Chronic exposure of cultured rat pancreatic islets to elevated concentrations of islet amyloid polypeptide (IAPP) causes a decrease in islet DNA content and medium insulin accumulation

ELSEVIER

Regulatory Peptldes 53 (1994) 103-109

Chronic exposure of cultured rat pancreatic islets to elevated concentrations of islet amyloid polypeptide (IAPP) causes a decrease in islet D N A content and medium insulin accumulation Stellan Sandier a,,, Mats Strldsbergb dDepartment oJ Medtcal Cell Btolog~ Btomedwal Center Uppvala Umverstt) S-751 23 Uppsala Sweden bDepartment of Chmcal Chemtgtn Umversttl Hospttal Uppsala Umver~lt) S-751 85 Uppsala, S~eden

Recewed 15 February 1994 revised version received 26 May 1994 accepted 16 June 1994

Abstract The biological action of islet amylold polypeptlde (lAPP) remains to be established, although a role for lAPP In causing fl-cell failure in diabetes has been proposed Acute in vitro experiments with IAPP have given controversial results as to fl-cell msuhn secretion We have studied the effect of a long-term exposure of pancreatic islets to elevated IAPP concentrations Thus, rat pancreatic islets were cultured for 4 days in medium supplemented with 0, 0 1, 1, 10, 100 or 1000 nM of synthetic amldated rat IAPP Islets exposed to the two highest IAPP concentrations contained about 20°0 less DNA, whereas the rate of DNA synthesis was unchanged Culture with 1000 nM IAPP, but not the lower concentrations of the peptlde, slightly decreased the glucose oxidation rate There was a correlation between increasing lAPP concentrations and and a dechne in the medium insulin accumulation The preceding IAPP exposure did, however, neither significantly affect basal and glucose-stimulated lnsuhn secretion nor (pro)insulin and total protein biosynthesis rates, when tested after the culture The finding of a decreased islet cell DNA content after culture with elevated lAPP concentrations suggests a toxic action to islet cells, whereas a putative inhibition of the islet insulin production appears to be transient K e i ~,ords Amyhn, Beta cell, Insulin secretion, Islet amylold polypeptlde, Pancreatic islet, Tissue culture

I. Introduction Islet amylold polypeptlde ( l A P P ) or amylln has been shown to be colocahzed with m s u h n in gran* Corresponding author Fax +46 18 556401 0167-0115/94/$7 00 © 1994 Elsevier Soence B V All rights reserved SSD1 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 5 7 - 5

ules of pancreatic fl-cells [5,14] Despite extensive investigations, the physiological role(s) of the peptide have not yet been established [22] Some experimental data suggest that I A P P may exert peripheral effects on muscle and liver cells, which could lead to decreased glycogen synthesis and

104

S Sandier 14 5tH&bere,, Reeulator~ Pepttde~ 53 (1994) 103-109

an increased glucose production [6] It has thus been suggested that hypersecretlon of lAPP may contribute to increased peripheral msuhn resistance and to the development of non-msuhn-dependent diabetes mellitus [6] Several studies suggest that lAPP cosecrete together with lnsuhn at an essentially constant molar ratio [9-11,18], but during pathological processes a relative hypersecretlon of lAPP in relation to lnsuhn might occur A direct effect of lAPP m the regulation of msuhn release from the pancreatic /3-cells, perhaps via paracrlne or autocrine influences, has also been proposed [2] This Issue and a putative toxic effect of lAPP has been addressed in the present communication In these experiments ~solated rat pancreatic islets have been exposed in tissue culture to different concentrations of rat lAPP. whereupon islet lnsuhn and D N A contents as well as rates of cell rephcatIon, glucose oxidation, (pro)lnsuhn and total protein biosynthesis and glucose-stimulated insuhn secretion have been measured

2. Materials and methods 2 1 Chemtcals Chemicals were purchased from BoehrlngerMannhelm (Mannhelm, Germany) collagenase from Clostrldmm htstoh'twum,type CLS (EC 3 4 24 3), Northumbria Blologlcals (Cramhngton, U K ) culture medium R P M I 1640 and fetal calf serum (FCS), Miles (Slough. U K ) bovine serum alumln (BSA), Amersham International (Amersham, U K ) L-[4 5-3H]leucme, D-[U-14C]glucose and [meth3l-3H]thymldlne, Sigma Chemicals (St Louis. MO, USA) antimycin A, Hepes and thymldlne, Novo Industries (Copenhagen, Denmark) a kit for lmmunoassay for msuhn, New England Nuclear (Boston, MA, USA) hyamlne h:ydroxide, Peninsula Laboratories, (Belmont, CA, USA) Synthesized amldated rat and human IAPP and a polyclonal rabbit antl-IAPP antiserum

2 2 Islet tsolatlon and culture Non-Inbred, adult male Sprague-Dawley rats were obtained from a local colony (Blome&cal Center, Uppsala, Sweden) Pancreatic islets were isolated by a collagenase digestion procedure [17] Groups of 150-200 Islets were precultured free-floating for 6-7 days in medium R P M I 1640 (11 1 mM glucose) supplemented with 10°o (v/v) FCS at 3 7 : C m an atmosphere of huml&fied air + 5° o CO2 Medium was changed every second day Then islets in groups of 50-75 were transferred to new culture dishes contaming 2 25 ml R P M I 1640 + 0 25 ml FCS ~lthout or with addition of rat lAPP at the concentrations 0 1, 1, 10, 100 and 1000 nM The Islets were further cultured for 4 days and the medium ~ as exchanged after two days After 4 days of culture the ~slets were subsequently treated according to one of the protocols given below 2 3 lAPP and msuhn dete.nmattons On da2y 4 the number of remaining islets were counted m a stereomlcroscope and subsequentl~ all islets were harvested and homogenlsed in 0 2 ml redlstllled water A fraction of the homogenate was mixed with acid ethanol and insulin was extracted overnight at 4°C The insulin concentration of the extract was measured by RIA [8] Another fraction of the aqueous homogenate was used for D N A measurement b~y fluorophotometry [ 17] Samples of the culture medium were collected for measurement of medium insuhn and IAPP concentrations on da) 2 and day 4 The RIA for lAPP has been described in detail elsewhere [19] H u m a n IAPP ~ a s used for standard and tracer preparations The detection hmlt of the assay was 0 125 fmol/tube No cross-reaction against calcltonln gene-related peptlde was observed 2 4 Islet msuhn telease After culture triplicate groups of 10 islets were transferred to sealed glass vials containing 0 25 ml

S Sandier M Stndsberg/Regulator>, Pepndes 53 (1994) 103-109

Krebs-Rlnger bicarbonate buffer supplemented with 2 mg/ml BSA and 10 mM Hepes, hereafter referred to as K R B H The islets were incubated at 37°C (95~o O2+ 5~o CO2) at 17 mM glucose for 1 h Then the incubation medium was gently removed and replaced by K R B H supplemented with 16 7 mM glucose, and the incubation was continued for a second hour The incubation media were collected and frozen at - 2 0 ° C prior to RIA for lnsuhn [8] 2 5 Islet glucose oxMatwn rate

For estimation of glucose oxidation rates groups of 10 islets in trlphcate were incubated for 90 mln In 100 /~1 K R B H without BSA supplemented with D-[U-14C]glucose and 16 7 mM non-radiactlve glucose, in glass vials at 37 °C in an atmosphere of 95 ~o 02 and 5~o CO2 The incubation was interrupted and 0 05 mM Antlmycm A added 14CO2 liberated was trapped in hyamme hydroxide and the radioactivity measured by liquid scintillation counting

105

aqueous homogenate were precipitated with 10% trlchloroacetlc acid, and the labelled DNA was separated from unbound radioactivity via filtering through glass microfibre filters, and the radioactivity on the filters were counted In two other ahquots of the homogenate the DNA content was determined 2 8 Statistical analysts

A mean was calculated from each duplicate or trlphcate group of islets and then considered as one separate observation Furthermore, every observation represented different rat islet donors Values were expressed as means + S E M and groups of data were compared, using Student's paired t-test The culture medium insulin and IAPP accumulation data were compared by ANOVA and Flsher's protected least statistical difference (PLSD) test, using StatVlew® (Abacus Concepts, Berkeley, CA, USA) These data were also analysed by linear regression test

2 6 Islet (pro)msuhn and totalprotem biosynthesis rate

(Pro)lnsuhn and total protein biosynthesis rates were measured in duplicate groups of 10 islets incubated for 2 h at 37°C at 16 7 mM glucose in 100/~l K R B H and 50/zC1/ml L-[4 5-3H]leuclne Islets were subsequently homogenlsed in water and the total protein biosynthesis rate was measured after trlchloroacetlc acid precipitation and the (pro)lnsuln biosynthesis rate was determined by an immune absorption technique [17] 2 7 Islet cell rephcatton rate

