Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1977 Sep 1;74(3):878–900. doi: 10.1083/jcb.74.3.878

Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes

D Bernaert, JC Wanson, P Drochmans, A Popowski
PMCID: PMC2110106  PMID: 409722

Abstract

Insulin in the presence of high concentrations of glucose has a beneficial trophic effect on the development of primary cultures of hepatocytes. Compared to the situation observed in hormone-free control cultures, the flattening of the reaggregated hepatocytes is enhanced, and the reconstituted cell trabeculae are enlarged and tend to form a confluent monolayer after 3 days; the survival time is prolonged from 3 to 5 or 6 days. Ultrastructural modifications are also initiated by insulin; numerous glycogen particles appear after 24 h, in between the cisternae of the proliferated smooth endoplasmic reticulum. After 48 h, large amounts of glycogen are stored, and numerous polysomes are present. A small number of cells showed an increased synthesis of lipid droplets in the lumen of the smooth endoplasmic reticulum and form liposomes at the same time. After 72 h, cytolysomes filled with glycogen develop, simulating glycogenosis type II. Simultaneously, microtubules and microfilaments, closely related to numerous polysomes, appear in cytoplasmic extensions constituting undulating membranes. The biochemical data demonstrate that, in the absence of insulin, a high concentration of glucose stimulates glycogenesis and hinders glycogenolysis. This effect of glucose on polysaccharide synthesis is progressively lost. The addition of insulin to the culture induces after 48 and 72 h, a three- to fivefold increase of the glucose incorporation into glycogen, as compared to the controls. The presence of insulin is required to maintain the hepatocyte's capacity to store glycogen. Glycogen synthetase is converted into its active form under the influence of glucose. Insulin increases the rate of activation.

Full Text

The Full Text of this article is available as a PDF (7.8 MB).

