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Distribution of immunoassayable glucagon in gastrointestinal tissues
Metabolism Clinical and Experimental VOL.
XV, NO.
10
PRELIMINARY
OCTOBER,
1966
REPORT
Distribution of Immunoassayable Glucagon in Gastrointestinal Tissues RI/ ROCER H. UNGER, HERMANN KETTERER AXD ANNA M. EISENTRAUT Glucagon has been identified by a specific radioimmunoassay in extracts of canine stomach and duodenum, rat stomach, duodenum, and jejunum, and human jejunum. Canine stomach glucagon appears to be similar to glucagon of
pancreatic origin with respect to its immunoreactivity, its gel filtration elution pattern, and its in vivo hyperglycemic activity. The implication of these findings are briefly discussed. (hletabolism 15: No. 16, October, 865-867, 1966)
LTHOUGH A glucagon-like hyperglycemic glycogenolytic factor was rcported by Sutherland and deDuvel to be present in acid-alcohol extrnctl; and more recently Makman and Sutherland” of canine gastric mucosa, have reported a glucagon-like effect of extracts of human gastrointestinal by liver, a careful survey of gastrotissues on cyclic 3’, S-AMP production intestinal glucagon using the specific radioimmunoassay for glucagon,” has not as yet been reported, although immunoassayable glucagon was reported to be present in both canine and human gastrointestinal tissues.“ The physiologic importance of such information has recently been enhanced by the reports by Lawrence” and Samols” that the ingestion of large doses of glucose induce a rise in plasma glucagon, and the suggestion by SamoW that this glucagon may originate from the gastrointestinal tract. 7-h e present studies were designed to determine by specific radioimmuno-
A
From the Department of Internal Medicine, Unioersity of Texas Southwestern Medical School and Veterans Administration Hospital, Dallas, Texas. This stud!/ was supported by U.S.P.H.S. Grant AM 0270048, and by the Upjohn Compamy, Kalamazoo, Michigan. Received for publication July 1, 1966. ROGER H. UNGER, M.D.: Associate Professor of Internal Medicine, University of Texas Southwestern Medical School, and Director of Research and Chief of Metabolism Section. Veterans Administration Hospital, Dallas, Texas. HERMAXN KETTERER, M.D. Research Fellow in Internal Medicine. University of Texas Southwestern Medical School, Dallas, TWXIY. ANNA hf. EISENTRAUT:Riologirt, Vrtc~rrtruAdministration Hospital. Dallas, Texas. 865
866
UNCER,
Table l.-Extractable ____~
lmmunoassayable (pg. equivalents/Cm. ____
__
______~_~
-
Rat Dog Human (surgical specimen Human (postmortem specimens)
KETTERER
AND
Glucagon of Gastrointestinal of wet weight)
EISENTRAUl
Tissues -___-
Stomach
Duodenum
Jejunum
0.0031 0.2-0.47
0.006 0.043
0.14 2.1
2.4 -1.5
O-O.004
-
0.061
0.4-9.0
0.004
0.009
-
Ile11m
0.032
Pancreas
~__~.
0.24” (O-0.8) -.____
‘Mean
of 50 pancreases.
assay the distribution and relative concentration testinal tissues of rat. dog and man.
