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The effects of glucagon, tolbutamide, and cholecystokinin octapeptide on plasma insulin and glucose concentrations in sheep
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
43, 405-408 (1981)
The Effects of Glucagon, Tolbutamide, and Cholecystokinin Octapeptide on Plasma Insulin and Glucose Concentrations in Sheep There are considerable differences between the insulin responses to specific stimuli in different species of mammals. Some of the variations may be related to the different end products of digestion. Ruminants, unlike monogastric species, absorb very little glucose from the gastrointestinal tract (Roe et al., 1966), relying instead upon gluconeogenesis (Lindsay, 1978). Glucagon and tolbutamide stimulate the release of insulin in monogastrics (Ohneda et al., 1975) and glucagon has also been shown to be effective in sheep (Bassett, 1971). Impure cholecystokinin preparations are insulinotrophic in several species (Gerich et al., 1976) including goats (Baile et al., 1969) and sheep (Trenkle, 1972) but the pure synthetic octapeptide (CCK-OP) has only been tested in monogastrics (Frame et al., 1975). This study investigated the effects of glucagon and tolbutamide injected separately or together, and CCK-OP on plasma’insulin and glucose concentrations in sheep.
food. At least 48 hr separated any two injections into one animal. Heparinized blood samples were collected and plasma stored at -20” for analysis. Plasma insulin concentrations were determined by radioimmunoassay (Fuller et al., 1977). Plasma glucase levels were measured using a glucose analyzer (Yellow Springs Instrument, Model 23A). The results were analyzed by analysis of variance (Steel and Torrie, 1960) and statistical differences determined using Kramer’s (1956) multiple range test for unequal num-’ bers of replications.
RESULTS AND DISCUSSION
Insulin and glucose concentrations rose within 2 and 5 min, respectively, of glucagon injection (P < 0.05 both cases, Fig. 1). Both parameters remained elevated until 70 min (P < 0.05, P < 0.01). A similar pattern of response has been reported in two sheep (Kamalu, 1970). The rapidity of the insulin response in sheep is comparable to that observed in man after a similar dose of glucagon and occurs before and probably’ independently of the rise in glucose. Peak insulin and glucose concentrations occur later; and are much larger than observed in man, METHODS (Samols et al., 1965; Adamson and Cerasi, Six mature male castrate sheep, penned individu1975). ally, were fed 1 kg barley and fishmeal pellets plus 0.1 Tolbutamide (14.3 mg/kg, Fig. 1) caused kg hay per day, as two daily feeds. One jugular catheter was implanted into each sheep at least 2 days be- a release of insulin after lo- 15 min (P < fore the start of the experiment. These were used for 0.05) and a fall in plasma glucose that was the administration of drugs and collection of blood only significant at 50 min (P < 0.05):. The’ samples. maximum insulin increment in sheep (112 At least three of the sheep were injected with glucagon (Nova Industri A/S, Copenhagen, Denmark; 14.3 pU/rnl) ,was within the range of values re‘ported for human studies using this dose CLgikg body wt), tolbutamide (Hoechst Pharmaceuti(Asano ,et al., 1978; Koncz et al., 1979) al-, cals, Hounslow, Middlesex, U.K.; 14.3 mg/kg), glucagon (14.3 pgikg) plus tolbutamide (7.15 or 14.30 though the hy,poglycemic response was mgikg), or CCK-OP (Squibb :sincalide SQ 19844, E. R. smaller, in agreement with another study in, Squibb and Sons Ltd., Twickenham, Middlesex, sheep (Kronfeld, et al., 1967). The area U.K.; 33,66, and 100 &kg). Saline, the vehicle for all injections, was used as the control injection. Each in- under the insulin response curve after tolbutamide injection to sheep was larger than jection was performed at 9 AM following 16 hr without 405 0016-6480/81/030405-04$01.00/0 Copyright @ 1981 by Academic Press, Inc. All rights of r&production in any form reserved
NOTES
0
30
FIG. 1. Effects of glucagon plasma concentrations of insulin used: (0) 14.3 pg glucagonikg tolbutamideikg (n = 4); (A) 14.3 mg tolbutamide/kg (n = 4); (0) 14.3 mg tolbutamide/kg (n = 4);
w
si,
and tolbutamide on and glucose. Doses (n = 5); (A) 14.3 mg pg glucagon plus 7.15 14.3 pg glucagon plus (X) saline.
