Regulatory Peptides, 31 (1990) 23-31
23
Elsevier REGPEP 00962
Inhibitory effect of rat amylin on the insulin responses to glucose and arginine in the perfused rat pancreas R a m o n a A. Silvestre, Elena Peirr, Pilar D r g a n o , P a l o m a Miralles, and Jos6 M a r c o Hospital Puerta de Hierro, Universidad Aut6noma de Madrid, Madrid (Spain)
(Received 30 March 1990; revised version received and accepted 16 July 1990) K e y words: Rat amylin; Rat pancreas; Insulin; Glucagon
Summary Amylin, a 37-amino acid polypeptide, is the main component of amyloid deposits in the islets of Langerhans, and has been identified in the B-cell secretory granules. We have investigated the effect of rat amylin "on the insulin and glucagon release by the isolated, perfused rat pancreas. Amylin infusion at 750 nM, markedly reduced unstimulated insulin release (ca. 50~o, P < 0.025), whereas it did not modify glucagon output. At the same concentration, amylin also blocked the insulin response to 9 mM glucose (ca. 80%, P < 0.025) without affecting the suppressor effect of glucose on glucagon release. The inhibitory effect of amylin on glucose-induced insulin secretion was confirmed by lowering the amylin concentration (500 nM) and increasing the glucose stimulus (11 mM); again, no effect of amylin on glucagon release was observed. Finally, amylin, at 500 nM, reduced the insulin response to 3.5 mM arglnine (ca. 40%, P < 0.025) without modifying the secretion of glucagon elicited by this amino acid. It can be concluded that, in the rat pancreas, the inhibitory effect of homologous amylin on unstimulated insulin secretion, as well as on the insulin responses to metabolic substrates (glucose and arginine), favours the concept of this novel peptide as a potential diabetogenic agent.
Correspondence: J. Marco, Hospital Puerta de Hierro, UniversidadAut6nomade Madrid, San Martin de Porres, 4, 28035 Madrid, Spain.
0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
24 Introduction
Recently, a 37-amino acid peptide which shows a close sequential homology to calcitonin gene-related peptide (CGRP) [ 1-4] has been isolated from amyloid deposits in the islets of Langerhans. This novel molecule was identified in different pancreatic specimens, such as a human insulinoma [1,2], normal pancreatic islets [2,5,6], and pancreatic islets from type2 diabetics [1,3-5,7] or from adult diabetic cats [2,7]. Hence, it was named according to its specific tissue origin, i.e., insulinoma-amyloid peptide [1 ], islet-amyloid polypeptide [2], and diabetes-associated peptide [3]. Cooper et al. [8] have recently proposed the term amylin to designate this peptide, a term which, for the sake of briefness, will be employed throughout this text. Immunocytochemical studies have revealed the presence of amylin in the B-cell secretory granules of a number of species [6,9,10]. Among these is the rat [6], in which no pancreatic amyloid deposits have been found [ 11 ]. The release of amylin has recently been reported in both isolated rat pancreatic islets [ 12] and in humans [ 13]. As for the biosynthesis of amylin, it has been shown that in human insulinoma [ 14] as well as in rat [ 15] and human pancreatic islets [ 16], amylin is generated by proteolytic processing similar to that of insulin and other islet hormones. Furthermore, RNA hybridization studies performed by Leffert et al. [ 15] have shown that, in the rat, amylin mRNA is abundant in the islets of Langerhans, but is not present in tissues such as brain, lung, heart, adrenal gland, testes, kidney, intestines and liver. This tissue specificity would point to a role for amylin in the control of islet function. Concerning this subject, Ohsawa et al. [17] have reported that human amylin, at 10 #M, reduced glucosestimulated insulin release in isolated rat pancreatic islets. However, Petterson and Ahr6n [18] found no effect of this peptide, in a range of 10 -5 #M to 1 #M, on glucose-induced insulin output by cultured rat pancreatic islets. To further explore the influence of amylin on islet cell secretion, we have investigated the effect of rat amylin on unstimulated insulin and glucagon release as well as on the responses of these hormones to glucose and to arginine in the isolated perfused rat pancreas.
