FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2082710199> ?p ?o ?g. }

• W2082710199 endingPage "34428" @default.
• W2082710199 startingPage "34422" @default.
• W2082710199 abstract "A previous study on the gene structure of rat pyruvate carboxylase revealed that two tissue-specific promoters are responsible for the production of multiple transcripts with 5′-end heterogeneity (Jitrapakdee, S., Booker, G. W., Cassady, A. I., and Wallace, J. C. (1997) J. Biol. Chem. 272, 20522–20530). Here we report transcription and translation regulation of pyruvate carboxylase (PC) expression during development and in genetically obese rats. The abundance of PC mRNAs was low in fetal liver but increased by 2–4-fold within 7 days after birth, concomitant with an 8-fold increase in the amount of immunoreactive PC and its activity and then decreased during the weaning period. Reverse transcriptase polymerase chain reaction analysis indicated that the proximal promoter was activated during the suckling period and reduced in activity at weaning. In genetically obese Zucker rats, adipose PC was 4–5-fold increased, concomitant with a 5–6-fold increase in mRNA level. Reverse transcriptase-polymerase chain reaction analysis also showed that the proximal promoter was activated in the hyperlipogenic condition. Conversely, transcription of the proximal promoter was not detectable in various liver cell lines, suggesting that this promoter was not functional under cell culture conditions. In rat pancreatic islets and insulinoma cells, only transcripts D and E, generated from the distal promoter of the PC gene, were expressed. Glucose increased PC transcripts from the distal promoter when the insulinoma cells were maintained in 10 mm glucose. We conclude that the proximal promoter of the rat PC gene plays a major role in gluconeogenesis and lipogenesis, whereas the distal promoter is necessary for anaplerosis.In vitro translation and in vivo polysome profile analysis indicated that transcripts C and E were translated with similar translational efficiencies that are substantially greater than that of transcript D, suggesting that 5′-untranslated regions play a role in translational control. A previous study on the gene structure of rat pyruvate carboxylase revealed that two tissue-specific promoters are responsible for the production of multiple transcripts with 5′-end heterogeneity (Jitrapakdee, S., Booker, G. W., Cassady, A. I., and Wallace, J. C. (1997) J. Biol. Chem. 272, 20522–20530). Here we report transcription and translation regulation of pyruvate carboxylase (PC) expression during development and in genetically obese rats. The abundance of PC mRNAs was low in fetal liver but increased by 2–4-fold within 7 days after birth, concomitant with an 8-fold increase in the amount of immunoreactive PC and its activity and then decreased during the weaning period. Reverse transcriptase polymerase chain reaction analysis indicated that the proximal promoter was activated during the suckling period and reduced in activity at weaning. In genetically obese Zucker rats, adipose PC was 4–5-fold increased, concomitant with a 5–6-fold increase in mRNA level. Reverse transcriptase-polymerase chain reaction analysis also showed that the proximal promoter was activated in the hyperlipogenic condition. Conversely, transcription of the proximal promoter was not detectable in various liver cell lines, suggesting that this promoter was not functional under cell culture conditions. In rat pancreatic islets and insulinoma cells, only transcripts D and E, generated from the distal promoter of the PC gene, were expressed. Glucose increased PC transcripts from the distal promoter when the insulinoma cells were maintained in 10 mm glucose. We conclude that the proximal promoter of the rat PC gene plays a major role in gluconeogenesis and lipogenesis, whereas the distal promoter is necessary for anaplerosis.In vitro translation and in vivo polysome profile analysis indicated that transcripts C and E were translated with similar translational efficiencies that are substantially greater than that of transcript D, suggesting that 5′-untranslated regions play a role in translational control. pyruvate carboxylase acetyl-CoA carboxylase untranslated region reverse transcriptase-polymerase