Abstract
The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2-3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling-weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was highly induced by fasting during the suckling, whereas no such change in mitochondrial HMG-CoA synthase mRNA levels has been observed in rat. The enzyme activity of mitochondrial HMG-CoA synthase increased 27-fold during starvation in piglets, but remained one order of magnitude lower than rats. These results indicate that post-transcriptional mechanism(s) and/or intrinsic differences in the encoded enzyme are responsible for the low activity of pig HMG-CoA synthase observed throughout development or after fasting.
Full Text
The Full Text of this article is available as a PDF (559.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arias G., Matas R., Asins G., Hegardt F. G., Serra D. The effect of fasting and insulin treatment on carnitine palmitoyl transferase I and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase mRNA levels in liver from suckling rats. Biochem Soc Trans. 1995 Aug;23(3):493S–493S. doi: 10.1042/bst023493s. [DOI] [PubMed] [Google Scholar]
- Asins G., Serra D., Arias G., Hegardt F. G. Developmental changes in carnitine palmitoyltransferases I and II gene expression in intestine and liver of suckling rats. Biochem J. 1995 Mar 1;306(Pt 2):379–384. doi: 10.1042/bj3060379. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Augenfeld J., Fritz I. B. Carnitine palmitolyltransferase activity and fatty acid oxidation by livers from fetal and neonatal rats. Can J Biochem. 1970 Mar;48(3):288–294. doi: 10.1139/o70-050. [DOI] [PubMed] [Google Scholar]
- Ayté J., Gil-Gómez G., Hegardt F. G. Nucleotide sequence of a rat liver cDNA encoding the cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase. Nucleic Acids Res. 1990 Jun 25;18(12):3642–3642. doi: 10.1093/nar/18.12.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bailey E., Lockwood E. A. Some aspects of fatty acid oxidation and ketone body formation and utilization during development of the rat. Enzyme. 1973;15(1):239–253. [PubMed] [Google Scholar]
- Baird G. D., Hibbitt K. G., Lee J. Enzymes involved in acetoacetate formation in various bovine tissues. Biochem J. 1970 May;117(4):703–709. doi: 10.1042/bj1170703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beaudry M. A., Chiasson J. L., Exton J. H. Gluconeogenesis in the suckling rat. Am J Physiol. 1977 Sep;233(3):E175–E180. doi: 10.1152/ajpendo.1977.233.3.E175. [DOI] [PubMed] [Google Scholar]
- Bengtsson G., Gentz J., Hakkarainen J., Hellström R., Persson B. Plasma levels of FFA, glycerol, beta-hydroxybutyrate and blood glucose during the postnatal development of the pig. J Nutr. 1969 Mar;97(3):311–315. doi: 10.1093/jn/97.3.311. [DOI] [PubMed] [Google Scholar]
- Bieber L. L., Markwell M. A., Blair M., Helmrath T. A. Studies on the development of carnitine palmitoyltransferase and fatty acid oxidation in liver mitochondria of neonatal pigs. Biochim Biophys Acta. 1973 Nov 29;326(2):145–154. doi: 10.1016/0005-2760(73)90240-3. [DOI] [PubMed] [Google Scholar]
- Buesa C., Martínez-Gonzalez J., Casals N., Haro D., Piulachs M. D., Bellés X., Hegardt F. G. Blattella germanica has two HMG-CoA synthase genes. Both are regulated in the ovary during the gonadotrophic cycle. J Biol Chem. 1994 Apr 22;269(16):11707–11713. [PubMed] [Google Scholar]
- Békési A., Williamson D. H. An explanation for ketogenesis by the intestine of the suckling rat: the presence of an active hydroxymethylglutaryl-coenzyme A pathway. Biol Neonate. 1990;58(3):160–165. doi: 10.1159/000243256. [DOI] [PubMed] [Google Scholar]