that found with growing pigs or cattle (Gregory et al., 1977, 1980). The difference may be due to the maturity of the sheep used (over 2 years) or interspecies variation. Glucagon plus tolbutamide (7.15 mg/kg) caused a greater release of insulin at 2 min (P < 0.05, Fig. 1) and lower glucose levels at 50-70 min (P < 0.05) than the injection of glucagon alone. Insulin and glucose levels were higher from 5 to 50 min after glucagon plus tolbutamide (14.3 mg/kg) than after this dose of tolbutamide alone (P < 0.01 both parameters at all times). The maximum incremental insulin responses to glucagon (14.3 &kg) and tolbutamide (14.3 mg/kg) were approximately additive. The greater initial insulin response was probably responsible for the smaller hyperglycemic response. Similar effects have been observed in man using the same dose of glucagon (14.3 pg/kg) and tolbutamide (7.15 mg/kg), although the hyperinsulinemic and hyperglycemic responses were considerably smaller in man (Ryan et al., 1967). All doses of CCK-OP produced a similar and transient hyperinsulinemic response (Table 1) without changing plasma glucose concentrations. The pattern of insulin response was comparable, although peak plasma insulin concentrations were considerably,greater than observed after a similar dose of CCK-OP was infused over 30 min into dogs (Frame et al., 1975). The intravenous injection of 66 ng CCK-OP/kg to humans failed to stimulate insulin release (Hedner et al., 1973). Insulin secretion in the ruminant is thus sensitive to CCK-OP, in confirmation of earlier studies using impure cholecystokinin (Baile et al., 1969; Trenkle, 1972). The physiological meaning of this finding is uncertain until circulating levels of cholecystokinin are known in sheep. In conclusion, the insulin-secreting mechanisms in the sheep are thus equally sensitive as in monogastric species to stimulation by tolbutamide, while glucagon, either alone or with tolbutamide, and
NOTES
407
TABLE 1 THE EFFECTOF CCK-OP ON PLASMAINSULIN CONCENTRATIONSIN SHEEP(~ = 3) Plasma insulin concentrations Dose of CCK-OP WW 0 (saline) 33 66 100
Mean preinjection” Mean SEM Mean SEM Mean SEM Mean SEM
31.3 8.83 37.4 10.75 27.3 4.00 44.8 7.56
Minutes after injection 2
5
10
20
30
32.7 7.00 129.2* 26.54 120.9* 22.67 141.9* 10.74
35.5 10.39 68.3 10.16 59.4 9.74 87.4 22.08
40.2 13.17 43.3 6.13 29.0 6.62 47.2 12.91
34.0 12.26 42.9 15.85 21.4 5.15 45.9 22.08
38.7 13.31 35*9 11.41 18.5 4.25 42.,9 20.09
a Insulin concentrations 30, 25, 20, 10, and 1 min before injection were averaged for each sheep, * Insulin concentrations significantly greater than preinjection value, P < 0.01.
