Summary
To determine if cGMP might function as a second messenger for insulin, anin situ liver perfusion system was established in which hepatic effects of insulin could be correlated with changes in cyclic nucleotides. Several combinations of insulin (10 mU/ml) and glucose (50 mg/ml) were infused (0.1 ml/min) for 30 min into fasted normal and diabetic rats with removal of a similar volume of blood. Samples of livers were removed at the beginning and end and at various times during the perfusion. In normal animals perfused with buffer alone, hepatic glycogen content fell. When glucose (with or without added insulin) was added to the perfusate, glycogen levels rose. With buffer alone, there was no change in the independent (I) form of glycogen synthase at 10 min but a modest increase at 30 min. With insulin and/or glucose, there was a large increase in the I-form of the enzyme at 10 min and a further rise at 30 min. Neither cGMP nor cAMP changed even though tissue samples were obtained at multiple times throughout the perfusion. Cyclic nucleotides were also measured in liver slices exposed to insulin (1 mU/ml) after 30 min of preincubation for stabilization. Although significant increases in cGMP were noted in the tissue exposed to insulin, similar significant rises also occurred in appropriately paired control slices. When glucagon was used in both thein situ perfusion and the paired liver slice systems, the expected rapid and large increases in cAMP levels occurred attesting to the validity of both approaches in evaluating hepatic cyclic nucleotide responses. These results plus the paucity of convincing data in the literature strongly suggest that cGMP can no longer be considered a candidate for the putative second messenger of insulin.
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References
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 isolated from normal fed rats - Biochem. biophys. Res. Commun.61, 222, 1974.
Bishop J. S., Goldberg N. D., Larner J.: Insulin regulation of hepatic glycogen metabolism in the dog - Amer. J. Physiol.220, 499, 1971.
Bishop J. S., Larner J.: Rapid activation-inactivation of liver uridine diphosphate glucoseglycogen transferase and phosphorylase by insulin and glucagonin vivo - J. biol. Chem.242, 1354, 1967.
Blatt L. M., Kim K. H.: Regulation of rat liver glycogen synthetase. Relationship of the hormonal activation and the time-dependentin vitro activation - J. biol. Chem.246, 7256, 1971.
Bruno G. A., Christian J. E.: Determination of carbon-14 in aqueous bicarbonate solutions by liquid scintillation counting techniques - Analyt. Chem.33, 1216, 1961.
Buschiazzo H., Exton J. H., Park C. R.: Effects of glucose on glycogen synthetase, phosphorylase, and glycogen deposition in the perfused rat liver - Proc. nat. Acad. Sci. (Wash.)65, 383, 1970.
Conn H. O., Kipnis D. M.: The effect of various 3′5′ cyclic nucleotides on gluconeogenesis and glycogenolysis in the perfused rat liver - Biochem. biophys. Res. Commun.37, 319, 1969.
Curnow R. T., Rayfield E. J., George D. T., Zenser T. V., De Rubertis F.: Control of hepatic glycogen metabolism in therhesus monkey: effect of glucose, insulin and glucagon administration - Amer. J. Physiol.228, 80, 1975.
Davidson M. B., Aoki V. S.: Fasting glucose homeostasis in rats after chronic exposure to hypoxia - Amer. J. Physiol.219, 378, 1970.
DeWulf H., Hers H. G.: The stimulation of glycogen synthesis and of glycogen synthetase in the liver by the administration of glucose - Europ. J. Biochem.2, 50, 1967.
DeWulf H., Hers H. G.: The interconversion of liver glycogen synthetasea andb in vitro - Europ. J. Biochem.6, 552, 1968.
Exton J. H., Hardman J. G., Williams T. F., Sutherland E. W., Park C. R.: Effects of guanosine 3′,5′-monophosphate on the perfused rat liver - J. biol. Chem.246, 2658, 1971.
Fain J. N., Butcher F. R.: Cyclic guanosine 3′:5′-monophosphate and the regulation of lipolysis in rat fat cells - J. cyclic Nucleotide Res.2, 71, 1976.
Freychet P.: Interactions of polypeptide hormones with cell membrane specific receptors: studies with insulin and glucagon - Diabetologia12, 83, 1976.
Friedmann B., Goodman E. H. Jr.,Weinhouse S.: Liver glycogen synthesis in intact alloxan diabetic rats - J. biol. Chem.238, 2899, 1963.
