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Glucose-insulin interactions on exocrine secretion from the perfused rat pancreas
GASTROENTEROLOGY
1984:87:1305-12
Glucose-Insulin Interactions on Exocrine Secretion From the Perfused Rat Pancreas ROBERTO BRUZZONE, ELISABETH R. TRIMBLE, ASLLAN GJINOVCI, and ALBERT E. RENOLD Institut de Biochimie Switzerland
Clinique.
University
of Geneva,
The effects of glucose and insulin on pancreatic enzyme release have been investigated using the isolated perfused rat pancreas. Basal and caeruleinstimulated secretion was significantly less in the presence of 15 mM glucose than with 5 mM glucose, except al. a supramaximal concentration of caerulein (10~’ M) where secretion was similar in both groups. Addition of exogenous insulin also caused a reduction in enzyme secretion, but the time of onset of the inhibitory action was delayed compared to that observed with glucose. Furthermore, it was found that the effects of 15 mM glucose and exogenous insulin were not additive at the concentration used in these experiments, and that the inhibitory action of insulin was glucose-dependent. Such glucase-insulin interactions must play an important role in the modulation of pancreatic enzyme secretion. Although much is known about the neurological and hormonal control of pancreatic enzyme secretion, less is known about the modulatory role played by the various products of digestion. For example, although quite a few studies have been directed at identifying the effects of glucose, there remains a great deal of uncertainty about the role played by glucose and the mechanisms involved in glucose modulation of enzyme secretion. Early in vivo studReceived December 21. 1983. Accepted June 25, 1984. Address requests for reprints to: Dr. R. Bruzzone, Institut de Biochimie Clinique, University of Geneva, Sentier de la Roseraie, 1211 Geneva 4. Switzerland. This project was supported by Grant No. 3.246-0.82 of the Swiss National Science Foundation. This work has been presented previously. in part, at the 19th Meeting of the European Association for the Study of Diabetes. Oslo, 1983, and published in abstract form (Diabetologia 1983: 25:144). The authors thank Ms. L. Cavillier, N. Challet, B. Christen, and T. Cuche for expert technical assistance: and Mr. C. Jorand for animal care. c 1984 by the American Gastroenterological Association onlli-.5n85/84/~.3.~ln
Sentier
de la Roseraie,
1211 Geneva
4
ies gave conflicting results. with hyperglycemia being reported as causing either increased (l-3) or decreased (4) enzyme secretion. More recent in vivo studies have tended to show a decreased enzyme output in the presence of hyperglycemia (5-7) [see Schapiro et al. (8) for a review]. Towne et al. (6) and MacGregor et al. (7) considered that the glucose effects on exocrine secretion might not be direct but mediated, at least in part, by neural mechanisms. In addition, recent in vitro studies have yielded conflicting results with glucose being shown to inhibit (9) or potentiate cholecystokinin (CCK)-induced enzyme release (10). We undertook the present study in an attempt to clarify further some actions of glucose on the exocrine pancreas using the isolated perfused rat pancreas. This preparation allowed us to preserve the normal anatomic architecture of the pancreas, while excluding possible effects of the central nervous system. The effects of glucose have been studied under basal and caerulein-stimulated conditions. Caerulein is believed to react with the same receptors as CCK (11). Receptors for this family of peptides are of two types, high and low affinity, the latter activated at only high (supramaximal) concentrations of CCK. It has been proposed that activation of the low affinity receptors leads to reduced enzyme release compared with that obtained when only high affinity receptors are maximally activated (12,13). For this reason we have investigated the effects of glucose at submaximal, (near) maximal, and supramaximal concentrations of caerulein. In addition, as the effects of glucose and insulin are often interrelated, in certain experiments the effects of insulin on enzyme output have also been studied. Materials
and Methods
Male Wistar rats weighing 250-300 g were fasted overnight but allowed water ad libitum. Rats were anestheAbbreviations
used
in this paper:
CCK, cholecystokinin.
GASTROENTEROLOGY Vol. 67, No. 6
1306 BRUZZONE ET AL.
tized with pentothal (100 mg/kg body weight, ip.). The procedure used to isolate and perfuse the pancreas was that described by Penhos et al. (14) with minor modifications as described in detail elsewhere (15). The pancreas was perfused with a modified Krebs-Ringer HEPES buffer (KRB-HEPES) containing: 12.5 mM HEPES, 121 mM NaCl, 4.8 mM KCl, 1.0 mM CaC12, 1.2 mM KH2P04, 1.2 mM MgS04, 24.6 mM NaHC03, 40 g/L dextran T-40, and 2.5 g/L human serum albumin. The perfusing buffer was continuously gassed with a mixture of 95% O2 and 5% COz, pH 7.4. Flow rate was kept constant at 6 ml/min and pressure was maintained between 40 and 60 mmHg throughout the experiment. The pancreatic effluent was collected continuously from a catheter in the portal vein and divided into 30-s aliquots in plastic tubes containing 250 KIUiml of Trasylol. These were immediately chilled (at 4°C) and stored at -20°C until assayed for the determination of insulin. Pancreatic juice was collected simultaneously from the duodenal end of Wirsung’s duct by means of a small steel cannula attached to a calibrated micropipette. (The hepatic end of the common bile duct was tied off .) The juice was immediately diluted in chilled (4°C) 0.9% NaCl and then stored at -20°C until assayed for protein and enzymes. In all experiments, after completion of surgery, the pancreas was perfused for a 30-min equilibration period during which no samples were collected. Then basal secretion was measured for 15 min (-15-O min) followed by a 60min stimulatory period (O-60 min).
Protocol
1
The pancreas was perfused with KRB-HEPES containing either 5 or 15 mM glucose or 10 mM pyruvate throughout the experiment. Caerulein (Farmitalia, Milan, Italy) was dissolved in perfusion buffer and infused via a side-arm at a concentration of lo-” M from 0 to 60 min. In some experiments, the effect of insulin was tested by the addition of pork insulin to the perfusion medium. The concentration of insulin used was 25 rig/ml during the equilibration and basal periods and 50 rig/ml during stimulation with caerulein.
Protocol
2
The pancreas was perfused with buffer containing either 5 or 15 mM glucose throughout the experiment. Caerulein was added successively at concentrations of lo-“, lo-‘0, and lo-’ M, each for a 20-min period. In some experiments, pork insulin was introduced as described for protocol 1.
Protocol
3
The pancreas was perfused with buffer containing 5 mM glucose during the equilibration and basal periods. Then caerulein (lo-” M) was added from 0 to 60 min. During the first part (O-20 min) of the stimulation, glucose concentration was kept at 5 mM. During the second part (20-60 min), either the glucose concentration was
changed to 15 mM or pork insulin continued presence of 5 mM glucose.
Protocol
was added
in the
4
During the equilibration period, the perfusate contained 5 mM glucose. Then glucose was removed for the rest of the experiment [basal and stimulatory periods]. In some experiments, pork insulin was added after 20 min of stimulation until the end of the experiment (i.e., from 20 to 60 min]. Assays Proteins were measured by the method of Bradford (16). The assay was carried out in 0.02 M phosphate buffer, pH 6.9, using human serum albumin as standard. The intra- and interassay coefficients of variation were 7.6% and 14.6%, respectively, at a protein concentration of 4 pgl ml. a-Amylase was measured by a modification of the method of Bernfeld (17), using soluble starch as the substrate and maltose monohydrate as the standard. One unit of amylase is defined as the amount of amylase that hydrolizes 1 pmol of maltose per minute per milliliter at 30°C. Intra- and interassay coefficients of variation were 12.4% and 15.7%, respectively, at a maltose concentration of 1 *mol/ml. Lipase was measured by means of a commercially available kit (Monotest 10 Lipase, Boehringer GmbH, Mannheim, Federal Republic of Germany). The assay was carried out at pH 8.8 in 26 mM Tris buffer containing 0.1 mM CaCl* and 0.1% human serum albumin. Porcine lipase was used as standard. One unit of lipase is the amount that hydrolizes 1 pmol of triolein per minute per milliliter at 25°C. The intra- and interassay coefficients of variation were 8.7% and 15.6%, respectively, at a lipase concentration of 150 U/ml. Chymotrypsinogen was activated to chymotrypsin before its assay by addition of bovine trypsin (16 ~1 of a l-mgi ml solution in 0.001 N HCl). The samples were previously diluted to -75-150 pglml of protein with 0.08 M Tris buffer plus 0.1 M CaCl*, pH 7.8. To ensure that maximal activation was reached, pilot experiments with different activation techniques were performed measuring either bovine chymotrypsinogen A or samples at various dilutions. No significant differences were found when using the following activation techniques: 1, 2, and 3 h at 37% 24 and 48 hat 4°C. Once activated, samples were cooled on ice and measured according to Hummel (18) within 2 h. aChymotrypsin was used as the standard and 1.07 mM Nbenzoyl tyrosine ethyl ester was used as the substrate. One unit of chymotrypsinogen is the amount that hydrolizes 1 pmol of substrate per minute per milliliter at pH 7.8 and 25°C. Intra- and interassay coefficients of variation were 4.7% and 9.8%, respectively, at a chymotrypsinogen concentration of 1.5 U/ml. Insulin was measured by radioimmunoassay, as detailed elsewhere (19).