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. [ 19] as adapted in our laboratory [20]. 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, FRG), and frozen at - 2 0 °C until the time of assay. Synthetic amydated rat amylin (Peninsula Laboratories, Inc., 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. The perfusion medium consisted of a Krebs-Henseleit buffer (gas phase 95:5, O2:CO2; pH 7.4) supplemented with 4 ~ (w/v) dextran T-70, 0.5~/o (w/v) bovine
25 albumin (Cohn fraction V) and glucose (5.5 mM). After a 35 min equilibration period, baseline samples were collected for 6 or 8 min. At zero time, amylin was infused through a sidearm cannula for 18-20 min. Two amylin concentrations were employed: (1) a priming bolus of 6.4 nmol, followed by constant infusion at a rate of 1.5 nmol/min (750 nM), and (2) a priming bolus of 4.7 nmol, followed by constant infusion at a rate of 1 nmol/min (500 nM). Additions to the perfusate - glucose or L-arginine hydrochloride (Sigma Chemical Co., St. Louis, MO) - were made as described in the corresponding figures. Radioimmunoassay was employed to measure insulin [21] and glucagon [22]. Antiglucagon serum (30 K) was kindly donated by Dr. R.H. Unger (University of Texas Health Sciences Center at Dallas, Texas). All samples for each series of experiments were analyzed in the same run. Results are presented as the mean + S.E.M. For each perfusion, hormone response was calculated as the integrated area of the curve above or below the mean preinfusion level (average of all its baseline levels), using the trapezoidal method. Differences between values were tested for significance by Student's t-test for unpaired samples and by analysis of variance. Results
Effect of rat amylin (750 nM) on unstimulated insulin and glucagon release by the perfused rat pancreas (Fig. 1) Incorporation of rat amylin into the perfusate was followed by a depression of insulin release (F1o,5o = 4.11; P < 0.01) which persisted upon cessation of amylin infusion. Furthermore, during amylin infusion, insulin output, as calculated by the integrated area under the response curve, was reduced as compared to control peffusions (33.2 _+ 4 ng/20 rain vs. 68.2 + 9.9 ng/20 rain; P < 0.025). Glucagnn levels from control and amylin experiments overlapped. Effect of rat amylin (750 nM) on the insulin and glucagon responses to glucose (9 raM) by the pe~fused rat pancreas (Fig. 2) The infusion of rat amylin virtually abolished the insulin response to glucose (incremental area: 15 +_ 5 ng/18 min vs. 74 +_ 21 ng/18 rain in control experiments; P < 0.05). This inhibition clearly affected both phases of insulin release. Amylin did not significantly affect the suppressor effect of glucose on glucagon secretion (decremental area: 1381 + 652pg/18 rain vs. 2266 _+ 639pg/18 min in control experiments; P : 0.36). Effect of rat amylin (500 nM) on the insulin and glucagon responses to glucose (I 1 mM) by the perfused rat pancreas (Fig. 3) The release of insulin elicited by 11 mM glucose (incremental area: 266 + 33 ng/20 min) was markedly inhibited by 500 nM rat amylin (incremental area: 81 + 31 ng/20 rain; P < 0.01). As in the preceding series of experiments, the blocking effect of glucose on glucagon output observed in the control experiments (decremental area: 1855 + 564 pg/20 min) was not significantly modified by amylin (decremental area: 2402 + 1015 pg/20 min; P = 0.65).
26 RAT-AMYLIN(750nM) ~SALINE; e-°
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M I N U T ES
Fig. 1. Effect of rat amylin (a priming bolus of 6.4 nmol followed by constant infusion at a rate of 1.5 nmol/min) on unstimulated insulin and glucagon release by the perfused rat pancreas. Solid lines represent control experiments• Broken lines represent rat amylin experiments (means + S.E.M.).
Effect of rat amylin (500 nM) on the insulin and glucagon responses to arginine (3.5 mM) by the perfused rat pancreas (Fig. 4) Rat amylin significantly reduced the insulin response to arginine (incremental area: 247 + 33 ng/20 min vs. 400 + 49 ng/20 min in control experiments; P < 0.025), this effect being more marked in the late phase of insulin release. The stimulatory effect of arglnine on glucagon output (incremental area: 10,534 + 2111 pg/20 min) was not affected by amylin (incremental area: 12,453 + 2314 pg/20 min; P = 0.6).
Discussion Our results show that, in the perfused rat pancreas, homologous amylin, at 750 nM, markedly blocked both phases of the insulin response to glucose (9 mM). The inhibitory effect of rat amylin on glucose-induced insulin output was confirmed by lowering the amylin concentration (500 nM) and increasing the glucose stimulus (11 mM). Employing a preparation of isolated rat pancreatic islets, Ohsawa et al. [ 17] found a suppressor effect of 10 # M human amylin on the insulin release induced by 16.7 m M glucose. This effect was not observed with 1 MM amylin. The difference between their
27
;LUCOSE(9 mM) GLUCOSE (9 raM) ; N:7 15 .
