Summary
A number of enzymatic methods have been developed to prepare hepatocytes using collagenase and hyaluronidase. However, best cell preparations are obtained by using only low concentrations of collagenase and exposing the liver to the enzyme for a very short period of time. These isolated cells with intact cell membranes and large numbers of microvilli on the cell surface respond to hormones at physiological concentrations suggesting that these microvilli contain hormone receptors. In addition, high glycogen content is essential to maintain thein vivo metabolic characteristics of the hepatocytes suggesting that intracellular glycogen plays an important role in the hormonal regulation of metabolism in hepatocytes. Studies with glucagon and insulin on carbohydrate metabolism show that the molar ratios of these hormones control gluconeogenesis and glycogenolysis. Furthermore,in vitro addition of insulin stimulates glycogen synthesis and activates glycogen synthase. Insulin also stimulates protein synthesis in cells containing high glycogen and maintains more normal parallel strands of polyribosomes. Studies with isolated hepatocytes from diabetic, hypophysectomized and adrenalectomized animals show a reduced glucagon response to glycogenolysis. This lack of glucagon response was not due to reduction in glycogen levels. Other hormones such as somatostatin and parathyroid also give rise to alterations in carbohydrate metabolism in isolated hepatocytes.
Similar content being viewed by others
References
Akapan 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 Isolated from Normal Fed Rats — Biochem. biophys. Res. Commun.61, 222, 1974.
Allen D. O., Largis E. E., Miller E. A., Ashmore J.: Continuous Monitoring of Lipolytic Rates in Perifused Isolated Fat Cells — J. appl. Physiol.34, 125, 1973.
Anderson N. G.: The Mass Isolation of Whole Cells from Rat Liver — Science151, 627, 1953.
Aubin St. P. M. G., Bucher N. L. R.: A Study of Binucleate Cell Counts in Resting and Regenerating Rat Liver Employing a Mechanical Method for the Separation of Liver Cells — Anat. Rec.112, 797, 1952.
Berry M. N., Friend D. S.: High-Yield Preparation of Isolated Rat Liver Parenchymal Cells: a Biochemical and Fine Structural Study — J. Cell Biol.43, 506, 1969.
Berry M. N., Kun E.: Rate Limiting Steps of Gluconeogenesis in Liver Cells as Determined with the Aid of Fluoro-Dicarboxylic Acids — Europ. J. Biochem.27, 395, 1972.
Billings R. E., Ashmore J., Wagle S. R., McMahon R. E.: Studies on Drug Metabolism in Isolated Hepatocytes — Fed. Proc.34, 727, 1975.
Branster M. V., Morton R. K.: Isolation of Intact Liver Cells — Nature (Lond.)180, 1283, 1957.
Bucher N. L. R., Scott J. F., Simpson E.: Preparation of Cell Suspensions from Liver and Investigations of the Properties of Free Cells — Cancer Res.11, 240, 1951.
Capuzzi D. M., Rothman V., Margolis S.: Regulation of Lipogenesis by Cyclic Nucleotides in Intact Hepatocytes Prepared by a Simplified Technique — J. biol. Chem.249, 1286, 1974.
Christoffersen T., Berg T.: Glucagon Control of Cyclic AMP Accumulation in Isolated Intact Rat Liver Parenchymal Cells in Vitro — Biochim. biophys. Acta (Amst.)338, 408, 1974.
Christoffersen T., Morland J., Osnes J. B., Berg T., Boman D., Seglen P. O.: Enrichment of Glucagon Sensitive Adenyl Cyclase upon Preparation of Parenchymal Cells from Rat Liver — Arch. Biochem.150, 807, 1972.
Crane L. F., Miller D. L.: Synthesis and Secretion of Fibrinogen and Albumin by Isolated Rat Hepatocytes — Biochem. biophys. Res. Commun.60, 1269, 1974.
Exton J. H., Park C. R.: Control of Gluconeogenesis in Liver. I. General Features of Gluconeogenesis in the Perfused Liver of Rats — J. biol. Chem.242, 2622, 1967.
Garrison J. C., Haynes R. C. Jr.: Hormonal Control of Glycogenolysis and Gluconeogenesis in Isolated Rat Liver Cells — J. biol. Chem.248, 5333, 1973.
Good C. A., Kramer H., Somogyi M.: The Determination of Glycogen — J. biol. Chem.100, 485, 1933.
Hems R., Ross B. D., Berry M. N., Krebs H. A.: Gluconeogenesis in the Perfused Rat Liver — Biochem. J.101, 284, 1966.
Hoshi M., Shreeve W. W.: Release and Production of Insulin by Isolated, Perifused Rat Pancreatic Islets. Control by Glucose — Diabetes22, 16, 1973.
Howard R. B., Christensen A. K., Gibbs F. A., Pesch L. A.: The Enzymatic Preparation of Isolated Intact Parenchymal Cells from Rat Liver — J. Cell Biol.35, 675, 1967.