December
1984
1307
GLUCOSE INHIBITION OF ENZYME SECRETION
600 -
Source of Materials Soluble starch and D( +)-maltose monohydrate were obtained from Fluka AG, Buchs, Switzerland; purified human serum albumin from Behringwerke AG, Marburg, Federal Republic of Germany; and N-benzoyl tyrosine ethyl ester from Calbiochem, Behring Corp., La Jolla, Calif. Bovine a-chymotrypsin CDS-7lA, bovine chymotrypsinogen A CGC, bovine trypsin, and cr-amylase AA 2BA were obtained from Worthington Biochemical Corp., Freehold, N.J. Porcine lipase and the lipase kit (Monotest 10) were generously donated by Professor F. H. Schmidt, Boehringer GmbH, Mannheim, Federal Republic of Germany. Caerulein was a generous gift of Dr. C. De Paolis, Farmitalia, Milan, Italy. Aprotinin (Trasylol) was obtained from Bayer AG, Wuppertal, Federal Republic of Germany. HEPES (N-2 hydroxyethylpiperazine, N’-2-ethanesulfonic acid) was obtained from Sigma Chemical Co., St. Louis, MO., and pork insulin from Novo Research Institute, Bagsvaerd, Denmark. The insulin tracer was a generous gift from Dr. Bruce Frank, Eli Lilly Co., Indianapolis, Ind., and the guinea pig antipork insulin serum was kindly donated by Dr. A. Lernmark, Hagedorn Research Laboratory, Gentofte, Denmark.
‘45
30
!
15 I 0+
o J i&d
0
160 1
10 5
r I
"T
1.2
*
08
Statistics Results are expressed as mean analyses were made using Student’s analysis of variance where appropriate. were considered significant.
2 SEM. Statistical unpaired t-test or p-Values of 50.05
Results Eflects of Glucose on Enzyme Secretion Juice Flow (Protocols 1 and 2)
and
In all these experiments, the glucose concentration was kept constant throughout the equilibration, basal, and stimulatory periods at either 5 or 15 mM glucose. Basal secretion, The output of amylase, lipase, and chymotrypsinogen were all -70% lower in the presence of 15 mM glucose than with 5 mM glucose (Figure 1, inserts, p < 0.005). The rates of juice flow with 5 and 15 mM glucose were 1.5 f 0.3 and 0.5 +- 0.1 $5 min, respectively; p < 0.05. Caerulein-stimulated secretion. The effects of caerulein were (i) first tested at a single dose that gave a marked, near-maximal stimulation and (ii) second at three separate concentrations which were submaximal, near-maximal, and supramaximal.
(i) When 10-l’ M caerulein
was introduced into the perfusate there was a prompt increase in enzyme secretion (Figure 1). The overall augmentations in enzyme secretion for the 60-min period of stimulation with caerulein were 12.6-, 13.9-, and 12.5-fold increases for amylase, li-
0.41 0+
0
20
io
0
6’0
minutes
Figure 1. Pancreatic enzyme release in the presence of either 5 mM glucose (closed circles, n = 6) or 15 mM glucose (open circles, n = 4). Caerulein (lo-" M) was added from 0 to 60 min. Inserts show enzyme output during the basal period. These values represent the mean of three consecutive samples collected every 5 min before the addition of caerulein, for each experiment. All results are expressed as mean 2 SEM.
pase, and chymotrypsinogen, respectively. The response to caerulein stimulation was less prompt in the presence of 15 mM glucose, with maximum values being attained during the second 5-min period of stimulation. The average enzyme secretory rates during the 60-min stimulation were increased with respect to basal values by 51.7-, 69-l-, and 40.7-fold for amylase, respectively. lipase, and chymotrypsinogen, However, in absolute terms total enzyme output stimulated by 10 -lo M caerulein was at least 50% lower in the presence of 15 mM glucose than that found with 5 mM glucose (amylase, F = 77.2, p < 0.001; lipase, F = 67.8, p < 0.001; chymotrypsinogen, F = 61.6, p < 0.001). During stimulation with 10-l’ M caerulein the rates of juice flow with 5 and 15 mM glucose were 12.7 5 1.6 and 5.9 + 1.1 ~115 min, respectively; p < 0.02.
1308
BRUZZONE
GASTROENTEROLOGY
ET AL.
(ii) The dose-response curve for caerulein with respect to pancreatic enzyme secretion is bellshaped; increasing concentrations of caerulein are usually described as submaximal, maximal, and supramaximal (12,13). In the presence of 5 mM glucose and in our perfused pancreas preparation, caerulein concentrations of 10pll, 10-l”, and lo-” M were submaximal, (near] maximal and supramaximal, respectively. Thus, with 1O-g M caerulein, enzyme secretion was less than with 10-l’ M caerulein (Figure 2, left-hand panel). As previously noted for 10p’o M caerulein, at lo-l1 M caerulein, enzyme secretion was lower with 15 than with 5 mM glucose (Figure 2: amylase, F = 17.9, p < 0.001; lipase, F = 8.1,p < 0.025;chymotrypsinogen, F = 4.6,p < 0.025). However, at 10 -’ M caerulein, enzyme secretion was similar in the presence of 5 and 15 mM glucose. In contrast to the finding with 5 mM glucose, in the presence of 15 mM glucose lo-’ M caerulein was not supramaximal with respect to stimulated enzyme secretion.
Vol.
87, No.
60
0 16 12 8 L 0
Effects of Exogenous Insulin on Enzyme Secretion, Protein Output, and Juice Flow (Protocols 1 and 2) As it was not clear from the above experiments whether the effect of glucose on enzyme secretion was a direct effect on the acinar cell or mediated, for example, through increased insulin secretion, further experiments were carried out to determine if exogenous insulin could alter enzyme secretion. The amount of added insulin was chosen arbitrarily and was approximately the amount secreted by the pancreas at 15 mM glucose. Insulin was infused at 25 ngiml during the equilibration and basal periods and at 50 ngiml during the stimulatory periods in all of the following experiments. Basal secretion. Insulin inhibited basal secretion of amylase, lipase, and chymotrypsinogen when the glucose concentration in the perfusate was 5 mM (Figure 3, extreme left-hand columns]. The addition of insulin to the perfusate did not have an additive effect on the inhibition caused by 15 mM glucose. Total protein output at 5 mM glucose was 10.5 f. 1.7 and 4.6 2 1.2 pg/min in the absence and presence of insulin, respectively, p < 0.025.With 15 mM glucose, the basal protein output was 1.3 + 0.7 and 0.33 k 0.29 pglmin in the presence and absence of insulin, respectively [not significant). With 5 mM glucose, juice flow was not inhibited by insulin (1.52 + 0.3VS. 1.00 ? 0.20 /d/5min, respectively). With 15 mM glucose, juice flow was 0.50 t 0.10 and 0.48 +0.16 &5 min, in the presence and absence of insulin, respectively.
