Effect of Cobalt Chloride on Blood Glucose, Plasma Insulin and Lipids in Rabbits.

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1 Effect of Cobalt Chloride on Blood Glucose, Plasma Insulin and Lipids in Rabbits. Masayoshi Ohmichi, Hiroyuki Morita, Yoko Onuma and Shigeo Koike Department of Hygiene, School of Medicine, Showa University, Tokyo A previous study in this laboratory has shown that rabbits injected with cobalt chloride for three days exhibited an increase in both plasma triglyceride and blood glucose.1) Cobalt chloride has been reported to produce varying degrees of alpha-cell degranulation and damage accompanied by transient hyperglycemia in rabbits.2-4) The transient hyperglycemia was explained as a result of the release of glucagon.5-8) Eaton9) observed an exaggerated elevation in serum glucagon but serum glucose concentration was normal in chronic cobalt-treated rats. Unger10,11) reported that plasma glucagon and insulin levels vary inversely with the need for endogenous glucose production. As contrasted to the damage evoked in alpha cells, beta cell damage was mild and the regranulation process started rapidly.12) From the opposite action of insulin and glucagon on hepatic glucose production, it is conceivable that alpha cell damage due to cobalt chloride may bring about some change in the level of insulin activity on the liver. The mechanism involved in hypertriglyceridemia induced by cobalt chloride in rabbits has remained obscure. It is well established that the major factors which determine the endogenous triglyceride concentration in plasma are blood levels of FFA, glucose and insulin.13) Cobalt chloride acts directly on hepatic cells to enhance the secretion of triglyceride or indirectly to stimulate secretion of glucagon, insulin14) or both, followed by hypertriglyceridemia. This result is a matter that remained to be clarified. The present study was undertaken to examine whether the elevation of blood glucose level and hypertriglyceridemia induced by cobalt chloride have any relationship to the basal insulin concentration as well as glucose-stimulated integrated insulin secretion level. MATERIAL AND METHODS White male rabbits weighing 3-4kg were fed laboratory chow (Oriental RC-4 solid food) and drinking water ad libitum for more than one month. Cobalt chloride was injected intramuscularly in a dose of 25mg/kg daily for three days. The control rabbits, which were pair fed to cobalt-treated, received the same volume of saline injection as the experimental animals. Blood samples were withdrawn from ear vein at 0 and the fourth day after the injection for estimation of the following substances: triglyceride, cholesterol, phospholipids, free fatty acids (FFA) and insulin. Each rabbit received 2g/kg of glucose (25% solution) by stomach tube. Blood glucose, Rlasma insulin and FFA concentration were measured at 0, 30, 60, 90, 120, 180, 240 and 300min. Glucose was measured by the hexokinase method.15) Plasma insulin was assayed by a double-antibody method of radioimmunoassay16) using RI KIT purchased from Dainabot RI Institute. Plasma triglyceride (TG) was measured by a modified method of Van Handel and Zilversmidt,17) cholesterol by a modified method of Zak,18) phospholipid by Hoeflmayr-Fried

2 method19) and FFA by the method of Laurell and Tibbling.20) Glucose-stimulated insulin secretion was expressed in microunit hours determined by planimetry of the area drew by the rise in concentration of plasma above the fasting baseline concentration during the 5 hours GTT. GTT glucose "SUM" represents the sum of the 0, 30, 60, 90, 120, 180, 240 and 300min plasma glucose values after an oral GTT. RESULTS The basal blood glucose level in cobalt-treated rabbits was significantly elevated as compared to pair-fed normal control animals (Table 1). The cobalt-treated rabbits demon- To define the secretory status of beta cell, the glucose was administered to stimulate insulin secretion as illustrated in Fig. 1. When 2g/kg body weight of glucose was adminis- observed. The response in the cobalt-treated animals had a more pronounced pattern and a insulin concentration and glucose-stimulated response of the beta cell were significantly increased in the cobalt-treated rabbits. In spite of the increase in basal blood glucose (Fig. 2), the sum of blood glucose concentration following oral glucose (GTT glucose "SUM") was not significantly different from that of control animals. In plasma from cobalt-treated rabbits, the concentration of triglyceride was more than 13 times higher than that in the saline treated control group. The concentration of phospholipid and cholesterol (total, free) in the plasma of cobalt-treated rabbits were also significantly increased (Table 1). Between basal insulin and triglyceride concentration, no significant correlation (r=4343, Table 1 Effect of cobalt on blood glucose, plasma FFA and lipids Table 2 Effect of cobalt on plasma insulin, GTT glucose "SUM" and glucose stimulated insulin secretion

