Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Aug 15;366(Pt 1):289–297. doi: 10.1042/BJ20020598

Down-regulation of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene by insulin: the role of the forkhead transcription factor FKHRL1.

Alícia Nadal 1, Pedro F Marrero 1, Diego Haro 1
PMCID: PMC1222772  PMID: 12027802

Abstract

Normal physiological responses to carbohydrate shortages cause the liver to increase the production of ketone bodies from the acetyl-CoA generated from fatty acid oxidation. This allows the use of ketone bodies for energy, thereby preserving the limited glucose for use by the brain. This adaptative response is switched off by insulin rapidly inhibiting the expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (HMGCS2) gene, which is a key control site of ketogenesis. We decided to investigate the molecular mechanism of this inhibition. In the present study, we show that FKHRL1, a member of the forkhead in rhabdosarcoma (FKHR) subclass of the Fox family of transcription factors, stimulates transcription from transfected 3-hydroxy-3-methylglutaryl-CoA synthase promoter-luciferase reporter constructs, and that this stimulation is repressed by insulin. An FKHRL1-responsive sequence AAAAATA, located 211 bp upstream of the HMGCS2 gene transcription start site, was identified by deletion analysis. It binds FKHRL1 in vivo and in vitro and confers FKHRL1 responsiveness on homologous and heterologous promoters. If it is mutated, it partially blocks the effect of insulin in HepG2 cells, both in the absence and presence of overexpressed FKHRL1. These results suggest that FKHRL1 contributes to the regulation of HMGCS2 gene expression by insulin.

Full Text

The Full Text of this article is available as a PDF (257.0 KB).

