Article
Effect of insulin on glucose- and arginine-stimulated somatostatin
secretion from the isolated perfused rat pancreas
GERBER, Pietro, et al.
Abstract
The effects of exogenous insulin on somatostatin secretion from the isolated perfused rat
pancreas have been investigated in the presence of 5.6 mM glucose and when somatostatin
secretion was stimulated by either glucose (16.7 mM) or arginine (20 mM). Insulin (15 mU/ml)
significantly and rapidly suppressed glucose- and arginine-stimulated somatostatin release.
However, at 5.6 mM glucose and, in the absence of other stimulators of somatostatin release,
insulin had no effect on the somatostatin secretion rate.
Reference
GERBER, Pietro, et al. Effect of insulin on glucose- and arginine-stimulated somatostatin
secretion from the isolated perfused rat pancreas. Endocrinology, 1981, vol. 109, no. 1, p.
279-83
DOI : 10.1210/endo-109-1-279
PMID : 6113132
Available at:
http://archive-ouverte.unige.ch/unige:40540
Disclaimer: layout of this document may differ from the published version.
0013-7227/81/1091-0279$02.00/0
Endocrinology
Copyright© 1981 by The Endocrine Society
I
Vol. 109, No. 1
Printed in U.S.A.
Effect of Insulin on Glucose- and Arginine-Stimulated
Somatostatin Secretion from the Isolated Perfused Rat
Pancreas*
PIETRO P. G. GERBER,t ELISABETH R. TRIMBLE, CLAES B. WOLLHEIM,
ALBERT E. RENOLD
AND
Institut de Biochimie Clinique, Universite de Geneve, Sentier de la Roseraie, 1211 Geneve 4, Switzerland
ABSTRACT. The effects of exogenous insulin on somatostatin
secretion from the isolated perfused rat pancreas have been
investigated in the presence of 5.6 mM glucose and when somatostatin secretion was stimulated by either glucose (16.7 mM)
or arginine (20 mM). Insulin (15 mU/ml) significantly and rapidly
suppressed glucose- and arginine-stimulated somatostatin
release. However, at 5.6 mM glucose and, in the absence of other
stimulators of somatostatin release, insulin had no effect on the
somatostatin secretion rate. (Endocrinology 109: 279, 1981)
;
Materials and Methods
T
HE PANCREATIC hormones, insulin, glucagon,
and somatostatin, appear to play a role in controlling nutrient homeostasis (1). In addition to the effects
of pancreatic hormones on target tissues, an intraislet
action of the hormones is possible. Thus, glucagon is
known to stimulate the release of insulin (2-4) and pancreatic somatostatin (5, 6), somatostatin to inhibit both
insulin (7-9) and glucagon (10-13) secretion, and insulin
to suppress glucagon secretion (14, 15). In these studies,
higher concentrations of the hormones than those found
in peripheral blood were employed. This may be necessary for the assessment of paracrine effects, since the
concentrations of the hormones to which the pancreatic
endocrine cells are exposed are not precisely known, but
they are certainly higher than the circulating levels. The
increased plasma concentrations of glucagon and somatostatin in dogs with experimental insulinopenic diabetes may suggest an inhibitory action of insulin on the
pancreatic a-cells (16) similar to that exerted on the acells (16, 17). However, all efforts to demonstrate a direct
action of insulin on somatostatin secretion from mammalian pancreas preparations have so far given negative
results (6, 18-20). In the present study, we investigated
the effect of insulin on somatostatin release from the
isolated perfused rat pancreas under basal conditions and
during stimulation with glucose or arginine.
The isolated pancreas was prepared from overnight fasted
male Wistar rats, weighing 200-250 g, by the method of Penhos
et al. (21) with minor modifications. The perfusate was modified
Krebs-Ringer bicarbonate buffer containing 1 mM Ca ++, 40 mg/
ml dextran, 2 mg/ml human serum albumin, and 400 kiU/ml
aprotinin. The buffer was continuously gassed with a mixture
of 95% 02 and 5% C02; the partial pressure of oxygen in the
buffer entering the pancreas was 450 mm Hg, and the perfusion
rate was 4 ml/min. Previous studies (22) have shown that
steady basal concentrations of somatostatin are achieved in the
effluent after the first 20 min of perfusion. Baseline samples
were accordingly obtained 28, 29, and 30 min after starting the
perfusion (designated -2, -1, and 0 min). The experimental
variables were introduced at 0 min (i.e. 30 min after the perfusion had begun) according to protocols that are explained in
the text. The somatostatin RIA used has been described in
detail previously (22); its sensitivity was 15-20 pg/ml. Statistical
analysis was made by Student's t test.
The following substances were used: Dextran T-40 (Pharmacia Fine Chemicals AB, Uppsala, Sweden); human serum
albumin (Swiss Red Cross, Bern, Switzerland); aprotinin (Trasylol) kindly provided by Prof. G. L. Haberland (Bayer A. G.,
Wuppertal, West Germany); cyclic somatostatin and N-tyrosylated somatostatin for standard and iodinated tracer, respectively (Serono, Freiburg, West Germany); somatostatin antiserum, a gift from Dr. J. Ardill (Queen's University, Belfast,
United Kingdom); and pork insulin (Actrapid; Novo Industri
A/S, Copenhagen, Denmark).