chain reaction insulinoma polymerase chain reaction base pair(s) rapid amplification of cDNA ends. Pyruvate carboxylase (PC1; EC 6.4.1.1) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, the first regulated step in the gluconeogenic pathway from pyruvate and its precursors. Mammalian PC is a mitochondrial enzyme that plays a vital role in gluconeogenesis in liver and kidney. PC is also involved in lipogenesis, enabling acetyl group export from mitochondria as citrate for the cytoplasmic biosynthesis of fatty acids in adipose tissue and lactating mammary gland (1Wallace J.C. Jitrapakdee S. Chapman-Smith A. Int. J. Biochem. Cell Biol. 1998; 30: 1-5Crossref PubMed Scopus (56) Google Scholar). In astrocytes, PC plays an anaplerotic role in replenishing citric acid cycle intermediates used in the biosynthesis of glutamine, which is subsequently converted to glutamate, aspartate, and γ-aminobutyric acid in neurons (2Gamberino W.C. Berkich D.A. Lynch C.J. Xu B. LaNoue K.F. J. Neurochem. 1997; 69: 2312-2325Crossref PubMed Scopus (93) Google Scholar). Recent studies have shown that PC contributes to glucose-induced insulin secretion from pancreatic islet cells (3MacDonald M.J. Arch. Biochem. Biophys. 1995; 319: 128-132Crossref PubMed Scopus (48) Google Scholar) by participating in a pyruvate-malate shuttle across the mitochondrial membrane to produce cytosolic NADPH (4MacDonald M.J. J. Biol. Chem. 1995; 270: 20051-20058Abstract Full Text Full Text PDF PubMed Google Scholar). Short term regulation of PC activity can be achieved by an allosteric regulator, acetyl-CoA, whereas long term regulation involves changes in the total amount of PC through alterations in the rate of enzyme synthesis in liver, kidney, and adipose tissue (5Barritt G.J. Keech D.B. Wallace J.C. Pyruvate Carboxylase. CRC Press, Inc., Boca Raton, FL1985: 147-177Google Scholar). It has long been demonstrated that PC activity is affected under different physiological and pathological circumstances, e.g. neonatal development, diabetes, nutritional alterations (5Barritt G.J. Keech D.B. Wallace J.C. Pyruvate Carboxylase. CRC Press, Inc., Boca Raton, FL1985: 147-177Google Scholar), and genetic obesity (6Lynch C.J. McCall K.M. Billingsley M.L. Bohlen L.M. Hreniuk S.P. Martin L.F. Witters L.A. Vannucci S.J. Am. J. Physiol. 1992; 262: E698Google Scholar). Although cDNA sequences encoding PC from mammals have been cloned (7Zhang J. Xia W.-L. Brew K. Ahmad F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1766-1770Crossref PubMed Scopus (32) Google Scholar, 8Wexler I.D. Du Y. Ligaris M.V. Mandal S.K. Freytag S.O. Yang B. Liu T. Hwon M. Patel M.S. Kerr D.S. Biochim. Biophys. Acta. 1994; 1227: 46-52Crossref PubMed Scopus (46) Google Scholar, 9MacKay N. Rigat B. Douglas C. Chen H-S. Robinson B.H. Biochem. Biophys. Res. Commun. 1994; 202: 1009-1014Crossref PubMed Scopus (19) Google Scholar, 10Lehn D.A. Moran S.M. MacDonald M.J. Gene. 1995; 165: 331-332Crossref PubMed Scopus (6) Google Scholar, 11Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. Biochem. J. 1996; 316: 630-637Crossref Scopus (31) Google Scholar, 12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar), little is known about the molecular basis of PC expressionin vivo. Cell culture studies have shown that the differentiation of mouse 3T3-L1 fibroblasts to mature adipocytes is accompanied by an increase in the level of PC mRNA and the rate of enzyme synthesis (13Angus C.W. Lane M.D. Biochem. Biophys. Res. Commun. 1981; 103: 1216-1222Crossref PubMed Scopus (11) Google Scholar, 14Freytag S.O. Collier K.J. J. Biol. Chem. 1984; 259: 12831-12837Abstract Full Text PDF PubMed Google Scholar, 15Zhang J. Xia W.-L. Ahmad F. Biochem. J. 1995; 306: 205-210Crossref PubMed Scopus (19) Google Scholar). Our studies on the isolation of cDNA (11Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. Biochem. J. 1996; 316: 630-637Crossref Scopus (31) Google Scholar) and the gene structure (16Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. J. Biol. Chem. 1997; 272: 20522-20530Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) of rat PC indicated that although there is only one copy of the PC gene (17Webb G.C. Jitrapakdee S. Bottema C.D.K. Wallace J.C. Cytogenet. Cell Genet. 1997; 79: 151-152Crossref PubMed Scopus (9) Google Scholar), it generates several forms of PC mRNA, which diverge at their 5′-untranslated regions (UTR) but share the same open reading frame encoding a 1178-residue polypeptide (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar). Two tissue-specific promoters are responsible for the production of two primary transcripts, which then are differentially spliced to five mature transcripts (16Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. J. Biol. Chem. 1997; 272: 20522-20530Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Transcripts generated from the proximal promoter are restricted to gluconeogenic and lipogenic tissues, whereas transcripts generated from the distal promoter are expressed in a wide variety of tissues (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar). Key questions that remain unanswered are which promoter of the rat PC gene is activated or repressed under different physiological conditions and whether the 5′-UTRs of different PC mRNAs can modulate their expression. To gain a further insight into the molecular mechanisms that regulate PC expression in vivo, we have assessed the role of trancriptional regulation during postnatal development and in genetically obese rats by means of a reverse-transcriptase polymerase chain reaction (RT-PCR) analysis that identifies each one of the multiple forms composing the PC mRNA population. We have also shown the 5′-UTRs of different PC mRNAs modulate translation of PC bothin vitro and in vivo. Sprague-Dawley rats were bred and housed at a constant temperature of 25 °C in the animal house, University of Adelaide, and were subjected to treatments approved by the Animal Ethics Committee of the University of Adelaide. Litters of 8- or 12-week-old Zucker rats were obtained from Monash University, Clayton, Victoria. Tissues were quickly removed from the sacrificed animals, snap-frozen in liquid nitrogen, and stored at −80 °C. The rat cell lines used were: BRL3A (ATCC: CRL 1442), Reuber H35 (ATCC: CRL 1548), McA-RH777 (ATCC: CRL 1601), L6 myoblast (ATCC: CRL 1458), rat mammary gland carcinoma cell line (obtained from Institute of Medical and Veterinary Science, Adelaide, Australia), FTO3 (obtained from the Institute of Cell and Tumor Biology, Heidelberg, Germany), fetal rat liver hepatoma cell line (FRL4.1; gift from Dr. G. Yeoh, Department of Biochemistry, University of Western Australia, Nedlands), all of which were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a humidified atmosphere of 5% CO2. Pancreatic islets were isolated from well fed Sprague-Dawley rats weighing 300 g as described previously (3MacDonald M.J. Arch. Biochem. Biophys. 1995; 319: 128-132Crossref PubMed Scopus (48) Google Scholar). Islets were immediately homogenized in RNAzol B (Tel-Test Inc., Friendswood, TX) or maintained for 24 h in RPMI 1640 culture medium containing 10% fetal bovine serum and homogenized in RNAzol B. Rat insulinoma (INS-1) cells were maintained in RPMI 1640 medium containing 50 μm β-mercaptoethanol, 1 mm pyruvate, 10 mm Hepes buffer, pH 7.35, and 10% fetal bovine serum. The pRPC3 plasmid was constructed by subcloning a 0.3-kilobase pairs SacI-PstI fragment of the RACE (Anchor/PC 2) PCR product amplified from rat PC cDNA (11Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. Biochem. J. 1996; 316: 630-637Crossref Scopus (31) Google Scholar) to SacI-PstI-digested pBluescript SK (Stratagene). The pβ-actin plasmid was constructed by RT-PCR as follows. Rat brain RNA was annealed to a cDNA synthesis primer designed from the coding region of rat β-actin mRNA (5′-TCTTCATGGTGCTAGGAGCCAG-3′) (18Nudel U. Zakut R. Shani M. Neuman S. Levy Z. Yaffe D. Nucleic Acids Res. 1983; 11: 1759-1771Crossref PubMed Scopus (1017) Google Scholar) and reverse-transcribed. The cDNA was subjected to PCR with a sense primer (β-actin (+)) (5′-AACTTGGATCCCCTGGAGAAGAGCTATGAGCTG-3′; with aBamHI restriction site attached at the 5′-end, underlined) and an antisense primer (β-actin (−)) (5′-TCCCGGGTACCAGACAGCACTGTGTTTGGCA-3′; with aKpnI restriction site attached at the 5′-end, underlined) (18Nudel U. Zakut R. Shani M. Neuman S. Levy Z. Yaffe D. Nucleic Acids Res. 1983; 11: 1759-1771Crossref PubMed Scopus (1017) Google Scholar). The PCR products were double-digested with KpnI andBamHI, cloned into