Regulatory Peptides, 34 (1991) 159-167

159

© 1991 Elsevier Science Publishers B.V. 0167-0115/91/$03.50 REGPEP 01053

Homologous pancreastatin inhibits insulin secretion without affecting glucagon and somatostatin release in the perfused rat pancreas Elena P e i r r , Pilar D r g a n o , P a l o m a Miralles, R a m o n a A. Silvestre and Jos6 M a r c o Hospital Puerta de Hierro and Departamento de Fisiologla, Universidad Autbnoma de Madrid, Madrid (Spain)

(Received 15 October 1990; revised version received and accepted 6 March 1991) K e y words." Rat pancreastatin; Rat pancreas; Insulin; Glucagon; Somatostatin

Summary The identification of pancreastatin in pancreatic extracts prompted the investigation of its effects on islet cell function. However, in most of the investigations to date, pig pancreastatin was tested in heterologous species. Since there is great interspecies variability in the amino acid sequence of pancreastatin, we have investigated the influence of rat pancreastatin on insulin, glucagon and somatostatin secretion in a homologous animal model, namely the perfused rat pancreas. During 5.5 mM glucose infusion, pancreastatin (40 nM) inhibited insulin secretion (ca. 4 0 ~ , P < 0.025) as well as the insulin responses to 10 mM arginine (ca. 50~o, P < 0.025) and to 1 nM vasoactive intestinal polypeptide (ca. 50~o ; P < 0.05). Pancreastatin failed to significantly modify glucagon or somatostatin release under any of the above experimental conditions. In addition, a lower pancreastatin concentration (15.7 nM) markedly suppressed the insulin release evoked by 11 mM glucose (ca. 85 ~o, P < 0.05). Our present observations reinforce the concept that pancreastatin is an effective inhibitor of insulin secretion, influencing the B-cell function directly and not through an A-cell or D-cell paracrine effect.

Correspondence: J. Marco, Hospital Puerta de Hierro, Universidad Aut6noma de Madrid, San Martin de Porres, 4, 28035 Madrid, Spain.

160

Introduction

The identification of pancreastatin, a 49-amino acid straight chain polypeptide, in pig pancreatic extracts [ 1] prompted the investigation of its effects on islet cell function. In fact, in the original description of pancreastatin, it was already reported that this molecule inhibited glucose-induced insulin secretion in the perfused rat pancreas [1 ]. A number of subsequent studies have also revealed an inhibitory effect of pancreastatin on insulin output both in vitro [2-6] and in vivo [7,8]. However, other investigators have observed no influence [9,10] or even a stimulatory effect of pancreastatin on B-cell function [ 11 ]. As for the effect of pancreastatin on glucagon and somatostatin release, results are also controversial [2-4,7-10]. It is noteworthy that in most of the aforementioned studies, pig pancreastatin was tested in a heterologous species. Since there is great interspecies variability in the amino acid sequence of pancreastatin, as is the case with its precursor chromogranin A [12,13 ], we have examined the influence of rat pancreastatin on insulin, glucagon and somatostatin secretion in a homologous animal model, i.e., the perfused rat pancreas. These hormones were measured during 5.5 mM glucose infusion as well as in response to 10 mM arginine and to 1 nM vasoactive intestinal polypeptide (VIP), the latter two substances being effective secretagogues of pancreatic B, A and D cells. In addition, the effect of rat pancreastatin on the insulin response to an increase in the glucose concentration of the perfusate (from 5.5 mM to 11 mM) was tested.