of glucagon
in the gastroin-
METHODS All animal tissues were obtained before death to avoid postmortem proteolysis which appears to reduce pancreatic glucagon in man. Small pieces of human gastrointestinal tissue were obtained at surgery, and an entire human small intestine was obtained shortly after an acute traumatic death of a previously healthy young man. All tissues were frozen in dry ict ar.d stored at -10 C. until extraction. Tissues were extracted by the method of Kenny.’ At the time of assay, 10 mg. of desiccated extract was reconstituted in 10 ml. of 0.2 M. glycine buffer (pH 8.8) containing 0.25 per cent albumin and 1:lOO rabbit serum. Higher dilutions were made as required with the same butier. Glucagon was assayed by a modification of the technic.” Insulin was assayed 1~). the method of previously reported radioinlmrlnoassa) Yalow and Berson.8 In other studies designed to compare the molecular size of gastric glucagon with glucagon11s’. 0.5 ml. of undiluted extract solution, to which 100 pc~g. plucagon-Ilsl had been added. was passed through a 110 X 0.7 cm. column of C-25 Sephadex gel and immunoassa!.able ghlcagon and the total radioactivity in each 2 ml. ehlate was measured. KESULTS
Immunoassayable glucagon was present in varying concentrations in extracts of dog stomach and small bowel, in rat jejunum and duodenum, and in a specimen of human jejunum obtained from a patient during a revision of gastrojejunostomy. Very low concentrations of extractable glucagon were found in the portions of human stomach surveyed. These results are summarized in Table 1. Insulin concentration, measured so as to exclude contamination with hormones of pancreatic origin, was negligible in all tissue extracts. Serial dilutions of canine gastric and pancreatic extracts were assayed fol glucagon to determine if gastric and pancreatic glucagon are immunologically similar. With each extract a proportional decline in measured glucagon concentration was observed, suggesting their similarity. To obtain a gross estimate of molecular size, eluates of canine stomach extract, to which pancreatic beef-pork glucagon-1131 had been added, were was collected after gel filtration. The elution curve of total radioactivity virtually identical to that of immunoassayable glucagon, suggesting a similal range of molecular size.
(:LUCACGN
DISTHIBUTION
IN
GASTROINTESTINAL
867
TISSUES
Finally, the biologic activity of gastric glucagon present in the glucagon peak of the Sephadex elution curve was determined. The injection into a nembutalized dog of an estimated 100 mpg. of immunoassayable gastric glucagon caused a prompt rise in arterial glucose concentration similar to that observed in other experiments with commercial beef-pork glucagon. DISCUSSION
The results clearly indicate that glucagon or a factor with the same immunochemical characteristics is present in acid-alcohol extracts of gastrointestinal tissues of rat, dog and man. Its similarity to pancreatic glucagon with respect to biologic activity and to range of molecular size is suggested by the studies of canine gastric glucagon. In all 3 species the tissue concentrations of glucagon present in the gastrointestinal tract are estimated to be well within the range required to induce physiologic effects, even if turnover of hormone is no more than 5 per cent per hour. Thus, the study both confirms the original conclusions of Sutherland and deDuve,’ and indicates that the suggestion of Samols” concerning a regulatory role of gastrointestinal glucagon, while in no sense substantiated, is within the realm of possibility. REFERENCES 1. Sutherland.
E. U:., and del)uve. C.: Origin and distribution of the hyperglycemic-glycogenolytic factor of the pancreas. J. Biol. Chem. 175663, 1948. 2. Makman, M. H., and Sutherland, E. u’.: Use of liver adenyl cyclase for assayof glucagon in human gastrointestinal tract and pancreas. Endocrinology 75: 127. 1961. :3. Linger, R. H., Eisentraut, A. M.. McCall. M. S.. and Madison. L. L.: Glucagon antibodies and an immunoassay for ghrcagon. J. Clin. Invest. 40:1280, 1961. 4. -, -, and Sims. Ii., McCall. M. S., and Madison. L. L.: Sites of origin of glucagon
in
dogs
and
humans.
Clin.
Kes. 9:53,
1961.
5. Lawrence, A. M.: Radioimmunoassayable glucagon levels in man: effects of starvation, hypoglycemia, and glucose administration. Proc. Nat. Acad. Sci. 55:317. 1966. 6. Samols. E., Tyler, J.. Marri, G., and Marks, V.: Stimulation of glucagon secretion by oral glucose. Lancet 7424: 1287, 7. Kenny,
1965. A. J.:
Extractable
the human pancreas. crinol. 15:1089. 1955. 8. Yalow.
R. S., and
Berson,
assay of endogenous man. J. Chn. Invest.
glucagon J.
