0013-7227/78/1035-1579$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 5 Printed in U.S.A.
Glucose and Insulin in the Regulation of Glucagon Release from the Isolated Perfused Dog Stomach* PIERRE J. LEFEBVRE AND ALFRED S. LUYCKXf Division of Diabetes, Institute of Medicine, University of Liege, B-4020 Liege, Belgium ABSTRACT. The respective roles of glucose and insulin in the regulation of glucagon release from the canine stomach were investigated using an isolated blood-perfused preparation. At normal blood glucose and plasma insulin levels, the stomach released small amounts of glucagon. Such basal gastric glucagon release was not modified by hyperglycemia. In contrast, gastric glucagon release was increased by hypoglycemia or 2-deoxy-D-glucose-induced cytoglycopenia. Antibody
neutralization of basal circulating concentrations of insulin (10 ± 1 juU/ml) doubled the stimulation induced by hypoglycemia alone. It is concluded that: 1) suppression of gastric glucagon release is observed with very low concentrations of insulin; 2) basal gastric glucagon release is not further suppressed by hyperglycemia; and 3) that hypoglycemia and cytoglycopenia stimulate gastric glucagon secretion. (Endocrinology 103: 1579, 1978)
I
T IS now accepted that the canine stomach possesses cells identical to pancreatic A cells (1-3) and that this organ contains (1, 3, 4) and releases (5-9) a polypeptide which is immunochemically, physicochemically, and biologically indistinguishable from pancreatic glucagon. However, recent in vivo investigations have suggested that the stomach is not a major source of circulating glucagon in normal dogs (7). In contrast, it is probably the main, if not the only, source of glucagon in the depancreatized dog (8); it is also a significant source of glucagon in the alloxan-diabetic dog (9). In these last two instances, exogenous insulin at low rates of infusion rapidly abolished gastric glucagon secretion. The present studies were designed to examine the respective roles of glucose and insulin in the regulation of basal glucagon release from the isolated perfused canine stomach, a system which already permitted us to study the kinetics of arginine-induced gastric glucagon release (5) as well as its modifications under various experimental conditions (6, 10).
Materials and Methods
Stomachs from overnight-fasted normal mongrel dogs of both sexes were isolated with their arterial and venous supply and perfused with whole blood collected from large blood-donor dogs according to a procedure described in detail elsewhere (6). The perfusing blood was supplemented with 1000 U/ml Trasylol (Bayer, Leverkusen, West Germany). Immediately after the beginning of the perfusion, the glucose concentration of the perfusing blood was measured (see below) and eventually adjusted to the level desired by addition to the blood reservoir of a given volume of a 10% (wt/vol) glucose solution in distilled water. The desired blood glucose concentration (about 100 or 300 mg/100 ml) was maintained throughout the experiment by infusing into the blood reservoir a 12 mg/ml glucose solution at the rate of 0.5 ml/min: this amount was calculated to compensate for glucose consumption by both the stomach and the blood cells. When hypoglycemia was desired, the blood was recirculated for 45-60 min without adding glucose; this procedure made it possible to attain blood glucose concentrations of 25-30 mg/100 ml. In some experiments, the following substances were added to the perfusing blood: 1) 2-deoxy-D-glucose (BDH Laboratory Chemicals, Poole, England) as a 10% (wt/vol) solution in disReceived October 11, 1977. Address reprint requests to: Dr. P. J. Lefebvre, Institut tilled water in a volume calculated to attain a final de Medecine, Hopital de Baviere, B-4020 Liege, Belgium. blood concentration of 100 mg/100 ml (in these * This work was supported by the Fonds National de experiments the blood glucose concentration was la Recherche Scientifique and the Fonds de la Recherche also maintained at about 100 mg/100 ml); 2) antiinScientifique Medicale of Belgium; presented in part at the sulin serum (guinea pig anti-insulin serum, GPAIS American Diabetes Association Meeting June 5-7, 1977, 567 from P. H. Wright, Indianapolis, IN, obtained St. Louis, MO. through the courtesy of W. Malaisse, Bruxelles). f Maitre de Recherches of the F.N.R.S. 1579
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 01:48 For personal use only. No other uses without permission. . All rights reserved.
