Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Oct 1;98(7):1575–1584. doi: 10.1172/JCI118951

Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.

H Shimano 1, J D Horton 1, R E Hammer 1, I Shimomura 1, M S Brown 1, J L Goldstein 1
PMCID: PMC507590  PMID: 8833906

Abstract

The NH2-terminal domain of sterol-regulatory element binding protein-1a (SREBP-1a) activates transcription of genes encoding enzymes of cholesterol and fatty acid biosynthesis in cultured cells. This domain is synthesized as part of a membrane-bound precursor that is attached to the nuclear envelope and endoplasmic reticulum. In sterol-depleted cells a two-step proteolytic process releases this NH2-terminal domain, which enters the nucleus and activates transcription. Proteolysis is suppressed by sterols, thereby suppressing transcription. In the current experiments we produce transgenic mice that overexpress a truncated version of human SREBP-1a that includes the NH2-terminal domain but lacks the membrane attachment site. This protein enters the nucleus without a requirement for proteolysis, and therefore it cannot be down-regulated. Expression was driven by the phosphoenolpyruvate carboxykinase (PEPCK) promoter, which gives high level expression in liver. When placed on a low carbohydrate/high protein diet to induce the PEPCK promoter, the transgenic mice developed progressive and massive enlargement of the liver, owing to the engorgement of hepatocytes with cholesterol and triglycerides. The mRNAs encoding 3-hydroxy-3-methylglutaryl CoA (HMG CoA) synthase, HMG CoA reductase, squalene synthase, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1 were all elevated markedly, as was the LDL receptor mRNA. The rates of cholesterol and fatty acid synthesis in liver were elevated 5- and 25-fold, respectively. Remarkably, plasma lipid levels were not elevated. The amount of white adipose tissue decreased progressively as the liver enlarged. These studies indicate that the NH2-terminal domain of SREBP-1a can produce major effects on lipid synthesis and storage in the liver.

Full Text

The Full Text of this article is available as a PDF (3.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amy C. M., Witkowski A., Naggert J., Williams B., Randhawa Z., Smith S. Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase. Proc Natl Acad Sci U S A. 1989 May;86(9):3114–3118. doi: 10.1073/pnas.86.9.3114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BUCHER N. L., OVERATH P., LYNEN F. beta-Hydroxy-beta-methyl-glutaryl coenzyme A reductase, cleavage and condensing enzymes in relation to cholesterol formation in rat liver. Biochim Biophys Acta. 1960 Jun 3;40:491–501. doi: 10.1016/0006-3002(60)91390-1. [DOI] [PubMed] [Google Scholar]
  3. Beisiegel U., Schneider W. J., Goldstein J. L., Anderson R. G., Brown M. S. Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia. J Biol Chem. 1981 Nov 25;256(22):11923–11931. [PubMed] [Google Scholar]
  4. Bennett M. K., Lopez J. M., Sanchez H. B., Osborne T. F. Sterol regulation of fatty acid synthase promoter. Coordinate feedback regulation of two major lipid pathways. J Biol Chem. 1995 Oct 27;270(43):25578–25583. doi: 10.1074/jbc.270.43.25578. [DOI] [PubMed] [Google Scholar]
  5. Bucolo G., David H. Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem. 1973 May;19(5):476–482. [PubMed] [Google Scholar]
  6. Chen C. W., Thomas C. A., Jr Recovery of DNA segments from agarose gels. Anal Biochem. 1980 Jan 15;101(2):339–341. doi: 10.1016/0003-2697(80)90197-9. [DOI] [PubMed] [Google Scholar]
  7. Chen W. J., Andres D. A., Goldstein J. L., Russell D. W., Brown M. S. cDNA cloning and expression of the peptide-binding beta subunit of rat p21ras farnesyltransferase, the counterpart of yeast DPR1/RAM1. Cell. 1991 Jul 26;66(2):327–334. doi: 10.1016/0092-8674(91)90622-6. [DOI] [PubMed] [Google Scholar]
  8. Chin D. J., Gil G., Russell D. W., Liscum L., Luskey K. L., Basu S. K., Okayama H., Berg P., Goldstein J. L., Brown M. S. Nucleotide sequence of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, a glycoprotein of endoplasmic reticulum. Nature. 1984 Apr 12;308(5960):613–617. doi: 10.1038/308613a0. [DOI] [PubMed] [Google Scholar]