Results
Received May 6, 1980.
• This work was supported by Research Grants 3,744-0.76 SR and
3.487-0.79 SR from the Swiss National Science Foundation.
t To whom requests for reprints should be addressed.
Throughout the experiments shown in Fig. 1, the glucose concentration was kept constant at 5.6 IllM. After ~0
279
GERBER ET AL.
280
n = 6,
Endo • 1981
Vol109 • No I
x:t SEM
n = 7,
GLUCOSE
5.6mM
GLUCOSE
x± SEM
Insulin
ARGININE 20 mM
Insulin
SRIF
SRIF
(pg/min)
(pg/min)
800
600
600
5.6mM
500
400
400
200
300
0
-10
0
10
20
30
40
50
minutes
200
FIG. 1. Effect of exogenous insulin (15 mU /ml) on somatostatin (SRIF)
secretion from the isolated perfused rat pancreas in the presence of 5.6
mM glucose. The addition of insulin (0-30 min) was without effect on
the SRIF secretion rate.
100+-----~---.----~----~--~
min of equilibration and the recording of three baseline
somatostatin secretion rates (-2, -1, and 0 min), insulin
was infused for 30 min (0-30 min) at a concentration of
15 mU/ml (0.63 ILg/ml). Sampling continued for 20 min
after insulin was withdrawn (30-50 min). The somatostatin secretion rate did not vary significantly from the
initial basal rate, either during the period of insulin
infusion or subsequently. At this nonstimulatory glucose
concentration, therefore, somatostatin secretion was not
affected by insulin.
The next experiments were designed to evaluate further whether insulin affects somatostatin secretion by
adding insulin after first inducing stimulation of somatostatin secretion by glucose or arginine. The basal perfusate contained 5.6 mM glucose (-30 to 0 min). Somatostatin secretion was then stimulated, either by increasing
glucose to 16.7 mM or by adding 20 mM arginine (in the
presence of 5.6 mM glucose) during the period from 0-40
min. After 20 min of stimulation with either glucose or
arginine, insulin was infused into a side-arm to give a
final concentration of 15 mU/ml insulin in the perfusate
during the second half of the stimulatory period (20-40
min) without modifying the concentration of any other
constituent. As shown in Fig. 2, 16.7 mM glucose caused
-10
0
10
20
30
40
minutes
FIG. 2. Effect of exogenous insulin (15 mU/ml) on glucose-stimulated
somatostatin (SRIF) secretion from the isolated perfused pancreas.
The glucose concentration was 5.6 mM from -30 to 0 min and 16.7 mM
from 0 min to the end of the experiment. Insulin was added from 20-40
min. SRIF secretion was significantly stimulated by 16.7 mM glucose
(P < 0.02 at all time points, compared with the basal secretion rate).
The effect of insulin on stimulated SRIF release was significant at 22,
26, 28, and 40 min (P < 0.05) and at 23, 24, 27, and 30 min (P < 0.025).
a significant increase in the somatostatin secretion rate
at all time points at which it was measured. The subsequent addition of insulin resulted in an inhibition of this
glucose-induced somatostatin release. At 8 of 12 time
points during the 20 min of insulin infusion, the somatostatin secretion rate was significantly less than the mean
somatostatin secretion rate for the previous 20 min when
16.7 mM glucose was infused in the absence of insulin.
Furthermore, the somatostatin secretion rate during insulin infusion with 16.7 mM glucose never significantly
exceeded that during the initial baseline period when 5.6
mM glucose was infused alone.
Arginine (20 mM) added to 5.6 mM glucose resulted in
an increase in somatostatin secretion (Fig. 3). The further
EFFECT OF INSULIN ON SOMATOSTATIN
addition of insulin caused a rapid inhibition of somatostatin secretion, so that the secretion rate during insulin
infusion was significantly less than the mean secretion
rate during the previous 20 min of arginine infusion at all
time points measured. In control experiments where 16.7
mM glucose or 20 mM arginine plus 5.6 mM glucose was
infused for the period from 0-40 min (without the addition of insulin at 20-40 min), the somatostatin secretion
rate during the second 20-min period of stimulation (2040 min) was similar to that during the first 20-min period
(0-20 min; Table 1). In addition, the results of all the
experiments have been expressed as integrated incremental somatostatin secretion for the period from 0-40 min
(Table 2). In control experiments, when glucose was
infused for 40 min without insulin, the integrated incremental somatostatin secretion from 0-20 min was similar
to that for the subsequent 20-40 min (Table 2A). By
contrast, in the experiments where insulin was added
during the period from 20-40 min, the integrated incremental somatostatin secretion was markedly reduced
compared to that during the 0-20 min period of the same
experiment (P < 0.05, paired statistics) and compared to
n =6,
G 5.6 mM
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x :t SEM
GLUCOSE
281
0
16.7 mM
Insulin
.,;:;"'
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.,
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SRIF
(pg/min)
400
..,.
18
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minutes
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<0
FIG. 3. Effect of exogenous insulin (15 mU/ml) on arginine-stimulated
somatostatin (SRIF) secretion from the isolated perfused pancreas.
Arginine stimulated SRIF secretion (P < 0.005 at 1 min and P < 0.05
at 3, 5, 6, 7, 10 and 20 min, compared with the basal secretion rate).
The effect of insulin on stimulated SRIF release was significant at all
time points (P < 0.02) . G, Glucose.
2
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a:>
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GERBER ET AL.
282
TABLE 2. Integrated incremental somatostatin secretion during stimulation with 16.7 mM glucose or 20 mM arginine in the presence or
absence of insulin during the second part of the stimulatory period
(picograms per 20 min; mean ± SEM)
A. Glucose (16.7 mM) without
insulin
p
B. Glucose (16.7 mM) with insulin (20-40 min)
C. Arginine (20 mM) without
insulin
p
D. Arginine (20 mM) with insulin (20-40 mM)
A + C, n
=
4; B, n
= 6; D, n =
0-20 min
p
20-40 min
2293 ± 353
NS
3318 ± 454
NS
2283 ± 479
<0.05
<0.001
185 ± 661
3708 ± 776
NS
2854 ± 497
<0.01
<0.005
- 746 ± 413
NS
2064 ± 678
7.
that during the 20- to 40-min period of the control
experiments carried out in the absence of insulin (P <
0.001, unpaired statistics). Similarly, when insulin was
introduced from 20-40 min in the arginine-stimulated
pancreas, there was a marked reduction in the integrated
incremental somatostatin secretion for this period compared with that during 0-20 min in the same experiment
(P < 0.01) or that during 20-40 min in control experiments (P < 0.005; see Table 2, C and D).
Discussion
The results presented here demonstrate that somatostatin secretion stimulated by either glucose or arginine
can be inhibited by an increase in the insulin concentration surrounding the pancreatic A-cells. Basal somatostatin release (i.e. 5.6 mM glucose without added stimuli)
was unaffected by insulin. This finding under basal conditions confirms previous work in the rat (18, 19) and dog
(6).
With respect to the inhibition by insulin of glucosestimulated somatostatin release, our results appear at
variance with those of Hermansen et al. (20), who failed
to observe any effect of 25 mU/ml insulin on somatostatin release. The reason for this difference is unclear; apart
from the species difference, the composition of the perfusate was also different (20). In the isolated perfused
chicken pancreas, Honey and Weir (23) observed a stimulation of somatostatin secretion by 20 mU/ml insulin.
While this finding contrasts markedly with the results
reported here, the interaction between hormones in the
pancreas of birds may be different from that occurring in
mammals. Indeed, in the duck, the infusion of somatostatin in vivo resulted in a stimulation of glucagon secretion rather than the expected inhibition (24).
The use of high concentrations of pancreatic hormones
is necessary when investigating their possible paracrine
effects. When insulin secretion was stimulated by 16.7
mM glucose, the insulin concentration measured in the
Endo • 1981
Vol109 • No I
mixed effluent of the isolated perfused pancreas preparation was 1 mU/ml (22). This concentration can be
increased by a factor of 10 under maximal stimulatory
conditions. It appears reasonable, therefore, to use a
concentration of insulin large enough to permit the Acells to recognize an increase.
Suppression of pancreatic somatostatin secretion by
insulin under physiological conditions may explain the
findings of increased somatostatin secretion in experimental insulinopenic diabetes in dogs (16) and rats (25).
In addition, insulin treatment of alloxan-diabetic dogs
partially normalized the increased somatostatin and glucagon levels measured in peripheral blood (16). In acute
in vivo experiments, insulin infused iv inhibited mealstimulated pancreatic somatostatin secretion; it also inhibited stimulated somatostatin release from the gastric
antrum but not from the fundus (26). However, it is not
possible to determine from in vivo studies whether insulin has a direct effect on the pancreatic A-cell. In in
vitro experiments using the perfused rat stomach, 5.6 mM "'
glucose, after a delay, caused a stimulation of somatostatin secretion, which could be prevented by simultaneous
insulin infusion (27).
In conclusion, the results presented here suggest that
insulin can exert a direct inhibitory influence on somatostatin secretion under certain conditions. It would seem
that each of the three principal hormones of the pancreatic islet, insulin, glucagon, and somatostatin, may
affect the secretion of the other two within the pancreatic
islet. The concentration of one of the hormones in the
pancreatic effluent may thus depend on the secretory
activity of the other hormone-secreting cells in the islets.
With regard to pancreatic polypeptide, knowledge is as
yet insufficient to show whether it may be similarly
involved in reciprocal paracrine regulation of pancreatic
hormone secretion.
Acknowledgment
The authors acknowledge with gratitude the expert technical assistance of Jarmila Slesinger.
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