3 Fig. 1 Mean plasma insulin concentrations following glucose ingestion in control Fig. 2 Mean blood glucose concentrations during oral tolerance tests in control P>0.1) could be observed, while a high correlation could be demonstrated between plasma triglyceride concentration and glucose-stimulated immunoreactive insulin secretion (r=0.672, P<0.01). Higher concentration of plasma FFA was found in the control pair-fed group as compared to the cobalt-treated animals. Plasma FFA level in both the cobalt-treated and the control rabbits, was not decreased, when glucose was given orally. DISCUSSION These studies and previous studies1,22) demonstrated that cobalt chloride produces a rise in blood glucose. Some authors have reported that a single injection of cobalt chloride evoked a transient hyperglycemia. The transitory hyperglycemia was followed by a return to normoglycemia within two to four hours after cobalt chloride injection, whereas an impairment of alpha cells of pancreatic islets persisted after ten days. A similar change of blood glucose level was observed in animals injected repeatedly with cobalt chloride.2-4) Volk et al2) postulated that the reestablishment and maintenance of normoglycemia in spite of persistent alpha cell damage suggest that blood sugar homeostasis does not depend on the integrity of these cells. Fodden reported daily intravenous injections of 25mg/kg of cobalt chloride into a rabbit caused hyperglycemia on the third day. Eaton21) found only a minimal rise of serum glucose concentration on the third day of injection in chronic cobalt-treated rats. If hyperglycemia was a function of the alpha cell damage, during which period an elevated glucagon level persisted as shown by Eaton9) in cobalt-treated rats, the rise of blood glucose level in the present study may be explained from the change of blood glucagon concentration, but we can not refer to this point, since there are no data available about glucagon concentration in this study. However, accompanied by the need for endogenous glucose production, antagonistic alteration in glucagon and insulin concentration might be expected. From the higher concentration of insulin and glucose in cobalt-treated rabbits in this study, it may be deduced that a reduction of plasma glucagon level, which is in disagreement with hyperglucagonemia in

4 chronic cobalt-treated rats suggested by Eaton.9) Conflicting supposition derived from the above-mentioned experimental data about the alteration of plasma glucagon levels must await for the experimental results. In the present study the increased levels were observed in basal insulin and glucosestimulated insulin secretion, while plasma glucose concentration following oral GTT remained at the similar level in the cobalt-treated rabbits as the control group. The increased insulin secretion can not be explained from the insulin resistance, since in this term the increase in glucose uptake and the inhibition of glucose from the liver are involved, and as the consequence of this there was a lowering of blood glucose and of serum plasma FFA concentration. It seems probable that the major factors which determine the endogenous triglyceride concentration in plasma were blood levels of FFA, glucose and insulin.13) In the present investigation a significant correlation was proved between plasma triglyceride concentration and glucose-stimulated insulin secretion. While such correlation study does not provide information relating to the cause of the hypertriglyceridemia, yet it is conceivable that an increased secretion of insulin is one factor contributing to the development of hyperlipemia. It was evidenced that elevated levels of insulin in the presence of increased glucose loads stimulate both hepatic triglyceride production and secretion in man23,24) and in the rat25). Plasma FFA that enters the liver in cobalt-treated rabbits is likely to be more established to form triglyceride than to be oxidized, since secretion of plasma triglyceride from the liver of cobalt-treated rabbits is much greater than those of control rabbits. It was proved recently in our laboratory26) that turnover rate of triglyceride in the plasma of rabbits treated with cobalt chloride was significantly elavated. Experiments to elucidate whether cobalt ion acts directly on liver cells to stimulate the production of triglyceride or indirectly via change of insulin and glucagon secretion to raise the heatic glucose production, or to enhance hepatic triglyceride secretion would need to be performed. SUMMARY Daily cobalt chloride injection for three days produced a rise of basal blood glucose concentration accompanied by the elevation of basal insulin levels as well as glucose-stimulated insulin secretion in rabbits, while the sum of blood glucose concentration following oral glucose remained normal. Hypertriglyceridemia observed in cobalt-treated rabbits correlated highly with glucose-stimulated insulin secretion. Higher concentration of plasma FFA was observed in pair-fed control rabbits as comparedto the cobalt-treated rabbits. REFERENCES 1) Ohyama, K., Morita, H., Hasegawa, T. and Koike, S.: Effect of cobalt chloride on the content of lipid and glycolitic intermediates in rabbit liver and heart. Jap. J. Hyg. 28, (1973). 2) Volk, B. W., Lazarus, S. S. and Goldner, M. G.: Alpha cell damage and blood sugar changes in rabbits after administration of cobalt. Proc. Soc. Exp. Biol. Med. 82, (1953). 3) Fooden, J. H.: Experiments with chemicals noxious to the pancreatic alpha cells. Am. J. Clin. Path. 23, (1953). 4) Goldner, M. G., Volk, B. W. and Lazarus, S. S.: The effect of cobaltous chloride on the blood sugar and alpha cells in the pancreatic islets of the rabbits. Metabolism, 1, (1952). 5) Fodden, J. H. and Read, W. O.: The activity of extracted pancreatic-glycogenolytic factor after cobaltous chloride and synthalin A. Endocrinol. 54, (1954). 6) Lochner, J. de V., Eisentraut, A. M. and Unger, R. H.: The effects of CoCl2 on glucagon levels in plasma and pancreas of the rat. Metabolism, 13, (1964). 7) Telib, M. and Schmidt, F. H.: Effects of cobaltous chloride in laboratory animals: II Effects on blood sugar, insulin and lipids in rabbits. Endokrinologie, 61, (1973). 8) Bromer, W. W. and Chance, R. E.: Zinc glucagon depression of blood amino acids in rabbits. Dia-

