- Email: [email protected]
Response of calcitonin cells, parathyroid, and serum electrolytes during glucagon-induced hypocalcemia in the mongoose, Herpestes edwardsi (Geoffroy)
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
37, 541-545 (1979)
NOTE Response of Calcitonin Cells, Parathyroid, and Serum Electrolytes During Glucagon-Induced Hypocalcemia in the Mongoose, Herpestes edwardsi (Geoffroy) In mongooses (Herpestes edwurdsi) hypocalcemia was induced by an intravenous injection of crystalline glucagon in a dosage of 0.2 mg/kg body wt. Blood samples from each specimen were taken before the injection (zero time) and after 30, 60, 90, 120, 240, and 360 min following the injection. The serum calcium and inorganic phosphate levels were determined. For histological study, animals were killed at the same time intervals after injection. Specific stains were used for the selective staining of calcitonin cells. Glucagon injection evokes a decreased level of serum calcium and inorganic phosphate. A progressive degranulation of the secretory material of calcitonin cells has been observed at 30 and 60 min after the injection. The parathyroids do not show any significant change.
In addition to its role in carbohydrate metabolism, glucagon (a hyperglycemicglycolytic polypeptide of molecular weight 3485), has also been reported to increase urinary excretion of calcium and other electrolytes in dogs (Charbon et al., 1963; Dewonck et ul., 1963; Pullman et al., 1967; Staub et al., 1957) and in man (Elrick et al., 1958), and to produce hypocalcemia in the rat (Morain and Aliapoulios, 1968; Stern and Bell, 1969; Williams et al., 1969), rabbit (Paloyan et al., 1967a), dog (Avioli et ul., 1969; Paloyan et al., 1967b), monkey (Raman, 1970), and man (Avioli et al., 1968; Birge and Avioli, 1969). The present study is aimed to study the impact of exogenously administered glucagon on microscopic structure of calcitonin (C) cells, parathyroid cells, and serum electrolytes of the mongoose. MATERIAL
AND METHODS
Twenty-four mongooses of the species Herpestes edwardsi were procured locally and maintained on live fish for 1 week. They were then divided into two numerically equal groups, i.e., diluent injected (control) and glucagon injected (experimental). They were injected with diluent (controls) and glucagon (experimentals) after an overnight fast.
An initial sample (fasting sample) of blood was collected from the femoral vein before the diluent or glucagon injections were administered (zero time). Crystalline glucagon (Eli Lilly and Co.) in a dosage of 0.2 mg/kg body wt was injected intravenously. It was dissolved in 0.005 N HCI (pH 2.6) and diluted with 0.9% sodium chloride solution containing 0. I% gelatin (diluent) so as to provide a concentration of 0.2 mg/ml of the solution. The controls were given intravenously 1 ml of diluentikg body wt. The blood samples were taken under light ether anesthesia from the femoral vein at 30. 60, 90, 120, 240, and 360 min after the initiation of the injection from both the groups. The analysis of calcium and inorganic phosphate was performed with the serum by Trinder’s (1960) and Fiske and SubbaRow’s (1925) method, respectively. In an effort to evaluate the hypocalcemic effect of glucagon on C cells and parathyroid, the animals were sacrificed immediately after the blood samples were taken in batches of two. The thyroid-parathyroid complex was removed and fixed in glutaraldehyde picric acid (GPA) mixture (Solcia et al.. 1968) and Bouin’s fluid. Sections were cut at 4-6 pm and processed with hematoxylin-eosin, lead-hematoxylin (Solcia ef al., 1969), and Davenport’s silver impregnation (Kameda, 1968).
RESULTS Serum Calcium
In the diluent-injected group only a nominal drop in serum calcium level has been recorded (Fig. 1). 541
00 16-6480179104054 l-05$0 1.0010 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
542
NOTE -
SERUM
CALCIUM
r
SERUM
INORGANIC
-
SERUM
CALCIUM
-
SERUM
INORGANIC
DILUENT
INJECTED
PHOSPHATE GLUCAGON
INJECTED
PHOSPHATE
f
0
60
I20
180
240
300
360
MINUTES
FIG. 1. Changes in the serum calcium and inorganic phosphate level after intravenous injection of diluent and glucagon.