KpnI-BamHI-digested pBluescript, and sequenced. The above plasmids were linearized by digestion with Ecl136 and BamHI, respectively, and full-length probes were gel-purified. The antisense riboprobes were synthesized using the MaxiScript kit (Ambion, TX) with T7 RNA polymerase in the presence of 50 μCi of [α-32P]UTP (8000 mCi/mmol). The full-length probe was gel-purified and eluted. Total RNA was isolated from frozen tissue using an RNaqueous kit (Ambion, TX), and ribonuclease protection analysis was performed using a ribonuclease protection assay kit (Ambion, TX). Briefly, 10 μg of total RNA were hybridized with approximately 1 × 105 cpm of each probe at 45 °C for 18 h. The probe and unhybridized RNAs were digested with RNase A and T1 for 30 min at 37 °C. The 201-bp protected fragment representing β-actin mRNA and the 341-bp protected fragment representing the common coding region of different PC mRNAs (see Fig. 1) were denatured and separated by electrophoresis on 6% acrylamide, 8 m urea gel at 350 V for 2 h. The gel was dried and placed in the PhosphorImager screen overnight. The intensities of hybridization bands were quantitated by PhosphorImager analysis using the ImageQuant Software (Molecular Dynamics) and corrected for uneven loading of the RNA samples by comparison with β-actin bands. The first strand cDNA for PCR was synthesized as follows. 10 μg of total RNA were hybridized with 100 ng each of PC-8 primer (5′-GGACCACTGGAACGCCTGT-3′) (positions +484 to +503) (11Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. Biochem. J. 1996; 316: 630-637Crossref Scopus (31) Google Scholar) and of β-actin 1 primer (as described above) and reverse-transcribed as described previously (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar). The PCR primers were as follows: 5′-GGCGAATTCCGATGGCAATCTCACCTCTGTTGGC-3′ (MRACE II), which was directed to a common coding sequence of both classes of PC mRNA (positions +127 to +150) and the specific primers 5′-GGCTTGAGGCGACGGGGCGAAG-3′ (U1) for class I mRNA and 5′-GGCTTGAGGCGACGGGGCGAAG-3′ (U2) for the class II mRNA (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar). The positions of PCR primers relative to the 5′-UTR of each class of PC mRNA and the sizes of PCR products are shown in Fig. 1. The PCR profile consisted of 30 cycles of amplification as described previously (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar). The β-actin cDNA, an internal control, was also amplified using the same conditions as described for PC except that the primers were β-actin (−) and β-actin (+) as described above. PCR profiles consist of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The PCR products were analyzed on 2.5% agarose gel electrophoresis. One g of frozen liver was ground to a powder in liquid nitrogen and homogenized in 4 volumes of extraction buffer (0.25 m sucrose, 0.1 mm EDTA, 20 mm Tris-HCl, pH 7.2, 0.5 mm dithiothreitol, and 0.5 mmphenylmethylsulfonyl fluoride) at 4 °C. The homogenate was freeze-dried and reconstituted in 3 ml of 50 mm Tris acetate, pH 7.0, 5 mm ATP, 5 mmMgCl2, and 0.5 mm EDTA. The homogenate was centrifuged at 20,000 × g for 20 min at 4 °C, and the supernatant-containing PC was kept at −80 °C. For adipose tissue, 1–2 g of epididymal fat pads were homogenized as described above, and fat was removed from the homogenate by centrifugation at 10,000 × g for 5 min at 4 °C. The aqueous extract was removed from the cell debris, freeze-dried, and subsequently reconstituted as described above. Twenty μg of total protein (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) were assayed for PC activity at 30 °C for 2 min by a radioactive CO2 fixation (20Ballard F.J. Hanson R.W. Biochem. J. 1967; 104: 866-871Crossref PubMed Scopus (302) Google Scholar), except that the reaction was coupled with the conversion of oxaloacetate to malate by 2 units of malic dehydrogenase (Sigma) in the presence of 0.1 mm NADH. One unit of PC is defined as the conversion of 1 μmol of NaH14CO3 to malate per min, and the PC activity is defined as milliunits/mg of total protein. Total protein from liver homogenate (20 μg) was subjected to reducing discontinuous SDS-polyacrylamide gel electrophoresis on a 4% stacking gel and 7.5% polyacrylamide-separating gel (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). The separated proteins were transferred to nitrocellulose filters using a semi-dry electroblotter (Multiphor II Novablot, Amersham Pharmacia Biotech). Membranes were blocked with 1% bovine serum albumin in 10 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.05% Tween 20. The membrane was reacted for 2 h with a 1:20,000 dilution of anti-chicken PC rabbit IgG polyclonal antibodies that cross-react with rat PC (22Rohde M. Lim F. Wallace J.C. Arch. Biochem. Biophys. 