- Email: [email protected]
Effects of pancreastatin on insulin, glucagon and somatostatin secretion by the perfused rat pancreas
Life Sciences, Vol. 42, pp. 1361-1367 Printed in the U.S.A.
EFFECTS R.A.
Pergamon Press
OF P A N C R E A S T A T I N ON INSULIN, G L U C A G O N AND S O M A T O S T A T I N S E C R E T I O N BY THE P E R F U S E D RAT P A N C R E A S Silvestre,
E. Peir6, P. M i r a l l e s , and J. M a r c o *
M.L.
Villanueva
H o s p i t a l P u e r t a de Hierro, U n i v e r s i d a d A u t 6 n o m a de Madrid, San M a r t i n de P o r r e s 4, 28035 Madrid, Spain (Received in final form February 4, 1988) Summar Z P a n c r e a s t a t i n is a novel peptide, i s o l a t e d from p o r c i n e p a n c r e a t i c extracts, w h i c h has b e e n shown to inhibit g l u c o s e - i n d u c e d i n s u l i n r e l e a s e "in vitro" To a c h i e v e further i n s i g h t into the i n f l u e n c e of p a n c r e a s t a t i n on p a n c r e a t i c h o r m o n e secretion, we have s t u d i e d the effects of this p e p t i d e on u n s t i m u l a t e d insulin, g l u c a g o n and s o m a t o s t a t i n output, as well as on the r e s p o n s e s of these h o r m o n e s to g l u c o s e and to t o l b u t a m i d e in the p e r f u s e d rat p a n c r e a s . P a n c r e a s t a t i n s t r o n g l y inhibited u n s t i m u l a t e d i n s u l i n r e l e a s e as well as the i n s u l i n r e s p o n s e s to g l u c o s e and to t o l b u t a m i d e . It did not s i g n i f i c a n t l y a f f e c t g l u c a g o n or s o m a t o s t a t i n o u t p u t u n d e r any of the a b o v e - m e n t i o n e d c o n d i t i o n s . These findings s u g g e s t that p a n c r e a s t a t i n inhibits B - c e l l s e c r e t o r y a c t i v i t y directly, and not t h r o u g h an A - c e l l or D - c e l l p a r a c r i n e effect. P a n c r e a s t a t i n is a 4 9 - a m i n o acid s t r a i g h t chain m o l e c u l e w h i c h was r e c e n t l y i s o l a t e d by T a t e m o t o et al. (i) from p o r c i n e p a n c r e a t i c extracts. In their o r i g i n a l report, these i n v e s t i g a tors also showed that p a n c r e a s t a t i n i n h i b i t s g l u c o s e - i n d u c e d insulin r e l e a s e "in vitro". To further i n v e s t i g a t e the i n f l u e n c e of p a n c r e a s t a t i n on p a n c r e a t i c h o r m o n e secretion, we have s t u d i e d the e f f e c t of this p e p t i d e on u n s t i m u l a t e d insulin, g l u c a g o n and s o m a t o s t a t i n output, as well as on the r e s p o n s e s of these h o r m o n e s to g l u c o s e and to t o l b u t a m i d e in the p e r f u s e d rat pancreas.
Materials
and M e t h o d s
F e d m a l e W i s t a r rats (200-250 g b o d y weight) from our inbred c o l o n y w e r e u s e d as donors. A f t e r a n e s t h e s i a of the rat with p e n t o b a r b i t a l s o d i u m (50 mg/kg, i n t r a p e r i t o n e a l l y ) , the p a n c r e a s was d i s s e c t e d and p e r f u s e d "in situ" a c c o r d i n g to the p r o c e d u r e of L e c l e r c q - M e y e r et al. (2); all a d j a c e n t organs, i n c l u d i n g duodenum, w e r e excluded. The p a n c r e a s was p e r f u s e d t h r o u g h the coeliac and s u p e r i o r m e s e n t e r i c a r t e r i e s via a c a n n u l a i n s e r t e d into
*To w h o m c o r r e s p o n d e n c e
should be addressed.