Selected References

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

  1. Akpan J. O., Gardner R., Wagle S. R. Studies on the effects of insulin and acetylcholine on activation of glycogen synthase and on glycogenesis in hepatocytes. Biochem Biophys Res Commun. 1974 Nov 6;61(1):222–229. doi: 10.1016/0006-291x(74)90556-7. [DOI] [PubMed] [Google Scholar]
  2. BAUDHUIN P., HERS H. G., LOEB H. AN ELECTRON MICROSCOPIC AND BIOCHEMICAL STUDY OF TYPE II GLYCOGENOSIS. Lab Invest. 1964 Sep;13:1139–1152. [PubMed] [Google Scholar]
  3. 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]
  4. Berthillier G., Azzar G. J., Got R. Etude de l'activité de transfert de glucose, à partir d'UDP-glucose, dans les membranes microsomiques des hépatocytes de rat. Eur J Biochem. 1975 Feb 3;51(1):275–282. doi: 10.1111/j.1432-1033.1975.tb03927.x. [DOI] [PubMed] [Google Scholar]
  5. Berthillier G., Got R. Biosynthèse de l'UDPglucose par les microsomes des hépatocytes de rat. Biochim Biophys Acta. 1974 Sep 5;362(2):390–402. [PubMed] [Google Scholar]
  6. Bishop J. S., Goldberg N. D., Larner J. Insulin regulation of hepatic glycogen metabolism in the dog. Am J Physiol. 1971 Feb;220(2):499–506. doi: 10.1152/ajplegacy.1971.220.2.499. [DOI] [PubMed] [Google Scholar]
  7. Bishop J. S., Larner J. Rapid activation-inactivation of liver uridine diphosphate glucose-glycogen transferase and phosphorylase by insulin and glucagon in vivo. J Biol Chem. 1967 Mar 25;242(6):1354–1356. [PubMed] [Google Scholar]
  8. Bissell D. M., Hammaker L. E., Meyer U. A. Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. J Cell Biol. 1973 Dec;59(3):722–734. doi: 10.1083/jcb.59.3.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Blatt L. M., Kim K. H. Regulation of rat liver glycogen synthetase. Relationship of the hormonal activation and the time-dependent in vitro activation. J Biol Chem. 1971 Dec 10;246(23):7256–7264. [PubMed] [Google Scholar]
  10. Bonney R. J., Becker J. E., Walker P. R., Potter V. R. Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of the regulation of enzyme synthesis. In Vitro. 1974 May-Jun;9(6):399–413. doi: 10.1007/BF02615992. [DOI] [PubMed] [Google Scholar]
  11. Bonney R. J., Hopkins H. A., Walker P. R., Potter V. R. Glycolytic isoenzymes and glycogen metabolism in regenerating liver from rats on controlled feeding schedules. Biochem J. 1973 Sep;136(1):115–124. doi: 10.1042/bj1360115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Buschiazzo H., Exton J. H., Park C. R. Effects of glucose on glycogen synthetase, phosphorylase, and glycogen deposition in the perfused rat liver. Proc Natl Acad Sci U S A. 1970 Feb;65(2):383–387. doi: 10.1073/pnas.65.2.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cardell R. R., Jr Action of metabolic hormones on the fine structure of rat liver cells. I. Effects of fasting on the ultrastructure of hepatocytes. Am J Anat. 1971 May;131(1):21–53. doi: 10.1002/aja.1001310103. [DOI] [PubMed] [Google Scholar]
  15. Chang A. Y., Schneider D. I. Rate of gluconeogenesis and levels of gluconeogenic enzymes in liver and kidney of diabetic and normal Chinese hamsters. Biochim Biophys Acta. 1970 Dec 29;222(3):587–592. doi: 10.1016/0304-4165(70)90184-4. [DOI] [PubMed] [Google Scholar]
  16. Claude A. Growth and differentiation of cytoplasmic membranes in the course of lipoprotein granule synthesis in the hepatic cell. I. Elaboration of elements of the Golgi complex. J Cell Biol. 1970 Dec;47(3):745–766. doi: 10.1083/jcb.47.3.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Crisp D. M., Pogson C. I. Glycolytic and gluconeogenic enzyme activities in parenchymal and non-parenchymal cells from mouse liver. Biochem J. 1972 Feb;126(4):1009–1023. doi: 10.1042/bj1261009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. De Wulf H., Hers H. G. The stimulation of glycogen synthesis and of glycogen synthetase in the liver by the administration of glucose. Eur J Biochem. 1967 Jul;2(1):50–56. doi: 10.1111/j.1432-1033.1967.tb00104.x. [DOI] [PubMed] [Google Scholar]
