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The effect of glucagon antibodies on plasma glucose and insulin levels
Biochimica et Biophysica Acta, 320 (1973) 741-744 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA R e p o r t BBA 21379 T h e e f f e c t o f g l u c a g o n a n t i b o d i e s o n p l a s m a g l u c o s e a n d insulin levels
RICHARD M. EPAND and R.J. DOUGLAS Departments of Chemistry and Microbiology, University of Guelph, Guelph, Ontario (Canada)
(Received August 7th, 1973)
SUMMARY The actions of glucagon and insulin are interrelated as the two hormones have opposite physiological effects and the secretion of insulin is regulated, at least in part, by the level of glucagon. We have found that rabbits which are immunized against glucagon have normal fasting levels of blood glucose but a lowered level of insulin. These rabbits are also able to rapidly utilize intravenously injected glucose but with a much lower plasma level of insulin. These results demonstrate that in the presence of glucagon antibodies, normal blood sugar levels can be maintained with a reduced supply of insulin. It is suggested that this finding may be useful in the treatment of diabetes.
Diabetic subjects not only have lower levels of effective insulin but also have higher levels of glucagon than do normal subjects during hyperglycemia 1'2. This may occur because of an insulin requirement for the hyperglycemic suppression of glucagon release 3 . Si~ace glucagon is known to have glycogenolytic and gluconeogenic activities which are opposite to the activities of insulin, diabetes is thus a bihormonal disease. The opposing effects of insulin and glucagon can be demonstrated by the simultaneous administration of the two hormones, which results in an increased rate of turnover of glucose without a significant effect on the level of glucose in the blood 4 . One of the mechanisms by which these hormones exert their opposing actions is by stimulation or inhibition of membrane bound adenyl cyclase thereby regulating the intracellular concentration of cyclic AMP s One method to depress the effects of glucagon is to produce antibodies against the hormone. Although bovine 6 , porcine 6 , rat 7 , human 8 and rabbit 9 glucagons appear to be chemically identical, rabbits are still able to produce antibodies to bovine glucagon when it is introduced admixed with an adjuvant 1° . In addition, a case of the production
742
BBA REPORT
of glucagon antibodies in a human subject has been reported 11 . Glucagon is known to stimulate the release of insulin 12, thus in rabbits whose effective glucagon concentration has been lowered by the presence of antibodies to glucagon the insulin level would also be expected to fall below the normal level. Because of the opposing effects of insulin and glucagon, this should have very little effect on the glucose level in blood. Antibodies to glucagon could thus be used in the treatment of diabetes to counteract the effects caused by the lowered levels of insulin activity. Immunization was carried out by subcutaneous and intramuscular injections of 2 ml each of a 1 mg/ml emulsion of glucagon (Sigma Chem. Co.) in complete Freund's adjuvant (Calbiochem.). Five male albino rabbits weighing 2.3 kg were immunized by injections, given twice a week for two consecutive weeks. One month after the last injection, a 4 ml booster injection containing 10 mg glucagon in emulsion with complete Freund's adjuvant was administered. About 5 ml of blood was drawn from a rabbit's ear into an anticoagulant tube containing oxalate and fluoride. The tubes were maintained in ice and were centrifuged within an hour. The plasma was used immediately for measurement of glucose and antiglucagon levels. The remaining plasma was frozen for subsequent measurement of insulin levels. The dextran coated charcoal assay 13 was used for the detection of glucagon antibodies. [12SI]Glucagon was made by the method of Burrows e t al. 14 and was; separated from unreacted iodine using a 1.5 cm X 100 cm column packed with Sephadex G-10 and eluting with ethanol-water (2: 1, v/v). 50/11 of immune plasma were, needed to bind 7 0 - 1 0 0 ng of the [12sI]glucagon. This corresponded to 40-60% of the label. The same amount of preimmune plasma from a control group bound about 5% of the labeled glucagon. The concentration of glucagon used for these assays was determined by its ability to activate adenyl cyclase from rat liver membranes in an in vitro system Is. Plasma insulin levels were determined with a Schwarz/Mann Radioimmunoassay kit which utilizes the technique in which complexes containing [l~sI]insulin are precipitated by antiglobulin. The assay was calibrated with the human hormone. We used this standard curve to measure rabbit insulin which differs from human insulin by the replacement of a threonine residue at position 30 of the B chain by a serine residue. This minor chemical change may give rise to a slight error in the insulin levels we report but would not affect the relative values for the various groups of rabbits. The coated charcoal radioimmunoassay of insulin 16 was used to detect the presence of insulin antibodies in the plasma samples. Levels of glucose in deproteinized plasma were determined by the glucose oxidase method (Sigma Chemical Co., Kit 510-DA). Plasma glucose, insulin and glucagon antibody levels of three groups of rabbits were measured. These groups consisted of: a control group which received no treatment; a group which was later immunized with glucagon (preimmune); a group which was immunized with glucagon (immune). Plasma samples from these latter rabbits (immune),
743
BBA REPORT
TABLE I F A S T I N G PLASMA GLUCOSE A N D I N S U L I N LEVELS Levels are the average value ± standard deviation.
