Acta Physiol Scand 1990, 138, 389-394
Failure of islet amyloid polypeptide t o inhibit basal and glucose-stimulated insulin secretion in model experiments in mice and rats M. PETTERSSON" and B. AHREN"? Departments of "Pharmacology and +Surgery, University of Lund, Sweden PETTERSSON, M. & AHREN,B. 1990.Failure of islet amyloid polypeptide to inhibit basal and glucose-stimulated insulin secretion in model experiments in mice and rats. Acta Physiol Scand 138, 389-394. Received 17 May 1989, accepted 2 October 1989. ISSN 00014772. Departments of Pharmacology and Surgery, Lund University, Sweden. Islet amyloid polypeptide (IAPP), also known as amylin, has previously been demonstrated to occur in amyloid deposits in pancreatic islets in type 2 diabetics, and, therefore, the peptide has been suggested to be involved in the pathogenesis of diabetes. The 37 amino acid peptide shows approximately 50% homology with the intrapancreatic neuropeptide calcitonin gene-related peptide (CGRP), a peptide that inhibits insulin secretion. We therefore examined, in model experiments in mice and rats, if IAPP also exerts this effect. IAPP was given intravenously, at dose levels at which CGRP previously has been shown to inhibit insulin secretion. Thus, in mice, IAPP was injected at 0.85 and 4.25 nmol kg-', and in rats IAPP was infused at 17 or 68 pmol min-'. However, neither basal nor glucose-stimulated insulin release was inhibited by IAPP under these experimental conditions. We also investigated if IAPP (10-'l to I O - ~M), when incubated in vitro with isolated, overnight-cultured rat islets, could affect insulin secretion induced by glucose (3.3, 8.3 or 11.7 mM). However, also in vitro no effect by IAPP on insulin release was observed. Hence, in mice and rats, IAPP does not inhibit insulin secretion under experimental conditions identical to those previously used to demonstrate an inhibition by CGRP. Therefore, we conclude ( I ) that the homologous amino acid sequence within IAPP and CGRP does not seem to be sufficient for inducing inhibition of insulin release in mice and rats and (2) that the possible involvement of IAPP in the pathogenesis of diabetes type 2 still remains speculative. Key mords: insulin secretion, in vitro, in vivo, islets of Langerhans, islet amyloid polypeptide, mice, nerve tissue protein, rats.
Islet amyloid polypeptide (IAPP), also known as amylin, is a 37 amino acid polypeptide that has been purified from amyloid deposits in islets of Langerhans in type z diabetics and from insulinoma tissue (Westermark et al. 1986,1987a, b, Clark et al. 1987, Cooper et al. 1987a). IAPP has also been demonstrated to occur within the normal p-cells (Cooper et al. 1987 b, Westermark et al. 1987 c). Analysis of the amino acid sequence of IAPP has demonstrated approximately 50 yo homology with the intrapancreatic neuropeptide, Correspondence : Dr M. Pettersson, Department of Pharmacology, Solvegatan 10, S-223 62 Lund, Sweden.
calcitonin gene-related peptide (CGRP) (Cooper et al. 1987a, Westermark et al. 1986). This latter peptide, CGRP, has earlier been shown to inhibit insulin secretion in mice, rats, and pigs (Pettersson et al. 1986, 1987, AhrCn et al. 1987, Pettersson & A h r h 1988 a, Ishizuka et al. 1988 a, b). Because of the occurrence of IAPP in islet amyloid, which is markedly increased in diabetic islets (Westermark et al. 1986, 1987a, b, Clark et al. 1987, Cooper et al. 1987b, Johnson et al. 1988), and its structural similarity to CGRP, which inhibits insulin secretion, suggestions have been made that IAPP might be involved in the pathogenesis of diabetes mellitus. One study recently showed that IAPP inhibited glucose-
389
390
.M. Pettersson and B. 24hre'n
stimulated insulin release from isolated rat islets, b u t onll- a t a h-ery high dose level, IO-' M (Ohsawa et al. 1989). We have investigated if IAPP affects insulin secretion in model experiments and a t dose levels at which we previously showed that CGRP inhibits insulin secretion (Pettersson rt ul. 1986, Pettersson & Ahren 1988a, b). T h u s , the effects of IAPP on insulin secretion in mice and rats and in isolated rat islets were examined.