Friedmann B., Goodman E. H. Jr.,Weinhouse S.: Effects of glucose feeding, cortisol, and insulin on liver glycogen synthesis in the rat - Endocrinology81, 486, 1967.
Glinsmann W. H., Hern E. P.: Inactivation of rat liver glycogen synthetase by 3′:5′-cyclic nucleotides - Biochem. biophys. Res. Commun.36, 931, 1969.
Glinsmann W. H., Hern W. H., Linarelli L. G., Farese R. V.: Similarities between effects of adenosine 3′,5′-monophosphate and guanosine 3′5′-monophosphate on liver and adrenal metabolism - Endocrinology85, 711, 1969.
Glinsmann W. H., Pauk G., Hern E.: Control of rat liver glycogen synthetase and phosphorylase activities by glucose - Biochem. biophys. Res. Commun.39, 774, 1970.
Gold A. H.: The effect of diabetes and insulin on liver glycogen synthetase activation - J. biol. Chem.245, 903, 1970.
Goldberg N. D., Dietz S. B., O'toole A. G. O.: Cyclic guanosine 3′,5′-monophosphate in mammalian tissues and urine - J. biol. Chem.244, 4458, 1969.
Golden S., Wals P. A., Katz J.: An improved procedure for the assay of glycogen synthase and phosphorylase in rat liver homogenates - Analyt. Biochem.77, 436, 1977.
Goldfine I. D.: Does insulin need a second messenger? - Diabetes26, 148, 1977.
Hardeland R., Holzapfel G.: Induction of tyrosine amino-transferase by insulin not mediated by cyclic GMP - Naturwissenschaften62, 397, 1975.
Hepp K. D.: Studies on the mechanism of insulin action: basic concepts and clinical implications - Diabetologia13, 177, 1977.
Hers H. G.: The control of glycogen metabolism in the liver - Ann. Rev. Biochem.45, 167, 1976.
Hornbrook K. R.: Synthesis of liver glycogen in starved alloxan diabetic rats - Diabetes19, 916, 1970.
Hue L., Bontemps F., Hers H. G.: The effect of glucose and potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations - Biochem. J.152, 105, 1975.
Illiano G., Tell G. P. E., Siegel M. I., Cuatrecasas P.: Guanosine 3′:5′-cyclic monophosphate and the action of insulin and acetycholine - Proc. nat. Acad. Sci. (Wash.)70, 2443, 1973.
Jarett L., Seals J. R., McDonald J. M.: Insulin effect on protein phosphorylation of plasma membranes and mitochondria in a subcellular preparation from rat adipocytes: generation of an insulin second messenger - Clin. Res.27, 486, 1979.
Jefferson L. S., Exton J. H., Butcher R. W., Sutherland E. W., Park C. R.: Role of adenosine 3′,5′-monophosphate on the effects of insulin and antiinsulin serum on liver metabolism - J. biol. Chem.243, 1031, 1968.
Ketterer H., Eisentraut A. M., Unger R. H.: Effect upon insulin secretion of physiologic doses of glucagon administered via the portal vein - Diabetes16, 283, 1967.
Kreutner W., Goldberg N. D.: Dependence on insulin of the apparent hydrocortisone activation of hepatic glycogen synthetase - Proc. nat. Acad. Sci. (Wash.)58, 1515, 1967.
Lawrence J. C. Jr.,Larner J., Kahn C. R., Roth J.: Auto-antibodies to the insulin receptor activate glycogen synthase in rat adipocytes - Molec. cell. Biochem.22, 153, 1978.
Miller T. B.: Effects of diabetes on glucose metabolism - Amer. J. Physiol.234, E13, 1978.
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.53, 466, 1973.
Miller T. B. Jr.,Larner J.: Mechanism of control of hepatic glycogenesis by insulin - J. biol. Chem.248, 3483, 1973.
Mulmed L. N., Gannon M. C., Gilboe D. P., Tan A. W. H., Nuttall F. Q.: Glycogen synthase, synthase phosphatase, and phosphorylase response to glucose in somatostatinpretreated intact rats - Diabetes28, 231, 1979.
Nichols W. K., Goldberg N. D.: The relationship between insulin and apparent glucocorticoid-promoted activation of hepatic glycogen synthetase - Biochim. biophys. Acta (Amst.)279, 245, 1972.