0024~3205/88 $3.00 + tOO Copyright (c) 1988 Pergamon Press plc
1362
Pancreastatin and Pancreatic Hormone Output
Vol. 42, No. 14, 1988
the aorta. E f f l u e n t samples were c o l l e c t e d from the portal vein, w i t h o u t recycling, at two m i n u t e intervals (flow rate, 2 ml/min) in tubes c o n t a i n i n g 2000 KIU T r a s y l o l (Bayer AG, Leverkusen, FRG), and frozen at -20°C until the time of assay. The p e r f u s i o n m e d i u m c o n s i s t e d of a K r e b s - R i n g e r b i c a r b o n a t e buffer (gas phase 95:5, 02:CO2; pH 7.4) s u p p l e m e n t e d w i t h 4% (wt/vol) d e x t r a n T-70, 0.5% (wt/vol) bovine a l b u m i n (Cohn fraction V) and glucose (5.5 mM). A f t e r an e q u i l i b r a t i o n period of 35 minutes, b a s e l i n e samples were c o l l e c t e d for 12 minutes. At zero time, s y n t h e t i c p o r c i n e p a n c r e a s t a t i n (Peninsula L a b o r a t o ries, Inc., Belmont, California) was infused through a sidearm cannula as a 2000 ng p r i m i n g dose, followed by c o n s t a n t infusion at a rate of 80 ng/ml (15.7 nM) for 22 or 24 minutes. As secretagogues of the e n d o c r i n e pancreas, ii m M glucose (Sigma C h e m i c a l Co. St. Louis, MO) and 100 m g / l t o l b u t a m i d e (Boehringer M a n n h e i m GmbH, Mannheim, FRG) w e r e employed. A d d i t i o n of these substances to the p e r f u s a t e was p e r f o r m e d as d e s c r i b e d in the c o r r e s p o n d i n g figures. In control experiments, an identical volume of perfusion m e d i u m was infused. R a d i o i m m u n o a s s a y was e m p l o y e d to m e a s u r e insulin (3), glucagon (4) and s o m a t o s t a t i n (5). A n t i g l u c a g o n serum (30K) and a n t i s o m a t o s t a t i n serum (80C) were kindly d o n a t e d by Dr. Roger H. U n g e r (University of Texas H e a l t h Sciences C e n t e r at Dallas, Texas). All samples for each given series of e x p e r i m e n t s were a n a l y z e d in the same run. Results are p r e s e n t e d as the m e a n ± SEM. Hormone response from zero time until the end of s e c r e t a g o g u e infusion was calculated as the i n t e g r a t e d area of the curve above or b e l o w the m e a n p r e i n f u s i o n level (average of all b a s e l i n e levels), using the t r a p e z o i d a l method. The s t a t i s t i c a l study was p e r f o r m e d by analysis of v a r i a n c e and by the S t u d e n t ' s t-test for u n p a i r e d observations.
Results Effects of p a n c r e a s t a t i n g l u c a g o n and s o m a t o s t a t i n (Figure i)
(15.7 ~LM) on u n s t i m u l a t e d insulin, release by the p e r f u s e d rat p a n c r e a s
As expected, in control s o m a t o s t a t i n release did not e x p e r i m e n t a l period.
perfusions, insulin, g l u c a g o n vary s i g n i f i c a n t l y throughout
and the
A d d i t i o n of p a n c r e a s t a t i n to the p e r f u s a t e m a r k e d l y inhibited u n s t i m u l a t e d insulin o u t p u t (F=2.43, p<0.05) . Furthermore, d u r i n g p a n c r e a s t a t i n infusion, insulin secretion, as c a l c u l a t e d by the i n t e g r a t e d area under the r e s p o n s e curve, was reduced as c o m p a r e d to control p e r f u s i o n s (37± 5 ng/24 min vs. 62 ± 7 ng/24 min, p<0.05). P a n c r e a s t a t i n did not m a t o s t a t i n release.
significantly
modify
glucagon
and so-
Vol. 42, No. 14, 1988
Pancreastatin and P~creatic Homone Output
PANCREASTATIN
1363
15.7nM
~CONTROL,N:7 PIT, N,6
. . . .
z
i
1-
C
~
200
~
.
Z
c
s
so
-12
0
24
32
MINUTES
FIG.
1
E f f e c t of p a n c r e a s t a t i n on u n s t i m u l a t e d insulin, g l u c a g o n and s o m a t o s t a t i n release by the p e r f u s e d rat pancreas. Solid and b r o k e n lines c o r r e s p o n d to control and p a n c r e a s t a t i n experiments, r e s p e c t i v e l y (means ±SEM).