- Casals N., Roca N., Guerrero M., Gil-Gómez G., Ayté J., Ciudad C. J., Hegardt F. G. Regulation of the expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene. Its role in the control of ketogenesis. Biochem J. 1992 Apr 1;283(Pt 1):261–264. doi: 10.1042/bj2830261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
- Clinkenbeard K. D., Reed W. D., Mooney R. A., Lane M. D. Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis. J Biol Chem. 1975 Apr 25;250(8):3108–3116. [PubMed] [Google Scholar]
- Dashti N., Ontko J. A. Rate-limiting function of 3-hydroxy-3-methylglutaryl-coenzyme A synthase in ketogenesis. Biochem Med. 1979 Dec;22(3):365–374. doi: 10.1016/0006-2944(79)90024-3. [DOI] [PubMed] [Google Scholar]
- Decaux J. F., Robin D., Robin P., Ferré P., Girard J. Intramitochondrial factors controlling hepatic fatty acid oxidation at weaning in the rat. FEBS Lett. 1988 May 9;232(1):156–158. doi: 10.1016/0014-5793(88)80407-1. [DOI] [PubMed] [Google Scholar]
- Duée P. H., Pégorier J. P., Quant P. A., Herbin C., Kohl C., Girard J. Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Biochem J. 1994 Feb 15;298(Pt 1):207–212. doi: 10.1042/bj2980207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Edwards J. B., Delort J., Mallet J. Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5' ends of mRNAs and for constructing cDNA libraries by in vitro amplification. Nucleic Acids Res. 1991 Oct 11;19(19):5227–5232. doi: 10.1093/nar/19.19.5227. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Escriva F., Ferre P., Robin D., Robin P., Decaux J. F., Girard J. Evidence that the development of hepatic fatty acid oxidation at birth in the rat is concomitant with an increased intramitochondrial CoA concentration. Eur J Biochem. 1986 May 2;156(3):603–607. doi: 10.1111/j.1432-1033.1986.tb09620.x. [DOI] [PubMed] [Google Scholar]
- Ferré P., Pégorier J. P., Williamson D. H., Girard J. R. The development of ketogenesis at birth in the rat. Biochem J. 1978 Dec 15;176(3):759–765. doi: 10.1042/bj1760759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ferré P., Satabin P., Decaux J. F., Escriva F., Girard J. Development and regulation of ketogenesis in hepatocytes isolated from newborn rats. Biochem J. 1983 Sep 15;214(3):937–942. doi: 10.1042/bj2140937. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Foster P. C., Bailey E. Changes in the activities of the enzymes of hepatic fatty acid oxidation during development of the rat. Biochem J. 1976 Jan 15;154(1):49–56. doi: 10.1042/bj1540049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gentz J., Bengtsson G., Hakkarainen J., Hellström R., Persson B. Metabolic effects of starvation during neonatal period in the piglet. Am J Physiol. 1970 Mar;218(3):662–668. doi: 10.1152/ajplegacy.1970.218.3.662. [DOI] [PubMed] [Google Scholar]
- Gil-Gómez G., Ayté J., Hegardt F. G. The rat mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme-A-synthase gene contains elements that mediate its multihormonal regulation and tissue specificity. Eur J Biochem. 1993 Apr 15;213(2):773–779. doi: 10.1111/j.1432-1033.1993.tb17819.x. [DOI] [PubMed] [Google Scholar]
- Gil G., Goldstein J. L., Slaughter C. A., Brown M. S. Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. I. Isolation and sequencing of a full-length cDNA. J Biol Chem. 1986 Mar 15;261(8):3710–3716. [PubMed] [Google Scholar]
- Girard J., Ferré P., Pégorier J. P., Duée P. H. Adaptations of glucose and fatty acid metabolism during perinatal period and suckling-weaning transition. Physiol Rev. 1992 Apr;72(2):507–562. doi: 10.1152/physrev.1992.72.2.507. [DOI] [PubMed] [Google Scholar]