1991; 290: 197-201Crossref PubMed Scopus (37) Google Scholar). The secondary anti-rabbit antibodies conjugated with alkaline phosphatase were then reacted with the primary antibodies for 2 h. The immunoprecipitate bands were visualized by adding nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate (Boehringer Mannheim) and quantitated by laser densitometer (Molecular Dynamics). Known amounts of purified rat PC (11Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. Biochem. J. 1996; 316: 630-637Crossref Scopus (31) Google Scholar) were run as internal standards so that the intensities of immunoprecipitated bands between different experiments could be compared. Using the RT-PCR conditions described above, cDNA encoding the biotin carboxylation domain of the rat PC was generated with a forward primer directed against residues 1 to 9 of rat PC having a BamHI restriction site attached at the 5′-end (underlined) (5′-CCAAAGGATCCATGCTGAAGTTCCAAACAGTTCGAGG-3′) and a reverse primer directed against residues 482 to 489 together with stop codons (bold) and KpnI restriction site (underlined) (5′-TCCCGGTACC TTATCACTGGAACAGCTCGGGGTTCTCATCG-3′) (11Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. Biochem. J. 1996; 316: 630-637Crossref Scopus (31) Google Scholar). The PCR product was then digested with BamHI andKpnI and cloned into BamHI and KpnI sites of pGEM3Zf (±) (Promega) and sequenced. The 5′-UTRs of transcripts C, D, and E, which have previously been cloned in pBluescript (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar), were then excised with EcoRI andBsu36I and inserted in-frame in front of the coding region of the above construct. Finally, the 3′-UTR and poly(A) tail of rat PC cDNA were excised from λRL 2.35 clones (11Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. Biochem. J. 1996; 316: 630-637Crossref Scopus (31) Google Scholar) and fused to the 3′-end of the above constructs at KpnI site. This resulted in the final constructs containing different 5′-UTRs of transcripts C, D, and E of the rat PC mRNAs (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar), respectively, fused to the biotin carboxylation domain, the 3′-UTR, and poly(A) tail. The RNAs were transcribed from 1.5 μg of either circular or linearized plasmids, with T7 RNA polymerase in the presence of m7Gppp(5′)G. Free m7Gppp(5′)G was removed by LiCl precipitation. Capped mRNAs (0.2 μg) were then subjected to in vitro translation with rabbit reticulocyte lysate (Promega), and protein was labeled with 20 μCi of [35S]methionine at 30 °C. The synthesized proteins were analyzed by reducing discontinuous SDS-polyacrylamide gel electrophoresis (12.5% separating gel, 4% stacking gel). The dried gel was exposed to a PhosphorImager screen overnight before being quantitated as above. Rat livers were homogenized in 3 ml of buffer A containing 10 mm Hepes, pH 7.4, 100 mm KCl, 5 mm MgCl2, 2% Triton X-100, 100 μg/ml cycloheximide, and 20,000 units of RNase inhibitor. Nuclei and unbroken cells were removed by centrifugation at 10,000 × g at 4 °C for 5 min. One ml of cytosolic supernatant was then overlaid on 11 ml of 15 to 45% sucrose gradient in buffer A and centrifuged at 40,000 rpm at 4 °C for 2 h in a Ti60 rotor (Beckman). One-ml fractions were collected manually from the bottom to the top of the tube. As a control, an equal portion of cytosolic supernatant was adjusted to 20 mm EDTA but lacking MgCl2. RNA in each fraction was extracted with phenol/chloroform and ethanol precipitation and subjected to RT-PCR analysis. Previous studies have shown that PC activity in fetal rat liver is barely detectable but increased markedly during the suckling period (20Ballard F.J. Hanson R.W. Biochem. J. 1967; 104: 866-871Crossref PubMed Scopus (302) Google Scholar, 23Yeung D. Stanley