Evidence for secretion of 7B2 by A- and B-cells of hamster pancreatic islets

Regulatory Peptides, 36 (1991) 407-414 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0167-0115/91/$03.50

407

REGPEP 01115

Evidence for secretion of 7B2 by A- and B-cells of hamster pancreatic islets H a r u o Iguchi 1, T o s h i m i t s u O k e d a 2 and R y o s a b u r o T a k a k i 2 ~Department of Biochemistry, National Kyushu Cancer Center, Fukuoka (Japan) and 2First Department of Medicine, Medical College of Oita, Oita (Japan) (Received 12 January 1991; revised version received 30 August 1991; accepted 5 September 1991)

Key words: 7B2; Pancreatic islet

Summary 7B2 is a neuroendocrine protein, and in the pancreatic islets the presence of 7B2 in A- and B-cells was immunohistochemically demonstrated. In order to examine 7B2 secretion by A- and B-cells of pancreatic islets, we prepared isolated hamster pancreatic islet cells as well as an A-cell-rich culture, and studied 7B2 secretion under certain stimulations. 7B2 was secreted by isolated hamster pancreatic islet cells. This secretion was stimulated by theophylline and arginine, but glucose had a weak effect on the 7B2 secretion. Such a response of 7B2 to the stimulations was different from that of insulin or glucagon. 7B2 secretion was also noted in the A-cell-rich culture. These results suggest that 7B2 is secreted by both A- and B-cells of the hamster pancreatic islets and its secretion is regulated under certain conditions.

Introduction 7B2, initially isolated from human and porcine pituitary gland [ 1,2], is distributed in the neuroendocrine tissues of the rat [3 ], and secretion of 7B2 by the rat pituitary gland [3], bovine adrenal medulla [4] and human GH-producing pituitary adenoma [5] was demonstrated. Although the function of 7B2 is unknown, the amino acid sequence of 7B2 is highly conserved between species [2,6], suggesting an important role of 7B2. The primary structure of human pituitary 7B2 cDNA was described in 1988 [7], and a Correspondence: H. Iguchi, Department of Biochemistry, National Kyushu Cancer Center, 3-1-1 Notame, Minami-ku, Fukuoka 815, Japan.

408 deduced amino acid sequence of 7B2 from cDNA revealed three regions sharing similarity with putative GTP-binding domains [7]. In the pancreas, the presence of 7B2 in A- and B-cells of islets was immunohistochemically demonstrated [8], however, secretion of 7B2 by these cells and/or its regulation have not been elucidated. In this paper, we studied the effects oftheophylline, arginine and glucose on 7B2 secretion using isolated hamster pancreatic islet cells.

Materials and Methods

Cell culture Pancreatic tissues were obtained from Syrian golden hamsters under nembutal anesthesia. Cultured islet cells were prepared according to the procedure described by Okeda et al. [9]. Briefly, islets were isolated from exocrine tissues by collagenase (type IV, Worthington Biochemical Co., Hallsmills, N J, U.S.A.) digestion followed by a Ficoll-Conray gradient centrifugation. Dissociation of isolated islets into single cells was performed using Dispase (Godo-Syusei Co., Tokyo, Japan) and mechanical digestion. The culture medium used was Dulbecco's modified Eagle's medium containing 60 or 100 mg/dl glucose, 250 units/ml aprotinin and 5~o fetal calf serum (DMEM). Viability of the cells was assessed by insulin secretion. Usually, cells (105 ) secrete 1.0-3.0 pmol insulin/h on day 3 after plating. The cells used in the present study secreted a similar quantity of insulin. Theophylline (10 -4, 10 -3, 10 -2 M), arginine (10-3, 10-2 M) and glucose (I00, 300 mg/dl) were used as secretagogues. The release experiment was performed by incubating 4 to 5 dishes (5.105 cells/dish) on day 3 and 4 after plating. Each dish was washed twice with 1 ml of Hanks' solution, and the medium was, then, changed to 1 ml of D M E M (control) or D M E M with the test substances. The cells were incubated for 60 min at 37 °C under 5 ~o COz-95 ~o air. The medium was collected at termination of the incubation and stored at - 2 0 °C until assay. To assess secretion of 7B2 by A-cells of pancreatic islets, the A-cell-rich culture was prepared according to Noguchi et al. [ 10]. Briefly, isolated islet cells, in which about 80~o were B-cells, were further treated with 10 mM alloxan (Wako Chemicals Co., Tokyo, Japan) for 10 min followed by washing with DMEM. After incubation for 12 h, the medium, containing floating dead cells (mostly B-cells), was removed and changed into fresh DMEM. Attached cells, mostly consisting of A-cells (about 80~o), were treated with trypsin and plated (1 • 105 cells/dish). The cells were further incubated for 24 h, and the medium was collected and stored at - 20 °C until assay. Gel permeation chromatography Gel permeation chromatography of culture medium was performed on a Sephadex G-100 column (95 x 1.4 cm) equilibrated with 1 M acetic acid. 2 ml of samples were layered onto the column and eluted with 1 M acetic acid at a flow rate of 7 ml/h at 4 °C. Fractions (1.3 ml) were collected, dried with a centrifugal concentrator (Taiyo VC-36, Taiyo Scientific Industrial Co., Tokyo, Japan) and reconstituted with RIA-buffer before assay. The column was calibrated with protein markers (Vo, catalase; 43K, ovalbumin ; 25K, chymotrypsinogen A; 13.7K, ribonuclease).