Group
No. of rabbits
Total No. of samples *
Glucose (rag %)
lnsulin (mierounits/ml)
Control Preimmune Immune
3 5 5
24 20 15
109.5 ± 2.2 113.5 ± 2.1 96.5 ± 2.6
27 ± 6 23 ~ 8 9 ~ 2
• Each sample analyzed in duplicate.
contained sufficient antibodies so that 50/A of plasma would bind at least 70 ng of the labeled glucagon. All animals immunized with glucagon produced sufficient antibodies to be classed in the immune group. The plasma from the immunized rabbits had no greater capacity to bind insulin than did the plasma from the control group which bound less than 0.4 microunits insulin per ml of plasma. The glucose level in plasma samples from the immune group which had been fasted overnight was also close to that for the control and preimmune groups although it appears slightly lower (Table I). However, the insulin levels of the immune group were considerably lower than those for the non-
30( 25(
o
20(
80 ~
150
3
.................................................. t
o
,~
6o
100
40 ~
JL
_1 5O
/." ~.."
rz
.... lb
2'o
20
30
60
120
C
TIME (minutes)
Fig. 1. Plasma glucose and insulin levels after the intravenous administration of 0.5 g glucose per kg. Zero time samples were taken after overnight fasting. Three rabbits from the control group and three from the immune group were used for this experiment. Plasma from each analyzed in duplicate. e--,, Glucose levels, control group; o o, Glucose levels, immune group; • . . . . =, Insulin levels, control group; ~- - - -n, Insulin levels, immune group. Bars indicate standard deviation of each measurement. Dashed curves refer to right hand ordinate.
744
BBA REPORT
immunized rabbits. These results demonstrate that rabbits immunized with glucagon are able to maintain normal blood sugar levels with a reduced level of plasma insulin. A plausible mechanism to explain this observation is that the presence of glucagon antibodies lowers the concentration of active glucagon in the plasma. The lowered glucagon concentration, in turn, decreases the secretion of insulin because of the insulinogenic activity of gtucagon. The combined reduction in the circulating levels of the two antagonistic hormones, insulin and glucagon has only a small effect on the blood glucose level. In addition, the slightly lower plasma glucose levels of the immune group may also contribute to the lowered insulin levels. The ability of the immune rabbits to utilize glucose despite the lower plasma levels of insulin is seen even more dramatically in the glucose tolerance test (Fig. 1). Here the immune rabbits can utilize glucose as rapidly as the control group but without as great an increase in plasma insulin levels. These results suggest the potential usefulness of glucagon antigenicity in diabetic therapy. Less insulin would be required to maintain normal blood glucose levels of a diabetic patient who had been immunized with glucagon. This possibility is presently being explored in our laboratory. The authors wish to express their gratitude to Mrs V. Grey for technical assistance and to Dr G.A. Robinson of the Biomedical Sciences Department for use of the gamma counter. This work was supported by the National Research Council of Canada, the Banting Research Foundation, and the University of Guelph Research Advisory Board.
REFERENCES 1 Unger, R.H., Aguilar-Parada, E., M/iller, W.A. and Eisentraut, A.M. (1970) J. Clin. Invest. 49, 837- 848 2 Heding, LG. and Rasmussen, S.M. (1972)Diabetologia 8, 408-411 3 M/iller, W.A., Faloona, G.R. and Unger, R.H. (1971)J. Clin. Invest. 50, 1992-1999 4 Cherrington, A.D. and Vranic, M. (1971)Metabolism 20, 625-628 5 llliano, G. and Cuatrecasas, P. (1972)Science 175, 906-908 6 Bromer, W.W., Boucher, M.E. and Koffenberger, Jr~ J.E. (1971) J. Biol. Chem. 246, 2822-2897 7 Sundby, F. and Markussen, J. (1971) Horm. Metab. Res. 3, 184-187 8 Thomsen, J., Kristiansen, K., Bmnfeldt, K. and Sundby, F. (1972)FEBS Lett. 21,315-319 9 Sundby, F. and Markussen, J. (1972)Herin. Metab. Res. 4, 56 10 Senyk, G., Nitecki, D. and Goodman, J.W. (1971) Science 171,407-408 11 Stahl, M., Nars, P.W., Herz, G., Baumann, J.B. and Girard, J. (1972)Herin. Metab. Res. 4, 224-225 12 Samols, E., Tyler, J.M. and Marks, V. (1972) in Glucagon, Molecular Physiology, Clinical and Therapeutic Implications (Lefebure, P.J. and Unger, R.H., eds), pp. 151-173, Pergamon Press, Oxford 13 Senyk, G., Williams, E.B., Niteeki, D.E. and Goodman, J.W. (1971)./. Exp. Med. 133, 1294-1308 14 Burrows, E.A., Peters, T., Lowell, F.C., Trakas, A.N. and Reilly, P. (1957) J. Clin. Invest. 36, 393-397 15 Epand, R.M. and Epand, R.F. (1973) Can. J. Biochem. 51,140-147 16 Herbert, V., Lau, K.-S., Gottlieb, C.W. and Bleicher, S.J. (1965)J. Clin. Endocrinol. 25, 1375-1384.
BBA R e p o r t BBA 21379 T h e e f f e c t o f g l u c a g o n a n t i b o d i e s o n p l a s m a g l u c o s e a n d insulin levels
RICHARD M. EPAND and R.J. DOUGLAS Departments of Chemistry and Microbiology, University of Guelph, Guelph, Ontario (Canada)
(Received August 7th, 1973)
SUMMARY The actions of glucagon and insulin are interrelated as the two hormones have opposite physiological effects and the secretion of insulin is regulated, at least in part, by the level of glucagon. We have found that rabbits which are immunized against glucagon have normal fasting levels of blood glucose but a lowered level of insulin. These rabbits are also able to rapidly utilize intravenously injected glucose but with a much lower plasma level of insulin. These results demonstrate that in the presence of glucagon antibodies, normal blood sugar levels can be maintained with a reduced supply of insulin. It is suggested that this finding may be useful in the treatment of diabetes.
