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Effect of enteric hormones on insulin secretion
Effect of Enteric Hormones on Insulin Secretion By MICHEL E.
KAHIL, GEORGE R. MCILHANEY AND PAUL H. JORDAN,JR.
The effects of glucagon, secretin, pancreozymin and tolbutamide upon the blood sugar and plasma insulin levels of patients with maturity-onset diabetes, normals and patients with subclinical diabetes were compared. Secretin administration caused a transient increase in peripheral insulin levels without concomitant blood glucose changes. No difference in the speed or magnitude of the insulin response was observed in the three groups of patients. Tolhutamide and glucagon produced a prompt elevation of the insulin levels in the normals and a diminished response in the diabetics. The plasma insulin response to tolbutamide was significantly greater than that observed after secretin. In four nor-
mal individuals in whom hyperglycemia (of about 190 mg. %) was produced by infusion of glucose, secretin did not elicit a further increase in the insulin levels. The rapid induction of insulin release in the diabetics following secretin and pancreozymin administration makes it unlikely that resistance on the part of the pancreas to the insulogenic effects of these hormones is responsible for the delayed insulin secretory response to post prandial hyperglycemia noted in these patients. The possibility has not been excluded, however, that an abnormal secretion of these enteric hormones is present in diabetes. (Metabolism 18: No. 1, January, 50-57, 1969)
HE RISE IN PLASMA INSULIN that follows a glucose load is greater when glucose is administered orally than when it is administered intravenous1y.l Noting this difference, McIntyre suggested that a hormonal factor released by the intestinal tract might act, along with the rise in blood glucose, to stimulate the release of insulin by the pancreatic islets.” Dupre supported this hypothesis by showing that extracts of porcine intestinal mucosa infused simultaneously with glucose greatly accelerated the rate of disposal of the infused glucose and effected higher serum levels of insulin-like activity than when glucose alone was administered intravenously.” It was subsequently proposed that in diabetics the delayed initial release of insulin that follows glucose ingestion may be the consequence of a altered responsiveness of the islet cells to enteric hormones.-’ To test this hypothesis. we studied the effects of intravenously administered secretin and pancreozymin on insulin release in normal subjects, diabetics, and
T
Veternm Adn7itlistrcrtio,z Hospittrl, Bet7 To7h From the Metabolic Resenrcl~ Laborntory, Gerrertrl Ho.cpitnl, md Deportments of Medicine m7d Surgery. Blrylor Colle,vc of Medicine, Houstotl. Texas. Supported by USPHS Grot7t.r AM 09399 tr17d FR00134. Received for publicntio,z JUW IO, 1969. MICHEL E. KAHIL, M.D.: Stcrff Physicim. Veteurrr7.s Admiuistrc~tiotz Hospirnl: Assistnrrt Professor of Medicine, Brrylor Coflege of Mcdicirte, Horirtou, Texm. GroRciE R. MCILHANEY, B.A.: Sejzior Medicnl Strldeut. Bo.vlor Collepc of Mcdicirv. Horlstou, Trsm. PAUL H. JORDAN, JR., M.D.: Chief-of-Stuff ntld Chief of Srrr,very. Vetcraru Administrrrtiot7 Te.ws. Hospital; Profesror of Surgery, Baylor Collc~~e of Medicitlc, Houstm, 50
METABOLISM.
VOL.
19, No.
1 (JANUARY),
1970
51
ENTERICHORMONES
patients with subclinical diabetes. The response to these agents was also compared to the insulinogenic response that follows tolbutamide and glucagon administration. MATERIALS
AND
METHODS
The patients studied fell into three groups: There were six nonobese maturity-onset diabetics whose fasting blood sugar was between 110 and 200 mg. lo. The average age of the patients was 4.5 (36-50). These patients (four men and two women) had been recently diagnosed and treated with diet and oral hypoglycemic drugs. These drugs were stopped 72 hours prior to the study. Six healthy male volunteers, mean age 34 (24-S2 1. with a negative family history of diabetes comprised our normal group. They were given a diet containing 300 Gm. of carbohydrate for two days and were then tested after an overnight fast. The four patients with subclinical diabetes had normal fasting blood glucose values and glucose tolerance tests (as defined by Fajans and Conn ): under normal circumstances but exhibited abnormal carbohydrate tolerance under stress ( cortisone administration. infection, acute myocardial infarction). Their mean age was 40 years ( 26-49 1. Following standard dietary preparation with 300 Gm. of carbohydrate a day for three days, the patients received intravenously on consecutive days 1 mg. of glucagon. I Cm. of Na tolbutamide, 75 Crick units of secretin (Sigma) diluted in 10 ml. saline and administered over a two-minute period. and 100 Crick units of pancreozymin (Sigma ) similarly diluted and administered. Peripheral venous blood samples for the determination of glucose” and immunoreactive insulin (1RI)r were collected in chilled tubes containing Na fluoride at 0, 1. 3, 5, 10. 1.5 and 30 minutes after administration of the drugs. After tolbutamide injection. additional samples were drawn at 60 and I20 minutes. To simulate the hyperglycemia observed in diabetic patients, five normal subjects received glucose infusions (7.5 mg./KG./min.) at B constant rate for 60 minutes. After 30 minutes of constant infusion, secretin was administered IV and hlood samples fat determination of immunoreactive insulin and glucose were ohtained as previourly outlined. To determine whether endogenous secretin production stimulates insulin release. experimentt were conducted on two mongrel dogs. Under sodium pentothal anesthesia. ;I polyethylene catheter was threaded through the splenic vein and guided into the superior pancreatico-duodenal vein in a retrograde manner to within 3-5 cm. from its junction with the portal vein. A Thomas Cannula was placed in the duodenum over the entrance of the pancreatic duct so that pancreatic juice could be collected. The animals were allowed to recover and the experiments performed with them awake. The dogs were fasted for 24 hours prior to each experiment. Pancreatic secretions and pancreatic vein blood were collected in the basal state. The duodenum was then perfused with 0.1 N HCl at a rate of 2.5 ml/min.ute throughout the experiment. Pancreatic vein blood was drawn at five and ten minutes and then every 20 minutes thereafter. Pancreatic juice collections were made at ten minutes and then at fifteen minute intervals. Plasma insulin was determined by the method of Morgan and Lazarow.7 and the level of hicarhonate was estimated by the method of Preshaw. Cooke and Gros\m;in.’
RESULTS
lmrlin
Secretion
Followirq
&cretin
Secretin infusion elicited a prompt but transient elevation of the insulin levels in the three groups of patients (Fig. 1 ). The maximal elevations (mean -t SEM ) above fasting levels of 15 2 4.2; 20 k 8 and 18 i. 2.9 !1U,/ml in the diabetics. normals and subclinical diabetics respectively were not significantly different Cp < 0.4 1. No detectable changes in blood glucose levels were observed in the three groups.
52
KAHIL,
NORMALS
MC ILHANEY
SUBCLINICAL
AND JORDAN
DIABETICS
$190
E 170 z 8 150 &30 n 110
8 690
5
IO
15
20
25
30
5
IO
15
20
25
30
5
IO
I5
20
25
30
Min.
Fig. l.- Effect of intravenous administration of 75 Crick Units of secretin on blood sugar and immunoreactive insulin levels in normals, diabetics and subjects with subclinical diabetes. Insulin values (mean ~lt SEM) represent rise above initial level.
Insulin Secretion Following Pancreozymin
Pancreozymin (Fig. 2) produced a minimal elevation of the insulin levels 1 min. after its administration to 9.0 + 1.0, 12 +- 4.0 and 8.0 + 3.0 ,JJ/ml in the normals, diabetics and subclinical diabetic patients respectively. The differences were not statistically significant (p < 0.5). There was no detectable change in the glucose values in any of the groups. Insulin Secretion Following Glucagon
In normal subjects, the maximal mean plasma insulin rise of 54 -t 7 @J/ml was noted five minutes after the glucagon administration, and in the diabetics there was a peak response of 3 1 -+ 8 pU/ml at the same time (Fig. 3 ) . The magnitude of the response was significantly different in the two groups (p < 0.5). It is noteworthy that the insulin elevation preceded any detectable change in the blood glucose concentration. Insulin Secretion Following Tolbutamide One minute after tolbutamide infusion, normal subjects showed a rise of the insulin level of 92 & 26 pU/ml. In contrast, the response in the diabetics was
blunted and delayed with a one minute elevation of 17 f 9 ,~U/ml and a peak rise at five minutes of 27 & 10 pU/ml. The mean values at 1, 3, and 5 minutes were significantly different (p < 0.025) in the normals and diabetics (Fig. 4). The patients with subclinical diabetes showed an insulin rise that was similar to
ENTERIC
HORMONES
!
DIABETICS
210 8 190 g
170
MCLINICAL
NORMALS
DIABETICS
I------
Min. Fig. 2.-Effect on blood sugar with subclinical
of intravenous administration of 100 Crick Units of pancreozymin and immunoreactive insulin levels in normals, diabetics and patients diabetes. Insulin values (mean 2 SEM) represent rise above initial
150 130
-
II0 90
-
DIABETICS
NORMALS
Fig. 3.-Effect of intravenous administration of 1 mg. of giucagon on blood sugar and immunoreactive insulin levels in normals and in diabetic subjects. Insulin values (mean 2 SEM) represent rise initial above level.
/HI
70 70
-
60
-
50
-
0
5
IO
I5
20 25 30 MINUTES
5
IO
15 20
25
30
54
KAHIL,
82
130 -
DIABETICS
NORMALS
MC ILHANEY
SUBCLINICAL
AND JORDAN
DbWEflCS
(J) 110 “s god
70/
50 120 I IO -
loo-
5 goX60g
70-
2
60-
;;~LJkJ
ii50-
k
0
IO
20?iO405060120
IO 2030405060120 MINUTES
IO 2030405D60120
Fig. 4.-Effect of intravenous administration of 1 Cm. of tolbutamide sugar and immunoreactive insulin levels in normals, diabetics and patients
clinical diabetes. Insulin values (mean t the normals but significantly different after tolbutamide administration. Insulin
Secretion
Following
Glucose
SEM) represent
on blood with sub-
rise above initial level.
from the diabetics
one and three minutes
Infusion
It is apparent from Fig. 5 that when hyperglycemia of a magnitude similar to that present in the diabetics was produced in five normal subjects, secretin failed to raise further the mean insulin level. In only one patient was a rise in insulin levels observed following the infusion of secretin, whereas there was a fall in the ,remaining four. Insulin
Secretion
in Response
to in vivo Release
of Secretin
The effects of continuous acid perfusion of the duodenum on the bicarbonate excretion by the pancreas and the insulin levels in the portal vein blood are shown in Fig. 6. The portal blood insulin rise after endogenous secretin production occurred within five minutes of the onset of duodenal stimulation. The insulin response was of short duration, however, and the levels returned to control values in spite of continued secretin production as evidenced by the prolonged pancreatic secretion.
ENTERICHORMONES
220 -
180 -
20 -
-10 GLUCOSE
INFUSION
I
:~~:~~:~.:.:.:~.~~~~~.: : _.z
7.5mp/kO/Min.
3
5
IO
15
30
MNUTES
Fig. S.-Effect of constant infusion of 7.5 mg./Kg./min. of glucose for 60 minutes together with intravenous administration of 75 Crick Units of secretin on the blood sugar and immunoreactive insulin levels in five normal subjects. Insulin values represent mean -C SEM. BICARBONATE
120 -
_. UY c 5 0 t E z
3 2z -
r--._
r /
2
,/’
loo-
,’
I
8060 -
40 -
4
Acid infusion started
1 - 140 - 120
__
_ 100
.g -_ 25 f
1’
I
20 -
-
-
80
-
60
-
40
'0" 2
Fig. 6-Effect of continuous acid perfusion of duodenum in two dogs on portal blood insulin response and bicarbonate excretion by pancreas.
TIME (minutes)
DISCUSSION Experiments in dogs and in man demonstrate that the rise in blood insulin concentration that follows glucose ingestion is greater than the rise provoked by glucose infusions. Secretin, pancreozymin and glucagon have been implicated as the humoral factors that enhance the insulin reieasing capacity of ingested glucosc.!‘~‘”
56
KAHIL.
MC ILHANEY
AND JORDAN
Several conflicting results, however, have raised doubts as to the physiological significance of these hormones. Dupre et al. have shown that duodenal instillation of hydrochloric acid in man resulted in a rise in the peripheral serum immunoreactive insulin concentration. l1 In our dog experiments, however, the stimulation of endogenous secretin production by duodenal acid instillation resulted in a very transient rise in portal venous blood insulin levels in spite of continued secretin production. Similarly, Boyns et al. showed that the induction of endogenous secretin production in two normal subjects by hydrochloric acid instillation in the duodenum did not affect the blood sugar or peripheral plasma insulin levels and that, when glucose plus acid and glucose alone were instilled in the duodenum, almost identical changes in the blood sugar and insulin levels were observed.” The role of glucagon in influencing the insulin secretory response to ingested glucose has been clarified by the studies of Unger et al.13 They showed that the rise in glucagon-like immunoreactivity that followed the ingestion of glucose was not of pancreatic origin since it was observed in the absence of the pancreas. This material, probably derived from the gut, was devoid of hyperglycemic and glycogenolytic activity but stimulated insulin release. Hyperglycemia, whether induced by intravenous or enteric glucose administration suppressed pancreatic glucagon secretion. Conflicting results on the in vitro effects of secretin and pancreozymin in causing insulin release from pancreatic tissue have been obtained by various mvestigators.14~15 In our studies, secretin administration resulted in a modest and transient increase in peripheral insulin levels without concomitant blood glucose changes. No difference in the speed or magnitude of the insulin response was observed in the normal, diabetic or subclinical diabetic subjects. It is of interest that an insulin rise was observed in the diabetics, whereas in four normal individuals in whom a comparable degree of hyperglycemia was induced by glucose infusions, a bolus injection of secretin did not elicit a further increase in the insulin levels. It should be pointed out that maintained stimulation of insulin secretion has been observed in man when simultaneous sustained infusions of secretin and glucose were administered.” Tolbutamide and pharmacological doses of glucagon produced a prompt outpouring of insulin in the normals and an obviously impaired response in the diabetics. The insulin response to secretin and pancreozymin in doses probably close to physiologic levels was similar in the normal and diabetic subjects but was far below the capacity of the islets in these patients to produce or release insulin in response to tolbutamide. By contrast, DecKert showed in eight diabetic patients that secretin was a much more potent stimulus for insulin secretion than intravenous glucose, suggesting that the defect in the diabetic may represent either a blockade of normal glucose transport into the p-cells or an abnormal metabolism of glucose by these cells. I6 The elevation of plasma insulin levels noted in our diabetic patients after secretin administration but not observed in normal subjects rendered hyperglycemic suggests that the islet cells in the diabetics though still containing insulin reserves may be hyporesponsive to rising blood glucose concentrations.
ENTERIC
57
HORMONES
REFERENCES 1. McIntyre, N., Holdsworth, C. D., and Turner, D. S.: New interpretation of oral glucose tolerance. Lancet 2:20, 1964. 3. -, - , and -: Intestinal factors in the control of insulin secretion. J. Clin. Endoer. 25: 1317, 1965. 3. Dupre. J .. and Beck, I. C.: Stimulation of release of insulin by an extract of intestinal mucosa. Diabetes 15:.555, 1966. 4. Brown, J.. and Straatsma. B. R.: The UCLA Interdepartmental Conference. Diabetes Mellitus: Current Concepts and Vascular Lesions (Renal and Retinal). Ann. Intern. Med. 68:634. 1968. 5. Fajans. S. S.: Diagnostic tests for diabetes mellitus. In Williams. R. H. (Ed.) : Diabetes. New York, Hoeber, 1960. 6. Nelson. N.: A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375, 1944. 7. Morgan. C. R.. and Lazarow, A.: Immunoassay of insulin: Two antibody system. Plasma insulin levels of normals, subdiabetic and diabetic rats. Diabetes 12: 115, 1963. X. Preshaw. R. M., Cooke, A. R.. and Grossman. M. I.: Quantitative aspects of response of canine pancreas to duodenal acidification. Amer. J. Physiol. 210:629, 1966. 9. Letterer. H., Ohneda, A.. Dupre. J., Eisentraut. A., and Unger. R. H.: Effect of enteric hormones on insulin and
glucagon secretion. J. Lab. Clin. Med. 6X: 888, 1966. 10. White, J. J., and Dupre, J.: Regulation of insulin secretion by the intestinal hormone secretin: Studies in man via transumbilical portal vein catheterization. Surgery 64:204, 1968. II. Dupre, J. J., Curtis. J. D.. Waddell. R. W., and Beck, J. C.: Regulation of pancreatic endocrine function by gastrointestinal hormones. Proc. Roy. Sot. Med. 61: 815. 1968. 12. Boyns. D. R., Jarrett. R. J., and Keen. H.: Intestinal hormones and plasma insulin. Lancet 1:409, 1966. 13. Unger, R. H., Ohneda. A., Valverde, I., Eisentraut. A. M.. and Extron, J.: Characterization of the responses of circulating glucagon-like immunoreactivity to intraduodenal and intravenous administration of glucose. J. Clin. Invest. 47:48, 196X. 14. Lazarus, N. R.. Voyles, N., Tanese. T., Devrim. S., and Recant, L.: Extra-gastrointestinal effects of secretin. gastrin and pancreozymin. Lancet 2:248. 1968. 15. Pfeiffer. E. F., Telib. M., Ammon, J.. Melani, F., and Ditschuneit, M.: The direct stimulation by secretin of insulin secretion in vitro. German Med. Monthly 11:432. 1966. 16. DecKert, T.: Insulin secretion following administration of secretin in patients with diabetes mellitus. Acta Endocr. 59: 150, 1968.
KAHIL, GEORGE R. MCILHANEY AND PAUL H. JORDAN,JR.
The effects of glucagon, secretin, pancreozymin and tolbutamide upon the blood sugar and plasma insulin levels of patients with maturity-onset diabetes, normals and patients with subclinical diabetes were compared. Secretin administration caused a transient increase in peripheral insulin levels without concomitant blood glucose changes. No difference in the speed or magnitude of the insulin response was observed in the three groups of patients. Tolhutamide and glucagon produced a prompt elevation of the insulin levels in the normals and a diminished response in the diabetics. The plasma insulin response to tolbutamide was significantly greater than that observed after secretin. In four nor-
mal individuals in whom hyperglycemia (of about 190 mg. %) was produced by infusion of glucose, secretin did not elicit a further increase in the insulin levels. The rapid induction of insulin release in the diabetics following secretin and pancreozymin administration makes it unlikely that resistance on the part of the pancreas to the insulogenic effects of these hormones is responsible for the delayed insulin secretory response to post prandial hyperglycemia noted in these patients. The possibility has not been excluded, however, that an abnormal secretion of these enteric hormones is present in diabetes. (Metabolism 18: No. 1, January, 50-57, 1969)
HE RISE IN PLASMA INSULIN that follows a glucose load is greater when glucose is administered orally than when it is administered intravenous1y.l Noting this difference, McIntyre suggested that a hormonal factor released by the intestinal tract might act, along with the rise in blood glucose, to stimulate the release of insulin by the pancreatic islets.” Dupre supported this hypothesis by showing that extracts of porcine intestinal mucosa infused simultaneously with glucose greatly accelerated the rate of disposal of the infused glucose and effected higher serum levels of insulin-like activity than when glucose alone was administered intravenously.” It was subsequently proposed that in diabetics the delayed initial release of insulin that follows glucose ingestion may be the consequence of a altered responsiveness of the islet cells to enteric hormones.-’ To test this hypothesis. we studied the effects of intravenously administered secretin and pancreozymin on insulin release in normal subjects, diabetics, and
T
Veternm Adn7itlistrcrtio,z Hospittrl, Bet7 To7h From the Metabolic Resenrcl~ Laborntory, Gerrertrl Ho.cpitnl, md Deportments of Medicine m7d Surgery. Blrylor Colle,vc of Medicine, Houstotl. Texas. Supported by USPHS Grot7t.r AM 09399 tr17d FR00134. Received for publicntio,z JUW IO, 1969. MICHEL E. KAHIL, M.D.: Stcrff Physicim. Veteurrr7.s Admiuistrc~tiotz Hospirnl: Assistnrrt Professor of Medicine, Brrylor Coflege of Mcdicirte, Horirtou, Texm. GroRciE R. MCILHANEY, B.A.: Sejzior Medicnl Strldeut. Bo.vlor Collepc of Mcdicirv. Horlstou, Trsm. PAUL H. JORDAN, JR., M.D.: Chief-of-Stuff ntld Chief of Srrr,very. Vetcraru Administrrrtiot7 Te.ws. Hospital; Profesror of Surgery, Baylor Collc~~e of Medicitlc, Houstm, 50
METABOLISM.
VOL.
19, No.
1 (JANUARY),
1970
51
ENTERICHORMONES
patients with subclinical diabetes. The response to these agents was also compared to the insulinogenic response that follows tolbutamide and glucagon administration. MATERIALS
AND
METHODS
The patients studied fell into three groups: There were six nonobese maturity-onset diabetics whose fasting blood sugar was between 110 and 200 mg. lo. The average age of the patients was 4.5 (36-50). These patients (four men and two women) had been recently diagnosed and treated with diet and oral hypoglycemic drugs. These drugs were stopped 72 hours prior to the study. Six healthy male volunteers, mean age 34 (24-S2 1. with a negative family history of diabetes comprised our normal group. They were given a diet containing 300 Gm. of carbohydrate for two days and were then tested after an overnight fast. The four patients with subclinical diabetes had normal fasting blood glucose values and glucose tolerance tests (as defined by Fajans and Conn ): under normal circumstances but exhibited abnormal carbohydrate tolerance under stress ( cortisone administration. infection, acute myocardial infarction). Their mean age was 40 years ( 26-49 1. Following standard dietary preparation with 300 Gm. of carbohydrate a day for three days, the patients received intravenously on consecutive days 1 mg. of glucagon. I Cm. of Na tolbutamide, 75 Crick units of secretin (Sigma) diluted in 10 ml. saline and administered over a two-minute period. and 100 Crick units of pancreozymin (Sigma ) similarly diluted and administered. Peripheral venous blood samples for the determination of glucose” and immunoreactive insulin (1RI)r were collected in chilled tubes containing Na fluoride at 0, 1. 3, 5, 10. 1.5 and 30 minutes after administration of the drugs. After tolbutamide injection. additional samples were drawn at 60 and I20 minutes. To simulate the hyperglycemia observed in diabetic patients, five normal subjects received glucose infusions (7.5 mg./KG./min.) at B constant rate for 60 minutes. After 30 minutes of constant infusion, secretin was administered IV and hlood samples fat determination of immunoreactive insulin and glucose were ohtained as previourly outlined. To determine whether endogenous secretin production stimulates insulin release. experimentt were conducted on two mongrel dogs. Under sodium pentothal anesthesia. ;I polyethylene catheter was threaded through the splenic vein and guided into the superior pancreatico-duodenal vein in a retrograde manner to within 3-5 cm. from its junction with the portal vein. A Thomas Cannula was placed in the duodenum over the entrance of the pancreatic duct so that pancreatic juice could be collected. The animals were allowed to recover and the experiments performed with them awake. The dogs were fasted for 24 hours prior to each experiment. Pancreatic secretions and pancreatic vein blood were collected in the basal state. The duodenum was then perfused with 0.1 N HCl at a rate of 2.5 ml/min.ute throughout the experiment. Pancreatic vein blood was drawn at five and ten minutes and then every 20 minutes thereafter. Pancreatic juice collections were made at ten minutes and then at fifteen minute intervals. Plasma insulin was determined by the method of Morgan and Lazarow.7 and the level of hicarhonate was estimated by the method of Preshaw. Cooke and Gros\m;in.’
RESULTS
lmrlin
Secretion
Followirq
&cretin
Secretin infusion elicited a prompt but transient elevation of the insulin levels in the three groups of patients (Fig. 1 ). The maximal elevations (mean -t SEM ) above fasting levels of 15 2 4.2; 20 k 8 and 18 i. 2.9 !1U,/ml in the diabetics. normals and subclinical diabetics respectively were not significantly different Cp < 0.4 1. No detectable changes in blood glucose levels were observed in the three groups.
52
KAHIL,
NORMALS
MC ILHANEY
SUBCLINICAL
AND JORDAN
DIABETICS
$190
E 170 z 8 150 &30 n 110
8 690
5
IO
15
20
25
30
5
IO
15
20
25
30
5
IO
I5
20
25
30
Min.
Fig. l.- Effect of intravenous administration of 75 Crick Units of secretin on blood sugar and immunoreactive insulin levels in normals, diabetics and subjects with subclinical diabetes. Insulin values (mean ~lt SEM) represent rise above initial level.
Insulin Secretion Following Pancreozymin
Pancreozymin (Fig. 2) produced a minimal elevation of the insulin levels 1 min. after its administration to 9.0 + 1.0, 12 +- 4.0 and 8.0 + 3.0 ,JJ/ml in the normals, diabetics and subclinical diabetic patients respectively. The differences were not statistically significant (p < 0.5). There was no detectable change in the glucose values in any of the groups. Insulin Secretion Following Glucagon
In normal subjects, the maximal mean plasma insulin rise of 54 -t 7 @J/ml was noted five minutes after the glucagon administration, and in the diabetics there was a peak response of 3 1 -+ 8 pU/ml at the same time (Fig. 3 ) . The magnitude of the response was significantly different in the two groups (p < 0.5). It is noteworthy that the insulin elevation preceded any detectable change in the blood glucose concentration. Insulin Secretion Following Tolbutamide One minute after tolbutamide infusion, normal subjects showed a rise of the insulin level of 92 & 26 pU/ml. In contrast, the response in the diabetics was
blunted and delayed with a one minute elevation of 17 f 9 ,~U/ml and a peak rise at five minutes of 27 & 10 pU/ml. The mean values at 1, 3, and 5 minutes were significantly different (p < 0.025) in the normals and diabetics (Fig. 4). The patients with subclinical diabetes showed an insulin rise that was similar to
ENTERIC
HORMONES
!
DIABETICS
210 8 190 g
170
MCLINICAL
NORMALS
DIABETICS
I------
Min. Fig. 2.-Effect on blood sugar with subclinical
of intravenous administration of 100 Crick Units of pancreozymin and immunoreactive insulin levels in normals, diabetics and patients diabetes. Insulin values (mean 2 SEM) represent rise above initial
150 130
-
II0 90
-
DIABETICS
NORMALS
Fig. 3.-Effect of intravenous administration of 1 mg. of giucagon on blood sugar and immunoreactive insulin levels in normals and in diabetic subjects. Insulin values (mean 2 SEM) represent rise initial above level.
/HI
70 70
-
60
-
50
-
0
5
IO
I5
20 25 30 MINUTES
5
IO
15 20
25
30
54
KAHIL,
82
130 -
DIABETICS
NORMALS
MC ILHANEY
SUBCLINICAL
AND JORDAN
DbWEflCS
(J) 110 “s god
70/
50 120 I IO -
loo-
5 goX60g
70-
2
60-
;;~LJkJ
ii50-
k
0
IO
20?iO405060120
IO 2030405060120 MINUTES
IO 2030405D60120
Fig. 4.-Effect of intravenous administration of 1 Cm. of tolbutamide sugar and immunoreactive insulin levels in normals, diabetics and patients
clinical diabetes. Insulin values (mean t the normals but significantly different after tolbutamide administration. Insulin
Secretion
Following
Glucose
SEM) represent
on blood with sub-
rise above initial level.
from the diabetics
one and three minutes
Infusion
It is apparent from Fig. 5 that when hyperglycemia of a magnitude similar to that present in the diabetics was produced in five normal subjects, secretin failed to raise further the mean insulin level. In only one patient was a rise in insulin levels observed following the infusion of secretin, whereas there was a fall in the ,remaining four. Insulin
Secretion
in Response
to in vivo Release
of Secretin
The effects of continuous acid perfusion of the duodenum on the bicarbonate excretion by the pancreas and the insulin levels in the portal vein blood are shown in Fig. 6. The portal blood insulin rise after endogenous secretin production occurred within five minutes of the onset of duodenal stimulation. The insulin response was of short duration, however, and the levels returned to control values in spite of continued secretin production as evidenced by the prolonged pancreatic secretion.
ENTERICHORMONES
220 -
180 -
20 -
-10 GLUCOSE
INFUSION
I
:~~:~~:~.:.:.:~.~~~~~.: : _.z
7.5mp/kO/Min.
3
5
IO
15
30
MNUTES
Fig. S.-Effect of constant infusion of 7.5 mg./Kg./min. of glucose for 60 minutes together with intravenous administration of 75 Crick Units of secretin on the blood sugar and immunoreactive insulin levels in five normal subjects. Insulin values represent mean -C SEM. BICARBONATE
120 -
_. UY c 5 0 t E z
3 2z -
r--._
r /
2
,/’
loo-
,’
I
8060 -
40 -
4
Acid infusion started
1 - 140 - 120
__
_ 100
.g -_ 25 f
1’
I
20 -
-
-
80
-
60
-
40
'0" 2
Fig. 6-Effect of continuous acid perfusion of duodenum in two dogs on portal blood insulin response and bicarbonate excretion by pancreas.
TIME (minutes)
DISCUSSION Experiments in dogs and in man demonstrate that the rise in blood insulin concentration that follows glucose ingestion is greater than the rise provoked by glucose infusions. Secretin, pancreozymin and glucagon have been implicated as the humoral factors that enhance the insulin reieasing capacity of ingested glucosc.!‘~‘”
56
KAHIL.