CCK-OP may be more potent insulin secretagogues in sheep. The hyperglycemic response to glucagon injection was greater while the hypoglycemic response to tolbutamide was relatively modest in sheep compared with monogastric species. Thus although the glucose turnover rate is only slightly less in ruminants than in monogastrics (reviewed by Weekes, 1979) the sheep is relatively resis$ant to rapid decreases in plasma glucose concentrations. ACKNOWLEDGMENT This work was supported by a Lord Rank Foundation Fellowship to P.M.M.G. REFERENCES Adamson, U., and Cerasi, E. (1975). Acute suppressive effect of human growth hormone on insulin release induced by glucagon and tolbutamide in man. Diabete Metab. 1, 51-56. Asano, T., Sasaki, H., and Okumura, M. (1978). Characterization of the effect of intravenous infusion of glucose and tolbutamide on insulin delivery rate in man. Diabetologia 1.5, 159-164. Baile, C. A., Glick, Z., and Mayer, J. (1969). Effects of secretin and cholecystokinin-pancreozymin on pancreatic juice and insulin secretion of goats. J. Dairy Sci. 52, 513-517. Bassett, J. M. (1971). The effects of glucagon on plasma concentrations of insulin, growth hormone, glucose, and free fatty acids in sheep:
Comparison with the effects of catecholamines. Aust. .I. Biol. Sri. 24, 311-320. Frame, C. M., Davidson, M. B., and Sturdevant, R. A. L. (1975). Effects of the octapeptide of cholecystokinin on insulin and glucagon secretion in the dog. Endocrinology 97, 549-553. Fuller, M. F., Weekes, T. E. C., Cadenhead, A.. and Bruce, J. B. (1977). The protein-sparing effect of carbohydrate. 2. The role of insulin. Brit. J. Nutr. 38,489-496. Gerich, J. E., Charles, M. A., and Grodsky, G. M. (1976). Regulation of pancreatic insulin and glucagon secretion. Annu. Rev. Physiol. 38, 353-388. Gregory, N. G., Lovell, R. D., Wood, J. D., and Lister, D. (1977). Insulin-secreting ability in Pietran and Large White Pigs. J. Agric. Sci. 89,407-413. Gregory, N. G., Truscott, T. G., and Wood,, J. D. (1980). Insulin secreting ability in relation to fatness in cattle. Proc. Nutr. Sot. 39, 7A. Hedner, P., Persson, G., and Ursing, D. (1973). Failure of intravenous cholecystokinin to elicit insulin release in man. Acta Endocrinol. (CopenFzagen) Suppl. 177, 327. Kamalu, T. N. (1970). Nutritional regulation of insulin and glucagon secretion in sheep. Ph.D. thesis, Iowa State University. Koncz, L., Soeldner, J. S., Otto, H., Smith T. M., and Gleason, R. E. (1979). Insulin secretory dynamics after two consecutive intravenous stimulations with glucose and/or tolbutamide. Mqtabolism 28, 1183 - 1197. Kramer, C. Y. (1956). Extension ,of muitiple range tests to group means with unequal numbers of replications. Biometrics 12, 307-310. Kronfeld, D. S., Raggi, F., and Frumin, A. M. (1967). Changing activity of erythrocyte glucose-6 phosphate dehydrogenase and tolerances to glu-
408
NOTES
case and tolbutamide in growing sheep. Proc. Sot. Exp. Biol. Med. 124, 1022-1025. Lindsay, D. B. (1978). Gluconeogenesis in ruminants. Biochem. Sot. Trans. 6, 1152-1156. Ohneda, A., Matsuda, K., Chiba, M., Iimura, Y., and Yamagata, S. (1975). Glucagon-induced insulin secretion in normal and diabetic subjects. Tohoku J. Exp. Med. 116, 103-110. Roe, W. E., Bergman, E. N., and Kon, K. (1966). Absorption of ketone bodies and other metabolites via the portal blood in sheep. Amer. J. Vet. Res. 27, 729-736. Ryan, W. G., Nibbe, A. F., and Schwartz, T. B. (1967). Beta-cytotrophic effects of glucose, glucagon and tolbutamide in man. Lancet 1, 1255-1256.
Samols, E., Marri, G., and Marks, V. (1965). Promotion of insulin secretion by glucagon. Lancet 2, 415-416. Steel, R. G. D., and Torrie, J. H. (1960). “Principles and Procedures of Statistics.” McGraw -Hill, London. Trenkle, A. (1972). Radioimmunoassay of plasma
hormones: Review of plasma insulin in ruminants. J. Dairy Sci. 55, 1200-1211. Weekes, T. E. C. (1979). Carbohydrate metabolism. In “Digestive Physiology and Nutrition of Ruminants,” 2nd ed. (D. C. Church, ed.), Vol. 2, pp. 187-209. 0 and B Books, Corvallis, Oreg.
M. M.
PATRICIA JANET
B.
GODDEN~
WARD
T. E. C. WEEKES The Department of Agricultural Biochemistry and Nutrition Faculty of Agriculture The University of Newcastle upon Tyne Newcastle upon Tyne NE1 7RU England Accepted July 16, 1980 * To whom correspondence should be addressed. Present address: Department of Biochemistry, University of Surrey, Guildford, Surrey, GU2 5XH, England.
AND
COMPARATIVE
ENDOCRINOLOGY
43, 405-408 (1981)
The Effects of Glucagon, Tolbutamide, and Cholecystokinin Octapeptide on Plasma Insulin and Glucose Concentrations in Sheep There are considerable differences between the insulin responses to specific stimuli in different species of mammals. Some of the variations may be related to the different end products of digestion. Ruminants, unlike monogastric species, absorb very little glucose from the gastrointestinal tract (Roe et al., 1966), relying instead upon gluconeogenesis (Lindsay, 1978). Glucagon and tolbutamide stimulate the release of insulin in monogastrics (Ohneda et al., 1975) and glucagon has also been shown to be effective in sheep (Bassett, 1971). Impure cholecystokinin preparations are insulinotrophic in several species (Gerich et al., 1976) including goats (Baile et al., 1969) and sheep (Trenkle, 1972) but the pure synthetic octapeptide (CCK-OP) has only been tested in monogastrics (Frame et al., 1975). This study investigated the effects of glucagon and tolbutamide injected separately or together, and CCK-OP on plasma’insulin and glucose concentrations in sheep.
food. At least 48 hr separated any two injections into one animal. Heparinized blood samples were collected and plasma stored at -20” for analysis. Plasma insulin concentrations were determined by radioimmunoassay (Fuller et al., 1977). Plasma glucase levels were measured using a glucose analyzer (Yellow Springs Instrument, Model 23A). The results were analyzed by analysis of variance (Steel and Torrie, 1960) and statistical differences determined using Kramer’s (1956) multiple range test for unequal num-’ bers of replications.
RESULTS AND DISCUSSION
Insulin and glucose concentrations rose within 2 and 5 min, respectively, of glucagon injection (P < 0.05 both cases, Fig. 1). Both parameters remained elevated until 70 min (P < 0.05, P < 0.01). A similar pattern of response has been reported in two sheep (Kamalu, 1970). The rapidity of the insulin response in sheep is comparable to that observed in man after a similar dose of glucagon and occurs before and probably’ independently of the rise in glucose. Peak insulin and glucose concentrations occur later; and are much larger than observed in man, METHODS (Samols et al., 1965; Adamson and Cerasi, Six mature male castrate sheep, penned individu1975). ally, were fed 1 kg barley and fishmeal pellets plus 0.1 Tolbutamide (14.3 mg/kg, Fig. 1) caused kg hay per day, as two daily feeds. One jugular catheter was implanted into each sheep at least 2 days be- a release of insulin after lo- 15 min (P < fore the start of the experiment. These were used for 0.05) and a fall in plasma glucose that was the administration of drugs and collection of blood only significant at 50 min (P < 0.05):. The’ samples. maximum insulin increment in sheep (112 At least three of the sheep were injected with glucagon (Nova Industri A/S, Copenhagen, Denmark; 14.3 pU/rnl) ,was within the range of values re‘ported for human studies using this dose CLgikg body wt), tolbutamide (Hoechst Pharmaceuti(Asano ,et al., 1978; Koncz et al., 1979) al-, cals, Hounslow, Middlesex, U.K.; 14.3 mg/kg), glucagon (14.3 pgikg) plus tolbutamide (7.15 or 14.30 though the hy,poglycemic response was mgikg), or CCK-OP (Squibb :sincalide SQ 19844, E. R. smaller, in agreement with another study in, Squibb and Sons Ltd., Twickenham, Middlesex, sheep (Kronfeld, et al., 1967). The area U.K.; 33,66, and 100 &kg). Saline, the vehicle for all injections, was used as the control injection. Each in- under the insulin response curve after tolbutamide injection to sheep was larger than jection was performed at 9 AM following 16 hr without 405 0016-6480/81/030405-04$01.00/0 Copyright @ 1981 by Academic Press, Inc. All rights of r&production in any form reserved
NOTES
0
30
FIG. 1. Effects of glucagon plasma concentrations of insulin used: (0) 14.3 pg glucagonikg tolbutamideikg (n = 4); (A) 14.3 mg tolbutamide/kg (n = 4); (0) 14.3 mg tolbutamide/kg (n = 4);
w
si,
and tolbutamide on and glucose. Doses (n = 5); (A) 14.3 mg pg glucagon plus 7.15 14.3 pg glucagon plus (X) saline.
that found with growing pigs or cattle (Gregory et al., 1977, 1980). The difference may be due to the maturity of the sheep used (over 2 years) or interspecies variation. Glucagon plus tolbutamide (7.15 mg/kg) caused a greater release of insulin at 2 min (P < 0.05, Fig. 1) and lower glucose levels at 50-70 min (P < 0.05) than the injection of glucagon alone. Insulin and glucose levels were higher from 5 to 50 min after glucagon plus tolbutamide (14.3 mg/kg) than after this dose of tolbutamide alone (P < 0.01 both parameters at all times). The maximum incremental insulin responses to glucagon (14.3 &kg) and tolbutamide (14.3 mg/kg) were approximately additive. The greater initial insulin response was probably responsible for the smaller hyperglycemic response. Similar effects have been observed in man using the same dose of glucagon (14.3 pg/kg) and tolbutamide (7.15 mg/kg), although the hyperinsulinemic and hyperglycemic responses were considerably smaller in man (Ryan et al., 1967). All doses of CCK-OP produced a similar and transient hyperinsulinemic response (Table 1) without changing plasma glucose concentrations. The pattern of insulin response was comparable, although peak plasma insulin concentrations were considerably,greater than observed after a similar dose of CCK-OP was infused over 30 min into dogs (Frame et al., 1975). The intravenous injection of 66 ng CCK-OP/kg to humans failed to stimulate insulin release (Hedner et al., 1973). Insulin secretion in the ruminant is thus sensitive to CCK-OP, in confirmation of earlier studies using impure cholecystokinin (Baile et al., 1969; Trenkle, 1972). The physiological meaning of this finding is uncertain until circulating levels of cholecystokinin are known in sheep. In conclusion, the insulin-secreting mechanisms in the sheep are thus equally sensitive as in monogastric species to stimulation by tolbutamide, while glucagon, either alone or with tolbutamide, and
NOTES
407
TABLE 1 THE EFFECTOF CCK-OP ON PLASMAINSULIN CONCENTRATIONSIN SHEEP(~ = 3) Plasma insulin concentrations Dose of CCK-OP WW 0 (saline) 33 66 100
Mean preinjection” Mean SEM Mean SEM Mean SEM Mean SEM
31.3 8.83 37.4 10.75 27.3 4.00 44.8 7.56
Minutes after injection 2
5
10
20
30
32.7 7.00 129.2* 26.54 120.9* 22.67 141.9* 10.74
35.5 10.39 68.3 10.16 59.4 9.74 87.4 22.08
40.2 13.17 43.3 6.13 29.0 6.62 47.2 12.91
34.0 12.26 42.9 15.85 21.4 5.15 45.9 22.08
38.7 13.31 35*9 11.41 18.5 4.25 42.,9 20.09
a Insulin concentrations 30, 25, 20, 10, and 1 min before injection were averaged for each sheep, * Insulin concentrations significantly greater than preinjection value, P < 0.01.
