Diabetes Research and Clinical Practice, 10 (1990)
189-192
189
Elsevier DIABET
0043 I
Brief Report
Amylin release from perfused rat pancreas in response to glucose and arginine Kaoru Inoue, Akitaka
Hisatomi,
Fumio Umeda
and Hajime Nawata
Third Department of Internal Medicine. Faculty of Medicine, Kyushu University, Fukuoka, Japan
(Received 29 March 1990) (Revision received 25 April 1990) (Accepted 27 April 1990)
Summary The effects of glucose and arginine on the release of amylin from the perfused rat pancreas were studied. Amylin, or islet amyloid polypeptide, is a 37-amino acid peptide isolated from pancreatic islet amyloid of patients with non-insulin-dependent diabetes mellitus (NIDDM). Glucose stimulated dose-dependently amylin release, showing a typical biphasic pattern. Additionally, 10 mM arginine in the presence of 5.5 mM glucose also stimulated amylin release. These findings suggest that amylin is a secretory protein and its release from the pancreas is regulated by glucose and other nutrients. Key words: Amylin; Islet amyloid polypeptide;
Pancreas
Introduction It has been thought that amyloid deposition in the pancreatic islets is a major pathological feature of non-insulin-dependent diabetes mellitus (NIDDM) [ 11. Recently, Westermark et al. [2,3] and Cooper et al. [4] independently purified and characterized the novel amyloid peptide in the deposit. This peptide, known as islet amyloid polypeptide (IAPP), or amylin, is a 37-amino acid peptide which is 46% identical to human calcitonin gene-related peptide (CGRP). Rat amylin has also been isolated from the normal rat panAddress for correspondence: Kaoru Inoue, MD, the Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-l-l Maidashi, Higashi-ku, Fukuoka 812, Japan. 0168-8227/90/$03.50
0 1990 Elsevier Science Publishers
perfusion;
Glucose
creas, and it is a 37-amino acid peptide which is 84% identical to human amylin and 5 1% identical to rat CGRP [ 51. cDNA clones corresponding to the rat amylin precursor have been isolated from an islet cDNA library [6]. The rat arnylin peptide is thought to be proteolytically processed from a 93-amino acid precursor. The pathophysiological significance of amylin has been proposed in relation with NIDDM [7,8]. Although amylin is co-localized and thought to be cosecreted with insulin [9,10], the regulation of amylin release from the pancreas by nutrients is still unclear. In the present study, using the perfused rat pancreas, we evaluated the effects of glucose and arginine on rat amylin release. The pathophysiological implications of the amylin response were discussed.
B.V. (Biomedical
Division)
190
Materials and Methods Isolated rat pancreas pe$usion
experiment
The pancreata of male Wistar-King albino rats weighing 350 to 400 g were isolated and perfused using the system of Grodsky and Fanska [ 1 l] with minor modifications [ 121. After overnight fasting, the rats were anesthetized with 50 mg/kg of sodium pentobarbital injected intraperitoneally. The pancreas with the adjacent proximal portion of the duodenum was isolated and transferred to a thermostatically controlled plexiglass perfusion chamber in which the celiac trunk and the portal vein were cannulated. Nonrecirculating perfusion was begun at a constant flow rate of 3.6 ml/min maintained by means of a peristaltic pump (Harvard Apparatus Co., Millis, MA, U.S.A.) using a Krebs-Ringer bicarbonate buffer supplemented with 4.5 y0 dextran T-70 (Pharmacia Fine Chemicals, Uppsala, Sweden), 1 y0 bovine serum albumin (Sigma Chemicals Co., St. Louis, MO, U.S.A.), 5 mM each of pyruvate, fumarate and glutamate, and 5.5 mM glucose. The perfusion medium was oxygenated with a 95% 0, and 5% CO, mixture and maintained at 37°C. The perfusion pressure was monitored continuously. An equilibration period of 20 min preceded each experimental period. Glucose or arginine HCl was dissolved in perfusate and added to the circulating perfusate through a sidearm infusion pump (Model 975; Harvard Apparatus Co.) to final concentrations of glucose (8.3, 11, 16.5 mM), and arginine (10 mM). Oneminute aliquots of the effluent were collected in chilled tubes containing 0.4 ml of an EDTAbenzamidine mixture (0.03 M/0.3 M). These were stored at - 20°C until assay. Rat amylin radioimmunoassay
Rat Amylin Radioimmunoassay kit was purchased from Peninsula Laboratories (Belmont, CA, U.S.A.) [ 131. RIA was performed by a double-antibody technique. Specific antiserum to rat amylin was produced in rabbits by immunization with synthetic rat amylin. The antiserum
exhibited 13.3% cross-reactivity with human amylin, but less than 0.01 y0 cross-reactivity with human CGRPs and rat CGRPs. The minimum sensitivity of the assay was 10 pg/ml. The dilution curve of the perfusate sample was parallel to the standard curve. Insulin radioimmunoassay
Immunoreactive dioimmunoassay purchased from (Tokyo, Japan)
insulin was measured by rausing a kit (Insulin RIA Bead) Dainabot Isotope Laboratory [ 141.
Statistical analysis
All data are expressed as mean ? SE. Statistical differences were determined by Student’s t-test with a 5% significance level.
Results Efect
of glucose on pancreatic amylin release
Glucose stimulated pancreatic amylin and insulin release (Fig. 1A). The amylin level during an equilibration period with a perfusate glucose concentration of 5.5 mM was below the assay limit. In response to 11 mM glucose, the amylin level rose from the base-line level to a peak of 591 + 102 pg/ml (m + SE) at 2 min. 16.5 mM glucose infusion induced a typical biphasic pattern of amylin release. In response to 16.5 mM glucose, the amylin level rose from the base-line level to a peak of 980 + 78 pg/ml at 2 min and remained above 100 pg/ml during the infusion. After cessation of the glucose infusion, the amylin release decreased rapidly to the basal level within 5 min. Maximum release both of amylin and insulin always occurred simultaneously. We also examined the effect of 8.3 mM glucose on amylin release. The cumulative output during the entire 10 min glucose infusion period was 3276 + 289 pg by 8.3 mM glucose (n = 4), 3700 k 590 pg by 11 mM glucose (n = 4), and 8616 + 735 pg by 16.5 mM glucose (n = 4). The cumulative output induced by 16.5 mM glucose increased signifi-
191
2
60 -
1.000 -
600 " E >
600 -
.!? 5
400 -
I 200 -
o-
-5
0
5
10
15
-5
0
min
5
10
15
min
-
-5
0
5
10
15
min
Fig. 1. (A) Release of amylin and insulin from the isolated perfused rat pancreas in response to 11 mM and 16.5 mM glucose. Data are expressed as mean f SE of four experiments. (B) Release of amylin and insulin from the isolated perfused rat pancreas in response to 10 mM arginine in the presence of 5.5 mM glucose. Data are expressed as mean f SE of four experiments.
compared with that by 8.3 mM cantly, (P < 0.001) and that by 11 mM glucose (P < O.Ol), respectively. Therefore, amylin response to glucose was dose-dependent. E#ect of arginine on pancreatic amylin release 10 mM arginine in the presence of 5.5 mM glucose stimulated pancreatic amylin and insulin release (Fig. 1B). The amylin level rose from the base-line level to a peak of 994 k 128 pg/ml at 2 min and remained above 100 pg/ml during the infusion. Immediately after cessation of the arginine infusion, the amylin release decreased rapidly to the basal level. Arginine infusion always induced maximum release of amylin and insulin simultaneously. The cumulative output during the entire 10 min arginine infusion period was 9298 f 1434 pg (n = 4).
Discussion The present study clearly demonstrates that amylin release is stimulated by glucose and arginine in the isolated perfused rat pancreas. Both nutrients elicit a typical biphasic pattern of amylin release. By the ultrastructural immunogold technique, it has been shown that amylin is co-localized with insulin in the secretory granules of the pancreatic /3-cells [ 9, lo]. mRNA of amylin has been expressed selectively in the pancreatic islets [6]. Furthermore, the analysis of rat amylin precursor sequences deduced from its cDNA has shown a hydrophobic amino-terminal sequence consistent with a signal peptide in a secretory protein [6]. These findings suggest that amylin may be co-secreted with insulin from the pancreatic islets. As to arginine stimulation, it was not clarified whether glucose dependency of
192
amylin secretion by arginine might exist, as reported on insulin [ 151. From co-localization of the two peptides within the same secretory granules, we speculated that physiological level of per&ate glucose might be necessary for amylin response to arginine as well as insulin. Concerning the biological action of this peptide, Leighton et al. [ 81 reported that amylin inhibits insulin-induced glycogen synthesis in skeletal muscle. Therefore, it is thought that amylin might be a putative endocrine hormone which contributes to glucose homeostasis through its opposing action against insulin. Amylin was originally purified and characterized from insular amyloid deposits in patients with NIDDM and diabetic cats. Overproduction of this peptide by feline pancreatic fi cells has recently been proven to antedate the onset of overt diabetes mellitus [ 161. From these data, this novel peptide might be a significant factor in the pathogenesis of NIDDM. Further study is needed to clarify the pathophysiological significance of amylin in diabetes. References Westermark, P. and Wilander, E. (1978) The influence of amyloid deposits on the islet volume in maturity onset diabetes mellitus. Diabetologia 15, 4 17-42 1. Westermark, P., Wernstedt, C., Wilander, E. and Sletten, K. (1986) A novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochem. Biophys. Res. Commun. 140, 827-831. Westermark, P., Wernstedt, C., Wilander, E., Hayden, D.W., O’Brien, T.D. and Johnson, K.H. (1987) Amyloid fibrils in human insuhnoma 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, 3881-3885. Cooper, G.J.S., Willis, A.C., Clark, A., Turner, R.C., Sim, R.B. and Reid, K.B.M. (1987) Purification and characterization of a peptide from amyloid-rich pancreases of
10
11 12
13
14
15
16
type 2 diabetic patients. Proc. Natl. Acad. Sci. USA. 84, 8628-8632. Asai, J., Nakazato, M., Kangawa, K., Matsukura, S. and Matsuo, H. (1989) Isolation and sequence determination of rat islet amyloid polypeptide. Biochem. Biophys. Res. Commun. 164, 400-405. Leffert, J.D., Newgard, C.B., Okamoto, H., Milburn, J.L. and Luskey, K.L. (1989) Rat amylin: cloning and tissuespecific expression in pancreatic islets. Proc. Natl. Acad. Sci. USA. 86, 3127-3130. Clark, A., Cooper, G.J.S., Lewis, C.E. et al. (1987) Islet amyloid formed from diabetes-associated peptide may be pathogenic in type-2 diabetes. Lancet ii, 231-234. Leighton, B. and Cooper, G.J.S. (1988) Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature 335, 632-635. Johnson, K.H., O’Brien,T.D., Hayden, D.W. et al. (1988) Immunolocalization of islet amyloid polypeptide (IAPP) ain pancreatic fi-cells by means of peroxidase-antiperoxidase (PAP) and protein A-gold techniques. Am. J. Pathol. 130, l-8. Lukinius, A., Wilander, E., Westermark, G.T., Engstriim, U. and Westermark, P. (1989) Co-localization of islet amyloid polypeptide and insulin in the fi-cell secretory granules of the human pancreatic islets. Diabetologia 32, 240-244. Grodsky, G.M. and Fanska, R.E. (1975) The in vitro perfused pancreas. Methods Enzymol. 39, 364-372. Hisatomi, A., Maruyama, H., Orci, L., Vasko, M. and Unger, R.H. (1985) Adrenergically mediated intrapancreatic control of the glucagon response to glucopenia in the isolated rat pancreas. J. Clin. Invest. 75, 420-426. Chang, J.K., Chou, J., Butler, P. et al. (1990) Determination of amylin concentration in rat and human plasma. FASEB J. 4, Part II, A 1226, No. 5575 (abstract). Hyodo, J., Nanba, O., Nakamura, K., Kaneko, H. and Irie, M. (1983) Clinical application of plasma IRI determination using solid phase bead method-with the special reference to the comparison with a previous double antibody method. Clin. Endocrinol. 3 1, 1129-l 138. Sorenson, R.L. and Elde, R.P. (1983) Dissociation of glucose stimulation of somatostatin and insulin release from glucose inhibition of glucagon release in the isolated perfused rat pancreas. Diabetes 32, 561-567. Johnson, K.H., O’Brien, T.D., Jordan, K. and Westermark, P. (1989) Impaired glucose tolerance is associated with increased islet amyloid polypeptide (IAPP) immunoreactivity in pancreatic /?-cells. Am. J. Pathol. 135,245-250.
189-192
189
Elsevier DIABET
0043 I
Brief Report
Amylin release from perfused rat pancreas in response to glucose and arginine Kaoru Inoue, Akitaka
Hisatomi,
Fumio Umeda
and Hajime Nawata
Third Department of Internal Medicine. Faculty of Medicine, Kyushu University, Fukuoka, Japan
(Received 29 March 1990) (Revision received 25 April 1990) (Accepted 27 April 1990)
Summary The effects of glucose and arginine on the release of amylin from the perfused rat pancreas were studied. Amylin, or islet amyloid polypeptide, is a 37-amino acid peptide isolated from pancreatic islet amyloid of patients with non-insulin-dependent diabetes mellitus (NIDDM). Glucose stimulated dose-dependently amylin release, showing a typical biphasic pattern. Additionally, 10 mM arginine in the presence of 5.5 mM glucose also stimulated amylin release. These findings suggest that amylin is a secretory protein and its release from the pancreas is regulated by glucose and other nutrients. Key words: Amylin; Islet amyloid polypeptide;
Pancreas
Introduction It has been thought that amyloid deposition in the pancreatic islets is a major pathological feature of non-insulin-dependent diabetes mellitus (NIDDM) [ 11. Recently, Westermark et al. [2,3] and Cooper et al. [4] independently purified and characterized the novel amyloid peptide in the deposit. This peptide, known as islet amyloid polypeptide (IAPP), or amylin, is a 37-amino acid peptide which is 46% identical to human calcitonin gene-related peptide (CGRP). Rat amylin has also been isolated from the normal rat panAddress for correspondence: Kaoru Inoue, MD, the Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-l-l Maidashi, Higashi-ku, Fukuoka 812, Japan. 0168-8227/90/$03.50
0 1990 Elsevier Science Publishers
perfusion;
Glucose
creas, and it is a 37-amino acid peptide which is 84% identical to human amylin and 5 1% identical to rat CGRP [ 51. cDNA clones corresponding to the rat amylin precursor have been isolated from an islet cDNA library [6]. The rat arnylin peptide is thought to be proteolytically processed from a 93-amino acid precursor. The pathophysiological significance of amylin has been proposed in relation with NIDDM [7,8]. Although amylin is co-localized and thought to be cosecreted with insulin [9,10], the regulation of amylin release from the pancreas by nutrients is still unclear. In the present study, using the perfused rat pancreas, we evaluated the effects of glucose and arginine on rat amylin release. The pathophysiological implications of the amylin response were discussed.
B.V. (Biomedical
Division)
190
Materials and Methods Isolated rat pancreas pe$usion
experiment
The pancreata of male Wistar-King albino rats weighing 350 to 400 g were isolated and perfused using the system of Grodsky and Fanska [ 1 l] with minor modifications [ 121. After overnight fasting, the rats were anesthetized with 50 mg/kg of sodium pentobarbital injected intraperitoneally. The pancreas with the adjacent proximal portion of the duodenum was isolated and transferred to a thermostatically controlled plexiglass perfusion chamber in which the celiac trunk and the portal vein were cannulated. Nonrecirculating perfusion was begun at a constant flow rate of 3.6 ml/min maintained by means of a peristaltic pump (Harvard Apparatus Co., Millis, MA, U.S.A.) using a Krebs-Ringer bicarbonate buffer supplemented with 4.5 y0 dextran T-70 (Pharmacia Fine Chemicals, Uppsala, Sweden), 1 y0 bovine serum albumin (Sigma Chemicals Co., St. Louis, MO, U.S.A.), 5 mM each of pyruvate, fumarate and glutamate, and 5.5 mM glucose. The perfusion medium was oxygenated with a 95% 0, and 5% CO, mixture and maintained at 37°C. The perfusion pressure was monitored continuously. An equilibration period of 20 min preceded each experimental period. Glucose or arginine HCl was dissolved in perfusate and added to the circulating perfusate through a sidearm infusion pump (Model 975; Harvard Apparatus Co.) to final concentrations of glucose (8.3, 11, 16.5 mM), and arginine (10 mM). Oneminute aliquots of the effluent were collected in chilled tubes containing 0.4 ml of an EDTAbenzamidine mixture (0.03 M/0.3 M). These were stored at - 20°C until assay. Rat amylin radioimmunoassay
Rat Amylin Radioimmunoassay kit was purchased from Peninsula Laboratories (Belmont, CA, U.S.A.) [ 131. RIA was performed by a double-antibody technique. Specific antiserum to rat amylin was produced in rabbits by immunization with synthetic rat amylin. The antiserum
exhibited 13.3% cross-reactivity with human amylin, but less than 0.01 y0 cross-reactivity with human CGRPs and rat CGRPs. The minimum sensitivity of the assay was 10 pg/ml. The dilution curve of the perfusate sample was parallel to the standard curve. Insulin radioimmunoassay
Immunoreactive dioimmunoassay purchased from (Tokyo, Japan)
insulin was measured by rausing a kit (Insulin RIA Bead) Dainabot Isotope Laboratory [ 141.