409

Determination of 7B2, insulin and glucagon 7B2 was m e a s u r e d by R I A according to the p r o c e d u r e described by Iguchi et al. [3]. A 7B2 antiserum used was raised against a synthetic fragment o f 7B2, c o r r e s p o n d i n g to amino acids 2 3 - 3 9 o f authentic porcine 7B2 (7B2 2 3 - 3 9 ) , coupled to bovine thyroglobulin in rabbits (a gift from Dr. Michel Chretien, Clinical Research Institute o f Montreal, Montreal, C a n a d a ) . The antiserum did not c r o s s r e a c t with P O M C - r e l a t e d peptides, G H , L H , F S H , T S H , P R L , AVP, oxytocin, neurophysin, C R F , G R F , s o m a t o s t a t i n 1-14, d y n o r p h i n 1-13, MetS-enkephalin, substance P, calcitonin, insulin, glucagon, PP, secretin and thyroglobulin. 7B2 2 3 - 3 9 and [~25I]7B2 2 3 - 3 9 were used as s t a n d a r d and tracer, respectively. Intra- and interassay coefficients o f variation were less than 10~o (n = 5) and the sensitivity o f the assay was 10 pmol/1. A dilution curve o f the culture m e d i u m revealed a parallel d i s p l a c e m e n t with an R I A s t a n d a r d curve ( d a t a not shown). Insulin and glucagon were m e a s u r e d using R I A kits ( D a i n a b o t Co., Tokyo, Japan).

Statistics S t u d e n t ' s t-test was performed to assess the significant difference between concentrations o f 7B2, insulin and glucagon in the control m e d i u m and those in the m e d i u m after stimulation by secretagogues. A 'P' value o f less than 0.05 was considered to be statistically significant.

Results M e d i u m c o n c e n t r a t i o n s o f 7B2, insulin and glucagon after 60 min incubation with or without theophylline, arginine and glucose are s u m m a r i z e d in Table I. Significant d o s e - r e s p o n s i v e increases of 7B2 and insulin concentrations were observed after an

TABLE I Medium concentrations of 7B2, insulin and glucagon after 60 min incubation

Theophylline (M) 0 10 - 4

10 -3 10 -2 Arginine (M) 0 10 3 10 2 Glucose (mg/dl) 60 100 300

7B2 (pmol/1)

Insulin (pmol/l)

Glucagon (pmol/1)

43 + 3.1 61 + 6.1 82 + 5.0

2720 + 230 3300 __+208 4290 + 186 4950 + 408

118 __+21 131 + 10 136 +__24 235 + 10

35 + 4.8 41 + 3.2 54_+5.6

1757 + 186 1614 + 186 1829 + 165

88 __+12 86 + 7.4 162 + 24

54 _+ 1.4 55 + 3.5 65 + 6.7

1765+ 78 2769 + 143 3271 + 337

88+ 8.0 19 + 7.7 27 + 6.3

51 + 4.4

410 (~) 20O

o q"

lOO

Control

10 - 4

10 - 3

10 2

l'hcophylline(M)

Fig. 1. Effects oftheophylline (10 -4, 10 -~, 10 2 M) on the secretion of 7B2 (open bars), insulin (stippled bars) and glucagon (filled bars) by isolated hamster pancreatic islet cells. Each bar represents the mean + S.D. value of five replicate dishes. Similar results were obtained in two independent experiments. (*P < 0.01 vs. control). a d d i t i o n o f t h e o p h y l l i n e (Fig. 1). T h e o p h y l l i n e also c a u s e d an increase o f the g l u c a g o n c o n c e n t r a t i o n , h o w e v e r , a significant i n c r e a s e was o b t a i n e d only after an a d d i t i o n o f 10 - 2 M t h e o p h y l l i n e (Fig. 1). A r g i n i n e (10 - 2 M ) c a u s e d significant i n c r e a s e s o f 7B2 and g l u c a g o n c o n c e n t r a t i o n s , but h a d n o effect on the insulin c o n c e n t r a t i o n (Fig. 2). A significant i n c r e a s e o f the (~) 2oo