R.S. Oliver I.T. Biochem. J. 1967; 105: 1219-1227Crossref PubMed Google Scholar). To determine whether this regulation occurs at the transcriptional, translational, or post-translational steps, we measured amounts of PC protein by Western immunoblot, PC activity assay, and PC transcripts using both ribonuclease protection assay and RT-PCR in different-aged rats i.e. late gestation (2 days before birth), suckling period (1-day and 7-day pups), weaned rats (28 days), and adults. As seen in Fig. 2, A andC, the levels of PC were very low in 20-day fetal rat liver but rapidly increased 4-fold within 1 day after birth and by 8-fold at day 7. However PC levels had decreased by the time of weaning to approximately adult levels. The increase in immunoreactive PC protein during the suckling period is concomitant with a similar increase in PC activity (4-fold and 8-fold increase) as indicated in Fig.2 C, indicating that post-translational modification is not responsible for the increase in PC activity. The amount of immunoreactive PC protein was also decreased to the same level in weaned rats and in adults as that seen in 1-day pups (see Fig. 2,A and C). Ribonuclease protection assay using a riboprobe synthesized from the common coding sequence of different forms of PC mRNA clearly showed that although both PC protein and PC activity were low in fetal liver, its transcripts were readily detectable (Fig. 2 B). The abundance of PC transcripts was increased 2-fold and 4-fold in 1-day and 7-day pups, respectively (Fig.2 C). However, the increases in the amounts of PC transcripts are not as dramatic as those observed with the increase in PC protein (4-fold and 8-fold), suggesting that translational control might play a role in accelerating the rate of enzyme synthesis. The abundance of PC transcripts is also decreased during weaning and in adults, as seen with the level of PC protein and PC activity (Fig. 2, B andC). Because the rat PC mRNAs are composed of multiple transcripts generated from alternate promoters (16Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. J. Biol. Chem. 1997; 272: 20522-20530Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), the RNase protection assay results only reflect an overall transcriptional activity of the gene. Since the sizes of the 5′-UTRs of different rat PC mRNA isoforms are relatively small compared with the common coding region of the messages (4.0 kilobase pairs), conventional Northern analysis using a probe synthesized from the coding region cannot differentiate between these subtle size differences. Therefore, semi-quantitative RT-PCR was also performed to identify which promoter of the PC gene was being used to control the expression. Two sense primers directed against the most 5′-untranslated region of class I (exon 1B) and class II (exon 1D) PC transcripts (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar, 16Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. J. Biol. Chem. 1997; 272: 20522-20530Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and an antisense primer directed to a common coding region of the different transcripts were used to selectively amplify by PCR the 5′-ends of the different PC cDNA species (see Fig. 1). We initially titrated amounts of RNAs in RT-PCR and found that using a 1:100 dilution of cDNA synthesized from 10 μg of starting RNA gave reliable results (data not shown). In 20-day fetal liver, transcript D, the major form transcribed from the distal promoter was detectable in a greater abundance than transcript C, transcribed from the proximal promoter (Fig. 3). The level of transcript C was increased in 1-day and 7-day pups, respectively. It is interesting to note that although transcript D was accumulated in adults concomitant with a decrease of transcript C (Fig. 3), the level of PC and its activity were decreased (Fig. 2, A andC). This suggested that the translation of transcript D produced during such a period is less efficient than transcript C. However, transcripts A and B, minor forms transcribed from the proximal promoter, were not detectable, and this is consistent with our previous study (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar). In genetically obese Zucker rats, it has been reported that PC levels are 2–5-fold-increased in the adipose tissue of obese animals (ob/ob) (6Lynch C.J. McCall K.M. Billingsley M.L. Bohlen L.M. Hreniuk S.P. Martin L.F. Witters L.A. Vannucci S.J. Am. J. Physiol. 