LEFEBVRE AND LUYCKX
1580
Fifty microliters the original serum diluted in 20 ml saline containing 1 g/100 ml albumin (lot PL77C24 from Institut Merieux, Lyon, France) were added to the blood reservoir (based on the neutralizing capacity of this antiserum, the quantity added represented an excess of 20 to 30 times the amount needed to neutralize the endogenous insulin present in the system). Blood sampling began after a 40-60 min equilibration period in each experimental condition. Each collection period comprised three 30sec consecutive venous collections immediately preceded and followed by one arterial sampling. Two to nine collection periods were carried out in each experiment. As previously described, gastric glucagon production (6) was calculated by multiplying the venoarterial difference in plasma glucagon concentration by the plasma flow (the latter being derived from the blood flow and the hematocrit). Blood glucose concentration was determined by the hexokinase method (11) adapted to the Technicon AutoAnalyzer (Technicon Instrument Co., Tarrytown, NY). For hormone assays, 0.4 ml of a solution containing Trasylol, 5000 U/ml, and Na2EDTA, 12 mg/ml, were added to 3.6 ml blood. The mixture was immediately centrifuged at 4 C, and the separated plasma was stored at -20 C. Plasma glucagon was determined in duplicate assays by a classical immunoassay procedure (12), using porcine [125I] iodoglucagon (NEN Chemicals, D 6072, Dreieichenhain, West Germany) as tracer, 30K antiserum (provided by Dr. R. H. Unger, Dallas, Texas) and dextran-charcoal separation of free and antibodybound hormone. Plasma insulin was determined as previously described (13) using human insulin as standard. Statistical evaluation included variance analysis and the Student's t test for unpaired data.
Results Changes in blood glucose concentration and gastric glucagon release When glucose was present at a "normal" concentration in the perfusing blood, 101 ± 5 (mean ± SE) mg/100 ml, mean gastric glucagon output was 279 ± 84 pg/100 g stomach/min (34 determinations in six perfusions); this mean value is statistically different from 0 and indicates a net modest glucagon output by the stomach. In some instances, a small but significant uptake of glucagon was recorded. Plasma insulin in these experiments was 8 ± 1 /xU/ml. When blood glucose was increased to 292 ± 4 mg/100 ml and plasma
Endo • 1978 Vol 103 • No 5
insulin kept at basal levels, 7 ± 1.5 juU/ml, gastric glucagon output was 359 ± 83 pg/100 g/min (30 determinations in five perfusions), a value not statistically different from the one obtained in normoglycemia. When the stomach was perfused with blood containing 32 ± 1 mg/100 ml glucose and 7 ± 1 juU insulin/ml plasma, gastric glucagon output was significantly increased: 610 ± 79 pg/100 g/min (73 determinations in nine perfusions; P < 0.02 versus release in normoglycemia). Effect of'2-deoxy-D -glucose When the blood perfusing the stomach contained equimolar concentrations of glucose (100 ± 6 mg/100 ml) and 2-deoxy-D-glucose (100 mg/100 ml), glucagon release was enhanced about 5 times: 1482 ± 142 pg/100 g/min (36 determinations in four perfusions; P < 0.001 versus release in normoglycemia). Figure 1 illustrates the changes in gastric glucagon production which occurred after 2deoxy-D-glucose was added to the perfusing blood. Effect of antibody neutralization of basal insulin levels The fact the hypoglycemia led to a rise in gastric glucagon release which was relatively modest when compared to that provoked by 2-deoxy-D-glucose-induced cytoglycopenia led us to suspect that basal insulin concentrations in the perfusing blood may cause some glucagon suppression. Therefore, stomachs were perfused with a hypoglycemic blood (31 ± 2 mg/100 ml) whose insulin content (10 it 1 T
rh
1000 500
fi. Glucose 100mg/100ml
Glucose 100mg/100 ml • 2-0eoxy-D-glucose lOOmg/IOOml
FIG. 1. Glucagon release from the isolated dog stomach perfused with blood containing 100 mg/100 ml glucose and supplemented, at time 0, by 100 mg/100 ml 2-deoxyD-glucose. Mean of four perfusions. Results are given as mean ± SEM.
