2,3-benzenetricarboxylic acid were purchased from Sigma. The protein assay kit was from Bio-Rad. The T3 RNA polymerase, RPAIII kit, RNase inhibitor, and β-actin antisense control template were obtained from Celbio (Milan, Italy). ANT2 antibody (D. B. A. Italia, Milan, Italy) was commercially available. All other reagents were of analytical grade. Animal Treatments—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 a.m. to 8:00 p.m.)-controlled room and randomly assigned to one of two different groups. The first one is the control group and will be further referred to as euthyroid. The second group was made chronically hypothyroid by continuous administration of 6-n-propyl-2-thiouracil (0.1% w/v, in drinking tap water) for 3–4 weeks (30Ruggiero F.M. Gnoni G.V. Quagliariello E. Lipids. 1987; 22: 148-151Crossref PubMed Scopus (22) Google Scholar). Both groups had free access to food that was a commercial mash (Morini Spa, Milan, Italy). The experimental design was in accordance with local and national guidelines covering animal experiments. Tricarboxylate Translocase Activity Assay—Rat liver mitochondria were prepared by differential centrifugation of the liver homogenates essentially as described previously (31Palmieri F. Stipani I. Quagliariello E. Klingenberg M. Eur. J. Biochem. 1972; 26: 587-594Crossref PubMed Scopus (173) Google Scholar). Mitochondrial integrity was monitored by measuring the respiratory control ratio. TCC activity was assayed in freshly isolated rat liver mitochondria as reported in Ref. 31Palmieri F. Stipani I. Quagliariello E. Klingenberg M. Eur. J. Biochem. 1972; 26: 587-594Crossref PubMed Scopus (173) Google Scholar. The malate/citrate exchange reaction was started by the addition to malate-loaded mitochondria (1–1.5 mg of protein) of 0.5 mm [14C]citrate and terminated by adding the inhibitor 1,2,3-benzenetricarboxylic acid at 12.5 mm (31Palmieri F. Stipani I. Quagliariello E. Klingenberg M. Eur. J. Biochem. 1972; 26: 587-594Crossref PubMed Scopus (173) Google Scholar). Immediately after the addition of the inhibitor, the tubes were rapidly centrifuged at 18,000 × g for 5 min at 2 °C, washed, and extracted with 20% HClO4. The mixture was then centrifuged, and the radioactivity in the supernatant was counted. Isolation of RNA and Northern Blot Analysis—Approximately 15 μg of total RNA, extracted from the livers of hypothyroid and euthyroid rats, according to Chomczynski and Sacchi (32Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar), was size-fractionated by electrophoresis through denaturing 1% formaldehyde-agarose gel and transferred to Hybond N+ nylon membrane. The RNA blots were hybridized with three α-32P-labeled probes corresponding to nucleotides 459–1421 of rat liver TCC cDNA (33Kaplan R.S. Mayor J.A. Wood D.O. J. Biol. Chem. 1993; 268: 13682-13690Abstract Full Text PDF PubMed Google Scholar), nucleotides 91–280 of rat liver adenine nucleotide translocase isoform 2 (ANT2) cDNA (GenBank™ accession number NM_057102), and nucleotides 613–726 of the ubiquitously expressed rat liver VDAC1 (voltage-dependent anion channel, isoform 1) (also known as mitochondrial porin) cDNA (GenBank™ accession number NM_031353), respectively. For normalization of the hybridization signals, the same membranes were hybridized using a probe encoding for part of the human β-actin. Membranes were exposed to x-ray film, and the intensity of the resulting bands was evaluated by densitometry with Molecular Analyst software. Immunoelectrophoretic Analysis—Western blots were carried out as reported in Ref. 29Giudetti A.M. Sabetta S. di Summa R. Leo M. Damiano F. Siculella L. Gnoni G.V. J. Lipid Res. 2003; 44: 2135-2141Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar. Membranes were submitted to the reaction with the first antibody directed against a C-terminal peptide of rat liver TCC (29Giudetti A.M. Sabetta S. di Summa R. Leo M. Damiano F. Siculella L. Gnoni G.V. J. Lipid Res. 2003; 44: 2135-2141Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), ANT2, or porin. Porin, the mitochondrial outer membrane channel, was used as a control, because its expression is not affected by thyroid hormones (34Yehuda-Shnaidman E. Kalderon B. Bar-Tana J. Endocrinology. 2005; 146: 2462-2472Crossref PubMed Scopus (21) Google Scholar). The bound antibody was revealed by peroxidase-conjugated anti-rabbit IgG antibody using 3,3′-diaminobenzidine and hydrogen peroxide as substrates. The blots were evaluated by densitometric analysis with Molecular Analyst software. Isolation of Nuclei and Nuclear Run-on Assay—Nuclei were isolated from hepatocytes obtained by liver perfusion and collagenase digestion as reported by Gnoni et al. (8Gnoni G.V. Geelen M.J.H. Bijleveld C. Quagliariello E. Van den Bergh S.G. Biochem. Biophys. Res. Commun. 1985; 128: 525-530Crossref PubMed Scopus (28) Google Scholar). After isolation, hepatocytes were washed twice with cold phosphate-buffered saline and then 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, 0.5% (v/v) Nonidet P40) by homogenization with a Dounce homogenizer. The crude nuclei were purified by centrifugation through a 2.0 m sucrose cushion as described by Siculella et al. (28Siculella L. Sabetta S. di Summa R. Leo M. Giudetti A.M. Palmieri F. Gnoni G.V. Biochem. Biophys. Res. Commun. 2002; 299: 418-423Crossref PubMed Scopus (23) Google Scholar) and stored at –80 °C in aliquots (210 μl) of storage buffer (50% (v/v) glycerol, 50 mm Hepes (pH 7.4), 150 mm NaCl, 0.1 mm EDTA, 10 mm dithiothreitol, 0.25 mm phenylmethylsulfonyl fluoride) containing ∼1 × 108 nuclei each for analysis later. Nuclei suspension was used in a nuclear run-on assay as described by Liu et al. (35Liu Y. Sun L. Jost J.P. Nucleic Acids Res. 1996; 24: 2718-2722Crossref PubMed Scopus (51) Google Scholar). The newly transcribed RNA was extracted as indicated above and hybridized to Hybond N+ nylon membranes as reported by Siculella et al. (28Siculella L. Sabetta S. di Summa R. Leo M. Giudetti A.M. Palmieri F. Gnoni G.V. Biochem. Biophys. Res. Commun. 2002; 299: 418-423Crossref PubMed Scopus (23) Google Scholar). Hybridization signals were quantified as described above. mRNA Turnover Assay—Hepatocytes from hypothyroid and euthyroid rats were maintained on plastic Petri dishes (60 mm) until monolayer formation (8Gnoni G.V. Geelen M.J.H. Bijleveld C. Quagliariello E. Van den Bergh S.G. Biochem. Biophys. Res. Commun. 1985; 128: 525-530Crossref PubMed Scopus (28) Google Scholar), and after 2-h plating, 4 μg/ml actinomycin D in Ham's F-12 medium was added. At different times, 10 plates (∼4 × 106 cells/plate) from each group were washed with cold phosphate-buffered saline, and total RNA was extracted as described above. For each time point, aliquots of 10 μg of RNA were loaded on a 1% formaldehyde-agarose gel, electrophoresed, and transferred onto nylon membranes. 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 and rehybridized with β-actin cDNA. The autoradiogram was quantified by densitometric scanning. Isolation of Nuclear RNA—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). RNA isolation was carried out as described by Chomczynski and Sacchi (32Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Probe Designed for the Ribonuclease Protection Assay—Several probes were designed for use in the ribonuclease protection assay. They were obtained by PCR amplification of a genomic clone p5B8 containing the TCC gene (data not shown) using specific primers as previously reported (36Siculella L. Damiano F. Sabetta S. Gnoni G.V. J. Lipid Res. 2004; 45: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Briefly, the first designed probe (see Fig. 5A), intron 3-exon 4 (I3-E4), was amplified using the following primers: rp1, 5′-GAATTCTGCTGCAGGAACGACCAGGA-3′ and rp2, 5′-AAGCTTCACGGTCTCCATGGG-3′. The second designed probe, exon 7-intron7, was amplified using the following primers: rp3, 5′-GAATTCGAGACAACCCCAACAAGCC-3′ and rp4, 5′-AAGCTTCTGCAGGACGCAGCAAGCC-3′. The third designed probe, exon 8-intron 8 (E8-I8) (see Fig. 5A) was obtained using the following primers: rp5, 5′-GAATTCGGCCTGGAGGCACACAAATAC-3′ and rp6, 5′-AAGCTTCTGGGTAGAGCAGAGAGCC-3. For subcloning purposes, an EcoRI site was added at the 5′-end of the primers rp1, rp3, and rp5, whereas a HindIII site was added at the 5′-end of the primers rp2, rp4, and rp6 (underlined). The amplified products were subcloned into pBlueskript II vector. After linearization, the recombinant plasmids were used in the in vitro transcription reactions. The β-actin probe, which was used as a control for RNA loading, was made from the pTRI-β-actin-125-Rat antisense