The influence of propranolol on catecholamine-induced changes in carbohydrate metabolism in the rabbit

European Journal of Pharmacology, 32 ( 1975 ) 186--194 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

THE INFLUENCE OF P R O P R A N O L O L ON CATECHOLAMINE-INDUCED CHANGES IN C A R B O H Y D R A T E METABOLISM IN THE RABBIT*,** JULIO MORATINOS***, DAVID E. POTTER and SYDNEY ELLIS

Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550, U.S.A Received 14 November 1974, revised MS received 25 February 1975, accepted 4 March 1975

J. MORATINOS, D.E. POTTER and S. ELLIS, The influence of propranolol on catecholamine-induced changes in carbohydrate metabolism in the rabbit, European J. Pharmacol. 32 (1975) 186--194. In vitro evidence has indicated that isoproterenol (ISO) is more potent than epinephrine (EPI) in releasing glucose from rabbit liver slices and that propranolol (PROP) is a competitive antagonist of ISO. In contrast, dose--response data from fasted rabbits have shown that EPI is more potent than ISO in increasing plasma glucose levels and in lowering hepatic glycogen levels. Pretreatment with PROP abolished ISO-induced hyperglycemia and changes in liver and muscle glycogen levels whereas only the muscle glycogen depleting effect of EPI was altered significantly. These results suggest that factors other than stimulation of hepatic ~-receptors must be involved in EPI-stimulated depletion of liver glycogen and hyperglycemia in the intact rabbit.

Tissue glycogen depletion Hyperglycemia

Epinephrine

Isoproterenol

1. Introduction McChesney et al. (1949) and Chen et al. (1951), reported that the order of hyperglycemia-producing potency of catecholamines by s.c. injection in conscious, fasted rabbit was 1-epinephrine > l-norepinephrine > d,l-isoproterenol. Recent work on rabbit liver slices from this laboratory (MiJhlbachovei et al., 1972) have raised some questions about the older reports on the order of potencies of catecholamines for producing hyperglycemia in rabbits. Miihlbachov~ et al. (1972 ) measuring glucose release from rabbit liver slices, found that the order of

* Presented in part at the meetings of the Federation of American Society for Experimental Biology, 1971. ** This work was supported in part by NIH Grant NS 07700. *** Present address: Departamento de Farmacologia, Facultad de Medicina, Valladolid, Spain.

Rabbit

Propranolol

potencies was 1-isoproterenol > 1-epinephrine > 1-norepinephrine. These in vitro data contradict the previously reported order of potencies in in vivo studies alluded to earlier. In addition, propranolol caused a parallel shift to the right of the log dose--response curves of both isoproterenol and norepinephrine, whereas phentolamine did not alter the epinephrine log dose-response curve. These in vitro results have strongly indicated that ~-receptors may be involved in catecholamine-induced glycogenolysis in rabbit liver. The present work was undertaken to examine the effects of 1-isoproterenol and 1-epinephrine on blood glucose and tissue glycogen levels in the intact rabbit and to determine if propranolol would antagonize these catecholamineinduced changes in carbohydrate homeostasis in vivo. In this way, we sought to examine possible reasons for the differences in the previously reported in vivo and in vitro p o t e n c y ratios for 1-isoproterenol and 1-epinephrine.

ADRENERGIC DRUGS AND GLUCOSE PRODUCTION 2. Materials and methods Unanesthetized New Zealand male rabbits weighing between 1.7 and 3.5 kg were fasted approximately 24 hr and then placed in well ventilated boxes. Rabbits were conditioned for restraint by placing them in boxes for a few hr every day during the two weeks preceding the experiment. Blood samples were taken from the central artery of the ear by means of an indwelling cannula. Two control samples were taken, one at 30 min and another immediately before drug treatment was started. Blood samples were collected at 15 min after the start of the infusion, at the end of the infusion (30 min) and then every half hour for an additional period of 2.5 hr. After s.c. injection of catecholamines, samples were taken at 30 min and at 1, 2, 3, 4 and 5 hr. For dose--response studies in comparative hyperglycemic potencies, 1-epinephrine and 1isoproterenol were either infused i.v. into the marginal vein of the contralateral ear or injected s.c. In the infusion experiments, a crossover design was employed with each animal serving as its own control and with an interval of one week between the infusion of saline (control) and each dose of either drug. Stock solutions of 1-epinephrine bitartrate and 1-isoproterenol • HC1 were prepared at a concentration of 1 mg/ml in acidified saline (pH = 4) for each experiment. Appropriate dilutions were made in 0.9% saline just before use. The duration of infusion was 30 min at a constant rote of 0.2 ml/min. In experiments with propranolol, 9 mg/kg was infused i.v. during a 30 min period just prior to the infusion of agonists. When catecholamines were injected s.c., a dose of propranolol, 9 mg/kg, was also injected s.c. 45 min preceding the injection of the agonist. The dose of each drug is expressed in terms of its free base. Plasma glucose was estimated by the modified m e t h o d of Hoffman (1937) using Technicon Autoanalyzer, or by the m e t h o d of Nelson (1944) with the copper reagent as modified by

187 Somogyi (1945). Liver and muscle samples for glycogen analysis were removed under pentobarbital anesthesia (30 mg/kg i.v.) an hour after the s.c. injection of either agonist. The upper left lobe of the liver and the left gastrocnemius muscle, in t h a t order, were quickly excised and placed in ice cold saline. The tissues were then prepared in a cold room at a temperature below 5°C. Tissue samples were trimmed and blotted on filter paper to remove excess moisture. A center strip of the left lobe of the liver was cut so that it weighed between 600 and 700 mg and a piece of gastrocnemius muscle was cut so that it weighed between 500 and 700 mg. The tissue samples were weighed on a torsion balance and then dropped into cold 30% KOH. The time for the entire process from the removal of the tissues to dropping them into 30% KOH for digestion was between 5 and 6 min. Glycogen was isolated and hydrolyzed according to the m e t h o d of Good et al. (1933) and the derived glucose analyzed by the method of Nelson and Somogyi as alluded to above.

3. Results

3.1. Hyperglycemic effects of l-epinephrine and l-isoproterenol infused i.v. The effects on plasma glucose levels of different doses of epinephrine and isoproterenol in fasted rabbits are compared with those seen after the infusion of saline in fig. 1. Epinephfine-induced hyperglycemic responses were linear to the log dose with the peak effect appearing at the end of the 30-min infusion period. Glucose levels gradually returned to the normal range so that 3 hr after the end of the infusion no differences were found between the blood sugar values after the infusion with epinephrine and after those with saline. The peak effects for the mean increases in blood sugar in fasted rabbits were 30 mg% at 0.1 pg/kg/min, 76 mg% at 0.3 pg/kg/min and 125 mg% at 1.0 pg/kg/min. When isoproterenol was infused, the peak effect also appeared at the end of the infusion

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ADRENERGIC DRUGS AND GLUCOSE PRODUCTION with complete recovery occurring within 3 hr as in the epinephrine studies. However, there was n o t a clear relationship between the magnitude of the response and the dose used, particularly at the higher concentrations. The maxim u m mean increase in glucose levels had a narrow range of 45--78 mg% in response to concentrations of isoproterenol ranging from 10 to 100 pg/kg/min i.v.

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3.2. Hyperglycemic responses to l-epinephrine and l-isopro terenol injected s. c. As shown in fig. 2, when epinephrine was injected s.c., the peak effect appeared between 1 and 3 hr, and the time to peak response was directly related to the dose. The magnitude of the peak responses in terms of change in blood glucose were 21, 59 and 189 mg%, at doses of 10, 30 and 100 pg/kg, respectively. The m a x i m u m increase in glucose levels after the s.c. injection of isoproterenol occurred between 2 and 3 hr. Doses of isoproterenol ranging from 10 to 300 pg/kg produced only small differences in the m a x i m u m response; increases in blood glucose ranged from about 25--60 mg%. The hyperglycemia produced by the highest dose (300 pg/kg) lasted the length of the experiment, whereas the hyperglycemias produced by doses of 100 pg/kg and lower doses returned to the control blood glucose level in 5 hr.

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administration; (b) The slopes of the curves are similar for the same drug injected by both routes of administration; ( c ) T h e spread between the two curves for epinephrine probably reflects a much slower absorption of epinephrine from the s.c. site due to vasoconstriction.

3.3. Comparison of dose-response curves to l-epinephrine and l-isoproterenol

3.4. Effects o f propranolol on catecholamineinduced hyperglycemias

Fig. 3 summarizes the dose--response curves plotted as the mean of the m a x i m u m changes in blood glucose in response to epinephrine and isoproterenol after intravenous infusion or subcutaneous injection. Epinephrine produced a well-defined dose--response relationship by both routes. In contrast, the dose--response curve to isoproterenol did n o t exhibit a clearcut discrimination between dose and effect. Three main features are apparent from these data: (a)1-Epinephrine is much more potent than 1-isoproterenol, regardless of the route of

For this study three groups of rabbits were used. The first group (propranolol--saline) received the ~-adrenergic antagonist, propranolol (0.3 mg/kg/min for 30 min), infused i.v. followed by an infusion of saline for 30 min. The second group (saline--catecholamine) received an infusion of saline for 30 min followed by an infusion of the catecholamine, either epinephrine (0.3 pg/kg/min) or isoproterenol (30 #g/kg/min) for 30 min. The infusion rates for the catecholamines were selected as those rates producing similar increases in plasma glucose.

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The third group (propranolol--catecholamine) was infused with propranolol for 30 min followed by one of the catecholamines for 30 min. A summary of the effects of propranolol on 1-epinephrine- and l-isoproterenol-induced hyperglycemias are depicted in fig. 4. Propranolol followed by a saline infusion produced small increases in plasma glucose (10--20 mg%) which could not be attributed solely to effects of the drug because changes of similar magnitude occurred in rabbits infused with only saline in other experiments. Pretreatment with propranolol produced a small, non-significant decrease in the peak effect evoked by epinephrine, however, a notably more rapid recovery of the blood sugar to the control level is evident in those animals pretreated with propranolol. Thus, 90 min after the end of the epinephrine infusion plasma glucose values had returned to the control range in rabbits previously infused with propranolol, whereas at that time the blood sugar values remained significantly higher (37 mg%) in those rabbits which received epinephrine alone. In marked contrast to the small antagonistic effect of propranolol on epinephrine-induced hyperglycemia, propranolol completely antagonized the isoproterenol-induced hyperglycemia (fig. 4).

3.5. Effects o f l-epinephrine and l-isoproterenol on liver and muscle glycogen In an attempt to better understand the differences in the hyperglycemic responses to epinephrine and to isoproterenol, the changes induced in liver and muscle glycogen by these catecholamines were investigated in fasted rabbits. In these studies the catecholamines were administered s.c. The rabbits were anesthetized with pentobarbital just prior to the 60-min blood sample and the removal of segments of liver and muscle for glycogen analyses. The changes in blood glucose and in the liver and muscle glycogen contents 1 hr after the s.c. injection of saline, epinephrine or isoproterenol are summarized in fig. 5. After the lower dose of epinephrine (0.1 mg/kg), blood glucose increased 80 mg% and muscle glycogen decreased 30%, but liver glycogen remained at the control level. The larger dose of epinephrine, 0.3 mg/kg, diminished markedly both liver and muscle glycogen;liver glycogen decreased 65% and muscle glycogen decreased 45%; blood glucose increased 122 mg%. A 45% reduction in muscle glycogen occurred after each of the three different doses of isoproterenol. Interestingly, the lowest dose of isoproterenol (0.1 mg/kg) induced a 65% in-

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crease in liver glycogen; no apparent change occurred with the intermediate dose (0.3 mg/kg), but the largest dose (1 mg/kg) produced a 40% decrease. These changes in tissue glycogen levels were accompanied by moderate hyperglycemias ranging from 30 mg% with the lowest dose to 60 mg% with the highest dose. LIVER

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For these experiments several groups of rabbits were required. Each of the animals in every group received a single s.c. injection of propranolol (9 mg/kg). 45 min after administration of propranolol the s.c. injections of individual groups were as follows: group one was treated with isoproterenol, 0.1 mg/kg; group two, isoproterenol, l mg/kg; group three, epinephrine, 0.1 mg/kg; group four, epinephrine, 0.3 mg/kg; group five, the control group, saline, 1 ml/kg. About 50 min after the catecholamine or saline injection the rabbits were anesthetized with pentobarbital and 10 min later blood samples and liver and muscle samples were obtained for determining the effects of propranolol on catecholamineinduced changes in blood glucose and tissue glycogen content• The results are represented graphically in fig. 6. Fig. 6 shows that propranolol alone did not exert significant influence upon the blood glucose concentration or liver and muscle glycogen. However, propranolol antagonized almost

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192

completely the hyperglycaemia and the decrease in muscle glycogen induced by 0.1 mg/kg isoproterenol. Pretreatment with propranolol partially antagonized the effect of isoo proterenol, 1.0 mg/kg, on muscle glycogen. In addition, propranolol prevented the increase in liver glycogen seen after the injection of isoprotereno!, 0.1 mg/kg, and abolished the reduction of liver glycogen observed with isoproterenol, 1 mg/kg. Fig. 6 also demonstrates the effect of pretreatment with propranolol on epinephrineinduced changes in tissue glycogen and plasma glucose levels. Whereas the muscle glycogendepleting effect of epinephrine was abolished by propranolol, the reduction in liver glycogen and the hyperglycemic response were not suppressed. Interestingly, ~-receptor blockade with propranolol enhanced the glycogenolytic effect of epinephrine on the liver. The latter effect must be considered in relation to the simultaneous changes, or lack thereof, in muscle glycogen in the absence or presence of propranolol.

4. Discussion These data confirm the previous reports of McCesney et al. (1949) and Chen et al. (1951) regarding the hyperglycemic potencies of epinephrine and isoproterenol in vivo in the rabbit. Likewise, the difference in the generation of glucose by isoproterenol in vitro (Miihlbachov~i et al., 1972) versus in vivo experiments was re-emphasized by this study. Hyperglycemic responses to increasing concentrations of epinephrine were clearly dose-related whether the catecholamine was administered by the s.c. route or by constant i.v. infusion. In contrast, increasing concentrations of isoproterenol produced a very flat dose--response curve indicative of the limited hyperglycemic activity of isoproterenol in the unanesthetized rabbit. The marked divergence in the potencies of epinephrine and isoproterenol in the rabbit probably reflects differences in activity at several organs that are important in glucose ho-

J. M O R A T I N O S E T AL.

meostasis. Since catecholamines appear to have multiple mechanisms involved in producing their hyperglycemic effect (Himms-Hagen, 1967), the relative importance of the contribution of liver and muscle glycogen to the catecholamine-induced hyperglycemia was evaluated in the absence and presence of propranolol. Our experimental results may be construed to mean that the most probable mechanisms for the production of hyperglycemia at lower doses of epinephrine involve: (1) increased gluconeogenesis from lactate (Cori, 1931) and (2) decreased peripheral utilization of glucose (Henneman and Shoemaker, 1961). Since there was no significant decrease in liver glycogen stores following low doses of epinephrine, it is assumed that either glucose release from the liver was not a significant contributing factor to the hyperglycemic response or that glucose release from the liver as measured by changes in glycogen content was buffered by gluconeogenesis from various metabolic substrates, particularly lactate from muscle. Epinephrine promoted significant liver glycogenolysis at higher doses and this effect is presumed to account for the further increase in plasma glucose that was observed. Intensification of liver glycogenolysis by low doses of epinephrine s.c., in the presence of propranolol, may reflect again the suppression of available glycogenic substrates (lactate, glycerol and amino acids) to the liver by the ~-blocking drug. In addition, the elimination of epinephrine-induced gluconeogenesis by propranolol may also explain the more rapid return of plasma glucose levels to control values following the i.v. administration of the catecholamine. A similar enhancement of epinephrine-induced hepatic glycogenolysis in mice in the presence of propranolol has been reported by Lundquist (1972). Failure of propranolol to reduce the peak elevation of plasma glucose after epinephrine i.v. may be related to glycogenolysis in the liver which is influenced indirectly by release of glucagon (Ezdinli and Sokal, 1966; Gerich et al., 1972; Taylor and Kajinuma, 1972) and augmented by the estabfished ability of epinephrine to inhibit the

ADRENERGIC DRUGS AND GLUCOSE PRODUCTION

release of insulin (Coore and Randle, 1964; Porte, 1967a). The limited ability of propranolol to suppress epinephrine-induced hyperglycemia has also been reported in man (Porte, 1967b) and in mice (Mennear et al., 1971; Lundquist, 1972). In an in vitro system, such as rabbit liver slices, only the direct effect of epinephrine on liver glycogen stores would be observed and this can be readily blocked by propranolol (Muhlbachova et al., 1972). The ability of propranolol to inhibit the glycogenolytic effect of epinephrine on the liver in vivo could be due to insufficient concentration of the ~-blocker within hepatic tissue (Newton and Hornbrook, 1972), however, it is interesting to note that the effects of high doses of isoproterenol on liver glycogen levels were completely negated. The proposal that an ~adrenergic mediated glycogenolysis in liver may be accentuated by ~-adrenergic blockade may explain the in vivo effect of epinephrine (Pinter et al., 1967). Only a dose of isoproterenol which approached toxic levels produced a significant reduction of liver glycogen levels. In contrast, smaller doses of isoproterenol caused an elevation in hepatic glycogen content; this observation suggests that either mobilization of glucogenic substrates to the liver and increased glucose production are occurring without appreciable loss of hepatic glycogen or that the concurrent production of hyperglycemia and release of insulin are resulting in increased hepatic glucose uptake and conversion to glycogen. Since the blockade of muscle glycogenolysis by propranolol occurred concomitantly with the elimination of isoproterenolinduced hyperglycemia, it is suggested that conversion of lactate to glucose in the liver (Exton et al., 1966) may account for the major portion of the change in plasma glucose following administration of isoproterenol. Porte (1967b) previously reported that propranolol could block the hyperglycemia induced by isoproterenol in human subjects. Recent evidence would also implicate the capability of isoproterenol to stimulate the release of insulin (Porte, 1967b; Malaisse et al.,

193

1967a,b; Potter et al., 1972). Insulin released into the portal system of the liver would tend to oppose any glycogenolytic effect of isoproterenol on the liver by lowering hepatic levels of cyclic AMP and by increasing the activity of hepatic glycogen synthetase (Exton and Park, 1968; Madison, 1969; Villar-Palasi et al., 1971). Interestingly, elimination of acute insulin release in rabbits produced an augmented hyperglycemic response to 1-isoproterenol whereas the response to 1-epinephrine did not change (Potter et al., 1972). These data give added support to the suggestion that the release of insulin by isoproterenol in normal rabbits obtunds the increased hepatic output of glucose which occurs in the absence of insulin release shown in diabetic rabbits. Moreover, recent evidence demonstrated that indeed isoproterenol does elevate insulin levels in the rabbit (Moratinos et al., 1972). Although the doses of propranolol employed in our work were large by comparison with the doses customarily used in other types of experiments, it should be pointed out that the doses of isoproterenol required to achieve elevations in blood glucose levels can be considered inordinately high as well. Also, it was considered necessary to expose the liver to as much propranolol as possible without producing overt signs of toxicity. The inability of propranolol to block glucagon-induced hyperglycemia attests to the fact that the integrity of the liver to respond to stimulation was not impaired by the high doses of propranolol (Potter et al., 1974). References Chen, G., R. Portman, D. Russell and C.R. Ensor, 1951, Comparative pharmacology of arterenol, epinephrine, and isopropylarterenol, J. Amer. Pharm. Assoc. 40, 273. Coore, H.G. and P.J. Randle, 1964, Regulation of insulin secretion studied with pieces of rabbit pancreas incubated in vitro, Biochem. J. 93, 66. Cori, C.F., 1931, Mammalian carbohydrate metabolism, Physiol. Rev. 11,143. Exton, J.H., L.S. Jefferson, R.W. Butcher and C.R. Park, 1966, Gluconeogenesis in the perfused liver.

194 The effects of fasting, alloxan diabetes, glucagon, epinephrine, adenosine 3',5'-monophosphate and insulin, Amer. J. Med. 4 0 , 7 0 9 . Exton, J.H. and C.R. Park, 1968, The role of cyclic AMP in the control of liver metabolism, Advan. Enzyme Regulation 6, 391. Ezdinli, E.Z. and J.E. Sokal, 1966, Comparison of glucagon and epinephrine effects in the dog, Endocrinology 78, 47. Gerich, J.E., J.H. Karam and P.H. Forsham, 1972, Reciprocal adrenergic control of pancreatic ~- and fl-cell function in man, (Abstract) Diabetes 21, 332. Good, C.G., H. Kramer and M. Somogyi, 1933, The determination of glycogen, J. Biol. Chem. 100, 485. Henneman, D.H. and W.C. Shoemaker, 1961, Effect of glucagon and epinephrine on regional metabolism of glucose pyruvate, lactate and citrate in normal, conscious dogs, Endocrinology 68, 889. Himms-Hagen, J., 1967, Sympathetic regulation of metabolism, Pharmacol. Rev. 1 9 , 3 6 7 . Hoffman, W.S., 1937, A rapid photoelectric method for the determination of glucose in blood and urine, J. Biol. Chem. 120, 51. Lundquist, I., 1972, Interaction of amines and aminergic blocking agents with blood glucose regulation. I. fl-Adrenergic Blockade, European J. Pharmacol. 18, 213. Madison, L.L., 1969, Role of insulin in the hepatic handling of glucose, Arch. Intern. Med. 123, 284. Malaisse, W., F. Malaisse-Lagae, P.H. Wright and J. Ashmore, 1967a, Effects of adrenergic and cholinergic agents upon insulin secretion in vitro, Endocrinology 80, 975. Malaisse, W.J., F. Malaisse-Lagae and D. Mayhew, 1967b, A possible role for adenyl cyclase in insulin secretion, J. Clin. Invest. 46, 1724. McChesney, E.W., J.P. McAuliff and H. Blumberg, 1949, The hyperglycemic actions of some analogs of epinephrine, Proc. Soc. Exptl. Biol. Med. 71, 220. Mennear, J.H., G.R. Spratto and T.S. Miyo, 1971, Failure of beta'-adrenergic blocking agents to antagonize the hyperglycemic effect of epinephrine in mice, Proc. Soc. Exptl. Biol. Med. 137, 88.

J. MORATINOS ET AL. Moratinos, J., D.E. Potter and S. Ellis, 1972, Metabolic effects of catecholamines in normal and alloxandiabetic rabbits, Fifth International Congress on Pharmacology, Abstract of Volunteer Papers, p. 161. M/ilbachov~i, E., P.S. Chan and S. Ellis, 1972, Quantitative studies of glucose release from rabbit liver slices by catecholamines and their antagonism by propranolol and phentolamine, J. Pharmacol. Exptl. Therap. 1 8 2 , 3 7 0 . Nelson, N., 1944, A photometric adaptation of the Somogyi method for the determination of glucose, J. Biol. Chem. 153, 375. Newton, N.E. and K.R. Hornbrook, 1972, Effects of adrenergic agents on carbohydrate metabolism of rat liver: Activities of adenyl cyclase and-glycogen phosphorylase, J. Pharmacol. Exptl. Therap. 181, 479. Pinter, E.J., C. Pattee, G. Petergy and J.M. Kleghorn, 1967, Metabolic effects of autonomic blockade, Lancet 2 , 1 0 1 . Porte, Jr., D., 1967a, A receptor mechanism for the inhibition of insulin release by epinephrine in man, J. Clin. Invest. 46, 86. Porte, Jr., D., 1967b, Beta adrenergic stimulation of insulin release in man, Diabetes 16, 150. Potter, D., J. Moratinos and S. Ellis, 1972, Isoproterenol- and epinephrine-induced hyperglycemias in rabbits: Effects of alloxan treatment and prandial state, Proc. Soc. Exptl. Biol. Med. 139, 1242. Potter, D.E., J. Moratinos and S. Ellis, 1974, Comparative hyperglycemic responses to norepinephrine, salbutamol and glucagon in normal and alloxan-diabetic rabbits, Arch. Intern. Pharmacodyn. 209, 358. Somogyi, M., 1945, Determination of blood sugar, J. Biol. Chem. 160, 69. Tayler, J.M. and H. Kajinuma, 1972, Influence of beta-adrenergic and cholinergic agents in vivo on pancreatic glucagon and insulin secretion (Abstract) Diabetes 2 1 , 3 3 2 . Villar-Palasi, C., J. Lamer and L.C. Shen, 1971, Glycogen metabolism and the mechanism of the action of cyclic AMP, Ann. N.Y. Acad. Sci. 185, 74.