Clin.
of
Endo-
S. A.: In~n~uno-
plasma 3931157.
insulin 1960.
in
XV, NO.
10
PRELIMINARY
OCTOBER,
1966
REPORT
Distribution of Immunoassayable Glucagon in Gastrointestinal Tissues RI/ ROCER H. UNGER, HERMANN KETTERER AXD ANNA M. EISENTRAUT Glucagon has been identified by a specific radioimmunoassay in extracts of canine stomach and duodenum, rat stomach, duodenum, and jejunum, and human jejunum. Canine stomach glucagon appears to be similar to glucagon of
pancreatic origin with respect to its immunoreactivity, its gel filtration elution pattern, and its in vivo hyperglycemic activity. The implication of these findings are briefly discussed. (hletabolism 15: No. 16, October, 865-867, 1966)
LTHOUGH A glucagon-like hyperglycemic glycogenolytic factor was rcported by Sutherland and deDuvel to be present in acid-alcohol extrnctl; and more recently Makman and Sutherland” of canine gastric mucosa, have reported a glucagon-like effect of extracts of human gastrointestinal by liver, a careful survey of gastrotissues on cyclic 3’, S-AMP production intestinal glucagon using the specific radioimmunoassay for glucagon,” has not as yet been reported, although immunoassayable glucagon was reported to be present in both canine and human gastrointestinal tissues.“ The physiologic importance of such information has recently been enhanced by the reports by Lawrence” and Samols” that the ingestion of large doses of glucose induce a rise in plasma glucagon, and the suggestion by SamoW that this glucagon may originate from the gastrointestinal tract. 7-h e present studies were designed to determine by specific radioimmuno-
A
From the Department of Internal Medicine, Unioersity of Texas Southwestern Medical School and Veterans Administration Hospital, Dallas, Texas. This stud!/ was supported by U.S.P.H.S. Grant AM 0270048, and by the Upjohn Compamy, Kalamazoo, Michigan. Received for publication July 1, 1966. ROGER H. UNGER, M.D.: Associate Professor of Internal Medicine, University of Texas Southwestern Medical School, and Director of Research and Chief of Metabolism Section. Veterans Administration Hospital, Dallas, Texas. HERMAXN KETTERER, M.D. Research Fellow in Internal Medicine. University of Texas Southwestern Medical School, Dallas, TWXIY. ANNA hf. EISENTRAUT:Riologirt, Vrtc~rrtruAdministration Hospital. Dallas, Texas. 865
866
UNCER,
Table l.-Extractable ____~
lmmunoassayable (pg. equivalents/Cm. ____
__
______~_~
-
Rat Dog Human (surgical specimen Human (postmortem specimens)
KETTERER
AND
Glucagon of Gastrointestinal of wet weight)
EISENTRAUl
Tissues -___-
Stomach
Duodenum
Jejunum
0.0031 0.2-0.47
0.006 0.043
0.14 2.1
2.4 -1.5
O-O.004
-
0.061
0.4-9.0
0.004
0.009
-
Ile11m
0.032
Pancreas
~__~.
0.24” (O-0.8) -.____
‘Mean
of 50 pancreases.
assay the distribution and relative concentration testinal tissues of rat. dog and man.
