Dual effects of hydrocortisone on exocrine rat pancreas

GASTROENTEROLOGY

Dual Effects of Hydrocortisone Exocrine Rat Pancreas

1987;93:1398-1403

on

MAKOTO OTSUKI, YOSHINORI OKABAYASHI, ATSUSHI OHKI, ITSUO SUEHIRO, and SHIGEAKI BABA Second Department of Internal Medicine, Kobe University Chuo-ku, Kobe, Japan

The acute and chronic effects of hydrocortisone.

on _ exocrine pancreatic function were examined in the isolated perfused rat pancreas. In the first part of this study, rats were given subcutaneous injections ofhydrocortisone at doses of 1.25,2.5,5, and 10 mgfkg body wt once daily for 7 days. Trypsin and lipase secretion in response to 100 pM cholecystokininoctapeptide was significantly increased in rats with the two highest doses of hydrocortisone compared with controls, irrespective of whether calculated as the total amount of stimulated output of enzymes or related to the secretion of enzyme to the pancreas content. On the other hand, the secretory responsiveness of amylase to 100 pM choiecystokinin-octapeptide was maxima1 at the 5-mg dose, and decreased with higher doses. In the second part, 100 PM hydrocortisone was superimposed for 20 min on 100 pM cholecystokinin-octapeptide stimulation to examine the acute effects of hydrocortisone on exocrine pancreatic function in the isolated perfused rat pancreas. Addition of hydrocortisone caused a significant inhibition of the secretion of pancreatic juice and amylase. The present study has clearly demonstrated the dual effects of glucocorticoids on the pancreas: inhibition and potentiation. There is a possibility that chronic treatment with large doses of glucocorticoid may sensitize the acinar cells and induce hypersecretion of trypsin and lipase, whereas acute treatment inhibits secretory function of exocrine pancreas. Previous in vivo and in vitro studies have suggested that glucocorticoids have a variety of important effects on the exocrine and endocrine pancreas. Received March 26, 1987. Accepted June 28, 1987. Address requests for reprints to: Dr. Makoto Otsuki, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. This work was supported in part by a grant from the Japanese Ministry of Health and Welfare (Intractable Diseases of the Pancreas). 0 1987 by the American Gastroenterological Association 0016-5085/87/$3.50

School

of Medicine,

Kusunoki-cho,

Adrenalectomy has been reported to cause a marked reduction in pancreatic weight and function, which can be prevented by glucocorticoid administration (1,Z). On the other hand, injections of glucocorticoids lead to an increase in amylase activity in young rats (3) and chick embryos (4). Moreover, it has been demonstrated that long-term glucocorticoid administration causes an increase in pancreatic weight and secretory activity (3,5,6) and hyperplasia and hypertrophy of both the duct epithelial (5) and acinar cells (7). For more than 30 yr corticosteroid administration has been suspected of initiating episodes of acute pancreatitis (for a review, see Reference 8). Experimental work in animals (9) and clinical studies in patients (8,lO) have added supportive evidence of varying degrees to this concept. However, in comparison to the tens of thousands of patients receiving steroids, the number of patients with acute pancreatitis is exceedingly small. The lack of convincing clinical and experimental reports casts doubt on the existence of steroid-induced pancreatitis as a clinically significant entity. In contrast, there are many case reports and experimental data suggesting that corticosteroids are effective in both the treatment and prevention of acute pancreatitis (l1,12). The contradictory results found in previous studies seem to be due, at least in part, to the difference in experimental design. Therefore, the study reported here was undertaken to determine a mechanism of action for the acute and chronic effects of glucocorticoids on exocrine pancreatic function in the isolated perfused rat pancreas. Materials

and Methods

Materials Hydrocortisone (Cortef; 1 ml contains 50 mg hydrocortisone, 4.8 mg sodium caboxymethylcellulose Abbreviations used in this paper: CCK, cholecystokinin; cholecystokinin-octapeptide.

CCK-8,

HYDROCORTISONE AND PANCREATIC FUNCTION

December 1987

low viscosity, 8.7 mg NaCl, 3.85 mg polysorbate, 6 mg benzyl alcohol, and water) was a gift from Upjohn Co., Kalamazoo, Mich. Synthetic COOH-terminal octapeptide of cholecystokinin (CCK-8) was a gift from Dr. Miguel Ondetti of Squibb Institute for Medical Research, Princeton, N.J. The following materials were purchased: enterokinase, cu-n-bezoyl-m-arginine-p-nitroanilide from Sigma Chemical Co., St. Louis, MO.; bovine plasma albumin (fraction V) from Armour Pharmaceutical Co., Phoenix, Ariz.; Dextran T-70 from Pharmacia Fine Chemicals, Uppsala, Sweden; Phadebas amylase test from Shionogi Pharmaceutical Co., Osaka, Japan; the glucose kit from Fujisawa Pharmaceutical Co., Osaka, Japan; and the lipase kit from Ono Pharmaceutical Co., Osaka, Japan.

Animals Male Wistar rats weighing 250-280 g were used throughout the experiments. The animals were kept at 23°C on a 12-h light-dark cycle and had free access to water and standard laboratory diet (Oriental Yeast Co., Tokyo, Japan). The hydrocortisone group received the steroid subcutaneously at doses of 1.25, 2.5, 5, and 10 mg/kg body wt, whereas the control group received saline. All injections were given at 9 AM on consecutive days for 7 days before death. Hydrocortisone was used because it was shown by Cohen and Kulka (13) to be equivalent to corticosterone in the induction of pancreatic chymotrypsinogen. It was also available in suspension, which facilitated the injections and ensured reproducibility from injection to injection. All animals were fed ad libitum until they were killed, and the last injection was always given 25 h before the start of experiments.

Isolated

Perfused

Pancreas

The isolated and perfused pancreas was prepared as previously reported (14). The perfusate was KrebsRinger bicarbonate solution containing 4.6% Dextran T-70, 0.25% bovine plasma albumin, and 2.8 mM glucose. The inlets of the vascular perfusion were the superior mesenteric and celiac arteries, and the outlet was the portal vein. The flow rate through the pancreas was kept constant at 2.0 ml/min by a roller pump. Pancreatic juice was collected at IO-min intervals by replacing a capillary tube that was attached to the free end of the pancreatic cannula inserted into the distal end of the common duct at a point shortly before its entrance into the duodenum. The rate of flow of pancreatic juice was measured and the juice was stored at -20°C for subsequent assay of amylase, trypsin, and lipase. After surgery, there was an equilibration period of 40 min before the start of an experiment. After a lo-min basal period in the presence of 2.8 mM glucose, the glucose concentration in the perfusate was changed to 8.3 mM by changing the medium reservoir. Ten minutes later, CCK-8 was added for 20 min at a concentration of 100 pM, which is the maximal effective concentration for stimulating exocrine pancreatic secretion from the isolated perfused rat pancreas (15). In another experiment, 100 PM hydrocortisone was superimposed for 20 min on 100 pM

1399

CCK-8 stimulation, which was started 20 min before hydrocortisone addition and continued for 60 min.

Assay A portion of pancreatic tissue was homogenized in 0.15 M NaCl using a motor-driven, Teflon-coated glass homogenizer. The amount of protein in the pancreatic homogenates was determined by the method of Lowry et al. (16) with bovine plasma albumin as a standard. Amylase activity in the pancreatic homogenates and pancreatic juice was determined by a chromogenic method with the Phadebas amylase test (17) and expressed as Somogyi units. Trypsinogen levels were determined after activation with enterokinase and measured by the method of Erlanger et al. (18) using cu-n-benzoyl-oL-arginine pnitroanilide hydrochloride as the substrate. Lipase activity was determined according to Whitaker (19) using Qnaphtyl palmitate as a substrate and expressed as international units. Serum glucose concentrations were determined by the hexokinase method (20) with the Stazyme glucose kit. The splenic portion of the pancreas was homogenized in acid alcohol in the aforementioned manner. Insulin was then extracted from this homogenate by a modification of the method of Davoren (21). Immunoreactive insulin concentration in the pancreatic extracts and serum from the jugular vein was measured by polyethyleneglycol radioimmunoassay (22) using crystalline rat insulin as a reference standard.

Data Analysis Comparison of the difference between the mean values of the various groups of experiments was made by analysis of variance. For statistical evaluation of the difference between the mean values of the various groups of experiments, the Wilcoxon rank sum test was used. A difference with p < 0.05 was considered statistically significant. Results are expressed as mean 5 SE.

Results Increasing

doses

of hydrocortisone

were asso-

of body weight gain. Rats given the two highest doses of hydrocortisone showed an initial body weight gain of -4-5 g/day and then tended to decline. In spite of the reduced body weight recovery or further loss of weight, the pancreatic wet weights of the rats receiving steroid injections were not significantly different from those of the control. Consequently, the injections of hydrocortisone led to a dose-dependent increase in the pancreatic wet weight per body weight. Hydrocortisone stimulated an increase in total protein content in the pancreas, with maximal effect observed at the 5-mg dose. Significant increase in pancreatic amylase content was seen only at the 5-mg dose of hydrocortisone, whereas trypsin content increased only at the lo-mg dose. Total lipase ciated

with constant

retardation

GASTROENTEROLOGY Vol. 93, No.6

1400 OTSUKI ET AL.

Table 1. Efiects of Hydrocortisone Treatment for 7 Days on Body and Pancreatic Weights, Pancreatic Protein, Enzyme and insulin Contents. and Serum Glucose and Insulin Concentrations Hydrocortisone

Parameters Body weight (g) Gain (g/7 days] Pancreas weight (mg] Pancreas content Protein (mg/pancreas) Amylase (lo3x W/pancreas) Trypsin (mglpancreas) Lipase (IUlpancreas) Insulin (/&pancreas) Serum concentration Glucose (mg/lOO ml) Insulin (@ml)

Control (5)

1.25(5)

308 2 5 29.0 2 1.0 986 t 23

306 t 6 30.3f.3.7 972 L 35

173 + a 128 rtii 11.4k 1.8 489 2 56 135 5 13 164 + 4 4.8-t0.2

2.5(6)

(mglkg body wt) 5 181

10 (ai

295 2 5 19.2IT1.7",b 1008 k 52

282 2 6".b 5.0+ 3.4"" 1081 2 27

271 t 4"< -11 .4 e 4 .la-d 995 C 38

175 2 7 103 t a 10.1I?0.9 437 * 39 126 f 11

187 2 15 106 +-4 10.92 0.9 440 2 51 145 + 11

228 k 171 2 11.92 467" 127 +

167 k 3 5.0f 1.3

155 * 9 7.02 0.4O

179 2 12 8.8-+1.6"

8"15a.b 0.9 36 9

209 k 118 2 13.32 513 k 101 2

6".b 7 0.9b 31 14

la2 2 13 10.9f 2.2a.b

IU, internationalunit; SU, Somogyiunit. Values are mean f SE of the numberof rats indicated in parenthesis.Hydrocortisonewas given once daily at 9 AMfor 7 days before death. Significant difference (p 5 0.05)vs.a control and t+d rats treated with hydrocortisone (b 1.25, ’ 2.5, and d 5 mg].

content in the pancreas was not significantly altered after hydrocortisone treatment for 7 days (Table 1). Subcutaneous injection of four different doses of hydrocortisone for 7 days led to a dose-dependent increase in serum immunoreactive insulin concentration, without altering total pancreatic immunoreactive insulin content and serum glucose concentration (Table 1). Hydrocortisone pretreatment with the two highest doses caused significant increases in the basal rate of flow of pancreatic juice and enzyme secretion (Table 2). However, there were no significant differences among the control and the treated groups when the basal outputs of these enzymes were calculated as concentrations in pancreatic juice. Trypsin and lipase secretion were significantly increased in rats

Table

with the two highest doses of hydrocortisone compared with the control group, irrespective of whether calculated as the total amount of stimulated output of enzymes or related to the secretion of enzyme to the pancreas content (Figure 1, Table 2). There were no significant differences in the total amount of stimulated output of amylase among the three hydrocortisone-pretreated groups and the control group. However, pancreata from the rats given the 5-mg dose of hydrocortisone secreted a significantly greater amount of amylase in response to 100 pM CCK-8 (Table 2). In contrast, CCK-8-stimulated pancreatic juice secretion from the rats pretreated with hydrocortisone was similar to that in the control rats (Figure 2, left; Table 2). Thus, the concentrations of trypsin and lipase in pancreatic juice secreted from

2. Effect of Hydrocortisone Treatment for 7 Days on Exocrine Pancreatic Secretion

Hydrocortisone(mg/kg body wt) Parameters Pancreatic juice (pUZ0 min) Basal CCK-8-stimulatede Amylase (W/Z0 min) Basal CCK-8-stimulatede Trypsin (pg/20min) Basal CCK-8-stimulatede Lipase (W/20 min) Basal CCK-a-stimulated”

Control (6)

1.25(6)

4.4?I0.5 71.4f 4.3

4.5* 0.4 69 2 8.5

321 f 70 8,483 f 973

273 -+72 a,737 f 1,049

2.5(7) 4.3-r0.4 81.6k 5 304 t 43 9,712 k 395

5 (61 5.9-t0.3a-c 71.3If5

10 (a) 5.82 0.5"< 74.5t 4.6

687 k 31"-" 13,411k 1,179a.b

484 k 62hd 9,8172 731

27.5f 4.2 671 -c 25

29.5f 6.1 681 !I 99

24.5* 6.7 844 k 112

55.9* 5.70-c 926 t 72'=

52.4 2 9.1”” 1,068" 42",b

0.45 -+ 0.1 10.4 2 1.7

0.45 If: 0.15 11.3k 3.9

0.44f.0.03 20.4+ 5.1

0.90-+0.25'+ 23.32 0.6",b

1.082 0.3"44.02 2.aaA

CCK-8, cholecystokinin-octapeptide; IU, international unit; SU, Somogyi unit. Values are mean ? SE of the number of experiments indicated in parenthesis. Significant difference (p 5 0.05) vs. ’ control and b--drats treated with hydrocortisone (b 1.25, ’ 2.5, and d 5.0 mg). e Pancreatic exocrine secretion during a 20-min perfusion with 100 pM CCK-8 (from 20 to 40 min) in similar experiments as shown in Figures 1 and 2.

HYDROCORTISONE AND PANCREATIC FUNCTION

December 1987

CCK8 UOOpM)

m

40 Time

Figure

(min)

Time

60

[min)

1. Time-course of trypsin (left) and lipase (right) outputs in response to a 20-min perfusion with 100 pM CCK-8. Pancreata were isolated and perfused from rats injected with 10 mg of hydrocortisone (solid lines; n = 8) or saline (dashed lines; n = 6) once daily for 7 days. Significant differences between hydrocortisonepretreated and control rat pancreata during the corresponding period at p 5 0.05 are indicated by asterisks. Results shown are mean 2 SE.

rats pretreated with the two highest doses of hydrocortisone were significantly greater than those in the control rats. In the second set of experiments, the acute effects of hydrocortisone on CCK-8-stimulated pancreatic exocrine secretion were examined in the isolated perfused rat pancreas (Figure 3). Addition of 100 PM hydrocortisone after a 20-min perfusion with 100 pM CCK-8 caused a significant inhibition of the secretion of pancreatic juice and amylase (Figure 3)+ The recovery of pancreatic exocrine responses after

CCK8(100pM)

I-

Hydrocortisone t

I-

,-

a Time

(mid

20 Time

40 (mid

60

80

Figure 3. Effect of 100 PM hydrocortisone on 100 pM CCK-8stimulated pancreatic juice (top) and amylase (bottom) outputs. Hydrocortisone was superimposed for 20 min on CCK-8 stimulation 20 min after the start of CCK-8 infusion. Significant differences between CCK-8 plus hydrocortisone (solid lines) and CCK-8 alone (dashed lines) during the correspondihg period at p s 0.05 are indicated by asterisks. Results shown are mean 2 SE of four separate experiments.

the removal of 100 PM hydrocortisone was slow. Both pancreatic juice and amylase outputs during the first 10 min after hydrocortisone removal were less than those in the controls during the corresponding period.

’

i_-r-

a

Oo

The present study has demonstrated the dual effects of glucocorticoids on exocrine pancreatic function. Glucocorticoids acutely and strongly inhibited exocrine response to CCK-8 stimulation in the isolated perfused pancreas, whereas isolated pancreata from rats pretreated with glucocorticoids for i’ days secreted greater amounts of digestive enzymes in response to CCK-8 compared with those from control rats. The pancreatic wet weights of the rats receiving steroid injections were not significantly different from those of the control rats, although increasing doses of hydrocortisone were associated with constant retardation of body weight gain or loss. Consequently, hydrocortisone treatment led to an increase in the pancreatic wet weight per body weight. The effect of prolonged administration of hydrocortisone on the pancreatic content of digestive enzymes is

/:L 6 .-Control

X 4 30. 1 3 0% 20. LL 8 T lo-

400

0

Dismission

r@Bz&!

40-

* o-0

1401

. . . .._ __

40

The

60

(mid

Figure 2. Time-course of pancreatic juice flow (left) and amylase output (right) in response to a 20-min perfusion with 100 pM CCK-8. Pancreata were isolated and perfused from rats injected with 10 mg of hydrocortisone (solid lines; n = 8) or saline (dashed iines; n = 6) once daily for 7 days. Results shown are mean 2 SE.

1402 OTSUKI ET AL.

very weak, especially for trypsin and lipase. This could be due to the short duration of administration. As compared with the control rats, basal secretion of the steroid-treated rats is characterized by a significant increase in volume and a greater increase in enzyme output. It is conceivable that entry of increased acid secretion into the duodenum after glucocorticoid treatment (23) might have, in part, contributed to the increased secretion of the pancreas (24). Pretreatment of rats with hydrocortisone dosedependently increased the secretory ability of digestive enzymes with the exception of amylase, which was maximal at the 5-mg dose of hydrocortisone. In contrast to the increased enzyme output, pancreatic juice secretion was not significantly altered by glucocorticoid pretreatment. Thus the functional modification characteristic in the steroid-treated rats is the increased concentration of digestive enzymes in stimulated pancreatic secretion. Recent in vitro study with the cultured AR42J cells has demonstrated that inclusion of dexamethasone in the culture medium leads to a significant increase in the number of CCK receptors on these cells and increases the sensitivity to CCK-8 (25,26). In isolated pancreatic acini prepared from hydrocortisonepretreated rats, we have observed a significant increase in the responsiveness and the sensitivity of amylase secretion in response to CCK-8 (Otsuki M, Okabayashi Y, Baba S, unpublished observation). Moreover, it has been shown that the addition of insulin or epidermal growth factor to the culture medium leads to a significant increase in basal as (27). well as CCK-g-stimulated amylase release Taken together, it is likely that hydrocortisone increases the responsiveness of amylase, trypsin, and lipase to CCK by modulating the affinity or receptor concentrations, or both, of other hormone receptors such as CCK (261,insulin (28), and epidermal growth factor (27,29) on acinar cells. One other possible mechanism is that in vivo administration of glucocorticoids increases the responsiveness of acinar cells by potentiating the insulin secretory function of B cells (l,30). In support of this hypothesis, addition of exogenous insulin or increase of endogenous insulin release by raising the glucose concentration is shown to potentiate the action of CCK in the isolated perfused rat pancreas (31). The secretory responsiveness of amylase is slightly different from that of trypsin and lipase. Hydrocortisone-pretreatment had a biphasic effect on the secretory ability of amylase in response to CCK-8. The secretory responsiveness of amylase to CCK correlates well with the changes of amylase content in the pancreas, being maximal at the 5-mg dose. It is likely that the responsiveness is deter-

GASTROENTEROLOGY

Vol. 93, No.6

mined not only by hydrocortisone itself but also by other factors modulated by hydrocortisone administration, most likely insulin. Of all the digestive enzymes in acinar cells, amylase is the most dependent on insulin levels (32,33). Glucocorticoid treatment dose-dependently increased plasma insulin concentration without altering the pancreatic insulin content. Insulin receptors are known to be upregulated by glucocorticoid in cultured AR42J cells (Logsdon CD, personal communication) and downregulated by insulin in rat pancreatic acinar cells (34). When up-regulation by glucocorticoids exceeds the down-regulation by elevated endogenous insulin levels, pancreatic amylase content and secretory function increase, whereas pancreatic amylase content and secretory function decrease when downregulation exceeds up-regulation. The direct inhibitory effects of glucocorticoids on pancreatic exocrine secretion are in seeming contradiction to evidence of acinar cell hyperactivity resulting from the chronic in vivo actions of glucocorticoid administration. In our preliminary study acute infusion of other steroid compounds, such as progesterone and androgen, had no significant influence on exocrine pancreas, which could exclude the possibility that the results obtained with hydrocortisone were nonspecific. The rapid inhibition of exocrine secretion suggests that the action of hydrocortisone is likely to involve regions of the acinar cells at or near the cell surface membrane. It is likely that glucocorticoid affects calcium fluxes, which could account for the inhibitory action of hydrocortisone on CCK-g-stimulated pancreatic enzyme secretion, as has been proposed in the case of corticosteroid and insulin secretion (35). The results of the present study have clearly demonstrated the dual effect of glucocorticoids on the pancreas: inhibition and potentiation of CCK-8stimulated exocrine pancreatic secretory function. Chronic glucocorticoid administration not only produces morphologic changes in the pancreas (1,3-5, 7,9,10)but also enhances the secretagogue-induced secretion of digestive enzymes by sensitizing the acinar cells and induces the hyperconcentration of pancreatic secretion. Recent studies have demonstrated that blockage of exocytosis is a major cause of experimental acute pancreatitis induced by infusing supramaximal doses of caerulein or by feeding a choline-deficient diet supplemented by ethionine (36),although inhibition of release of pancreatic enzymes from the acinar cells has previously been believed to be beneficial for the treatment of acute pancreatitis. Thus, though our findings with perfused rat pancreas demonstrate that prolonged administration of small doses of glucocorticoids have effects opposite to acute administration of large

December 1987

doses, both could be a cause of acute pancreatitis by increasing enzyme secretion or by blocking exocytosis.

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HCt3-

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