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REGULATION OF GASTRIC GLUCAGON RELEASE juU/ml plasma) was neutralized by an excess of insulin antibodies (see Materials and Methods). Under these conditions, gastric glucagon release was increased to 1122 ± 280 pg/100 g/min (2l determinations in four perfusions), a value significantly greater (P < 0.02) than the one obtained by hypoglycemia alone and of the same order of magnitude as the one observed under the influence of 2-deoxy-D-glucose. Discussion In their study of gastric A cell function in normal conscious dogs, Munoz-Barragan et al. (7) concluded that the stomach is probably not a major source of circulating glucagon. In contrast, Blazquez et al. showed that in both insulin-deprived depancreatized (8) or alloxan-diabetic dogs (9), the stomach contributed much to the increased glucagon circulating levels; in both conditions, insulin rapidly suppressed gastric glucagon release. The isolated perfused dog stomach provides a unique tool permitting investigation of A cell function in the absence of endogenously released insulin (5, 6). In the present investigation, we have demonstrated that, at basal concentrations of glucose and insulin, the release of glucagon by the dog stomach was indeed very modest (and in some experiments was even replaced by a discrete but significant glucagon uptake or degradation), thus supporting the conclusions reached by MunozBarragan et al. (7). In this isolated system, hyperglycemia alone did not further reduce this small basal gastric glucagon release. On the contrary, hypoglycemia slightly but significantly increased gastric glucagon release; its effect was doubled when the low concentration of insulin present in the perfusing blood (collected from overnight-fasted normal blood donor dogs) was neutralized by an excess of anti-insulin serum. This last finding demonstrates that even the presence of very small quantities of insulin is sufficient to suppress gastric glucagon release. Insulin may affect glucagon release either directly, as suggested by some observations made on the pancreatic A cell (see review, Ref. 14), or indirectly, by
1581
permitting glucose, even at low concentrations, to enter the A cell and suppress glucagon secretion (15-17). This exquisite sensitivity of the gastric A cell to insulin explains the dramatic fall in gastric glucagon release reported in pancreatectomized (8) or alloxan-diabetic dogs (9) when small amounts of insulin (0.0015 U/kg/min) are infused in these animals. Our data suggest that concentrations of insulin as low as 5-10 /xU/ml are sufficient to inhibit hypoglycemia-induced gastric glucagon release, although higher insulin concentrations may be required to inhibit arginine-stimulated gastric glucagon release (6). Cytoglycopenia, induced by 2-deoxy-D-glucose, a nonmetabolizable glucose analog which blocks intracellular glucose metabolism, is also a potent stimulus of gastric glucagon release as it is for pancreatic glucagon (18). The stimulation induced by 2-deoxyglucose is of the same order of magnitude as that obtained by simultaneous hypoglycemia and antibody-induced insulin withdrawal; it remains, however, below the maximal rate of gastric glucagon release obtained under the influence of 10 mM arginine (6). These results emphasize the critical role of insulin, even at very low concentrations, in the control of gastric glucagon secretion in dogs; they suggest that no conclusion can be reached about the absence of extrapancreatic sources of circulating glucagon in any species, including man, as long as a complete insulin deficiency is not produced. Acknowledgments We thank D. Binder for his help in the preparation of this manuscript. We are indebted to Professor A. Nizet for providing laboratory facilities, to Professor F. Monfort for his help in the statistical analyses, to Dr. Wald (Bayer, Brussels) for the generous gift of Trasylol, and to Dr. W. Malaisse (Brussels) for the generous gift of anti-insulin antiserum. We acknowledge with thanks the skillful technical assistance of Mr. and Mrs. H. Thoumsin, A. Rombeaux, C. Cartenstadt, C. Borremans, and R. Lallemand. We are indebted also to E. Vaessen-Petit for her secretarial assistance.