Howard R. B., Pesch L. A.: Respiratory Activity of Intact, Isolated Parenchymal Cells from Rat Liver — J. biol. Chem.243, 3105, 1968.
Hugget A. S., Nixon D. A.: Use of Glucose Oxidase, Peroxidase, and O-Dianisidine in Determination of Blood and Urinary Glucose — Lancet2, 368, 1957.
Ingebretsen W. R. Jr., Moxley M. A., Allen D. O., Wagle S. R.: Studies on Gluconeogenesis, Protein Synthesis and Cyclic AMP Levels in Isolated Parenchymal Cells Following Insulin Withdrawal from Alloxan Diabetic Rats — Biochem. biophys. Res. Commun.49, 601, 1972.
Ingebretsen W. R. Jr., Wagle S. R.: Rapid Method for the Isolation of Large Quantities of Rat Liver Parenchymal Cells with High Anabolic Rates — Biochem. biophys. Res. Commun.47, 403, 1972.
Ingebretsen W. R. Jr., Wagle S. R.: Studies on the Effects of Trycaprylin on Gluconeogenesis and Ketogenesis in Isolated Perfused Liver — Proc. Soc. exp. Biol. (N. Y.)147, 578, 1974.
Jacob S. T., Bhargava P. M.: A New Method for the Preparation of Liver Cell Suspensions — Exp. Cell Res.27, 453, 1962.
Johnson R. E. M., Das N. M., Butcher F. R., Fain J. N.: The Regulation of Gluconeogenesis in Isolated Rat Liver Cells by Glucagon, Insulin, Dibutyryl Cyclic Adenosine Monophosphate and Fatty Acids — J. biol. Chem.247, 3229, 1972.
Kaltenbach J. P.: Preparation of Suspensions of Intact Liver Cells — Fed. Proc.11, 237, 1952.
Kaltenbach J. P.: The Preparation and Utilization of Whole Cell Suspensions Obtained from Solid Mammalian Tissues — Exp. Cell Res.7, 568, 1954.
Kono T., Barham F. W.: Insulin-Like Effects of Trypsin on Fat Cells. Localization of the Metabolic Steps and the Cellular Site Affected by the Enzyme — J. biol. Chem.246, 6204, 1971.
Longmuir I. S., Rees W. A.: Preparation of Cell Suspensions from Rat Livers — Nature (Lond.)177, 997, 1956.
Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J.: Protein Measurements with the Folin Phenol Reagent — J. biol. Chem.193, 265, 1951.
Manchester K. L.: Effect of Insulin on Protein Synthesis — Diabetes21 (Suppl. 2), 447, 1972.
Monier D., Santhanam K., Wagle S. R.: Studies on the Inhibition of Amino Acid Incorporation into Protein by Isolated Rat Liver Ribosomes by Protein Kinase — Biochem. biophys. Res. Commun.46, 1881, 1972.
Moxley M. A., Bell N. H., Wagle S. R., Allen D. O., Ashmore J. A.: Parathyroid Hormone Stimulation of Glucose and Urea Production in Isolated Liver Cells — Amer. J. Physiol.227, 1058, 1974.
Oliver J. R., Wagle S. R.: Studies on the Inhibition of Insulin Release, Glycogenolysis and Gluconeogenesis by Somatostatin in the Rat Islets of Langerhans and Isolated Hepatocytes — Biochem. biophys. Res. Commun.62, 772, 1975.
Ontko J. A.: Chylomicron, Free Fatty Acid and Ketone Body Metabolism of Isolated Liver Cells and Liver Homogenates — Biochim. biophys. Acta (Amst.)137, 13, 1967.
Pain V. M., Lanoix J., Bergerson J. J. M., Clemens M. J.: Effect of Diabetes on the Ultrastructure of the Hepatocytes and on the Distribution and Activity of Ribosomes in the Free and Membrane Bound Populations — Biochim. biophys. Acta (Amst.)353, 487, 1974.
Penhos J. C., Krahl M. E.: Insulin Stimulus of Leucine Incorporation in Rat Liver Protein — Amer. J. Physiol.202, 349, 1962.
Peters T. Jr., Peters J. C.: Biosynthesis of Rat Serum Albumin. V. Intracellular Transport of Albumin and Rates of Albumin and Liver Protein Synthesis in Vivo under Various Physiological Conditions — J. biol. Chem.247, 3858, 1972.
Pilkis S. J., Exton J. H., Johnson R. A., Park C. R.: Effects du glucagon sur le métabolisme de l’AMP cyclique et des glucides dans le foie de rats diabétiques — Biochim. biophys. Acta (Amst.)343, 205, 1974.
Rappaport C., Howze G. B.: Dissociation of Adult Mouse Liver by Sodium Tetraphenylboron, a Potassium Complexing Agent — Proc. Soc. exp. Biol. (N. Y.)121, 1010, 1966.