short, each pancreas was cut into small pieces and rinsed twice in Hanks' buffer, supplemented with glucose (5.6 mM). After rinsing, j ml Hanks' buffer, supplemented with collagenase (type IV, 216 m U mg-'; Boehringer Mannheim GmbH, F R G ; 40 mg), was added to the pancreas, and the solution was S at 37 "C. shaken vigorously by hand for I ~ I min The digestion was stopped by adding 90 ml Hanks' buffer and islets were collected under a stereomicroscope. Islets from two rats were mixed in two petrishells and then cultured over night in 5 ml RPMI 1640 medium supplemented with 109; NU-serum, I O O I U ml-' of penicillin, IOO p g ml-I streptomycin Sf .%'I- E R I A 1, S AND M E T H 0 D S and 0 . 2 5 p g ml-' amphotericin B (Labassco, .4nima/s. The experiments were performed either in Goteborg, Sweden). The cells were cultured at 37 "C male Sprague-Dawley rats (zoo-~.+o g body a t ) or in in an atmosphere saturated with a mixture of 5 yo CO, in 0,. T h e following day, the islets were divided into female NMRI mice (25-3j g body wt) purchased from hticimex, Stockholm, Sweden. .ill animals groups of 10 islets. Each group was preincubated in dishes (Multidish Nunclon, Nunc, Roskilde, Denwere kept on a standard pellet diet (.\ma-Ewos, Siidertalje, Sweden) and tap water ad libitum before mark) in 1.0 ml modified KRB-Hepes medium with 3.3 mM glucose for 45 min at 37 "C. T h e medium the experiments. consisted of (in mM): I 15 NaCI, 4.4 KCI, 1.5 KHPO,, I n - ~ i z ot,.rperiments in mire. In the first experimental series, s!-nthetic human L4PP (Peninsula, Belmont, 0.8 MgSO,, 24 NaHCO, and 1 0 Hepes. After C.4? CS~4;dissolved in saline 0.I O 0 gelatine) was preincubation, eight islets were selected from each injected intravenousll- at 0.8j or 4 . z j nmol kg-' in group and transferred as single islets into new three different groups of unanaesthetized mice, and chambers (Microwell Module F-8, Irnmunoquality, blood was sampled from the retro-orbital venous hledium binding capacity, Nunc, Roskilde, Denmark). plexus at 2, 6, or 1 0 min after the injections. The One islet was incubated per chamber in 0 . 1 ml of the I-olume load was 10 11 ' g-'. Controls were given saline. modified KRB medium supplemented with 3.3 mM In the second experimental series, IAPP (0.85 or glucose. The single islets were then incubated for 90 min in 3.3, 8.3, or I 1.7 mM glucose, to which IAPP 4.2jnmol kg-') was injected alone or together with D-glucose (British Drug Houses, Poole, UK; 2.8 mmol (10-'' M or I O - ~M) was added according to the kg-') and blood samples were taken 2 min later. This protocols. After incubation, 50 ,ul medium from each time point was chosen since the peak plasma insulin 'chamber were collected for analysis of insulin. All level after intravenous glucose injection occurs after chemicals were from British Drug Houses Ltd, Poole, UK, except when otherwise stated. 2 min (r2hren 8i Lundquist 1981). The volume load Determination of insulin and glucose. Insulin concen~ o / t g l '. Controls were injected with was, again, trations in plasma and medium were measured by saline. In-?wo rrperiments in rats. Rats were anaesthetized radioimmunoassay (Heding 1966) using commercially v-ith thiopenthal (-4bbot SPA, Campo\erde, Itall-), available '"I-labelled porcine insulin (Novo Industri 90 mg kg-' i.p. Poll-ethylene catheters were inserted .%/S, Bagsvaerd, Denmark) and guinea-pig antiinto the right femoral vessels. The venous catheter was porcine insulin (Milab AB, Malmo, Sweden). Porcine connected to an infusion pump and used for infusion insulin was used as standard. Plasma glucose concenof the different substances at a rate of 0.07 rnl min-l. trations were measured by the glucose oxidase method Blood samples of 150 pl were collected through the (Bruss & Black 1978). Statistics. Two-way analysis of variance (two-way arterial catheter. After drawing two baseline blood samples, the ANOVA) was employed for the statistical evaluations of infusions, lasting for 30 or 70 min, were started. In the the in-zwo experiments in the rats. For evaluations of first experimental series, IAPP was infused alone at 17 the in-z9iz.o experiments in the mice and the in-vitro or 68 pmol min-' for 30 min. These dose levels equal experiments, Student's t-test was used. All results are approximately 77 and 306 pmol kg ' min-'. Controls expressed as means k SEM. were infused with saline. In the second experimental series, n-glucose (7 mg inin-') was infused for 70 min. R E S U L T S In one group ofrats, L l P P (17 or 68 pmol min-') was added to the glucose infusion after 30 min. In-riro experiments in mice (Figs. I a n d 2) In-virro erperiments. Pancreatic islets were isolated bj- the collagenase digestion method of Lacy 8i IAPP was injected intravenously a t 0.85 or 4.25 Kostianovsky (1967), with slight modifications. In nmol kg-' to unanaesthetized mice. I t was found
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that the peptide did not alter the basal plasma insulin concentrations in samples taken at 2 , 6, or 10min after injection (Fig. I). Injection of glucose (2.8 mmol kg-') elevated plasma insulin levels from 33 f 4 to 82 f2 pU ml-' (P < 0.001). This increase was not altered by a concomitant injection of IAPP (Fig. 2).
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In-vivo experiments in rats (Figs. 3 and 4) As in mice, intravenous administration of IAPP (17 or 68 pmol min-l) did not affect plasma insulin levels in rats, either under basal conditions (Fig. 3) or during glucose (7 mg min-l) infusion (Fig. 4). However, IAPP significantly, though slightly, lowered plasma glucose levels both under basal conditions and during glucose infusion (P < 0.001)(Figs. 3 and 4).
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Fig. 2. Plasma levels of insulin and glucose levels at In-vitro experiments in rats (Fig. 5) 2 min after the intravenous injection in mice of IAPP at 0.85 nmol kg-' (H)or 4.25 nmol kg-' (m), alone or Isolated, overnight-cultured rat islets were intogether with glucose (2.8 mmol kg-'). Controls (0)cubated with I A P P (10-l' to I O - ~M) at different were injected with saline. There were 20 animals in glucose concentrations (3.3, 8.3, or 11.7 mM). each group. MeansfSEM are shown. However, IAPP did not significantly alter the
392
M . Pettersson and B. Ahrin
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Fig. 4. Changes in plasma levels of insulin and glucose in rats during infusion of D-glucose (7 mg min-') together with IAPP at 17 pmol min-' (0-0; n = 7 ) or 68 pmol min-' (0-0 ; n = 7 ) . Shaded areas denote the controls infused with saline instead of IAPP (n = 7 ) . After infusion for 30 min of pglucose alone, plasma levels were : insulin 80 2 ,uU ml-' and glucose 17.4& 0.6 mmol I-'. Means fSEM are shown. Asterisks indicate the probability level of random
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Fig.
insulin release at any of these glucose concentrations. DISCUSSION
IAPP has been demonstrated to occur both within normal islet /?-cells (Cooper et al. 1987 b, Westermark et af. 1 9 8 7 ~ )and in islet amyloid
deposits, which are increased during the development of type 2 diabetes mellitus (Westermark et al. 1986, 1987a, b, Clark et al. 1987, Cooper et al. 1987b, Johnson et al. 1988). Therefore, the peptide might be suggested to be involved in the pathogenesis of the disease. T h i s suggestion is also inferred from its 50% structural homology with CGRP (Westermark et al.
IAPP fails to inhibit insulin secretion 1986, Cooper et al. 1987a), which is a peptide that inhibits insulin secretion (Pettersson et al. 1986, 1987, AhrCn et al. 1987, Ishizuka e t al. 1988a, b, Pettersson & AhrCn 1988a). I t could thus be speculated that IAPP, by mimicking the effect of CGRP, inhibits insulin secretion and thereby is involved in the impaired insulin secretion seen in diabetes. However, no inhibition of basal or glucose-stimulated insulin secretion was observed in our present study. W e used several different experimental conditions in the mouse and the rat, and studied the effect of IAPP also on isolated rat islets. Under all these conditions, C G R P has previously been shown to inhibit insulin secretion (Pettersson et al. 1986, 1987, Pettersson & Ahrin 1988a, b). Hence, the effects of C G R P and IAPP clearly differ, which indicates that the homologous portion of the two peptide molecules is not sufficient for the inhibitory action on insulin secretion induced by CGRP. Previously, IAPP was shown to inhibit glucose-stimulated insulin release from isolated rat islets at the very high dose level of IO-’ M (Ohsawa et al. 1989). Our study thus confirms the failure of IAPP to affect insulin secretion at lower dose levels. Hence, it is unlikely that IAPP physiologically affects insulin secretion when administered extracellularly. T h e possible function of the intracellular IAPP remains, however, to be determined. IAPP has recently been shown to inhibit basal and insulin-stimulated glycogen synthesis in isolated rat soleus muscle in vitro (Leighton & Cooper 1988). T h i s could hypothetically be an alternative mechanism by which the peptide takes part in the development of diabetes. However, we did not observe any hyperglycaemia during intravenous administration of IAPP, but rather a slight hypoglycaemia. This suggests that the net effect of IAPP on the glycaemia is dependent on different, perhaps opposing, actions, and during a short-term infusion a slight hypoglycaemia develops. We conclude that, although having approximately 50 % structural homology with CGRP, IAPP does not seem to exert any effect on the release of insulin under conditions where C G R P is known to be inhibitory. Hence, our results d o not support the hypothesis of a possible diabetogenic action of IAPP on insulin secretion. The technical assistance of Lena Kvist and the statistical advice of Claw Rerup PhD are gratefully 16
393
acknowledged. This study was supported by the Swedish Medical Research Council, Stockholm, Sweden (14X-6834 and 17P-8453), Nordisk Insulinfond, Gentofte, Denmark, the Swedish Diabetes Association, Stockholm, Sweden, Diabetesforeningen i Malmo, Sweden, Svenska Hoechst Diabetesfond, Stockholm, Sweden, Magnus Bergvalls and Albert Pdhlssons Stiftelser, Stockholm and Malmo, Sweden, Crafoordska Stiftelsen, Lund, Sweden, and by grants from the Medical Faculty, Lund University, Sweden. REFERENCES I. 1981. Effects of selective AHR~N B., & LUNDQUIST, and non-selective P-adrenergic agents on insulin secretion in vivo. Eur 3 Pharmacol 71, 93-104. A H R ~ NB., , M~RTENSSON, H. & NOBIN, A. 1987. Effects of calcitonin gene-related peptide (CGRP) on islet hormone secretion in the pig. Diabetologiu 30, 354-359. BRUSS,M.L. & BLACK,A.L. 1978. Enzymatic microdetermination of glycogen. Anal Biochem 84, 309-312. CLARK,A,, COOPER,G.J.S., LEWIS,C.E., MORRIS, J.F., WILLIS,A.C., REID,K.B.M. &TURNER, R.C. 1987. Islet amyloid formed from diabetes-associated peptide may be pathogeneic in type-2 diabetes. Lancet 2 , 23 1-234. COOPER,G.J.S., WILLIS,A.C., CLARK,A,, TURNER, R.C. & REID, K.B.M. 1987a. Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc Natl Acad Sci USA 84, 8628-8632. COOPER,G.J.S., WILIS,A.C., REID,K.B.M., CLARK, R.C., LEWIS,C.E., A., BAKER,C.A., TURNER, MORRIS,J.F., HOWLAND, K. & ROUTHBARD, J.B. 1987b. Diabetes-associated peptide. Lancet 2 , 966. HEDING,L.G. 1966. A simplified insulin radioimmunoassay method. In : L.Donato, G.Milhaud & JSirchis (eds.) LabeNed Proteins in Tracer Studies, pp. 345-350. Euratom, Brussels. J., GREELEY JR, G.H., COOPER,C.W. & ISHIZUKA, THOMPSON, J.C. 1988a. Effect of calcitonin generelated peptide on glucose and gastric inhibitory polypeptide-stimulated insulin release from cultured newborn and adult rat islet cells. Regul Pept 20, 73-82. ISHIZUKA, J., SINGH,P., GREELEY JR,G.H., TOWNSEND JR, C.M., COOPER, C.W., TATEMOTO, K. & THOMPSON,J.C. 1988b. A comparison of the insulinotropic and insulin-inhibitory actions of gut peptides on newborn and adult rat islet cells. Pancreas 3,77-82. JOHNSON, K.H., O’BRIEN,T.D., HAYDEN,D.W., JORDAN, K., GHOBRIAL, H.K.G., MAHONEY, W.C. & WESTERMARK, P. 1988. Immunolocalization of islet amyloid polypeptide (IAPP) in pancreatic beta cells by means of peroxidaseantiperoxidase (PAP) and protein A-gold techniques. Am 3 Puthol 130, 1-8. ACT 138
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I,ac.v, P . E . '& ~ O S T I ~ N O L S K 11. Y , 1967. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diahrtrs 16, 3 j-jq. I,EICHTOZ, B. & COOPER,G.J.S. 1988. Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in ritro. . X ~ i t i i r r33 j,
632-63.5.
Ai., ~ - . A M . ~ G L ' C H IT., , ~ I A K I S OH., & TO iS H I D ~ S. , 1989. Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion from isolated rat pancreatic islets. Bioi/wm Binph),s Rrs Cummiin 16n, 961 -67. PETTERSSOS, 31. & AHRE\, B. 1988a. Insulin and glucagon secretion in rats: efects of calcitonin gene-related peptide. Regid P e p 23, 37-jo. PETTERSSOS, XI. & I H R E S , B. 1988 b. Calcitonin generelated peptide inhibits glucose-stimulated insulin secretion in the rat in \-ivo and in 1-itro. Diubrtologio 3 1 %.;.31.1, P k ~ r ~ ~ . ~ s sl io. s, , .AHRE\, B., B ~ T T C H E RG., & St \ I X . L R , F. 1986. Calcitonin gene-related peptide : Occurrence in pancreatic islets in the mouse and thc rat and inhibition of insulin secretion in the mouse. Endnc.rrno/ogy I 19. 86j-869. PETT~RSSOS. Xi., L L S ~ L I S T I. .& .\HRE\, B. 1987.
Otti.iW.\,
13..
KASATSL-KA,
Neuropeptide Y and calcitonin gene-related peptide: Effects on glucagon and insulin secretion in the mouse. Endocr Res 13, 407-417.
~VESTERMARK,P., WERNSTEDT, C., WILANDER,E. & SLETTES, K. 1986. A novel peptide in the calcitonin gene-related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochem Biophys Rrs Commun 140, 827-831. \~ESTERXIAHK, P., WILANDER, E., WESTERMARK, G.T. &JOHNSON, K.H. 1987a. Islet amyloid polypeptidelike immunoreactivity in the islet B cells of type z (non-insulin-dependent) diabetic and non-diabetic individuals. Diubetologia 30, 887-892. \VESTERMARK, P., WERNSTEDT, C., OIBRIEN,T.D., IIAYDEN, D.W. & JOHNSON, K.H. 1987b. Islet am)-loid in type z human diabetes mellitus and adult diabetic cats contains a novel putative polypeptide hormone. A m 3 Pathol 127, 414-417. \\'ESTER.\IARK, P., WERNSTEDT, c., WILANDER, E., HAYDES,D.W., OBRIEN,T.D. & JOHNSON, K.H. 1 9 8 7 ~ .4m!loid . fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a novel neuropeptide-like protein also present Nut/ Acad Sci, C'SA 84, in normal islet cells. PYOC 3881-388 j.