References 1. Sasaki, H., B. Rubalcava, D. Baetens, E. Blazquez, C. B. Srikant, L. Orci, and R. H. Unger, Identification of glucagon in the gastrointestinal tract, J Clin Invest
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 01:48 For personal use only. No other uses without permission. . All rights reserved.
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56: 135, 1975. 2. Baetens, D., C. Rufener, C. B. Srikant, R. Dobbs, R. H. Unger, and L. Orci, Identification of glucagonproducing cells (A cells) in dog gastric mucosa, J Cell Biol 69: 455, 1976. 3. Larason, L. I., J. Hoist, R. Hakanson, and F. Sundler, Distribution and properties of glucagon immunoreactivity in the digestive tract of various mammals: an immunohistochemical and immunochemical study, Histochemistry 44: 281, 1975. 4. Morita, S., K. Doi, C. C. Yip, and M. Vranic, Measurements and partial characterization of immunoreactive glucagon in gastrointestinal tissues of dogs, Diabetes 25: 1018,1976. 5. Lefebvre, P. J., A. S. Luyckx, A. H. Brassinne, and A. H. Nizet, Glucagon and gastrin release by the isolated perfused dog stomach in response to arginine, Metabolism (Suppl. 7)25: 1477, 1976. 6. Lefebvre, P. J., and A. S. Luyckx, Factors controlling gastric glucagon release, J Clin Invest 59: 716, 1976. 7. Munoz-Barragan, M., E. Blazquez, G. S. Patton, R. E. Dobbs, and R. H. Unger, Gastric A-cell function in normal dogs, Am J Physiol 231:1057, 1976. 8. Blazquez, E., L. Munoz-Barragan, G. S. Patton, L. Oric, R. E. Dobbs, and R. H. Unger, Gastric A-cell function in insulin-deprived depancreatized dogs, Endocrinology 99: 1182, 1976. 9. Blazquez, E., L. Munoz-Barragan, G. S. Patton, R. E. Dobbs, and R. H. Unger, Demonstration of gastric glucagon hypersecretion in insulin-deprived alloxandiabetic dogs, J Lab Clin Med 89: 971, 1977. 10. Lefebvre, P. J., A. S. Luyckx, and A. H. Brassinne,
Endo • 1978 Vol 103 • No 5
Vagal stimulation and its role in eliciting gastrin but not glucagon release from the isolated perfused dog stomach, Gut 19: 185, 1978. 11. Schmidt, F. H., Enzymatic determination of glucose and fructose simultaneously. Klin Wschr 39: 1244, 1961. 12. Luyckx, A. S., Immunoassays for glucagon, In Lefebvre, P. J., and R. H. Unger (eds.), Glucagon: Molecular Physiology, Clinical and Therapeutic Implications, Pergamon Press, Oxford, 1972, p. 285. 13. Quabbe, H. J., Modifikation der radioimmunologischen Insulin-bestimmung nach Hales und Randle, Diabetologia 5: 107, 1969. 14. Samols, E., J. Tyler, and V. Marks, Glucagon-Insulin Interrelationships, In Lefebvre, P. J., and R. H. Unger (eds.), Glucagon: Molecular Physiology, Clinical and Therapeutic Implications, Pergamon Press, Oxford, 1972, p. 151. 15. Unger, R. H., E. Aguilar-Parada, W. A. Muller, and A. M. Eisentraut, Studies of pancreatic alpha-cell function in normal and diabetic subjects, J Clin Invest 49: 837, 1970. 16. Massi-Benedetti, F., A. Falorni, A. Luyckx and P. Lefebvre, Inhibition of glucagon in the human newborn by simultaneous administration of glucose and insulin, Horm Metab Res 6: 392, 1974. 17. Ostenson, C.-G., and C. Hellerstrom, Effect of insulin on the glucose utilization of the pancreatic A2-cell of the guinea-pig, Diabetologia 12: 413, 1976. 18. Muller, W. A., G. R. Faloona, and R. H. Unger, The effect of experimental insulin deficiency on glucagon secretion, J Clin Invest 50: 1992, 1971.