  9. Clarke C. F., Tanaka R. D., Svenson K., Wamsley M., Fogelman A. M., Edwards P. A. Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway. Mol Cell Biol. 1987 Sep;7(9):3138–3146. doi: 10.1128/mcb.7.9.3138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dietschy J. M., Spady D. K. Measurement of rates of cholesterol synthesis using tritiated water. J Lipid Res. 1984 Dec 15;25(13):1469–1476. [PubMed] [Google Scholar]
  11. Ericsson J., Jackson S. M., Lee B. C., Edwards P. A. Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene. Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):945–950. doi: 10.1073/pnas.93.2.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. GOULD R. G. Lipid metabolism and atherosclerosis. Am J Med. 1951 Aug;11(2):209–227. doi: 10.1016/0002-9343(51)90107-6. [DOI] [PubMed] [Google Scholar]
  13. GOULD R. G., TAYLOR C. B., HAGERMAN J. S., WARNER I., CAMPBELL D. J. Cholesterol metabolism. I. Effect of dietary cholesterol on the synthesis of cholesterol in dog tissue in vitro. J Biol Chem. 1953 Apr;201(2):519–528. [PubMed] [Google Scholar]
  14. 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]
  15. Ginsberg H. N. Synthesis and secretion of apolipoprotein B from cultured liver cells. Curr Opin Lipidol. 1995 Oct;6(5):275–280. doi: 10.1097/00041433-199510000-00006. [DOI] [PubMed] [Google Scholar]
  16. Gould R. G., Swyryd E. A. Sites of control of hepatic cholesterol biosynthesis. J Lipid Res. 1966 Sep;7(5):698–707. [PubMed] [Google Scholar]
  17. Guan G., Jiang G., Koch R. L., Shechter I. Molecular cloning and functional analysis of the promoter of the human squalene synthase gene. J Biol Chem. 1995 Sep 15;270(37):21958–21965. doi: 10.1074/jbc.270.37.21958. [DOI] [PubMed] [Google Scholar]
  18. Ha J., Daniel S., Kong I. S., Park C. K., Tae H. J., Kim K. H. Cloning of human acetyl-CoA carboxylase cDNA. Eur J Biochem. 1994 Jan 15;219(1-2):297–306. doi: 10.1111/j.1432-1033.1994.tb19941.x. [DOI] [PubMed] [Google Scholar]
  19. Hofmann S. L., Russell D. W., Brown M. S., Goldstein J. L., Hammer R. E. Overexpression of low density lipoprotein (LDL) receptor eliminates LDL from plasma in transgenic mice. Science. 1988 Mar 11;239(4845):1277–1281. doi: 10.1126/science.3344433. [DOI] [PubMed] [Google Scholar]
  20. Horton J. D., Cuthbert J. A., Spady D. K. Regulation of hepatic 7 alpha-hydroxylase expression and response to dietary cholesterol in the rat and hamster. J Biol Chem. 1995 Mar 10;270(10):5381–5387. doi: 10.1074/jbc.270.10.5381. [DOI] [PubMed] [Google Scholar]
  21. Hua X., Sakai J., Brown M. S., Goldstein J. L. Regulated cleavage of sterol regulatory element binding proteins requires sequences on both sides of the endoplasmic reticulum membrane. J Biol Chem. 1996 Apr 26;271(17):10379–10384. doi: 10.1074/jbc.271.17.10379. [DOI] [PubMed] [Google Scholar]
  22. Hua X., Sakai J., Ho Y. K., Goldstein J. L., Brown M. S. Hairpin orientation of sterol regulatory element-binding protein-2 in cell membranes as determined by protease protection. J Biol Chem. 1995 Dec 8;270(49):29422–29427. doi: 10.1074/jbc.270.49.29422. [DOI] [PubMed] [Google Scholar]
  23. Hua X., Wu J., Goldstein J. L., Brown M. S., Hobbs H. H. Structure of the human gene encoding sterol regulatory element binding protein-1 (SREBF1) and localization of SREBF1 and SREBF2 to chromosomes 17p11.2 and 22q13. Genomics. 1995 Feb 10;25(3):667–673. doi: 10.1016/0888-7543(95)80009-b. [DOI] [PubMed] [Google Scholar]
  24. Hua X., Yokoyama C., Wu J., Briggs M. R., Brown M. S., Goldstein J. L., Wang X. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11603–11607. doi: 10.1073/pnas.90.24.11603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Inoue T., Osumi T., Hata S. Molecular cloning and functional expression of a cDNA for mouse squalene synthase. Biochim Biophys Acta. 1995 Jan 2;1260(1):49–54. doi: 10.1016/0167-4781(94)00178-6. [DOI] [PubMed] [Google Scholar]
  26. Ishibashi S., Brown M. S., Goldstein J. L., Gerard R. D., Hammer R. E., Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest. 1993 Aug;92(2):883–893. doi: 10.1172/JCI116663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ishibashi S., Schwarz M., Frykman P. K., Herz J., Russell D. W. Disruption of cholesterol 7alpha-hydroxylase gene in mice. I. Postnatal lethality reversed by bile acid and vitamin supplementation. J Biol Chem. 1996 Jul 26;271(30):18017–18023. doi: 10.1074/jbc.271.30.18017. [DOI] [PubMed] [Google Scholar]
