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The influence of oral glucose loading on the insulin response to i.v. glucagon in children and adolescents
The Influence of Oral Glucose Loading on the Insulin Response to I.V. Glucagon in Children and Adolescents Z. Josefsberg, E. Flatau, M. Doron, and Z. Laron Fourteen children and
adolescents with
slight constitutional growth retardation ( 12 males and two females) aged from 71_to 1st yr underwent an oral glucose tolerance test (OGfl 1.75 g/Kg followed at 180 min by on i.v. glucagon injection (0.03 mg/Kg). On a separate occasion these children underwent a simple i.v. glucagon test. Comparing the glucose and insulin response in the two glucagon tests for each child we found that whereos in the single test the blood glucose rose
slowly with o peak at 30 min, in the combined test the peak was at 5 min. The mean peak values were similar (129 and 121 mg/lOO ml). The mean peak insulin response in the single test was 70 pU/ml (at 2 min) as compared to 253 pU/ml (at 2 min) in the combined test. Our studies provide further evidence for o direct effect of glucagon on insulin release and that glucose preloading augments this effect, without relation to the concomitant blood glucose concentrations.
A
NUMBER of studies in animals and man have shown that the insulin response to glucagon stimulation is augmented by concomitant hyperand blunted by hypoglycemia.5 The human studies were perglycemiai formed in adults, and glucose was administered by constant infusion and the glucagon administered mainly at hyperglycemic levels. The following study was planned to determine the effect of i.v. glucagon upon insulin secretion when it is administered 180 min after glucose loading, when glucose levels have returned to normal. MATERIALS AND
METHODS
The subject material comprised 17 children and adolescents with slight constitutional growth retardation, (i.e., height between 2-3 SD below the mean height for age) without any evident endocrine disorders.6 Fourteen subjects, with an age range from 7) to 184 yr (mean age 12.4 f 3.3 yr) underwent a standard oral glucose tolerance test-OGTT-( 1.75 g/kg of body weight) after an overnight fast and a high carbohydrate diet during the 3 days prior to the test. An indwelling needle was inserted into the antecubital vein and after the subject had rested for an hour blood was drawn at 0, 30, 60, 90, 120, 150, 180, 182, 185, 190, 200, and 210 min into heparinized tubes. The glucose intake lasted about 3 min. At 180 min glucagon (Lilly) was administered in one intravenous bolus at a dose of 0.03 mg/kg body weight. Blood for glucose was separated, the tubes centrifuged and the plasma frozen at -20°C until assayed. Within an interval of 1-3 wk all 14 subjects also underwent a separate intravenous glucagon test, without any special dietary preparation. Blood glucose was measured with a Technicon Autoanalyzer by Ferricyanid Colorimetry. Plasma insulin was assayed using a double antibody radioimmunoassay.’
From the Institute of Pediatric and Adolescent Endocrinology, Beilinson Medical Center. Petah Tikva and Sackler School of Medicine. Tel Aviv University, Israel. Received for publication June 19.1975. Dr. Laron is an Established Investigator of the Chief Scientist’s Bureau Ministry of Health. Reprint requests should be addressed IO Z. Laron. M.D., Professor of Pediatric Endocrinology, Director, Institute of Pediatric and Adolescenr Endocrinology. Beilinson Medicial Center, Tel Aviv Universily Medical School, Israel. 0 1976 by Grune & Stratton, Inc. &fob&m,
Vol. 25, No. 3 (March), 1976
277
278
JOSEFSBERG
150 INSULIN
IVGLUCAGON I
I v GLUCPGCN
GLUCOSE 1
1
ET AL
i
I
.UU/d ?.SEM
‘0 t d 0I___, 30
60
90 I
120 1
150 I
180q--165 El0
200
210
MINUTES
Fig. I. Blood glucose and plasma insulin response to oml glucose and i.v. glucagon iniocted at 180 min. as compared to a sepamte i.v. glucagon test in 14 children and adolescents with slight constitutional growth retardation. TRS: total reducing subtance as measured with a Technican Auteanalyxer by Ferriyanid colorimety.
RESULTS
Figure 1 and Table 1 present the effect upon blood glucose and plasma insulin of oral glucose followed by i.v. glucagon as compared,with that of the separate intravenous glucagon test in fourteen subjects. Glucose Response The shape of the glucose curves differs in the two i.v. glucagon tests. While basal values were identical, the rise in glucose was more rapid when glucagon was administered following the OGTT than when it was injected separately. The mean peak concentrations were at 5 and 30 min, respectively. The mean peak values as calculated from the individual responses were similar in both glucagon tests, as was the mean peak value during the OGTT.
ORAL GLUCOSE
279
LOADING
Table 1. The Mean Raw1 ond Peak Blood Glucose levels and the Plasma lnwlin Response in 14 Children ond Adelesconts With Constitutional Growth Retardation During on Oml Tolerance Followed by on I.V. Gluqon
lest and After o kpamte
OGll + I.V. Glucagon+ Fasting
Peak
BOWI
Glucose
I.V. Glucagon Test I.V. Glucogon
Peak
Peak
Fasting
Blood glucose mg/lOOml
f SEM
04.7 f 2.8
124.9 f 5.1
86.7 f 2.9
121.3 f
7.8
58.1 f 6.6
24.5 f 5.0
253.4 f 47.9
87.5 f 2.1
129.5 f
4.2
79.2 f
13.8
Plasma insulin pU/ml f SEM
11.7 f
1.9
8.9 f
1.3
*Glucagon injected at 180 min.
Insulin Response
Here the basal levels differed. The mean basal level of the separate glucagon test was 8.9 pU/ml, similar to the fasting levels of the OGTT (the slightly higher mean may be due to the rich pretesting carbohydrate diet), while the mean basal value at 180 min after oral glucose loading was 24.5 PI-J/ml. The insulin response was much more pronounced when glucagon was injected following the OGTT than it was in the separate glucagon test. The mean peak values were 213.4 PI-l/ml and 79.2 pU/ml, respectively, almost all of the peaks occur& at 2 min. There was no correlation between the insulin response and the concomitant blood glucose levels. DISCUSSION
Most studies previously reported have dealt with the effect of glucagon on insulin and glucose levels during hyperglycemic states induced by oral or iv. glucose: ‘e3 In our investigation the insulinogenic effect of i.v. glucagon was studied under two conditions: following oral glucose loading (OGTT) at a time when blood glucose had returned to basal levels and separately after overnight fasting with no special dietary preparation. It is of note that in both tests the basal glucose concentrations were normal and similar. Two striking differences were observed, however, in the shape of the glucose curve and the intensity of the insulin response. In the separate i.v. glucagon test, the blood glucose rose gradually with a peak level at 30 min after injection, whereas in the test performed after glucose loading it rose much more rapidly, with the peak reached after only 5 min. This observation can be explained by the increase in glycogen stores resulting from the high carbohydrate diet and oral glucose load administered prior to the test. Insulin was found to rise immediately after i.v. glucagon administration in both tests, with peak values being registered at 2 min, but the response was much greater when the glucagon was administered after glucose loading, with no correlation to the concomitant glucose concentration. This observation is supported by findings in previous studies carried out by us,” and differs from the view of Cerasi (1975) who wrote that potentiation of insulin release by glucose is obtained only if the preceding glycemia reaches a level of 250-300 mg/ 100 ml. When the basal and fasting insulin levels are compared in the two tests, it can be seen that at 180 min after glucose loading, the basal levels are higher
JOSEFSBERG
280
ET Al.
than the fasting levels recorded in the separate i.v. ghtcogon test. We consider this difference in initial levels to be due to the preload of glucose in the first test. The instantaneous increase of insulin upon i.v. glucagon administration indicates that it has a releasing effect on preformed stores in the &cell. These stores are augmented by the preceding glucose load,4 with no relation to the glucose concentration and can as we have shown also be elicited when the concomitant blood glucose levels are normal. Our findings provide additional evidence for the direct effect of glucagon on the B-cell, with induction of insulin release, possibly by a separate cell membrane receptor from glucose and show that a combination of preloading glucose and i.v. glucagon constitute an acute stimulus for insulin secretion. The mechanism which mediates the glucose potentiation of the insulin in the j3-cell remains unexplained so far. REFERENCES 1. Garcia MJ, Czerwinski C, DeSantis R, Lan VV, Ramoy E, Penhos JC: Hyperglycemia and insulinogenic effects of intravenous glucagon at different blood glucose levels. Proc Sot Exp Med 143:707, 1973 2. Ryan WG, Nibbe AE: Beta-cytotrophic effects of glucose, glucagon and tolbutamide in man. Lancet 1:1255, 1967 3. Ryan WG, Schwartz TB, Nibbe AF: Serum immunoreactive insulin levels during glucose tolerance and intensive islet stimulation. Diabetes 20:404, 1971 4. Cerasi S: Mechanisms of glucose stimulated insulin secretion in health and in diabetes: Some reevaluations and proposals. Diabetologia 1I:l, 1975
5. Goldfine ID, Cerasi S, Luft R: Glucagon stimulation of insulin release in man: inhibition during hypoglycemia. J Clin Endocrinol Metab 35312, 1972 6. Laron A: The hypothalamus and the pituitary gland (Hypophysis) in, Hubble DW (ed): Paediatric Endocrinology. Oxford, Blackwell Sci Publ, 1969, p 35 7. Hales CN, Randle RJ: Immunoassay of insulin with insulin-antibody precipitate. Biochem J 88: 137, 1963 8. Karp, M, Laron Z, Doron M: Insulin response to intravenous glucagon in children with familial growth retardation. Arch Dis Child (in press)
adolescents with
slight constitutional growth retardation ( 12 males and two females) aged from 71_to 1st yr underwent an oral glucose tolerance test (OGfl 1.75 g/Kg followed at 180 min by on i.v. glucagon injection (0.03 mg/Kg). On a separate occasion these children underwent a simple i.v. glucagon test. Comparing the glucose and insulin response in the two glucagon tests for each child we found that whereos in the single test the blood glucose rose
slowly with o peak at 30 min, in the combined test the peak was at 5 min. The mean peak values were similar (129 and 121 mg/lOO ml). The mean peak insulin response in the single test was 70 pU/ml (at 2 min) as compared to 253 pU/ml (at 2 min) in the combined test. Our studies provide further evidence for o direct effect of glucagon on insulin release and that glucose preloading augments this effect, without relation to the concomitant blood glucose concentrations.