Selected References

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

  1. Anderson M. J., Viars C. S., Czekay S., Cavenee W. K., Arden K. C. Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics. 1998 Jan 15;47(2):187–199. doi: 10.1006/geno.1997.5122. [DOI] [PubMed] [Google Scholar]
  2. Ayala J. E., Streeper R. S., Desgrosellier J. S., Durham S. K., Suwanichkul A., Svitek C. A., Goldman J. K., Barr F. G., Powell D. R., O'Brien R. M. Conservation of an insulin response unit between mouse and human glucose-6-phosphatase catalytic subunit gene promoters: transcription factor FKHR binds the insulin response sequence. Diabetes. 1999 Sep;48(9):1885–1889. doi: 10.2337/diabetes.48.9.1885. [DOI] [PubMed] [Google Scholar]
  3. Ayté J., Gil-Gómez G., Haro D., Marrero P. F., Hegardt F. G. Rat mitochondrial and cytosolic 3-hydroxy-3-methylglutaryl-CoA synthases are encoded by two different genes. Proc Natl Acad Sci U S A. 1990 May;87(10):3874–3878. doi: 10.1073/pnas.87.10.3874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Brunet A., Bonni A., Zigmond M. J., Lin M. Z., Juo P., Hu L. S., Anderson M. J., Arden K. C., Blenis J., Greenberg M. E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 1999 Mar 19;96(6):857–868. doi: 10.1016/s0092-8674(00)80595-4. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Cichy S. B., Uddin S., Danilkovich A., Guo S., Klippel A., Unterman T. G. Protein kinase B/Akt mediates effects of insulin on hepatic insulin-like growth factor-binding protein-1 gene expression through a conserved insulin response sequence. J Biol Chem. 1998 Mar 13;273(11):6482–6487. doi: 10.1074/jbc.273.11.6482. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Durham S. K., Suwanichkul A., Scheimann A. O., Yee D., Jackson J. G., Barr F. G., Powell D. R. FKHR binds the insulin response element in the insulin-like growth factor binding protein-1 promoter. Endocrinology. 1999 Jul;140(7):3140–3146. doi: 10.1210/endo.140.7.6856. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Goswami R., Lacson R., Yang E., Sam R., Unterman T. Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter. Endocrinology. 1994 Feb;134(2):736–743. doi: 10.1210/endo.134.2.7507835. [DOI] [PubMed] [Google Scholar]
  12. Guo S., Rena G., Cichy S., He X., Cohen P., Unterman T. Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J Biol Chem. 1999 Jun 11;274(24):17184–17192. doi: 10.1074/jbc.274.24.17184. [DOI] [PubMed] [Google Scholar]
  13. Hall R. K., Yamasaki T., Kucera T., Waltner-Law M., O'Brien R., Granner D. K. Regulation of phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein-1 gene expression by insulin. The role of winged helix/forkhead proteins. J Biol Chem. 2000 Sep 29;275(39):30169–30175. doi: 10.1074/jbc.M004898200. [DOI] [PubMed] [Google Scholar]
  14. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  15. Lin K., Dorman J. B., Rodan A., Kenyon C. daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science. 1997 Nov 14;278(5341):1319–1322. doi: 10.1126/science.278.5341.1319. [DOI] [PubMed] [Google Scholar]
  16. Lowe D. M., Tubbs P. K. Succinylation and inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase by succinyl-CoA and its possible relevance to the control of ketogenesis. Biochem J. 1985 Nov 15;232(1):37–42. doi: 10.1042/bj2320037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nakae J., Barr V., Accili D. Differential regulation of gene expression by insulin and IGF-1 receptors correlates with phosphorylation of a single amino acid residue in the forkhead transcription factor FKHR. EMBO J. 2000 Mar 1;19(5):989–996. doi: 10.1093/emboj/19.5.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. O'Brien R. M., Lucas P. C., Forest C. D., Magnuson M. A., Granner D. K. Identification of a sequence in the PEPCK gene that mediates a negative effect of insulin on transcription. Science. 1990 Aug 3;249(4968):533–537. doi: 10.1126/science.2166335. [DOI] [PubMed] [Google Scholar]
  19. Ogg S., Paradis S., Gottlieb S., Patterson G. I., Lee L., Tissenbaum H. A., Ruvkun G. The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature. 1997 Oct 30;389(6654):994–999. doi: 10.1038/40194. [DOI] [PubMed] [Google Scholar]
  20. Patel Satish, Lochhead Pamela A., Rena Graham, Fumagalli Stefano, Pende Mario, Kozma Sara C., Thomas George, Sutherland Calum. Insulin regulation of insulin-like growth factor-binding protein-1 gene expression is dependent on the mammalian target of rapamycin, but independent of ribosomal S6 kinase activity. J Biol Chem. 2002 Jan 9;277(12):9889–9895. doi: 10.1074/jbc.M109870200. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Rodríguez J. C., Ortiz J. A., Hegardt F. G., Haro D. Chicken ovalbumin upstream-promoter transcription factor (COUP-TF) could act as a transcriptional activator or repressor of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene. Biochem J. 1997 Sep 1;326(Pt 2):587–592. doi: 10.1042/bj3260587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Schmoll D., Walker K. S., Alessi D. R., Grempler R., Burchell A., Guo S., Walther R., Unterman T. G. Regulation of glucose-6-phosphatase gene expression by protein kinase Balpha and the forkhead transcription factor FKHR. Evidence for insulin response unit-dependent and -independent effects of insulin on promoter activity. J Biol Chem. 2000 Nov 17;275(46):36324–36333. doi: 10.1074/jbc.M003616200. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Serra D., Fillat C., Matas R., Bosch F., Hegardt F. G. Tissue-specific expression and dietary regulation of chimeric mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase/human growth hormone gene in transgenic mice. J Biol Chem. 1996 Mar 29;271(13):7529–7534. doi: 10.1074/jbc.271.13.7529. [DOI] [PubMed] [Google Scholar]
  29. Streeper R. S., Svitek C. A., Chapman S., Greenbaum L. E., Taub R., O'Brien R. M. A multicomponent insulin response sequence mediates a strong repression of mouse glucose-6-phosphatase gene transcription by insulin. J Biol Chem. 1997 May 2;272(18):11698–11701. doi: 10.1074/jbc.272.18.11698. [DOI] [PubMed] [Google Scholar]
  30. Suwanickul A., Morris S. L., Powell D. R. Identification of an insulin-responsive element in the promoter of the human gene for insulin-like growth factor binding protein-1. J Biol Chem. 1993 Aug 15;268(23):17063–17068. [PubMed] [Google Scholar]
  31. Tang E. D., Nuñez G., Barr F. G., Guan K. L. Negative regulation of the forkhead transcription factor FKHR by Akt. J Biol Chem. 1999 Jun 11;274(24):16741–16746. doi: 10.1074/jbc.274.24.16741. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Tomizawa M., Kumar A., Perrot V., Nakae J., Accili D., Rechler M. M., Kumaro A. Insulin inhibits the activation of transcription by a C-terminal fragment of the forkhead transcription factor FKHR. A mechanism for insulin inhibition of insulin-like growth factor-binding protein-1 transcription. J Biol Chem. 2000 Mar 10;275(10):7289–7295. doi: 10.1074/jbc.275.10.7289. [DOI] [PubMed] [Google Scholar]
  34. 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]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES