GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
In Vitro Insulin
32, 495-504 (1977)
Release from Chicken
Pancreas
STEPHEN P. NABER' AND ROBERT L. HAZELWOOD~ Department
of Biology,
University
of Houston,
Received February
Houston,
Texas
77004
I, 1977
Insulin release from the chicken pancreas (adult hens) was studied in vitro by incubating pancreatic pieces (20 + 5 mg) for four successive 15-min experimental periods preceded by a 30-min preincubation period. Release of immunoreactive insulin (IRI) into the medium was measured in response to glucose (50, 150, 300, 500, and 700 mg/dl), sulfonylureas, and in the presence or absence of extracellular calcium. The results obtained demonstrate that glucose stimulated insulin release only at concentrations of 500 and 700 mg/dl. At these concentrations, the rate of insulin release was maintained at approximately twice basal levels for only 15 to 30 min before returning to the preincubation value (45 nglgimin) irrespective of the prolonged (I-hr) glucose stimulation. Clucagon (5 /.&ml) effected a twofold increase in the rate of insulin release in the presence of 50 mg of glucose/d1 of medium. Three concentrations (3.1, 6.1, and 12.2 mg/dl) of tolbutamide stimulated a twofold increase of insulin release; however, these avian pancreatic pieces were refractory to a second tolbutamide stimulus. In contrast to mammals, the major metabolite of tolbutamide, carboxytolbutamide, was inert in this system. Glucose- and tolbutamide-induced insulin secretion both were inhibited in the absence of extracellular calcium; this inhibition was abolished by replacement of normal amounts of calcium in the incubation medium. The results of this study suggest that the chicken /3 cell differs from the mammalian counterpart by being insensitive to all but very high glucose concentrations, and by being incapable of a sustained increase in insulin release in response to prolonged or repetitive insulinotropic stimuli. Additionally, the insulin response to tolbutamide in vitro indicates that the insulin releasing action of this compound may underlie the hypoglycemic effect observed in birds. Dependence on extracellular calcium suggests that the functional mechanism for avian insulin release may be similar in birds and mammals, although birds may differ from mammals in their pattern of insulin release.
INTRODUCTION
Studies of avian insulin secretion in viva and in vitro have demonstrated patterns of release different from those of similar mammalian systems. Langslow et al. (1970) reported that plasma immunoreactive insulin (IRI) levels in young chickens increased rapidly in response to intracardiac glucose injection, only to return to normal levels within 30 min despite the persisting hyperglycemia (5 hr). Similar results were reported by Samols et al. (1969) following ’ Present address: Department of Pathology, Washington University School of Medicine, 660 S. Euclid Avenue., St. Louis, MO. 63110. ’ To whom reprint requests should be addressed.
intravenous glucose injection in ducks. Measurements of insulin release in vitro from avian pancreatic preparations have been accomplished in ducks (R-Candela et al. 1968), in penguins (Basabe et al. 1975), and in chickens (King and Hazelwood, 1976). Basabe et al. (1975) reported that pieces of penguin pancreas exhibited little sensitivity to glucose stimulation and that arginine was a more effective insulin secretogogue than glucose. King and Hazelwood (1976) demonstrated that glucose stimulated a biphasic insulin release from an isolated chicken pancreas perfused in siru only at concentrations at least twice those found in normal chicken plasma. The pattern of the second phase of insulin re495
Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 0016-6480
496
NABER
AND HAZELWOOD
lease was markedly diminished in comparison to the pattern from similarly perfused mammalian pancreas (Curry, 1971). Glucagon has been established as potentiator of glucose-induced insulin release in mammals both in vivo and in vitro (Samols et al., 196.5; Turner and McIntyre, 1966). However, what little information is available concerning the influence of glucagon on avian insulin release appears contradictory. Thus, while R-Candela et al. (1968) did not observe insulin release upon the addition of glucagon to pieces of duck pancreas in vitro, more recently Basabe et al. (1975) reported that glucagon stimulated insulin release from penguin pancreatic pieces even in the absence of nonstimulating glucose levels. Observations on the efficacy of glucagon in effecting insulin release in vivo also yield markedly different results (Mialhe, 1969; Langslow et al., 1970). The action of tolbutamide on avian carbohydrate metabolism also differs from that observed in mammals. No definite explanation for the acute hypoglycemic action of sulfonylureas in birds is available, whereas, in mammals, it has been well established that these compounds act by stimulating insulin release from the p cells (Grodsky et al., 1967). Studies by Hazelwood (1958) and by Mirsky and Gitelson (1958) suggested the possible existence of a nonpancreatic site of sulfonylurea action in chickens and ducks. Colca and Hazelwood (1976) suggested the existence of an extrapancreatic source of insulin in chickens when they observed increased circulating IRI levels prior to tolbutamide-induced hypoglycemia in depancreatized chickens. Similarly, in an in vitro study, Basabe et al. (1975) reported that the sulfonylurea, glibenclamide, was ineffective in eliciting insulin release from pieces of penguin pancreas. Thus, it is evident that the secretion of insulin by the avian pancreas differs from that observed with similarly prepared