.
.
.
GLUCOSE(grnM) RAT-AM~LIN (750 nM) ; N = 6
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100
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20
30
MINUTES
Fig. 2. Effect of rat amylin (a priming bolus of 6.4 nmol followed by constant infusion at a rate of 1.5 nmol/min)on the insulin and glucagonresponses to glucose(9 mM) by the perfusedrat pancreas. Solid lines represent glucose experiments. Broken lines represent glucose plus rat amylin experiments (means + S.E.M.). results and ours with respect to sensitivity might be due to the higher glucose concentration used by these investigators. More likely, however, it might reflect less responsiveness of collagenase-treated islets to amylin, and a species difference. With regard to the latter, it has been shown that although the N-terminal and C-terminal amino acid sequences of amylin are highly conserved among the mammals [ 11 ], there are 6 variations in the amino acid sequence of residues 18-29 of rat and human amylin [11,15,23]. Furthermore, Oshawa et al. [ 17] did not mention whether the synthetic amylin they tested was amydated at the C-terminus, and the C-terminal amidation seems to be necessary for amylin to exert its biological effects [16]. A similar interpretation could apply to the lack of effect of amylin on glucose-stimulated insulin release found by Pettersson and Ahr6n [ 18]. Rat amylin also inhibited unstimulated insulin output as well as the secretion of insulin elicited by arginine, observation not previously reported. This inhibition mainly affected the late phase of insulin release. It appears that the suppressor effect of rat amylin (500 nM) on arginine-induced insulin output (ca. 4 0 ~ ) was less marked than that observed during B-cell stimulation by glucose (ca. 70 ~o). This might be due to the different mechanisms by which these two metabolites stimulate insulin release [24]. Nevertheless, the mechanism of action of amylin on the B-ceU secretion remains unknown, as does that of its structural congener CGRP, another inhibitor of insulin
28 GLUGOSE ¢11mM) 25
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Fig. 3. Effect of rat amylin (a priming bolus of 4.7 nmol followed by constant infusion at a rate of 1 nmol/min) on the insulin and glucagon responses to glucose (11 mM) by the perfused rat pancreas. Solid lines represent glucose experiments. Broken lines represent glucose plus rat amylin experiments (means 5: S.E.M.).
release [25,26]. Interestingly, in the rat B cell subjected to amylin infusion, the differential insulin response to arginine and to glucose resembles that observed in mild type 2 diabetes [27], in which arginine-induced insulin secretion is maintained while the release of insulin evoked by glucose appears attenuated. Finally, in our rat pancreas preparation, homologous amylin had no effect on glucagon secretion, whether in unstimulated conditions, inhibited by glucose or stimulated by arginine. This observation suggests that amylin does not influence A-cell function. The possible implication of augmented amyloid deposits in the pathogenesis of type 2 diabetes is a matter of current interest [28,29]. In adult cats, impaired glucose tolerance is associated with increased amylin immunoreactivity in pancreatic B cells [30]. Amylin has been shown to inhibit both glucose uptake [8 ] and glycogen synthesis [8,31 ] in the rat soleus muscle 'in vitro'. Our finding that, in the isolated rat pancreas, homologous amylin inhibits unstimulated insulin release as well as the insulin responses to metabolic substrates, i.e., glucose and arglnine, favours the concept of this peptide as a potential diabetogenic agent.
29
ARGININE ( 3 . 5 r a M )
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ARGININE(3.S raM); N,=5
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ARGININE13.SmM) ;N=7 RAT- AMYLIN (5OOnM)
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.
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32
MINUTES Fig. 4. Effect of rat amylin (a priming bolus of 4.7 nmol followed by constant infusion at a rate of 1 nmol/min) on the insulin and glucagon responses to arginine (3.5 m M ) by the perfused rat pancreas. Solid lines represent arginine experiments. Broken lines represent arginine plus rat amylin experiments
Acknowledgments
E.P., P.D. and P.M. are Research Fellows from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Ministerio de Sanidad y Consumo, Spain. This work has been supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (89-0102, 89-0109 and 90-0043), and from the Comisi6n Interministerial de Ciencia y Tecnologia (PB-86003), Spain. The expert technical assistance of Ms. Pilar Garcia-Mufloz and Ms. Encarnaci6n Guti6rrez is gratefully acknowledged. We thank Ms. Martha Messman for her editorial help and Mr. Pablo R6spide for his assistance in processing the manuscript.
30
References 1 Westermark, P., Wernstedt, C., Wilander, E. and Sletten, K., A novel peptide in the calcitonin generelated peptide family as an amyloid fibril protein in the endocrine pancreas, Biochem. Biophys. Res. Commun., 140 (1986) 827-831. 2 Westermark, P., Wernstedt, C., Wilander, E., Hayden, D.W., O'Brien, T.D. and Johnson, K.H., Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells, Proc. Natl. Acad. Sci. USA, 84 (1987) 3881-3885. 3 Clark, A., Cooper, G.J.S., Lewis, C. E., Morris, J. F., Willis, A. C., Reid, K. B.M. and Turner, R. C., Islet amyloid formed from diabetes-associated peptide may be pathogenic in type-2 diabetes, Lancet, ii (1987) 231-234. 4 Cooper, G.J.S., Willis, A.C., Clark, A., Turner, R.C., Sim, R.B. and Reid, K. B. M., Purification and characterization ofa peptide from amyloid-rich pancreases of type 2 diabetic patients, Proc. Natl. Acad. Sci. USA, 84 (1987) 8628-8632. 5 Westermark, P., Wilander, E., Westermark, G.T. and Johnson, K.H., Islet amyloid polypeptide-like immunoreactivity in the islet B cells of Type 2 (non-insulin-dependent) diabetic and non-diabetic individuals, Diabetologia, 30 (1987) 887-892. 6 Johnson, K.H., O'Brien, T.D., Hayden, D.W., Jordan, K., Ghobrial, H.K.G., Mahoney, WC. and Westermark, P. Immunolocaiization of islet amyloid polypeptide (IAPP) in pancreatic beta cells by means of peroxidase-antiperoxidase (PAP) and protein A-gold techniques, Am. J. Pathol., 130 (1988) 1-8. 7 Westermark, P., Wernstedt, C., O'Brien, T. D., Hayden, D.W. and Johnson, K. H., Islet amyloid in type 2 human diabetes mellitus and adult diabetic cats contains a novel putative polypeptide hormone, Am. J. Pathol., 127 (1987) 414-417. 8 Cooper, G.J.S., Leighton, B., Dimitriadis, G.D., Parry-Billings, M., Kowalchuk, J.M., Howland, K., Rothbard, J.B., Willis, A.C. and Reid, K. B.M., Amylin found in amyloid deposits in human type 2 diabetes mellitus may be a hormone that regulates glycogen metabolism in skeletal muscle, Proc. Natl. Acad. Sci. USA, 85 (1988) 7763-7766. 9 Clark, A., Edwards, C.A, Ostle, L.R., Sutton, R., Rothbard, J.B., Morris, J.F. and Turner, R.C., Localisation of islet amyloid peptide in lipofuscin bodies and secretory granules of human B-cells and in islets of type-2 diabetic subjects, Cell. Tissue Res., 257 (1989) 179-185. 10 Lukinius, A., Wilander, E., Westermark, G. T., Engstr6m, U. and Westermark, P., Co-localization of islet amyloid polypeptide and insulin in the B cell secretory granules of the human pancreatic islets, Diabetologia, 32 (1989) 240-244. 11 Nishi, M., Chart, S.J., Nagamatsu, S., Bell, G. I. and Steiner, D. F., Conservation of the sequence of islet amyloid polypeptide in five mammals is consistent with its putative role as an islet hormone, Proc. Natl. Acad. Sci. USA, 86 (1989) 5738-5742. 12 Kanatsuka, A., Makino, H., Ohsawa, H., Tokuyama, Y., Yamaguchi, T., Yoshida, S. and Adachi, M., Secretion of islet amyloid polypeptide in response to glucose, FEBS Lett., 259 (1989) 199-201. 13 Van Jaarsveld, B. C., Hackeng, W. H. L., Nieuwenhuis, M. G., Erkelens, D. W., Geerdink, R.A. and Lips, C.J.M., Islet-amyloid polypeptide in human plasma, Lancet, i (1990) 60 (letter). 14 Sanke, T., Bell, G. I., Sample, C., Rubenstein, A. H. and Steiner, D. F., An islet amyloid peptide is derived from an 89-amino acid precursor by proteolytic processing, J. Biol. Chem., 263 (1988) 17243-17246. 15 Leffert, J.D., Newgard, C. B., Okamoto, H., Milburn, J. L. and Luskey, K.L., Rat amylin: Cloning and tissue-specific expression in pancreatic islets, Proc. Natl. Acad. Sci. USA, 86 (1989) 3127-3130. 16 Roberts, A. N., Leighton, B., Todd, J. A., Cockburn, D., Schofield, P.N., Sutton, R., Holt, S., Boyd, Y., Day, A.J., Foot, E.A., Willis, A.C., Reid, K.B.M. and Cooper, G.J.S., Molecular and functional characterization of amylin, a peptide associated with type 2 diabetes mellitus, Proc. Natl. Acad. Sci. USA, 86 (1989) 9662-9666. 17 Ohsawa, H., Kanatsuka, A., Yamaguchi, T., Makino, H. and Yoshida, S., Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion from isolated rat pancreatic islets, Biochem. Biophys. Res. Commun., 160 (1989) 961-967. 18 Pettersson, M. and Ahrgn, B., Calcitonin gene-related peptide inhibits insulin secretion: studies on its
