Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1991 May 15;276(Pt 1):245–250. doi: 10.1042/bj2760245

Regulation of glucose-6-phosphate dehydrogenase synthesis and mRNA abundance in cultured rat hepatocytes.

P Manos 1, R Nakayama 1, D Holten 1
PMCID: PMC1151171  PMID: 2039474

Abstract

Conditions were identified which, for the first time, demonstrate that primary hepatocytes can express the same range of glucose-6-phosphate dehydrogenase (G6PD) synthesis and mRNA as in live rats. Primary hepatocytes were cultured without prior exposure to serum, hormones or carbohydrates. Five modulators implicated in G6PD induction in vivo were examined: insulin, dexamethasone, tri-iodothyronine (T3), glucose and fructose, T3 did not affect G6PD activity, and did not interact with carbohydrate to affect the activity of G6PD. Neither glucose nor fructose alone affected G6PD activity, and they did not interact with insulin to increase G6PD activity. Hepatocytes isolated from fasted rats and cultured in serum-free media with amino acids ad the only energy source how a 12-fold increase in G6PD synthesis and mRNA (measured by a solution-hybridization assay). This induction does not require added hormones or carbohydrate. The addition of insulin alone caused another increase in G6PD synthesis and mRNA. There are at least three distinct phases to G6PD induction under these conditions. The largest increase in G6PD synthesis (12-fold) occurs in the absence of any hormones and with amino acids as the only energy source. This phase is due to increased G6PD mRNA. Insulin causes an additional 2-3-fold increase in G6PD synthesis and mRNA. However, dexamethasone and insulin are both required before G6PD synthesis is equal to that in rats which are fasted and refed on a high-carbohydrate diet.

Full text

PDF
249

Selected References

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

  1. Acosta D., Anuforo D. C., Smith R. V. Primary monolayer cultures of postnatal rat liver cells with extended differentiated functions. In Vitro. 1978 May;14(5):428–436. doi: 10.1007/BF02616104. [DOI] [PubMed] [Google Scholar]
  2. Battistuzzi G., D'Urso M., Toniolo D., Persico G. M., Luzzatto L. Tissue-specific levels of human glucose-6-phosphate dehydrogenase correlate with methylation of specific sites at the 3' end of the gene. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1465–1469. doi: 10.1073/pnas.82.5.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berdanier C. D., Shubeck D. Interaction of glucocorticoid and insulin in the responses of rats to starvation-refeeding. J Nutr. 1979 Oct;109(10):1766–1771. doi: 10.1093/jn/109.10.1766. [DOI] [PubMed] [Google Scholar]
  4. Berry M. N., Friend D. S. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec;43(3):506–520. doi: 10.1083/jcb.43.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bonney R. J., Walker P. R., Potter V. R. Isoenzyme patterns in parenchymal and non-parenchymal cells isolated from regenerating and regenerated rat liver. Biochem J. 1973 Dec;136(4):947–954. doi: 10.1042/bj1360947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burch H. B., Max P., Jr, Ghyu K., Lowry O. H. Metabolic intermediates in liver of rats given large amounts of fructose or dihydroxyacetone. Biochem Biophys Res Commun. 1969 Mar 10;34(5):619–626. doi: 10.1016/0006-291x(69)90783-9. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Decaux J. F., Antoine B., Kahn A. Regulation of the expression of the L-type pyruvate kinase gene in adult rat hepatocytes in primary culture. J Biol Chem. 1989 Jul 15;264(20):11584–11590. [PubMed] [Google Scholar]
  9. Diamant S., Gorin E., Shafrir E. Enzyme activities related to fatty-acid synthesis in liver and adipose tissue of rats treated with triiodothyronine. Eur J Biochem. 1972 Apr 24;26(4):553–559. doi: 10.1111/j.1432-1033.1972.tb01798.x. [DOI] [PubMed] [Google Scholar]
  10. Fritz R. S., Kletzien R. F. Regulation of glucose-6-phosphate dehydrogenase by diet and thyroid hormone. Mol Cell Endocrinol. 1987 May;51(1-2):13–17. doi: 10.1016/0303-7207(87)90113-4. [DOI] [PubMed] [Google Scholar]
  11. Fritz R. S., Stumpo D. J., Kletzien R. F. Glucose-6-phosphate dehydrogenase mRNA sequence abundance in primary cultures of rat hepatocytes. Effect of insulin and dexamethasone. Biochem J. 1986 Jul 15;237(2):617–619. doi: 10.1042/bj2370617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. GLOCK G. E., MCLEAN P. A preliminary investigation of the hormonal control of the hexose monophosphate oxidative pathway. Biochem J. 1955 Nov;61(3):390–397. doi: 10.1042/bj0610390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. GLOCK G. E., McLEAN P. Further studies on the properties and assay of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver. Biochem J. 1953 Oct;55(3):400–408. doi: 10.1042/bj0550400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hamblin P. S., Ozawa Y., Jefferds A., Mariash C. N. Interaction between fructose and glucose on the regulation of the nuclear precursor for mRNA-S14. J Biol Chem. 1989 Dec 25;264(36):21646–21651. [PubMed] [Google Scholar]
  15. Ho Y. S., Howard A. J., Crapo J. D. Cloning and sequence of a cDNA encoding rat glucose-6-phosphate dehydrogenase. Nucleic Acids Res. 1988 Aug 11;16(15):7746–7746. doi: 10.1093/nar/16.15.7746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Katsurada A., Iritani N., Fukuda H., Matsumura Y., Noguchi T., Tanaka T. Effects of nutrients and insulin on transcriptional and post-transcriptional regulation of glucose-6-phosphate dehydrogenase synthesis in rat liver. Biochim Biophys Acta. 1989 Nov 6;1006(1):104–110. doi: 10.1016/0005-2760(89)90329-9. [DOI] [PubMed] [Google Scholar]
  17. Kelley D. S., Kletzien R. F. Ethanol modulation of the hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase activity in primary cultures of rat hepatocytes. Biochem J. 1984 Jan 15;217(2):543–549. doi: 10.1042/bj2170543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kelley D. S., Nelson G. J., Hunt J. E. Effect of prior nutritional status on the activity of lipogenic enzymes in primary monolayer cultures of rat hepatocytes. Biochem J. 1986 Apr 1;235(1):87–90. doi: 10.1042/bj2350087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim M. H., Nakayama R., Holten D. Quantitation of glucose-6-phosphate dehydrogenase mRNA by solution hybridization: correlation with rates of synthesis. Biochim Biophys Acta. 1990 Jun 21;1049(2):177–181. doi: 10.1016/0167-4781(90)90038-4. [DOI] [PubMed] [Google Scholar]
  20. Kletzien R. F., Prostko C. R., Stumpo D. J., McClung J. K., Dreher K. L. Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels. J Biol Chem. 1985 May 10;260(9):5621–5624. [PubMed] [Google Scholar]
  21. Kurtz J. W., Wells W. W. Induction of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes. Requirement for insulin and dexamethasone. J Biol Chem. 1981 Nov 10;256(21):10870–10875. [PubMed] [Google Scholar]
  22. Leffert H. L., Paul D. Studies on primary cultures of differentiated fetal liver cells. J Cell Biol. 1972 Mar;52(3):559–568. doi: 10.1083/jcb.52.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Louie P., Nakayama R., Holten D. Solution hybridization quantitation of G6PD mRNA in rat epididymal fat pads. Biochim Biophys Acta. 1990 Sep 10;1087(1):25–30. doi: 10.1016/0167-4781(90)90116-j. [DOI] [PubMed] [Google Scholar]
  24. Manos P., Holten D. Primary cultures of hepatocytes in serum and hormone-free medium: identification of conditions which stimulate an in vivo-like induction of G6PD. In Vitro Cell Dev Biol. 1987 May;23(5):367–373. doi: 10.1007/BF02620994. [DOI] [PubMed] [Google Scholar]
  25. Mariash C. N. A novel hepatic nucleotide is correlated with the carbohydrate induction of messenger ribonucleic acid-S14. Endocrinology. 1989 Jan;124(1):212–217. doi: 10.1210/endo-124-1-212. [DOI] [PubMed] [Google Scholar]
  26. Mariash C. N., Kaiser F. E., Schwartz H. L., Towle H. C., Oppenheimer J. H. Synergism of thyroid hormone and high carbohydrate diet in the induction of lipogenic enzymes in the rat. Mechanisms and implications. J Clin Invest. 1980 May;65(5):1126–1134. doi: 10.1172/JCI109766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Michaelis O. E., 4th, Szepesi B. Effect of various sugars on hepatic glucose-6-phosphate dehydrogenase, malic enzyme and total liver lipid of the rat. J Nutr. 1973 May;103(5):697–705. doi: 10.1093/jn/103.5.697. [DOI] [PubMed] [Google Scholar]
  28. Miksicek R. J., Towle H. C. Changes in the rates of synthesis and messenger RNA levels of hepatic glucose-6-phosphate and 6-phosphogluconate dehydrogenases following induction by diet or thyroid hormone. J Biol Chem. 1982 Oct 10;257(19):11829–11835. [PubMed] [Google Scholar]
  29. Morikawa N., Nakayama R., Holten D. Dietary induction of glucose-6-phosphate dehydrogenase synthesis. Biochem Biophys Res Commun. 1984 May 16;120(3):1022–1029. doi: 10.1016/s0006-291x(84)80209-0. [DOI] [PubMed] [Google Scholar]
  30. Newgard C. B., Hirsch L. J., Foster D. W., McGarry J. D. Studies on the mechanism by which exogenous glucose is converted into liver glycogen in the rat. A direct or an indirect pathway? J Biol Chem. 1983 Jul 10;258(13):8046–8052. [PubMed] [Google Scholar]
  31. Noguchi T., Inoue H., Tanaka T. Regulation of rat liver L-type pyruvate kinase mRNA by insulin and by fructose. Eur J Biochem. 1982 Nov 15;128(2-3):583–588. doi: 10.1111/j.1432-1033.1982.tb07004.x. [DOI] [PubMed] [Google Scholar]
  32. Novello F., Gumaa J. A., McLean P. The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver. Biochem J. 1969 Mar;111(5):713–725. doi: 10.1042/bj1110713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Oliver I. T., Edwards A. M., Pitot H. C. Hormonal regulation of phosphoenolpyruvate carboxykinase in primary cultures of adult-rat liver parenchymal cells. Eur J Biochem. 1978 Jun 15;87(2):221–227. doi: 10.1111/j.1432-1033.1978.tb12369.x. [DOI] [PubMed] [Google Scholar]
  34. Oppenheimer J. H., Schwartz H. L. Factors determining the level of activity of 3,5,3'-triiodothyronine-responsive hepatic enzymes in the starved rat. Endocrinology. 1980 Nov;107(5):1460–1468. doi: 10.1210/endo-107-5-1460. [DOI] [PubMed] [Google Scholar]
  35. Oppenheimer J. H. Thyroid hormone action at the cellular level. Science. 1979 Mar 9;203(4384):971–979. doi: 10.1126/science.218285. [DOI] [PubMed] [Google Scholar]
  36. Rognstad R., Katz J. Effects of 2,4-dihydroxybutyrate on lipogenesis in rat hepatocytes. J Biol Chem. 1979 Dec 10;254(23):11969–11972. [PubMed] [Google Scholar]
  37. Rudack D., Chisholm E. M., Holten D. Rat liver glucose 6-phosphate dehydrogenase. Regulation by carbohydrate diet and insulin. J Biol Chem. 1971 Mar 10;246(5):1249–1254. [PubMed] [Google Scholar]
  38. Rudack D., Davie B., Holten D. Regulation of rat liver glucose 6- phosphate dehydrogenase levels by adenosine 3', 5' -monophosphate. J Biol Chem. 1971 Dec 25;246(24):7823–7824. [PubMed] [Google Scholar]
  39. Salati L. M., Adkins-Finke B., Clarke S. D. Free fatty acid inhibition of the insulin induction of glucose-6-phosphate dehydrogenase in rat hepatocyte monolayers. Lipids. 1988 Jan;23(1):36–41. doi: 10.1007/BF02535302. [DOI] [PubMed] [Google Scholar]
  40. Smedsrød B., Pertoft H., Gustafson S., Laurent T. C. Scavenger functions of the liver endothelial cell. Biochem J. 1990 Mar 1;266(2):313–327. doi: 10.1042/bj2660313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Spence J. T., Pitot H. C. Induction of lipogenic enzymes in primary cultures of rat hepatocytes. Relationship between lipogenesis and carbohydrate metabolism. Eur J Biochem. 1982 Nov;128(1):15–20. doi: 10.1111/j.1432-1033.1982.tb06924.x. [DOI] [PubMed] [Google Scholar]
  42. Stumpo D. J., Kletzien R. F. The effect of ethanol, alone and in combination with the glucocorticoids and insulin, on glucose-6-phosphate dehydrogenase synthesis and mRNA in primary cultures of hepatocytes. Biochem J. 1985 Feb 15;226(1):123–130. doi: 10.1042/bj2260123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sun J. D., Holten D. Levels of rat glucose-6-phosphate dehydrogenase messenger RNA. J Biol Chem. 1978 Oct 10;253(19):6832–6836. [PubMed] [Google Scholar]
  44. Tomlinson J. E., Nakayama R., Holten D. Repression of pentose phosphate pathway dehydrogenase synthesis and mRNA by dietary fat in rats. J Nutr. 1988 Mar;118(3):408–415. doi: 10.1093/jn/118.3.408. [DOI] [PubMed] [Google Scholar]
  45. Van den Berghe G. Metabolic effects of fructose in the liver. Curr Top Cell Regul. 1978;13:97–135. doi: 10.1016/b978-0-12-152813-3.50008-2. [DOI] [PubMed] [Google Scholar]
  46. Weber G., Convery H. J. Insulin: inducer of glucose 6-phosphate dehydrogenase. Life Sci. 1966 Jun;5(12):1139–1146. doi: 10.1016/0024-3205(66)90098-1. [DOI] [PubMed] [Google Scholar]
  47. Williams D. L., Newman T. C., Shelness G. S., Gordon D. A. Measurement of apolipoprotein mRNA by DNA-excess solution hybridization with single-stranded probes. Methods Enzymol. 1986;128:671–689. doi: 10.1016/0076-6879(86)28099-4. [DOI] [PubMed] [Google Scholar]
  48. Winberry L., Holten D. Rat liver glucose-6-p dehydrogenase. Dietary regulation of the rate of synthesis. J Biol Chem. 1977 Nov 10;252(21):7796–7801. [PubMed] [Google Scholar]
  49. Winberry L., Nakayama R., Wolfe R., Holten D. Regulation of glucose-6-P dehydrogenase activity in primary rat hepatocyte cultures. Biochem Biophys Res Commun. 1980 Sep 30;96(2):748–755. doi: 10.1016/0006-291x(80)91418-7. [DOI] [PubMed] [Google Scholar]
  50. Wolfe R. G., Holten D. The effect of dietary fat or cholesterol and cholic acid on the rate of synthesis of rat liver glucose-6-P dehydrogenase. J Nutr. 1978 Oct;108(10):1708–1717. doi: 10.1093/jn/108.10.1708. [DOI] [PubMed] [Google Scholar]

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

RESOURCES