Materials and Methods

Fed male Wistar rats (200-225 g body weight) were used as donors. After anesthesia of the rat with pentobarbital sodium (50 mg/kg, i.p.), the pancreas was dissected and perfused in situ according to the procedure of Leclercq-Meyer et al. [ 14] as adapted in our laboratory [15]. Effluent samples were collected from the portal vein, without recycling, at 2-min intervals (flow rate, 2 ml/min) in tubes containing 2000 KIU Trasylol (Bayer, AG, Leverkusen, F.R.G.), and frozen at - 2 0 °C until the time of assay. The perfusion medium consisted of a Krebs-Henseleit buffer (gas phase 95:5, O2/CO2; pH 7.4) supplemented with 4~o (w/v) dextran T-70, 0.5~o (w/v) bovine albumin (Cohn fraction V) and glucose (5.5 mM). Synthetic amydated rat pancreastatin (Peninsula Laboratories, Belmont, CA) was dissolved in 0.9 ~o NaC1 containing 0.1 ~o bovine albumin (Cohn Fraction V). This solution was prepared daily, immediately before experiments. After a 35 min equilibration period, baseline samples were collected for 8-12 min. At zero time, rat pancreastatin was infused through a sidearm cannula as a priming dose (250 pmol), followed by constant infusion (40 nM) for 16 or 20 min. As secretagogues of the endocrine pancreas, 10 mM L-arginine hydrochloride (Sigma Chemical Co., St. Louis, MO) and 1 nM VIP (Peninsula) were used. In another series of experiments, the effect of rat pancreastatin (a priming dose of 400 pmol, followed by constant infusion (15.7 nM)) on the insulin response to 11 mM glucose was tested. Additions to the perfusate were performed as described in the corresponding figures.

161

Insulin [16], glucagon [ 17] and somatostatin [18] were measured by radioimmunoassay. Antiglucagon serum (30K) and antisomatostatin serum (80C) were kindly donated by Dr. R.H. Unger (University of Texas Health Sciences Center, Dallas). All samples for each series of experiments were analysed in the same run. Results are presented as means + S.E.M. For each perfusion, hormone response was 5.5 mM

GLUCOSE

RAT- PST(4OnM)

z.~

~----

SALINE

; N=9

---

RAT-PANCREASTIN

; N=IO

3

l.l,i-i-rl± •

w

@

I

'~.I_LA"£I

i

400 Z

e-

0'~ 100

Z

40

I-.£ 20

0

-12

0

20

32

MINUTES Fig. 1. Effect of rat pancreastatin (a priming bolus of 250 pmol followed by constant infusion at a rate of 80 pmol/min) on insulin, glucagon and somatostatin release during 5.5 mM glucose infusion in the perfused rat pancreas. Solid lines represent control experiments. Broken lines represent rat pancreastatin experiments (means +_ S.E.M.).

162 calculated as the integrated area of the curve above or below the mean preinfusion level (average of all its baseline levels) with the trapezoidal method. Differences between values were tested for significance by analysis of variance and the Student's t-test for unpaired samples.

5.5 mM

GLUCOSE

ARGININE(IOmM) ARGININE

I

A RGI,N I N E

Bo

"---

--

I

RAT-

; N:8

• N--6

PANCRE*~STATIN

L

I

e

3000,

0 '

2000

(5 -nm 1000 ,..J Q.

L9

120'

Z

80

O I.-- ,, ~

40,

0

0

. . . . . . . . . .

-12

0

!

. . . .

10

,

20



,



i



30

MINUTES

Fig. 2. Effect of rat pancreastatin (a priming bolus of 250 pmol followed by constant infusion at a rate of 80 pmol/min) on the insulin, glucagon and somatostatin responses to arginine (10 mM) by the perfused rat pancreas. Solid lines represent arginine experiments. Broken lines represent arginine plus rat pancreastatin experiments (means + S.E.M.).

163

5.5 mM GLUCOSE VIP

( 1 nM

1

)

VIP ; N = 7 VIP + ; N=6 RAT-PA NCREASTATIN

15,

~=

lo

-

800 t

~.~c e

600 t

200]

J

"L J

/

!,

,, 80}

~1

~

"

4o 0

.

-12

0

10

!

20

,

.

.

.





30

MINUTES Fig. 3. Effect of rat pancreastatin (a priming bolus of 250 pmol followed by constant infusion at a rate of 80 pmol/min) on the insulin, glucagon and somatostatin responses to VIP (1 nM) by the perfused rat pancreas. Solid lines represent VIP experiments. Broken lines represent VIP plus rat pancreastatin experiments (means +_ S.E.M.)