In glucagon-injected animals, the hormone (glucagon) produces a hypocalcemic response which reaches its maximum at 90 min. The serum calcium decreases from a preinjection level of 10.424 c 0.096 mg/lOO ml to 9.420 2 0.113 mg/lOO ml at 90 min and thereafter tends to return toward the normal value so that at 360 min the serum calcium level is not significantly different from the preinjection level (Fig. 1). Phosphate The diluent-injected animals exhibit a slightly raised level of serum inorganic phosphate at 30 min and after that no sig-
Smun
Inorganic
nificant alteration has been recorded (Fig. 1). In glucagon-injected animals the serum inorganic phosphate shows a progressive fall up to 90 min followed by a tendency to become normal (Fig. 1). The Morphological Changes in Calcitonin Cells and Parathyroid The morphological and histochemical details of C cells (Fig. 2) and parathyroid of diluent-injected animals do not show differences from the account given by Swarup and Das (1974, 76) for normal specimens. The C cells of the glucagon-injected ani-
FIG. 2. C; 450. FIG. 3. Ca Iegranulation. LeadFIG. 4. Cakitonin cell from the animal treated for 360 min displaying pycnosis (arrow ,9. Lead hematoxylin x 450. 543
544
NOTE
mals exhibit marked degranulation of their secretory material at 30 and 60 min (Fig. 3) but afterward a large number of them are seen to possess these granules and only a meager percentage of them are in degranulated phase. In the animals treated for 360 min a few calcitonin cells display pycnosis (Fig. 4). The parathyroid of glucagon-injected group has not shown any significant difference from those of d&tent-injected group. DISCUSSION
The results obtained here are consistent with the earlier reports stating that glucagon produces a rather modest but significant hypocalcemia in animals (Charbon et al., 1963) and man (Paloyan, 1967). This hypocalcemic and hypophosphatemic action of glucagon in the mongoose is rapid and short lived and lasts only up to 90 min after a single injection and by 360 min the values return to preinjection levels. This is in agreement with the previous reports which describe the hypocalcemic action of glucagon in animals and in man. The increased urinary excretion of calcium and other electrolytes in dog and in man after glucagon treatment strengthens this observation. While working on the comparative effects of calcitonin and glucagon in rat, Morain and Aliapoulios (1968) have observed that the hypocalcemic response to glucagon is not abolished by prior acute nephrectomy. This has been confirmed by Avioli et al. (1969) and Williams et al. (1969). Thus, the fall in serum calcium could not be attributed to the glucagon-induced calciuria. The present finding in mongooses that produces hypocalcemia by glucagon stimulating the release of calcitonin from the calcitonin cells derives support from the studies in dog (Avioli et al., 1969) and sheep (quoted by Stern and Bell, 1969) as in these studies the hypocalcemic effects of glucagon are stated to have been abolished by thyroidectomy. But glucagon administra-
tion in rats (Bowser et al., 1968; Stern and Bell, 1969; Williams et al., 1969) gives contradictory results as its action in thyroparathyroidectomized and parathyroidectomized rats is similar to that in the intact rats. The latter studies indicate that the glucagon action is not dependent upon the presence or absence of the thyroid and parathyroid. This disparity in findings suggests that there may be species differences in the actions of glucagon. However, parathyroid independence of glucagon action in these reports is similar to the present report as we have not detected any histological change in the mongoose parathyroid gland. The pycnosis of some calcitonin cells observed in the present study may be due to the continued release of calcitonin caused by administration of glucagon. The results obtained from the mongoose are consistent with the earlier view that glucagon acts as secretogogue for calcitonin and might normally be involved in the physiological regulation of secretion of calcitonin. REFERENCES Avioli, L. V., Birge, S. J.. Kanagawa, H., and Shieber, W. (1968). Glucagon-induced hypocalcemia in man. .I. C/in. Znvesf. 47, 3a. Avioli, L. V., Birge, S. J., Scott, S., and Shieber, W. (1969). Role of thyroid gland during glucagoninduced hypocalcemia in the dog. Amer. J. Physiol. 216, 939-945. Avioli, L. V., Shieber, W., and Kipnis, D. M. (1971). Role of glucagon and adrenergic receptors in . thyrocalcitonin release in the dog. Endocrinology 88, 1337-1340. Birge, S., and Avioli, L. V. (1969). Glucagon-induced hypocalcaemia in man. J. C/in. Endocrinol. Metabol. 29, 213. Bowser, E. N., Henderson, W. J., and Williams, G. A. (1968). Glucagon-induced hypocalcaemia in rat. J. Lab.