Failure of islet amyloid polypeptide t o inhibit basal and glucose-stimulated insulin secretion in model experiments in mice and rats M. PETTERSSON" and B. AHREN"? Departments of "Pharmacology and +Surgery, University of Lund, Sweden PETTERSSON, M. & AHREN,B. 1990.Failure of islet amyloid polypeptide to inhibit basal and glucose-stimulated insulin secretion in model experiments in mice and rats. Acta Physiol Scand 138, 389-394. Received 17 May 1989, accepted 2 October 1989. ISSN 00014772. Departments of Pharmacology and Surgery, Lund University, Sweden. Islet amyloid polypeptide (IAPP), also known as amylin, has previously been demonstrated to occur in amyloid deposits in pancreatic islets in type 2 diabetics, and, therefore, the peptide has been suggested to be involved in the pathogenesis of diabetes. The 37 amino acid peptide shows approximately 50% homology with the intrapancreatic neuropeptide calcitonin gene-related peptide (CGRP), a peptide that inhibits insulin secretion. We therefore examined, in model experiments in mice and rats, if IAPP also exerts this effect. IAPP was given intravenously, at dose levels at which CGRP previously has been shown to inhibit insulin secretion. Thus, in mice, IAPP was injected at 0.85 and 4.25 nmol kg-', and in rats IAPP was infused at 17 or 68 pmol min-'. However, neither basal nor glucose-stimulated insulin release was inhibited by IAPP under these experimental conditions. We also investigated if IAPP (10-'l to I O - ~M), when incubated in vitro with isolated, overnight-cultured rat islets, could affect insulin secretion induced by glucose (3.3, 8.3 or 11.7 mM). However, also in vitro no effect by IAPP on insulin release was observed. Hence, in mice and rats, IAPP does not inhibit insulin secretion under experimental conditions identical to those previously used to demonstrate an inhibition by CGRP. Therefore, we conclude ( I ) that the homologous amino acid sequence within IAPP and CGRP does not seem to be sufficient for inducing inhibition of insulin release in mice and rats and (2) that the possible involvement of IAPP in the pathogenesis of diabetes type 2 still remains speculative. Key mords: insulin secretion, in vitro, in vivo, islets of Langerhans, islet amyloid polypeptide, mice, nerve tissue protein, rats.
Islet amyloid polypeptide (IAPP), also known as amylin, is a 37 amino acid polypeptide that has been purified from amyloid deposits in islets of Langerhans in type z diabetics and from insulinoma tissue (Westermark et al. 1986,1987a, b, Clark et al. 1987, Cooper et al. 1987a). IAPP has also been demonstrated to occur within the normal p-cells (Cooper et al. 1987 b, Westermark et al. 1987 c). Analysis of the amino acid sequence of IAPP has demonstrated approximately 50 yo homology with the intrapancreatic neuropeptide, Correspondence : Dr M. Pettersson, Department of Pharmacology, Solvegatan 10, S-223 62 Lund, Sweden.
calcitonin gene-related peptide (CGRP) (Cooper et al. 1987a, Westermark et al. 1986). This latter peptide, CGRP, has earlier been shown to inhibit insulin secretion in mice, rats, and pigs (Pettersson et al. 1986, 1987, AhrCn et al. 1987, Pettersson & A h r h 1988 a, Ishizuka et al. 1988 a, b). Because of the occurrence of IAPP in islet amyloid, which is markedly increased in diabetic islets (Westermark et al. 1986, 1987a, b, Clark et al. 1987, Cooper et al. 1987b, Johnson et al. 1988), and its structural similarity to CGRP, which inhibits insulin secretion, suggestions have been made that IAPP might be involved in the pathogenesis of diabetes mellitus. One study recently showed that IAPP inhibited glucose-
389
390
.M. Pettersson and B. 24hre'n
stimulated insulin release from isolated rat islets, b u t onll- a t a h-ery high dose level, IO-' M (Ohsawa et al. 1989). We have investigated if IAPP affects insulin secretion in model experiments and a t dose levels at which we previously showed that CGRP inhibits insulin secretion (Pettersson rt ul. 1986, Pettersson & Ahren 1988a, b). T h u s , the effects of IAPP on insulin secretion in mice and rats and in isolated rat islets were examined.