E f f e c t of p a n c r e a s t a t i n (15.7 nM) s o m a t o s t a t i n r e s p o n s e s to g l u c o s e p a n c r e a s (Figure 2)
on the insulin, @ l u c a @ o n and (ii ~I) by the p e r f u s e d rat
The release of insulin e l i c i t e d by glucose (incremental response: 261 z 69 ng/22 min) was c o n s i s t e n t l y b l o c k e d by pancrea s t a t i n (incremental response: 85 ± 13 ng/22 min, p<0.05). Panc r e a s t a t i n did not s i g n i f i c a n t l y affect the s u p p r e s s o r effect of g l u c o s e on g l u c a g o n s e c r e t i o n (decremental response: 4540 ± 5 6 8 pg/22 m i n vs. 3848±1295 pg/22 min in control experiments, p=0.6).
1364
Pancreastatin and Pancreatic Hormone Output
Vol. 42, No. 14, 1988
Glucose, at the c o n c e n t r a t i o n e m p l o y e d in these p e r f u s i o n s , did not s t i m u l a t e s o m a t o s t a t i n release; w h e n p a n c r e a s t a t i n was s i m u l t a n e o u s l y infused, no c h a n g e in the s e c r e t i o n of s o m a t o s t a t in was observed.
GLUCOSE 11raM -
CONTROL , N , 4
-
---
Z
_
20
PST, N,4
"
4o-._r,..,~_T.~"
,,
800Z-0
600-
°3~4oo-
) "~' I '
o E
~.,,
,.../
200. -t-
°
~ '~ I,-- Ol N
.
'
loo-
-12
0
22
32
MINUTES
FIG.
2
E f f e c t of p a n c r e a s t a t i n on the insulin, glucagon and s o m a t o s t a t l n r e s p o n s e s to g l u c o s e by the p e r f u s e d rat p a n c z e a s (means +_SEM) . Solid lines r e p r e s e n t g l u c o s e e x p e r i m e n t s . Broken lines r e p r e s e n t g l u c o s e plus p a n c r e a s t a t i n experiments.
E f f e c t of p a n c r e a s t a t i n (15.7 nM) on s o m a t o s t a t i n r e s p o n s e s to t o l b u t a m i d e rat p a n c r e a s (Figure 3). Pancreastatin
also
reduced
the insulin, g l u c a g o n and (I00 m y / l ) _ b Y ~he p e r f u s e d
tolbutamide-induced
i~suiin
re-
Vol. 42, No. 14, 1988
Pancreastatin and Pancreatic Hormone Output
1365
lease (incremental response: 27 + ii ng/22 min vs. 110-+ 23 ng/22 min in control experiments, p<0.025). The d e c r e a s e of g l u c a g o n output o b s e r v e d d u r i n g t o l b u t a m i d e infusion (decremental response: 3073 _+ 615 pg/22 min) was not sign i f i c a n t l y m o d i f i e d by p a n c r e a s t a t i n (decremental response: 3254 + 707 pg/22 min, p=0.9). Nor was t o l b u t a m i d e - i n d u c e d somatostatin release (incremental response: 1910 _+ 476 pg/22 min) s i g n i f i c a n t l y a f f e c t e d by p a n c r e a s t a t i n (incremental response: 1657 _+ 154 pg/22 rain, p=0.6). TOLBUTAMIDE
lOOmgll CONTROL, N=5 ---
15
PST, N=8
z E
z~
s
=~~°°l _~,
,
,-
T
1
T
~ J.,!%' T ,l
300"
~
250
o-
)I 150
12
0
20
10
30
MINUTES
FIG.
3
E f f e c t of p a n c r e a s t a t i n on the insulin, glucagon and s o m a t o s t a t i n r e s p o n s e s to t o l b u t a m i d e by the p e r f u s e d rat p a n c r e a s (means + SEM) . Solid lines r e p r e s e n t t 4 1 b u t a m i d e experiments. B r o k e n lines r e p r e s e n t t o l b u t a m i d e plus pancreastat±neexperiments.