164 Results

Effect of rat pancreastatin (40 nM) on insulin, glucagon and somatostatin secretion during 5.5 mM glucose infusion in the perfused rat pancreas (Fig. 1) Addition of pancreastatin to the perfusate markedly inhibited insulin secretion (Flo,9 o = 4.5; P < 0.01). Insulin release, as calculated by the integrated area under the response curve, was also reduced during pancreastatin infusion (43 + 5 ng/20 min) as compared to control perfusions (71 + 9 ng/20 min; P < 0.025). Pancreastatin did not significantly modify glucagon and somatostatin output. Effect of rat pancreastatin (40 nM) on the insulin, glucagon and somatostatin responses to arginine (10 mM) in the perfused rat pancreas (Fig. 2) The secretion of insulin elicited by arginine (incremental area: 911 + 95 ng/20 min) was significantly reduced by pancreastatin (incremental area: 480 + 103 ng/20 min; P < 0.025), this effect being more marked in the late phase of insulin release. The glucagon and somatostatin responses to arginine with or without pancreastatin were comparable (incremental areas: 23.4 + 1.3 vs. 26.4 + 2.4 ng/20 min, and 381 + 96 vs. 560 + 161 pg/20 min, respectively; NS). Effect of rat pancreastatin (40 nM) on the insulin, glucagon and somatostatin responses to VIP (1 nM) in the pe~fused rat pancreas (Fig. 3) Pancreastatin also inhibited VIP-induced insulin output (incremental areas: 47 + 15 vs. 96 + 13 ng/16 min in control experiments; P < 0.05), whereas it did not significantly alter the glucagon and somatostatin responses to this neuropeptide (incre-

GLUCOSE

5 . 5 mM

5.5

m M

20

15

....

z . _c -;E

GLUCOSE;

N= 8

GLUCOSE +

; N=5

RAT- P S T ( 1 5 . 7 n M )

10 CON Zm -- E

5

t

"

-8

"

i

0

.

.

.

.

I

.

.

.

.

10

!

20

I



.

,

!



,

,

3O

MINUTES

Fig. 4. Effect of rat pancreastatin (a priming bolus of 400 pmol followed by constant infusion at a rate of 31.4 pmol/min) on the insulin response to glucose (I1 mM) by the perfused rat pancreas. Solid lines represent glucose experiments. Broken lines represent glucose plus rat pancreastatin experiments (means + S.E.M,).

165

mental areas: 5.1 + 1.1 vs. 4.6 + 0.5 ng/16 min, and 236 + 108 vs. 465 + 283 pg/16 min, respectively; NS). Effect of rat pancreastatin (15.7 nM) on the insulin response to glucose (11 mM) in the perfused rat pancreas (Fig. 4) The infusion of rat pancreastatin virtually abolished the insulin release elicited by 11 mM glucose (incremental area: 25 + 17 ng/22 min vs. 167 + 48 ng/22 min in control experiments; P < 0.05).

Discussion

Our results demonstrate that in the perfused rat pancreas, homologous pancreastatin consistently inhibited the insulin secretion evoked by constant infusion of 5.5 mM glucose, as well as the insulin response to arginine and to VIP. These findings are in agreement with most data obtained employing pig pancreastatin and a variety of stimuli of insulin output in heterologous animal models [1-5,7,8]. It is noteworthy that Ostenson et al. [5], employing pig pancreastatin in a preparation of isolated rat pancreatic islets, found no suppressor effect of pancreastatin on insulin release when the glucose concentration of the medium was lowered to 3.3 mM. Rat pancreastatin also evoked a reduction of the insulin response to a higher glucose concentration (11 mM), comparable to that previously found in our laboratory [3] for an equimolar concentration of pig pancreastatin (ca. 80~o and ca. 70~o, respectively). The amino acid sequences of rat and pig pancreastatin differ in ca. 40~o [12]; however, the C-terminal fragment of both molecules is well preserved [ 19], and this region seems to be responsible for the biological activity exerted by pancreastatin in the rat [ 1,19,20]. Such a structural analogy might explain the similar effect of rat and pig pancreastatin on B-cell secretion in the rat pancreas. Accordingly, Miyasaka et al. have reported that, in the rat, a synthetic rat pancreastatin C-terminal fragment inhibits the insulin response to glucose [21]. Recently, Hoist et al. [ 10] have reported that pig pancreastatin does not affect insulin release in the perfused pig pancreas. The reason for the discrepancy between these authors' findings and ours is not known. Rat pancreastatin had no effect on either glucagon or somatostatin secretion, whether in unstimulated conditions or when stimulated by arginine or VIP. Previous work from our laboratory, performed in the perfused rat pancreas [3,4], also showed that pig pancreastatin failed to modify unstimulated glucagon and somatostatin release as well as the response of these hormones to glucose, tolbutamide, VIP, gastric inhibitory peptide (GIP), and the 26-33 octapeptide form of cholecystokinin (8-CCK). Results from other in vitro studies also point to a lack of statistically significant effects of pancreastatin on A-cell [2,9,10] and D-cell [2,10] secretion. Ahr6n et al. [7] have observed that in mice pancreastatin injection is followed by a slight and short-lived increment of basal plasma glucagon. This effect was not seen during adrenergic blockade by combined propanolol and phentolamine pretreatment, thus suggesting an indirect influence of pancreastatin on A-cell function.