short, each pancreas was cut into small pieces and rinsed twice in Hanks' buffer, supplemented with glucose (5.6 mM). After rinsing, j ml Hanks' buffer, supplemented with collagenase (type IV, 216 m U mg-'; Boehringer Mannheim GmbH, F R G ; 40 mg), was added to the pancreas, and the solution was S at 37 "C. shaken vigorously by hand for I ~ I min The digestion was stopped by adding 90 ml Hanks' buffer and islets were collected under a stereomicroscope. Islets from two rats were mixed in two petrishells and then cultured over night in 5 ml RPMI 1640 medium supplemented with 109; NU-serum, I O O I U ml-' of penicillin, IOO p g ml-I streptomycin Sf .%'I- E R I A 1, S AND M E T H 0 D S and 0 . 2 5 p g ml-' amphotericin B (Labassco, .4nima/s. The experiments were performed either in Goteborg, Sweden). The cells were cultured at 37 "C male Sprague-Dawley rats (zoo-~.+o g body a t ) or in in an atmosphere saturated with a mixture of 5 yo CO, in 0,. T h e following day, the islets were divided into female NMRI mice (25-3j g body wt) purchased from hticimex, Stockholm, Sweden. .ill animals groups of 10 islets. Each group was preincubated in dishes (Multidish Nunclon, Nunc, Roskilde, Denwere kept on a standard pellet diet (.\ma-Ewos, Siidertalje, Sweden) and tap water ad libitum before mark) in 1.0 ml modified KRB-Hepes medium with 3.3 mM glucose for 45 min at 37 "C. T h e medium the experiments. consisted of (in mM): I 15 NaCI, 4.4 KCI, 1.5 KHPO,, I n - ~ i z ot,.rperiments in mire. In the first experimental series, s!-nthetic human L4PP (Peninsula, Belmont, 0.8 MgSO,, 24 NaHCO, and 1 0 Hepes. After C.4? CS~4;dissolved in saline 0.I O 0 gelatine) was preincubation, eight islets were selected from each injected intravenousll- at 0.8j or 4 . z j nmol kg-' in group and transferred as single islets into new three different groups of unanaesthetized mice, and chambers (Microwell Module F-8, Irnmunoquality, blood was sampled from the retro-orbital venous hledium binding capacity, Nunc, Roskilde, Denmark). plexus at 2, 6, or 1 0 min after the injections. The One islet was incubated per chamber in 0 . 1 ml of the I-olume load was 10 11 ' g-'. Controls were given saline. modified KRB medium supplemented with 3.3 mM In the second experimental series, IAPP (0.85 or glucose. The single islets were then incubated for 90 min in 3.3, 8.3, or I 1.7 mM glucose, to which IAPP 4.2jnmol kg-') was injected alone or together with D-glucose (British Drug Houses, Poole, UK; 2.8 mmol (10-'' M or I O - ~M) was added according to the kg-') and blood samples were taken 2 min later. This protocols. After incubation, 50 ,ul medium from each time point was chosen since the peak plasma insulin 'chamber were collected for analysis of insulin. All level after intravenous glucose injection occurs after chemicals were from British Drug Houses Ltd, Poole, UK, except when otherwise stated. 2 min (r2hren 8i Lundquist 1981). The volume load Determination of insulin and glucose. Insulin concen~ o / t g l '. Controls were injected with was, again, trations in plasma and medium were measured by saline. In-?wo rrperiments in rats. Rats were anaesthetized radioimmunoassay (Heding 1966) using commercially v-ith thiopenthal (-4bbot SPA, Campo\erde, Itall-), available '"I-labelled porcine insulin (Novo Industri 90 mg kg-' i.p. Poll-ethylene catheters were inserted .%/S, Bagsvaerd, Denmark) and guinea-pig antiinto the right femoral vessels. The venous catheter was porcine insulin (Milab AB, Malmo, Sweden). Porcine connected to an infusion pump and used for infusion insulin was used as standard. Plasma glucose concenof the different substances at a rate of 0.07 rnl min-l. trations were measured by the glucose oxidase method Blood samples of 150 pl were collected through the (Bruss & Black 1978). Statistics. Two-way analysis of variance (two-way arterial catheter. After drawing two baseline blood samples, the ANOVA) was employed for the statistical evaluations of infusions, lasting for 30 or 70 min, were started. In the the in-zwo experiments in the rats. For evaluations of first experimental series, IAPP was infused alone at 17 the in-z9iz.o experiments in the mice and the in-vitro or 68 pmol min-' for 30 min. These dose levels equal experiments, Student's t-test was used. All results are approximately 77 and 306 pmol kg ' min-'. Controls expressed as means k SEM. were infused with saline. In the second experimental series, n-glucose (7 mg inin-') was infused for 70 min. R E S U L T S In one group ofrats, L l P P (17 or 68 pmol min-') was added to the glucose infusion after 30 min. In-riro experiments in mice (Figs. I a n d 2) In-virro erperiments. Pancreatic islets were isolated bj- the collagenase digestion method of Lacy 8i IAPP was injected intravenously a t 0.85 or 4.25 Kostianovsky (1967), with slight modifications. In nmol kg-' to unanaesthetized mice. I t was found
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Fig. 3. Changes in plasma levels of insulin and glucose in rats during intravenous infusion of IAPP at E 17 pmol min-' ;.--.( n = 6) or 68 pmol min-' ;.--.( n = 6). Shaded areas denote the controls c5 0 (n = 6). At the start of infusions, plasma levels were Fig. I. Plasma insulin levels at 2, 6 or 10min after insulin 29 f I pU ml-' and glucose 7.9 f o . 3 mmol intravenous administration of IAPP at 0.85 nmol kg-' I-'. MeansfSEM are shown. Crosses indicate the (B) or 4.25 nmol kg-' (m) in mice. Controls (0) probability level of random difference between the were injected with saline. There were 10 animals in respective curves and controls as analysed by ANOVA. each group. MeansfSEM are shown. 2
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that the peptide did not alter the basal plasma insulin concentrations in samples taken at 2 , 6, or 10min after injection (Fig. I). Injection of glucose (2.8 mmol kg-') elevated plasma insulin levels from 33 f 4 to 82 f2 pU ml-' (P < 0.001). This increase was not altered by a concomitant injection of IAPP (Fig. 2).
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In-vivo experiments in rats (Figs. 3 and 4) As in mice, intravenous administration of IAPP (17 or 68 pmol min-l) did not affect plasma insulin levels in rats, either under basal conditions (Fig. 3) or during glucose (7 mg min-l) infusion (Fig. 4). However, IAPP significantly, though slightly, lowered plasma glucose levels both under basal conditions and during glucose infusion (P < 0.001)(Figs. 3 and 4).