5 betes, 18, (1969). 9) Eaton, P.: Glucagon secretion and activity in the cobalt chloride-treated rat. Am. J. Physiol., 225, (1973). 10) Unger, R. H.: Glucagon and the insulin: gucagon ratio in diabetes and other catabolic illness. Diabetes, 20, (1971). 11) Unger, R. H.: Glucagon physiology and pathophysiology New Engl. J. Med. 285, (1971). 12) Telib, M.: Effects of cobaltous chloride in laboratory animals: I The histological and electron microscopical changes in the islets of rabbits. Endokrinologie, 60, (1972). 13) Nikkila, E. A.: Control of plasma and liver triglyceride kinetics by carbohydrate metabolism and insulin. A review. Adv. Lipid Res. vol. 7 pp , Academic Press, New York (1969). 14) Telib, M. and Pfeiffer, E. F.: Enhancement of reactive insulin secretion in vitro by cobalt-chloride (CoCl2). Horm. Metab. Res. 1, 44 (1969). 15) Schmidt, F. H.: Die enzymatische Bestimmungen von Glucose und Fructose nebeneinander. Klin. Wschr. 39, (1961). 16) Morgan, C. R. and Lazarow, A.: Immunoassay of insulin. Two antibody system: plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes, 12; (1963). 17) Kawade, M.: Microdetermination of surum triglyceride. Nihon Rinsho, 85, (1962). 18) Yoshikawa, H. et al.: Assessment on the quantitative analysis of serum total cholesterol using reagent containg ferric ion. Igaku no Ayumi, 33, (1960). 19) Hoeflemayr, J. and Fried, R.: Eine Methode zur routinemassigen Bestimmung des Lipid Phosphors 20) Laurell, S. and Tibbling, G.: Colorimetric microdetermination of free fatty acids in plasma, Clin. Chim. Acta, 16, (1967). 21) Eaton, R. P.: Cobalt chloride-induced hyperlipemia in the rat: effects on intermediary metabolism. Am. J. Physiol. 222, (1972). 22) Asano, S., Ohyama, K. and Koike, S.: Accumulation of triglyceride in rabbit liver induced by injection of cobalt chloride. Jap. J. Hyg., 27, 105 (1972). 23) Reaven, G. M., Lerner, R. L. Stern, M. P. and Farquhar, J. W.: Role of insulin in endogenous hypertriglyceridemia. J. Clin. Invest. 46, (1967). 24) Farquhar, J. W., Frank, A., Gross. R. C. and Reaven, G. M.: Glucose, insulin and triglyceride responses to high and low carbohydrate diets in man. J. Clin. Invest., 45, (1966). 25) Salans, L. B. and Reaven, G. M.: Effect of insulin pretreatment on glucose and lipid metabolism of liver slices from normal rats, Proc. Soc. Exp. Biol. Med., 122, (1966). 26) Morita, H., Ohmichi, M., Inami, I. and Koike, S.: Plasma triglyceride turnover in cobalt-treated rabbits. Jap. J. Hyg. (1974) in press.