1366
Pancreastatin and Pancreatic Hormone Output
Vol. 42, No. 14, 1988
Discussion The f o r e g o i n g results d e m o n s t r a t e that in the p e r f u s e d rat pancreas, p a n c r e a s t a t i n r e v e r s i b l y reduces u n s t i m u l a t e d insulin r e l e a s e and s t r o n g l y blocks the insulin response to tolbutamide. In addition, they c o n f i r m the inhibitory effect of p a n c r e a s t a t i n on g l u c o s e - i n d u c e d insulin output (i). R e c e n t data from our l a b o r a t o r y indicate that p a n c r e a s t a t i n also blocks insulin s e c r e t i o n as e l i c i t e d by gastric inhibitory peptide and by v a s o a c t i v e intestinal peptide (unpublished). Thus, it could be p o s t u l a t e d that p a n c r e a s t a t i n behaves as a general i n h i b i t o r of insulin release, perhaps by i n t e r f e r i n g w i t h a common and e s s e n t i a l step in this process. T a t e m o t o et al. (i) have mentioned, w i t h o u t p r e s e n t i n g data, that p a n c r e a s t a t i n inhibits g l u c o s e - i n d u c e d s o m a t o s t a t i n secretion by the p e r f u s e d rat pancreas. In our study, however, it failed to s i g n i f i c a n t l y affect u n s t i m u l a t e d or t o l b u t a m i d e induced s o m a t o s t a t i n release. We have also found a lack of effect of p a n c r e a s t a t i n on g l u c a g o n output under any of the conditions examined. These findings suggest that p a n c r e a s t a t i n inhibits B-cell s e c r e t o r y a c t i v i t y directly, and not through an A-cell or D-cell p a r a c r i n e effect. In the rat pancreas, p a n c r e a s t a t i n affects hormone output s i m i l a r l y to galanin, a n o t h e r r e c e n t l y i d e n t i f i e d peptide (6), p r e s e n t in nerve endings s u r r o u n d i n g the p a n c r e a t i c islets (7). G a l a n i n inhibits insulin release w i t h o u t a f f e c t i n g g l u c a g o n or somatostatin secretion (8,9). C o m p a r i s o n of the p r e s e n t data w i t h p r e v i o u s results from our l a b o r a t o r y (8) seems to indicate that the s u p p r e s s o r effect of p a n c r e a s t a t i n on insulin secretion is w e a k e r than that of galanin. The p h y s i o l o g i c a l r e l e v a n c e of these novel p e p t i d e s in the control of p a n c r e a t i c islet function remains to be elucidated.
Acknowledgements The e x p e r t technical work of Ms. A m a l i a MartEn, Ms. Paloma N i e t o and Ms. Pilar G a r c l a - M u ~ o z is g r a t e f u l l y acknowledged. We thank Ms. M a r t h a M e s s m a n for her secretarial help. This study was s u p p o r t e d by grants from the F o n d o de I n v e s t i g a c i o n e s Sanitarias de la S e g u r i d a d Social, M i n i s t e r i o de S a n i d a d y Consumo, (86/1972 and 87/858) and from the C o m i s i 6 n A s e s o r a de Investigaci6n C i e n t i f i c a y T~cnica, M i n i s t e r i o de E d u c a c i 6 n y C i e n c i a (PB86-0003), Spain. References I. K. TATEMOTO, S. EFENDIC, V. MUTT, G. MAKK, G.J. F E I S T N E R and J.D. BARCHAS, Nature 324, 476-478 (1986). 2. V. L E C L E R C Q - M E Y E R , J. MARCHAND, R. L E C L E R C Q and W.J. MALAISSE, D i a b e t e Metab. ~, 57-65 (1976). 3. V. HERBERT, K.-S. LAO, C.W. G O T T L I E B and S.J. BLEICHER, J. Clin. Endocrinol. Metab. 25, 1375-1384 (1965). 4. G.R. F A L O O N A and R.H. UNGER, M e t h o d s of H o r m o n e R a d i o i m m u n o a s say, pp. 317-330, A c a d e m i c Press, N e w York (1974).
Vol. 42, No. 14, 1988
Pancreastatin and Pancreatic Hormone Output
1367
5. V. HARRIS, J.M. CONLON, C.B. SRIKANT, K. McCORKLE, V. SCHUSDZIARRA, E. IPP and R.H. UNGER, Clin. Chim. A c t a 8_/7, 275-283 (1978) . 6. K. TATEMOTO, A. ROKAEUS, H. J~RNVALL, T.J. M c D O N A L D and V. MUTT, FEBS Lett. 3.6.4,.124-128 (1983). 7. B.E. DUNNING, B. AHREN, R.C. VEITH, G. BOTTCHER, F. S U N D L E R and G.J. TABORSKY, Jr., Am. J. Physiol. 251, E I 2 7 - E I 3 3 (1986). 8. R.A. SILVESTRE, P. MIRALLES, L. MONGE, P. MORENO, M.L. VILLAN U E V A and J. MARCO, E n d o c r i n o l o g y 121, 378-383 (1987). 9. P. MIRALLES, E. PEIR0, R.A. SILVESTRE, M.L. V I L L A N U E V A and J. MARCO, M e t a b o l i s m (in press).