  28. Ishitani K., Niitsu Y., Listowsky I. Characterization of the different polypeptide components and analysis of subunit assembly in ferritin. J Biol Chem. 1975 Apr 25;250(8):3124–3128. [PubMed] [Google Scholar]
  29. Kim J. B., Spiegelman B. M. ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev. 1996 May 1;10(9):1096–1107. doi: 10.1101/gad.10.9.1096. [DOI] [PubMed] [Google Scholar]
  30. Kirchgessner T. G., Svenson K. L., Lusis A. J., Schotz M. C. The sequence of cDNA encoding lipoprotein lipase. A member of a lipase gene family. J Biol Chem. 1987 Jun 25;262(18):8463–8466. [PubMed] [Google Scholar]
  31. Lopez J. M., Bennett M. K., Sanchez H. B., Rosenfeld J. M., Osborne T. F. Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1049–1053. doi: 10.1073/pnas.93.3.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lowenstein J. M., Brunengraber H., Wadke M. Measurement of rates of lipogenesis with deuterated and tritiated water. Methods Enzymol. 1975;35:279–287. doi: 10.1016/0076-6879(75)35165-3. [DOI] [PubMed] [Google Scholar]
  33. López-Casillas F., Bai D. H., Luo X. C., Kong I. S., Hermodson M. A., Kim K. H. Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5784–5788. doi: 10.1073/pnas.85.16.5784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Matsumoto A., Aburatani H., Shibasaki Y., Kodama T., Takaku F., Itakura H. Cloning and regulation of rat apolipoprotein B mRNA. Biochem Biophys Res Commun. 1987 Jan 15;142(1):92–99. doi: 10.1016/0006-291x(87)90455-4. [DOI] [PubMed] [Google Scholar]
  35. Ntambi J. M., Buhrow S. A., Kaestner K. H., Christy R. J., Sibley E., Kelly T. J., Jr, Lane M. D. Differentiation-induced gene expression in 3T3-L1 preadipocytes. Characterization of a differentially expressed gene encoding stearoyl-CoA desaturase. J Biol Chem. 1988 Nov 25;263(33):17291–17300. [PubMed] [Google Scholar]
  36. Osono Y., Woollett L. A., Herz J., Dietschy J. M. Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse. J Clin Invest. 1995 Mar;95(3):1124–1132. doi: 10.1172/JCI117760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Poncin J. E., Martial J. A., Gielen J. E. Cloning and structure analysis of the rat apolipoprotein A-I cDNA. Eur J Biochem. 1984 May 2;140(3):493–498. doi: 10.1111/j.1432-1033.1984.tb08129.x. [DOI] [PubMed] [Google Scholar]
  38. Rajavashisth T. B., Kaptein J. S., Reue K. L., Lusis A. J. Evolution of apolipoprotein E: mouse sequence and evidence for an 11-nucleotide ancestral unit. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8085–8089. doi: 10.1073/pnas.82.23.8085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reynolds G. A., Goldstein J. L., Brown M. S. Multiple mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase determined by multiple transcription initiation sites and intron splicing sites in the 5'-untranslated region. J Biol Chem. 1985 Aug 25;260(18):10369–10377. [PubMed] [Google Scholar]
  40. Rokosz L. L., Boulton D. A., Butkiewicz E. A., Sanyal G., Cueto M. A., Lachance P. A., Hermes J. D. Human cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase: expression, purification, and characterization of recombinant wild-type and Cys129 mutant enzymes. Arch Biochem Biophys. 1994 Jul;312(1):1–13. doi: 10.1006/abbi.1994.1273. [DOI] [PubMed] [Google Scholar]
  41. SIPERSTEIN M. D., GUEST M. J. Studies on the site of the feedback control of cholesterol synthesis. J Clin Invest. 1960 Apr;39:642–652. doi: 10.1172/JCI104079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sakai J., Duncan E. A., Rawson R. B., Hua X., Brown M. S., Goldstein J. L. Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment. Cell. 1996 Jun 28;85(7):1037–1046. doi: 10.1016/s0092-8674(00)81304-5. [DOI] [PubMed] [Google Scholar]
  43. Sato R., Yang J., Wang X., Evans M. J., Ho Y. K., Goldstein J. L., Brown M. S. Assignment of the membrane attachment, DNA binding, and transcriptional activation domains of sterol regulatory element-binding protein-1 (SREBP-1). J Biol Chem. 1994 Jun 24;269(25):17267–17273. [PubMed] [Google Scholar]