31
19 20
21 22 23 24 25
26
27 28 29 30
31
mechanism of action and possible similar influence of islet amyloid polypeptide, Diabetologia, 32 (1989) 529A (Abstract). Leclercq-Meyer, V., Marchand, J. Leclercq, R. and Malaisse, W.J., Glucagon and insulin release by the 'in vitro' perfused rat pancreas, Diabete Metab., 2 (1976) 57-65. Silvestre, R.A., Miralles, P., Moreno, P., Villanueva, M.L. and Marco, J., Somatostatin, insulin and 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. Asai, J., Nakazato, M., Kangawa, K., Matsukura, S. and Matsuo, H., Isolation and sequence determination of rat islet amyloid polypeptide, Biochem. Biophys. Res. Commun., 164 (1989) 400-405. Henquin, J.C., Regulation of insulin release by ionic and electrical events in B cells, Hormone Res., 27 (1987) 168-178. Pettersson, M., Ahr6n, B., BOttcher, G. and Sundler, F., Calcitonin gene-related peptide: occurrence in pancreatic islets in the mouse and the rat and inhibition of insulin secretion in the mouse, Endocrinology, 119 (1986) 865-869. Lewis, C.E., Clark, A., Ashcroft, S.J.H., Cooper, G.J.S. and Morris, J.F., Calcitonin gene-related peptide and somatostatin inhibit insulin release from individual rat B cells, Mol. Cell. Endocrinol., 57 (1988) 41-49. Pfeiffer, M.A., Halter, J.B., and Porte, D., Jr., Insulin secretion in diabetes mellitus, Am. J. Med., 70 (1981) 579-588. Johnson, K.H., 0'Brien, T.D., Betsholtz, C. and Westermark, P., Islet amyloid, islet-amyloid polypeptide, and diabetes mellitus, N. Engl. J. Med., 321 (1989) 513-518. Porte, D., Jr. and Kahn, S.E., Hyperproinsulinemia and amyloid in NIDDM. Clues to etiology if islet fl-ceU dysfunction?, Diabetes, 38 (1989) 1333-1336. Johnson, K. H., O'Brien, T. D., Jordan, K. and Westermark, P., Impaired glucose tolerance is associated with increased islet amyloid polypeptide (lAPP) immunoreactivity in pancreatic beta cells, Am. J. Pathol., 135 (1989) 245-250. Leighton, B. and Cooper, G.J.S., Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro, Nature, 335 (1988) 632-635.
23
Elsevier REGPEP 00962
Inhibitory effect of rat amylin on the insulin responses to glucose and arginine in the perfused rat pancreas R a m o n a A. Silvestre, Elena Peirr, Pilar D r g a n o , P a l o m a Miralles, and Jos6 M a r c o Hospital Puerta de Hierro, Universidad Aut6noma de Madrid, Madrid (Spain)
(Received 30 March 1990; revised version received and accepted 16 July 1990) K e y words: Rat amylin; Rat pancreas; Insulin; Glucagon
Summary Amylin, a 37-amino acid polypeptide, is the main component of amyloid deposits in the islets of Langerhans, and has been identified in the B-cell secretory granules. We have investigated the effect of rat amylin "on the insulin and glucagon release by the isolated, perfused rat pancreas. Amylin infusion at 750 nM, markedly reduced unstimulated insulin release (ca. 50~o, P < 0.025), whereas it did not modify glucagon output. At the same concentration, amylin also blocked the insulin response to 9 mM glucose (ca. 80%, P < 0.025) without affecting the suppressor effect of glucose on glucagon release. The inhibitory effect of amylin on glucose-induced insulin secretion was confirmed by lowering the amylin concentration (500 nM) and increasing the glucose stimulus (11 mM); again, no effect of amylin on glucagon release was observed. Finally, amylin, at 500 nM, reduced the insulin response to 3.5 mM arglnine (ca. 40%, P < 0.025) without modifying the secretion of glucagon elicited by this amino acid. It can be concluded that, in the rat pancreas, the inhibitory effect of homologous amylin on unstimulated insulin secretion, as well as on the insulin responses to metabolic substrates (glucose and arginine), favours the concept of this novel peptide as a potential diabetogenic agent.