0
Fig. 2. Plasma levels of insulin and glucose levels at In-vitro experiments in rats (Fig. 5) 2 min after the intravenous injection in mice of IAPP at 0.85 nmol kg-' (H)or 4.25 nmol kg-' (m), alone or Isolated, overnight-cultured rat islets were intogether with glucose (2.8 mmol kg-'). Controls (0)cubated with I A P P (10-l' to I O - ~M) at different were injected with saline. There were 20 animals in glucose concentrations (3.3, 8.3, or 11.7 mM). each group. MeansfSEM are shown. However, IAPP did not significantly alter the
392
M . Pettersson and B. Ahrin
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Fig. 4. Changes in plasma levels of insulin and glucose in rats during infusion of D-glucose (7 mg min-') together with IAPP at 17 pmol min-' (0-0; n = 7 ) or 68 pmol min-' (0-0 ; n = 7 ) . Shaded areas denote the controls infused with saline instead of IAPP (n = 7 ) . After infusion for 30 min of pglucose alone, plasma levels were : insulin 80 2 ,uU ml-' and glucose 17.4& 0.6 mmol I-'. Means fSEM are shown. Asterisks indicate the probability level of random
"'P < 0.001. difference between the respective curves and controls as analysed by ANOVA.
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Fig.
insulin release at any of these glucose concentrations. DISCUSSION
IAPP has been demonstrated to occur both within normal islet /?-cells (Cooper et al. 1987 b, Westermark et af. 1 9 8 7 ~ )and in islet amyloid
deposits, which are increased during the development of type 2 diabetes mellitus (Westermark et al. 1986, 1987a, b, Clark et al. 1987, Cooper et al. 1987b, Johnson et al. 1988). Therefore, the peptide might be suggested to be involved in the pathogenesis of the disease. T h i s suggestion is also inferred from its 50% structural homology with CGRP (Westermark et al.
IAPP fails to inhibit insulin secretion 1986, Cooper et al. 1987a), which is a peptide that inhibits insulin secretion (Pettersson et al. 1986, 1987, AhrCn et al. 1987, Ishizuka e t al. 1988a, b, Pettersson & AhrCn 1988a). I t could thus be speculated that IAPP, by mimicking the effect of CGRP, inhibits insulin secretion and thereby is involved in the impaired insulin secretion seen in diabetes. However, no inhibition of basal or glucose-stimulated insulin secretion was observed in our present study. W e used several different experimental conditions in the mouse and the rat, and studied the effect of IAPP also on isolated rat islets. Under all these conditions, C G R P has previously been shown to inhibit insulin secretion (Pettersson et al. 1986, 1987, Pettersson & Ahrin 1988a, b). Hence, the effects of C G R P and IAPP clearly differ, which indicates that the homologous portion of the two peptide molecules is not sufficient for the inhibitory action on insulin secretion induced by CGRP. Previously, IAPP was shown to inhibit glucose-stimulated insulin release from isolated rat islets at the very high dose level of IO-’ M (Ohsawa et al. 1989). Our study thus confirms the failure of IAPP to affect insulin secretion at lower dose levels. Hence, it is unlikely that IAPP physiologically affects insulin secretion when administered extracellularly. T h e possible function of the intracellular IAPP remains, however, to be determined. IAPP has recently been shown to inhibit basal and insulin-stimulated glycogen synthesis in isolated rat soleus muscle in vitro (Leighton & Cooper 1988). T h i s could hypothetically be an alternative mechanism by which the peptide takes part in the development of diabetes. However, we did not observe any hyperglycaemia during intravenous administration of IAPP, but rather a slight hypoglycaemia. This suggests that the net effect of IAPP on the glycaemia is dependent on different, perhaps opposing, actions, and during a short-term infusion a slight hypoglycaemia develops. We conclude that, although having approximately 50 % structural homology with CGRP, IAPP does not seem to exert any effect on the release of insulin under conditions where C G R P is known to be inhibitory. Hence, our results d o not support the hypothesis of a possible diabetogenic action of IAPP on insulin secretion. The technical assistance of Lena Kvist and the statistical advice of Claw Rerup PhD are gratefully 16
393