of glucagon
in the gastroin-
METHODS All animal tissues were obtained before death to avoid postmortem proteolysis which appears to reduce pancreatic glucagon in man. Small pieces of human gastrointestinal tissue were obtained at surgery, and an entire human small intestine was obtained shortly after an acute traumatic death of a previously healthy young man. All tissues were frozen in dry ict ar.d stored at -10 C. until extraction. Tissues were extracted by the method of Kenny.’ At the time of assay, 10 mg. of desiccated extract was reconstituted in 10 ml. of 0.2 M. glycine buffer (pH 8.8) containing 0.25 per cent albumin and 1:lOO rabbit serum. Higher dilutions were made as required with the same butier. Glucagon was assayed by a modification of the technic.” Insulin was assayed 1~). the method of previously reported radioinlmrlnoassa) Yalow and Berson.8 In other studies designed to compare the molecular size of gastric glucagon with glucagon11s’. 0.5 ml. of undiluted extract solution, to which 100 pc~g. plucagon-Ilsl had been added. was passed through a 110 X 0.7 cm. column of C-25 Sephadex gel and immunoassa!.able ghlcagon and the total radioactivity in each 2 ml. ehlate was measured. KESULTS
Immunoassayable glucagon was present in varying concentrations in extracts of dog stomach and small bowel, in rat jejunum and duodenum, and in a specimen of human jejunum obtained from a patient during a revision of gastrojejunostomy. Very low concentrations of extractable glucagon were found in the portions of human stomach surveyed. These results are summarized in Table 1. Insulin concentration, measured so as to exclude contamination with hormones of pancreatic origin, was negligible in all tissue extracts. Serial dilutions of canine gastric and pancreatic extracts were assayed fol glucagon to determine if gastric and pancreatic glucagon are immunologically similar. With each extract a proportional decline in measured glucagon concentration was observed, suggesting their similarity. To obtain a gross estimate of molecular size, eluates of canine stomach extract, to which pancreatic beef-pork glucagon-1131 had been added, were was collected after gel filtration. The elution curve of total radioactivity virtually identical to that of immunoassayable glucagon, suggesting a similal range of molecular size.
(:LUCACGN
DISTHIBUTION
IN
GASTROINTESTINAL
867
TISSUES
Finally, the biologic activity of gastric glucagon present in the glucagon peak of the Sephadex elution curve was determined. The injection into a nembutalized dog of an estimated 100 mpg. of immunoassayable gastric glucagon caused a prompt rise in arterial glucose concentration similar to that observed in other experiments with commercial beef-pork glucagon. DISCUSSION
The results clearly indicate that glucagon or a factor with the same immunochemical characteristics is present in acid-alcohol extracts of gastrointestinal tissues of rat, dog and man. Its similarity to pancreatic glucagon with respect to biologic activity and to range of molecular size is suggested by the studies of canine gastric glucagon. In all 3 species the tissue concentrations of glucagon present in the gastrointestinal tract are estimated to be well within the range required to induce physiologic effects, even if turnover of hormone is no more than 5 per cent per hour. Thus, the study both confirms the original conclusions of Sutherland and deDuve,’ and indicates that the suggestion of Samols” concerning a regulatory role of gastrointestinal glucagon, while in no sense substantiated, is within the realm of possibility. REFERENCES 1. Sutherland.
E. U:., and del)uve. C.: Origin and distribution of the hyperglycemic-glycogenolytic factor of the pancreas. J. Biol. Chem. 175663, 1948. 2. Makman, M. H., and Sutherland, E. u’.: Use of liver adenyl cyclase for assayof glucagon in human gastrointestinal tract and pancreas. Endocrinology 75: 127. 1961. :3. Linger, R. H., Eisentraut, A. M.. McCall. M. S.. and Madison. L. L.: Glucagon antibodies and an immunoassay for ghrcagon. J. Clin. Invest. 40:1280, 1961. 4. -, -, and Sims. Ii., McCall. M. S., and Madison. L. L.: Sites of origin of glucagon
in
dogs
and
humans.
Clin.
Kes. 9:53,
1961.
5. Lawrence, A. M.: Radioimmunoassayable glucagon levels in man: effects of starvation, hypoglycemia, and glucose administration. Proc. Nat. Acad. Sci. 55:317. 1966. 6. Samols. E., Tyler, J.. Marri, G., and Marks, V.: Stimulation of glucagon secretion by oral glucose. Lancet 7424: 1287, 7. Kenny,
1965. A. J.:
Extractable
the human pancreas. crinol. 15:1089. 1955. 8. Yalow.
R. S., and
Berson,
assay of endogenous man. J. Chn. Invest.
glucagon J.
Clin.
of
Endo-
S. A.: In~n~uno-
plasma 3931157.
insulin 1960.
in