In separate experiments groups of islets were cultured under the conditions described above for 4 days with or without addition of 100 nM IAPP On the last day of culture 1 #C1/ml of [methyl3H]thymldlne was added to the culture dishes After 2 h, duplicate gropus 50 islets were disrupted in 250 /~1 redlstllled water Ahquots of 50 /zl of the

3. Results

The concentration of lnsuhn in the culture medium tended to decline with higher concentrations of IAPP, and this attained statistical significance at 1000 nM IAPP on day 2, but not on day 4, using ANOVA (Table 1) However, both on day 2 ( P < 0 01) and day 4 ( P < 0 05) there was a negative linear correlation between increasing IAPP concentrations and the medium insulin accumulation The concentration of IAPP in the culture media of the control group, not supplemented with the polypeptide, was notable (Table 1) After medium supplementatlon with 0 1-10 nM IAPP the medium concentration of IAPP was not significantly changed compared to the control group Whether an influence of IAPP secretion to the culture medium occured following 10, 100 and 1000 nM of IAPP addition is unclear, since at these concentrations the

S SandleJ ~,1 Strut~hetg Re~,mlatorl PeptMe~ ~3 f1994)103-109

106 Table 1

Medluln I APP and insulin concentrations alter supplementation of rat islet cultures ~lth rat s)nthetlc I APP lAPP added (nM)

lAPP concentration (nM)

0 0 1 10 10 ll)O 1000

lnsuhn concentration (nM)

Da~ 2

Da\ 4

Da~ 2

Da\ 4

411_+ I 2 2 6 _+0 7 25+1) 6 6 1-+07 369_+73*** 422 + 11 7***

36+03 2 1 + 0 ~s 26-+(14 60+05 4 2 0 + 4 3 *~ 428 _+20 2***

709-+ ~,9 5 65q _+ 125 ~70-+12s 72~_+ 123 4 9 _ + 6 _~6 3~7 ~ 6 ~, 8*

715-+ 66(} 635 _+b5 3 695+955 656+1711 _~ ~ 7 _+ 53 6 4~6 + 54 7

Rat pancreatic islets in groups of 50 x~ere cultured in 2 25 ml ol medium RPMI 1640 (11 1 mM glucose)+ 0 25 ml FCS with or x~lthout addition ol lAPP, as indicated Medmm x~as changed after t ~ o da~s and fresh similar medmm x~as added Medium samples x~ere taken after 2 and 4 days of culture Values are means + S E M for 6 experiments *, ** and *** denote P < 0 05 P < 0 01 and P
released amounts

of IAPP

than the supplemented IAPP

supplementatton

tion of lAPP

from the Islets were less

amounts

Following 1000 nM

the measurable

concentra-

t~o day's after addition ~ as around 400

nM, whmh could indicate the degree of degradation of IAPP

occurrmg

Separate IAPP

expertments

1100 and Then

were

1000 nM)

dishes containing islets

in t h e m e d a a

RPMI

the IAPP

was

a c u t e l y (0 h ) a n d concentration

added

to culture

1640 + 10°o FCS,

concentration

where but no

was measured

48 h

The lAPP

35 7 + 7 7°,, (n = 5) a t 4 8 h, w h e n t h e l A P P

concen-

tration at 0 h was set to 100°,, C u l t u r e f o r 4 d a y s in t h e p r e s e n c e o f I A P P ( 1 0 a n d 100 n M ) c a u s e d

performed

after 24 h and

w a s 73 1 + 18 9 ° 0 (n = 5) at 2 4 h a n d

the number

a shght, but significant reduction

o f i s l e t s r e t r i e v e d ( T a b l e 2)

there was about 20 oo less DNA cultured

with addition

m

Moreover,

p e r ~slet in t h e g r o u p s

o f 100 n M

and

1000 nM,

s u g g e s t i n g a cell l o s s ( T a b l e 2) T h e l n s u h n c o n t e n t

Table 2 lslct retrlc'~dl D N A and msuhn contents of rat pancreatic islets cultured tor 4 days x~lth or ,,~lthout addition of lAPP I A PP (nM)

Islet retrle~ al ("o)

(Itg DNA,' 10 islets)

DN ,~ content

Insulin content tng lnsuhn 10 islets)

lnsuhn DN A (ng insulin ~g D N A )

0 0 1 1 10 100 1001)

95 0 + 0 8 920+ 1 8 92 6 + 2 8 8 9 4 + 1 8* 88 0 + 2 0* 91) 6 + 2 6

0 31 + 0 01 1129+002 I) 27 +_0 02 031_+002 [) 25 _+0 03* 0 24 + 0 02**

68b + 174 733_+914 635 + 121 687+94 1 606 + 93 3 639 -+ 82 8

2252 + 575 2648+351 2385-+ 431 2229+265 2361 + 263 270 ~,+ 346

The islets had been cultured as described m Table 1 Values are means + S E M for 6 experiments * and ** denote P < 0 05 and P < I"101 compared to the control group of islets with no IAPP added using Student's paired t-test

S Sandier M Smdsberg/Regulator), Pepndes 53 (1994,) 103-109

107

Table 3 lnsuhn release and glucose oxidation rate of rat pancreanc islets cultured for 4 days with or without addmon of IAPP lAPP (nM)

0 01 1 10 100 1000

Insuhn release (ng lnsuhn/10 islets x 60 mm)

Glucose oxidation (pmol glucose/10 islets × 90 mm)

l 7 mM glucose

16 7 mM glucose

16 7 mM glucose

13 6_+ 1 7 120+26 160+27 15 4+ 1 9 11 1_+2 1 13 6 _+2 4

58 7_+ 100 596+89 724+ 123 68 3 + 164 679+ 12 1 7l 3 _+16 5

556_+61 9 498+326 479+543 457+45 9 446_+656 449 _+60 0*

The islets had been cultured as described m Table 1 lAPP was not present dunng the acute msuhn release or glucose oxldanon mcubanons, after the culture period During the msuhn release experiments the islets were incubated dunng the first hour at 1 7 mM glucose and during the second hour at 16 7 mM glucose Values are means _+ S E M for 6-7 experiments * denotes P<0 05 vs islets w~th no IAPP added, using Student's pmred t-test

o f the islets was not significantly affected by any concentration o f I A P P (Table 2) W h e n glucose-stimulated insulin release was examined after culture, it was found that the b a s a l islet insulin release at 1 7 m M glucose was not altered by I A P P (Table 3) After stimulation with 16 7 m M glucose, all gropus o f islets r e s p o n d e d with an about 4 - 5 - f o l d increase in insulin secretion similar to the control group In line with the latter finding, the Islet glucose oxidation rate at 16 7 m M glucose was similar to the control group, except for islets cultured with addition o f 1000 n M l A P P which exhibited an approximately 20 °/o lowered oxidative rate (Table 3) A d d i t i o n o f l A P P (1000 n M ) for 4 days did neither affect the islet rate o f (pro)insulin biosynthesis (control 6 8 9 + 3 8 vs l A P P 683+38 kdpm/10 islets x 2 h, n = 9, P > 0 05) nor the total protein biosynthesis rate (control 2 0 5 + 15 2 vs l A P P 214 + 19 3 k d p m / 1 0 islets × 2 h, n = 9, P > 0 05) As a result also the percentage fraction o f synthesized (pro)insuhn o f the total protein biosynthesis rate was u n c h a n g e d (control 34 3 + 4 3 vs I A P P 34 5 + 3 5, n = 9, P > 0 05) Finally, the D N A replication rate on d a y 4, as m e a s u r e d by a tritlated thymidlne Incorp o r a n o n procedure, in M e t cells cultured with

supplementation o f 100 n M I A P P was equal to the control group (control 162 + l l 2 vs 100 n M I A P P 157 + 10 0 dpm//ag D N A × 2 h, n = 11, P > 0 05)

4. Discussion The current findings suggest that long-term in vitro exposure to I A P P affected some islet cell functions These functions were, however, not necessesarlly confined to the fl-cells The m o s t c o n s p i c u o u s observation was the d e c r e a s e d D N A content in the islets cultured in the presence o f high concentrations o f I A P P The experiments where t n t i a t e d thymldlne i n c o r p o r a t i o n was studied on d a y 4 did not reveal any d e c r e a s e d D N A synthesis, which otherwise could have suggested that a lowered Islet cell replication was contributing to this effect Thus, the red u c e d islet D N A content is m o s t likely due to an Increased loss o f islet cells in culture In this context it is o f great interest that h u m a n fibrlllar amyhn was recently r e p o r t e d to be toxic to b o t h h u m a n and rat islets in vitro [13] However, it is currently unclear by which cellular m e c h a n i s m ( s ) l A P P might have induced cell death in the present study L o r e n z o et al m a d e observations suggesting that a p o p t o s i s