EFFECTS R.A.
Pergamon Press
OF P A N C R E A S T A T I N ON INSULIN, G L U C A G O N AND S O M A T O S T A T I N S E C R E T I O N BY THE P E R F U S E D RAT P A N C R E A S Silvestre,
E. Peir6, P. M i r a l l e s , and J. M a r c o *
M.L.
Villanueva
H o s p i t a l P u e r t a de Hierro, U n i v e r s i d a d A u t 6 n o m a de Madrid, San M a r t i n de P o r r e s 4, 28035 Madrid, Spain (Received in final form February 4, 1988) Summar Z P a n c r e a s t a t i n is a novel peptide, i s o l a t e d from p o r c i n e p a n c r e a t i c extracts, w h i c h has b e e n shown to inhibit g l u c o s e - i n d u c e d i n s u l i n r e l e a s e "in vitro" To a c h i e v e further i n s i g h t into the i n f l u e n c e of p a n c r e a s t a t i n on p a n c r e a t i c h o r m o n e secretion, we have s t u d i e d the effects of this p e p t i d e on u n s t i m u l a t e d insulin, g l u c a g o n and s o m a t o s t a t i n output, as well as on the r e s p o n s e s of these h o r m o n e s to g l u c o s e and to t o l b u t a m i d e in the p e r f u s e d rat p a n c r e a s . P a n c r e a s t a t i n s t r o n g l y inhibited u n s t i m u l a t e d i n s u l i n r e l e a s e as well as the i n s u l i n r e s p o n s e s to g l u c o s e and to t o l b u t a m i d e . It did not s i g n i f i c a n t l y a f f e c t g l u c a g o n or s o m a t o s t a t i n o u t p u t u n d e r any of the a b o v e - m e n t i o n e d c o n d i t i o n s . These findings s u g g e s t that p a n c r e a s t a t i n inhibits B - c e l l s e c r e t o r y a c t i v i t y directly, and not t h r o u g h an A - c e l l or D - c e l l p a r a c r i n e effect. P a n c r e a s t a t i n is a 4 9 - a m i n o acid s t r a i g h t chain m o l e c u l e w h i c h was r e c e n t l y i s o l a t e d by T a t e m o t o et al. (i) from p o r c i n e p a n c r e a t i c extracts. In their o r i g i n a l report, these i n v e s t i g a tors also showed that p a n c r e a s t a t i n i n h i b i t s g l u c o s e - i n d u c e d insulin r e l e a s e "in vitro". To further i n v e s t i g a t e the i n f l u e n c e of p a n c r e a s t a t i n on p a n c r e a t i c h o r m o n e secretion, we have s t u d i e d the e f f e c t of this p e p t i d e on u n s t i m u l a t e d insulin, g l u c a g o n and s o m a t o s t a t i n output, as well as on the r e s p o n s e s of these h o r m o n e s to g l u c o s e and to t o l b u t a m i d e in the p e r f u s e d rat pancreas.
Materials
and M e t h o d s
F e d m a l e W i s t a r rats (200-250 g b o d y weight) from our inbred c o l o n y w e r e u s e d as donors. A f t e r a n e s t h e s i a of the rat with p e n t o b a r b i t a l s o d i u m (50 mg/kg, i n t r a p e r i t o n e a l l y ) , the p a n c r e a s was d i s s e c t e d and p e r f u s e d "in situ" a c c o r d i n g to the p r o c e d u r e of L e c l e r c q - M e y e r et al. (2); all a d j a c e n t organs, i n c l u d i n g duodenum, w e r e excluded. The p a n c r e a s was p e r f u s e d t h r o u g h the coeliac and s u p e r i o r m e s e n t e r i c a r t e r i e s via a c a n n u l a i n s e r t e d into
*To w h o m c o r r e s p o n d e n c e
should be addressed.
0024~3205/88 $3.00 + tOO Copyright (c) 1988 Pergamon Press plc
1362
Pancreastatin and Pancreatic Hormone Output
Vol. 42, No. 14, 1988
the aorta. E f f l u e n t samples were c o l l e c t e d from the portal vein, w i t h o u t recycling, at two m i n u t e intervals (flow rate, 2 ml/min) in tubes c o n t a i n i n g 2000 KIU T r a s y l o l (Bayer AG, Leverkusen, FRG), and frozen at -20°C until the time of assay. The p e r f u s i o n m e d i u m c o n s i s t e d of a K r e b s - R i n g e r b i c a r b o n a t e buffer (gas phase 95:5, 02:CO2; pH 7.4) s u p p l e m e n t e d w i t h 4% (wt/vol) d e x t r a n T-70, 0.5% (wt/vol) bovine a l b u m i n (Cohn fraction V) and glucose (5.5 mM). A f t e r an e q u i l i b r a t i o n period of 35 minutes, b a s e l i n e samples were c o l l e c t e d for 12 minutes. At zero time, s y n t h e t i c p o r c i n e p a n c r e a s t a t i n (Peninsula L a b o r a t o ries, Inc., Belmont, California) was infused through a sidearm cannula as a 2000 ng p r i m i n g dose, followed by c o n s t a n t infusion at a rate of 80 ng/ml (15.7 nM) for 22 or 24 minutes. As secretagogues of the e n d o c r i n e pancreas, ii m M glucose (Sigma C h e m i c a l Co. St. Louis, MO) and 100 m g / l t o l b u t a m i d e (Boehringer M a n n h e i m GmbH, Mannheim, FRG) w e r e employed. A d d i t i o n of these substances to the p e r f u s a t e was p e r f o r m e d as d e s c r i b e d in the c o r r e s p o n d i n g figures. In control experiments, an identical volume of perfusion m e d i u m was infused. R a d i o i m m u n o a s s a y was e m p l o y e d to m e a s u r e insulin (3), glucagon (4) and s o m a t o s t a t i n (5). A n t i g l u c a g o n serum (30K) and a n t i s o m a t o s t a t i n serum (80C) were kindly d o n a t e d by Dr. Roger H. U n g e r (University of Texas H e a l t h Sciences C e n t e r at Dallas, Texas). All samples for each given series of e x p e r i m e n t s were a n a l y z e d in the same run. Results are p r e s e n t e d as the m e a n ± SEM. Hormone response from zero time until the end of s e c r e t a g o g u e infusion was calculated as the i n t e g r a t e d area of the curve above or b e l o w the m e a n p r e i n f u s i o n level (average of all b a s e l i n e levels), using the t r a p e z o i d a l method. The s t a t i s t i c a l study was p e r f o r m e d by analysis of v a r i a n c e and by the S t u d e n t ' s t-test for u n p a i r e d observations.
Results Effects of p a n c r e a s t a t i n g l u c a g o n and s o m a t o s t a t i n (Figure i)
(15.7 ~LM) on u n s t i m u l a t e d insulin, release by the p e r f u s e d rat p a n c r e a s
As expected, in control s o m a t o s t a t i n release did not e x p e r i m e n t a l period.
perfusions, insulin, g l u c a g o n vary s i g n i f i c a n t l y throughout
and the
A d d i t i o n of p a n c r e a s t a t i n to the p e r f u s a t e m a r k e d l y inhibited u n s t i m u l a t e d insulin o u t p u t (F=2.43, p<0.05) . Furthermore, d u r i n g p a n c r e a s t a t i n infusion, insulin secretion, as c a l c u l a t e d by the i n t e g r a t e d area under the r e s p o n s e curve, was reduced as c o m p a r e d to control p e r f u s i o n s (37± 5 ng/24 min vs. 62 ± 7 ng/24 min, p<0.05). P a n c r e a s t a t i n did not m a t o s t a t i n release.
significantly
modify
glucagon
and so-
Vol. 42, No. 14, 1988
Pancreastatin and P~creatic Homone Output
PANCREASTATIN
1363
15.7nM
~CONTROL,N:7 PIT, N,6
. . . .
z
i
1-
C
~
200
~
.
Z
c
s
so
-12
0
24
32
MINUTES
FIG.
1
E f f e c t of p a n c r e a s t a t i n on u n s t i m u l a t e d insulin, g l u c a g o n and s o m a t o s t a t i n release by the p e r f u s e d rat pancreas. Solid and b r o k e n lines c o r r e s p o n d to control and p a n c r e a s t a t i n experiments, r e s p e c t i v e l y (means ±SEM).
E f f e c t of p a n c r e a s t a t i n (15.7 nM) s o m a t o s t a t i n r e s p o n s e s to g l u c o s e p a n c r e a s (Figure 2)
on the insulin, @ l u c a @ o n and (ii ~I) by the p e r f u s e d rat
The release of insulin e l i c i t e d by glucose (incremental response: 261 z 69 ng/22 min) was c o n s i s t e n t l y b l o c k e d by pancrea s t a t i n (incremental response: 85 ± 13 ng/22 min, p<0.05). Panc r e a s t a t i n did not s i g n i f i c a n t l y affect the s u p p r e s s o r effect of g l u c o s e on g l u c a g o n s e c r e t i o n (decremental response: 4540 ± 5 6 8 pg/22 m i n vs. 3848±1295 pg/22 min in control experiments, p=0.6).
1364
Pancreastatin and Pancreatic Hormone Output
Vol. 42, No. 14, 1988
Glucose, at the c o n c e n t r a t i o n e m p l o y e d in these p e r f u s i o n s , did not s t i m u l a t e s o m a t o s t a t i n release; w h e n p a n c r e a s t a t i n was s i m u l t a n e o u s l y infused, no c h a n g e in the s e c r e t i o n of s o m a t o s t a t in was observed.
GLUCOSE 11raM -
CONTROL , N , 4
-
---
Z
_
20
PST, N,4
"
4o-._r,..,~_T.~"
,,
800Z-0
600-
°3~4oo-
) "~' I '
o E
~.,,
,.../
200. -t-
°
~ '~ I,-- Ol N
.
'
loo-
-12
0
22
32
MINUTES
FIG.
2
E f f e c t of p a n c r e a s t a t i n on the insulin, glucagon and s o m a t o s t a t l n r e s p o n s e s to g l u c o s e by the p e r f u s e d rat p a n c z e a s (means +_SEM) . Solid lines r e p r e s e n t g l u c o s e e x p e r i m e n t s . Broken lines r e p r e s e n t g l u c o s e plus p a n c r e a s t a t i n experiments.
E f f e c t of p a n c r e a s t a t i n (15.7 nM) on s o m a t o s t a t i n r e s p o n s e s to t o l b u t a m i d e rat p a n c r e a s (Figure 3). Pancreastatin
also
reduced
the insulin, g l u c a g o n and (I00 m y / l ) _ b Y ~he p e r f u s e d
tolbutamide-induced
i~suiin
re-
Vol. 42, No. 14, 1988
Pancreastatin and Pancreatic Hormone Output
1365
lease (incremental response: 27 + ii ng/22 min vs. 110-+ 23 ng/22 min in control experiments, p<0.025). The d e c r e a s e of g l u c a g o n output o b s e r v e d d u r i n g t o l b u t a m i d e infusion (decremental response: 3073 _+ 615 pg/22 min) was not sign i f i c a n t l y m o d i f i e d by p a n c r e a s t a t i n (decremental response: 3254 + 707 pg/22 min, p=0.9). Nor was t o l b u t a m i d e - i n d u c e d somatostatin release (incremental response: 1910 _+ 476 pg/22 min) s i g n i f i c a n t l y a f f e c t e d by p a n c r e a s t a t i n (incremental response: 1657 _+ 154 pg/22 rain, p=0.6). TOLBUTAMIDE
lOOmgll CONTROL, N=5 ---
15
PST, N=8
z E
z~
s
=~~°°l _~,
,
,-
T
1
T
~ J.,!%' T ,l
300"
~
250
o-
)I 150
12
0
20
10
30
MINUTES
FIG.
3
E f f e c t of p a n c r e a s t a t i n on the insulin, glucagon and s o m a t o s t a t i n r e s p o n s e s to t o l b u t a m i d e by the p e r f u s e d rat p a n c r e a s (means + SEM) . Solid lines r e p r e s e n t t 4 1 b u t a m i d e experiments. B r o k e n lines r e p r e s e n t t o l b u t a m i d e plus pancreastat±neexperiments.