- Granner D., Pilkis S. The genes of hepatic glucose metabolism. J Biol Chem. 1990 Jun 25;265(18):10173–10176. [PubMed] [Google Scholar]
- Guzmán M., Bijleveld C., Geelen M. J. Flexibility of zonation of fatty acid oxidation in rat liver. Biochem J. 1995 Nov 1;311(Pt 3):853–860. doi: 10.1042/bj3110853. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hahn P., Taller M. Ketone formation in the intestinal mucosa of infant rats. Life Sci. 1987 Sep 21;41(12):1525–1528. doi: 10.1016/0024-3205(87)90718-1. [DOI] [PubMed] [Google Scholar]
- Kattar-Cooley P. A., Wang H. H., Mende-Mueller L. M., Miziorko H. M. Avian liver 3-hydroxy-3-methylglutaryl-CoA synthase: distinct genes encode the cholesterogenic and ketogenic isozymes. Arch Biochem Biophys. 1990 Dec;283(2):523–529. doi: 10.1016/0003-9861(90)90677-q. [DOI] [PubMed] [Google Scholar]
- Lepine A. J., Boyd R. D., Welch J. Effect of colostrum intake on plasma glucose, non-esterified fatty acid and glucoregulatory hormone patterns in the neonatal pig. Domest Anim Endocrinol. 1989 Jul;6(3):231–241. doi: 10.1016/0739-7240(89)90017-9. [DOI] [PubMed] [Google Scholar]
- Lin X., Adams S. H., Odle J. Acetate represents a major product of heptanoate and octanoate beta-oxidation in hepatocytes isolated from neonatal piglets. Biochem J. 1996 Aug 15;318(Pt 1):235–240. doi: 10.1042/bj3180235. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Martínez-González J., Buesa C., Piulachs M. D., Bellés X., Hegardt F. G. 3-Hydroxy-3-methylglutaryl-coenzyme-A synthase from Blattella germanica. Cloning, expression, developmental pattern and tissue expression. Eur J Biochem. 1993 Oct 15;217(2):691–699. doi: 10.1111/j.1432-1033.1993.tb18295.x. [DOI] [PubMed] [Google Scholar]
- Mascaró C., Buesa C., Ortiz J. A., Haro D., Hegardt F. G. Molecular cloning and tissue expression of human mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. Arch Biochem Biophys. 1995 Mar 10;317(2):385–390. doi: 10.1006/abbi.1995.1178. [DOI] [PubMed] [Google Scholar]
- McGarry J. D., Foster D. W. Regulation of hepatic fatty acid oxidation and ketone body production. Annu Rev Biochem. 1980;49:395–420. doi: 10.1146/annurev.bi.49.070180.002143. [DOI] [PubMed] [Google Scholar]
- Misra I., Narasimhan C., Miziorko H. M. Avian 3-hydroxy-3-methylglutaryl-CoA synthase. Characterization of a recombinant cholesterogenic isozyme and demonstration of the requirement for a sulfhydryl functionality in formation of the acetyl-enzyme reaction intermediate. J Biol Chem. 1993 Jun 5;268(16):12129–12135. [PubMed] [Google Scholar]
- Mitchell G. A., Robert M. F., Hruz P. W., Wang S., Fontaine G., Behnke C. E., Mende-Mueller L. M., Schappert K., Lee C., Gibson K. M. 3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL). Cloning of human and chicken liver HL cDNAs and characterization of a mutation causing human HL deficiency. J Biol Chem. 1993 Feb 25;268(6):4376–4381. [PubMed] [Google Scholar]
- Odle J., Lin X., van Kempen T. A., Drackley J. K., Adams S. H. Carnitine palmitoyltransferase modulation of hepatic fatty acid metabolism and radio-HPLC evidence for low ketogenesis in neonatal pigs. J Nutr. 1995 Oct;125(10):2541–2549. doi: 10.1093/jn/125.10.2541. [DOI] [PubMed] [Google Scholar]
- Pégorier J. P., Duée P. H., Assan R., Peret J., Girard J. Changes in circulating fuels, pancreatic hormones and liver glycogen concentration in fasting or suckling newborn pigs. J Dev Physiol. 1981 Jun;3(3):203–217. [PubMed] [Google Scholar]