A
NUMBER of studies in animals and man have shown that the insulin response to glucagon stimulation is augmented by concomitant hyperand blunted by hypoglycemia.5 The human studies were perglycemiai formed in adults, and glucose was administered by constant infusion and the glucagon administered mainly at hyperglycemic levels. The following study was planned to determine the effect of i.v. glucagon upon insulin secretion when it is administered 180 min after glucose loading, when glucose levels have returned to normal. MATERIALS AND
METHODS
The subject material comprised 17 children and adolescents with slight constitutional growth retardation, (i.e., height between 2-3 SD below the mean height for age) without any evident endocrine disorders.6 Fourteen subjects, with an age range from 7) to 184 yr (mean age 12.4 f 3.3 yr) underwent a standard oral glucose tolerance test-OGTT-( 1.75 g/kg of body weight) after an overnight fast and a high carbohydrate diet during the 3 days prior to the test. An indwelling needle was inserted into the antecubital vein and after the subject had rested for an hour blood was drawn at 0, 30, 60, 90, 120, 150, 180, 182, 185, 190, 200, and 210 min into heparinized tubes. The glucose intake lasted about 3 min. At 180 min glucagon (Lilly) was administered in one intravenous bolus at a dose of 0.03 mg/kg body weight. Blood for glucose was separated, the tubes centrifuged and the plasma frozen at -20°C until assayed. Within an interval of 1-3 wk all 14 subjects also underwent a separate intravenous glucagon test, without any special dietary preparation. Blood glucose was measured with a Technicon Autoanalyzer by Ferricyanid Colorimetry. Plasma insulin was assayed using a double antibody radioimmunoassay.’
From the Institute of Pediatric and Adolescent Endocrinology, Beilinson Medical Center. Petah Tikva and Sackler School of Medicine. Tel Aviv University, Israel. Received for publication June 19.1975. Dr. Laron is an Established Investigator of the Chief Scientist’s Bureau Ministry of Health. Reprint requests should be addressed IO Z. Laron. M.D., Professor of Pediatric Endocrinology, Director, Institute of Pediatric and Adolescenr Endocrinology. Beilinson Medicial Center, Tel Aviv Universily Medical School, Israel. 0 1976 by Grune & Stratton, Inc. &fob&m,
Vol. 25, No. 3 (March), 1976
277
278
JOSEFSBERG
150 INSULIN
IVGLUCAGON I
I v GLUCPGCN
GLUCOSE 1
1
ET AL
i
I
.UU/d ?.SEM
‘0 t d 0I___, 30
60
90 I
120 1
150 I
180q--165 El0
200
210
MINUTES
Fig. I. Blood glucose and plasma insulin response to oml glucose and i.v. glucagon iniocted at 180 min. as compared to a sepamte i.v. glucagon test in 14 children and adolescents with slight constitutional growth retardation. TRS: total reducing subtance as measured with a Technican Auteanalyxer by Ferriyanid colorimety.
RESULTS
Figure 1 and Table 1 present the effect upon blood glucose and plasma insulin of oral glucose followed by i.v. glucagon as compared,with that of the separate intravenous glucagon test in fourteen subjects. Glucose Response The shape of the glucose curves differs in the two i.v. glucagon tests. While basal values were identical, the rise in glucose was more rapid when glucagon was administered following the OGTT than when it was injected separately. The mean peak concentrations were at 5 and 30 min, respectively. The mean peak values as calculated from the individual responses were similar in both glucagon tests, as was the mean peak value during the OGTT.