mammalian systems. It appeared desirable, therefore, to investigate the response of the avian p cell to potential insulin secretogogues to help clarify the role of this organ in avian carbohydrate metabolism. By using pancreatic pieces in vitro, it was possible to isolate the release of insulin from other metabolic processes occurring simultaneously in the chicken and, therefore, to investigate the direct effects of various agents on insulin release. The present study was designed to evaluate the sensitivity of the chicken j? cell to varied glucose concentrations and to glucagon, and to determine whether or not the sulfonylureas and a principal metabolite were capable of eliciting pancreatic insulin release directly. The dependence of the insulin-release process on the presence of extracellular calcium also was investigated. MATERIALS
AND METHODS
Chemicals. All materials for insulin radioimmunoassay (IRI) were obtained from the Amersham-Searle Co., Chicago, Illinois, except the chicken insulin standards provided by Dr. J. R. Kimmel, University of Kansas Medical Center, Kansas City, Kansas. Sodium tolbutamide (Orinase Diagnostic) and carboxytolbutamide were gifts from Dr. W. E. Dulin, the Upjohn Co., Kalamazoo, Michigan, and glucagon was made available as a gift from Dr. Mary A. Root, Eli Lilly Co., Indianapolis, Indiana. Trayslol (Aprotinin) was purchased from FBA Pharmaceuticals, New York, New York. All other chemicals were of reagent grade. Animals. All chickens were adult, single-comb White Leghorn hens, weighing between 1200 and 1800 g, and were obtained from a local commercial source. The birds were allowed free access to feed (Purina) and water and were maintained under a 12-hr light/dark cycle for at least 3 days prior to the experiments. Incubation medium. The basic medium used for all experiments was a modified Krebs-Ringer bicarbonate buffer (Krebs, 1950) with the following composition in meq/liter: Na+, 141.0; K+, 5.93; Ca2+, 5.08; Mg*+, 2.36; Cl-, 104.8; PO,*-, 2.22: SOd2-, 2.36; HCOI-, 24.9; pyruvate, 4.9; glutamate, 4.9; and fumarate, 5.4. Bovine serum albumin, Fraction V (Sigma Chemical Corp., St. Louis, MO.) was added at a concentration of I mg/ml. D-Glucose was present in concentrations ranging from 50 to 700 mg/dl as dictated by the experimental design. The medium was
INSULIN
RELEASE
FROM
equilibrated with a 95% 02:5% CO, gas mixture to maintain the pH at 7.4. Experimental protocol. The basic protocol followed for all experiments designed to measure insulin release was a modification of the pancreatic piece method of Coore and Randle (1964). Chickens were decapitated, the abdomen was opened quickly, and the duodenal loop containing the pancreas was excised (within I min of death) and placed in ice-cold preincubated medium. The dorsal lobe of the pancreas was separated and eight contiguous pieces of 20 2 5 mg were cut from the middle one-third of the lobe and transferred to another flask containing preincubation medium. This region of the chicken pancreas has been shown to contain the highest density of insulinsecreting /I-islet tissue (Mikami and Ono, 1962). The pieces were weighed to the nearest 0.1 mg on a torsion balance and transferred to individual incubation flasks containing 4 ml of preincubation medium. The pieces were preincubated for 30 min at 42” (avian core body temperature) under a continuous gas (95% 0,:5% COJ flow in a Dubnoff metabolic shaker (75 cyclesimin). Following preincubation, each piece was transferred to a Buchner funnel, rinsed twice with 42” preincubation medium, and then carried through four successive 15-min incubations, each in 4 ml of fresh experimental incubation medium. At the completion of the preincubation and of each experimental incubation period, a sample of the medium was removed from each flask and immediately frozen (-20”) prior to assay for immunoreactive insulin content by the double-antibody Method C of Hales and Randle (1963). Insulin secretory rates were expressed as nanograms of chicken insulin released per gram of wet weight of pancreas per minute. Statistical sig-
AVIAN
PANCREAS
497
nificance of the results was calculated using a singleclassification analysis of variance (Sokal and Rohlf, 1969).
RESULTS
The possibility of insulin degradation by endogenous pancreatic enzymes was evaluated by incubating pancreatic pieces for 15 or 30 min as described above. At the conclusion of this period the tissue was removed from the flasks and the incubation of the medium was continued for an additional 15 or 30 min. The immunoreactive insulin (IRI) content of the medium following incubation of the medium alone was expressed as a percentage of that found at the end of the tissue preincubation period. The degradation studies indicated that 31% of the IRI released by the tissue samples was lost in 15 min of incubation and 66% of it was lost following 30 min of incubation. This loss was reduced in the presence of Trasylol, a protease inhibitor (Fig. 1). Thus, the rate of insulin degradation was time dependent and proceeded at a constant rate of approximately 2.1% per minute. Additionally, the rate of insulin release from pancreas pieces incubated in a medium containing 50 mg of glucose/d1 remained constant, as seen in Fig. 2 (first two
* 15 MINUTE INCUBATION
30 MINUTE INCUBATION
FIG. 1. Degradation of chicken insulin in vitro during incubations of pancreatic pieces. Incubations were carried out in a medium containing 225 mg of glucose/dl. Insulin content of the medium at completion of the 15 or 30-min preincubation period (not shown) was considered to be 100%. The bars represent degradation during IS or 30 min of incubation of the medium after removal of tissue. N = the number of observations; mean + SEM.
498
NABER
PERIOD:PRE-INCUB.
I ’
I .OOl
AND
HAZELWOOD 2
I
3
I
1
4
I
P-VALUE: - 100 NS
NS
NS -80
T
INCUBATION
T
(MINUTES)
FIG. 2. Effect of glucose concentration on insulin release from chicken pancreatic pieces. All pancreatic pieces were preincubated in a medium containing 50 mg of glucoseidl. Each piece was transferred to fresh incubation medium at the conclusion of each period. N = the number of observations; mean ? SEM.
bars). From these results it was concluded that insulin-release rates could be determined in this system without interference from insulin degradation and that the addition of Trasylol was not necessary.
only when the concentration of glucose in the medium was increased to 500 or 700 mg/dl. This insulinotropic response occurred only during the first period of incubation. During Periods 2 through 4, the rate of insulin release at all glucose concenEffect of Glucose and Glucagon vs Insulin trations returned to values which were not Release significantly different from the basal rates The sensitivity of pancreatic tissue to observed at 50 mg of glucose/d1 (P > 0.05). The response of chicken pancreatic various glucose concentrations, measured by preincubating the pieces for 30 min in a pieces to glucagon at levels of 5 pg of medium containing 50 mg of glucose/dl, and glucagon/ml of medium was investigated in (50 mgidl) followed by four successive 15min incuba- the presence of nonstimulating tions (Periods 1 through 4) in media con- and stimulating (500 mg/dl) concentrations taining either 50, 150, 300,500, or 700 mg of of glucose. As shown in Fig. 3, a marked glucose/dl, is illustrated in Fig. 2. Glucose, response was observed during the first 15 min of incubation (Period 1) when the mean at a concentration of 50 mg of glucose/dl, resulted in a response ranging from 45 to 58 insulin-release rate increased from a basal ng of IRI/g/min throughout 90 min of incu- level of 73 to 145 ngigimin (P < 0.01). The bation (Fig. 2). This response was considhigher basal rate of insulin release observed ered basal. Exposure of pancreatic pieces in these experiments is attributable to the presence of Trasylol (1000 kIU/ml) added to increasing glucose concentrations showed a statistically significant response to the medium to prevent degradation of the
INSULIN PERIOD:
Pra-Ins
RELEASE ,
FROM
I
t--
AVIAN
PANCREAS
(5ug/ml)
h
2 GLUCAGON Q< 001
3
mln
O-15 INCUBATION
FIG. 3. &ml) was to prevent correlative
,
4
T
a I.-l 30
499
15-30
8
500
501
30-45
45-60
(minutes)
Effect of glucagon on insulin release from pieces of chicken pancreas. Crystalline porcine glucagon (5 present in Periods 1 through 4. A protease inhibitor (Trasylol, 1000 klU/ml) was added to the medium excessive glucagon degradation. Trasylol was used during preincubation in order to ensure that basal release rates were established. N = the number of observations: mean 2 SEM.
exogenous glucagon. When the concentration of glucose was elevated to 500 mg/dl (Periods 2 through 4) in the presence of glucagon, the increased rate of insulin release persisted for an additional 15 min (Period 2, P < O.OOl), and then declined to near-basal preincubation rates in Periods 3 and 4. Effect of Tolbutamide Carboxytolbutamide
and on Insulin Release
The insulin response to sodium tolbutamide in vitro was investigated at concentrations of 3.1,6.1, and 12.2 mg/dl in the presence of 225 mg of glucose/dl, a level which corresponds to the fasting plasma glucose level of the chicken (Hazelwood, 1976) and which does not stimulate insulin release (Fig. 2). The highest tolbutamide concentration (12.2 mg/dl) was selected by estimating the concentration of tolbutamide expected in the extracellular space of an adult chicken following the intravenous injection of an amount of tolbutamide known to produce a 35-40% hypoglycemia in vivo (Pittman and Hazelwood, 1973). Following
a 30-min preincubation in medium containing 225 mg of glucose/d1 the sulfonylurea was added in Period 1. All three tolbutamide concentrations elicited an approximately twofold elevation in insulin release during this period (Fig. 4). When the pieces were returned to the drug-free medium (Periods 2 and 3), the insulinrelease rates returned to levels which did not differ significantly from the preincubation control rates (P > 0.05). When the tolbutamide challenge was repeated in Period 4, the pieces which had previously been exposed to 12.2 mg of tolbutamide/dl in Period 1, the insulin-release rate increased to 72 ng/g/min, a level not significantly different from the preincubation value of 54 ng/g/min . In order to test the possibility that the lack of response to tolbutamide in Period 4 (Fig. 4) may have been due to functional deterioration of the pancreatic tissue, rather than to a reduced capacity to respond to a second challenge, in another experiment tolbutamide was added to the incubation medium only in Period 4 (Fig.