- Pégorier J. P., Duée P. H., Girard J., Peret J. Metabolic fate of non-esterified fatty acids in isolated hepatocytes from newborn and young pigs. Evidence for a limited capacity for oxidation and increased capacity for esterification. Biochem J. 1983 Apr 15;212(1):93–97. doi: 10.1042/bj2120093. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Quant P. A. Activity and expression of hepatic mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase during the starved-to-fed transition. Biochem Soc Trans. 1990 Oct;18(5):994–995. doi: 10.1042/bst0180994. [DOI] [PubMed] [Google Scholar]
- Quant P. A., Tubbs P. K., Brand M. D. Glucagon activates mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in vivo by decreasing the extent of succinylation of the enzyme. Eur J Biochem. 1990 Jan 12;187(1):169–174. doi: 10.1111/j.1432-1033.1990.tb15291.x. [DOI] [PubMed] [Google Scholar]
- Rodríguez J. C., Gil-Gómez G., Hegardt F. G., Haro D. Peroxisome proliferator-activated receptor mediates induction of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene by fatty acids. J Biol Chem. 1994 Jul 22;269(29):18767–18772. [PubMed] [Google Scholar]
- Royo T., Pedragosa M. J., Ayté J., Gil-Gómez G., Vilaró S., Hegardt F. G. Testis and ovary express the gene for the ketogenic mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. J Lipid Res. 1993 Jun;34(6):867–874. [PubMed] [Google Scholar]
- Russ A. P., Ruzicka V., Maerz W., Appelhans H., Gross W. Amplification and direct sequencing of a cDNA encoding human cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase. Biochim Biophys Acta. 1992 Oct 20;1132(3):329–331. doi: 10.1016/0167-4781(92)90172-v. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Serra D., Asins G., Hegardt F. G. Ketogenic mitochondrial 3-hydroxy 3-methylglutaryl-CoA synthase gene expression in intestine and liver of suckling rats. Arch Biochem Biophys. 1993 Mar;301(2):445–448. doi: 10.1006/abbi.1993.1169. [DOI] [PubMed] [Google Scholar]
- Serra D., Casals N., Asins G., Royo T., Ciudad C. J., Hegardt F. G. Regulation of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase protein by starvation, fat feeding, and diabetes. Arch Biochem Biophys. 1993 Nov 15;307(1):40–45. doi: 10.1006/abbi.1993.1557. [DOI] [PubMed] [Google Scholar]
- Swiatek K. R., Kipnis D. M., Mason G., Chao K. L., Cornblath M. Starvation hypoglycemia in newborn pigs. Am J Physiol. 1968 Feb;214(2):400–405. doi: 10.1152/ajplegacy.1968.214.2.400. [DOI] [PubMed] [Google Scholar]
- Thumelin S., Esser V., Charvy D., Kolodziej M., Zammit V. A., McGarry D., Girard J., Pegorier J. P. Expression of liver carnitine palmitoyltransferase I and II genes during development in the rat. Biochem J. 1994 Jun 1;300(Pt 2):583–587. doi: 10.1042/bj3000583. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thumelin S., Forestier M., Girard J., Pegorier J. P. Developmental changes in mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene expression in rat liver, intestine and kidney. Biochem J. 1993 Jun 1;292(Pt 2):493–496. doi: 10.1042/bj2920493. [DOI] [PMC free article] [PubMed] [Google Scholar]
- UNGER R. H., EISENTRAUT A. M., McCALL M. S., MADISON L. L. Glucagon antibodies and an immunoassay for glucagon. J Clin Invest. 1961 Jul;40:1280–1289. doi: 10.1172/JCI104357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Williamson D. H., Bates M. W., Krebs H. A. Activity and intracellular distribution of enzymes of ketone-body metabolism in rat liver. Biochem J. 1968 Jul;108(3):353–361. doi: 10.1042/bj1080353. [DOI] [PMC free article] [PubMed] [Google Scholar]
- von Heijne G. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 1986 Jun;5(6):1335–1342. doi: 10.1002/j.1460-2075.1986.tb04364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]