MC ILHANEY
AND JORDAN
Several conflicting results, however, have raised doubts as to the physiological significance of these hormones. Dupre et al. have shown that duodenal instillation of hydrochloric acid in man resulted in a rise in the peripheral serum immunoreactive insulin concentration. l1 In our dog experiments, however, the stimulation of endogenous secretin production by duodenal acid instillation resulted in a very transient rise in portal venous blood insulin levels in spite of continued secretin production. Similarly, Boyns et al. showed that the induction of endogenous secretin production in two normal subjects by hydrochloric acid instillation in the duodenum did not affect the blood sugar or peripheral plasma insulin levels and that, when glucose plus acid and glucose alone were instilled in the duodenum, almost identical changes in the blood sugar and insulin levels were observed.” The role of glucagon in influencing the insulin secretory response to ingested glucose has been clarified by the studies of Unger et al.13 They showed that the rise in glucagon-like immunoreactivity that followed the ingestion of glucose was not of pancreatic origin since it was observed in the absence of the pancreas. This material, probably derived from the gut, was devoid of hyperglycemic and glycogenolytic activity but stimulated insulin release. Hyperglycemia, whether induced by intravenous or enteric glucose administration suppressed pancreatic glucagon secretion. Conflicting results on the in vitro effects of secretin and pancreozymin in causing insulin release from pancreatic tissue have been obtained by various mvestigators.14~15 In our studies, secretin administration resulted in a modest and transient increase in peripheral insulin levels without concomitant blood glucose changes. No difference in the speed or magnitude of the insulin response was observed in the normal, diabetic or subclinical diabetic subjects. It is of interest that an insulin rise was observed in the diabetics, whereas in four normal individuals in whom a comparable degree of hyperglycemia was induced by glucose infusions, a bolus injection of secretin did not elicit a further increase in the insulin levels. It should be pointed out that maintained stimulation of insulin secretion has been observed in man when simultaneous sustained infusions of secretin and glucose were administered.” Tolbutamide and pharmacological doses of glucagon produced a prompt outpouring of insulin in the normals and an obviously impaired response in the diabetics. The insulin response to secretin and pancreozymin in doses probably close to physiologic levels was similar in the normal and diabetic subjects but was far below the capacity of the islets in these patients to produce or release insulin in response to tolbutamide. By contrast, DecKert showed in eight diabetic patients that secretin was a much more potent stimulus for insulin secretion than intravenous glucose, suggesting that the defect in the diabetic may represent either a blockade of normal glucose transport into the p-cells or an abnormal metabolism of glucose by these cells. I6 The elevation of plasma insulin levels noted in our diabetic patients after secretin administration but not observed in normal subjects rendered hyperglycemic suggests that the islet cells in the diabetics though still containing insulin reserves may be hyporesponsive to rising blood glucose concentrations.
ENTERIC
57
HORMONES
REFERENCES 1. McIntyre, N., Holdsworth, C. D., and Turner, D. S.: New interpretation of oral glucose tolerance. Lancet 2:20, 1964. 3. -, - , and -: Intestinal factors in the control of insulin secretion. J. Clin. Endoer. 25: 1317, 1965. 3. Dupre. J .. and Beck, I. C.: Stimulation of release of insulin by an extract of intestinal mucosa. Diabetes 15:.555, 1966. 4. Brown, J.. and Straatsma. B. R.: The UCLA Interdepartmental Conference. Diabetes Mellitus: Current Concepts and Vascular Lesions (Renal and Retinal). Ann. Intern. Med. 68:634. 1968. 5. Fajans. S. S.: Diagnostic tests for diabetes mellitus. In Williams. R. H. (Ed.) : Diabetes. New York, Hoeber, 1960. 6. Nelson. N.: A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375, 1944. 7. Morgan. C. R.. and Lazarow, A.: Immunoassay of insulin: Two antibody system. Plasma insulin levels of normals, subdiabetic and diabetic rats. Diabetes 12: 115, 1963. X. Preshaw. R. M., Cooke, A. R.. and Grossman. M. I.: Quantitative aspects of response of canine pancreas to duodenal acidification. Amer. J. Physiol. 210:629, 1966. 9. Letterer. H., Ohneda, A.. Dupre. J., Eisentraut. A., and Unger. R. H.: Effect of enteric hormones on insulin and
glucagon secretion. J. Lab. Clin. Med. 6X: 888, 1966. 10. White, J. J., and Dupre, J.: Regulation of insulin secretion by the intestinal hormone secretin: Studies in man via transumbilical portal vein catheterization. Surgery 64:204, 1968. II. Dupre, J. J., Curtis. J. D.. Waddell. R. W., and Beck, J. C.: Regulation of pancreatic endocrine function by gastrointestinal hormones. Proc. Roy. Sot. Med. 61: 815. 1968. 12. Boyns. D. R., Jarrett. R. J., and Keen. H.: Intestinal hormones and plasma insulin. Lancet 1:409, 1966. 13. Unger, R. H., Ohneda. A., Valverde, I., Eisentraut. A. M.. and Extron, J.: Characterization of the responses of circulating glucagon-like immunoreactivity to intraduodenal and intravenous administration of glucose. J. Clin. Invest. 47:48, 196X. 14. Lazarus, N. R.. Voyles, N., Tanese. T., Devrim. S., and Recant, L.: Extra-gastrointestinal effects of secretin. gastrin and pancreozymin. Lancet 2:248. 1968. 15. Pfeiffer. E. F., Telib. M., Ammon, J.. Melani, F., and Ditschuneit, M.: The direct stimulation by secretin of insulin secretion in vitro. German Med. Monthly 11:432. 1966. 16. DecKert, T.: Insulin secretion following administration of secretin in patients with diabetes mellitus. Acta Endocr. 59: 150, 1968.