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Vol. 103, No. 5 Printed in U.S.A.
Glucose and Insulin in the Regulation of Glucagon Release from the Isolated Perfused Dog Stomach* PIERRE J. LEFEBVRE AND ALFRED S. LUYCKXf Division of Diabetes, Institute of Medicine, University of Liege, B-4020 Liege, Belgium ABSTRACT. The respective roles of glucose and insulin in the regulation of glucagon release from the canine stomach were investigated using an isolated blood-perfused preparation. At normal blood glucose and plasma insulin levels, the stomach released small amounts of glucagon. Such basal gastric glucagon release was not modified by hyperglycemia. In contrast, gastric glucagon release was increased by hypoglycemia or 2-deoxy-D-glucose-induced cytoglycopenia. Antibody
neutralization of basal circulating concentrations of insulin (10 ± 1 juU/ml) doubled the stimulation induced by hypoglycemia alone. It is concluded that: 1) suppression of gastric glucagon release is observed with very low concentrations of insulin; 2) basal gastric glucagon release is not further suppressed by hyperglycemia; and 3) that hypoglycemia and cytoglycopenia stimulate gastric glucagon secretion. (Endocrinology 103: 1579, 1978)
I
T IS now accepted that the canine stomach possesses cells identical to pancreatic A cells (1-3) and that this organ contains (1, 3, 4) and releases (5-9) a polypeptide which is immunochemically, physicochemically, and biologically indistinguishable from pancreatic glucagon. However, recent in vivo investigations have suggested that the stomach is not a major source of circulating glucagon in normal dogs (7). In contrast, it is probably the main, if not the only, source of glucagon in the depancreatized dog (8); it is also a significant source of glucagon in the alloxan-diabetic dog (9). In these last two instances, exogenous insulin at low rates of infusion rapidly abolished gastric glucagon secretion. The present studies were designed to examine the respective roles of glucose and insulin in the regulation of basal glucagon release from the isolated perfused canine stomach, a system which already permitted us to study the kinetics of arginine-induced gastric glucagon release (5) as well as its modifications under various experimental conditions (6, 10).
Materials and Methods
Stomachs from overnight-fasted normal mongrel dogs of both sexes were isolated with their arterial and venous supply and perfused with whole blood collected from large blood-donor dogs according to a procedure described in detail elsewhere (6). The perfusing blood was supplemented with 1000 U/ml Trasylol (Bayer, Leverkusen, West Germany). Immediately after the beginning of the perfusion, the glucose concentration of the perfusing blood was measured (see below) and eventually adjusted to the level desired by addition to the blood reservoir of a given volume of a 10% (wt/vol) glucose solution in distilled water. The desired blood glucose concentration (about 100 or 300 mg/100 ml) was maintained throughout the experiment by infusing into the blood reservoir a 12 mg/ml glucose solution at the rate of 0.5 ml/min: this amount was calculated to compensate for glucose consumption by both the stomach and the blood cells. When hypoglycemia was desired, the blood was recirculated for 45-60 min without adding glucose; this procedure made it possible to attain blood glucose concentrations of 25-30 mg/100 ml. In some experiments, the following substances were added to the perfusing blood: 1) 2-deoxy-D-glucose (BDH Laboratory Chemicals, Poole, England) as a 10% (wt/vol) solution in disReceived October 11, 1977. Address reprint requests to: Dr. P. J. Lefebvre, Institut tilled water in a volume calculated to attain a final de Medecine, Hopital de Baviere, B-4020 Liege, Belgium. blood concentration of 100 mg/100 ml (in these * This work was supported by the Fonds National de experiments the blood glucose concentration was la Recherche Scientifique and the Fonds de la Recherche also maintained at about 100 mg/100 ml); 2) antiinScientifique Medicale of Belgium; presented in part at the sulin serum (guinea pig anti-insulin serum, GPAIS American Diabetes Association Meeting June 5-7, 1977, 567 from P. H. Wright, Indianapolis, IN, obtained St. Louis, MO. through the courtesy of W. Malaisse, Bruxelles). f Maitre de Recherches of the F.N.R.S. 1579
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 01:48 For personal use only. No other uses without permission. . All rights reserved.
LEFEBVRE AND LUYCKX
1580
Fifty microliters the original serum diluted in 20 ml saline containing 1 g/100 ml albumin (lot PL77C24 from Institut Merieux, Lyon, France) were added to the blood reservoir (based on the neutralizing capacity of this antiserum, the quantity added represented an excess of 20 to 30 times the amount needed to neutralize the endogenous insulin present in the system). Blood sampling began after a 40-60 min equilibration period in each experimental condition. Each collection period comprised three 30sec consecutive venous collections immediately preceded and followed by one arterial sampling. Two to nine collection periods were carried out in each experiment. As previously described, gastric glucagon production (6) was calculated by multiplying the venoarterial difference in plasma glucagon concentration by the plasma flow (the latter being derived from the blood flow and the hematocrit). Blood glucose concentration was determined by the hexokinase method (11) adapted to the Technicon AutoAnalyzer (Technicon Instrument Co., Tarrytown, NY). For hormone assays, 0.4 ml of a solution containing Trasylol, 5000 U/ml, and Na2EDTA, 12 mg/ml, were added to 3.6 ml blood. The mixture was immediately centrifuged at 4 C, and the separated plasma was stored at -20 C. Plasma glucagon was determined in duplicate assays by a classical immunoassay procedure (12), using porcine [125I] iodoglucagon (NEN Chemicals, D 6072, Dreieichenhain, West Germany) as tracer, 30K antiserum (provided by Dr. R. H. Unger, Dallas, Texas) and dextran-charcoal separation of free and antibodybound hormone. Plasma insulin was determined as previously described (13) using human insulin as standard. Statistical evaluation included variance analysis and the Student's t test for unpaired data.
Results Changes in blood glucose concentration and gastric glucagon release When glucose was present at a "normal" concentration in the perfusing blood, 101 ± 5 (mean ± SE) mg/100 ml, mean gastric glucagon output was 279 ± 84 pg/100 g stomach/min (34 determinations in six perfusions); this mean value is statistically different from 0 and indicates a net modest glucagon output by the stomach. In some instances, a small but significant uptake of glucagon was recorded. Plasma insulin in these experiments was 8 ± 1 /xU/ml. When blood glucose was increased to 292 ± 4 mg/100 ml and plasma
Endo • 1978 Vol 103 • No 5
insulin kept at basal levels, 7 ± 1.5 juU/ml, gastric glucagon output was 359 ± 83 pg/100 g/min (30 determinations in five perfusions), a value not statistically different from the one obtained in normoglycemia. When the stomach was perfused with blood containing 32 ± 1 mg/100 ml glucose and 7 ± 1 juU insulin/ml plasma, gastric glucagon output was significantly increased: 610 ± 79 pg/100 g/min (73 determinations in nine perfusions; P < 0.02 versus release in normoglycemia). Effect of'2-deoxy-D -glucose When the blood perfusing the stomach contained equimolar concentrations of glucose (100 ± 6 mg/100 ml) and 2-deoxy-D-glucose (100 mg/100 ml), glucagon release was enhanced about 5 times: 1482 ± 142 pg/100 g/min (36 determinations in four perfusions; P < 0.001 versus release in normoglycemia). Figure 1 illustrates the changes in gastric glucagon production which occurred after 2deoxy-D-glucose was added to the perfusing blood. Effect of antibody neutralization of basal insulin levels The fact the hypoglycemia led to a rise in gastric glucagon release which was relatively modest when compared to that provoked by 2-deoxy-D-glucose-induced cytoglycopenia led us to suspect that basal insulin concentrations in the perfusing blood may cause some glucagon suppression. Therefore, stomachs were perfused with a hypoglycemic blood (31 ± 2 mg/100 ml) whose insulin content (10 it 1 T
rh
1000 500
fi. Glucose 100mg/100ml
Glucose 100mg/100 ml • 2-0eoxy-D-glucose lOOmg/IOOml
FIG. 1. Glucagon release from the isolated dog stomach perfused with blood containing 100 mg/100 ml glucose and supplemented, at time 0, by 100 mg/100 ml 2-deoxyD-glucose. Mean of four perfusions. Results are given as mean ± SEM.