Correspondence: J. Marco, Hospital Puerta de Hierro, UniversidadAut6nomade Madrid, San Martin de Porres, 4, 28035 Madrid, Spain.
0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
24 Introduction
Recently, a 37-amino acid peptide which shows a close sequential homology to calcitonin gene-related peptide (CGRP) [ 1-4] has been isolated from amyloid deposits in the islets of Langerhans. This novel molecule was identified in different pancreatic specimens, such as a human insulinoma [1,2], normal pancreatic islets [2,5,6], and pancreatic islets from type2 diabetics [1,3-5,7] or from adult diabetic cats [2,7]. Hence, it was named according to its specific tissue origin, i.e., insulinoma-amyloid peptide [1 ], islet-amyloid polypeptide [2], and diabetes-associated peptide [3]. Cooper et al. [8] have recently proposed the term amylin to designate this peptide, a term which, for the sake of briefness, will be employed throughout this text. Immunocytochemical studies have revealed the presence of amylin in the B-cell secretory granules of a number of species [6,9,10]. Among these is the rat [6], in which no pancreatic amyloid deposits have been found [ 11 ]. The release of amylin has recently been reported in both isolated rat pancreatic islets [ 12] and in humans [ 13]. As for the biosynthesis of amylin, it has been shown that in human insulinoma [ 14] as well as in rat [ 15] and human pancreatic islets [ 16], amylin is generated by proteolytic processing similar to that of insulin and other islet hormones. Furthermore, RNA hybridization studies performed by Leffert et al. [ 15] have shown that, in the rat, amylin mRNA is abundant in the islets of Langerhans, but is not present in tissues such as brain, lung, heart, adrenal gland, testes, kidney, intestines and liver. This tissue specificity would point to a role for amylin in the control of islet function. Concerning this subject, Ohsawa et al. [17] have reported that human amylin, at 10 #M, reduced glucosestimulated insulin release in isolated rat pancreatic islets. However, Petterson and Ahr6n [18] found no effect of this peptide, in a range of 10 -5 #M to 1 #M, on glucose-induced insulin output by cultured rat pancreatic islets. To further explore the influence of amylin on islet cell secretion, we have investigated the effect of rat amylin on unstimulated insulin and glucagon release as well as on the responses of these hormones to glucose and to arginine in the isolated perfused rat pancreas.