Diabetic subjects not only have lower levels of effective insulin but also have higher levels of glucagon than do normal subjects during hyperglycemia 1'2. This may occur because of an insulin requirement for the hyperglycemic suppression of glucagon release 3 . Si~ace glucagon is known to have glycogenolytic and gluconeogenic activities which are opposite to the activities of insulin, diabetes is thus a bihormonal disease. The opposing effects of insulin and glucagon can be demonstrated by the simultaneous administration of the two hormones, which results in an increased rate of turnover of glucose without a significant effect on the level of glucose in the blood 4 . One of the mechanisms by which these hormones exert their opposing actions is by stimulation or inhibition of membrane bound adenyl cyclase thereby regulating the intracellular concentration of cyclic AMP s One method to depress the effects of glucagon is to produce antibodies against the hormone. Although bovine 6 , porcine 6 , rat 7 , human 8 and rabbit 9 glucagons appear to be chemically identical, rabbits are still able to produce antibodies to bovine glucagon when it is introduced admixed with an adjuvant 1° . In addition, a case of the production
742
BBA REPORT
of glucagon antibodies in a human subject has been reported 11 . Glucagon is known to stimulate the release of insulin 12, thus in rabbits whose effective glucagon concentration has been lowered by the presence of antibodies to glucagon the insulin level would also be expected to fall below the normal level. Because of the opposing effects of insulin and glucagon, this should have very little effect on the glucose level in blood. Antibodies to glucagon could thus be used in the treatment of diabetes to counteract the effects caused by the lowered levels of insulin activity. Immunization was carried out by subcutaneous and intramuscular injections of 2 ml each of a 1 mg/ml emulsion of glucagon (Sigma Chem. Co.) in complete Freund's adjuvant (Calbiochem.). Five male albino rabbits weighing 2.3 kg were immunized by injections, given twice a week for two consecutive weeks. One month after the last injection, a 4 ml booster injection containing 10 mg glucagon in emulsion with complete Freund's adjuvant was administered. About 5 ml of blood was drawn from a rabbit's ear into an anticoagulant tube containing oxalate and fluoride. The tubes were maintained in ice and were centrifuged within an hour. The plasma was used immediately for measurement of glucose and antiglucagon levels. The remaining plasma was frozen for subsequent measurement of insulin levels. The dextran coated charcoal assay 13 was used for the detection of glucagon antibodies. [12SI]Glucagon was made by the method of Burrows e t al. 14 and was; separated from unreacted iodine using a 1.5 cm X 100 cm column packed with Sephadex G-10 and eluting with ethanol-water (2: 1, v/v). 50/11 of immune plasma were, needed to bind 7 0 - 1 0 0 ng of the [12sI]glucagon. This corresponded to 40-60% of the label. The same amount of preimmune plasma from a control group bound about 5% of the labeled glucagon. The concentration of glucagon used for these assays was determined by its ability to activate adenyl cyclase from rat liver membranes in an in vitro system Is. Plasma insulin levels were determined with a Schwarz/Mann Radioimmunoassay kit which utilizes the technique in which complexes containing [l~sI]insulin are precipitated by antiglobulin. The assay was calibrated with the human hormone. We used this standard curve to measure rabbit insulin which differs from human insulin by the replacement of a threonine residue at position 30 of the B chain by a serine residue. This minor chemical change may give rise to a slight error in the insulin levels we report but would not affect the relative values for the various groups of rabbits. The coated charcoal radioimmunoassay of insulin 16 was used to detect the presence of insulin antibodies in the plasma samples. Levels of glucose in deproteinized plasma were determined by the glucose oxidase method (Sigma Chemical Co., Kit 510-DA). Plasma glucose, insulin and glucagon antibody levels of three groups of rabbits were measured. These groups consisted of: a control group which received no treatment; a group which was later immunized with glucagon (preimmune); a group which was immunized with glucagon (immune). Plasma samples from these latter rabbits (immune),
743
BBA REPORT
TABLE I F A S T I N G PLASMA GLUCOSE A N D I N S U L I N LEVELS Levels are the average value ± standard deviation.
Group
No. of rabbits
Total No. of samples *
Glucose (rag %)
lnsulin (mierounits/ml)
Control Preimmune Immune
3 5 5
24 20 15
109.5 ± 2.2 113.5 ± 2.1 96.5 ± 2.6
27 ± 6 23 ~ 8 9 ~ 2
• Each sample analyzed in duplicate.
contained sufficient antibodies so that 50/A of plasma would bind at least 70 ng of the labeled glucagon. All animals immunized with glucagon produced sufficient antibodies to be classed in the immune group. The plasma from the immunized rabbits had no greater capacity to bind insulin than did the plasma from the control group which bound less than 0.4 microunits insulin per ml of plasma. The glucose level in plasma samples from the immune group which had been fasted overnight was also close to that for the control and preimmune groups although it appears slightly lower (Table I). However, the insulin levels of the immune group were considerably lower than those for the non-
30( 25(
o
20(
80 ~
150
3
.................................................. t
o
,~
6o
100
40 ~
JL
_1 5O
/." ~.."
rz
.... lb
2'o
20
30
60
120
C
TIME (minutes)
Fig. 1. Plasma glucose and insulin levels after the intravenous administration of 0.5 g glucose per kg. Zero time samples were taken after overnight fasting. Three rabbits from the control group and three from the immune group were used for this experiment. Plasma from each analyzed in duplicate. e--,, Glucose levels, control group; o o, Glucose levels, immune group; • . . . . =, Insulin levels, control group; ~- - - -n, Insulin levels, immune group. Bars indicate standard deviation of each measurement. Dashed curves refer to right hand ordinate.