acknowledged. This study was supported by the Swedish Medical Research Council, Stockholm, Sweden (14X-6834 and 17P-8453), Nordisk Insulinfond, Gentofte, Denmark, the Swedish Diabetes Association, Stockholm, Sweden, Diabetesforeningen i Malmo, Sweden, Svenska Hoechst Diabetesfond, Stockholm, Sweden, Magnus Bergvalls and Albert Pdhlssons Stiftelser, Stockholm and Malmo, Sweden, Crafoordska Stiftelsen, Lund, Sweden, and by grants from the Medical Faculty, Lund University, Sweden. REFERENCES I. 1981. Effects of selective AHR~N B., & LUNDQUIST, and non-selective P-adrenergic agents on insulin secretion in vivo. Eur 3 Pharmacol 71, 93-104. A H R ~ NB., , M~RTENSSON, H. & NOBIN, A. 1987. Effects of calcitonin gene-related peptide (CGRP) on islet hormone secretion in the pig. Diabetologiu 30, 354-359. BRUSS,M.L. & BLACK,A.L. 1978. Enzymatic microdetermination of glycogen. Anal Biochem 84, 309-312. CLARK,A,, COOPER,G.J.S., LEWIS,C.E., MORRIS, J.F., WILLIS,A.C., REID,K.B.M. &TURNER, R.C. 1987. Islet amyloid formed from diabetes-associated peptide may be pathogeneic in type-2 diabetes. Lancet 2 , 23 1-234. COOPER,G.J.S., WILLIS,A.C., CLARK,A,, TURNER, R.C. & REID, K.B.M. 1987a. Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc Natl Acad Sci USA 84, 8628-8632. COOPER,G.J.S., WILIS,A.C., REID,K.B.M., CLARK, R.C., LEWIS,C.E., A., BAKER,C.A., TURNER, MORRIS,J.F., HOWLAND, K. & ROUTHBARD, J.B. 1987b. Diabetes-associated peptide. Lancet 2 , 966. HEDING,L.G. 1966. A simplified insulin radioimmunoassay method. In : L.Donato, G.Milhaud & JSirchis (eds.) LabeNed Proteins in Tracer Studies, pp. 345-350. Euratom, Brussels. J., GREELEY JR, G.H., COOPER,C.W. & ISHIZUKA, THOMPSON, J.C. 1988a. Effect of calcitonin generelated peptide on glucose and gastric inhibitory polypeptide-stimulated insulin release from cultured newborn and adult rat islet cells. Regul Pept 20, 73-82. ISHIZUKA, J., SINGH,P., GREELEY JR,G.H., TOWNSEND JR, C.M., COOPER, C.W., TATEMOTO, K. & THOMPSON,J.C. 1988b. A comparison of the insulinotropic and insulin-inhibitory actions of gut peptides on newborn and adult rat islet cells. Pancreas 3,77-82. JOHNSON, K.H., O’BRIEN,T.D., HAYDEN,D.W., JORDAN, K., GHOBRIAL, H.K.G., MAHONEY, W.C. & WESTERMARK, P. 1988. Immunolocalization of islet amyloid polypeptide (IAPP) in pancreatic beta cells by means of peroxidaseantiperoxidase (PAP) and protein A-gold techniques. Am 3 Puthol 130, 1-8. ACT 138
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I,ac.v, P . E . '& ~ O S T I ~ N O L S K 11. Y , 1967. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diahrtrs 16, 3 j-jq. I,EICHTOZ, B. & COOPER,G.J.S. 1988. Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in ritro. . X ~ i t i i r r33 j,
632-63.5.
Ai., ~ - . A M . ~ G L ' C H IT., , ~ I A K I S OH., & TO iS H I D ~ S. , 1989. Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion from isolated rat pancreatic islets. Bioi/wm Binph),s Rrs Cummiin 16n, 961 -67. PETTERSSOS, 31. & AHRE\, B. 1988a. Insulin and glucagon secretion in rats: efects of calcitonin gene-related peptide. Regid P e p 23, 37-jo. PETTERSSOS, XI. & I H R E S , B. 1988 b. Calcitonin generelated peptide inhibits glucose-stimulated insulin secretion in the rat in \-ivo and in 1-itro. Diubrtologio 3 1 %.;.31.1, P k ~ r ~ ~ . ~ s sl io. s, , .AHRE\, B., B ~ T T C H E RG., & St \ I X . L R , F. 1986. Calcitonin gene-related peptide : Occurrence in pancreatic islets in the mouse and thc rat and inhibition of insulin secretion in the mouse. Endnc.rrno/ogy I 19. 86j-869. PETT~RSSOS. Xi., L L S ~ L I S T I. .& .\HRE\, B. 1987.
Otti.iW.\,
13..
KASATSL-KA,
Neuropeptide Y and calcitonin gene-related peptide: Effects on glucagon and insulin secretion in the mouse. Endocr Res 13, 407-417.
~VESTERMARK,P., WERNSTEDT, C., WILANDER,E. & SLETTES, K. 1986. A novel peptide in the calcitonin gene-related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochem Biophys Rrs Commun 140, 827-831. \~ESTERXIAHK, P., WILANDER, E., WESTERMARK, G.T. &JOHNSON, K.H. 1987a. Islet amyloid polypeptidelike immunoreactivity in the islet B cells of type z (non-insulin-dependent) diabetic and non-diabetic individuals. Diubetologia 30, 887-892. \VESTERMARK, P., WERNSTEDT, C., OIBRIEN,T.D., IIAYDEN, D.W. & JOHNSON, K.H. 1987b. Islet am)-loid in type z human diabetes mellitus and adult diabetic cats contains a novel putative polypeptide hormone. A m 3 Pathol 127, 414-417. \\'ESTER.\IARK, P., WERNSTEDT, c., WILANDER, E., HAYDES,D.W., OBRIEN,T.D. & JOHNSON, K.H. 1 9 8 7 ~ .4m!loid . fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a novel neuropeptide-like protein also present Nut/ Acad Sci, C'SA 84, in normal islet cells. PYOC 3881-388 j.