166 O u r p r e s e n t o b s e r v a t i o n s that, in the rat p a n c r e a s , h o m o l o g o u s p a n c r e a s t a t i n b l o c k s i n s u l i n release w i t h o u t affecting g l u c a g o n or s o m a t o s t a t i n secretion reinforce o u r p r o p o s e d c o n c e p t that p a n c r e a s t a t i n inhibits B-cell f u n c t i o n directly, a n d n o t t h r o u g h a n A-cell or D-cell p a r a c r i n e effect [4].

Acknowledgements E . P . a n d P . D . are R e s e a r c h Fellows from the F o n d o de I n v e s t i g a c i o n e s S a n i t a r i a s 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 C o n s u m o , S p a i n . This w o r k has b e e n s u p p o r t e d by g r a n t s from the F o n d o de I n v e s t i g a c i o n e s S a n i t a r i a s de la S e g u r i d a d Social (89-0102, 89-0109, 90-0043 a n d 90-0894), a n d f r o m the C o m i s i 6 n I n t e r m i n i s t e r i a l de C i e n c i a y T e c n o l o g l a (PB-86003), S p a i n . T h e expert t e c h n i c a l a s s i s t a n c e o f Ms. Pilar G a r c i a M u f l o z a n d Ms. E n c a r n a c i 6 n G u t i ~ r r e z is gratefully a c k n o w l e d g e d . W e t h a n k Ms. M a r t h a M e s s m a n for her editorial help.