Clin.
Med.
72, 857.
Charbon, J. A., Hoekstra, M. H., and Kool, D. S. (1963). The influence of glucagon on the urinary excretion of water, sodium, potassium, calcium, magnesium, chloride and inorganic phosphate. Acta Physiol. Pharmacol. Neerl. 12, 48-56. Dewonck, G., Bacq, Z. M.. and Barac, J. (1963). Influence du alucaaon - sur l’excretion urinaire du
calcium chez le chien. C. R. Sot. Biol. 157, 897899. Elrick, H., Huffman, E. R., Hlad, C. J. Jr., Whipple. N., and Staub, A. (1958). Effects of glucagon on renal function in man. J. C/in. Endocrine/. Merabol. 18, 813-824. Fiske, C. H.. and SubbaRow, Y. (1925). The colorimetric determination of phosphorous. J. Biol. Chem.
66, 375-400.
Kameda, Y. (1968). Parafollicular cells of the thyroid as studied with Davenport’s silver impregnation. Arch.
Histol.
Japan
30, 83-94.
Morain, W. D., and Aliapoulios, M. A. (1968). Comparative hypocalcemic effects of thyrocalcitonin and glucagon. in “Abstr. Third Intern. Cong. Endocrinol., Mexico. D. F., June 30-July 5,” No. 157, p. 6. Excerpta Medica Intern. Cong. Series, Amsterdam. Paloyan, E. (1967). Recent developments in the early diagnosis of hyperparathyroidism. Surg. C/in. N. Amer. 47, 61. Paloyan, E., Paloyan, D.. and Harper, P. V. (1%7a). Glucagon-induced hypocalcemia. Metabolism 16, 35-39. Paloyan, E., Paloyan, D., and Harper, P. V. (1967b). The role of glucagon hypersecretion in the relationship of pancreatitis and hyperparathyroidism. Surgery 62, 167-173. Pullman, T. N., Lavender, A. R., and Aho, I. (1967). Direct effects of glucagon on renal hemodynamics and excretion of inorganic ions. Metabolism 16, 358-373. Raman, A. (1970). The effect of glucagon on plasma calcium ion activity in the monkey. Quart. J. Exp. Physiol.
tive staining of endocrine cells by basic dyes after acid hydrolysis. Stain Technol. 43, 257-263. Staub, A.. Springes, V., Stall, F.. and Elrick, H. (1957). A renal action of glucagon. Proc. Sot. Exp.
Biol.
Med.
94, 57-60.
Stern, P. H., and Bell, N. H. (1969). Effects of glucagon on serum calcium in the rat and on bone resorption in tissue culture. Endocrinology 84, 111-117. Swarup, K., and Das, V. K. (1974). Effect of experimental hypercalcaemia on the thyroid calcitonin cells of Indian grey mongoose, Herpestes edwardsi (Geoffroy). Arch. Histol. Japan 37, 121131. Swarup, K.. and Das, V. K. (1976). Calcitonin cells and unusual follicles of the thyroid of the Indian grey mongoose. Herpestes edwardsi (Geoffroy). Acta
Anat.
95, 384-398.
Trinder, P. (1960). In “Microanalysis in Medical Biochemistry” (I. D. P. Wootton, ed.). Churchill, London. Williams, G. A., Bowser. E. N., and Henderson, W. J. (1969). Mode of hypocalcemic action of glucagon in the rat. Endocrinology 85, 537-541. KRISHNA SWARUP AJAI K. SRIVASTAV~ NAGENDRA PRASAD
TEWARI
Department of Zoology University of Gorakhpar Gorakhpur-273001, India Accepted
January
14, 1479
55, 271-274.
Solcia. E., Capella, C., and Vassallo, G. (1969). Lead-haematoxylin as a stain for endocrine cells. Histochemie 20, 116-126. Solcia, E., Vassallo, G.. and Capella, C. (1968). Selec-
I I wish to thank the University Grants Commission, New Delhi for the award of a research fellowship and to Dr. W. W. Bromer of Eli Lilly and Company for the generous gift of porcine glucagon.