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REGULATION OF GASTRIC GLUCAGON RELEASE juU/ml plasma) was neutralized by an excess of insulin antibodies (see Materials and Methods). Under these conditions, gastric glucagon release was increased to 1122 ± 280 pg/100 g/min (2l determinations in four perfusions), a value significantly greater (P < 0.02) than the one obtained by hypoglycemia alone and of the same order of magnitude as the one observed under the influence of 2-deoxy-D-glucose. Discussion In their study of gastric A cell function in normal conscious dogs, Munoz-Barragan et al. (7) concluded that the stomach is probably not a major source of circulating glucagon. In contrast, Blazquez et al. showed that in both insulin-deprived depancreatized (8) or alloxan-diabetic dogs (9), the stomach contributed much to the increased glucagon circulating levels; in both conditions, insulin rapidly suppressed gastric glucagon release. The isolated perfused dog stomach provides a unique tool permitting investigation of A cell function in the absence of endogenously released insulin (5, 6). In the present investigation, we have demonstrated that, at basal concentrations of glucose and insulin, the release of glucagon by the dog stomach was indeed very modest (and in some experiments was even replaced by a discrete but significant glucagon uptake or degradation), thus supporting the conclusions reached by MunozBarragan et al. (7). In this isolated system, hyperglycemia alone did not further reduce this small basal gastric glucagon release. On the contrary, hypoglycemia slightly but significantly increased gastric glucagon release; its effect was doubled when the low concentration of insulin present in the perfusing blood (collected from overnight-fasted normal blood donor dogs) was neutralized by an excess of anti-insulin serum. This last finding demonstrates that even the presence of very small quantities of insulin is sufficient to suppress gastric glucagon release. Insulin may affect glucagon release either directly, as suggested by some observations made on the pancreatic A cell (see review, Ref. 14), or indirectly, by
1581
permitting glucose, even at low concentrations, to enter the A cell and suppress glucagon secretion (15-17). This exquisite sensitivity of the gastric A cell to insulin explains the dramatic fall in gastric glucagon release reported in pancreatectomized (8) or alloxan-diabetic dogs (9) when small amounts of insulin (0.0015 U/kg/min) are infused in these animals. Our data suggest that concentrations of insulin as low as 5-10 /xU/ml are sufficient to inhibit hypoglycemia-induced gastric glucagon release, although higher insulin concentrations may be required to inhibit arginine-stimulated gastric glucagon release (6). Cytoglycopenia, induced by 2-deoxy-D-glucose, a nonmetabolizable glucose analog which blocks intracellular glucose metabolism, is also a potent stimulus of gastric glucagon release as it is for pancreatic glucagon (18). The stimulation induced by 2-deoxyglucose is of the same order of magnitude as that obtained by simultaneous hypoglycemia and antibody-induced insulin withdrawal; it remains, however, below the maximal rate of gastric glucagon release obtained under the influence of 10 mM arginine (6). These results emphasize the critical role of insulin, even at very low concentrations, in the control of gastric glucagon secretion in dogs; they suggest that no conclusion can be reached about the absence of extrapancreatic sources of circulating glucagon in any species, including man, as long as a complete insulin deficiency is not produced. Acknowledgments We thank D. Binder for his help in the preparation of this manuscript. We are indebted to Professor A. Nizet for providing laboratory facilities, to Professor F. Monfort for his help in the statistical analyses, to Dr. Wald (Bayer, Brussels) for the generous gift of Trasylol, and to Dr. W. Malaisse (Brussels) for the generous gift of anti-insulin antiserum. We acknowledge with thanks the skillful technical assistance of Mr. and Mrs. H. Thoumsin, A. Rombeaux, C. Cartenstadt, C. Borremans, and R. Lallemand. We are indebted also to E. Vaessen-Petit for her secretarial assistance.