108

S Sandier M Strtdsberg/ Regulatom Pepndes 53 11994)103-109

was reduced by IAPP [ 13 ] Another Interesting morphological observation an human pancreatic glands is that islet/~-cells close to even very small amylold deposits showed quite extensive disruption of the cell membrane [21] Since we did not measure glucagon and somatostatm contents in the islets and the insuhn content was not significantly reduced, an obwous posslblhty is that the islet non-/~-cells had been reduced in number following culture with IAPP The present glucose oxidation data makes it unhkely that an impaired islet glucose metabolism, which for instance is the case in rat pancreatic islets exposed to the cytokme anterleukm-lb [17], is the cause of the putative cell death The shghtly lowered Islet glucose oxidation rate observed after culture with 1000 nM IAPP, may rather reflect a reduced cell content per islet, as indicated b~ the D N A measurements The medmm msuhn concentration was decreased both on days 2 and 4 after addition 1000 nM IAPP to the cultures, but It appeared as if this suppression tended to become less pronounced on da~ 4 It can thus be speculated that IAPP induced Inhibition of lnsuhn release was only transient This notion is supported by the finding that the islet glucoseshmulated lnsuhn release, when tested in the absence of IAPP acutely after four days of culture, was not impaired It can nevertheless not be excluded that a putative IAPP effect on the lnsuhn secretion at 16 7 m M glucose was reversed by the 1 h incubation at 1 7 mM glucose m the insulin release experiments The fact that no change of the basal lnsuhn secrehon at 1 7 mM glucose was observed, however, argues against this notion Acute add~uon of IAPP m lnsuhn secretion expemments has indicated varmble results Thus, 0 1-1000 nM of rat IAPP was found not to affect glucose-stimulated insuhn release of isolated rat islets [1,3] and in pancreas perfusion experiments of the rat using 100 nM IAPP [ 12], whereas 10,000 nM IAPP slightly inhabited glucose-stimulated insulin release of rat islets in vitro [1,15,16] On the other hand, an inhibition of insulin secretion was recently reported at a lob con-

centration as 0 075 nM of rat IAPP in the perfused rat pancreas [7] Moreover, data suggest that the /~-cell sensitivity to IAPP in vitro might be higher in single cell preparations [20] This might explain wh~ rat msuhnoma cells, growing an a monolayer, showed an inhibited proansuhn biosynthesis, after exposure for 6 h to 10-100 nM of human IAPP [4] In conclusion the present study suggests that IAPP might to some extent affect islet cell vlablllt~ and some/~-cell functions Since IAPP appears to be the major constituent of Islet amylold deposits [22] It seems important to further elucidate the possible role of IAPP in processes of islet cell destrucuon or under condmons of increased functional demands on the/~-cell, e g , in the period preceding the outbreak of diabetes

5. Acknowledgements We are grateful for the excellent technical assistance by Margareta Engkvtst, Eva Forsbeck, UllaBrItt Jonsson, Astrid Nordln and Lisa Palhn This study was supported b) grants from the Swedish Medical Research Council (12P-10739, 12X-109, 12X-8273, 12X-9884) the Swedish Diabetes Association, the Nordic Insulin Fund, the Aage LomsHansen Fund, the Ernfors Family Fund, the Juvenile Diabetes Foundation International and the Erlk, Karln and G o s t a Selander's Fund

References [ 1] ~rRajab ~X and Ahren B Effects of amldated rat islet amvlold polypetlde on glucose-stimulated msuhn secretion m ~lVO and m ~ltro m rats, Eur J Pharmacol 192 (199l) 443-445 [ 2] Bretherton-Watt, D and Bloom S R Islet amvlold pol~peptlde The cause of t)pe-2 d~abetes ) Trends Endocrmol Metab 2 (1991) 203-206 [ 3] Brodemck, C L , B r o o k e G S , D i M a r c h l R D and Gold G , Human and rat amyhn have no effects on msuhn secretion in isolated rat pancreatic ~slets B~ochem Blophvs Re~ C o m m u n , 177 (1991) 932-938

