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Some explanations for species differences in hyperglycemic responses to adrenergic agents
Life Sciences, Vol . 22, pp . 1229-1236 Printed in the U .S .A .
Pergamon Press
SOME EXPLANATIONS FOR SPECIES DIFFERENCES IN HYPERGLYCEMIC RESPONSES TO ADRENERGIC AGENTS Sydney Ellis Department of Pharmacology and Toxicology University of Texas Medical Branch Galveston, Texas 77550
SUMMARY There are species differences in the metabolic responses to catecholamines and their antagonists . In one group (dog, cat) isoproterenol equals or surpasses the potency of epinephrine for increasing plasma glucose and propranolol and other ß-adrenergic antagonists in moderate doses suppress these responses . In the second group (rat, rabbit, baboon, human) isoproterenol is considerably less potent than epinephrine for increasing plasma glucose and for reducing liver glycogen and the ß-adrenergic antagonists are poor inhibitors of epinephrine-induced hyperglycemia or liver glycogen depletion . In all these species isoproterenol is more potent than epinephrine for increasing muscle glycogenolysis and plasma lactic acid and these effects are sensitive to inhibition by ß-adrenergic antagonists . The adrenergic receptors involved in the activation of hepatic glucose release by epinephrine in rats and rabbits differ from those in the dog . In rats and rabbits evidence has been obtained which implicates the release of insulin by isoproterenol as an important factor in limiting the hyperglycemic response to isoproterenol . Alloxan-diabetic rats and rabbits, radioimmunoassay of insulin and glucagon, and the use of somatostatin as an inhibitor of glucagon and insulin release have been applied to produce the evidence which implicates glucagon release as a large factor in the epinephrine-induced hyperglycemia and hepatic glycogenolysis in the rat, rabbit and baboon, but not in the dog . A relatively simple investigation many years ago of the structure-activity-relationship for the epinephrine-induced hyperglycemic response in the rat produced data that raised a series of broad questions, some of which continue to be challenging . The first unexpected finding was that isoproterenol in the fed rat produced no increase in blood glucose (1) . When sufficient species were investigated, it became evident that there 0300-9653/78/0410-1229$02 .00/0 Copyright © 1978 Pergamon Press
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were two types of responses . In a few species (dog(2), cat(3)) isoproterenol is a quite potent hyperglycemic agent and may be more potent than epinephrine ; however, in several species (rat, rabbit, monkey, baboon, man) isoproterenol proves to be a much less potent agent than epinephrine (2) . The explanation for these species differences continues to be a challenge despite the substantial progress that has been made . ADRENERGIC RECEPTOR TYPES MEDIATING HYPERGLYCEMIA A question that was raised early related to the type of receptor involved in the hyperglycemic response to catecholamines . That this answer was sought long before the discovery of suitable ß-adrenergic antagonists led to some interesting, but puzzling observations . The only generally effective antagonist for epinephrine-hyperglycemia in many species was ergotamine, which is pharmacologically classified as an a-adrenergic antagonist . Ergotamine and dihydroergotamine are effective in the rat, but most other a-adrenergic antagonists were ineffective (3) . In addition, ephedrine was found to be a poor agent for producing hyperglycemia in the rat, but it was quite effective for antagonizing epinephrine-hyperglycemia (1) . The introduction of the ß-adrenergic antagonists into this area of research has further emphasized the characteristic species differences in epinephrine-hyperglycemia . The ß-adrener gic antagonists are very effective for inhibiting epinephrinehyperglycemia in some species (dog, cat), but in other species (mouse, rat, rabbit, baboon, man) their antagonistic effect is, at best, limited (4) . It is interesting that it is in those species in which isoproterenol is a potent hyperglycemia agent that the ß-adrenergic antagonists are effective inhibitors of epinephrine-hyperglycemia, whereas, in those species in which isoproterenol is a poor hyperglycemic agent, the ß-antagonists are ineffective . Indeed, there have been periods when it was proposed that determining the characteristics of the adrenergic receptor mediating glycogenolysis and glucose production in the liver would be all that is needed to explain the species differences . When the question of the type of adrenergic receptor was attacked more directly by experiments in vitro on rabbit liver slices (5), the results were quite unexpect~in view of the results in the intact rabbit . In rabbit liver slices, glucose production was increased by the catecholamines with the order of relative potencies characteristic of the ß-adrenergic receptor ; that is, isoproterenol is more potent than epinephrine which, in turn, is more potent than norepinephrine . The receptor in the rabbit liver has the further essential properties of a ß-adrenergic receptor, namely, it was not inhibited by the aadrenergic antagonist, phentolamine, but was relatively sensitive to inhibition by propranolol . Noteworthy in the propranolol results was that the observation that a ten-fold increase in propranolol concentration displaced to the right the concentration-response curves of norepinephrine or isoproterenol only about a half log unit rather than a full log unit expected in the simplest case in which one agonist molecule and one antagonist molecule compete for a receptor site . The kinetics of
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this interaction may indicate that to produce sufficient antagonism in the liver an excessively large dose of propranolol may be required in the intact rabbit which is unable to tolerate propranolol in a dose greater than 10 mg/kg . The rat liver presents a more complex adrenergic receptor system . The activation of adenyl cyclase in the rat liver shows the responses to catecholamines and adrenergic antagonists con sistent with the involvement of a ß-receptor (6,7), but the activation of phosphorylase may occur through this ß-receptor system (the only one activated by isoproterenol) or through another system (also activated by epinephrine or other a-agoniste) which does not involve cyclic AMP and which is insensitive to inhibition by propranolol and by most a-adrenergic antagonists, but is sensitive to inhibition by dihydroergotamine . The demonstrated actions of isoproterenol which increase cyclic AMP and phosphorylase do not appear to result in large increases in hepatic glucose production through either glycogenolysis or gluconeogenesis . The fact that isoproterenol in vivo may inhibit its own effect, or those of epinephrine and glucagon, on phosphorylase activation has been attributed to the production of an inhibitory metabolic product (6), but the demonstration that this phenomenon does not occur in the alloxan-diabetic rat (8) suggests that it is the release of insulin by isoproterenol which suppresses epinephrine-induced hyperglycemia in the normal rat . In other species direct or indirect evidence indicates that liver glucose production can be increased by an effect on aadrenergic receptors in addition to an effect on ß-adrenergic receptors . Evidence for an a-receptor mediated effect is particularly well demonstrated in the guinea pig liver (9) . The response is produced by several selective a-agonists and inhibIn the cat liver also there ited by selective a-antagonists . appears to be an a-receptor system activating glucose release in addition to the ß-adrenergic receptor system (10) . The adrenergic antagonists have been helpful in analyzing the mechanisms of the metabolic responses to the catecholamines . The results obtained with adrenergic antagonists have been quite important in defining the types of receptors involved in certain responses, but some of the data obtained has only made it more evident that much further research is necessary . The first of the so called "ß-blockers" demonstrated in the . dog (14) that the adrenergic receptors involved in increasing plasma glucose, lactic acid and free fatty acids are clearly ß adrenergic receptors . The later use of the "pure" ß-adrenergic antagonists have supported these early results and have added the catecholamine-induced increase in oxygen consumption (15) to the liver, muscle and adipose tissue effects which are "ß-receptor functions" . The adrenergic receptors for metabolic responses in the cat also appeared to involve only ß-adrenergic receptors (16), but a recent report contained evidence for an a-receptor mechanism, as well as the ß-receptors, for increasing hepatic glucose production (10) . As indicated above, the definition of the receptors controlling the hyperglycemic response and liver glycogen depletion in rats, rabbits, baboons and humans is less certain . A review of the findings in these latter species will define th~a pertinent problems .
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The metabolic responses which define the characteristics of the group of animals which includes rats, rabbits, baboons and humans are that these species are relatively resistant to the hyperglycemic response to isoproterenol in contrast to their sensitivity to epinephrine-induced hyperglycemia and in these species the epinephrine-induced increase in blood sugar is resistant to blockade by ß-adrenergic antagonists . The resistance of epinephrine-hyperglycemia to inhibition by the available ß-adrenergic antagonists must be examined in detail . The published data is quite clear in demonstrating that the ß-adrenergic antagonists, at dose levels which suppress cardiovascular ß-adrenergic receptor responses and muscle glycogenolysis, do not produce much inhibition of the hyperglycemic response to epinephrine . Nonetheless, it has been claimed that a high dose of a "ß-blocker" in the rat can suppress epinephrinehyperglycemia . When maximally tolerated doses of propranolol were tested in rats (17) and rabbits (13) and a very large dose of sotalol in rats (17), there was indeed considerable inhibition of the epinephrine-induced hyperglycemia, but the responsé was certainly not completely suppressed . Measurement of the glycogen changes in liver and muscle made it clear that the ß-adrenergic antagonists prevented muscle glycogenolysis and thus reduced the supply of lactate and other substrates for hepatic gluconeogenesis in rats (17) and rabbits (13), but that even the highest levels of the ß-antagonists did not reduce the epinephrineinduced depletion of hepatic glycogen . In the rabbit, pretreatment with propranolol produced complex hepatic effects ; the glycogen-depleting effect of epinephrine was potentiated and the biphasic response to