CCK-OP may be more potent insulin secretagogues in sheep. The hyperglycemic response to glucagon injection was greater while the hypoglycemic response to tolbutamide was relatively modest in sheep compared with monogastric species. Thus although the glucose turnover rate is only slightly less in ruminants than in monogastrics (reviewed by Weekes, 1979) the sheep is relatively resis$ant to rapid decreases in plasma glucose concentrations. ACKNOWLEDGMENT This work was supported by a Lord Rank Foundation Fellowship to P.M.M.G. REFERENCES Adamson, U., and Cerasi, E. (1975). Acute suppressive effect of human growth hormone on insulin release induced by glucagon and tolbutamide in man. Diabete Metab. 1, 51-56. Asano, T., Sasaki, H., and Okumura, M. (1978). Characterization of the effect of intravenous infusion of glucose and tolbutamide on insulin delivery rate in man. Diabetologia 1.5, 159-164. Baile, C. A., Glick, Z., and Mayer, J. (1969). Effects of secretin and cholecystokinin-pancreozymin on pancreatic juice and insulin secretion of goats. J. Dairy Sci. 52, 513-517. Bassett, J. M. (1971). The effects of glucagon on plasma concentrations of insulin, growth hormone, glucose, and free fatty acids in sheep:
Comparison with the effects of catecholamines. Aust. .I. Biol. Sri. 24, 311-320. Frame, C. M., Davidson, M. B., and Sturdevant, R. A. L. (1975). Effects of the octapeptide of cholecystokinin on insulin and glucagon secretion in the dog. Endocrinology 97, 549-553. Fuller, M. F., Weekes, T. E. C., Cadenhead, A.. and Bruce, J. B. (1977). The protein-sparing effect of carbohydrate. 2. The role of insulin. Brit. J. Nutr. 38,489-496. Gerich, J. E., Charles, M. A., and Grodsky, G. M. (1976). Regulation of pancreatic insulin and glucagon secretion. Annu. Rev. Physiol. 38, 353-388. Gregory, N. G., Lovell, R. D., Wood, J. D., and Lister, D. (1977). Insulin-secreting ability in Pietran and Large White Pigs. J. Agric. Sci. 89,407-413. Gregory, N. G., Truscott, T. G., and Wood,, J. D. (1980). Insulin secreting ability in relation to fatness in cattle. Proc. Nutr. Sot. 39, 7A. Hedner, P., Persson, G., and Ursing, D. (1973). Failure of intravenous cholecystokinin to elicit insulin release in man. Acta Endocrinol. (CopenFzagen) Suppl. 177, 327. Kamalu, T. N. (1970). Nutritional regulation of insulin and glucagon secretion in sheep. Ph.D. thesis, Iowa State University. Koncz, L., Soeldner, J. S., Otto, H., Smith T. M., and Gleason, R. E. (1979). Insulin secretory dynamics after two consecutive intravenous stimulations with glucose and/or tolbutamide. Mqtabolism 28, 1183 - 1197. Kramer, C. Y. (1956). Extension ,of muitiple range tests to group means with unequal numbers of replications. Biometrics 12, 307-310. Kronfeld, D. S., Raggi, F., and Frumin, A. M. (1967). Changing activity of erythrocyte glucose-6 phosphate dehydrogenase and tolerances to glu-
408
NOTES
case and tolbutamide in growing sheep. Proc. Sot. Exp. Biol. Med. 124, 1022-1025. Lindsay, D. B. (1978). Gluconeogenesis in ruminants. Biochem. Sot. Trans. 6, 1152-1156. Ohneda, A., Matsuda, K., Chiba, M., Iimura, Y., and Yamagata, S. (1975). Glucagon-induced insulin secretion in normal and diabetic subjects. Tohoku J. Exp. Med. 116, 103-110. Roe, W. E., Bergman, E. N., and Kon, K. (1966). Absorption of ketone bodies and other metabolites via the portal blood in sheep. Amer. J. Vet. Res. 27, 729-736. Ryan, W. G., Nibbe, A. F., and Schwartz, T. B. (1967). Beta-cytotrophic effects of glucose, glucagon and tolbutamide in man. Lancet 1, 1255-1256.
Samols, E., Marri, G., and Marks, V. (1965). Promotion of insulin secretion by glucagon. Lancet 2, 415-416. Steel, R. G. D., and Torrie, J. H. (1960). “Principles and Procedures of Statistics.” McGraw -Hill, London. Trenkle, A. (1972). Radioimmunoassay of plasma
hormones: Review of plasma insulin in ruminants. J. Dairy Sci. 55, 1200-1211. Weekes, T. E. C. (1979). Carbohydrate metabolism. In “Digestive Physiology and Nutrition of Ruminants,” 2nd ed. (D. C. Church, ed.), Vol. 2, pp. 187-209. 0 and B Books, Corvallis, Oreg.
M. M.
PATRICIA JANET
B.
GODDEN~
WARD
T. E. C. WEEKES The Department of Agricultural Biochemistry and Nutrition Faculty of Agriculture The University of Newcastle upon Tyne Newcastle upon Tyne NE1 7RU England Accepted July 16, 1980 * To whom correspondence should be addressed. Present address: Department of Biochemistry, University of Surrey, Guildford, Surrey, GU2 5XH, England.