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. [ 19] as adapted in our laboratory [20]. 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, FRG), and frozen at - 2 0 °C until the time of assay. Synthetic amydated rat amylin (Peninsula Laboratories, Inc., 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. The perfusion medium consisted of a Krebs-Henseleit buffer (gas phase 95:5, O2:CO2; pH 7.4) supplemented with 4 ~ (w/v) dextran T-70, 0.5~/o (w/v) bovine
25 albumin (Cohn fraction V) and glucose (5.5 mM). After a 35 min equilibration period, baseline samples were collected for 6 or 8 min. At zero time, amylin was infused through a sidearm cannula for 18-20 min. Two amylin concentrations were employed: (1) a priming bolus of 6.4 nmol, followed by constant infusion at a rate of 1.5 nmol/min (750 nM), and (2) a priming bolus of 4.7 nmol, followed by constant infusion at a rate of 1 nmol/min (500 nM). Additions to the perfusate - glucose or L-arginine hydrochloride (Sigma Chemical Co., St. Louis, MO) - were made as described in the corresponding figures. Radioimmunoassay was employed to measure insulin [21] and glucagon [22]. Antiglucagon serum (30 K) was kindly donated by Dr. R.H. Unger (University of Texas Health Sciences Center at Dallas, Texas). All samples for each series of experiments were analyzed in the same run. Results are presented as the mean + S.E.M. For each perfusion, hormone response was calculated as the integrated area of the curve above or below the mean preinfusion level (average of all its baseline levels), using the trapezoidal method. Differences between values were tested for significance by Student's t-test for unpaired samples and by analysis of variance. Results
Effect of rat amylin (750 nM) on unstimulated insulin and glucagon release by the perfused rat pancreas (Fig. 1) Incorporation of rat amylin into the perfusate was followed by a depression of insulin release (F1o,5o = 4.11; P < 0.01) which persisted upon cessation of amylin infusion. Furthermore, during amylin infusion, insulin output, as calculated by the integrated area under the response curve, was reduced as compared to control peffusions (33.2 _+ 4 ng/20 rain vs. 68.2 + 9.9 ng/20 rain; P < 0.025). Glucagnn levels from control and amylin experiments overlapped. Effect of rat amylin (750 nM) on the insulin and glucagon responses to glucose (9 raM) by the pe~fused rat pancreas (Fig. 2) The infusion of rat amylin virtually abolished the insulin response to glucose (incremental area: 15 +_ 5 ng/18 min vs. 74 +_ 21 ng/18 rain in control experiments; P < 0.05). This inhibition clearly affected both phases of insulin release. Amylin did not significantly affect the suppressor effect of glucose on glucagon secretion (decremental area: 1381 + 652pg/18 rain vs. 2266 _+ 639pg/18 min in control experiments; P : 0.36). Effect of rat amylin (500 nM) on the insulin and glucagon responses to glucose (I 1 mM) by the perfused rat pancreas (Fig. 3) The release of insulin elicited by 11 mM glucose (incremental area: 266 + 33 ng/20 min) was markedly inhibited by 500 nM rat amylin (incremental area: 81 + 31 ng/20 rain; P < 0.01). As in the preceding series of experiments, the blocking effect of glucose on glucagon output observed in the control experiments (decremental area: 1855 + 564 pg/20 min) was not significantly modified by amylin (decremental area: 2402 + 1015 pg/20 min; P = 0.65).
26 RAT-AMYLIN(750nM) ~SALINE; e-°
N=8
RAT-AMYLIN
: N=6
6" c
_z~ ,,J'~
C
.
.
.
.
i
.
.
.
.
i
.
.
.
.
i
•
.
600 e-
zE O~
400
3". og
200
-6
0
10
20
34
M I N U T ES
Fig. 1. Effect of rat amylin (a priming bolus of 6.4 nmol followed by constant infusion at a rate of 1.5 nmol/min) on unstimulated insulin and glucagon release by the perfused rat pancreas. Solid lines represent control experiments• Broken lines represent rat amylin experiments (means + S.E.M.).
Effect of rat amylin (500 nM) on the insulin and glucagon responses to arginine (3.5 mM) by the perfused rat pancreas (Fig. 4) Rat amylin significantly reduced the insulin response to arginine (incremental area: 247 + 33 ng/20 min vs. 400 + 49 ng/20 min in control experiments; P < 0.025), this effect being more marked in the late phase of insulin release. The stimulatory effect of arglnine on glucagon output (incremental area: 10,534 + 2111 pg/20 min) was not affected by amylin (incremental area: 12,453 + 2314 pg/20 min; P = 0.6).
Discussion Our results show that, in the perfused rat pancreas, homologous amylin, at 750 nM, markedly blocked both phases of the insulin response to glucose (9 mM). The inhibitory effect of rat amylin on glucose-induced insulin output was confirmed by lowering the amylin concentration (500 nM) and increasing the glucose stimulus (11 mM). Employing a preparation of isolated rat pancreatic islets, Ohsawa et al. [ 17] found a suppressor effect of 10 # M human amylin on the insulin release induced by 16.7 m M glucose. This effect was not observed with 1 MM amylin. The difference between their
27
;LUCOSE(9 mM) GLUCOSE (9 raM) ; N:7 15 .
.
.
.