  19. Drochmans P., Wanson J. C., Mosselmans R. Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions. J Cell Biol. 1975 Jul;66(1):1–22. doi: 10.1083/jcb.66.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ehrenreich J. H., Bergeron J. J., Siekevitz P., Palade G. E. Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J Cell Biol. 1973 Oct;59(1):45–72. doi: 10.1083/jcb.59.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Farquhar J. W., Frank A., Gross R. C., Reaven G. M. Glucose, insulin, and triglyceride responses to high and low carbohydrate diets in man. J Clin Invest. 1966 Oct;45(10):1648–1656. doi: 10.1172/JCI105472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Friedmann B., Goodman E. H., Jr, Weinhouse S. Effects of glucose feeding, cortisol, and insulin on liver glycogen synthesis in the rat. Endocrinology. 1967 Sep;81(3):486–496. doi: 10.1210/endo-81-3-486. [DOI] [PubMed] [Google Scholar]
  23. Geelen M. J., Gibson D. M. Lipogenesis in maintenance cultures of rat hepatocytes. FEBS Lett. 1975 Oct 15;58(1):334–339. doi: 10.1016/0014-5793(75)80292-4. [DOI] [PubMed] [Google Scholar]
  24. Gerschenson L. E., Casanello D. Metabolism of rat liver cells cultured in suspension: insulin and glucagon effects on glycogen level. Biochem Biophys Res Commun. 1968 Nov 25;33(4):584–589. doi: 10.1016/0006-291x(68)90335-5. [DOI] [PubMed] [Google Scholar]
  25. Gerschenson L. E., Okigaki T., Andersson M., Molson J., Davidson M. B. Fine structural and growth characteristics of cultured rat liver cells. Insulin effects. Exp Cell Res. 1972 Mar;71(1):49–58. doi: 10.1016/0014-4827(72)90262-5. [DOI] [PubMed] [Google Scholar]
  26. Gibson D. M., Lyons R. T., Scott D. F., Muto Y. Synthesis and degradation of the lipogenic enzymes of rat liver. Adv Enzyme Regul. 1972;10:187–204. doi: 10.1016/0065-2571(72)90014-3. [DOI] [PubMed] [Google Scholar]
  27. Glaumann H., Bergstrand A., Ericsson J. L. Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver. J Cell Biol. 1975 Feb;64(2):356–377. doi: 10.1083/jcb.64.2.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Glinsmann W. H., Mortimore G. E. Influence of glucagon and 3', 5'-AMP on insulin responsiveness of the perfused rat liver. Am J Physiol. 1968 Sep;215(3):553–559. doi: 10.1152/ajplegacy.1968.215.3.553. [DOI] [PubMed] [Google Scholar]
  29. Glinsmann W., Pauk G., Hern E. Control of rat liver glycogen synthetase and phosphorylase activities by glucose. Biochem Biophys Res Commun. 1970 May 22;39(4):774–782. doi: 10.1016/0006-291x(70)90272-x. [DOI] [PubMed] [Google Scholar]
  30. Gold A. H. The effect of diabetes and insulin on liver glycogen synthetase activation. J Biol Chem. 1970 Feb 25;245(4):903–905. [PubMed] [Google Scholar]
  31. Griffiths J. B. The effect of insulin on the growth and metabolism of the human diploid cell, WI-38. J Cell Sci. 1970 Sep;7(2):575–585. doi: 10.1242/jcs.7.2.575. [DOI] [PubMed] [Google Scholar]
  32. Hamilton R. L., Regen D. M., Gray M. E., LeQuire V. S. Lipid transport in liver. I. Electron microscopic identification of very low density lipoproteins in perfused rat liver. Lab Invest. 1967 Feb;16(2):305–319. [PubMed] [Google Scholar]
  33. Heilmeyer L. M., Jr, Meyer F., Haschke R. H., Fischer E. H. Control of phosphorylase activity in a muscle glycogen particle. II. Activation by calcium. J Biol Chem. 1970 Dec 25;245(24):6649–6656. [PubMed] [Google Scholar]
  34. Hers H. G., De Wulf H., Stalmans W. The control of glycogen metabolism in the liver. FEBS Lett. 1970 Dec 28;12(2):73–82. doi: 10.1016/0014-5793(70)80569-5. [DOI] [PubMed] [Google Scholar]
  35. Hers H. G., De Wulf H., Stalmans W., van den Berghe G. The control of glycogen synthesis in the liver. Adv Enzyme Regul. 1970;8:171–190. doi: 10.1016/0065-2571(70)90016-6. [DOI] [PubMed] [Google Scholar]
  36. Hershko A., Mamont P., Shields R., Tomkins G. M. "Pleiotypic response". Nat New Biol. 1971 Aug;232(33):206–211. [PubMed] [Google Scholar]
  37. Hollenberg M. D., Cuatrecasas P. Insulin: interaction with membrane receprots and relationship to cyclic purine nucleotides and cell growth. Fed Proc. 1975 Jun;34(7):1556–1563. [PubMed] [Google Scholar]