744
BBA REPORT
immunized rabbits. These results demonstrate that rabbits immunized with glucagon are able to maintain normal blood sugar levels with a reduced level of plasma insulin. A plausible mechanism to explain this observation is that the presence of glucagon antibodies lowers the concentration of active glucagon in the plasma. The lowered glucagon concentration, in turn, decreases the secretion of insulin because of the insulinogenic activity of gtucagon. The combined reduction in the circulating levels of the two antagonistic hormones, insulin and glucagon has only a small effect on the blood glucose level. In addition, the slightly lower plasma glucose levels of the immune group may also contribute to the lowered insulin levels. The ability of the immune rabbits to utilize glucose despite the lower plasma levels of insulin is seen even more dramatically in the glucose tolerance test (Fig. 1). Here the immune rabbits can utilize glucose as rapidly as the control group but without as great an increase in plasma insulin levels. These results suggest the potential usefulness of glucagon antigenicity in diabetic therapy. Less insulin would be required to maintain normal blood glucose levels of a diabetic patient who had been immunized with glucagon. This possibility is presently being explored in our laboratory. The authors wish to express their gratitude to Mrs V. Grey for technical assistance and to Dr G.A. Robinson of the Biomedical Sciences Department for use of the gamma counter. This work was supported by the National Research Council of Canada, the Banting Research Foundation, and the University of Guelph Research Advisory Board.
REFERENCES 1 Unger, R.H., Aguilar-Parada, E., M/iller, W.A. and Eisentraut, A.M. (1970) J. Clin. Invest. 49, 837- 848 2 Heding, LG. and Rasmussen, S.M. (1972)Diabetologia 8, 408-411 3 M/iller, W.A., Faloona, G.R. and Unger, R.H. (1971)J. Clin. Invest. 50, 1992-1999 4 Cherrington, A.D. and Vranic, M. (1971)Metabolism 20, 625-628 5 llliano, G. and Cuatrecasas, P. (1972)Science 175, 906-908 6 Bromer, W.W., Boucher, M.E. and Koffenberger, Jr~ J.E. (1971) J. Biol. Chem. 246, 2822-2897 7 Sundby, F. and Markussen, J. (1971) Horm. Metab. Res. 3, 184-187 8 Thomsen, J., Kristiansen, K., Bmnfeldt, K. and Sundby, F. (1972)FEBS Lett. 21,315-319 9 Sundby, F. and Markussen, J. (1972)Herin. Metab. Res. 4, 56 10 Senyk, G., Nitecki, D. and Goodman, J.W. (1971) Science 171,407-408 11 Stahl, M., Nars, P.W., Herz, G., Baumann, J.B. and Girard, J. (1972)Herin. Metab. Res. 4, 224-225 12 Samols, E., Tyler, J.M. and Marks, V. (1972) in Glucagon, Molecular Physiology, Clinical and Therapeutic Implications (Lefebure, P.J. and Unger, R.H., eds), pp. 151-173, Pergamon Press, Oxford 13 Senyk, G., Williams, E.B., Niteeki, D.E. and Goodman, J.W. (1971)./. Exp. Med. 133, 1294-1308 14 Burrows, E.A., Peters, T., Lowell, F.C., Trakas, A.N. and Reilly, P. (1957) J. Clin. Invest. 36, 393-397 15 Epand, R.M. and Epand, R.F. (1973) Can. J. Biochem. 51,140-147 16 Herbert, V., Lau, K.-S., Gottlieb, C.W. and Bleicher, S.J. (1965)J. Clin. Endocrinol. 25, 1375-1384.