100

0 Control

10 . 3

10 2 Arffinine(~l)

Fig. 2. Effects of arginine (10 3, 10 2 M) on the secretion of 7B2 (open bars), insulin (stippled bars) and glucagon (filled bars) by isolated hamster pancreatic islet cells. Each bar represents the mean + S.D. value of four replicate dishes. Similar results were obtained in two independent experiments. (*P < 0.01 vs. control).

411

(t)

L

200

100

::::::::::::::: :!:!:!:!:[:3!:! ::~:!:!:!:!:!:!:

........ ::::::::::::: ~:~:[:!:[:!:! <+:+:+ ::::::::::::::::::::: :+:.:.:.:.: +:+x+ ....... !:[:!:3!:[:[ ::::::::::::: :!:!:!:!:!:!: ::::::::::::: !:3!:!:!:[:! :~:~:~:~:~:~: ::::::::::::: !:!:!:!:!:!:! .:.:.:.:.:.:. ~:~:~:~:!:~:!

-7

!:!:!:!:[:!:[:! :!:!:!:!:!:!:!:

::::::::::::::: .::+:+:.:+: !:~:~:~$~:!: :!:!:!:!:!:!:!: +:+x+:, ~:~:!:~:~:~:~:i :~:!:~:~:~:~:~: !:!:!:!:3!:[:! .:+x.x+ :!:[¢[$!:!: x+x+:.:

::::::::::::::: ::::::::::::::: :~:~:~:!:~:~:~: ::::::::::::::: :[:!:~:~:~:~:~:

:::::::::::: ::::5::::::: :::::::::::::

iiiiiiii[iiiiii

:[:~:~:~:~:~:

1

~:~:i:~:~:~:~:i

N 60

300

100

(; I ucose ( l g / d l ) Fig. 3. Effects o f g l u c o s e (60 m g / d l (control), 100, 300 m g / d l ) o n t h e s e c r e t i o n o f 7B2 ( o p e n b a r s ) , i n s u l i n ( s t i p p l e d b a r s ) a n d g l u c a g o n (filled b a r s ) by i s o l a t e d h a m s t e r p a n c r e a t i c islet cells. E a c h b a r r e p r e s e n t s t h e m e a n + S.D. v a l u e o f four r e p l i c a t e d i s h e s . S i m i l a r r e s u l t s w e r e o b t a i n e d in t w o i n d e p e n d e n t e x p e r i m e n t s . ( * P < 0.05, * * P < 0.01 vs. control).

insulin concentration and a significant decrease of the glucagon concentration were observed in the presence of 100 and 300 mg/dl of glucose (Fig. 3). The 7B2 concentration was also significantly increased in the presence of 300 mg/dl of glucose, but the magnitude of this increase of 7B2 was low as compared to that of insulin (Fig. 3). 100 mg/dl of glucose had no effect on the 7B2 concentration (Fig. 3).

Vo

43K

25K

13.7K

i00

Z

¢q e~

50

10

i

4'0

8'0 Fraction

120

number

Fig. 4. G e l p e r m e a t i o n c h r o m a t o g r a p h y o f t h e c u l t u r e m e d i u m o f i s o l a t e d h a m s t e r p a n c r e a t i c islet cells o n a S e p h a d e x G - 1 0 0 c o l u m n (95 x 1.4 cm). T h e c o l u m n w a s c a l i b r a t e d by p r o t e i n m a r k e r s (Vo, c a t a l a s e ; 4 3 K , o v a l b u m i n ; 2 5 K , c h y m o t r y p s i n o g e n A ; 13.7K, r i b o n u c l e a s e ) .

412 In the A-cell-rich culture, 7B2 and glucagon were detected in the medium after 24 h incubation, and their concentrations were 211 + 3 2 and 7362_+ 1345pmol/1 mean _+ SD, n = 8), respectively. Insulin was not detected in this culture medium. Fig. 4 depicts an elution profile of the culture medium of the normal islet cells on Sephadex G-100. The majority of 7B2 was eluted at a position with an apparent molecular mass of more than 43K, while a minor peak with a smaller molecular mass was noted.