References 1. Sasaki, H., B. Rubalcava, D. Baetens, E. Blazquez, C. B. Srikant, L. Orci, and R. H. Unger, Identification of glucagon in the gastrointestinal tract, J Clin Invest
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 01:48 For personal use only. No other uses without permission. . All rights reserved.
1582
LEFEBVRE AND LUYCKX
56: 135, 1975. 2. Baetens, D., C. Rufener, C. B. Srikant, R. Dobbs, R. H. Unger, and L. Orci, Identification of glucagonproducing cells (A cells) in dog gastric mucosa, J Cell Biol 69: 455, 1976. 3. Larason, L. I., J. Hoist, R. Hakanson, and F. Sundler, Distribution and properties of glucagon immunoreactivity in the digestive tract of various mammals: an immunohistochemical and immunochemical study, Histochemistry 44: 281, 1975. 4. Morita, S., K. Doi, C. C. Yip, and M. Vranic, Measurements and partial characterization of immunoreactive glucagon in gastrointestinal tissues of dogs, Diabetes 25: 1018,1976. 5. Lefebvre, P. J., A. S. Luyckx, A. H. Brassinne, and A. H. Nizet, Glucagon and gastrin release by the isolated perfused dog stomach in response to arginine, Metabolism (Suppl. 7)25: 1477, 1976. 6. Lefebvre, P. J., and A. S. Luyckx, Factors controlling gastric glucagon release, J Clin Invest 59: 716, 1976. 7. Munoz-Barragan, M., E. Blazquez, G. S. Patton, R. E. Dobbs, and R. H. Unger, Gastric A-cell function in normal dogs, Am J Physiol 231:1057, 1976. 8. Blazquez, E., L. Munoz-Barragan, G. S. Patton, L. Oric, R. E. Dobbs, and R. H. Unger, Gastric A-cell function in insulin-deprived depancreatized dogs, Endocrinology 99: 1182, 1976. 9. Blazquez, E., L. Munoz-Barragan, G. S. Patton, R. E. Dobbs, and R. H. Unger, Demonstration of gastric glucagon hypersecretion in insulin-deprived alloxandiabetic dogs, J Lab Clin Med 89: 971, 1977. 10. Lefebvre, P. J., A. S. Luyckx, and A. H. Brassinne,
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Vagal stimulation and its role in eliciting gastrin but not glucagon release from the isolated perfused dog stomach, Gut 19: 185, 1978. 11. Schmidt, F. H., Enzymatic determination of glucose and fructose simultaneously. Klin Wschr 39: 1244, 1961. 12. Luyckx, A. S., Immunoassays for glucagon, In Lefebvre, P. J., and R. H. Unger (eds.), Glucagon: Molecular Physiology, Clinical and Therapeutic Implications, Pergamon Press, Oxford, 1972, p. 285. 13. Quabbe, H. J., Modifikation der radioimmunologischen Insulin-bestimmung nach Hales und Randle, Diabetologia 5: 107, 1969. 14. Samols, E., J. Tyler, and V. Marks, Glucagon-Insulin Interrelationships, In Lefebvre, P. J., and R. H. Unger (eds.), Glucagon: Molecular Physiology, Clinical and Therapeutic Implications, Pergamon Press, Oxford, 1972, p. 151. 15. Unger, R. H., E. Aguilar-Parada, W. A. Muller, and A. M. Eisentraut, Studies of pancreatic alpha-cell function in normal and diabetic subjects, J Clin Invest 49: 837, 1970. 16. Massi-Benedetti, F., A. Falorni, A. Luyckx and P. Lefebvre, Inhibition of glucagon in the human newborn by simultaneous administration of glucose and insulin, Horm Metab Res 6: 392, 1974. 17. Ostenson, C.-G., and C. Hellerstrom, Effect of insulin on the glucose utilization of the pancreatic A2-cell of the guinea-pig, Diabetologia 12: 413, 1976. 18. Muller, W. A., G. R. Faloona, and R. H. Unger, The effect of experimental insulin deficiency on glucagon secretion, J Clin Invest 50: 1992, 1971.
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