S Sandier, M Stndsberg/Regulator) Pepndes 53 (1994) 103-109 [ 4] Chuang, L -M, Wu, H -P, Jou, T -S, Tal, T -Y and LIn, B J , Inhibitory effect of islet amylold polypeptlde of glucose-reduced prolnsuhn biosynthesis m rat lnsuhnoma cells. Pancreas, 7 (1992) 472-476 [ 5] Clark, A , Edwards, C A , Ostle, L R , Sutton, R , Rothbard, J R , Morris, J F and Turner, R C , Locahsatlon of islet amylold peptlde in hpofuscm bodies and secretory granules of human B-cells and islets of type-2 diabetic subjects, Cell T~ssue Res, 257 (1989) 179-185 [ 6] Cooper, G J S, Leighton, B , Dlmltrmdls, G D , ParryBllhngs, M , Kowalchuk, J M , Howland, K , Rothbard, J B , Wdhs, A C and Reid, K B M , Amyhn found m amylold deposits m human type 2 diabetic patients, Proc Natl Acad SCl USA, 85 (1988) 7763-7766 [ 7] Degano, P , Sllvestre, R A , Salas, M , Pelro, E and Marco, J , Amyhn inhibits glucose-induced lnsuhn secretion in a dose-dependent manner Study in the perfused rat pancreas, Regul Pept, 43 (1993) 91-96 [ 8] Hedlng, L G , Determination of total serum msuhn (IRI) in insuhn-treated patients, Dlabetologla, 8 (1972) 260-266 [ 9] Fehmann, H C , Weber, V, G0ke, R , GOke, B and Arnold, R, Cosecretlon of amyhn and msuhn from isolated rat pancreas, FEBS Lett, 262 (1990) 279-281 [10] Kahn, S E , D'Alesslo, D A , Schwartz, M W , Fujimoto, W Y, Enslck, J W , Taborsky Jr G T , and Porte Jr, D , Evidence of secretion of islet amylold polypeptlde and lnsuhn by fl-cells, Diabetes, 39 (1990) 634-638 [11] Kanatsuka, A , Maklno, H , Ohsawa, H , Tokuyama, Y, Yamaguchl, T , Yoshlda, S, and Adachl, M , Secretion of islet amylold polypeptlde and insuhn by fl-cells, FEBS Lett, 259 (1989) 199-201 [12] Koglre, M , Ishlzuka, J , Thompson, J C and Greely J r , G H , Inhibitory action of islet amylold polypeptlde and calcltonm gene-related peptlde on release of lnsuhn from isolated perfused rat pancreas, Pancreas, 6 (1991) 459-463 [13] Lorenzo, A , Razzabonl, B , Weir, G C and Yanker, B A ,

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Pancreatic islet cell toxlclty of amyhn associated with type-2 &abetes melhtus, Nature, 368 (1994) 756-760 LuklnlUS, A , Wllander, E , Westermark, G T , EngstrOm U and Westermark, P , Co-localization of islet amylold polypeptlde and msuhn m the B cell granules of the human pancreatic islets, Dlabetologla, 32 (1989) 240-244 Nagamatsu, S, Carrol, R J , Grodsky, G M , and Sterner, D F , Lack of islet amylold polypetlde regulation of msuhn biosynthesis or secretion m normal rat islets, Diabetes, 39 (1990) 871-874 Ohsawa, H , Kanatsuka, A , Yamaguchl, T , Maklno, H and Yoshlda, S, Islet amylold polypetlde inhibits glucosestimulated insulin secretion from ~solated rat pancreatic islets, Blochem Blophys Res Commun, 160 (1989) 961967 Sandier, S, Andersson, A and Hellerstr0m, C , Inhibitory effects of interleukln 1 on insulin secretion, lnsuhn biosynthesis and oxldatwe metabolism of isolated rat pancreatic islets, Endocrinology 121 (1987) 1424-1431 Strldsberg, M , Sandier, S and Wllander, E, Cosecretlon of islet amylold polypeptlde (IAPP) and msuhn from isolated rat pancreatic islets following stimulation or mhlbltlOn of fl-cell function, Regul Pept, 45 (1993) 363-370 Stndsberg, M , Wllander, E , Oberg, K , Lundqvlst G and Enksson, B, Islet amylold polypeptlde (IAPP) producing pancreatic islet tumor A clinical and biochemical characterization, Scand J Gastroenterol, 27 (1992) 381-387 Wagoner, P K , Chen, C, Worley, J F , Dukes, I D and Oxford, G S, Amyhn modulates fl-cell glucose sensing vm effects on stimulus-secretion coupling, Proc Natl Acad So USA, 90 (1993) 9145-9149 Westermark, P , Free structure of islets of Langerhans in insular amyloldosls, Vlrchows Arch A, 359 (1973) 1-18 Westermark, P , Johnson, K H , O'Brlen, T D and Betsholtz, C , Islet amylold polypetlde - a novel controversy m dmbetes research, Dlabetologla, 35 (1992) 297-303