Discussion

The amino acid sequence of 7B2 is highly conserved between species [2,6], suggesting that 7B2 could have an important role. However, its function is still unknown. Martens [7] revealed the primary structure of human pituitary 7B2 cDNA and suggested that 7B2 is a G T P binding protein based on the amino acid sequence deduced from cDNA. 7B2 is distributed mainly in the neuroendocrine tissues of the rat [3], and in the rat and human pancreatic islet the presence of 7B2 in A- and B-cells was immunohistochemicaUy demonstrated [8]. In this report, we found secretion of 7B2 by the isolated hamster pancreatic islet cells as well as the A-cell-rich culture. This is consistent with the findings demonstrated by immunohistochemical studies [8]. In the isolated hamster pancreatic islet cells, secretion of 7B2 was stimulated by theophylline, arginine and glucose. The responses of 7B2 to these stimulus were different from those of insulin or glucagon. Immunohistochemical studies coupled with electromicroscope studies revealed that 7B2 was localized in the secretory granules of A- and B-cells [8]. The different responses of 7B2, insulin and glucagon to these stimulus may be due, in part, to localization of 7B2 in secretory granules of both A- and B-cells. The molecular weight of 7B2 deduced from the human pituitary 7B2-cDNA is 20,793 [7]. However, in the gel permeation chromatography of various samples obtained from several species using acetic acid as an eluate, the majority of 7B2 was eluted at the position with an apparent molecular mass of more than 43K, and this peak of 7B2 was shifted to 20-21K in the following analysis of SDS-PAGE in the reduced condition [3,11,12]. Similarly, the major peak of 7B2 was observed at an apparent molecular mass of more than 43K in the present gel permeation chromatography. A molecular form of 7B2, secreted by hamster pancreatic islets, appears to be the same as was seen in other tissues or biological fluids of several species. The majority of 7B2 may be eluted as a dimer in this gel permeation chromatography system. The minor peak with an apparent molecular mass of less than 13.7K was also noted in the present study. A 7B2 molecule contains two sites of paired basic amino acids [7], suggesting proteolytic cleavage of 7B2 into small fragments. In fact, processing of a 7B2 molecule was demonstrated in the pulse-chase experiment using an amphibian pituitary neurointermediate lobe [13 ]. The minor peak of 7B2 may be a processing product of 7B2. As to the function of 7B2, a precursor protein for biologically active peptides and/or a GTP-binding capacity were suggested [7,13]. A processing product of chromogranin A, which is secreted from chromaffin cells, has been shown to inhibit catecholamine secretion from chromaffin cells [14,15]. This is an example of the autocrine

413 regulation. A similar p h e n o m e n o n of c h r o m o g r a n i n A - d e r i v e d p e p t i d e s was also o b s e r v e d in the p a r a t h y r o i d g l a n d [16]. 7B2 is a m e m b e r o f the c h r o m o g r a n i n / s e c r e t o g r a n i n family [17]. 7B2 m a y f u n c t i o n in the p a n c r e a t i c islets t h r o u g h a p a r a c r i n e / a u t o c r i n e fashion. In c o n c l u s i o n , 7B2 is a secretory protein o f the h a m s t e r p a n c r e a t i c islets, a n d its secretion is u n d e r a certain control.

Acknowledgements W e t h a n k Dr. Michel C h r e t i e n (Clinical R e s e a r c h I n s t i t u t e o f M o n t r e a l , M o n t r e a l , C a n a d a ) for p r o v i d i n g a 7B2 a n t i s e r u m a n d Mrs. H i t o m i N a k a m u r a for secretarial services. This w o r k was s u p p o r t e d , in part, by a G r a n t - i n - A i d for C a n c e r R e s e a r c h (2-17) from the M i n i s t r y o f H e a l t h a n d Welfare o f J a p a n .

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414 14 Simon, J.-P., Bader, M.-F. and Aunis, D., Secretion from chromaffin cells is controlled by chromogranin A-derived peptides, Proc. Natl. Acad. Sci. USA, 85 (1988) 1712-1716. 15 Galindo, E., Rill, A., Bader, M.-F. and Aunis, D., Chromostatin, a 20-amino acid peptide derived from chromogranin A, inhibits chromaffin cell secretion, Proc. Natl. Acad. Sci. USA, 88 (1991) 1426-1430. 16 Fasciotto, B.H., Gorr, S.-U., Bourdeau, A.M. and Cohn, D.V., Autocrine regulation of parathyroid secretion: inhibition of secretion by chromogranin-A (secretory protein-I) and potentiation of secretion by chromogranin-A and pancreastatin antibodies, Endocrinology, 127 (1990) 1329-1335. 17 Huttner, W.B., Gerdes, H.H. and Rosa, P., The granin (chromogranin/secretogranin) family, Trends Biochem. Sci., 16 (1991) 27-30.