GLUCOSE(grnM) RAT-AM~LIN (750 nM) ; N = 6
•
Z
10-
100
-8
0
10
20
30
MINUTES
Fig. 2. Effect of rat amylin (a priming bolus of 6.4 nmol followed by constant infusion at a rate of 1.5 nmol/min)on the insulin and glucagonresponses to glucose(9 mM) by the perfusedrat pancreas. Solid lines represent glucose experiments. Broken lines represent glucose plus rat amylin experiments (means + S.E.M.). results and ours with respect to sensitivity might be due to the higher glucose concentration used by these investigators. More likely, however, it might reflect less responsiveness of collagenase-treated islets to amylin, and a species difference. With regard to the latter, it has been shown that although the N-terminal and C-terminal amino acid sequences of amylin are highly conserved among the mammals [ 11 ], there are 6 variations in the amino acid sequence of residues 18-29 of rat and human amylin [11,15,23]. Furthermore, Oshawa et al. [ 17] did not mention whether the synthetic amylin they tested was amydated at the C-terminus, and the C-terminal amidation seems to be necessary for amylin to exert its biological effects [16]. A similar interpretation could apply to the lack of effect of amylin on glucose-stimulated insulin release found by Pettersson and Ahr6n [ 18]. Rat amylin also inhibited unstimulated insulin output as well as the secretion of insulin elicited by arginine, observation not previously reported. This inhibition mainly affected the late phase of insulin release. It appears that the suppressor effect of rat amylin (500 nM) on arginine-induced insulin output (ca. 4 0 ~ ) was less marked than that observed during B-cell stimulation by glucose (ca. 70 ~o). This might be due to the different mechanisms by which these two metabolites stimulate insulin release [24]. Nevertheless, the mechanism of action of amylin on the B-ceU secretion remains unknown, as does that of its structural congener CGRP, another inhibitor of insulin
28 GLUGOSE ¢11mM) 25
c
°"
GLUCO6E (11 raM) ; N ~ 6 GLUCOSE(llmM)
;N=6
RAT-AMYLIN¢5OO nM )
2O
!.;
~ ~.
i 500' 400.
S
ii3°° ~
200 lOO0
10
20
32
MINUTES
Fig. 3. Effect of rat amylin (a priming bolus of 4.7 nmol followed by constant infusion at a rate of 1 nmol/min) on the insulin and glucagon responses to glucose (11 mM) by the perfused rat pancreas. Solid lines represent glucose experiments. Broken lines represent glucose plus rat amylin experiments (means 5: S.E.M.).
release [25,26]. Interestingly, in the rat B cell subjected to amylin infusion, the differential insulin response to arginine and to glucose resembles that observed in mild type 2 diabetes [27], in which arginine-induced insulin secretion is maintained while the release of insulin evoked by glucose appears attenuated. Finally, in our rat pancreas preparation, homologous amylin had no effect on glucagon secretion, whether in unstimulated conditions, inhibited by glucose or stimulated by arginine. This observation suggests that amylin does not influence A-cell function. The possible implication of augmented amyloid deposits in the pathogenesis of type 2 diabetes is a matter of current interest [28,29]. In adult cats, impaired glucose tolerance is associated with increased amylin immunoreactivity in pancreatic B cells [30]. Amylin has been shown to inhibit both glucose uptake [8 ] and glycogen synthesis [8,31 ] in the rat soleus muscle 'in vitro'. Our finding that, in the isolated rat pancreas, homologous amylin inhibits unstimulated insulin release as well as the insulin responses to metabolic substrates, i.e., glucose and arglnine, favours the concept of this peptide as a potential diabetogenic agent.
29
ARGININE ( 3 . 5 r a M )
60. 50 -
)=
~
ARGININE(3.S raM); N,=5
..
ARGININE13.SmM) ;N=7 RAT- AMYLIN (5OOnM)
40 -
E g-~
30-
c
20-
10-
.
.
.
.
1600 -
.
.
.
.
e~
.
.
I
,,oo
I ........... -8
0
L ....... 10
20
32
MINUTES Fig. 4. Effect of rat amylin (a priming bolus of 4.7 nmol followed by constant infusion at a rate of 1 nmol/min) on the insulin and glucagon responses to arginine (3.5 m M ) by the perfused rat pancreas. Solid lines represent arginine experiments. Broken lines represent arginine plus rat amylin experiments
Acknowledgments
E.P., P.D. and P.M. are Research Fellows from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Ministerio de Sanidad y Consumo, Spain. This work has been supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (89-0102, 89-0109 and 90-0043), and from the Comisi6n Interministerial de Ciencia y Tecnologia (PB-86003), Spain. The expert technical assistance of Ms. Pilar Garcia-Mufloz and Ms. Encarnaci6n Guti6rrez is gratefully acknowledged. We thank Ms. Martha Messman for her editorial help and Mr. Pablo R6spide for his assistance in processing the manuscript.
30
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31
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