AND
COMPARATIVE
ENDOCRINOLOGY
37, 541-545 (1979)
NOTE Response of Calcitonin Cells, Parathyroid, and Serum Electrolytes During Glucagon-Induced Hypocalcemia in the Mongoose, Herpestes edwardsi (Geoffroy) In mongooses (Herpestes edwurdsi) hypocalcemia was induced by an intravenous injection of crystalline glucagon in a dosage of 0.2 mg/kg body wt. Blood samples from each specimen were taken before the injection (zero time) and after 30, 60, 90, 120, 240, and 360 min following the injection. The serum calcium and inorganic phosphate levels were determined. For histological study, animals were killed at the same time intervals after injection. Specific stains were used for the selective staining of calcitonin cells. Glucagon injection evokes a decreased level of serum calcium and inorganic phosphate. A progressive degranulation of the secretory material of calcitonin cells has been observed at 30 and 60 min after the injection. The parathyroids do not show any significant change.
In addition to its role in carbohydrate metabolism, glucagon (a hyperglycemicglycolytic polypeptide of molecular weight 3485), has also been reported to increase urinary excretion of calcium and other electrolytes in dogs (Charbon et al., 1963; Dewonck et ul., 1963; Pullman et al., 1967; Staub et al., 1957) and in man (Elrick et al., 1958), and to produce hypocalcemia in the rat (Morain and Aliapoulios, 1968; Stern and Bell, 1969; Williams et al., 1969), rabbit (Paloyan et al., 1967a), dog (Avioli et ul., 1969; Paloyan et al., 1967b), monkey (Raman, 1970), and man (Avioli et al., 1968; Birge and Avioli, 1969). The present study is aimed to study the impact of exogenously administered glucagon on microscopic structure of calcitonin (C) cells, parathyroid cells, and serum electrolytes of the mongoose. MATERIAL
AND METHODS
Twenty-four mongooses of the species Herpestes edwardsi were procured locally and maintained on live fish for 1 week. They were then divided into two numerically equal groups, i.e., diluent injected (control) and glucagon injected (experimental). They were injected with diluent (controls) and glucagon (experimentals) after an overnight fast.
An initial sample (fasting sample) of blood was collected from the femoral vein before the diluent or glucagon injections were administered (zero time). Crystalline glucagon (Eli Lilly and Co.) in a dosage of 0.2 mg/kg body wt was injected intravenously. It was dissolved in 0.005 N HCI (pH 2.6) and diluted with 0.9% sodium chloride solution containing 0. I% gelatin (diluent) so as to provide a concentration of 0.2 mg/ml of the solution. The controls were given intravenously 1 ml of diluentikg body wt. The blood samples were taken under light ether anesthesia from the femoral vein at 30. 60, 90, 120, 240, and 360 min after the initiation of the injection from both the groups. The analysis of calcium and inorganic phosphate was performed with the serum by Trinder’s (1960) and Fiske and SubbaRow’s (1925) method, respectively. In an effort to evaluate the hypocalcemic effect of glucagon on C cells and parathyroid, the animals were sacrificed immediately after the blood samples were taken in batches of two. The thyroid-parathyroid complex was removed and fixed in glutaraldehyde picric acid (GPA) mixture (Solcia et al.. 1968) and Bouin’s fluid. Sections were cut at 4-6 pm and processed with hematoxylin-eosin, lead-hematoxylin (Solcia ef al., 1969), and Davenport’s silver impregnation (Kameda, 1968).
RESULTS Serum Calcium
In the diluent-injected group only a nominal drop in serum calcium level has been recorded (Fig. 1). 541
00 16-6480179104054 l-05$0 1.0010 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
542
NOTE -
SERUM
CALCIUM
r
SERUM
INORGANIC
-
SERUM
CALCIUM
-
SERUM
INORGANIC
DILUENT
INJECTED
PHOSPHATE GLUCAGON
INJECTED
PHOSPHATE
f
0
60
I20
180
240
300
360
MINUTES
FIG. 1. Changes in the serum calcium and inorganic phosphate level after intravenous injection of diluent and glucagon.
In glucagon-injected animals, the hormone (glucagon) produces a hypocalcemic response which reaches its maximum at 90 min. The serum calcium decreases from a preinjection level of 10.424 c 0.096 mg/lOO ml to 9.420 2 0.113 mg/lOO ml at 90 min and thereafter tends to return toward the normal value so that at 360 min the serum calcium level is not significantly different from the preinjection level (Fig. 1). Phosphate The diluent-injected animals exhibit a slightly raised level of serum inorganic phosphate at 30 min and after that no sig-
Smun
Inorganic
nificant alteration has been recorded (Fig. 1). In glucagon-injected animals the serum inorganic phosphate shows a progressive fall up to 90 min followed by a tendency to become normal (Fig. 1). The Morphological Changes in Calcitonin Cells and Parathyroid The morphological and histochemical details of C cells (Fig. 2) and parathyroid of diluent-injected animals do not show differences from the account given by Swarup and Das (1974, 76) for normal specimens. The C cells of the glucagon-injected ani-
FIG. 2. C; 450. FIG. 3. Ca Iegranulation. LeadFIG. 4. Cakitonin cell from the animal treated for 360 min displaying pycnosis (arrow ,9. Lead hematoxylin x 450. 543
544
NOTE
mals exhibit marked degranulation of their secretory material at 30 and 60 min (Fig. 3) but afterward a large number of them are seen to possess these granules and only a meager percentage of them are in degranulated phase. In the animals treated for 360 min a few calcitonin cells display pycnosis (Fig. 4). The parathyroid of glucagon-injected group has not shown any significant difference from those of d&tent-injected group. DISCUSSION
The results obtained here are consistent with the earlier reports stating that glucagon produces a rather modest but significant hypocalcemia in animals (Charbon et al., 1963) and man (Paloyan, 1967). This hypocalcemic and hypophosphatemic action of glucagon in the mongoose is rapid and short lived and lasts only up to 90 min after a single injection and by 360 min the values return to preinjection levels. This is in agreement with the previous reports which describe the hypocalcemic action of glucagon in animals and in man. The increased urinary excretion of calcium and other electrolytes in dog and in man after glucagon treatment strengthens this observation. While working on the comparative effects of calcitonin and glucagon in rat, Morain and Aliapoulios (1968) have observed that the hypocalcemic response to glucagon is not abolished by prior acute nephrectomy. This has been confirmed by Avioli et al. (1969) and Williams et al. (1969). Thus, the fall in serum calcium could not be attributed to the glucagon-induced calciuria. The present finding in mongooses that produces hypocalcemia by glucagon stimulating the release of calcitonin from the calcitonin cells derives support from the studies in dog (Avioli et al., 1969) and sheep (quoted by Stern and Bell, 1969) as in these studies the hypocalcemic effects of glucagon are stated to have been abolished by thyroidectomy. But glucagon administra-
tion in rats (Bowser et al., 1968; Stern and Bell, 1969; Williams et al., 1969) gives contradictory results as its action in thyroparathyroidectomized and parathyroidectomized rats is similar to that in the intact rats. The latter studies indicate that the glucagon action is not dependent upon the presence or absence of the thyroid and parathyroid. This disparity in findings suggests that there may be species differences in the actions of glucagon. However, parathyroid independence of glucagon action in these reports is similar to the present report as we have not detected any histological change in the mongoose parathyroid gland. The pycnosis of some calcitonin cells observed in the present study may be due to the continued release of calcitonin caused by administration of glucagon. The results obtained from the mongoose are consistent with the earlier view that glucagon acts as secretogogue for calcitonin and might normally be involved in the physiological regulation of secretion of calcitonin. REFERENCES Avioli, L. V., Birge, S. J.. Kanagawa, H., and Shieber, W. (1968). Glucagon-induced hypocalcemia in man. .I. C/in. Znvesf. 47, 3a. Avioli, L. V., Birge, S. J., Scott, S., and Shieber, W. (1969). Role of thyroid gland during glucagoninduced hypocalcemia in the dog. Amer. J. Physiol. 216, 939-945. Avioli, L. V., Shieber, W., and Kipnis, D. M. (1971). Role of glucagon and adrenergic receptors in . thyrocalcitonin release in the dog. Endocrinology 88, 1337-1340. Birge, S., and Avioli, L. V. (1969). Glucagon-induced hypocalcaemia in man. J. C/in. Endocrinol. Metabol. 29, 213. Bowser, E. N., Henderson, W. J., and Williams, G. A. (1968). Glucagon-induced hypocalcaemia in rat. J. Lab.