1992; 262: E698Google Scholar). To investigate whether this increase in the amount of PC is because of an enhanced transcriptional activity of the gene, we performed ribonuclease protection assays and RT-PCR as described above. In adipose tissues of obese rats, PC transcripts were increased 5- to 6-fold higher than those of their lean litter mates, whereas β-actin message was not affected (Fig.4 A). RT-PCR clearly showed that the increase in mRNA was mainly because of an increase in transcript C with a decrease in transcripts D and E between the two groups of rats (Fig. 4 B). In livers from the obese rats, PC transcripts were also increased. A 4-fold difference was observed between the two groups of animals (see Fig. 4 A). RT-PCR analysis revealed the increase in hepatic PC mRNA again resulted from an increase in transcript C (see Fig. 4 B) with little change in transcripts D and E. The increase in PC transcripts in both adipose tissue and liver was concomitant with an increase in the amount of PC detected by Western immunoblot. As shown in Fig. 4 C, the levels of adipose PC in obese rats were 4–5-fold higher than those of their lean litter mates, but immunoreactive hepatic PC in obese rats was only 2–3-fold higher than in their lean litter mates. Although PC was detected by Western immunoblot in every cell line (data not shown), the expression patterns of PC transcripts in liver cell lines were different from that of an intact liver. Transcript C generated from the proximal promoter was not detectable even though 100 times more cDNAs were used in RT-PCR, but transcripts D and E transcribed from the distal promoter (12Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar) were detectable by RT-PCR (Fig.5 A). It is unlikely that the primer binding site located within exon 1D (Fig. 1) was absent or deleted because all of these cell lines were independently derived from different sources. It appears that the proximal promoter driving expression of transcript C in liver is not functional under these cell culture conditions. PC expression has been shown to be activated upon glucose-induced insulin secretion in pancreatic islets (3MacDonald M.J. Arch. Biochem. Biophys. 1995; 319: 128-132Crossref PubMed Scopus (48) Google Scholar) and insulinoma (INS-1) cells (24Brun T. Roche E. Assimacopoulos-Jeannet F. Corkey B.E. Kim K.-H. Prentki M. Diabetes. 1996; 45: 190-198Crossref PubMed Scopus (135) Google Scholar). RT-PCR was performed on RNA extracted from these two cell types. Only transcripts D and E were detected in both cell types (see Fig.5 B), suggesting that the distal promoter plays a role in anaplerosis. To test whether glucose can increase transcription from the distal promoter, INS-1 cells were initially maintained in RPMI medium containing 1 mm glucose for 48 days and then maintained in the same glucose-free media supplemented with 1, 10, and 20 mm glucose. Glucose (10 or 20 mm) dramatically increased the level of transcripts D and E in INS-1 cells (Fig. 5 B)." @default.
• W2082710199 created "2016-06-24" @default.
• W2082710199 creator A5005909432 @default.
• W2082710199 creator A5006957202 @default.
• W2082710199 creator A5026986628 @default.
• W2082710199 creator A5037504318 @default.
• W2082710199 date "1998-12-01" @default.
• W2082710199 modified "2023-10-11" @default.
• W2082710199 title "Regulation of Rat Pyruvate Carboxylase Gene Expression by Alternate Promoters during Development, in Genetically Obese Rats and in Insulin-secreting Cells" @default.
• W2082710199 cites W1525779234 @default.
• W2082710199 cites W1555617925 @default.
• W2082710199 cites W1588593248 @default.
• W2082710199 cites W1639883523 @default.
• W2082710199 cites W1764017277 @default.
• W2082710199 cites W1776931561 @default.
• W2082710199 cites W1915827411 @default.
• W2082710199 cites W1967404050 @default.
• W2082710199 cites W1970882557 @default.
• W2082710199 cites W1979638982 @default.
• W2082710199 cites W1987417071 @default.
• W2082710199 cites W1999916384 @default.
• W2082710199 cites W2000292982 @default.
• W2082710199 cites W2000396454 @default.
• W2082710199 cites W2017272722 @default.
• W2082710199 cites W2020221566 @default.
• W2082710199 cites W2024220274 @default.
• W2082710199 cites W2037620727 @default.
• W2082710199 cites W2049360609 @default.
• W2082710199 cites W2049812112 @default.
• W2082710199 cites W2064028860 @default.
• W2082710199 cites W2073827764 @default.
• W2082710199 cites W2074546186 @default.
• W2082710199 cites W2082903399 @default.
• W2082710199 cites W2087664265 @default.
• W2082710199 cites W2090331145 @default.
• W2082710199 cites W2091655349 @default.
• W2082710199 cites W2094385264 @default.
• W2082710199 cites W2096573235 @default.
• W2082710199 cites W2098544099 @default.
• W2082710199 cites W2100837269 @default.
• W2082710199 cites W2114882367 @default.
• W2082710199 cites W2116872973 @default.
• W2082710199 cites W2315981238 @default.
• W2082710199 cites W4245381392 @default.
• W2082710199 cites W4293247451 @default.
• W2082710199 cites W44935923 @default.
• W2082710199 cites W65635151 @default.
• W2082710199 doi "https://doi.org/10.1074/jbc.273.51.34422" @default.
• W2082710199 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9852109" @default.
• W2082710199 hasPublicationYear "1998" @default.
• W2082710199 type Work @default.
• W2082710199 sameAs 2082710199 @default.
• W2082710199 citedByCount "53" @default.
• W2082710199 countsByYear W20827101992012 @default.
• W2082710199 countsByYear W20827101992013 @default.
• W2082710199 countsByYear W20827101992014 @default.
• W2082710199 countsByYear W20827101992015 @default.
• W2082710199 countsByYear W20827101992016 @default.
• W2082710199 countsByYear W20827101992017 @default.
• W2082710199 countsByYear W20827101992019 @default.
• W2082710199 countsByYear W20827101992021 @default.
• W2082710199 countsByYear W20827101992022 @default.
• W2082710199 crossrefType "journal-article" @default.
• W2082710199 hasAuthorship W2082710199A5005909432 @default.
• W2082710199 hasAuthorship W2082710199A5006957202 @default.
• W2082710199 hasAuthorship W2082710199A5026986628 @default.
• W2082710199 hasAuthorship W2082710199A5037504318 @default.
• W2082710199 hasBestOaLocation W20827101991 @default.
• W2082710199 hasConcept C101762097 @default.
• W2082710199 hasConcept C104317684 @default.
• W2082710199 hasConcept C126322002 @default.
• W2082710199 hasConcept C134018914 @default.
• W2082710199 hasConcept C140137476 @default.
• W2082710199 hasConcept C150194340 @default.
• W2082710199 hasConcept C153911025 @default.
• W2082710199 hasConcept C181199279 @default.
• W2082710199 hasConcept C185592680 @default.
• W2082710199 hasConcept C2779306644 @default.
• W2082710199 hasConcept C55493867 @default.
• W2082710199 hasConcept C71924100 @default.
• W2082710199 hasConcept C86803240 @default.
• W2082710199 hasConceptScore W2082710199C101762097 @default.
• W2082710199 hasConceptScore W2082710199C104317684 @default.
• W2082710199 hasConceptScore W2082710199C126322002 @default.
• W2082710199 hasConceptScore W2082710199C134018914 @default.
• W2082710199 hasConceptScore W2082710199C140137476 @default.
• W2082710199 hasConceptScore W2082710199C150194340 @default.
• W2082710199 hasConceptScore W2082710199C153911025 @default.
• W2082710199 hasConceptScore W2082710199C181199279 @default.
• W2082710199 hasConceptScore W2082710199C185592680 @default.
• W2082710199 hasConceptScore W2082710199C2779306644 @default.
• W2082710199 hasConceptScore W2082710199C55493867 @default.
• W2082710199 hasConceptScore W2082710199C71924100 @default.
• W2082710199 hasConceptScore W2082710199C86803240 @default.
• W2082710199 hasIssue "51" @default.
• W2082710199 hasLocation W20827101991 @default.
• W2082710199 hasOpenAccess W2082710199 @default.
• W2082710199 hasPrimaryLocation W20827101991 @default.

• next