Nuttall F. Q., Gannon M. C.: Regulation of glycogen synthetase in liver - Clin. Res.20, 777, 1972.
Nuttall F. Q., Gannon M. C., Larner J.: Oral glucose effect on glycogen synthetase and phosphorylase in heart, muscle and liver - Physiol. chem. Phys.4, 497, 1972.
Pilkis S. J., Park C. R.: Mechanism of action of insulin - Ann. Rev. Pharmacol.14, 365, 1974.
Pointer R. H., Butcher F. R., Fain J. N.: Studies on the role of cyclic guanosine 3′:5′-monophosphate and extracellular Ca+2 in the regulation of glycogenolysis in rat liver cells - J. biol. Chem.251, 2987, 1976.
Shimazu T.: Glycogen synthetase activity in liver: regulation by the autonomie nerves - Science156, 1256, 1967.
Shimazu T., Matsushita H., Ishikawa K.: Cholinergic stimulation of the rat hypothalamus: effects on liver glycogen synthesis - Science194, 535, 1976.
Soeldner J. S., Slone D.: Critical variables in the radioimmunoassay of serum insulin using the double antibody technique - Diabetes14, 771, 1965.
Sparks J. W., Lynch A., Glinsmann W. H.: Regulation of rat liver glycogen synthesis and activities of glycogen cycle enzymes by glucose and galactose - Metabolism25, 47, 1976.
Steiner A. L., Parder C. W., Kipnis D. M.: Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cycle nucleotides - J. biol. Chem.247, 1106, 1972.
Steiner D. F.: Mechanisms of regulation of hepatic glycogen synthesis - Nature (Lond.)24, 1171, 1964.
Steiner D. F., Chan S. J., Terris S., Hofmann C.: Insulin as a cellular growth regulator - In: Hepatotrophic Factors. Ciba Foundation Symposium 55 (new series) Excerpta Medica/Elsevier, Amsterdam; 1978; p. 217.
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.239, 1292, 1964.
Steiner D. F., Rauda V., Williams R. H.: Effects of insulin, glucagon, and glucocorticoids upon hepatic glycogen synthesis from uridine diphosphate glucose - J. biol. Chem.236, 299, 1961.
Tauri S., Saito Y., Fujimoto M., Okabayashi T.: Effects of insulin on diaphragm muscle independent of the variation of tissue levels of cyclic AMP and cyclic GMP - Arch. Biochem.174, 192, 1976.
van Handel E.: Estimation of glycogen in small amounts of tissue - Analyt. Biochem.11, 256, 1965.
Vesely D. L., Castro A., Levey G. S.: Decreased rat hepatic guanylate cyclase activity in streptozotocin-induced diabetes mellitus - Diabetes26, 308, 1977.
Walaas O., Walaas E., Wick A. N.: The stimulatory effect by insulin on the incorporation of32P radioactive inorganic phosphate into intracellular inorganic phosphate, adenine nucleotides and guanine nucleotides of the intact isolated rat diaphragm - Diabetologia5, 79, 1969.
Walli A. K., Birkmann L., Schimassek H.: The effects of glucose and fructose on glycogen metabolism in the isolated perfused rat liver - Biochem. Soc. Trans.3, 1039, 1975.
Walli A. K., Siebler G., Zepf E., Schimassek H.: Glycogen metabolism in isolated perfused rat liver - Hoppe-Seyler's Z. physiol. Chem.355, 353, 1974.
Whitton P. D., Hems D. A.: Glycogen synthesis in the perfused liver of streptozotocin-diabetic rats - Biochem. J.150, 153, 1975.
Witters L. A., Alberico L., Avruch J.: Insulin regulation of glycogen synthase in the isolated rat hepatocyte - Biochem. biophys. Res. Commun.69, 997, 1976.
Witters L. A., Avruch J.: Insulin regulation of hepatic glycogen synthase and phosphorylase - Biochemistry (Wash.)17, 406, 1978.
Worcester J.: The statistical method - New Engl. J. Med.274, 27, 1966.
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Davidson, M.B., Yoshii, V. Hepatic effects of glucose and insulin independent of cyclic nucleotide changes. Acta diabet. lat 18, 297–310 (1981). https://doi.org/10.1007/BF02042813
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DOI: https://doi.org/10.1007/BF02042813