control template (Ambion). When this plasmid was transcribed with T3 RNA polymerase antisense, transcripts of 160 nt are produced, and their hybridization to rat total RNA protects a 126-nt fragment derived from exon 5 of rat β-actin. The fourth TCC probe spans the polyadenylation/cleavage site in the 3′-untranslated region (UTR) so that it can detect the abundance of polyadenylated RNA after cleavage, as well as uncleaved RNA in a RNase protection assay. DNA was amplified by PCR of a genomic clone p5B8, using the following primers: rp7, 5′-GTGCAGAAGCTTACCGCATTCCAGGGGCTATAG-3′; rp8, 5′-TAAAGGAGCTCTCACTAGGG-3′ located 2759 bp and 3023 bp downstream of the ATG codon, respectively, and designed on the basis of the published rat genome sequence (GenBank™ NW_047358). The amplified sequence was ligated into pBlueskript II vector, and the plasmid was linearized prior to use in the riboprobe synthesis reaction. Ribonuclease Protection Assay—Antisense RNAs were synthesized by an in vitro transcription reaction as reported in Ref. 36Siculella L. Damiano F. Sabetta S. Gnoni G.V. J. Lipid Res. 2004; 45: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar. RNA samples were subjected to ribonuclease protection assay using the RPAIII kit and following the instructions given by the supplier. Nuclear RNA (10 μg) was hybridized with 2 × 105 counts/min of 32P-labeled-specific antisense probe in 20 μl of hybridization solution at 50 °C for 16 h. For the normalization, a 2 × 103 counts/min of antisense β-actin 32P-labeled RNA probe with a 100-fold lower activity was added in each hybridization reaction to obtain a signal comparable with the test mRNAs. Probes were also hybridized with 10 μg of yeast RNA used as a control for testing the RNase activity (data not shown). After digestion with RNase A/T1, the protected fragments were separated onto a 6% denaturing polyacrylamide gel. Gels were dried, exposed for autoradiography, and the intensity of the bands was evaluated by densitometry with Molecular Analyst software. RNase H Blot—RNase H analysis was carried out according to Ref. 37Amir-Ahmady B. Salati L.M. J. Biol. Chem. 2001; 276: 10514-10523Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar. Briefly 15 μg of nuclear RNA was mixed with 50 pmol of a TCC-specific oligonucleotide (5′-CTATTGTATCTGTGTCCAGCCTGGCCGT-3′, nucleotides located 2517 bp downstream ATG codon of the rat genome sequence). The mixture was heated to 70 °C and then, in 10 min, cooled to room temperature (37Amir-Ahmady B. Salati L.M. J. Biol. Chem. 2001; 276: 10514-10523Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Digestion with RNase H was for 20 min at 37 °C. The samples were electrophoresed through denaturing 1% formaldehyde-agarose gel, transferred to Hybond N+ nylon membrane, and hybridized with an amplified probe (366 nt) corresponding to the 3′-end of the TCC mRNA, precisely from 2526 to 2892 bp downstream of the ATG codon of the rat genome sequence. Statistical Analysis—All data are presented as means ± S.E. 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. Time Course of Citrate/Malate Exchange—Fig. 1 shows the time course of [14C]citrate exchange by malate-loaded mitochondria from the livers of euthyroid and hypothyroid rats. Citrate/malate exchange was strongly reduced in liver mitochondria from hypothyroid versus euthyroid animals. As shown in this figure, during the linear range (15–30 s) of the reaction, the decrease of the transport activity was reproducibly found to be ∼50% in hypothyroid versus euthyroid animals. Effect of Hypothyroid State on TCC mRNA and Protein Levels in Rat Hepatocytes—To establish whether hypothyroidism affected TCC mRNA and protein levels, Northern and Western blot analyses were carried out. Quantitation of the signals showed that the hypothyroid state caused an ∼30% reduction of the hepatic TCC mRNA level compared with euthyroid rats (Fig. 2A). This figure also shows that, in agreement with a previous report (12Blenneman B. Lehay P. Kim T.S. Freake H.C. Mol. Cell. Endocrinol. 