500
NABER PERIOD, Pm-lncub.
1 I
1
AND
HAZELWOOD
I
2
I
3
I
i-
1
15
30-45 INCUBATION
4
15
45-60
(IWIUtO8]
FIG. 4. Effect of tolbutamide on insulin release from chicken pancreatic pieces. Hatched bars indicate the presence of tolbutamide. Glucose concentration was 225 mg/dl in all periods. N = the number of observations; mean rt SEM; * = P < 0.001; ** = P < 0.025
5). The pancreatic pieces responded dramatically with a release rate of 202 ng/ g/min as compared to a preincubation rate of 97 ng/g/min (P < 0.001). This response is comparable to the twofold increase in release rate observed in the experiments in which tolbutamide was present only in Period 1 (Fig. 4). It has been reported that carboxytolbutamide, the major metabolite of tolbutamide, causes insulin release in the mammalian pancreas (Feldman and Lebovitz, 1969). When this metabolite was added to the medium at a concentration of 12.2 mg/dl in both Periods 1 and 4 in the presence of 225 mg glucose/dl, it did not stimulate insulin release (Fig. 5). Dependence of Insulin Release on Extracellular Calcium The necessity of extracellular calcium for in vitro insulin release by mammalian pancreas is well documented (e.g., Milner and Hales, 1967; Curry et al. 1968). The present
experiments were designed to examine whether the presence of extracellular calcium is necessary for glucoseand tolbutamide-induced insulin release from chicken pancreatic tissue as well. For this purpose the tissue was depleted of its calcium content by preincubation in a calcium-free and glucose-free medium for 60 min as suggested by Malaisse et al. (1973). Control insulin release was then measured for 15 min in the absence of extracellular calcium; in the subsequent 15-min incubation the rate of insulin release was measured in calcium-free medium containing either glucose (500 mg/dl) or tolbutamide (12.2 mgdl). The pieces were then incubated in a medium containing normal extracellular calcium concentration (5.08 meq/liter) for 60 min, followed by measurements of insulin released during a 15-min control period and a 15-min period of stimulation with either glucose (500 mg/dl) or tolbutamide (12.2 mg/dl) in the
INSULIN
RELEASE
PERIOD’ Pro-lncub. I I
1
FROM
I
AVIAN
2
I
INCUBATION
I I
501
PANCREAS
5
I I
4
(mIntIt.
FIG. 5. Effect of carboxytolbutamide on insulin release from pieces of chicken pancreas. Hatched columns represent either Period 4 in which tolbutamide was present, or the Periods I and 4 in which carboxytoibutamide was present. Glucose concentration was 225 mg/dl in all periods. N = number of observations; mean + SEM; * = P < 0.001; NS = nonsignificant.
presence of extracellular calcium (5.08 meq/liter). During the tolbutamide experiments the glucose concentration was maintained at 225 mg/dl throughout the four periods. Incubation in a calcium-free medium decreased the response to glucose and tolbutamide stimulation to basal levels. When calcium was added to the incubation medium, the insulin-releasing effect of glucose and tolbutamide was restored. DISCUSSION
Measurements of insulin-release rates over a wide range of glucose concentrations (50-700 mg/dl) indicated that the avian p cell is insensitive to glucose until a concentration of 500 mg/dl is reached. This concentration of glucose is at least twice the normal physiologic (fed or fasted) chicken plasma glucose level. A similar insensitivity to glucose has been reported by Rhoten (1974) for the perifused islets of the lizard Anolis carolinensis, whose normal blood glucose level usually exceeds 200 mg/dl.
These observations are to be contrasted with those on mammals in which glucose triggers insulin release at concentrations close to their physiological plasma levels of 90-100 mg/dl (Malaisse et al., 1967). Moreover, in pieces of rat pancreas the rate of insulin release increases (sigmoidally) ninefold when the concentration of glucose in the medium is increased from 50 to 500 mg/dl (Malaisse et al., 1967). However, the insulin release from chicken pancreatic pieces increased only twofold when the glucose concentration was increased from 50 to 700 mg/dl. The rapid decline in insulin release observed following 15 to 30 min of glucose stimulation indicated that the chicken /3 cell may possess a poor insulinogenic reserve. Furthermore, when chicken pancreatic pieces were exposed to two 15-min periods of tolbutamide stimulation separated by 30 min without stimulation, the release of insulin was elevated significantly only during the first experimental period. These findings are supported by the observations
502
NABER
AND
HAZELWOOD
GLUCOSE (500mg/~l)
I
Co
1
FREE 1 WlTHCa
TOL6UlAMlM Ca
FREE i WITH Co
r)
I _ xg%?‘t
!I- I tP
AND
COMPARATIVE
ENDOCRINOLOGY
In Vitro Insulin
32, 495-504 (1977)
Release from Chicken
Pancreas
STEPHEN P. NABER' AND ROBERT L. HAZELWOOD~ Department
of Biology,
University
of Houston,
Received February
Houston,
Texas
77004
I, 1977
Insulin release from the chicken pancreas (adult hens) was studied in vitro by incubating pancreatic pieces (20 + 5 mg) for four successive 15-min experimental periods preceded by a 30-min preincubation period. Release of immunoreactive insulin (IRI) into the medium was measured in response to glucose (50, 150, 300, 500, and 700 mg/dl), sulfonylureas, and in the presence or absence of extracellular calcium. The results obtained demonstrate that glucose stimulated insulin release only at concentrations of 500 and 700 mg/dl. At these concentrations, the rate of insulin release was maintained at approximately twice basal levels for only 15 to 30 min before returning to the preincubation value (45 nglgimin) irrespective of the prolonged (I-hr) glucose stimulation. Clucagon (5 /.&ml) effected a twofold increase in the rate of insulin release in the presence of 50 mg of glucose/d1 of medium. Three concentrations (3.1, 6.1, and 12.2 mg/dl) of tolbutamide stimulated a twofold increase of insulin release; however, these avian pancreatic pieces were refractory to a second tolbutamide stimulus. In contrast to mammals, the major metabolite of tolbutamide, carboxytolbutamide, was inert in this system. Glucose- and tolbutamide-induced insulin secretion both were inhibited in the absence of extracellular calcium; this inhibition was abolished by replacement of normal amounts of calcium in the incubation medium. The results of this study suggest that the chicken /3 cell differs from the mammalian counterpart by being insensitive to all but very high glucose concentrations, and by being incapable of a sustained increase in insulin release in response to prolonged or repetitive insulinotropic stimuli. Additionally, the insulin response to tolbutamide in vitro indicates that the insulin releasing action of this compound may underlie the hypoglycemic effect observed in birds. Dependence on extracellular calcium suggests that the functional mechanism for avian insulin release may be similar in birds and mammals, although birds may differ from mammals in their pattern of insulin release.
INTRODUCTION
Studies of avian insulin secretion in viva and in vitro have demonstrated patterns of release different from those of similar mammalian systems. Langslow et al. (1970) reported that plasma immunoreactive insulin (IRI) levels in young chickens increased rapidly in response to intracardiac glucose injection, only to return to normal levels within 30 min despite the persisting hyperglycemia (5 hr). Similar results were reported by Samols et al. (1969) following ’ Present address: Department of Pathology, Washington University School of Medicine, 660 S. Euclid Avenue., St. Louis, MO. 63110. ’ To whom reprint requests should be addressed.
intravenous glucose injection in ducks. Measurements of insulin release in vitro from avian pancreatic preparations have been accomplished in ducks (R-Candela et al. 1968), in penguins (Basabe et al. 1975), and in chickens (King and Hazelwood, 1976). Basabe et al. (1975) reported that pieces of penguin pancreas exhibited little sensitivity to glucose stimulation and that arginine was a more effective insulin secretogogue than glucose. King and Hazelwood (1976) demonstrated that glucose stimulated a biphasic insulin release from an isolated chicken pancreas perfused in siru only at concentrations at least twice those found in normal chicken plasma. The pattern of the second phase of insulin re495
Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 0016-6480
496
NABER
AND HAZELWOOD
lease was markedly diminished in comparison to the pattern from similarly perfused mammalian pancreas (Curry, 1971). Glucagon has been established as potentiator of glucose-induced insulin release in mammals both in vivo and in vitro (Samols et al., 196.5; Turner and McIntyre, 1966). However, what little information is available concerning the influence of glucagon on avian insulin release appears contradictory. Thus, while R-Candela et al. (1968) did not observe insulin release upon the addition of glucagon to pieces of duck pancreas in vitro, more recently Basabe et al. (1975) reported that glucagon stimulated insulin release from penguin pancreatic pieces even in the absence of nonstimulating glucose levels. Observations on the efficacy of glucagon in effecting insulin release in vivo also yield markedly different results (Mialhe, 1969; Langslow et al., 1970). The action of tolbutamide on avian carbohydrate metabolism also differs from that observed in mammals. No definite explanation for the acute hypoglycemic action of sulfonylureas in birds is available, whereas, in mammals, it has been well established that these compounds act by stimulating insulin release from the p cells (Grodsky et al., 1967). Studies by Hazelwood (1958) and by Mirsky and Gitelson (1958) suggested the possible existence of a nonpancreatic site of sulfonylurea action in chickens and ducks. Colca and Hazelwood (1976) suggested the existence of an extrapancreatic source of insulin in chickens when they observed increased circulating IRI levels prior to tolbutamide-induced hypoglycemia in depancreatized chickens. Similarly, in an in vitro study, Basabe et al. (1975) reported that the sulfonylurea, glibenclamide, was ineffective in eliciting insulin release from pieces of penguin pancreas. Thus, it is evident that the secretion of insulin by the avian pancreas differs from that observed with similarly prepared
mammalian systems. It appeared desirable, therefore, to investigate the response of the avian p cell to potential insulin secretogogues to help clarify the role of this organ in avian carbohydrate metabolism. By using pancreatic pieces in vitro, it was possible to isolate the release of insulin from other metabolic processes occurring simultaneously in the chicken and, therefore, to investigate the direct effects of various agents on insulin release. The present study was designed to evaluate the sensitivity of the chicken j? cell to varied glucose concentrations and to glucagon, and to determine whether or not the sulfonylureas and a principal metabolite were capable of eliciting pancreatic insulin release directly. The dependence of the insulin-release process on the presence of extracellular calcium also was investigated. MATERIALS
AND METHODS
Chemicals. All materials for insulin radioimmunoassay (IRI) were obtained from the Amersham-Searle Co., Chicago, Illinois, except the chicken insulin standards provided by Dr. J. R. Kimmel, University of Kansas Medical Center, Kansas City, Kansas. Sodium tolbutamide (Orinase Diagnostic) and carboxytolbutamide were gifts from Dr. W. E. Dulin, the Upjohn Co., Kalamazoo, Michigan, and glucagon was made available as a gift from Dr. Mary A. Root, Eli Lilly Co., Indianapolis, Indiana. Trayslol (Aprotinin) was purchased from FBA Pharmaceuticals, New York, New York. All other chemicals were of reagent grade. Animals. All chickens were adult, single-comb White Leghorn hens, weighing between 1200 and 1800 g, and were obtained from a local commercial source. The birds were allowed free access to feed (Purina) and water and were maintained under a 12-hr light/dark cycle for at least 3 days prior to the experiments. Incubation medium. The basic medium used for all experiments was a modified Krebs-Ringer bicarbonate buffer (Krebs, 1950) with the following composition in meq/liter: Na+, 141.0; K+, 5.93; Ca2+, 5.08; Mg*+, 2.36; Cl-, 104.8; PO,*-, 2.22: SOd2-, 2.36; HCOI-, 24.9; pyruvate, 4.9; glutamate, 4.9; and fumarate, 5.4. Bovine serum albumin, Fraction V (Sigma Chemical Corp., St. Louis, MO.) was added at a concentration of I mg/ml. D-Glucose was present in concentrations ranging from 50 to 700 mg/dl as dictated by the experimental design. The medium was
INSULIN
RELEASE
FROM
equilibrated with a 95% 02:5% CO, gas mixture to maintain the pH at 7.4. Experimental protocol. The basic protocol followed for all experiments designed to measure insulin release was a modification of the pancreatic piece method of Coore and Randle (1964). Chickens were decapitated, the abdomen was opened quickly, and the duodenal loop containing the pancreas was excised (within I min of death) and placed in ice-cold preincubated medium. The dorsal lobe of the pancreas was separated and eight contiguous pieces of 20 2 5 mg were cut from the middle one-third of the lobe and transferred to another flask containing preincubation medium. This region of the chicken pancreas has been shown to contain the highest density of insulinsecreting /I-islet tissue (Mikami and Ono, 1962). The pieces were weighed to the nearest 0.1 mg on a torsion balance and transferred to individual incubation flasks containing 4 ml of preincubation medium. The pieces were preincubated for 30 min at 42” (avian core body temperature) under a continuous gas (95% 0,:5% COJ flow in a Dubnoff metabolic shaker (75 cyclesimin). Following preincubation, each piece was transferred to a Buchner funnel, rinsed twice with 42” preincubation medium, and then carried through four successive 15-min incubations, each in 4 ml of fresh experimental incubation medium. At the completion of the preincubation and of each experimental incubation period, a sample of the medium was removed from each flask and immediately frozen (-20”) prior to assay for immunoreactive insulin content by the double-antibody Method C of Hales and Randle (1963). Insulin secretory rates were expressed as nanograms of chicken insulin released per gram of wet weight of pancreas per minute. Statistical sig-
AVIAN
PANCREAS
497
nificance of the results was calculated using a singleclassification analysis of variance (Sokal and Rohlf, 1969).
RESULTS
The possibility of insulin degradation by endogenous pancreatic enzymes was evaluated by incubating pancreatic pieces for 15 or 30 min as described above. At the conclusion of this period the tissue was removed from the flasks and the incubation of the medium was continued for an additional 15 or 30 min. The immunoreactive insulin (IRI) content of the medium following incubation of the medium alone was expressed as a percentage of that found at the end of the tissue preincubation period. The degradation studies indicated that 31% of the IRI released by the tissue samples was lost in 15 min of incubation and 66% of it was lost following 30 min of incubation. This loss was reduced in the presence of Trasylol, a protease inhibitor (Fig. 1). Thus, the rate of insulin degradation was time dependent and proceeded at a constant rate of approximately 2.1% per minute. Additionally, the rate of insulin release from pancreas pieces incubated in a medium containing 50 mg of glucose/d1 remained constant, as seen in Fig. 2 (first two
* 15 MINUTE INCUBATION
30 MINUTE INCUBATION
FIG. 1. Degradation of chicken insulin in vitro during incubations of pancreatic pieces. Incubations were carried out in a medium containing 225 mg of glucose/dl. Insulin content of the medium at completion of the 15 or 30-min preincubation period (not shown) was considered to be 100%. The bars represent degradation during IS or 30 min of incubation of the medium after removal of tissue. N = the number of observations; mean + SEM.
498
NABER
PERIOD:PRE-INCUB.
I ’
I .OOl
AND
HAZELWOOD 2
I
3
I
1
4
I
P-VALUE: - 100 NS
NS
NS -80
T
INCUBATION
T
(MINUTES)
FIG. 2. Effect of glucose concentration on insulin release from chicken pancreatic pieces. All pancreatic pieces were preincubated in a medium containing 50 mg of glucoseidl. Each piece was transferred to fresh incubation medium at the conclusion of each period. N = the number of observations; mean ? SEM.
bars). From these results it was concluded that insulin-release rates could be determined in this system without interference from insulin degradation and that the addition of Trasylol was not necessary.
only when the concentration of glucose in the medium was increased to 500 or 700 mg/dl. This insulinotropic response occurred only during the first period of incubation. During Periods 2 through 4, the rate of insulin release at all glucose concenEffect of Glucose and Glucagon vs Insulin trations returned to values which were not Release significantly different from the basal rates The sensitivity of pancreatic tissue to observed at 50 mg of glucose/d1 (P > 0.05). The response of chicken pancreatic various glucose concentrations, measured by preincubating the pieces for 30 min in a pieces to glucagon at levels of 5 pg of medium containing 50 mg of glucose/dl, and glucagon/ml of medium was investigated in (50 mgidl) followed by four successive 15min incuba- the presence of nonstimulating tions (Periods 1 through 4) in media con- and stimulating (500 mg/dl) concentrations taining either 50, 150, 300,500, or 700 mg of of glucose. As shown in Fig. 3, a marked glucose/dl, is illustrated in Fig. 2. Glucose, response was observed during the first 15 min of incubation (Period 1) when the mean at a concentration of 50 mg of glucose/dl, resulted in a response ranging from 45 to 58 insulin-release rate increased from a basal ng of IRI/g/min throughout 90 min of incu- level of 73 to 145 ngigimin (P < 0.01). The bation (Fig. 2). This response was considhigher basal rate of insulin release observed ered basal. Exposure of pancreatic pieces in these experiments is attributable to the presence of Trasylol (1000 kIU/ml) added to increasing glucose concentrations showed a statistically significant response to the medium to prevent degradation of the
INSULIN PERIOD:
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501
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Effect of glucagon on insulin release from pieces of chicken pancreas. Crystalline porcine glucagon (5 present in Periods 1 through 4. A protease inhibitor (Trasylol, 1000 klU/ml) was added to the medium excessive glucagon degradation. Trasylol was used during preincubation in order to ensure that basal release rates were established. N = the number of observations: mean 2 SEM.
exogenous glucagon. When the concentration of glucose was elevated to 500 mg/dl (Periods 2 through 4) in the presence of glucagon, the increased rate of insulin release persisted for an additional 15 min (Period 2, P < O.OOl), and then declined to near-basal preincubation rates in Periods 3 and 4. Effect of Tolbutamide Carboxytolbutamide
and on Insulin Release
The insulin response to sodium tolbutamide in vitro was investigated at concentrations of 3.1,6.1, and 12.2 mg/dl in the presence of 225 mg of glucose/dl, a level which corresponds to the fasting plasma glucose level of the chicken (Hazelwood, 1976) and which does not stimulate insulin release (Fig. 2). The highest tolbutamide concentration (12.2 mg/dl) was selected by estimating the concentration of tolbutamide expected in the extracellular space of an adult chicken following the intravenous injection of an amount of tolbutamide known to produce a 35-40% hypoglycemia in vivo (Pittman and Hazelwood, 1973). Following
a 30-min preincubation in medium containing 225 mg of glucose/d1 the sulfonylurea was added in Period 1. All three tolbutamide concentrations elicited an approximately twofold elevation in insulin release during this period (Fig. 4). When the pieces were returned to the drug-free medium (Periods 2 and 3), the insulinrelease rates returned to levels which did not differ significantly from the preincubation control rates (P > 0.05). When the tolbutamide challenge was repeated in Period 4, the pieces which had previously been exposed to 12.2 mg of tolbutamide/dl in Period 1, the insulin-release rate increased to 72 ng/g/min, a level not significantly different from the preincubation value of 54 ng/g/min . In order to test the possibility that the lack of response to tolbutamide in Period 4 (Fig. 4) may have been due to functional deterioration of the pancreatic tissue, rather than to a reduced capacity to respond to a second challenge, in another experiment tolbutamide was added to the incubation medium only in Period 4 (Fig.
500
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45-60
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FIG. 4. Effect of tolbutamide on insulin release from chicken pancreatic pieces. Hatched bars indicate the presence of tolbutamide. Glucose concentration was 225 mg/dl in all periods. N = the number of observations; mean rt SEM; * = P < 0.001; ** = P < 0.025
5). The pancreatic pieces responded dramatically with a release rate of 202 ng/ g/min as compared to a preincubation rate of 97 ng/g/min (P < 0.001). This response is comparable to the twofold increase in release rate observed in the experiments in which tolbutamide was present only in Period 1 (Fig. 4). It has been reported that carboxytolbutamide, the major metabolite of tolbutamide, causes insulin release in the mammalian pancreas (Feldman and Lebovitz, 1969). When this metabolite was added to the medium at a concentration of 12.2 mg/dl in both Periods 1 and 4 in the presence of 225 mg glucose/dl, it did not stimulate insulin release (Fig. 5). Dependence of Insulin Release on Extracellular Calcium The necessity of extracellular calcium for in vitro insulin release by mammalian pancreas is well documented (e.g., Milner and Hales, 1967; Curry et al. 1968). The present
experiments were designed to examine whether the presence of extracellular calcium is necessary for glucoseand tolbutamide-induced insulin release from chicken pancreatic tissue as well. For this purpose the tissue was depleted of its calcium content by preincubation in a calcium-free and glucose-free medium for 60 min as suggested by Malaisse et al. (1973). Control insulin release was then measured for 15 min in the absence of extracellular calcium; in the subsequent 15-min incubation the rate of insulin release was measured in calcium-free medium containing either glucose (500 mg/dl) or tolbutamide (12.2 mgdl). The pieces were then incubated in a medium containing normal extracellular calcium concentration (5.08 meq/liter) for 60 min, followed by measurements of insulin released during a 15-min control period and a 15-min period of stimulation with either glucose (500 mg/dl) or tolbutamide (12.2 mg/dl) in the
INSULIN
RELEASE
PERIOD’ Pro-lncub. I I
1
FROM
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AVIAN
2
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INCUBATION
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501
PANCREAS
5
I I
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FIG. 5. Effect of carboxytolbutamide on insulin release from pieces of chicken pancreas. Hatched columns represent either Period 4 in which tolbutamide was present, or the Periods I and 4 in which carboxytoibutamide was present. Glucose concentration was 225 mg/dl in all periods. N = number of observations; mean + SEM; * = P < 0.001; NS = nonsignificant.
presence of extracellular calcium (5.08 meq/liter). During the tolbutamide experiments the glucose concentration was maintained at 225 mg/dl throughout the four periods. Incubation in a calcium-free medium decreased the response to glucose and tolbutamide stimulation to basal levels. When calcium was added to the incubation medium, the insulin-releasing effect of glucose and tolbutamide was restored. DISCUSSION
Measurements of insulin-release rates over a wide range of glucose concentrations (50-700 mg/dl) indicated that the avian p cell is insensitive to glucose until a concentration of 500 mg/dl is reached. This concentration of glucose is at least twice the normal physiologic (fed or fasted) chicken plasma glucose level. A similar insensitivity to glucose has been reported by Rhoten (1974) for the perifused islets of the lizard Anolis carolinensis, whose normal blood glucose level usually exceeds 200 mg/dl.
These observations are to be contrasted with those on mammals in which glucose triggers insulin release at concentrations close to their physiological plasma levels of 90-100 mg/dl (Malaisse et al., 1967). Moreover, in pieces of rat pancreas the rate of insulin release increases (sigmoidally) ninefold when the concentration of glucose in the medium is increased from 50 to 500 mg/dl (Malaisse et al., 1967). However, the insulin release from chicken pancreatic pieces increased only twofold when the glucose concentration was increased from 50 to 700 mg/dl. The rapid decline in insulin release observed following 15 to 30 min of glucose stimulation indicated that the chicken /3 cell may possess a poor insulinogenic reserve. Furthermore, when chicken pancreatic pieces were exposed to two 15-min periods of tolbutamide stimulation separated by 30 min without stimulation, the release of insulin was elevated significantly only during the first experimental period. These findings are supported by the observations
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