-15 0 20 LO 60minuti;50 20 LO 60
CRL (Ml Figure
0
10-J'lo-'010-s
0
10-J'lo-'0 10-9
2. Effect of increasing concentrations of caerulein (CHL) on amylase, lipase, and chymotrypsinogen secretion in the presence of either 5 mM (hatched bars. n = 6) or 15 mM (open bars, n = 5) glucose. Three concentrations of CRL were infused successively during the stimulatory period, each for 20 min. Results are presented as mean 2 SEM.
Caerulein-Stimulated
Secretion
(i) With 5 mM glucose, insulin had an inhibitory effect on amylase and lipase secretion stimulated by lo- ” M caerulein (F = 77.2, p < 0.001 and F = 54.1, p < 0.001, respectively). In these experiments, insulin did not have a significant effect on chymotrypsinogen secretion (Figure 3). In the presence of 15 mM glucose, insulin had no effect on stimulated enzyme secretion. Insulin inhibited protein output with 5 mM glucose (120 -+ 7.1 vs. 79.0 2 15.6 pg/min, F = 61.1, p < O.OOl), whereas it had no effect with 15 mM glucose (47.6 k 9.2 and 39.6 +- 4.0 pgimin, without and with added insulin, respectively). Furthermore, insulin did not inhibit caeruleinstimulated (lo-” M) juice flow with either 5 mM glucose (12.7 k 1.6 vs. 8.5 t 1.4 pl/5 min, in the absence and presence of exogenous insulin, respectively] or with 15 mM glucose (5.9 t 1.1
6
December
1984
vs 6.1 -C0.5 &5 min, without and with added insulin, respectively). (ii) The effect of insulin at different caerulein concentrations was investigated only in the presence of 5 mM glucose. Enzyme secretion stimulat.ed by lOPro M caerulein was significantly inhibited by insulin (amylase, F = 35.8, p < 0.001; lipase, F = 6.9, p < 0.025; chymotrypsinogen, F = 15.0, p < 0.001; Figure 4). As in the case of glucose, insulin had no inhibitory effect on enzyme output at a caerulein concentration of lo-” M.
GLUCOSE INHIBITIONOF ENZYME SECRETION 1309
GLUCOSE 5 mM
GLUCOSE 15 mM
90
P E
2 Z 4 f
60
3o
Time Dependence of the Effects of Glucose and Insulin on Enzyme Secretion (Protocol 31 Further experiments were undertaken to assess whether the prolonged exposure time to high concentrations of glucose and to exogenous insulin during the equilibration period could be of importance in determining the degree to which caeruleinstimulated enzyme output was inhibited by these agents in the above experiments. Glucose (15 mM) or insulin (50 ngiml, in the continued presence of 5 mM glucose) was introduced 20 min after the addition of caerulein (lo-” M), and their effects were followed for 40 min. The results are shown in Table 1. In control experiments, amylase secretion was constant during the three successive 20-min periods of caerulein stimulation. When 15 mM glucose was added after the first 20 min (O-20 min) of caerulein stimulation, a significant decrease of amylase output was noted during both the second (20-40 min) and third (40-60 min) periods. The addition of insulin also reduced amylase release but, in contrast to the effect of 15 mM glucose, inhibition of amylase secretion was significant only during the third period (40-60 min). However, for both glucose and insulin the inhibitory effect on exocrine secretion after 40 min was considerably less pronounced when compared with experiments where they had been present 45 min before the introduction of caerulein. i.e., during the equilibration and basal periods (compare with Figures 1 and 3). Effect of Insulin on Enzyme Secretion in the Absence of Glucose (Protocols 4 and 1) These experiments were carried out to test whether the inhibitory effect of insulin on enzyme output could be due indirectly to an effect of insulin on glucose metabolism in pancreatic tissue. (i) In the first series of experiments, 5 mM glucose was present in the equilibration period and was
INSULIN CRL 04 Figure
- +
- +
0
10-10
-+
-+ 0
10-10
3. Effect of exogenous insulin on basal and caerulein (CHL)-stimulated enzyme secretion in the presence of either 5 mM (hatched bars] or 15 mM [open hors] glucose. Results are expressed as mean -C SEM. The number of experiments for each protocol was as follows: 5 mM glucose, n = 6: 5 mM glucose + insulin. II = 4; 15 mM glucose, n = 4: 15 mM glucose + insulin. n = 3. Insulin concentration was 25 @ml during the basal and 50 ngiml during the stimulatory period, as indicated in Materials and Methods.
withdrawn 15 min before the introduction of lOPro M caerulein at 0 min. In one group of experiments (control experiments), no further changes were made during the 60-min stimulatory period. In the second group of experiments, 50 ng/ml insulin was added after 20 min of caerulein stimulation. Insulin did not have an inhibitory effect on amylase secretion in these experiments [Table 2). (ii) A second series of experiments was carried out where 5 mM glucose was replaced by 10 mM pyruvate. In this case, pyruvate was present throughout the experiment, including the equilibration period. Although a stable secretory rate was not achieved in these experiments, amylase
GASTROENTEROLOGY Vol. 87, No. 6
BRUZZONE ET AL.
Table 1. Glucose and Insulin Modulation of CaeruleinStimulated Amylase Secretion From the Isolated Perfused Rat Pancreas: Effect of Time
60 -.
im
Controls (n = 6) Glucose (n = 6) Insulin (n = 4)
O-20 min
20-40 min
40-40 min
1ooa 100 100
99.9 * 9.6 77.1 + 5.6+ 87.9 2 7.6
100.4 2 9.0
76.2 2 7.2” 72.2 2 3.3”
All experiments had the usual equilibration and basal periods in the presence of 5 mM glucose. In all three experimental groups the perfusate contained 5 mM glucose and lo-” M caerulein during the period O-20 min. In protocol 1, this was kept constant without change until 60 min. In protocols 2 and 3, there was one alteration in the perfusate from 20-60 min; in protocol 2, the glucose concentration was increased to 15 mM; and in protocol 3, 50 ngi ml insulin was added. o Only the stimulatory period is shown here. The data were normalized, with the first 20 min being expressed as 100%. For each protocol, the output of amylase in the periods 20-40 and 40-60 min is expressed as a percentage of the output during o-20 min for the same experiment (mean 2 SEM). The number of experiments is indicated in parentheses. b Glucose inhibited amylase secretion during 20-40 min (F = 14.7; p < 0.001) and during 40-60 min (F = 10.3; p < 0.005). Insulin inhibited amylase secretion only during 40-60 min (F = 10.0; p < 0.005).
Discussion ”
-15
0
20 LO 60
-15
minutes
CRL (M)
0
lo-" lo-'0w
0
0
20 LO 60
lo-" 10-M 1w
Figure 4. Basal and caerulein (CAL)-stimulated enzyme output in the absence (hatched bars, n = 6) and in the presence (open bars, n = 7) of exogenously added insulin (details in Materials and Methods]. Glucose concentration was 5 mM in both series of experiments. Caerulein was added as described in Figure 2. Results are presented as mean ? SEM.
output was totally unaffected by the presence of insulin introduced at -45 min (data not shown).
Thus, insulin had no inhibitory effect on amylase secretion in the absence of glucose or when 5 mM glucose was replaced by 10 mM pyruvate.
These results show that, at the level of the pancreas, the ambient glucose concentration has a profound effect on pancreatic enzyme secretion. Glucose inhibited basal and caerulein-stimulated enzyme secretion except for high concentrations of caerulein where it had no effect (i.e., lo-’ M caerulein, which is a supramaximal concentration with 5 mM glucose). The inhibitory action of high glucose concentrations found in these experiments would confirm the results of Danielsson (9) who also found inhibition of enzyme release when pancreatic pieces from fasted animals were incubated with 16.7 mM glucose, the effect of glucose being grossly attenuated when fed animals were used. With the same preparation, Robberecht and Christophe (20) found that 10 mM glucose had a delayed inhibitory action Table 2. Insulin ModuJation of Caerulein-Stimulated Amylase Secretion: Effect of the Absence of Glucose
Insulin Secretion Basal insulin secretion was --loo-fold higher in the presence of 15 mM glucose than with 5 mM glucose. Addition of caerulein stimulated insulin output but this effect was more pronounced at high concentrations of glucose. Insulin levels were also measured both in the absence of glucose and when 5 mM glucose was replaced by 10 mM pyruvate. Under these conditions caerulein did not modify the insulin secretory rate (Table 3).
Controls (n = 4) Insulin (n = 6)
O-20 min
20-40 min
40-60 min
100
105.4 2 9.7 94.5 + 10.5
118.5 2 19.5 87.5 t 8.8
100
In both protocols, during the equilibration period, the perfusate contained 5 mM glucose; it was then removed for the rest of the experiment. Caerulein (lo- lo M) was added from 0 to 60 min in both experimental groups. In addition, in the insulin experiments, 50 ngiml pork insulin was added from 20 to 60 min. Basal amylase secretion was 4.9 + 2.3 and 3.4 ‘-’ 0.3 Uimin for the first and second group of experiments, respectively. Results are expressed as mean 5 SEM; the number of experiments is indicated in parentheses.
December
Table
3.
1984
Basal
GLUCOSE
and
Caerulein-Stimulated
Insulin
Output From the Isolated
INHIBITION
Perfused
OF ENZYME
SECRETION
1311
Rat Pancreas
Substrate Glucose (mM1 5 15 0 0
Pyruvate (mM1 0 0 0 10
Insulin n 6 4 4 5
Basal 0.37 44.0 1.14 0.15
2 k k k
10 min 0.16 6.0 0.19 0.05
1.79 ?z 0.95 150 t 21.7 1.08 I 0.17 0.21 + 0.09
secretion
20 min
(ngimin)
30 min
1.45 2 0.50 141 + 40.6 1.25 i- 0.25 0.19 2 0.05
3.67 208 1.15 0.15
+ + k f
2.43 23.2 0.28 0.03
40 min 1.68 2 202 t 1.11 + 0.23 t
1.07 48.6 0.28 0.04
50 min 1.25 223 1.32 0.17
+ 0.88 t 54.2 +- 0.33 + 0.06
60 min 1.90 199 1.17 0.24
+ t k ?
1.38 65.3 0.35 0.10
Mean f SEM. The substrate concentration was kept constant throughout the entire experiment in protocols 1,2, and 4. In protocol 3, 5 mM glucose was present in the equilibration period and, thereafter, removed. For all experiments. basal output represents a mean of three samples collected during the basal period at 5-min intervals. Caerulein was then infused at the constant rate of lo-” M from 0 to 60 min.
on enzyme secretion. By contrast, Saito et al. (10) using the isolated perfused rat pancreas, although finding no consistent effect of glucose on basal enzyme secretion, described a potentiating effect of glucose on CCK-induced secretion. The reasons for these discrepancies are not obvious; they may be due to differences in concentration and purity of the secretagogue used in their experiments. An interesting point in the present results is the fact that the effect of high glucose concentrations on caeruleinstimulated enzyme secretion is restricted to those concentrations of caerulein that give submaximal or (near) maximal stimulation with 5 mM glucose. Thus, the alteration in caerulein-stimulated release is possibly the result of interference between caerulein and its high affinity receptors (12,13) or of metabolic events activated by this receptor, or both. The question remained as to whether the effect of glucose was a direct one on the acinar cell or whether it was mediated by another factor, e.g., glucose-induced insulin release. We, therefore, tested the effect of exogenous insulin and found that it also had an inhibitory action on enzyme secretion. However, this was not additive with the effect of glucose when tested with lo-” M caerulein. In the presence of 5 mM glucose, the effect of the single insulin concentration tested was less pronounced than that of 15 mM glucose. Basal enzyme secretion and both amylase and lipase secretion stimulated by lo-lo M caerulein were inhibited. However, the effects of insulin on caerulein-stimulated chymotrypsinogen secretion were inconstant (compare Figures 3 and 4). In addition, in another series of experiments, we showed that the time of onset of inhibition by insulin was delayed with respect to that of glucose. Despite these differences, it is clear that the inhibitory action of insulin demonstrated in these experiments is dependent on the presence of glucose, as insulin had no effect on secretion in the absence of glucose or when 5 mM glucose was replaced by 10 mM pyruvate. As was the case with glucose, insulin had no effect on enzyme secretion in the presence of lo-" M caerulein. This, and the
glucose dependence of the insulin inhibitory effect, would be consistent with the inhibitory actions of glucose and insulin acting through a common mechanism. Danielsson (9), using much higher concentrations of insulin, found it had an inhibitory effect on basal and caerulein-stimulated amylase secretion from pancreatic fragments. By contrast, both Kanno and Saito (21) and Saito et al. (10) found that insulin had a stimulatory effect on amylase secretion stimulated by half-maximal concentrations of CCK-pancreozymin. However, in another paper where isolated acini from diabetic rats were studied at a high concentration of glucose (11.1mM), Otsuki and Williams (22) found that 100 nM insulin had a potentiating effect on CCK-induced amylase release only after at least 1 h of preincubation with insulin and only at supramaximal CCK concentrations. It is clear, therefore, that the action of insulin on enzyme secretion is more complex than hitherto imagined. In the present experiments, although insulin had an inhibitory action at intermediate glucose concentrations (5 mM), it had no effect in the absence of glucose or at high glucose concentrations (15 mM). It is, therefore, evident that insulin dose-responses at each of many glucose concentrations will be needed to achieve a fuller understanding of interactions between glucose and insulin at the level of the acinar cell. However, in the experiments presented here, high concentrations of glucose were shown to inhibit basal and caerulein-stimulated enzyme secretion if the caerulein concentration was not supramaximal. Furthermore, it has been demonstrated that insulin may also decrease enzyme secretion by a glucose-dependent mechanism. Although the amounts of exogenous insulin used in this study may appear rather high, it has been proposed that the actual insulin concentrations reaching the exocrine tissue via the so-called insuloacinar portal vessels could be much higher than those measured in the peripheral circulation (23). Thus, both glucose and insulin creatic
play important roles enzyme secretion.
in modulation
of pan-
1312
BRUZZONE
ET AL.
References 1. La Barre J. The role of the central nervous system in the control of pancreatic secretion II. The external secretion of the pancreas during hyperglycemia. Am J Physiol 1930;94:17-21. 2. Hebb CO. Relation between the blood sugar concentration and the exocrine function of the pancreas. Arch Intern Pharmacodyn 1935;52:33-47. 3. Crider JO, Conly SS, Dorchester JEC. Thomas JE. Effect of intravenous injection of hypertonic glucose solution on external secretion of the pancreas. Am J Physiol 1956:186:187-g. 4. Okada S, Kuramochi K. Tsukahara T. Ooinoue T. Pancreatic function IV. The humoroneural regulation of the gastric, pancreatic and biliary secretions. Arch Intern Med 1929; 49:446-71. on pancreatic 5. Dyck WP. Influence of intrajejunal glucose exocrine function in man. Gastroenterology 1971:60:864-9. 6. Towne JB. Hamilton RE. Stephenson DV. Mechanism of hyperalimentation in the suppression of upper gastrointestinal secretions. Am J Surg 1973;126:716-8. 7. MacGregor IL, Deveney C. Way LW. Meyer JH. The effect of acute hyperglycemia on meal-stimulated gastric, biliary and pancreatic secretion, and serum gastrin. Gastroenterology 1976;70:197-202. 8. Schapiro H. Faulconer RJ. Lind JF, Dreiling DA. The effect of insulin on the exocrine pancreas: a review. Mt Sinai J Med 1981;48:95-110. 9. Danielsson A. Effects of glucose, insulin and glucagon on amylase secretion from incubated mouse pancreas. Pflugers Arch 1974:348:333-42. 10. Saito A, Williams JA. Kanno T. Potentiation of cholecystokinin-induced exocrine secretion by both exogenous and endogenous insulin in isolated and perfused rat pancreata. J Chn Invest 1980;65:777-82. 11. Jensen RT. Gardner JD. Identification and characterization of receptors for secretagogues on pancreatic acinar cells. Fed Proc 1981;40:2486-96. 12. Sankaran H. Goldfine ID, Deveney CW. Wong KY. Williams JA. Binding of cholecystokinin to high affinity receptors on
GASTROENTEROLOGY
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isolated rat pancreatic acini. J Biol Chem 1980;255:1849-53. 13. Sankaran H, Goldfine ID, Bailey A, Licko V, Williams JA. Relationship of cholecystokinin receptor binding to regulation of biological functions in pancreatic acini. Am J Physiol 1982;242:G250-7. 14. Penhos JC. Wu C, Basabe JC, Lopez N. Wolff FW. A rat pancreas-small gut preparation for the study of intestinal factor(s) and insulin release. Diabetes 1969;18:733-8. 15. Gerber PPG, Trimble ER, Wollheim CB, Renold AE. Miller RB. Glucose and cyclic AMP as stimulators of somatostatin and insulin secretion from the isolated, perfused rat pancreas. Diabetes 1981;30:40-4. 16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. 17. Bernfeld P. Amylases. a and /% In: Colowick SP. Kaplan NO. eds. Methods in enzymology. New York: Academic, 1955: 149-58. 18. Hummel BCW. A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can J Biochem Physiol 1959;37:1393-9. 19. Trimble ER, Halban PA, Wollheim CB. Renold AR. Functional differences between rat islets of ventral and dorsal pancreatic origin. J Clin Invest 1982;69:405-13. 20. Robberecht P. Christophe J. Effets du glucose sur la secretion d’amylase par des fragments perifuses de pancreas du rat. Arch Int Physiol Biochim 1971;79:1037-8. 21. Kanno T, Saito A. The potentiating influences of insulin on pancreozymin-induced hyperpolarization and amylase release in the pancreatic acinar cell. J Physiol [London) 1976: 261:505-21. 22. Otsuki M, Williams JA. Direct modulation of pancreatic CCK receptors and enzyme secretion by insulin in isolated pancreatic acini from diabetic rats. Diabetes 1983;32:241-6. 23. Fujita T, Yanatori Y. Murakami T. Insulo-acinar axis, its vascular basis and its functional and morphological changes caused by CCK-PZ and caerulein. In: Fujita T. ed. Endocrine gut and pancreas. Amsterdam: Elsevier Scientific Publishing. 1976:347-57.
1984:87:1305-12
Glucose-Insulin Interactions on Exocrine Secretion From the Perfused Rat Pancreas ROBERTO BRUZZONE, ELISABETH R. TRIMBLE, ASLLAN GJINOVCI, and ALBERT E. RENOLD Institut de Biochimie Switzerland
Clinique.
University
of Geneva,
The effects of glucose and insulin on pancreatic enzyme release have been investigated using the isolated perfused rat pancreas. Basal and caeruleinstimulated secretion was significantly less in the presence of 15 mM glucose than with 5 mM glucose, except al. a supramaximal concentration of caerulein (10~’ M) where secretion was similar in both groups. Addition of exogenous insulin also caused a reduction in enzyme secretion, but the time of onset of the inhibitory action was delayed compared to that observed with glucose. Furthermore, it was found that the effects of 15 mM glucose and exogenous insulin were not additive at the concentration used in these experiments, and that the inhibitory action of insulin was glucose-dependent. Such glucase-insulin interactions must play an important role in the modulation of pancreatic enzyme secretion. Although much is known about the neurological and hormonal control of pancreatic enzyme secretion, less is known about the modulatory role played by the various products of digestion. For example, although quite a few studies have been directed at identifying the effects of glucose, there remains a great deal of uncertainty about the role played by glucose and the mechanisms involved in glucose modulation of enzyme secretion. Early in vivo studReceived December 21. 1983. Accepted June 25, 1984. Address requests for reprints to: Dr. R. Bruzzone, Institut de Biochimie Clinique, University of Geneva, Sentier de la Roseraie, 1211 Geneva 4. Switzerland. This project was supported by Grant No. 3.246-0.82 of the Swiss National Science Foundation. This work has been presented previously. in part, at the 19th Meeting of the European Association for the Study of Diabetes. Oslo, 1983, and published in abstract form (Diabetologia 1983: 25:144). The authors thank Ms. L. Cavillier, N. Challet, B. Christen, and T. Cuche for expert technical assistance: and Mr. C. Jorand for animal care. c 1984 by the American Gastroenterological Association onlli-.5n85/84/~.3.~ln
Sentier
de la Roseraie,
1211 Geneva
4
ies gave conflicting results. with hyperglycemia being reported as causing either increased (l-3) or decreased (4) enzyme secretion. More recent in vivo studies have tended to show a decreased enzyme output in the presence of hyperglycemia (5-7) [see Schapiro et al. (8) for a review]. Towne et al. (6) and MacGregor et al. (7) considered that the glucose effects on exocrine secretion might not be direct but mediated, at least in part, by neural mechanisms. In addition, recent in vitro studies have yielded conflicting results with glucose being shown to inhibit (9) or potentiate cholecystokinin (CCK)-induced enzyme release (10). We undertook the present study in an attempt to clarify further some actions of glucose on the exocrine pancreas using the isolated perfused rat pancreas. This preparation allowed us to preserve the normal anatomic architecture of the pancreas, while excluding possible effects of the central nervous system. The effects of glucose have been studied under basal and caerulein-stimulated conditions. Caerulein is believed to react with the same receptors as CCK (11). Receptors for this family of peptides are of two types, high and low affinity, the latter activated at only high (supramaximal) concentrations of CCK. It has been proposed that activation of the low affinity receptors leads to reduced enzyme release compared with that obtained when only high affinity receptors are maximally activated (12,13). For this reason we have investigated the effects of glucose at submaximal, (near) maximal, and supramaximal concentrations of caerulein. In addition, as the effects of glucose and insulin are often interrelated, in certain experiments the effects of insulin on enzyme output have also been studied. Materials
and Methods
Male Wistar rats weighing 250-300 g were fasted overnight but allowed water ad libitum. Rats were anestheAbbreviations
used
in this paper:
CCK, cholecystokinin.
GASTROENTEROLOGY Vol. 67, No. 6
1306 BRUZZONE ET AL.
tized with pentothal (100 mg/kg body weight, ip.). The procedure used to isolate and perfuse the pancreas was that described by Penhos et al. (14) with minor modifications as described in detail elsewhere (15). The pancreas was perfused with a modified Krebs-Ringer HEPES buffer (KRB-HEPES) containing: 12.5 mM HEPES, 121 mM NaCl, 4.8 mM KCl, 1.0 mM CaC12, 1.2 mM KH2P04, 1.2 mM MgS04, 24.6 mM NaHC03, 40 g/L dextran T-40, and 2.5 g/L human serum albumin. The perfusing buffer was continuously gassed with a mixture of 95% O2 and 5% COz, pH 7.4. Flow rate was kept constant at 6 ml/min and pressure was maintained between 40 and 60 mmHg throughout the experiment. The pancreatic effluent was collected continuously from a catheter in the portal vein and divided into 30-s aliquots in plastic tubes containing 250 KIUiml of Trasylol. These were immediately chilled (at 4°C) and stored at -20°C until assayed for the determination of insulin. Pancreatic juice was collected simultaneously from the duodenal end of Wirsung’s duct by means of a small steel cannula attached to a calibrated micropipette. (The hepatic end of the common bile duct was tied off .) The juice was immediately diluted in chilled (4°C) 0.9% NaCl and then stored at -20°C until assayed for protein and enzymes. In all experiments, after completion of surgery, the pancreas was perfused for a 30-min equilibration period during which no samples were collected. Then basal secretion was measured for 15 min (-15-O min) followed by a 60min stimulatory period (O-60 min).
Protocol
1
The pancreas was perfused with KRB-HEPES containing either 5 or 15 mM glucose or 10 mM pyruvate throughout the experiment. Caerulein (Farmitalia, Milan, Italy) was dissolved in perfusion buffer and infused via a side-arm at a concentration of lo-” M from 0 to 60 min. In some experiments, the effect of insulin was tested by the addition of pork insulin to the perfusion medium. The concentration of insulin used was 25 rig/ml during the equilibration and basal periods and 50 rig/ml during stimulation with caerulein.
Protocol
2
The pancreas was perfused with buffer containing either 5 or 15 mM glucose throughout the experiment. Caerulein was added successively at concentrations of lo-“, lo-‘0, and lo-’ M, each for a 20-min period. In some experiments, pork insulin was introduced as described for protocol 1.
Protocol
3
The pancreas was perfused with buffer containing 5 mM glucose during the equilibration and basal periods. Then caerulein (lo-” M) was added from 0 to 60 min. During the first part (O-20 min) of the stimulation, glucose concentration was kept at 5 mM. During the second part (20-60 min), either the glucose concentration was
changed to 15 mM or pork insulin continued presence of 5 mM glucose.
Protocol
was added
in the
4
During the equilibration period, the perfusate contained 5 mM glucose. Then glucose was removed for the rest of the experiment [basal and stimulatory periods]. In some experiments, pork insulin was added after 20 min of stimulation until the end of the experiment (i.e., from 20 to 60 min]. Assays Proteins were measured by the method of Bradford (16). The assay was carried out in 0.02 M phosphate buffer, pH 6.9, using human serum albumin as standard. The intra- and interassay coefficients of variation were 7.6% and 14.6%, respectively, at a protein concentration of 4 pgl ml. a-Amylase was measured by a modification of the method of Bernfeld (17), using soluble starch as the substrate and maltose monohydrate as the standard. One unit of amylase is defined as the amount of amylase that hydrolizes 1 pmol of maltose per minute per milliliter at 30°C. Intra- and interassay coefficients of variation were 12.4% and 15.7%, respectively, at a maltose concentration of 1 *mol/ml. Lipase was measured by means of a commercially available kit (Monotest 10 Lipase, Boehringer GmbH, Mannheim, Federal Republic of Germany). The assay was carried out at pH 8.8 in 26 mM Tris buffer containing 0.1 mM CaCl* and 0.1% human serum albumin. Porcine lipase was used as standard. One unit of lipase is the amount that hydrolizes 1 pmol of triolein per minute per milliliter at 25°C. The intra- and interassay coefficients of variation were 8.7% and 15.6%, respectively, at a lipase concentration of 150 U/ml. Chymotrypsinogen was activated to chymotrypsin before its assay by addition of bovine trypsin (16 ~1 of a l-mgi ml solution in 0.001 N HCl). The samples were previously diluted to -75-150 pglml of protein with 0.08 M Tris buffer plus 0.1 M CaCl*, pH 7.8. To ensure that maximal activation was reached, pilot experiments with different activation techniques were performed measuring either bovine chymotrypsinogen A or samples at various dilutions. No significant differences were found when using the following activation techniques: 1, 2, and 3 h at 37% 24 and 48 hat 4°C. Once activated, samples were cooled on ice and measured according to Hummel (18) within 2 h. aChymotrypsin was used as the standard and 1.07 mM Nbenzoyl tyrosine ethyl ester was used as the substrate. One unit of chymotrypsinogen is the amount that hydrolizes 1 pmol of substrate per minute per milliliter at pH 7.8 and 25°C. Intra- and interassay coefficients of variation were 4.7% and 9.8%, respectively, at a chymotrypsinogen concentration of 1.5 U/ml. Insulin was measured by radioimmunoassay, as detailed elsewhere (19).
December
1984
1307
GLUCOSE INHIBITION OF ENZYME SECRETION
600 -
Source of Materials Soluble starch and D( +)-maltose monohydrate were obtained from Fluka AG, Buchs, Switzerland; purified human serum albumin from Behringwerke AG, Marburg, Federal Republic of Germany; and N-benzoyl tyrosine ethyl ester from Calbiochem, Behring Corp., La Jolla, Calif. Bovine a-chymotrypsin CDS-7lA, bovine chymotrypsinogen A CGC, bovine trypsin, and cr-amylase AA 2BA were obtained from Worthington Biochemical Corp., Freehold, N.J. Porcine lipase and the lipase kit (Monotest 10) were generously donated by Professor F. H. Schmidt, Boehringer GmbH, Mannheim, Federal Republic of Germany. Caerulein was a generous gift of Dr. C. De Paolis, Farmitalia, Milan, Italy. Aprotinin (Trasylol) was obtained from Bayer AG, Wuppertal, Federal Republic of Germany. HEPES (N-2 hydroxyethylpiperazine, N’-2-ethanesulfonic acid) was obtained from Sigma Chemical Co., St. Louis, MO., and pork insulin from Novo Research Institute, Bagsvaerd, Denmark. The insulin tracer was a generous gift from Dr. Bruce Frank, Eli Lilly Co., Indianapolis, Ind., and the guinea pig antipork insulin serum was kindly donated by Dr. A. Lernmark, Hagedorn Research Laboratory, Gentofte, Denmark.
‘45
30
!
15 I 0+
o J i&d
0
160 1
10 5
r I
"T
1.2
*
08
Statistics Results are expressed as mean analyses were made using Student’s analysis of variance where appropriate. were considered significant.
2 SEM. Statistical unpaired t-test or p-Values of 50.05
Results Eflects of Glucose on Enzyme Secretion Juice Flow (Protocols 1 and 2)
and
In all these experiments, the glucose concentration was kept constant throughout the equilibration, basal, and stimulatory periods at either 5 or 15 mM glucose. Basal secretion, The output of amylase, lipase, and chymotrypsinogen were all -70% lower in the presence of 15 mM glucose than with 5 mM glucose (Figure 1, inserts, p < 0.005). The rates of juice flow with 5 and 15 mM glucose were 1.5 f 0.3 and 0.5 +- 0.1 $5 min, respectively; p < 0.05. Caerulein-stimulated secretion. The effects of caerulein were (i) first tested at a single dose that gave a marked, near-maximal stimulation and (ii) second at three separate concentrations which were submaximal, near-maximal, and supramaximal.
(i) When 10-l’ M caerulein
was introduced into the perfusate there was a prompt increase in enzyme secretion (Figure 1). The overall augmentations in enzyme secretion for the 60-min period of stimulation with caerulein were 12.6-, 13.9-, and 12.5-fold increases for amylase, li-
0.41 0+
0
20
io
0
6’0
minutes
Figure 1. Pancreatic enzyme release in the presence of either 5 mM glucose (closed circles, n = 6) or 15 mM glucose (open circles, n = 4). Caerulein (lo-" M) was added from 0 to 60 min. Inserts show enzyme output during the basal period. These values represent the mean of three consecutive samples collected every 5 min before the addition of caerulein, for each experiment. All results are expressed as mean 2 SEM.
pase, and chymotrypsinogen, respectively. The response to caerulein stimulation was less prompt in the presence of 15 mM glucose, with maximum values being attained during the second 5-min period of stimulation. The average enzyme secretory rates during the 60-min stimulation were increased with respect to basal values by 51.7-, 69-l-, and 40.7-fold for amylase, respectively. lipase, and chymotrypsinogen, However, in absolute terms total enzyme output stimulated by 10 -lo M caerulein was at least 50% lower in the presence of 15 mM glucose than that found with 5 mM glucose (amylase, F = 77.2, p < 0.001; lipase, F = 67.8, p < 0.001; chymotrypsinogen, F = 61.6, p < 0.001). During stimulation with 10-l’ M caerulein the rates of juice flow with 5 and 15 mM glucose were 12.7 5 1.6 and 5.9 + 1.1 ~115 min, respectively; p < 0.02.
1308
BRUZZONE
GASTROENTEROLOGY
ET AL.
(ii) The dose-response curve for caerulein with respect to pancreatic enzyme secretion is bellshaped; increasing concentrations of caerulein are usually described as submaximal, maximal, and supramaximal (12,13). In the presence of 5 mM glucose and in our perfused pancreas preparation, caerulein concentrations of 10pll, 10-l”, and lo-” M were submaximal, (near] maximal and supramaximal, respectively. Thus, with 1O-g M caerulein, enzyme secretion was less than with 10-l’ M caerulein (Figure 2, left-hand panel). As previously noted for 10p’o M caerulein, at lo-l1 M caerulein, enzyme secretion was lower with 15 than with 5 mM glucose (Figure 2: amylase, F = 17.9, p < 0.001; lipase, F = 8.1,p < 0.025;chymotrypsinogen, F = 4.6,p < 0.025). However, at 10 -’ M caerulein, enzyme secretion was similar in the presence of 5 and 15 mM glucose. In contrast to the finding with 5 mM glucose, in the presence of 15 mM glucose lo-’ M caerulein was not supramaximal with respect to stimulated enzyme secretion.
Vol.
87, No.
60
0 16 12 8 L 0
Effects of Exogenous Insulin on Enzyme Secretion, Protein Output, and Juice Flow (Protocols 1 and 2) As it was not clear from the above experiments whether the effect of glucose on enzyme secretion was a direct effect on the acinar cell or mediated, for example, through increased insulin secretion, further experiments were carried out to determine if exogenous insulin could alter enzyme secretion. The amount of added insulin was chosen arbitrarily and was approximately the amount secreted by the pancreas at 15 mM glucose. Insulin was infused at 25 ngiml during the equilibration and basal periods and at 50 ngiml during the stimulatory periods in all of the following experiments. Basal secretion. Insulin inhibited basal secretion of amylase, lipase, and chymotrypsinogen when the glucose concentration in the perfusate was 5 mM (Figure 3, extreme left-hand columns]. The addition of insulin to the perfusate did not have an additive effect on the inhibition caused by 15 mM glucose. Total protein output at 5 mM glucose was 10.5 f. 1.7 and 4.6 2 1.2 pg/min in the absence and presence of insulin, respectively, p < 0.025.With 15 mM glucose, the basal protein output was 1.3 + 0.7 and 0.33 k 0.29 pglmin in the presence and absence of insulin, respectively [not significant). With 5 mM glucose, juice flow was not inhibited by insulin (1.52 + 0.3VS. 1.00 ? 0.20 /d/5min, respectively). With 15 mM glucose, juice flow was 0.50 t 0.10 and 0.48 +0.16 &5 min, in the presence and absence of insulin, respectively.
-15 0 20 LO 60minuti;50 20 LO 60
CRL (Ml Figure
0
10-J'lo-'010-s
0
10-J'lo-'0 10-9
2. Effect of increasing concentrations of caerulein (CHL) on amylase, lipase, and chymotrypsinogen secretion in the presence of either 5 mM (hatched bars. n = 6) or 15 mM (open bars, n = 5) glucose. Three concentrations of CRL were infused successively during the stimulatory period, each for 20 min. Results are presented as mean 2 SEM.
Caerulein-Stimulated
Secretion
(i) With 5 mM glucose, insulin had an inhibitory effect on amylase and lipase secretion stimulated by lo- ” M caerulein (F = 77.2, p < 0.001 and F = 54.1, p < 0.001, respectively). In these experiments, insulin did not have a significant effect on chymotrypsinogen secretion (Figure 3). In the presence of 15 mM glucose, insulin had no effect on stimulated enzyme secretion. Insulin inhibited protein output with 5 mM glucose (120 -+ 7.1 vs. 79.0 2 15.6 pg/min, F = 61.1, p < O.OOl), whereas it had no effect with 15 mM glucose (47.6 k 9.2 and 39.6 +- 4.0 pgimin, without and with added insulin, respectively). Furthermore, insulin did not inhibit caeruleinstimulated (lo-” M) juice flow with either 5 mM glucose (12.7 k 1.6 vs. 8.5 t 1.4 pl/5 min, in the absence and presence of exogenous insulin, respectively] or with 15 mM glucose (5.9 t 1.1
6
December
1984
vs 6.1 -C0.5 &5 min, without and with added insulin, respectively). (ii) The effect of insulin at different caerulein concentrations was investigated only in the presence of 5 mM glucose. Enzyme secretion stimulat.ed by lOPro M caerulein was significantly inhibited by insulin (amylase, F = 35.8, p < 0.001; lipase, F = 6.9, p < 0.025; chymotrypsinogen, F = 15.0, p < 0.001; Figure 4). As in the case of glucose, insulin had no inhibitory effect on enzyme output at a caerulein concentration of lo-” M.
GLUCOSE INHIBITIONOF ENZYME SECRETION 1309
GLUCOSE 5 mM
GLUCOSE 15 mM
90
P E
2 Z 4 f
60
3o
Time Dependence of the Effects of Glucose and Insulin on Enzyme Secretion (Protocol 31 Further experiments were undertaken to assess whether the prolonged exposure time to high concentrations of glucose and to exogenous insulin during the equilibration period could be of importance in determining the degree to which caeruleinstimulated enzyme output was inhibited by these agents in the above experiments. Glucose (15 mM) or insulin (50 ngiml, in the continued presence of 5 mM glucose) was introduced 20 min after the addition of caerulein (lo-” M), and their effects were followed for 40 min. The results are shown in Table 1. In control experiments, amylase secretion was constant during the three successive 20-min periods of caerulein stimulation. When 15 mM glucose was added after the first 20 min (O-20 min) of caerulein stimulation, a significant decrease of amylase output was noted during both the second (20-40 min) and third (40-60 min) periods. The addition of insulin also reduced amylase release but, in contrast to the effect of 15 mM glucose, inhibition of amylase secretion was significant only during the third period (40-60 min). However, for both glucose and insulin the inhibitory effect on exocrine secretion after 40 min was considerably less pronounced when compared with experiments where they had been present 45 min before the introduction of caerulein. i.e., during the equilibration and basal periods (compare with Figures 1 and 3). Effect of Insulin on Enzyme Secretion in the Absence of Glucose (Protocols 4 and 1) These experiments were carried out to test whether the inhibitory effect of insulin on enzyme output could be due indirectly to an effect of insulin on glucose metabolism in pancreatic tissue. (i) In the first series of experiments, 5 mM glucose was present in the equilibration period and was
INSULIN CRL 04 Figure
- +
- +
0
10-10
-+
-+ 0
10-10
3. Effect of exogenous insulin on basal and caerulein (CHL)-stimulated enzyme secretion in the presence of either 5 mM (hatched bars] or 15 mM [open hors] glucose. Results are expressed as mean -C SEM. The number of experiments for each protocol was as follows: 5 mM glucose, n = 6: 5 mM glucose + insulin. II = 4; 15 mM glucose, n = 4: 15 mM glucose + insulin. n = 3. Insulin concentration was 25 @ml during the basal and 50 ngiml during the stimulatory period, as indicated in Materials and Methods.
withdrawn 15 min before the introduction of lOPro M caerulein at 0 min. In one group of experiments (control experiments), no further changes were made during the 60-min stimulatory period. In the second group of experiments, 50 ng/ml insulin was added after 20 min of caerulein stimulation. Insulin did not have an inhibitory effect on amylase secretion in these experiments [Table 2). (ii) A second series of experiments was carried out where 5 mM glucose was replaced by 10 mM pyruvate. In this case, pyruvate was present throughout the experiment, including the equilibration period. Although a stable secretory rate was not achieved in these experiments, amylase
GASTROENTEROLOGY Vol. 87, No. 6
BRUZZONE ET AL.
Table 1. Glucose and Insulin Modulation of CaeruleinStimulated Amylase Secretion From the Isolated Perfused Rat Pancreas: Effect of Time
60 -.
im
Controls (n = 6) Glucose (n = 6) Insulin (n = 4)
O-20 min
20-40 min
40-40 min
1ooa 100 100
99.9 * 9.6 77.1 + 5.6+ 87.9 2 7.6
100.4 2 9.0
76.2 2 7.2” 72.2 2 3.3”
All experiments had the usual equilibration and basal periods in the presence of 5 mM glucose. In all three experimental groups the perfusate contained 5 mM glucose and lo-” M caerulein during the period O-20 min. In protocol 1, this was kept constant without change until 60 min. In protocols 2 and 3, there was one alteration in the perfusate from 20-60 min; in protocol 2, the glucose concentration was increased to 15 mM; and in protocol 3, 50 ngi ml insulin was added. o Only the stimulatory period is shown here. The data were normalized, with the first 20 min being expressed as 100%. For each protocol, the output of amylase in the periods 20-40 and 40-60 min is expressed as a percentage of the output during o-20 min for the same experiment (mean 2 SEM). The number of experiments is indicated in parentheses. b Glucose inhibited amylase secretion during 20-40 min (F = 14.7; p < 0.001) and during 40-60 min (F = 10.3; p < 0.005). Insulin inhibited amylase secretion only during 40-60 min (F = 10.0; p < 0.005).
Discussion ”
-15
0
20 LO 60
-15
minutes
CRL (M)
0
lo-" lo-'0w
0
0
20 LO 60
lo-" 10-M 1w
Figure 4. Basal and caerulein (CAL)-stimulated enzyme output in the absence (hatched bars, n = 6) and in the presence (open bars, n = 7) of exogenously added insulin (details in Materials and Methods]. Glucose concentration was 5 mM in both series of experiments. Caerulein was added as described in Figure 2. Results are presented as mean ? SEM.
output was totally unaffected by the presence of insulin introduced at -45 min (data not shown).
Thus, insulin had no inhibitory effect on amylase secretion in the absence of glucose or when 5 mM glucose was replaced by 10 mM pyruvate.
These results show that, at the level of the pancreas, the ambient glucose concentration has a profound effect on pancreatic enzyme secretion. Glucose inhibited basal and caerulein-stimulated enzyme secretion except for high concentrations of caerulein where it had no effect (i.e., lo-’ M caerulein, which is a supramaximal concentration with 5 mM glucose). The inhibitory action of high glucose concentrations found in these experiments would confirm the results of Danielsson (9) who also found inhibition of enzyme release when pancreatic pieces from fasted animals were incubated with 16.7 mM glucose, the effect of glucose being grossly attenuated when fed animals were used. With the same preparation, Robberecht and Christophe (20) found that 10 mM glucose had a delayed inhibitory action Table 2. Insulin ModuJation of Caerulein-Stimulated Amylase Secretion: Effect of the Absence of Glucose
Insulin Secretion Basal insulin secretion was --loo-fold higher in the presence of 15 mM glucose than with 5 mM glucose. Addition of caerulein stimulated insulin output but this effect was more pronounced at high concentrations of glucose. Insulin levels were also measured both in the absence of glucose and when 5 mM glucose was replaced by 10 mM pyruvate. Under these conditions caerulein did not modify the insulin secretory rate (Table 3).
Controls (n = 4) Insulin (n = 6)
O-20 min
20-40 min
40-60 min
100
105.4 2 9.7 94.5 + 10.5
118.5 2 19.5 87.5 t 8.8
100
In both protocols, during the equilibration period, the perfusate contained 5 mM glucose; it was then removed for the rest of the experiment. Caerulein (lo- lo M) was added from 0 to 60 min in both experimental groups. In addition, in the insulin experiments, 50 ngiml pork insulin was added from 20 to 60 min. Basal amylase secretion was 4.9 + 2.3 and 3.4 ‘-’ 0.3 Uimin for the first and second group of experiments, respectively. Results are expressed as mean 5 SEM; the number of experiments is indicated in parentheses.
December
Table
3.
1984
Basal
GLUCOSE
and
Caerulein-Stimulated
Insulin
Output From the Isolated
INHIBITION
Perfused
OF ENZYME
SECRETION
1311
Rat Pancreas
Substrate Glucose (mM1 5 15 0 0
Pyruvate (mM1 0 0 0 10
Insulin n 6 4 4 5
Basal 0.37 44.0 1.14 0.15
2 k k k
10 min 0.16 6.0 0.19 0.05
1.79 ?z 0.95 150 t 21.7 1.08 I 0.17 0.21 + 0.09
secretion
20 min
(ngimin)
30 min
1.45 2 0.50 141 + 40.6 1.25 i- 0.25 0.19 2 0.05
3.67 208 1.15 0.15
+ + k f
2.43 23.2 0.28 0.03
40 min 1.68 2 202 t 1.11 + 0.23 t
1.07 48.6 0.28 0.04
50 min 1.25 223 1.32 0.17
+ 0.88 t 54.2 +- 0.33 + 0.06
60 min 1.90 199 1.17 0.24
+ t k ?
1.38 65.3 0.35 0.10
Mean f SEM. The substrate concentration was kept constant throughout the entire experiment in protocols 1,2, and 4. In protocol 3, 5 mM glucose was present in the equilibration period and, thereafter, removed. For all experiments. basal output represents a mean of three samples collected during the basal period at 5-min intervals. Caerulein was then infused at the constant rate of lo-” M from 0 to 60 min.
on enzyme secretion. By contrast, Saito et al. (10) using the isolated perfused rat pancreas, although finding no consistent effect of glucose on basal enzyme secretion, described a potentiating effect of glucose on CCK-induced secretion. The reasons for these discrepancies are not obvious; they may be due to differences in concentration and purity of the secretagogue used in their experiments. An interesting point in the present results is the fact that the effect of high glucose concentrations on caeruleinstimulated enzyme secretion is restricted to those concentrations of caerulein that give submaximal or (near) maximal stimulation with 5 mM glucose. Thus, the alteration in caerulein-stimulated release is possibly the result of interference between caerulein and its high affinity receptors (12,13) or of metabolic events activated by this receptor, or both. The question remained as to whether the effect of glucose was a direct one on the acinar cell or whether it was mediated by another factor, e.g., glucose-induced insulin release. We, therefore, tested the effect of exogenous insulin and found that it also had an inhibitory action on enzyme secretion. However, this was not additive with the effect of glucose when tested with lo-” M caerulein. In the presence of 5 mM glucose, the effect of the single insulin concentration tested was less pronounced than that of 15 mM glucose. Basal enzyme secretion and both amylase and lipase secretion stimulated by lo-lo M caerulein were inhibited. However, the effects of insulin on caerulein-stimulated chymotrypsinogen secretion were inconstant (compare Figures 3 and 4). In addition, in another series of experiments, we showed that the time of onset of inhibition by insulin was delayed with respect to that of glucose. Despite these differences, it is clear that the inhibitory action of insulin demonstrated in these experiments is dependent on the presence of glucose, as insulin had no effect on secretion in the absence of glucose or when 5 mM glucose was replaced by 10 mM pyruvate. As was the case with glucose, insulin had no effect on enzyme secretion in the presence of lo-" M caerulein. This, and the
glucose dependence of the insulin inhibitory effect, would be consistent with the inhibitory actions of glucose and insulin acting through a common mechanism. Danielsson (9), using much higher concentrations of insulin, found it had an inhibitory effect on basal and caerulein-stimulated amylase secretion from pancreatic fragments. By contrast, both Kanno and Saito (21) and Saito et al. (10) found that insulin had a stimulatory effect on amylase secretion stimulated by half-maximal concentrations of CCK-pancreozymin. However, in another paper where isolated acini from diabetic rats were studied at a high concentration of glucose (11.1mM), Otsuki and Williams (22) found that 100 nM insulin had a potentiating effect on CCK-induced amylase release only after at least 1 h of preincubation with insulin and only at supramaximal CCK concentrations. It is clear, therefore, that the action of insulin on enzyme secretion is more complex than hitherto imagined. In the present experiments, although insulin had an inhibitory action at intermediate glucose concentrations (5 mM), it had no effect in the absence of glucose or at high glucose concentrations (15 mM). It is, therefore, evident that insulin dose-responses at each of many glucose concentrations will be needed to achieve a fuller understanding of interactions between glucose and insulin at the level of the acinar cell. However, in the experiments presented here, high concentrations of glucose were shown to inhibit basal and caerulein-stimulated enzyme secretion if the caerulein concentration was not supramaximal. Furthermore, it has been demonstrated that insulin may also decrease enzyme secretion by a glucose-dependent mechanism. Although the amounts of exogenous insulin used in this study may appear rather high, it has been proposed that the actual insulin concentrations reaching the exocrine tissue via the so-called insuloacinar portal vessels could be much higher than those measured in the peripheral circulation (23). Thus, both glucose and insulin creatic
play important roles enzyme secretion.
in modulation
of pan-
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