1366
Pancreastatin and Pancreatic Hormone Output
Vol. 42, No. 14, 1988
Discussion The f o r e g o i n g results d e m o n s t r a t e that in the p e r f u s e d rat pancreas, p a n c r e a s t a t i n r e v e r s i b l y reduces u n s t i m u l a t e d insulin r e l e a s e and s t r o n g l y blocks the insulin response to tolbutamide. In addition, they c o n f i r m the inhibitory effect of p a n c r e a s t a t i n on g l u c o s e - i n d u c e d insulin output (i). R e c e n t data from our l a b o r a t o r y indicate that p a n c r e a s t a t i n also blocks insulin s e c r e t i o n as e l i c i t e d by gastric inhibitory peptide and by v a s o a c t i v e intestinal peptide (unpublished). Thus, it could be p o s t u l a t e d that p a n c r e a s t a t i n behaves as a general i n h i b i t o r of insulin release, perhaps by i n t e r f e r i n g w i t h a common and e s s e n t i a l step in this process. T a t e m o t o et al. (i) have mentioned, w i t h o u t p r e s e n t i n g data, that p a n c r e a s t a t i n inhibits g l u c o s e - i n d u c e d s o m a t o s t a t i n secretion by the p e r f u s e d rat pancreas. In our study, however, it failed to s i g n i f i c a n t l y affect u n s t i m u l a t e d or t o l b u t a m i d e induced s o m a t o s t a t i n release. We have also found a lack of effect of p a n c r e a s t a t i n on g l u c a g o n output under any of the conditions examined. These findings suggest that p a n c r e a s t a t i n inhibits B-cell s e c r e t o r y a c t i v i t y directly, and not through an A-cell or D-cell p a r a c r i n e effect. In the rat pancreas, p a n c r e a s t a t i n affects hormone output s i m i l a r l y to galanin, a n o t h e r r e c e n t l y i d e n t i f i e d peptide (6), p r e s e n t in nerve endings s u r r o u n d i n g the p a n c r e a t i c islets (7). G a l a n i n inhibits insulin release w i t h o u t a f f e c t i n g g l u c a g o n or somatostatin secretion (8,9). C o m p a r i s o n of the p r e s e n t data w i t h p r e v i o u s results from our l a b o r a t o r y (8) seems to indicate that the s u p p r e s s o r effect of p a n c r e a s t a t i n on insulin secretion is w e a k e r than that of galanin. The p h y s i o l o g i c a l r e l e v a n c e of these novel p e p t i d e s in the control of p a n c r e a t i c islet function remains to be elucidated.
Acknowledgements The e x p e r t technical work of Ms. A m a l i a MartEn, Ms. Paloma N i e t o and Ms. Pilar G a r c l a - M u ~ o z is g r a t e f u l l y acknowledged. We thank Ms. M a r t h a M e s s m a n for her secretarial help. This study was s u p p o r t e d by grants from the F o n d o de I n v e s t i g a c i o n e s Sanitarias de la S e g u r i d a d Social, M i n i s t e r i o de S a n i d a d y Consumo, (86/1972 and 87/858) and from the C o m i s i 6 n A s e s o r a de Investigaci6n C i e n t i f i c a y T~cnica, M i n i s t e r i o de E d u c a c i 6 n y C i e n c i a (PB86-0003), Spain. References I. K. TATEMOTO, S. EFENDIC, V. MUTT, G. MAKK, G.J. F E I S T N E R and J.D. BARCHAS, Nature 324, 476-478 (1986). 2. V. L E C L E R C Q - M E Y E R , J. MARCHAND, R. L E C L E R C Q and W.J. MALAISSE, D i a b e t e Metab. ~, 57-65 (1976). 3. V. HERBERT, K.-S. LAO, C.W. G O T T L I E B and S.J. BLEICHER, J. Clin. Endocrinol. Metab. 25, 1375-1384 (1965). 4. G.R. F A L O O N A and R.H. UNGER, M e t h o d s of H o r m o n e R a d i o i m m u n o a s say, pp. 317-330, A c a d e m i c Press, N e w York (1974).
Vol. 42, No. 14, 1988
Pancreastatin and Pancreatic Hormone Output
1367
5. V. HARRIS, J.M. CONLON, C.B. SRIKANT, K. McCORKLE, V. SCHUSDZIARRA, E. IPP and R.H. UNGER, Clin. Chim. A c t a 8_/7, 275-283 (1978) . 6. K. TATEMOTO, A. ROKAEUS, H. J~RNVALL, T.J. M c D O N A L D and V. MUTT, FEBS Lett. 3.6.4,.124-128 (1983). 7. B.E. DUNNING, B. AHREN, R.C. VEITH, G. BOTTCHER, F. S U N D L E R and G.J. TABORSKY, Jr., Am. J. Physiol. 251, E I 2 7 - E I 3 3 (1986). 8. R.A. SILVESTRE, P. MIRALLES, L. MONGE, P. MORENO, M.L. VILLAN U E V A and J. MARCO, E n d o c r i n o l o g y 121, 378-383 (1987). 9. P. MIRALLES, E. PEIR0, R.A. SILVESTRE, M.L. V I L L A N U E V A and J. MARCO, M e t a b o l i s m (in press).