Inhibition by Somatostatin of Glucagon and Insulin Release from the Perfused Rat Pancreas in Response to Arginine, Isoproterenol and Theophylline: Evidence for a Preferential Effect on Glucagon Secretion JOHN E. GERICH, ROBERT LOVINGER, AND GEROLD M. GRODSKY The Metabolic Research Unit and Departments of Pediatrics and Biochemistry and Biophysics, University of California Medical Center, San Francisco, California gon responses to isoproterenol, an activator of adenylate cyclase, and to theophylline, a phosphodiesterase inhibitor, were completely abolished by 100 ng/ml somatostatin. Isoproterenol did not cause insulin release in this system, but insulin responses to theophylline were diminished by somatostatin. The present studies thus indicate that somatostatin is a potent inhibitor of both glucagon and insulin secretion and indicate that it acts directly on the pancreatic alpha and beta cells. Glucagon secretion is approximately 20 times more sensitive to the inhibitory effects of somatostatin than is insulin secretion. Furthermore, the present results suggest that somatostatin may act by modifying cAMPdependent systems rather than by altering cAMP levels. (Endocrinology 96: 749, 1975)
ABSTRACT. To determine whether somatostatin inhibits glucagon secretion directly at the pancreatic level and to study quantitatively the relative effects of somatostatin on glucagon and insulin secretion, the effects of various concentrations of somatostatin on glucagon and insulin release from the in vitro perfused rat pancreas in response to arginine (14.2 mM), isoproterenol (2 mg/ml) and theophylline (10 ITIM) were studied. Glucagon and insulin responses to arginine were progressively inhibited by somatostatin over a concentration range from 0.1-100 ng/ml. At all doses, somatostatin caused greater inhibition of glucagon secretion than of insulin secretion. Approximately 4 ng/ml somatostatin reduced glucagon responses 50%, whereas 90 ng/ml was required to produce comparable inhibition of insulin responses. Gluca-
S
OMATOSTATIN, a hypothalamic peptide (1) originally found to inhibit pituitary growth hormone and TSH secretion (2-5), has recently been shown to lower plasma glucose, insulin and glucagon levels in the baboon (6) and to inhibit plasma insulin and glucagon responses to arginine in man (7). Since somatostatin inhibits glucose-induced insulin release from the canine pancreas perfused in vitro (8), it seems likely that a direct effect on the pancreatic beta cell is involved. However, comparable studies concerning the action of somatostatin on glucagon release have not been reported. Therefore, the present investigation was undertaken to characterize further the pancreatic effects of somatostatin by comparing its relative
Received May 29, 1974. Supported in part by Grant AM-01410, NIAMD; the Levi J. and Mary Skaggs Foundation; and the Kroc Foundation.
ability to inhibit glucagon and insulin release from the in vitro perfused rat pancreas in response to arginine, isoproterenol and theophylline. Materials and Methods Animals. Overnight-fasted, male Long-Evans rats, approximately 30 weeks old and 250 g in weight were used in all experiments. Perfusion technique and apparatus. The techniques for dissection of the rat pancreas and its perfusion have been previously described in detail (9,10). In brief, fasted rats were anesthetized with sodium pentobarbital; the pancreas with adjacent stomach, spleen and part of the duodenum was removed and subsequently perfused with a nonrecirculating medium consisting of 1% albumin, 3% dextran, Krebs bicarbonate buffer, pH 7.4. Perfusate was introduced into the celiac artery and total portal vein effluent (10 ml/min) was collected every 60 s. Flow rate and pressure were monitored and remained constant throughout experiments. 749
Endo • 1975 Vol96«No3
GERICH, LOVINGER AND GRODSKY
750
Experimental protocol. After an equilibration period of at least 20 min, stimuli were introduced via side arm syringe. In arginine experiments, perfusate contained 100 mg/100 ml glucose; during isoproterenol and theophylline studies, perfusate contained 50 mg/100 ml glucose. Linear somatostatin (2) (synthesized by Dr. Jean Rivier and kindly supplied by Dr. Roger Guillemin, Salk Institute, San Diego, Ca.) was dissolved in 2M glycine buffer, pH 3.0, and appropriately diluted in perfusate immediately before use. In dose-response experiments, different concentrations of somatostatin were introduced via separate side arm syringe for 10 min during stimulation with arginine. An 8-min interval was allowed between each somatostatin infusion. In studies involving sequential stimulation by isoproterenol, theophylline and arginine, somatostatin infusion was initiated 10 min prior to the initial stimulation and continued throughout experimental periods. Tenmin "rest" periods were allowed between stimuli. Parallel control experiments for these latter studies were performed without infusion of somatostatin. Glucagon and insulin determinations. Perfusate was collected in chilled tubes containing 15%
5.5 mM
EDTA and immediately frozen for subsequent determination of glucagon and insulin. Glucagon was measured by a modification of the Unger and Eisentraut immunoassay (11) using the highly specific Unger 30K antiserum. Duplicate 0.2 ml samples of perfusate were added to 0.5 ml glycine buffer, pH 8.8, containing 0.25% human serum albumin, and to this mixture was added 0.4 ml antiglucagon antiserum (final dilution 1:40,000). After a 72-h incubation, bound and free glucagon were separated with dextran-coated charcoal, and the free fraction was counted. The sensitivity of this assay is 20 pg/ml. The intra-assay coefficient of variation is 10% and the interassay coefficient is 15%. Insulin was measured by a solid phase modification of the method of Grodsky and Forsham (12). Calculations. Hormone secretion was calculated from areas under the curve during individual perfusion experiments. Percent inhibition was determined according to the formula: . observed \ 100 - I expected x 100 . The expected hormone secretion for each perfusion experiment was interpolated from the mean perfusate hormone of two points immediately prior to and after infusion of somatostatin. Statistical significance was determined using two-sided paired t tests.
GLUCOSE
Results
Effect of somatostatin on glucagon and insulin responses to arginine (Figs. 1 -3). To GIUCAGON c determine whether somatostatin directly Z o inhibited pancreatic alpha and beta cell o < responses to arginine, somatostatin was infused at a rate calculated to produce a d final concentration of 100 ng/ml during stimulation of glucagon and insulin secreN=3 tion by arginine in the presence of 100 mg/ml glucose (Fig. 1). Within 1-2 min of •7: 30 introduction of somatostatin, perfusate glucagon and insulin levels fell and remained suppressed. With discontinuation of somatostatin, both glucagon and insulin levels rose abruptly to levels seen prior to somatostatin infusion. There was no de8 12 16 20 24 28 32 36 tected overshoot. Inhibition of glucagon MINUTES secretion occurred to a greater extent than did inhibition of insulin release (86 ± 2% FIG. 1. Effect of 100 ng/ml somatostatin on glucagon vs 53 ± 4% respectively, P < 0.001). and insulin responses to arginine (mean ± SE). E a
751
PANCREATIC EFFECTS OF SOMATOSTATIN
5.5mM
_
GLUCOSE'
2.0
1.5
GLUCAGON
1.0
FIG. 2. Effects of various concentrations (0.1, 1, 10 ng/ml) of somatostatin on glucagon and insulin responses to arginine (mean ± SE).
0.5
0 25
N=6
12
9 6 3 0
To determine further whether pancreatic alpha and beta cells differed in their sensitivity to the inhibitory effects of somatostatin, insulin and glucagon release in response to arginine stimulation was studied during sequential infusion of somatostatin at 3 different concentrations: 0.1, 1, and 10 ng/ml (Fig. 2 and Table 1). Each concentration of somatostatin caused significant inhibition of glucagon and insulin release, followed by a rapid return of hormone secretion toward control levels after discontinuation of somatostatin infusion. No overshoot was observed. In fact, after termination of the last infusion of somatostatin (10 ng/ml), both glucagon and insulin release were less than before the introduction of somatostatin. Although possibly due to residual effects of somatostatin, this appears merely to represent the characteristic waning hormonal response to prolonged arginine infusion previously described in this system (13). As shown in Table 1, somatostatin caused significantly greater suppression of glucagon responses than of insulin responses at all concentrations studied. Calculation of per cent inhibition by another method (dividing mean observed responses by those expected from
interpolation of mean data prior to and after infusion of somatostatin) also indicates that somatostatin inhibited glucagon secretion to a greater degree than insulin secretion (9.9 vs 5.9, 34.9 vs 14.4, 63.4 vs 37.4, and 76.6 vs 46.8%, respectively, for 0.1, 1, 10 and 100 ng/ml somatostatin). Extrapolation of these data indicate that approximately 4 ng/ml somatostatin would reduce glucagon
secretion 50%, whereas comparable inhibition of insulin release would require approximately 90-100 ng/ml somatostatin. Thus, it appears that in this system the pancreatic alpha cell is approximately 20 times more sensitive to the effects of somatostatin than is the pancreatic beta cell. Effects of somatostatin on glucagon and insulin responses to isoproterenol, theophylline, and arginine (Fig. 3). To determine whether somatostatin inhibited pancreatic alpha and beta cell responses to other stimuli, glucagon and insulin release during sequential 10-min infusions of isoproterenol, theophylline and arginine, alone and during concomitant infusion of somatostatin (100 ng/ml), was studied. Isoproterenol stimulated biphasic gluca-
GERICH, LOVINGER AND GRODSKY
752 lOOng/ml
SOMATOSTATIN
2000r 1200
Endo ' 1975 Vol 96 i No 3
caused biphasic glucagon and insulin release. Somatostatin inhibited both phases of glucagon responses (P < 0.01). Total insulin responses were consistently lower than those observed without somatostatin, but only second phase release was significantly inhibited (P < 0.05). Discussion
800 600 400 200
0 30 25 20 15
N=4 -
10 8 6 4 2 0
FIG. 3. Effect of somatostatin (100 ng/ml) on glucagon and insulin responses to isoproterenol (2 ng/ml), theophylline (10 HIM) and arginine (19.2 HIM) (mean ± SE).
gon release, but at this concentration (2 ng/ml) and in the presence of 50 mg/100 ml glucose it had no effect on insulin secretion. Somatostatin completely inhibited both phases of the glucagon responses (P < 0.01). Theophylline stimulated nonphasic secretion of both glucagon and insulin. Somatostatin completely abolished glucagon responses and significantly diminished insulin responses. Arginine in the presence of 100 mg/100 ml glucose
The present investigations were undertaken to ascertain whether somatostatin inhibited glucagon secretion directly at the pancreatic level and also to quantify the relative effect of somatostatin on glucagon and insulin secretion. Accordingly, we studied the effects of somatostatin on hormone release from the in vitro perfused rat pancreas in response to arginine, isoproterenol and theophylline. Somatostatin at a concentration of 100 ng/ml diminished glucagon and insulin responses to all stimuli. In addition, somatostatin appeared to be a more effective inhibitor of glucagon secretion than of insulin release at all 4 concentrations studied. Although this differential effect may be dependent to some extent on the present experimental design and thus may not reflect absolute quantitative differences in sensitivity to somatostatin under all conditions, nevertheless, we found that it required about 20 times more somatostatin to diminish insulin responses to arginine 50% than for comparable suppression of glucagon release. The mechanism by which somatostatin inhibits pancreatic hormone secretion is unclear. However, somatostatin inhibited glucagon responses to isoproterenol, an activator of adenylate cyclase, and responses to theophylline, an agent that elevates intracellular cAMP by diminishing its destruction. These results suggest that somatostatin may act on cAMP-dependent systems rather than by directly modifying intracellular cyclic AMP levels.* The lack * Somatostatin (100 ng/ml) also inhibits glucagon responses to epinephrine in vitro. (Weir, G., personal communication).
PANCREATIC EFFECTS OF SOMATOSTATIN
753
TABLE 1. Comparison of inhibitory effects of various concentrations of somatostatin on glucagon and insulin secretion Glucagon secretion pg/min Somatostatin ng/ml 0.1
Mean SE
P
ABSTRACT. To determine whether somatostatin inhibits glucagon secretion directly at the pancreatic level and to study quantitatively the relative effects of somatostatin on glucagon and insulin secretion, the effects of various concentrations of somatostatin on glucagon and insulin release from the in vitro perfused rat pancreas in response to arginine (14.2 mM), isoproterenol (2 mg/ml) and theophylline (10 ITIM) were studied. Glucagon and insulin responses to arginine were progressively inhibited by somatostatin over a concentration range from 0.1-100 ng/ml. At all doses, somatostatin caused greater inhibition of glucagon secretion than of insulin secretion. Approximately 4 ng/ml somatostatin reduced glucagon responses 50%, whereas 90 ng/ml was required to produce comparable inhibition of insulin responses. Gluca-
S
OMATOSTATIN, a hypothalamic peptide (1) originally found to inhibit pituitary growth hormone and TSH secretion (2-5), has recently been shown to lower plasma glucose, insulin and glucagon levels in the baboon (6) and to inhibit plasma insulin and glucagon responses to arginine in man (7). Since somatostatin inhibits glucose-induced insulin release from the canine pancreas perfused in vitro (8), it seems likely that a direct effect on the pancreatic beta cell is involved. However, comparable studies concerning the action of somatostatin on glucagon release have not been reported. Therefore, the present investigation was undertaken to characterize further the pancreatic effects of somatostatin by comparing its relative
Received May 29, 1974. Supported in part by Grant AM-01410, NIAMD; the Levi J. and Mary Skaggs Foundation; and the Kroc Foundation.
ability to inhibit glucagon and insulin release from the in vitro perfused rat pancreas in response to arginine, isoproterenol and theophylline. Materials and Methods Animals. Overnight-fasted, male Long-Evans rats, approximately 30 weeks old and 250 g in weight were used in all experiments. Perfusion technique and apparatus. The techniques for dissection of the rat pancreas and its perfusion have been previously described in detail (9,10). In brief, fasted rats were anesthetized with sodium pentobarbital; the pancreas with adjacent stomach, spleen and part of the duodenum was removed and subsequently perfused with a nonrecirculating medium consisting of 1% albumin, 3% dextran, Krebs bicarbonate buffer, pH 7.4. Perfusate was introduced into the celiac artery and total portal vein effluent (10 ml/min) was collected every 60 s. Flow rate and pressure were monitored and remained constant throughout experiments. 749
Endo • 1975 Vol96«No3
GERICH, LOVINGER AND GRODSKY
750
Experimental protocol. After an equilibration period of at least 20 min, stimuli were introduced via side arm syringe. In arginine experiments, perfusate contained 100 mg/100 ml glucose; during isoproterenol and theophylline studies, perfusate contained 50 mg/100 ml glucose. Linear somatostatin (2) (synthesized by Dr. Jean Rivier and kindly supplied by Dr. Roger Guillemin, Salk Institute, San Diego, Ca.) was dissolved in 2M glycine buffer, pH 3.0, and appropriately diluted in perfusate immediately before use. In dose-response experiments, different concentrations of somatostatin were introduced via separate side arm syringe for 10 min during stimulation with arginine. An 8-min interval was allowed between each somatostatin infusion. In studies involving sequential stimulation by isoproterenol, theophylline and arginine, somatostatin infusion was initiated 10 min prior to the initial stimulation and continued throughout experimental periods. Tenmin "rest" periods were allowed between stimuli. Parallel control experiments for these latter studies were performed without infusion of somatostatin. Glucagon and insulin determinations. Perfusate was collected in chilled tubes containing 15%
5.5 mM
EDTA and immediately frozen for subsequent determination of glucagon and insulin. Glucagon was measured by a modification of the Unger and Eisentraut immunoassay (11) using the highly specific Unger 30K antiserum. Duplicate 0.2 ml samples of perfusate were added to 0.5 ml glycine buffer, pH 8.8, containing 0.25% human serum albumin, and to this mixture was added 0.4 ml antiglucagon antiserum (final dilution 1:40,000). After a 72-h incubation, bound and free glucagon were separated with dextran-coated charcoal, and the free fraction was counted. The sensitivity of this assay is 20 pg/ml. The intra-assay coefficient of variation is 10% and the interassay coefficient is 15%. Insulin was measured by a solid phase modification of the method of Grodsky and Forsham (12). Calculations. Hormone secretion was calculated from areas under the curve during individual perfusion experiments. Percent inhibition was determined according to the formula: . observed \ 100 - I expected x 100 . The expected hormone secretion for each perfusion experiment was interpolated from the mean perfusate hormone of two points immediately prior to and after infusion of somatostatin. Statistical significance was determined using two-sided paired t tests.
GLUCOSE
Results
Effect of somatostatin on glucagon and insulin responses to arginine (Figs. 1 -3). To GIUCAGON c determine whether somatostatin directly Z o inhibited pancreatic alpha and beta cell o < responses to arginine, somatostatin was infused at a rate calculated to produce a d final concentration of 100 ng/ml during stimulation of glucagon and insulin secreN=3 tion by arginine in the presence of 100 mg/ml glucose (Fig. 1). Within 1-2 min of •7: 30 introduction of somatostatin, perfusate glucagon and insulin levels fell and remained suppressed. With discontinuation of somatostatin, both glucagon and insulin levels rose abruptly to levels seen prior to somatostatin infusion. There was no de8 12 16 20 24 28 32 36 tected overshoot. Inhibition of glucagon MINUTES secretion occurred to a greater extent than did inhibition of insulin release (86 ± 2% FIG. 1. Effect of 100 ng/ml somatostatin on glucagon vs 53 ± 4% respectively, P < 0.001). and insulin responses to arginine (mean ± SE). E a
751
PANCREATIC EFFECTS OF SOMATOSTATIN
5.5mM
_
GLUCOSE'
2.0
1.5
GLUCAGON
1.0
FIG. 2. Effects of various concentrations (0.1, 1, 10 ng/ml) of somatostatin on glucagon and insulin responses to arginine (mean ± SE).
0.5
0 25
N=6
12
9 6 3 0
To determine further whether pancreatic alpha and beta cells differed in their sensitivity to the inhibitory effects of somatostatin, insulin and glucagon release in response to arginine stimulation was studied during sequential infusion of somatostatin at 3 different concentrations: 0.1, 1, and 10 ng/ml (Fig. 2 and Table 1). Each concentration of somatostatin caused significant inhibition of glucagon and insulin release, followed by a rapid return of hormone secretion toward control levels after discontinuation of somatostatin infusion. No overshoot was observed. In fact, after termination of the last infusion of somatostatin (10 ng/ml), both glucagon and insulin release were less than before the introduction of somatostatin. Although possibly due to residual effects of somatostatin, this appears merely to represent the characteristic waning hormonal response to prolonged arginine infusion previously described in this system (13). As shown in Table 1, somatostatin caused significantly greater suppression of glucagon responses than of insulin responses at all concentrations studied. Calculation of per cent inhibition by another method (dividing mean observed responses by those expected from
interpolation of mean data prior to and after infusion of somatostatin) also indicates that somatostatin inhibited glucagon secretion to a greater degree than insulin secretion (9.9 vs 5.9, 34.9 vs 14.4, 63.4 vs 37.4, and 76.6 vs 46.8%, respectively, for 0.1, 1, 10 and 100 ng/ml somatostatin). Extrapolation of these data indicate that approximately 4 ng/ml somatostatin would reduce glucagon
secretion 50%, whereas comparable inhibition of insulin release would require approximately 90-100 ng/ml somatostatin. Thus, it appears that in this system the pancreatic alpha cell is approximately 20 times more sensitive to the effects of somatostatin than is the pancreatic beta cell. Effects of somatostatin on glucagon and insulin responses to isoproterenol, theophylline, and arginine (Fig. 3). To determine whether somatostatin inhibited pancreatic alpha and beta cell responses to other stimuli, glucagon and insulin release during sequential 10-min infusions of isoproterenol, theophylline and arginine, alone and during concomitant infusion of somatostatin (100 ng/ml), was studied. Isoproterenol stimulated biphasic gluca-
GERICH, LOVINGER AND GRODSKY
752 lOOng/ml
SOMATOSTATIN
2000r 1200
Endo ' 1975 Vol 96 i No 3
caused biphasic glucagon and insulin release. Somatostatin inhibited both phases of glucagon responses (P < 0.01). Total insulin responses were consistently lower than those observed without somatostatin, but only second phase release was significantly inhibited (P < 0.05). Discussion
800 600 400 200
0 30 25 20 15
N=4 -
10 8 6 4 2 0
FIG. 3. Effect of somatostatin (100 ng/ml) on glucagon and insulin responses to isoproterenol (2 ng/ml), theophylline (10 HIM) and arginine (19.2 HIM) (mean ± SE).
gon release, but at this concentration (2 ng/ml) and in the presence of 50 mg/100 ml glucose it had no effect on insulin secretion. Somatostatin completely inhibited both phases of the glucagon responses (P < 0.01). Theophylline stimulated nonphasic secretion of both glucagon and insulin. Somatostatin completely abolished glucagon responses and significantly diminished insulin responses. Arginine in the presence of 100 mg/100 ml glucose
The present investigations were undertaken to ascertain whether somatostatin inhibited glucagon secretion directly at the pancreatic level and also to quantify the relative effect of somatostatin on glucagon and insulin secretion. Accordingly, we studied the effects of somatostatin on hormone release from the in vitro perfused rat pancreas in response to arginine, isoproterenol and theophylline. Somatostatin at a concentration of 100 ng/ml diminished glucagon and insulin responses to all stimuli. In addition, somatostatin appeared to be a more effective inhibitor of glucagon secretion than of insulin release at all 4 concentrations studied. Although this differential effect may be dependent to some extent on the present experimental design and thus may not reflect absolute quantitative differences in sensitivity to somatostatin under all conditions, nevertheless, we found that it required about 20 times more somatostatin to diminish insulin responses to arginine 50% than for comparable suppression of glucagon release. The mechanism by which somatostatin inhibits pancreatic hormone secretion is unclear. However, somatostatin inhibited glucagon responses to isoproterenol, an activator of adenylate cyclase, and responses to theophylline, an agent that elevates intracellular cAMP by diminishing its destruction. These results suggest that somatostatin may act on cAMP-dependent systems rather than by directly modifying intracellular cyclic AMP levels.* The lack * Somatostatin (100 ng/ml) also inhibits glucagon responses to epinephrine in vitro. (Weir, G., personal communication).
PANCREATIC EFFECTS OF SOMATOSTATIN
753
TABLE 1. Comparison of inhibitory effects of various concentrations of somatostatin on glucagon and insulin secretion Glucagon secretion pg/min Somatostatin ng/ml 0.1
Mean SE
P