Clin.
Med.
72, 857.
Charbon, J. A., Hoekstra, M. H., and Kool, D. S. (1963). The influence of glucagon on the urinary excretion of water, sodium, potassium, calcium, magnesium, chloride and inorganic phosphate. Acta Physiol. Pharmacol. Neerl. 12, 48-56. Dewonck, G., Bacq, Z. M.. and Barac, J. (1963). Influence du alucaaon - sur l’excretion urinaire du
calcium chez le chien. C. R. Sot. Biol. 157, 897899. Elrick, H., Huffman, E. R., Hlad, C. J. Jr., Whipple. N., and Staub, A. (1958). Effects of glucagon on renal function in man. J. C/in. Endocrine/. Merabol. 18, 813-824. Fiske, C. H.. and SubbaRow, Y. (1925). The colorimetric determination of phosphorous. J. Biol. Chem.
66, 375-400.
Kameda, Y. (1968). Parafollicular cells of the thyroid as studied with Davenport’s silver impregnation. Arch.
Histol.
Japan
30, 83-94.
Morain, W. D., and Aliapoulios, M. A. (1968). Comparative hypocalcemic effects of thyrocalcitonin and glucagon. in “Abstr. Third Intern. Cong. Endocrinol., Mexico. D. F., June 30-July 5,” No. 157, p. 6. Excerpta Medica Intern. Cong. Series, Amsterdam. Paloyan, E. (1967). Recent developments in the early diagnosis of hyperparathyroidism. Surg. C/in. N. Amer. 47, 61. Paloyan, E., Paloyan, D.. and Harper, P. V. (1%7a). Glucagon-induced hypocalcemia. Metabolism 16, 35-39. Paloyan, E., Paloyan, D., and Harper, P. V. (1967b). The role of glucagon hypersecretion in the relationship of pancreatitis and hyperparathyroidism. Surgery 62, 167-173. Pullman, T. N., Lavender, A. R., and Aho, I. (1967). Direct effects of glucagon on renal hemodynamics and excretion of inorganic ions. Metabolism 16, 358-373. Raman, A. (1970). The effect of glucagon on plasma calcium ion activity in the monkey. Quart. J. Exp. Physiol.
tive staining of endocrine cells by basic dyes after acid hydrolysis. Stain Technol. 43, 257-263. Staub, A.. Springes, V., Stall, F.. and Elrick, H. (1957). A renal action of glucagon. Proc. Sot. Exp.
Biol.
Med.
94, 57-60.
Stern, P. H., and Bell, N. H. (1969). Effects of glucagon on serum calcium in the rat and on bone resorption in tissue culture. Endocrinology 84, 111-117. Swarup, K., and Das, V. K. (1974). Effect of experimental hypercalcaemia on the thyroid calcitonin cells of Indian grey mongoose, Herpestes edwardsi (Geoffroy). Arch. Histol. Japan 37, 121131. Swarup, K.. and Das, V. K. (1976). Calcitonin cells and unusual follicles of the thyroid of the Indian grey mongoose. Herpestes edwardsi (Geoffroy). Acta
Anat.
95, 384-398.
Trinder, P. (1960). In “Microanalysis in Medical Biochemistry” (I. D. P. Wootton, ed.). Churchill, London. Williams, G. A., Bowser. E. N., and Henderson, W. J. (1969). Mode of hypocalcemic action of glucagon in the rat. Endocrinology 85, 537-541. KRISHNA SWARUP AJAI K. SRIVASTAV~ NAGENDRA PRASAD
TEWARI
Department of Zoology University of Gorakhpar Gorakhpur-273001, India Accepted
January
14, 1479
55, 271-274.
Solcia. E., Capella, C., and Vassallo, G. (1969). Lead-haematoxylin as a stain for endocrine cells. Histochemie 20, 116-126. Solcia, E., Vassallo, G.. and Capella, C. (1968). Selec-
I I wish to thank the University Grants Commission, New Delhi for the award of a research fellowship and to Dr. W. W. Bromer of Eli Lilly and Company for the generous gift of porcine glucagon.