  44. Shechter I., Klinger E., Rucker M. L., Engstrom R. G., Spirito J. A., Islam M. A., Boettcher B. R., Weinstein D. B. Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase. J Biol Chem. 1992 Apr 25;267(12):8628–8635. [PubMed] [Google Scholar]
  45. Sheng Z., Otani H., Brown M. S., Goldstein J. L. Independent regulation of sterol regulatory element-binding proteins 1 and 2 in hamster liver. Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):935–938. doi: 10.1073/pnas.92.4.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Short M. K., Clouthier D. E., Schaefer I. M., Hammer R. E., Magnuson M. A., Beale E. G. Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice. Mol Cell Biol. 1992 Mar;12(3):1007–1020. doi: 10.1128/mcb.12.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shoulders C. C., Kornblihtt A. R., Munro B. S., Baralle F. E. Gene structure of human apolipoprotein A1. Nucleic Acids Res. 1983 May 11;11(9):2827–2837. doi: 10.1093/nar/11.9.2827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Siperstein M. D., Fagan V. M. Feedback control of mevalonate synthesis by dietary cholesterol. J Biol Chem. 1966 Feb 10;241(3):602–609. [PubMed] [Google Scholar]
  49. Sorci-Thomas M., Wilson M. D., Johnson F. L., Williams D. L., Rudel L. L. Studies on the expression of genes encoding apolipoproteins B100 and B48 and the low density lipoprotein receptor in nonhuman primates. Comparison of dietary fat and cholesterol. J Biol Chem. 1989 May 25;264(15):9039–9045. [PubMed] [Google Scholar]
  50. Spady D. K., Dietschy J. M. Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster, and rat. J Lipid Res. 1983 Mar;24(3):303–315. [PubMed] [Google Scholar]
  51. Tokunaga K., Taniguchi H., Yoda K., Shimizu M., Sakiyama S. Nucleotide sequence of a full-length cDNA for mouse cytoskeletal beta-actin mRNA. Nucleic Acids Res. 1986 Mar 25;14(6):2829–2829. doi: 10.1093/nar/14.6.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vallett S. M., Sanchez H. B., Rosenfeld J. M., Osborne T. F. A direct role for sterol regulatory element binding protein in activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene. J Biol Chem. 1996 May 24;271(21):12247–12253. doi: 10.1074/jbc.271.21.12247. [DOI] [PubMed] [Google Scholar]
  53. Wang X., Briggs M. R., Hua X., Yokoyama C., Goldstein J. L., Brown M. S. Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. J Biol Chem. 1993 Jul 5;268(19):14497–14504. [PubMed] [Google Scholar]
  54. Wang X., Sato R., Brown M. S., Hua X., Goldstein J. L. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell. 1994 Apr 8;77(1):53–62. doi: 10.1016/0092-8674(94)90234-8. [DOI] [PubMed] [Google Scholar]
  55. Yang C. Y., Chen S. H., Gianturco S. H., Bradley W. A., Sparrow J. T., Tanimura M., Li W. H., Sparrow D. A., DeLoof H., Rosseneu M. Sequence, structure, receptor-binding domains and internal repeats of human apolipoprotein B-100. Nature. 1986 Oct 23;323(6090):738–742. doi: 10.1038/323738a0. [DOI] [PubMed] [Google Scholar]
  56. Yang J., Brown M. S., Ho Y. K., Goldstein J. L. Three different rearrangements in a single intron truncate sterol regulatory element binding protein-2 and produce sterol-resistant phenotype in three cell lines. Role of introns in protein evolution. J Biol Chem. 1995 May 19;270(20):12152–12161. doi: 10.1074/jbc.270.20.12152. [DOI] [PubMed] [Google Scholar]
  57. Yang J., Sato R., Goldstein J. L., Brown M. S. Sterol-resistant transcription in CHO cells caused by gene rearrangement that truncates SREBP-2. Genes Dev. 1994 Aug 15;8(16):1910–1919. doi: 10.1101/gad.8.16.1910. [DOI] [PubMed] [Google Scholar]
  58. Yokode M., Hammer R. E., Ishibashi S., Brown M. S., Goldstein J. L. Diet-induced hypercholesterolemia in mice: prevention by overexpression of LDL receptors. Science. 1990 Nov 30;250(4985):1273–1275. doi: 10.1126/science.2244210. [DOI] [PubMed] [Google Scholar]
  59. Yokoyama C., Wang X., Briggs M. R., Admon A., Wu J., Hua X., Goldstein J. L., Brown M. S. SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell. 1993 Oct 8;75(1):187–197. [PubMed] [Google Scholar]
  60. Yuan Z. Y., Liu W., Hammes G. G. Molecular cloning and sequencing of DNA complementary to chicken liver fatty acid synthase mRNA. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6328–6331. doi: 10.1073/pnas.85.17.6328. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES