Influence of Repeated Administration of Cholecystokinin and Secretin on the Pancreas of the Rat u. R. F ~ L S C HK. , WINCKLER
& K. G. WORMSLEY Division of Gastroenterology and Metabolism, Dept. of Medicine, University of Gottingen, Gottingen, West Germany, and Ninewells Hospital, Dept. of Therapeutics, University of Dundee, Dundee, Scotland
Folsch, U. R., Winckler, K. & Wormsley, K. G. Influence of repeated administration of cholecystokinin and secretin on the pancreas of the rat. Scund. J. Gastroent. 1978, 13,
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Otago on 01/04/15 For personal use only.
663-67 I.
Repeated injections of cholecystokinin (CCK) during a period of up to 3 weeks significantly increased the weight of the pancreas in rats. This was associated with an increase in the amount of protein per unit weight of DNA, suggesting hypertrophy of the acinar cells. and with increase in the total amount of pancreatic DNA, indicating additional hyperplasia of the gland. CCK administration also increased the pancreatic content of amylase and trypsin, but the content of lipase remained unchanged. The rate of secretion of the two enzymes increased in the CCK-treated rats, although it appeared that the functional capacity of the individual pancreatic acinar cells was not increased. CCK injections had no effect on the insulin content of the pancreas or on the composition of the parotid glands. Repeated injections of secretin in the doses used in this study had no effect on the pancreas. Key-words: Amylase; cholecystokinin-pancreozymin;DNA; insulin; lipase; pancreas;
parotid gland; protein; secretin; secretion; trypsin
U.R. Folsch, M.D., Dept. of Medicine, University of Gottingen, Robert-Koch-Str.40, 3400 Gottingen, West Germany
In previous studies, we have shown that administration of a soybean diet containing active trypsin inhibitors resulted in enlargement of the rat pancreas (4) and an increased output of enzymes after stimulation with cholecystokinin (CCK) (5). It has been suggested that the chronic release of excessive amounts of CCK is the mediator of the pancreatic effect produced by the soybean diet ( l o ) , since CCK is known t o exert trophic effects on the exocrine pancreas (1, 14, 18, 2 1). This hypothesis has been questioned (1 1). The pancreas and parotid gland of rats have therefore been investigated morphologically and biochemically after repeated injections of CCK for 10 or 20 days. For comparison with CCK, the effect o f a similar course of injections of secretin was also studied. A preliminary report of this study has been published (6).
METHODS
Animals and diet For the duration of the study male Wistar rats (230-260 g ) were given a diet of heat-inactivated soybean flour, in which the trypsin inhibitors are destroyed (Soya Foods Ltd., London). The flour was supplemented with vitamins, minerals and trace elements to provide a diet which is both completely adequate for the rats and yet provides a satisfactory control for comparison with the effects of the diet of raw soybean flour. The detailed composition of the diet has been described ( 5 ) . A . Studies of structural and enzymic changes in the rat pancreas Nine rats were injected subcutaneously every 12 h for 10 days with 30 Ivy dog units (IU) CCK
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Otago on 01/04/15 For personal use only.
664
U.R . Folsch, K . Winckler & K . G. Worrnsley
per kg body weight. The control group of seven rats received 150 mmol/l sodium chloride. In these and following experiments all injections were made up in a volume of 0.5 ml. All animals were given their last injection 24 h before study. Following a 24-h fast, the animals were killed by a blow to the head and exsanguination. The pancreas was rapidly removed from the rat, freed from fat and lymph nodes, and weighed. Most of the pancreas was homogenised in 150 mmol/l sodium chloride with an Ultra-Turrax homogeniser (Jahnke & Kunkel, Stauffen, FRG) at 20,000 rpm for 4 x 15 sec at 0°C. The homogenate was sonicated with a Branson Sonifier at setting 5 for 4 x 15 sec at O°C and, after an interruption of about 10 min, for another 45 sec (3 x 15 sec). The homogenate was further treated, as described below, for the determination of enzyme activities, protein and DNA. Small pieces of the intact pancreas were fixed for light microscopy in Bouin’s solution and for electron microscopy in ice-cold Karnovsk y’s solution, postfixed in 1% osmium tetroxide, buffered with sodium cacodylate and embedded in Epon. Thick sections were stained with toluidine blue for light microscopy; thin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss 9s electron microscope. B. Secretory studies The rats were injected twice daily for 20 days either with 150 mmol/l sodium chloride (n= 13); with 20 clinical units (CU) secretin per kg (n = 6); with 7.5 IU C C K per kg (n = 6) or with 30 IU C C K per kg (n = 6, both for the experiments with submaximal and those with maximal stimulation with CCK). Secretin and CCK (20% pure) were purchased from the GIH Laboratory, Karolinska Institutet, Stockholm, Sweden. The body weight of each animal was checked daily and expressed as percent increase compared with the starting weight. The rats were studied following a 24-h fast. As mentioned in section A, all animals received their last subcutaneous injection 24 h before the secretory studies were started. The animals were anaesthetised at 0 7 15 with urethane (total of 0.15 g/100 g body weight) given by two intraperitoneal injections, with an interval of 2 0 min. Surgery was started 3 h later. A glass cannula was placed in the trachea. Pancreatic enzymes were
collected by perfusing the duodenum with 0.15 mmol/l sodium chloride at a rate of 7.5 m1/15 min, as described previously (3,7). The perfusates which contained the pancreatic secretion were collected on ice in 15-min batches. Body temperature of the animals was kept constant at 37OC by a heated pad and infrared light. Fifteen or 6 0 IU C C K per kg-h were infused intravenously in 0.15 mmol/l sodium chloride for 3 h after basal secretion had been collected for four successive 15-min periods. Intravenous administration was effected through a nylon catheter (0.63 mm external diameter) (Portex) into a jugular vein at a rate of 1.65 mlh. Immediately at the end of the test rats were killed and the pancreas removed as described in section A for estimation of pancreatic weight and DNA content.
C. Parotid gland For the purpose of comparison with the effect of C C K on the pancreas, and because it has been claimed that C C K has stimulatory effects on the parotid glands ( 1 6), these glands were removed from the rats of group A at the time of sacrifice. Each gland was freed from fat and lymph nodes and weighed. The gland was homogenised as mentioned in section A, in order to estimate protein, amylase and DNA. D. Analytical methods 1. Trypsin. The pancreatic homogenates or the duodenal perfusates were activated with enterokinase (Miles Laboratories, Inc., Kankakee, USA) and then mixed with an equal amount of glycerol, as described previously (3). Tryptic activity was measured photometrically (1 7), using benzoyl-argininenitroanilide as substrate. 2. The activity ofamylase was measured with 3 3 dinitro-salicylic acid, using Zulkovsky starch as substrate (20). 3. The activity of lipase was measured titrimetrically, using an emusion of olive oil as substrate ( 1 9). 4. Protein concentrations were determined according to the method of Lowry et al. ( 13), with the Folin-phenol reagent. Bovine albumin (Behringwerke, Marburg, FRG) was used as standard. The quantities of protein were expressed in terms of mg per kg DNA.
i0.04
2 13
k0.23 1.25'
8
1.07
402'
2
298
67.1
p p p p
k1.5
16.9'
k 1.8
10.9
+_
139
1387d
2121
738
mg DNA g
k 415
4308'
k 447
2703
pancreas
Amylase (U) per mg protein
< 0.025. < 0.01. < 0.005. < 0.001.
k2.7
81.6d
? 1.9
Values are means ? S.E.M. Significant differences versus controls: Significant differences versus controls: Significant differences versus controls: Significant differences versus controls:
60 IU CCK-PZ (n=9)
0.15M NaC I (n=7)
Injection Wet weight DNA, Protein per pancreas, mg/100g ___ kg x day mg/100g BW DNA S.C. BW
Table I. Pancreatic weight and composition after injection of 6 0 IU CCK-PZ/kg X
4
5 4.7
101.2d
?
59
mg protein g
25493' k 1103
? 335
& 1006
14507
pancreas
8177'
& 349
3993
mg DNA
Trypsin (mu)per
Pancreatic content of enzymes
day for 10 days
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Otago on 01/04/15 For personal use only.
2 5.3
52.2
f 4.8
55.3
mg protein
4265
268
k 462
+_
3637
mg DNA
Lipase (U) per g
? 1418
13223
k 1130
13548
pancreas
a
Q
3
3
a
5
R
a
z %
302
303 6
1915c k353 624 k171
1.. 12 k0.09
k 205
llMb
606 k122
1.75' -1.0.17
1.93e -1.0.19
1.12 k0.06
Significant differences versus controls: p
& K. G. WORMSLEY Division of Gastroenterology and Metabolism, Dept. of Medicine, University of Gottingen, Gottingen, West Germany, and Ninewells Hospital, Dept. of Therapeutics, University of Dundee, Dundee, Scotland
Folsch, U. R., Winckler, K. & Wormsley, K. G. Influence of repeated administration of cholecystokinin and secretin on the pancreas of the rat. Scund. J. Gastroent. 1978, 13,
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Otago on 01/04/15 For personal use only.
663-67 I.
Repeated injections of cholecystokinin (CCK) during a period of up to 3 weeks significantly increased the weight of the pancreas in rats. This was associated with an increase in the amount of protein per unit weight of DNA, suggesting hypertrophy of the acinar cells. and with increase in the total amount of pancreatic DNA, indicating additional hyperplasia of the gland. CCK administration also increased the pancreatic content of amylase and trypsin, but the content of lipase remained unchanged. The rate of secretion of the two enzymes increased in the CCK-treated rats, although it appeared that the functional capacity of the individual pancreatic acinar cells was not increased. CCK injections had no effect on the insulin content of the pancreas or on the composition of the parotid glands. Repeated injections of secretin in the doses used in this study had no effect on the pancreas. Key-words: Amylase; cholecystokinin-pancreozymin;DNA; insulin; lipase; pancreas;
parotid gland; protein; secretin; secretion; trypsin
U.R. Folsch, M.D., Dept. of Medicine, University of Gottingen, Robert-Koch-Str.40, 3400 Gottingen, West Germany
In previous studies, we have shown that administration of a soybean diet containing active trypsin inhibitors resulted in enlargement of the rat pancreas (4) and an increased output of enzymes after stimulation with cholecystokinin (CCK) (5). It has been suggested that the chronic release of excessive amounts of CCK is the mediator of the pancreatic effect produced by the soybean diet ( l o ) , since CCK is known t o exert trophic effects on the exocrine pancreas (1, 14, 18, 2 1). This hypothesis has been questioned (1 1). The pancreas and parotid gland of rats have therefore been investigated morphologically and biochemically after repeated injections of CCK for 10 or 20 days. For comparison with CCK, the effect o f a similar course of injections of secretin was also studied. A preliminary report of this study has been published (6).
METHODS
Animals and diet For the duration of the study male Wistar rats (230-260 g ) were given a diet of heat-inactivated soybean flour, in which the trypsin inhibitors are destroyed (Soya Foods Ltd., London). The flour was supplemented with vitamins, minerals and trace elements to provide a diet which is both completely adequate for the rats and yet provides a satisfactory control for comparison with the effects of the diet of raw soybean flour. The detailed composition of the diet has been described ( 5 ) . A . Studies of structural and enzymic changes in the rat pancreas Nine rats were injected subcutaneously every 12 h for 10 days with 30 Ivy dog units (IU) CCK
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Otago on 01/04/15 For personal use only.
664
U.R . Folsch, K . Winckler & K . G. Worrnsley
per kg body weight. The control group of seven rats received 150 mmol/l sodium chloride. In these and following experiments all injections were made up in a volume of 0.5 ml. All animals were given their last injection 24 h before study. Following a 24-h fast, the animals were killed by a blow to the head and exsanguination. The pancreas was rapidly removed from the rat, freed from fat and lymph nodes, and weighed. Most of the pancreas was homogenised in 150 mmol/l sodium chloride with an Ultra-Turrax homogeniser (Jahnke & Kunkel, Stauffen, FRG) at 20,000 rpm for 4 x 15 sec at 0°C. The homogenate was sonicated with a Branson Sonifier at setting 5 for 4 x 15 sec at O°C and, after an interruption of about 10 min, for another 45 sec (3 x 15 sec). The homogenate was further treated, as described below, for the determination of enzyme activities, protein and DNA. Small pieces of the intact pancreas were fixed for light microscopy in Bouin’s solution and for electron microscopy in ice-cold Karnovsk y’s solution, postfixed in 1% osmium tetroxide, buffered with sodium cacodylate and embedded in Epon. Thick sections were stained with toluidine blue for light microscopy; thin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss 9s electron microscope. B. Secretory studies The rats were injected twice daily for 20 days either with 150 mmol/l sodium chloride (n= 13); with 20 clinical units (CU) secretin per kg (n = 6); with 7.5 IU C C K per kg (n = 6) or with 30 IU C C K per kg (n = 6, both for the experiments with submaximal and those with maximal stimulation with CCK). Secretin and CCK (20% pure) were purchased from the GIH Laboratory, Karolinska Institutet, Stockholm, Sweden. The body weight of each animal was checked daily and expressed as percent increase compared with the starting weight. The rats were studied following a 24-h fast. As mentioned in section A, all animals received their last subcutaneous injection 24 h before the secretory studies were started. The animals were anaesthetised at 0 7 15 with urethane (total of 0.15 g/100 g body weight) given by two intraperitoneal injections, with an interval of 2 0 min. Surgery was started 3 h later. A glass cannula was placed in the trachea. Pancreatic enzymes were
collected by perfusing the duodenum with 0.15 mmol/l sodium chloride at a rate of 7.5 m1/15 min, as described previously (3,7). The perfusates which contained the pancreatic secretion were collected on ice in 15-min batches. Body temperature of the animals was kept constant at 37OC by a heated pad and infrared light. Fifteen or 6 0 IU C C K per kg-h were infused intravenously in 0.15 mmol/l sodium chloride for 3 h after basal secretion had been collected for four successive 15-min periods. Intravenous administration was effected through a nylon catheter (0.63 mm external diameter) (Portex) into a jugular vein at a rate of 1.65 mlh. Immediately at the end of the test rats were killed and the pancreas removed as described in section A for estimation of pancreatic weight and DNA content.
C. Parotid gland For the purpose of comparison with the effect of C C K on the pancreas, and because it has been claimed that C C K has stimulatory effects on the parotid glands ( 1 6), these glands were removed from the rats of group A at the time of sacrifice. Each gland was freed from fat and lymph nodes and weighed. The gland was homogenised as mentioned in section A, in order to estimate protein, amylase and DNA. D. Analytical methods 1. Trypsin. The pancreatic homogenates or the duodenal perfusates were activated with enterokinase (Miles Laboratories, Inc., Kankakee, USA) and then mixed with an equal amount of glycerol, as described previously (3). Tryptic activity was measured photometrically (1 7), using benzoyl-argininenitroanilide as substrate. 2. The activity ofamylase was measured with 3 3 dinitro-salicylic acid, using Zulkovsky starch as substrate (20). 3. The activity of lipase was measured titrimetrically, using an emusion of olive oil as substrate ( 1 9). 4. Protein concentrations were determined according to the method of Lowry et al. ( 13), with the Folin-phenol reagent. Bovine albumin (Behringwerke, Marburg, FRG) was used as standard. The quantities of protein were expressed in terms of mg per kg DNA.
i0.04
2 13
k0.23 1.25'
8
1.07
402'
2
298
67.1
p p p p
k1.5
16.9'
k 1.8
10.9
+_
139
1387d
2121
738
mg DNA g
k 415
4308'
k 447
2703
pancreas
Amylase (U) per mg protein
< 0.025. < 0.01. < 0.005. < 0.001.
k2.7
81.6d
? 1.9
Values are means ? S.E.M. Significant differences versus controls: Significant differences versus controls: Significant differences versus controls: Significant differences versus controls:
60 IU CCK-PZ (n=9)
0.15M NaC I (n=7)
Injection Wet weight DNA, Protein per pancreas, mg/100g ___ kg x day mg/100g BW DNA S.C. BW
Table I. Pancreatic weight and composition after injection of 6 0 IU CCK-PZ/kg X
4
5 4.7
101.2d
?
59
mg protein g
25493' k 1103
? 335
& 1006
14507
pancreas
8177'
& 349
3993
mg DNA
Trypsin (mu)per
Pancreatic content of enzymes
day for 10 days
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Otago on 01/04/15 For personal use only.
2 5.3
52.2
f 4.8
55.3
mg protein
4265
268
k 462
+_
3637
mg DNA
Lipase (U) per g
? 1418
13223
k 1130
13548
pancreas
a
Q
3
3
a
5
R
a
z %
302
303 6
1915c k353 624 k171
1.. 12 k0.09
k 205
llMb
606 k122
1.75' -1.0.17
1.93e -1.0.19
1.12 k0.06
Significant differences versus controls: p