  38. Hue L., Bontemps F., Hers H. The effects of glucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations. Biochem J. 1975 Oct;152(1):105–114. doi: 10.1042/bj1520105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Jeejeebhoy K. N., Ho J., Greenberg G. R., Phillips M. J., Bruce-Robertson A., Sodtke U. Albumin, fibrinogen and transferrin synthesis in isolated rat hepatocyte suspensions. A model for the study of plasma protein synthesis. Biochem J. 1975 Jan;146(1):141–155. doi: 10.1042/bj1460141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Jones A. L., Ruderman N. B., Herrera M. G. Electron microscopic and biochemical study of lipoprotein synthesis in the isolated perfused rat liver. J Lipid Res. 1967 Sep;8(5):429–446. [PubMed] [Google Scholar]
  41. KRISMAN C. R. A method for the colorimetric estimation of glycogen with iodine. Anal Biochem. 1962 Jul;4:17–23. doi: 10.1016/0003-2697(62)90014-3. [DOI] [PubMed] [Google Scholar]
  42. Kreutner W., Goldberg N. D. Dependence on insulin of the apparent hydrocortisone activation of hepatic glycogen synthetase. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1515–1519. doi: 10.1073/pnas.58.4.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  44. Lombardi B. Considerations on the pathogenesis of fatty liver. Lab Invest. 1966 Jan;15(1 Pt 1):1–20. [PubMed] [Google Scholar]
  45. Mahley R. W., Hamilton R. L., Lequire V. S. Characterization of lipoprotein particles isolated from the Golgi apparatus of rat liver. J Lipid Res. 1969 Jul;10(4):433–439. [PubMed] [Google Scholar]
  46. Miller T. B., Jr, Hazen R., Larner J. An absolute requirement for insulin in the control of hepatic glycogenesis by glucose. Biochem Biophys Res Commun. 1973 Jul 17;53(2):466–474. doi: 10.1016/0006-291x(73)90685-2. [DOI] [PubMed] [Google Scholar]
  47. Miller T. B., Jr, Larner J. Mechanism of control of hepatic glycogenesis by insulin. J Biol Chem. 1973 May 25;248(10):3483–3488. [PubMed] [Google Scholar]
  48. Morgan C. R., Jersild R. A., Jr Alterations in the morphology of rat liver cells influenced by insulin. Anat Rec. 1970 Apr;166(4):575–585. doi: 10.1002/ar.1091660404. [DOI] [PubMed] [Google Scholar]
  49. Pilkis S. J., Korner A. Effect of diabetes and insulin treatment on protein synthetic activity of rat liver ribosomes. Biochim Biophys Acta. 1971 Nov 19;247(4):597–608. doi: 10.1016/0005-2787(71)90695-2. [DOI] [PubMed] [Google Scholar]
  50. Reaven G. M., Lerner R. L., Stern M. P., Farquhar J. W. Role of insulin in endogenous hypertriglyceridemia. J Clin Invest. 1967 Nov;46(11):1756–1767. doi: 10.1172/JCI105666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. SALAS M., VINUELA E., SOLS A. INSULIN-DEPENDENT SYNTHESIS OF LIVER GLUCOKINASE IN THE RAT. J Biol Chem. 1963 Nov;238:3535–3538. [PubMed] [Google Scholar]
  52. STEINER D. F., KING J. INDUCED SYNTHESIS OF HEPATIC URIDINE DIPHOSPHATE GLUCOSE-GLYCOGEN GLUCOSYLTRANSFERASE AFTER ADMINISTRATION OF INSULIN TO ALLOXAN-DIABETIC RATS. J Biol Chem. 1964 May;239:1292–1298. [PubMed] [Google Scholar]
  53. Schwartz A. G., Amos H. Insulin dependence of cells in primary culture: influence on ribo-some integrity. Nature. 1968 Sep 28;219(5161):1366–1367. doi: 10.1038/2191366a0. [DOI] [PubMed] [Google Scholar]
  54. Scott R. B., Cooper L. W. Glycogenolysis and glycogen synthesis in a cell-free system from rat liver. Biochem Biophys Res Commun. 1971 Sep;44(5):1071–1076. doi: 10.1016/s0006-291x(71)80194-8. [DOI] [PubMed] [Google Scholar]
  55. Seglen P. O. Effects of anaerobiosis, glucose, insulin and glucagon on glycogen metabolism in isolated parenchymal rat liver cells. FEBS Lett. 1973 Nov 1;36(3):309–312. doi: 10.1016/0014-5793(73)80398-9. [DOI] [PubMed] [Google Scholar]
  56. Seglen Per O. Glycogen synthesis in isolated parenchymal rat liver cells. FEBS Lett. 1973 Feb 15;30(1):25–28. doi: 10.1016/0014-5793(73)80611-8. [DOI] [PubMed] [Google Scholar]
  57. Stalmans W., De Wulf H., Hue L., Hers H. G. The sequential inactivation of glycogen phosphorylase and activation of glycogen synthetase in liver after the administration of glucose to mice and rats. The mechanism of the hepatic threshold to glucose. Eur J Biochem. 1974 Jan 3;41(1):127–134. doi: 10.1111/j.1432-1033.1974.tb03252.x. [DOI] [PubMed] [Google Scholar]
  58. Stalmans W., de Wulf H., Hers H. G. The control of liver glycogen synthetase phosphatase by phosphorylase. Eur J Biochem. 1971 Feb;18(4):582–587. doi: 10.1111/j.1432-1033.1971.tb01279.x. [DOI] [PubMed] [Google Scholar]
  59. Stein O., Stein Y. Visualization of intravenously injected 9, 10-3-H2-palmitic acid in rat liver by electronmicroscopic autoradiography. Isr J Med Sci. 1966 Mar-Apr;2(2):239–242. [PubMed] [Google Scholar]
  60. Temin H. M. Studies on carcinogenesis by avian sarcoma viruses. VI. Differential multiplication of uninfected and of converted cells in response to insulin. J Cell Physiol. 1967 Jun;69(3):377–384. doi: 10.1002/jcp.1040690314. [DOI] [PubMed] [Google Scholar]
  61. Thomas J. A., Schlender K. K., Larner J. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal Biochem. 1968 Oct 24;25(1):486–499. doi: 10.1016/0003-2697(68)90127-9. [DOI] [PubMed] [Google Scholar]
  62. Trotter N. L. Electron-opaque, lipid-containing bodies in mouse liver at early intervals after partial hepatectomy and sham operation. J Cell Biol. 1965 Jun;25(3 Suppl):41–52. doi: 10.1083/jcb.25.3.41. [DOI] [PubMed] [Google Scholar]
  63. Turkington R. W., Riddle M. Hormone-dependent formation of polysomes in mammary cells in vitro. J Biol Chem. 1970 Oct 10;245(19):5145–5152. [PubMed] [Google Scholar]
  64. Vardanis A. Glycogen-bound enzymes: a new method of isolation. Arch Biochem Biophys. 1969 Mar;130(1):408–412. doi: 10.1016/0003-9861(69)90052-6. [DOI] [PubMed] [Google Scholar]
  65. Villar-Palasi C. Oligo- and polysaccharide inhibition of muscle transferase D phosphatase. Ann N Y Acad Sci. 1969 Oct 14;166(2):719–730. doi: 10.1111/j.1749-6632.1969.tb46429.x. [DOI] [PubMed] [Google Scholar]
  66. Wanson J. C., Drochmans P., Mosselmans R., Ronveaux M. F. Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations. J Cell Biol. 1977 Sep;74(3):858–877. doi: 10.1083/jcb.74.3.858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wanson J. C., Drochmans P. Rabbit skeletal muscle glycogen. A morphological and biochemical study of glycogen beta-particles isolated by the precipitation-centrifugation method. J Cell Biol. 1968 Jul;38(1):130–150. doi: 10.1083/jcb.38.1.130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wanson J. C., Drochmans P. Role of the sarcoplasmic reticulum in glycogen metabolism. Binding of phosphorylase, phosphorylase kinase, and primer complexes to the sarcovesicles of rabbit skeletal muscle. J Cell Biol. 1972 Aug;54(2):206–224. doi: 10.1083/jcb.54.2.206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Weber G. Integrative action of insulin at the molecular level. Isr J Med Sci. 1972 Mar;8(3):325–343. [PubMed] [Google Scholar]
  70. Witters L. A., Alberico L., Avruch J. Insulin regulation of glycogen synthase in the isolated rat hepatocyte. Biochem Biophys Res Commun. 1976 Apr 19;69(4):997–1003. doi: 10.1016/0006-291x(76)90471-x. [DOI] [PubMed] [Google Scholar]
  71. Younger L. R., King J., Steiner D. F. Hepatic proliferative response to insulin in severe alloxan diabetes. Cancer Res. 1966 Jul;26(7):1408–1414. [PubMed] [Google Scholar]
  72. de Barsy T., Hers H. G. Biochemical and ultrastructural study of leucocytes in type II glycogenosis. Arch Int Physiol Biochim. 1975 Dec;83(5):954–955. [PubMed] [Google Scholar]
  73. de Man J. C., Blok A. P. Relationship between glycogen and agranular endoplasmic reticulum in rat hepatic cells. J Histochem Cytochem. 1966 Feb;14(2):135–146. doi: 10.1177/14.2.135. [DOI] [PubMed] [Google Scholar]
  74. de Wulf H., Hers H. G. The interconversion of liver glycogen synthetase a and b in vitro. Eur J Biochem. 1968 Dec 5;6(4):552–557. doi: 10.1111/j.1432-1033.1968.tb00480.x. [DOI] [PubMed] [Google Scholar]
  75. de Wulf H., Hers H. G. The role of glucose, glucagon and glucocorticoids in the regulation of liver glycogen synthesis. Eur J Biochem. 1968 Dec 5;6(4):558–564. doi: 10.1111/j.1432-1033.1968.tb00481.x. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES