EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
24, 105-109
( 1976)
lncreose in Pancreatic Amylase mediated YEE S. KIM, Department
LAWRENCE
of
J. BAUDENDISTEL,
Pharmacology,
by ~~ucocorticoid z
RENE WEGRIA, AND RUTH E. KIM
St. Louis University Missouri 63104
Received fuEy 3, 1975; and (n re&ed
School of Medicine,
St. Louis,
form J&J 28, I975
The amylase activity of the pancreas in young adult rats is increased by pharmacological doses of glucocorticoid. The increase in the enzyme activity is qualitatively proportional to the dosage. Cycloheximide precludes the glucocorticoid-mediated increase in the enzyme, but insulin has little effect on the enzyme in nondiabetic animals. After the first day of glucocorticoid treatment, the increase in amylase activity is significant but the blood sugar level remains unchanged. These results suggest that the increase in pancreatic amylase is caused by a direct action of glucocorticoid on the pancreatic tissue similar to the effect of the steroids on liver enzymes.
INTRODUCTION Glucocorticoid induction of enzymes in the liver has been studied extensively, and mechanisms underlying the enzyme induction have been proposed (Kim, 1968, 1969; Segal and Kim, 1963; Tomkins et al.. 1969; Sekeris and Van der Meufen, 1974). In contrast to these studies on enzyme induction in liver, very little work has been done concerning the possible gfucocorticoid induction of enzymes in other tissues. Adrenalectomy decreases the pancreatic content of amylase (Snook, 1968; Sesso and Migliorini, 1959), and glucocorticoid is required in the developmental regulation of pancreatic enzymes in the later stages of d~erentiation process of the embryonic pancreas (Cohen and Kulka, 1974; Sanders and Rutter, 1974). Insulin- and diet-mediated increase in pancreatic amylase have also been described ( Abdeljlil et al., 1963, 1965). We have studied the effect of ghrcocorticoid on pancreatic amylase in the intact ad&t rat, and evidence is presented here which suggests that gIu~ocorticoid stimulates the synthesis of amylase in this organ in a manner similar to that observed in the liver tissue. MATERIALS
AND
METHODS
Male Sprague-Hawley rats weigh~g 10~2~ gm were used. The body weight difference among the animals used in one experiment was less than 5 gm. Animals were maintained on Wayne laboratory animal feed and water ad lib&m. Amylochrome reagents for the amylase assay were purchased from Hoffman-La Roche. 1 Supported in part by Research Grant No. AM IX280 from the National Institute of Arthritis and Metabolic Diseases, USPHS, and in part by the General Research Support Grant, St. Louis University School of Medicine. 105 Copyright
All
rights
1976 by Academic Press,Inc. aox form reserved.
o P reproduction in
106
KIM
ET AL.
Prednisolone acetate was obtained from Pfizer Laboratories and was diluted in 0.9% NaCl solution before it was given to animals. Protamine zinc insulin and regular insulin were obtained from Eli Lilly and Squibb, respectively. Cycloheximide was a generous gift of Dr. Stubbs of Upjohn Co. The specific schedules and dosages of administration of each drug are described in the appropriate figure and tables of Results. The pancreas isolated from each animal was rinsed thoroughly in cold 0.9% NaCl solution and, after being blotted free of excess solution, was weighed, frozen in liquid nitrogen, and stored at -30°C until used. The frozen pancreas was homogenized in a saline solution in a Lourdes high speed Multi-Mixer for 2 min, and the final concentration was adjusted to 1 gm of pancreas per 20 ml of homogenate. The DNA concentration of the homogenate was estimated by the method of Schneider (195’). An aliquot of the homogenate was centrifuged at 10,OOOg for 10 min, and the arnylase activities in the supernatant fluids were assayed according to the method described by Klein et al. (1970). In order to determine the blood sugar level, the blood was drawn by cardiac puncture and was kept at room temperature for 1 hr, after which the blood sample was centrifuged twice at 10,000~ for 10 min. The clear supernatant fluid was obtained, and the glucose concentration in the serum was estimated by the hexokinase method ( Slein, 1965). RESULTS Arm&se Activities of Pancreas i?r G~ucoco~~co~d-~Teated Animals The time course of changes in pancreatic amylase activity in control and glucocorticoid-treated animals is shown in Fig. 1. The amylase activity in control animals remained unchanged during the 4-day period of the experiment. The amylase activity in the glucocorticoid-treated animals increased to a maximum level in approximately 3 days. At the maximum, the enzyme content in the tissue of the glucocorticoid-treated pancreas was three times that in the normal pancreas. Effects of Prednisolone, Cycloheximide, and Insulin on the Amylase Activity The amylase activities in the pancrease of animals that had received these different drugs for 3 days are compared with the amylase activity in control animals in Table I. The enzyme activities in animals given 4 mgm/lOO gm of body weight of prednisolone were greater than the enzyme activities in animals that had received 2 mgm/lOO gm of body weight of the steroid. The increase in the amylase activity was thus dose dependent. Amylase activity in the pancrease of animals given cycloheximide was 23y0 lower than the enzyme activity in control animals. The amylase content in the pancreas of animals that had received both prednisolone and cycloheximide was 31% lower than the control value. Thus, the protein inhibitor precludes the glucocorticoid-mediated increase in the tissue content of amylase. The dosage and duration of insulin used in this experiment were the same as the dosage and duration of insulin administration that have been shown to restore the enzyme level in diabetic animals (Abdeljlil et al., 1965). In nondiabetic animals, administration of insulin had little effect on pancreatic amylase. In fact, insulin caused a slight decrease in the tissue activities of amylase.
INCREASE
IN
PANCREATIC
107
AMYLASE
Time (day)
FIG. 1. Time course of changes in pancreatic amylase activity in normal and glucocorticoidtreated rats. Animals were divided into control ( 0) and prednisolone ( l ) groups, and animals in the prednisolone group were given subcutaneously 4 mgm/lOO gm of body weight of prednisolone acetate daily. Five animals from each group were killed every 24 hr. The last injection of steroid was given 2 hr before killing. The methods of assaying amylase and DNA in the pancreas are described in the text.
Relationship
between
Blood Sugar Level and Pancreatic
Amylase
In order to determine the chronological sequence of events, the effect of prednisolone on the pancreatic amylase and on the blood sugar level was examined. In an attempt to detect small changes and to try to separate the two events, a smaller dosage of prednisolone and a shorter duration of the glucocorticoid treatment were used in this experiment. The results are shown in Table II. Under the conditions of these experiments, the amylase activity inTABLE EFFECTS
Status
OF PREDNISOLONE, AMYL~SE
I
CYCLOHEXIMIDE AND INSULIN IN RAT PANCRE.~S
of animala
Amylase
t
activity
-
mgm mgm 50 rg 50 rg, and 4 mgm
test (P)
units/mgm of DNA f SE ( x 109 Control Prednisolone, 4 Prednisolone, 2 Cycloheximide, Cycloheximide, prednisolone, Insulin, 10 units
ON THE
14.9 40.9 28.8 11.5
Ratio (Control = 100%)
i f i i
0.5 3.9 3.5 2.6
100 271 193 77
AND
MOLECULAR
PATHOLOGY
24, 105-109
( 1976)
lncreose in Pancreatic Amylase mediated YEE S. KIM, Department
LAWRENCE
of
J. BAUDENDISTEL,
Pharmacology,
by ~~ucocorticoid z
RENE WEGRIA, AND RUTH E. KIM
St. Louis University Missouri 63104
Received fuEy 3, 1975; and (n re&ed
School of Medicine,
St. Louis,
form J&J 28, I975
The amylase activity of the pancreas in young adult rats is increased by pharmacological doses of glucocorticoid. The increase in the enzyme activity is qualitatively proportional to the dosage. Cycloheximide precludes the glucocorticoid-mediated increase in the enzyme, but insulin has little effect on the enzyme in nondiabetic animals. After the first day of glucocorticoid treatment, the increase in amylase activity is significant but the blood sugar level remains unchanged. These results suggest that the increase in pancreatic amylase is caused by a direct action of glucocorticoid on the pancreatic tissue similar to the effect of the steroids on liver enzymes.
INTRODUCTION Glucocorticoid induction of enzymes in the liver has been studied extensively, and mechanisms underlying the enzyme induction have been proposed (Kim, 1968, 1969; Segal and Kim, 1963; Tomkins et al.. 1969; Sekeris and Van der Meufen, 1974). In contrast to these studies on enzyme induction in liver, very little work has been done concerning the possible gfucocorticoid induction of enzymes in other tissues. Adrenalectomy decreases the pancreatic content of amylase (Snook, 1968; Sesso and Migliorini, 1959), and glucocorticoid is required in the developmental regulation of pancreatic enzymes in the later stages of d~erentiation process of the embryonic pancreas (Cohen and Kulka, 1974; Sanders and Rutter, 1974). Insulin- and diet-mediated increase in pancreatic amylase have also been described ( Abdeljlil et al., 1963, 1965). We have studied the effect of ghrcocorticoid on pancreatic amylase in the intact ad&t rat, and evidence is presented here which suggests that gIu~ocorticoid stimulates the synthesis of amylase in this organ in a manner similar to that observed in the liver tissue. MATERIALS
AND
METHODS
Male Sprague-Hawley rats weigh~g 10~2~ gm were used. The body weight difference among the animals used in one experiment was less than 5 gm. Animals were maintained on Wayne laboratory animal feed and water ad lib&m. Amylochrome reagents for the amylase assay were purchased from Hoffman-La Roche. 1 Supported in part by Research Grant No. AM IX280 from the National Institute of Arthritis and Metabolic Diseases, USPHS, and in part by the General Research Support Grant, St. Louis University School of Medicine. 105 Copyright
All
rights
1976 by Academic Press,Inc. aox form reserved.
o P reproduction in
106
KIM
ET AL.
Prednisolone acetate was obtained from Pfizer Laboratories and was diluted in 0.9% NaCl solution before it was given to animals. Protamine zinc insulin and regular insulin were obtained from Eli Lilly and Squibb, respectively. Cycloheximide was a generous gift of Dr. Stubbs of Upjohn Co. The specific schedules and dosages of administration of each drug are described in the appropriate figure and tables of Results. The pancreas isolated from each animal was rinsed thoroughly in cold 0.9% NaCl solution and, after being blotted free of excess solution, was weighed, frozen in liquid nitrogen, and stored at -30°C until used. The frozen pancreas was homogenized in a saline solution in a Lourdes high speed Multi-Mixer for 2 min, and the final concentration was adjusted to 1 gm of pancreas per 20 ml of homogenate. The DNA concentration of the homogenate was estimated by the method of Schneider (195’). An aliquot of the homogenate was centrifuged at 10,OOOg for 10 min, and the arnylase activities in the supernatant fluids were assayed according to the method described by Klein et al. (1970). In order to determine the blood sugar level, the blood was drawn by cardiac puncture and was kept at room temperature for 1 hr, after which the blood sample was centrifuged twice at 10,000~ for 10 min. The clear supernatant fluid was obtained, and the glucose concentration in the serum was estimated by the hexokinase method ( Slein, 1965). RESULTS Arm&se Activities of Pancreas i?r G~ucoco~~co~d-~Teated Animals The time course of changes in pancreatic amylase activity in control and glucocorticoid-treated animals is shown in Fig. 1. The amylase activity in control animals remained unchanged during the 4-day period of the experiment. The amylase activity in the glucocorticoid-treated animals increased to a maximum level in approximately 3 days. At the maximum, the enzyme content in the tissue of the glucocorticoid-treated pancreas was three times that in the normal pancreas. Effects of Prednisolone, Cycloheximide, and Insulin on the Amylase Activity The amylase activities in the pancrease of animals that had received these different drugs for 3 days are compared with the amylase activity in control animals in Table I. The enzyme activities in animals given 4 mgm/lOO gm of body weight of prednisolone were greater than the enzyme activities in animals that had received 2 mgm/lOO gm of body weight of the steroid. The increase in the amylase activity was thus dose dependent. Amylase activity in the pancrease of animals given cycloheximide was 23y0 lower than the enzyme activity in control animals. The amylase content in the pancreas of animals that had received both prednisolone and cycloheximide was 31% lower than the control value. Thus, the protein inhibitor precludes the glucocorticoid-mediated increase in the tissue content of amylase. The dosage and duration of insulin used in this experiment were the same as the dosage and duration of insulin administration that have been shown to restore the enzyme level in diabetic animals (Abdeljlil et al., 1965). In nondiabetic animals, administration of insulin had little effect on pancreatic amylase. In fact, insulin caused a slight decrease in the tissue activities of amylase.
INCREASE
IN
PANCREATIC
107
AMYLASE
Time (day)
FIG. 1. Time course of changes in pancreatic amylase activity in normal and glucocorticoidtreated rats. Animals were divided into control ( 0) and prednisolone ( l ) groups, and animals in the prednisolone group were given subcutaneously 4 mgm/lOO gm of body weight of prednisolone acetate daily. Five animals from each group were killed every 24 hr. The last injection of steroid was given 2 hr before killing. The methods of assaying amylase and DNA in the pancreas are described in the text.
Relationship
between
Blood Sugar Level and Pancreatic
Amylase
In order to determine the chronological sequence of events, the effect of prednisolone on the pancreatic amylase and on the blood sugar level was examined. In an attempt to detect small changes and to try to separate the two events, a smaller dosage of prednisolone and a shorter duration of the glucocorticoid treatment were used in this experiment. The results are shown in Table II. Under the conditions of these experiments, the amylase activity inTABLE EFFECTS
Status
OF PREDNISOLONE, AMYL~SE
I
CYCLOHEXIMIDE AND INSULIN IN RAT PANCRE.~S
of animala
Amylase
t
activity
-
mgm mgm 50 rg 50 rg, and 4 mgm
test (P)
units/mgm of DNA f SE ( x 109 Control Prednisolone, 4 Prednisolone, 2 Cycloheximide, Cycloheximide, prednisolone, Insulin, 10 units
ON THE
14.9 40.9 28.8 11.5
Ratio (Control = 100%)
i f i i
0.5 3.9 3.5 2.6
100 271 193 77