isoproterenol (an increase in hepatic glycogen at lower doses and a reduction at higher doses of isoproterenol) was completely suppressed so that no changes in hepatic glycogen occurred when isoproterenol was administered over the same range of doses (13) . When it was found that the common ß-adrenergic antagonists has only a limited antagonism against epinephrine-induced hyperglycemia and liver glycogen depletion, the old observation (1) that ephedrine was a good antagonist against epinephrine-hyperglycemia in the rat suggested that ephedrine might be a new type of antagonist . When ephedrine was explored in more detail (18) as a potential antagonist, it was found, unhappily, that the antagonistic effect of ephedrine was quite like that of propranolol ; ephedrine inhibited the depletion of muscle glycogen by epinephrine, but did not inhibit the liver glycogen depletion better than did propranolol . Apparently the ethanolamine side chain is essential for this action of ephedrine since amphetamine (18) was ineffective as an epinephrine antagonist . Further exploration of the mechanism of antagonism of epinephrine-induced hyperglycemia by dihydroergotamine has demonIn the strated that this antagonist has interesting effects . intact rat it inhibited epinephrine-induced hyperglycemia and liver glycogen depletion, but did not inhibit muscle or heart glycogenolysis (3) . The action in the liver was found to be selective since both glucagon and cyclic adenosine-3',5'-phosphate decreased hepatic glycogen in the presence of a dose of dihydroergotamine which inhibited the hepatic action of epinephrine (19) . In a study of the site of the antagonistic effect
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of dihydroergotamine on rabbit liver slices (20) it was found that dihydroergotamine antagonized epinephrine non-competitively, but there was no antagonism against glucagon or cyclic AMP at a concentration of dihydroergotamine markedly antagonizing epinephrine . Although propranolol has not been a completely effective epinephrine antagonist in rats and rabbits, the effects of propranolol as an antagonist against isoproterenol have been those expected of a non-selective ß-adrenergic antagonist interacting with a "pure" ß-adrenergic agonist . The antagonist inhibited or suppressed the hyperglycemia-producing effect of isoproterenol in alloxan-diabetic rats (21) and in normal and alloxan-diabetic rabbits (13,22) . Since the hyperglycemic effect of isoproterenol is accompanied by a large reduction in muscle glycogen but not a concomitant change in liver glycogen, the suppression of the isoproterenol-induced increase in blood glucose may be related to the suppression of the input of substrates for hepatic gluconeogenesis by muscle and other tissues . It is also pertinent than ethanol and 3-mercaptopicolinic acid, two substances which reduce lactic acid use in gluconeogenesis, suppressed the isoproterenol-induced hyperglycemias in fasted normal rats and in alloxan-diabetic rats while not greatly modifying the epinephrine-induced hyperglycemias (23) . IMPORTANCE OF INSULIN SECRETION The results of the rabbit liver slice experiments indicated that isoproterenol should be~inore potent than epinephrine in the activation of glucose production . It is also known that isoproterenol is more effective in vivo for increasing plasma lactic acid and glycerol, both excél~ent substrates for hepatic gluconeogenesis (11) . Why, then, is isoproterenol a weak hyperglycemic agent in the rabbit (and also in several other species, including man)7 One proposed explanation for the marked differences in the hyperglycemic effects of epinephrine and isoproterenol in some animals involves the fact that these two catecholamines produce opposite effects on insulin release from the pancreatic islet cells (12) . It is now well established that epinephrine, through a-adrenergic receptors, inhibits insulin release, whereas isoproterenol, through its action on ß-adrenergic receptors, markedly increases insulin release . Since insulin opposes adrenergic effects on glucose metabolism in liver, muscle and other tissues, the release of insulin by isoproterenol might be sufficiently effective in opposing the direct effect of isoproterenol on the liver (and possibly on other tissues) to modulate the hyperglycemic response to isoproterenol . In support of this proposal are the observations in rats (3) and rabbits (13) that isoproterenol, in comparison with epinephrine, has little effect on liver glycogen but a more potent effect on muscle glycogen . This hypothesis has been tested extensively in rabbits and rats by indirect and direct methods . An indirect method for determining the importance of insulin release to explain the differences in the hyperglycemic responses to epinephrine and isoproterenol is to limit the re lease of insulin . This was done by producing alloxan-diabetic
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animals which could be controlled with a daily dose of protamine zinc insulin . Studies have been done with alloxan-diabetic rata and rabbits . A more direct method is to use a radioimmunoassay procedure to determine the changes produced in plasma pancreatic hormones under the influences of the catecholamines and other procedures . In the fed alloxan-diabetic rat isoproterenol was a more potent hyperglycemic agent than epinephrine, whereas in the fed normal rat epinephrine-hyperglycemia was increased but isoproterenôl produced no increase in blood sugar (8) . The hyperglycemic response to epinephrine was essentially the same in the diabetic as in the normal rat . The diabetic state in the rabbit also potentiated the hyperglycemic responses to a wide range of doses of isoproterenol, but did not modify the response to epinephrine (12) . The direct measurement of changes in plasma immunoreactive insulin after a range of doses of isoproterenol in fed and in fasted rats indicated that isoproterenol induced a dose-related increase in insulin with a significantly greater increase in fed rats than in fasted rats (8) . These data again supported the concept that insulin release by isoproterenol minimized its total effect on blood sugar . The fact that the fed rats had higher plasma insulin than fasted rats at each dose of isoproterenol was relatable to the complete absence of a rise in blood glucose in response to isoproterenol in the fed rat while a modest increase in blood glucose occurred in the fasted rat . There is compelling evidence now that the opposite effects of isoproterenol and epinephrine on insulin release must play a major role in the differences observed in those animals in which isoproterenol is a much poorer hyperglycemic agent than epinephrine . There are, albeit, many additional questions which must be explored . IMPORTANCE OF GLUCAGON SECRETION Glucagon release was proposed many years ago (24) as the indirect mechanism through which epinephrine caused hepatic glycogenolysis and hyperglycemia . This hypothesis has been examined with two general approaches : (1) by determining the changes in plasma immunoreactive glucagon (and insulin) induced by epinephrine and isoproterenol in normal rats, rabbits, baboons and dogs and by isoproterenol in diabetic rats and rabbits ; and (2) by administering somatostatin (SRIF) to inhibit pancreatic hormone release in order to determine whether inhibition of glucagon release reduced catecholamine-induced hyperglycemia . Somatostatin is an interesting analytical tool for explorations in this area . It completely suppressed catecholamine-induced glucagon-release and markedly reduced insulin-release without interfering with the catecholamine-induced increases in plasma lactate and free fatty acids . In rats, rabbits, dogs and baboons epinephrine was much more potent than isoproterenol in raising plasma glucagon levels . Part of this difference may be related to the known suppression of glucagon release by insulin which is released by isoproterenol . This may also be the explanation for the amplified plasma glucagon
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response to isoproterenol in diabetic animals . Furthermore, the increased hyperglycemic response to isoproterenol in diabetic animals is suppressed by pretreatment with propranolol or with somatostatin, both of which inhibit release of glucagon . These results strongly implicate glucagon in the hyperglycemic responses to catecholamines in certain species (25) . in the dog somatostatin prevented the increase in plasma glucagon in response to epinephrine and to isoproterenol without inhibiting the hyperglycemic responses (26) . The results in the rat were quite different . Somatostatin suppressed the elevation of plasma glucagon by epinephrine in normal rats and by isoproterenol in diabetic rats and also greatly inhibited the hyperglycemic responses (26) . Somatostatin also greatly depressed the hyperglycemia and the hyperglucagonemia in response to epinephrine in the baboon (27) . These observations show that glucagon release is a major factor in catecholamine-induced hyperglycemia in the rat and the baboon but in the dog glucagon release is of little significance in the hyperglycemic responses . CONCLUSIONS The species differences in the metabolic responses to catecholamines has defined characteristics . Isoproterenol has a much less potent hyperglycemic effect than epinephrine in rats, rabbits, baboons and humans, but in dogs and cats isoproterenol is as potent as, or more potent than, epinephrine . In the species in which isoproterenol is the poorer hyperglycemic agent, it also has less influence on liver glycogen and, in these species, propranolol and other common ß-adrenergic antagonists have a limited effect against epinephrine-induced hyperglycemia and hepatic glycogen depletion . The release of insulin by isoproterenol is an important factor which limits the effect of isoproterenol on blood glucose and hepatic glycogen in the rat and the rabbit . Glucagon release, in the normal or diabetic rat and rabbit and in the baboon, appears to play a considerable role in the catecholamine-induced hyperglycemias, whereas in the dog glucagon release is of minor importance . ACKNOWLEDGEMENT The research accomplished in the author's laboratories was largely supported by grants from the National Institutes of Health . A grant from the Smith Kline & French Laboratories, Philadelphia, supported principally the research on~baboons and monkeys . REFERENCES 1. 2. 3.
4.
S . ELLIS and H . L . ANDERSON, J . Pharmacol . exp . Ther . _101 92-100 (1951) . S . ELLIS, Ph siolo ical Pharmacolo Academic Press, New Yor 1967 .
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B . L . KENNEDY and S . ELLIS, Archs int . Pharmacodyn . Thér . 177 390-406
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. S ELLIS, B . L . KENNEDY, A . J . EUSEBI, and N . H . VINCENT, Ann . N . Y . Aced . Sci . 139(3) 826-832 (1967) .
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E . MUHLHACHOVÂ, P . S . CHAN, and S . ELLIS, J . Pharmacol . exp . Ther . 18 2 370-377 (1972) . N . E . NEWTON ând K . R . HORNBROOK, J . Pharmacol . exp . Ther . 18 1 479-488 (1972) . .E . M . TOLBERT, F . R . BUTCHER, and J . N . FAIN, J . Biol . M Chem . 248 5686-5692 (1973) . D . E . PÔTTER and S . ELLIS, J . Pharmacol . exp . Ther . 19 3 576-584 (1975) . D . G . HAYLETT and D . H . JENKINSON, J . Physiol . (London) 225 751-772 (1972) . .H . KUO, J . R . KAMAKA, and B . K . B . LUM, J . Pharmacol . S exp . Ther . 20 2 301-309 (1977) . D . E . POTTER,J . MORATINOS, and S . ELLIS, Biochem . Pharma cology 26 1065-1069 (1977) . D . E . POTTER, J . MORATINOS, and S . ELLIS, Proc . Soc . exp . Biol . Med . 13 9 1242-1245 (1972) . J . MORATINOS,D . E . POTTER, and S . ELLIS, Europ . J . Pharmacol . 32 186-194 (1975) S. E . MAYER, N . C . MORAN, and J . FAIN, J . Pharmacol . exp . Ther . 13 4 18-27 (1961) . B . L . KÉNNEDY and S . ELLIS, J . Pharmacol . exp . Ther . _l68 137-145 (1969) . A . J . EUSEBI and S . ELLIS, Pharmacologist 7 136 (1965) . H . GOTHELF and S . ELLIS, Proc . Soc . exp . Biol . Med . 14 7 259-262 (1974) . S . ELLIS, Actual .pharmacol . 25 91-114 (1972) . B . GOTHELF and S . ELLIS, V Int. Cong . Pharmacol . Abst . p . 8 6 (1972) . P . S . CHAN and S . ELLIS, Fedn Proc . 28 742 (1969) . D . E . POTTER, L . M . WILSON, a~n S - ELLIS, Proc . Soc . exp . Biol . Med . 15 4 337-340 (1977) . J . MORATINOS, S . ELLIS, and D . E . POTTER, Pharmacology 15 407-414 (1977) . D. E . POTTER, Bîochem . Pharmacol . _26 2231-2235 (1977) . J . E . SORAL and E . J . SARCIONE, Nature, Lond . _2O4 881-883 (1964) . L . C . WOODSON, J . W. BARNETT, and D . E . POTTER, Pharmacologist 18 248 (1976) . L . C . WOODSON, J . W . BARNETT, and D. E . POTTER, Fedn Proc . 36 339 (1977) . L. C . WOODSON, D . E . POTTER, R . R . REMPEN, and S . ELLIS, Fedn Proc . 35 688 (1976) .
Pergamon Press
SOME EXPLANATIONS FOR SPECIES DIFFERENCES IN HYPERGLYCEMIC RESPONSES TO ADRENERGIC AGENTS Sydney Ellis Department of Pharmacology and Toxicology University of Texas Medical Branch Galveston, Texas 77550
SUMMARY There are species differences in the metabolic responses to catecholamines and their antagonists . In one group (dog, cat) isoproterenol equals or surpasses the potency of epinephrine for increasing plasma glucose and propranolol and other ß-adrenergic antagonists in moderate doses suppress these responses . In the second group (rat, rabbit, baboon, human) isoproterenol is considerably less potent than epinephrine for increasing plasma glucose and for reducing liver glycogen and the ß-adrenergic antagonists are poor inhibitors of epinephrine-induced hyperglycemia or liver glycogen depletion . In all these species isoproterenol is more potent than epinephrine for increasing muscle glycogenolysis and plasma lactic acid and these effects are sensitive to inhibition by ß-adrenergic antagonists . The adrenergic receptors involved in the activation of hepatic glucose release by epinephrine in rats and rabbits differ from those in the dog . In rats and rabbits evidence has been obtained which implicates the release of insulin by isoproterenol as an important factor in limiting the hyperglycemic response to isoproterenol . Alloxan-diabetic rats and rabbits, radioimmunoassay of insulin and glucagon, and the use of somatostatin as an inhibitor of glucagon and insulin release have been applied to produce the evidence which implicates glucagon release as a large factor in the epinephrine-induced hyperglycemia and hepatic glycogenolysis in the rat, rabbit and baboon, but not in the dog . A relatively simple investigation many years ago of the structure-activity-relationship for the epinephrine-induced hyperglycemic response in the rat produced data that raised a series of broad questions, some of which continue to be challenging . The first unexpected finding was that isoproterenol in the fed rat produced no increase in blood glucose (1) . When sufficient species were investigated, it became evident that there 0300-9653/78/0410-1229$02 .00/0 Copyright © 1978 Pergamon Press
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were two types of responses . In a few species (dog(2), cat(3)) isoproterenol is a quite potent hyperglycemic agent and may be more potent than epinephrine ; however, in several species (rat, rabbit, monkey, baboon, man) isoproterenol proves to be a much less potent agent than epinephrine (2) . The explanation for these species differences continues to be a challenge despite the substantial progress that has been made . ADRENERGIC RECEPTOR TYPES MEDIATING HYPERGLYCEMIA A question that was raised early related to the type of receptor involved in the hyperglycemic response to catecholamines . That this answer was sought long before the discovery of suitable ß-adrenergic antagonists led to some interesting, but puzzling observations . The only generally effective antagonist for epinephrine-hyperglycemia in many species was ergotamine, which is pharmacologically classified as an a-adrenergic antagonist . Ergotamine and dihydroergotamine are effective in the rat, but most other a-adrenergic antagonists were ineffective (3) . In addition, ephedrine was found to be a poor agent for producing hyperglycemia in the rat, but it was quite effective for antagonizing epinephrine-hyperglycemia (1) . The introduction of the ß-adrenergic antagonists into this area of research has further emphasized the characteristic species differences in epinephrine-hyperglycemia . The ß-adrener gic antagonists are very effective for inhibiting epinephrinehyperglycemia in some species (dog, cat), but in other species (mouse, rat, rabbit, baboon, man) their antagonistic effect is, at best, limited (4) . It is interesting that it is in those species in which isoproterenol is a potent hyperglycemia agent that the ß-adrenergic antagonists are effective inhibitors of epinephrine-hyperglycemia, whereas, in those species in which isoproterenol is a poor hyperglycemic agent, the ß-antagonists are ineffective . Indeed, there have been periods when it was proposed that determining the characteristics of the adrenergic receptor mediating glycogenolysis and glucose production in the liver would be all that is needed to explain the species differences . When the question of the type of adrenergic receptor was attacked more directly by experiments in vitro on rabbit liver slices (5), the results were quite unexpect~in view of the results in the intact rabbit . In rabbit liver slices, glucose production was increased by the catecholamines with the order of relative potencies characteristic of the ß-adrenergic receptor ; that is, isoproterenol is more potent than epinephrine which, in turn, is more potent than norepinephrine . The receptor in the rabbit liver has the further essential properties of a ß-adrenergic receptor, namely, it was not inhibited by the aadrenergic antagonist, phentolamine, but was relatively sensitive to inhibition by propranolol . Noteworthy in the propranolol results was that the observation that a ten-fold increase in propranolol concentration displaced to the right the concentration-response curves of norepinephrine or isoproterenol only about a half log unit rather than a full log unit expected in the simplest case in which one agonist molecule and one antagonist molecule compete for a receptor site . The kinetics of
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this interaction may indicate that to produce sufficient antagonism in the liver an excessively large dose of propranolol may be required in the intact rabbit which is unable to tolerate propranolol in a dose greater than 10 mg/kg . The rat liver presents a more complex adrenergic receptor system . The activation of adenyl cyclase in the rat liver shows the responses to catecholamines and adrenergic antagonists con sistent with the involvement of a ß-receptor (6,7), but the activation of phosphorylase may occur through this ß-receptor system (the only one activated by isoproterenol) or through another system (also activated by epinephrine or other a-agoniste) which does not involve cyclic AMP and which is insensitive to inhibition by propranolol and by most a-adrenergic antagonists, but is sensitive to inhibition by dihydroergotamine . The demonstrated actions of isoproterenol which increase cyclic AMP and phosphorylase do not appear to result in large increases in hepatic glucose production through either glycogenolysis or gluconeogenesis . The fact that isoproterenol in vivo may inhibit its own effect, or those of epinephrine and glucagon, on phosphorylase activation has been attributed to the production of an inhibitory metabolic product (6), but the demonstration that this phenomenon does not occur in the alloxan-diabetic rat (8) suggests that it is the release of insulin by isoproterenol which suppresses epinephrine-induced hyperglycemia in the normal rat . In other species direct or indirect evidence indicates that liver glucose production can be increased by an effect on aadrenergic receptors in addition to an effect on ß-adrenergic receptors . Evidence for an a-receptor mediated effect is particularly well demonstrated in the guinea pig liver (9) . The response is produced by several selective a-agonists and inhibIn the cat liver also there ited by selective a-antagonists . appears to be an a-receptor system activating glucose release in addition to the ß-adrenergic receptor system (10) . The adrenergic antagonists have been helpful in analyzing the mechanisms of the metabolic responses to the catecholamines . The results obtained with adrenergic antagonists have been quite important in defining the types of receptors involved in certain responses, but some of the data obtained has only made it more evident that much further research is necessary . The first of the so called "ß-blockers" demonstrated in the . dog (14) that the adrenergic receptors involved in increasing plasma glucose, lactic acid and free fatty acids are clearly ß adrenergic receptors . The later use of the "pure" ß-adrenergic antagonists have supported these early results and have added the catecholamine-induced increase in oxygen consumption (15) to the liver, muscle and adipose tissue effects which are "ß-receptor functions" . The adrenergic receptors for metabolic responses in the cat also appeared to involve only ß-adrenergic receptors (16), but a recent report contained evidence for an a-receptor mechanism, as well as the ß-receptors, for increasing hepatic glucose production (10) . As indicated above, the definition of the receptors controlling the hyperglycemic response and liver glycogen depletion in rats, rabbits, baboons and humans is less certain . A review of the findings in these latter species will define th~a pertinent problems .
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The metabolic responses which define the characteristics of the group of animals which includes rats, rabbits, baboons and humans are that these species are relatively resistant to the hyperglycemic response to isoproterenol in contrast to their sensitivity to epinephrine-induced hyperglycemia and in these species the epinephrine-induced increase in blood sugar is resistant to blockade by ß-adrenergic antagonists . The resistance of epinephrine-hyperglycemia to inhibition by the available ß-adrenergic antagonists must be examined in detail . The published data is quite clear in demonstrating that the ß-adrenergic antagonists, at dose levels which suppress cardiovascular ß-adrenergic receptor responses and muscle glycogenolysis, do not produce much inhibition of the hyperglycemic response to epinephrine . Nonetheless, it has been claimed that a high dose of a "ß-blocker" in the rat can suppress epinephrinehyperglycemia . When maximally tolerated doses of propranolol were tested in rats (17) and rabbits (13) and a very large dose of sotalol in rats (17), there was indeed considerable inhibition of the epinephrine-induced hyperglycemia, but the responsé was certainly not completely suppressed . Measurement of the glycogen changes in liver and muscle made it clear that the ß-adrenergic antagonists prevented muscle glycogenolysis and thus reduced the supply of lactate and other substrates for hepatic gluconeogenesis in rats (17) and rabbits (13), but that even the highest levels of the ß-antagonists did not reduce the epinephrineinduced depletion of hepatic glycogen . In the rabbit, pretreatment with propranolol produced complex hepatic effects ; the glycogen-depleting effect of epinephrine was potentiated and the biphasic response to isoproterenol (an increase in hepatic glycogen at lower doses and a reduction at higher doses of isoproterenol) was completely suppressed so that no changes in hepatic glycogen occurred when isoproterenol was administered over the same range of doses (13) . When it was found that the common ß-adrenergic antagonists has only a limited antagonism against epinephrine-induced hyperglycemia and liver glycogen depletion, the old observation (1) that ephedrine was a good antagonist against epinephrine-hyperglycemia in the rat suggested that ephedrine might be a new type of antagonist . When ephedrine was explored in more detail (18) as a potential antagonist, it was found, unhappily, that the antagonistic effect of ephedrine was quite like that of propranolol ; ephedrine inhibited the depletion of muscle glycogen by epinephrine, but did not inhibit the liver glycogen depletion better than did propranolol . Apparently the ethanolamine side chain is essential for this action of ephedrine since amphetamine (18) was ineffective as an epinephrine antagonist . Further exploration of the mechanism of antagonism of epinephrine-induced hyperglycemia by dihydroergotamine has demonIn the strated that this antagonist has interesting effects . intact rat it inhibited epinephrine-induced hyperglycemia and liver glycogen depletion, but did not inhibit muscle or heart glycogenolysis (3) . The action in the liver was found to be selective since both glucagon and cyclic adenosine-3',5'-phosphate decreased hepatic glycogen in the presence of a dose of dihydroergotamine which inhibited the hepatic action of epinephrine (19) . In a study of the site of the antagonistic effect
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of dihydroergotamine on rabbit liver slices (20) it was found that dihydroergotamine antagonized epinephrine non-competitively, but there was no antagonism against glucagon or cyclic AMP at a concentration of dihydroergotamine markedly antagonizing epinephrine . Although propranolol has not been a completely effective epinephrine antagonist in rats and rabbits, the effects of propranolol as an antagonist against isoproterenol have been those expected of a non-selective ß-adrenergic antagonist interacting with a "pure" ß-adrenergic agonist . The antagonist inhibited or suppressed the hyperglycemia-producing effect of isoproterenol in alloxan-diabetic rats (21) and in normal and alloxan-diabetic rabbits (13,22) . Since the hyperglycemic effect of isoproterenol is accompanied by a large reduction in muscle glycogen but not a concomitant change in liver glycogen, the suppression of the isoproterenol-induced increase in blood glucose may be related to the suppression of the input of substrates for hepatic gluconeogenesis by muscle and other tissues . It is also pertinent than ethanol and 3-mercaptopicolinic acid, two substances which reduce lactic acid use in gluconeogenesis, suppressed the isoproterenol-induced hyperglycemias in fasted normal rats and in alloxan-diabetic rats while not greatly modifying the epinephrine-induced hyperglycemias (23) . IMPORTANCE OF INSULIN SECRETION The results of the rabbit liver slice experiments indicated that isoproterenol should be~inore potent than epinephrine in the activation of glucose production . It is also known that isoproterenol is more effective in vivo for increasing plasma lactic acid and glycerol, both excél~ent substrates for hepatic gluconeogenesis (11) . Why, then, is isoproterenol a weak hyperglycemic agent in the rabbit (and also in several other species, including man)7 One proposed explanation for the marked differences in the hyperglycemic effects of epinephrine and isoproterenol in some animals involves the fact that these two catecholamines produce opposite effects on insulin release from the pancreatic islet cells (12) . It is now well established that epinephrine, through a-adrenergic receptors, inhibits insulin release, whereas isoproterenol, through its action on ß-adrenergic receptors, markedly increases insulin release . Since insulin opposes adrenergic effects on glucose metabolism in liver, muscle and other tissues, the release of insulin by isoproterenol might be sufficiently effective in opposing the direct effect of isoproterenol on the liver (and possibly on other tissues) to modulate the hyperglycemic response to isoproterenol . In support of this proposal are the observations in rats (3) and rabbits (13) that isoproterenol, in comparison with epinephrine, has little effect on liver glycogen but a more potent effect on muscle glycogen . This hypothesis has been tested extensively in rabbits and rats by indirect and direct methods . An indirect method for determining the importance of insulin release to explain the differences in the hyperglycemic responses to epinephrine and isoproterenol is to limit the re lease of insulin . This was done by producing alloxan-diabetic
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animals which could be controlled with a daily dose of protamine zinc insulin . Studies have been done with alloxan-diabetic rata and rabbits . A more direct method is to use a radioimmunoassay procedure to determine the changes produced in plasma pancreatic hormones under the influences of the catecholamines and other procedures . In the fed alloxan-diabetic rat isoproterenol was a more potent hyperglycemic agent than epinephrine, whereas in the fed normal rat epinephrine-hyperglycemia was increased but isoproterenôl produced no increase in blood sugar (8) . The hyperglycemic response to epinephrine was essentially the same in the diabetic as in the normal rat . The diabetic state in the rabbit also potentiated the hyperglycemic responses to a wide range of doses of isoproterenol, but did not modify the response to epinephrine (12) . The direct measurement of changes in plasma immunoreactive insulin after a range of doses of isoproterenol in fed and in fasted rats indicated that isoproterenol induced a dose-related increase in insulin with a significantly greater increase in fed rats than in fasted rats (8) . These data again supported the concept that insulin release by isoproterenol minimized its total effect on blood sugar . The fact that the fed rats had higher plasma insulin than fasted rats at each dose of isoproterenol was relatable to the complete absence of a rise in blood glucose in response to isoproterenol in the fed rat while a modest increase in blood glucose occurred in the fasted rat . There is compelling evidence now that the opposite effects of isoproterenol and epinephrine on insulin release must play a major role in the differences observed in those animals in which isoproterenol is a much poorer hyperglycemic agent than epinephrine . There are, albeit, many additional questions which must be explored . IMPORTANCE OF GLUCAGON SECRETION Glucagon release was proposed many years ago (24) as the indirect mechanism through which epinephrine caused hepatic glycogenolysis and hyperglycemia . This hypothesis has been examined with two general approaches : (1) by determining the changes in plasma immunoreactive glucagon (and insulin) induced by epinephrine and isoproterenol in normal rats, rabbits, baboons and dogs and by isoproterenol in diabetic rats and rabbits ; and (2) by administering somatostatin (SRIF) to inhibit pancreatic hormone release in order to determine whether inhibition of glucagon release reduced catecholamine-induced hyperglycemia . Somatostatin is an interesting analytical tool for explorations in this area . It completely suppressed catecholamine-induced glucagon-release and markedly reduced insulin-release without interfering with the catecholamine-induced increases in plasma lactate and free fatty acids . In rats, rabbits, dogs and baboons epinephrine was much more potent than isoproterenol in raising plasma glucagon levels . Part of this difference may be related to the known suppression of glucagon release by insulin which is released by isoproterenol . This may also be the explanation for the amplified plasma glucagon
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response to isoproterenol in diabetic animals . Furthermore, the increased hyperglycemic response to isoproterenol in diabetic animals is suppressed by pretreatment with propranolol or with somatostatin, both of which inhibit release of glucagon . These results strongly implicate glucagon in the hyperglycemic responses to catecholamines in certain species (25) . in the dog somatostatin prevented the increase in plasma glucagon in response to epinephrine and to isoproterenol without inhibiting the hyperglycemic responses (26) . The results in the rat were quite different . Somatostatin suppressed the elevation of plasma glucagon by epinephrine in normal rats and by isoproterenol in diabetic rats and also greatly inhibited the hyperglycemic responses (26) . Somatostatin also greatly depressed the hyperglycemia and the hyperglucagonemia in response to epinephrine in the baboon (27) . These observations show that glucagon release is a major factor in catecholamine-induced hyperglycemia in the rat and the baboon but in the dog glucagon release is of little significance in the hyperglycemic responses . CONCLUSIONS The species differences in the metabolic responses to catecholamines has defined characteristics . Isoproterenol has a much less potent hyperglycemic effect than epinephrine in rats, rabbits, baboons and humans, but in dogs and cats isoproterenol is as potent as, or more potent than, epinephrine . In the species in which isoproterenol is the poorer hyperglycemic agent, it also has less influence on liver glycogen and, in these species, propranolol and other common ß-adrenergic antagonists have a limited effect against epinephrine-induced hyperglycemia and hepatic glycogen depletion . The release of insulin by isoproterenol is an important factor which limits the effect of isoproterenol on blood glucose and hepatic glycogen in the rat and the rabbit . Glucagon release, in the normal or diabetic rat and rabbit and in the baboon, appears to play a considerable role in the catecholamine-induced hyperglycemias, whereas in the dog glucagon release is of minor importance . ACKNOWLEDGEMENT The research accomplished in the author's laboratories was largely supported by grants from the National Institutes of Health . A grant from the Smith Kline & French Laboratories, Philadelphia, supported principally the research on~baboons and monkeys . REFERENCES 1. 2. 3.
4.
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