References 1 Tatemoto, K., Efendic, S., Mutt, V., Makk, G., Feistner, G.J. and Barchas, J.D., Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion, Nature, 324 (1986) 476-478. 2 Efendic, S., Tatemoto, K., Mutt, V., Quan, C., Chang, D. and Ostenson, C.G., Pancreastatin and islet hormone release, Proc. Natl. Acad. Sci. USA, 84 (1987) 7257-7260. 3 Silvestre, R. A., Peir6, E., Miralles, P., Vilanueva, M. L. and Marco, J., Effects ofpancreastatin on insulin, glucagon and somatostatin secretion by the perfused rat pancreas, Life Sci., 42 (1988) 1361-1367. 4 Peir6, E., Miralles, P., Silvestre, R.A., Villanueva, M.L. and Marco, J., Pancreastatin inhibits insulin secretion as induced by glucagon, vasoactive intestinal peptide, gastric inhibitory peptide, and 8-cholecystokinin in the perfused rat pancreas, Metabolism, 38 (1989) 679-682. 50stenson, C.G., Sandier, S. and Efendi¢, S., Effects of porcine pancreastatin on secretion and biosynthesis of insulin and glucose oxidation of isolated rat pancreatic islets, Pancreas, 4 (1989) 441-446. 6 Funakoshi, A., Jimi, A., Yasunami, Y., Tateishi, K., Funakoshi, S., Tamamura, H. and Yajima, H., Bioactivity of human pancreastatin and its localization in pancreas, Biochem. Biophys. Res. Commun., 159 (1989) 913-918. 7 Ahr6n, B., Lindskog, S., Tatemoto, K. and Efendic, S., Pancreastatin inhibits insulin secretion and stimulates glucagon secretion in mice, Diabetes, 37 (1988) 281-285. 8 Funakoshi, A., Miyasaka, K., Kitani, K. and Tatemoto, K., Effect of pancreastatin on pancreatic endocrine function in the conscious rat, Regul. Pept., 24 (1989) 225-231. 9 Ohneda, A., Koizumi, F. and Ohneda, M., Effect of p0rcine pancreastatin on endocrine function of canine pancreas, Tohoku J. Exp. Med., 159 (1989) 291-298. 10 Hoist, J.J., Ostenson, C,G., Harling, H. and Messell, T., Porcine pancreastatin has no effect on endocrine secretion from the pig pancreas, Diabetologia, 33 (1990) 403-406. ii Ishizuka, J.~ Asada, I., Poston, G.J(, Lluis, F., Tatemoto, K., Greeley, G.H., Jr. and Thompson, J.C., Effect of pancreastatin on pancreaticendocrine and exocrine secretion. Pancreas, 4 (1989) 277-281. 12 lacangelo, A., Okayama, H. and Eiden, L. E., Primary structure of rat chromogranin A and distribution of its mRNA, FEBS Lett., 227 (1988) 115-121. 13 Iacangelo, A. L., Fischer-Colbrie, R., Koller, K.J., Brownstein, M.J. and Eiden, L. E., The sequence of porcine chromogranin A messenger RNA demonstrates chromogranin A can serve as the precursor for the biologically active hormone, pancreastatin, Endocrinology, 122 (1988) 2339-23411 14 Leclercq-Meyer, V., Marchand, J., Leclercq, R. and Malaisse, W. J., Glucagon and insulin release by the "in vitro' perfused rat pancreas, Diab. Metab.; 2 (1976) 57-65. 15 Silvestre, R.A., Miralles, P., Moreno, P., Villanueva, M.L. and Marco, J., Somatostatin, insulin and

167

16 17 18 19

20

21

glucagon secretion by the perfused pancreas from the cysteamine-treated rat, Biochem. Biophys. Res. Commun., 134 (1986) 1291-1297. Herbert, V., Lau, K. S., Gottlieb, C.W. and Bleicher, S.J., Coated charcoal immunoassay of insulin, J. Clin. Endocrinol. Metab., 25 (1965) 1375-1384. Faloona, G.R. and Unger, R.H., Glucagon. In B.M. Jaffe and H.R. Behrman (Eds.), Methods of Hormone Radioimmunoassay, Academic Press, New York, 1974, pp. 317-330. Harris, V., Conlon, J.M., Srikant, C.B., McCorkle, K., Schusdziarra, V., Ipp, E. and Unger, R.H., Measurements of somatostatin-like immunoreactivity in plasma, Clin. Chim. Acta, 87 (1978) 275-283. Funakoshi, A., Miyasaka, K., Kitani, K., Tamamura, H., Funakoshi, S. and Yajima, H., Bioactivity of synthetic C-terminal fragment of rat pancreastatin on endocrine pancreas, Biochem. Biophys. Res. Commun., 158 (1989) 844-849. Miyasaka, K., Funakoshi, A., Kitani, K., Tamamura, H., Fujii, N. and Funakoshi, S., The importance of the C-terminal amide structure of rat pancreastatin to inhibit pancreatic exocrine secretion, FEBS Lett., 263 (1990) 279-280. Miyasaka, K., Funakoshi, A., Yasunami, Y., Nakamura, R., Kitani, K., Tamamura, H., Funakoshi, S. and Fujii, N., Rat pancreastatin inhibits both pancreatic exocrine and endocrine secretions in rats, Regul. Pept., 28 (1990) 189-198.

Homologous pancreastatin inhibits insulin secretion without affecting glucagon and somatostatin release in the perfused rat pancreas.

The identification of pancreastatin in pancreatic extracts prompted the investigation of its effects on islet cell function. However, in most of the i...
401KB Sizes 0 Downloads 0 Views