ORAL GLUCOSE
279
LOADING
Table 1. The Mean Raw1 ond Peak Blood Glucose levels and the Plasma lnwlin Response in 14 Children ond Adelesconts With Constitutional Growth Retardation During on Oml Tolerance Followed by on I.V. Gluqon
lest and After o kpamte
OGll + I.V. Glucagon+ Fasting
Peak
BOWI
Glucose
I.V. Glucagon Test I.V. Glucogon
Peak
Peak
Fasting
Blood glucose mg/lOOml
f SEM
04.7 f 2.8
124.9 f 5.1
86.7 f 2.9
121.3 f
7.8
58.1 f 6.6
24.5 f 5.0
253.4 f 47.9
87.5 f 2.1
129.5 f
4.2
79.2 f
13.8
Plasma insulin pU/ml f SEM
11.7 f
1.9
8.9 f
1.3
*Glucagon injected at 180 min.
Insulin Response
Here the basal levels differed. The mean basal level of the separate glucagon test was 8.9 pU/ml, similar to the fasting levels of the OGTT (the slightly higher mean may be due to the rich pretesting carbohydrate diet), while the mean basal value at 180 min after oral glucose loading was 24.5 PI-J/ml. The insulin response was much more pronounced when glucagon was injected following the OGTT than it was in the separate glucagon test. The mean peak values were 213.4 PI-l/ml and 79.2 pU/ml, respectively, almost all of the peaks occur& at 2 min. There was no correlation between the insulin response and the concomitant blood glucose levels. DISCUSSION
Most studies previously reported have dealt with the effect of glucagon on insulin and glucose levels during hyperglycemic states induced by oral or iv. glucose: ‘e3 In our investigation the insulinogenic effect of i.v. glucagon was studied under two conditions: following oral glucose loading (OGTT) at a time when blood glucose had returned to basal levels and separately after overnight fasting with no special dietary preparation. It is of note that in both tests the basal glucose concentrations were normal and similar. Two striking differences were observed, however, in the shape of the glucose curve and the intensity of the insulin response. In the separate i.v. glucagon test, the blood glucose rose gradually with a peak level at 30 min after injection, whereas in the test performed after glucose loading it rose much more rapidly, with the peak reached after only 5 min. This observation can be explained by the increase in glycogen stores resulting from the high carbohydrate diet and oral glucose load administered prior to the test. Insulin was found to rise immediately after i.v. glucagon administration in both tests, with peak values being registered at 2 min, but the response was much greater when the glucagon was administered after glucose loading, with no correlation to the concomitant glucose concentration. This observation is supported by findings in previous studies carried out by us,” and differs from the view of Cerasi (1975) who wrote that potentiation of insulin release by glucose is obtained only if the preceding glycemia reaches a level of 250-300 mg/ 100 ml. When the basal and fasting insulin levels are compared in the two tests, it can be seen that at 180 min after glucose loading, the basal levels are higher
JOSEFSBERG
280
ET Al.
than the fasting levels recorded in the separate i.v. ghtcogon test. We consider this difference in initial levels to be due to the preload of glucose in the first test. The instantaneous increase of insulin upon i.v. glucagon administration indicates that it has a releasing effect on preformed stores in the &cell. These stores are augmented by the preceding glucose load,4 with no relation to the glucose concentration and can as we have shown also be elicited when the concomitant blood glucose levels are normal. Our findings provide additional evidence for the direct effect of glucagon on the B-cell, with induction of insulin release, possibly by a separate cell membrane receptor from glucose and show that a combination of preloading glucose and i.v. glucagon constitute an acute stimulus for insulin secretion. The mechanism which mediates the glucose potentiation of the insulin in the j3-cell remains unexplained so far. REFERENCES 1. Garcia MJ, Czerwinski C, DeSantis R, Lan VV, Ramoy E, Penhos JC: Hyperglycemia and insulinogenic effects of intravenous glucagon at different blood glucose levels. Proc Sot Exp Med 143:707, 1973 2. Ryan WG, Nibbe AE: Beta-cytotrophic effects of glucose, glucagon and tolbutamide in man. Lancet 1:1255, 1967 3. Ryan WG, Schwartz TB, Nibbe AF: Serum immunoreactive insulin levels during glucose tolerance and intensive islet stimulation. Diabetes 20:404, 1971 4. Cerasi S: Mechanisms of glucose stimulated insulin secretion in health and in diabetes: Some reevaluations and proposals. Diabetologia 1I:l, 1975
5. Goldfine ID, Cerasi S, Luft R: Glucagon stimulation of insulin release in man: inhibition during hypoglycemia. J Clin Endocrinol Metab 35312, 1972 6. Laron A: The hypothalamus and the pituitary gland (Hypophysis) in, Hubble DW (ed): Paediatric Endocrinology. Oxford, Blackwell Sci Publ, 1969, p 35 7. Hales CN, Randle RJ: Immunoassay of insulin with insulin-antibody precipitate. Biochem J 88: 137, 1963 8. Karp, M, Laron Z, Doron M: Insulin response to intravenous glucagon in children with familial growth retardation. Arch Dis Child (in press)