1995; 110: 1-8Crossref PubMed Scopus (87) Google Scholar), the expression of the housekeeping gene for β-actin was unmodified by hypothyroidism. Furthermore, the hepatic abundance of the mRNA for ANT2, the ubiquitous adenine nucleotide translocase isoform 2, was lowered in liver from hypothyroid rats, as reported by Dummler et al. (38Dummler K. Muller S. Seitz H.J. Biochem. J. 1996; 317: 913-918Crossref PubMed Scopus (107) Google Scholar), whereas in the liver from the same group of animals, no alteration in the expression of VDAC1 mRNA, coding for porin, was observed. Interestingly, the immunodecoration reported in Fig. 2B revealed also that the TCC and ANT2 protein levels of mitochondria from hypothyroid rats were lowered by ∼35 and 45%, respectively, as compared with euthyroid animals. By contrast, the amount of porin, the mitochondrial outer membrane channel, coded by VDAC mRNA and used as a control, was unaffected by the hypothyroid state. The immunoelectrophoretic results for TCC and ANT2 protein content correlate well with the observed alterations in the corresponding mRNA levels. Turnover of TCC mRNA—We then asked whether the reduction in the hepatic TCC mRNA accumulation in hypothyroid rats could reflect changes in the TCC mRNA stability. To address this question, the apparent half-life of TCC mRNA was estimated. Fig. 3 shows the semilog plot representing the decay curve of TCC mRNA (upper panel) and β-actin mRNA (lower panel) from hypothyroid and euthyroid rat hepatocytes. The apparent half-life of TCC mRNA from both groups of rats was similar (10.7 ± 1.1 h in hypothyroid versus 11.2 ± 0.7 h in euthyroid rats). In the same RNA preparation, the relative rate of degradation of the β-actin mRNA remained constant. Run-on Assay—To investigate whether the decrease in the amount of TCC mRNA observed in the hypothyroid state was controlled at the transcriptional level, a nuclear run-on assay was carried out. Nuclei isolated from hypothyroid and euthyroid rat hepatocyte suspensions were allowed to incorporate [α-32P]UTP. The labeled RNA was extracted and hybridized to dots (5 μg) of TCC cDNA, β-actin cDNA, and pUC19 applied to the filters. β-actin and pUC19 were used for normalization and as a negative control, respectively. Incorporation of [α-32P]UTP into TCC and β-actin transcripts was specifically suppressed by α-amanitin (4 μg/ml) (data not shown), confirming that the labeled RNAs were transcribed by RNA polymerase II. The dot blot hybridization revealed a decrease of ∼40% in the transcriptional rate of TCC mRNA from hypothyroid versus euthyroid rats (Fig. 4). The transcriptional rate of β-actin remained constant in the two groups of rats. Processing of TCC Precursor RNA—To study the effects of the hypothyroid state on the splicing of TCC RNA, we compared the amount of unspliced and spliced TCC RNA in the nuclei of hepatocytes from hypothyroid versus euthyroid rats. For this purpose, we used a ribonuclease protection assay and three probes (I3-E4, E7-I7, and E8-I8) to investigate the processing of the precursor-mRNA (pre-mRNA) (Fig. 5A). The three probes represent discrete locations within the primary transcript and permit quantitative analysis of the amount of pre-mRNA. I3-E4 hybridized across a 3′-splice site, and E7-I7 and E8-I8 hybridized across a 5′-splice site. RNase digestion of each hybrid of probe and target RNA resulted in two types of protected fragments, the longer fragments (intron3-exon4, exon7-intron7, and exon8-intron8) corresponding to unspliced RNA that contains both the exon and the intron sequences and the smaller fragments (exons 4, 7, and 8) corresponding to spliced RNA that contains only the exon sequences. The terms "unspliced" and "spliced" RNA refer to an RNA mix containing one or more of all of the TCC introns, and each probe provides information about the splicing of only 1 of the 8 introns in the TCC gene. Using the E8-I8 probe in the nuclei from hypothyroid rats, the amount of spliced RNA (E8-protected fragment) was ∼2.5-fold greater than the amount of unspliced RNA. To note that, in the nuclei from euthyroid rats, the amount of spliced RNA was ∼5-fold greater than that of unspliced RNA (Fig. 5). Furthermore, the reduction in the amount of spliced RNA in the nuclei of hypothyroid rats compared with euthyroid animals was ∼30%, which is similar to the decrease in the accumulation of the mature TCC RNA measured in the cytoplasm (Fig. 2A). By contrast, the amount of unspliced RNA was similar in both rats, despite the ∼40% reduction in
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