Rodbell M.: Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis — J. biol. Chem.239, 375, 1964.
Rous P., Beard J. W.: Selection with the Magnet and Cultivation of Reticulo-Endothelial Cells (Kupffer Cells) — J. exp. Med.59, 577, 1934.
Schneider W. C., Potter V. R.: The Assay of Animal Tissue for Respiratory Enzymes. 2. Succinic Dehydrogenase and Cytochrome Oxidase — J. biol. Chem.149, 217, 1943.
Schreiber M., Schreiber G., Kartenbeck J.: Protein and Ribonucleic and Synthesis in Single-Cell Suspensions from Morris Hepatoma 5123 c and from Normal Rat Liver — Cancer Res.34, 2143, 1974.
Stern M., Wagle S. R., Sweeney M. J., Ashmore J.: Studies on Experimental Diabetes. I. Effects of Anti-Insulin Serum on14C-Glucose Metabolism — J. biol. Chem.238, 12, 1963.
Unger R. H.: Insulin-Glucagon Ratio — Israel J. med. Sci.8, 252, 1972.
Unger R. H.: Alpha- and Beta-Cell Interrelationships in Health and Disease — Metabolism23, 581, 1974.
Wagle S. R.: Studies on the in Vitro Effects of Insulin on Glycogen Synthesis and Ultrastructure in Isolated Rat Liver Hepatocytes — Biochem. biophys. Res. Commun.53, 937, 1973.
Wagle S. R.: Studies on the Differential Response to Glucagon on Gluconeogenesis in Isolated Hepatocytes Containing High and Low Glycogen — Biochem. biophys. Res. Commun.59, 1366, 1974.
Wagle S. R.: Studies on the Lack of Glucagon Response to Glycogenolysis and Gluconeogenesis and Decrease in Protein Synthesis in Isolated Hepatocytes from Hypophysectomized Animals — Biochem. biophys. Res. Commun.64, 1169, 1975.
Wagle S. R., Ashmore J.: Interrelations between Amino Acid Metabolism and Carbohydrate Formation in Insulin Deficiency — J. biol. Chem.236, 2868, 1961.
Wagle S. R., Ashmore J.: Studies on Experimental Diabetes. 2. Carbon Dioxide Fixation — J. biol. Chem.238, 17, 1963.
Wagle S. R., Ashmore J.: Studies on Experimental Diabetes. 3. Effects of Acute Insulin Insufficiency on14C-Glucose Formation from Labeled Substrates — J. biol. Chem.239, 1289, 1964.
Wagle S. R., Gaskins R. K., Jacoby A., Ashmore J.: Studies on Glucose Synthesis by Rat Liver and Kidney Cortex Slices — Life Sci.5, 655, 1966.
Wagle S. R., Ingebretsen W. R. Jr.: Stimulation of Glycogenolysis by Epinephrine and Glucagon and its Inhibition by Insulin in Isolated Rat Liver Hepatocytes — Biochem. biophys. Res. Commun.52, 125, 1973.
Wagle S. R., Ingebretsen W. R. Jr.: Studies on the Effects of Collagenase and Hyaluronidase on the Glycogen Content of the Isolated Rat Liver Parenchymal Cells — Proc. Soc. exp. Biol. (N. Y.)147, 581, 1974.
Wagle S. R., Ingebretsen W. R. Jr.: Studies on Gluconeogenesis and Protein Synthesis in Isolated Rat Liver Hepatocytes — Proc. Soc. exp. Biol. (N. Y.)149, 480, 1975.
Wagle S. R., Ingebretsen W. R. Jr.: Isolation, Purification and Metabolic Characteristics of Rat Liver Hepatocytes — In:Lowenstein J. M. (Ed.): Methods in Enzymology. Vol. 35. Lipids, Part B. Academic Press, New York, 1975; p. 579.
Wagle S. R., Sampson L.: Studies on Differential Response to Insulin on the Stimulation of Amino Acid Incorporation into Protein in Isolated Hepatocytes Containing Different Levels of Glycogen — Biochem. biophys. Res. Commun.64, 72, 1975.
Zahlten R. N., Stratman F. W., Lardy H. A.: Regulation of Glucose Synthesis in Hormone-Sensitive Isolated Rat Hepatocytes — Proc. nat. Acad. Sci. (Wash.)70, 3213, 1973.
Author information
Authors and Affiliations
Additional information
Part of this review was written while on sabatical leave at Department of Biochemistry, University of Freiburg as a visiting guest professor.
Supported by U.S.P.H.S. grant No. AM 14340.
Rights and permissions
About this article
Cite this article
Wagle, S.R. Studies on hormonal regulation of metabolism using isolated hepatocytes. Acta diabet. lat 13, 186–201 (1976). https://doi.org/10.1007/BF02581117
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02581117