Statistical analysis
All data are expressed as mean ? SE. Statistical differences were determined by Student’s t-test with a 5% significance level.
Results Efect
of glucose on pancreatic amylin release
Glucose stimulated pancreatic amylin and insulin release (Fig. 1A). The amylin level during an equilibration period with a perfusate glucose concentration of 5.5 mM was below the assay limit. In response to 11 mM glucose, the amylin level rose from the base-line level to a peak of 591 + 102 pg/ml (m + SE) at 2 min. 16.5 mM glucose infusion induced a typical biphasic pattern of amylin release. In response to 16.5 mM glucose, the amylin level rose from the base-line level to a peak of 980 + 78 pg/ml at 2 min and remained above 100 pg/ml during the infusion. After cessation of the glucose infusion, the amylin release decreased rapidly to the basal level within 5 min. Maximum release both of amylin and insulin always occurred simultaneously. We also examined the effect of 8.3 mM glucose on amylin release. The cumulative output during the entire 10 min glucose infusion period was 3276 + 289 pg by 8.3 mM glucose (n = 4), 3700 k 590 pg by 11 mM glucose (n = 4), and 8616 + 735 pg by 16.5 mM glucose (n = 4). The cumulative output induced by 16.5 mM glucose increased signifi-
191
2
60 -
1.000 -
600 " E >
600 -
.!? 5
400 -
I 200 -
o-
-5
0
5
10
15
-5
0
min
5
10
15
min
-
-5
0
5
10
15
min
Fig. 1. (A) Release of amylin and insulin from the isolated perfused rat pancreas in response to 11 mM and 16.5 mM glucose. Data are expressed as mean f SE of four experiments. (B) Release of amylin and insulin from the isolated perfused rat pancreas in response to 10 mM arginine in the presence of 5.5 mM glucose. Data are expressed as mean f SE of four experiments.
compared with that by 8.3 mM cantly, (P < 0.001) and that by 11 mM glucose (P < O.Ol), respectively. Therefore, amylin response to glucose was dose-dependent. E#ect of arginine on pancreatic amylin release 10 mM arginine in the presence of 5.5 mM glucose stimulated pancreatic amylin and insulin release (Fig. 1B). The amylin level rose from the base-line level to a peak of 994 k 128 pg/ml at 2 min and remained above 100 pg/ml during the infusion. Immediately after cessation of the arginine infusion, the amylin release decreased rapidly to the basal level. Arginine infusion always induced maximum release of amylin and insulin simultaneously. The cumulative output during the entire 10 min arginine infusion period was 9298 f 1434 pg (n = 4).
Discussion The present study clearly demonstrates that amylin release is stimulated by glucose and arginine in the isolated perfused rat pancreas. Both nutrients elicit a typical biphasic pattern of amylin release. By the ultrastructural immunogold technique, it has been shown that amylin is co-localized with insulin in the secretory granules of the pancreatic /3-cells [ 9, lo]. mRNA of amylin has been expressed selectively in the pancreatic islets [6]. Furthermore, the analysis of rat amylin precursor sequences deduced from its cDNA has shown a hydrophobic amino-terminal sequence consistent with a signal peptide in a secretory protein [6]. These findings suggest that amylin may be co-secreted with insulin from the pancreatic islets. As to arginine stimulation, it was not clarified whether glucose dependency of
192
amylin secretion by arginine might exist, as reported on insulin [ 151. From co-localization of the two peptides within the same secretory granules, we speculated that physiological level of per&ate glucose might be necessary for amylin response to arginine as well as insulin. Concerning the biological action of this peptide, Leighton et al. [ 81 reported that amylin inhibits insulin-induced glycogen synthesis in skeletal muscle. Therefore, it is thought that amylin might be a putative endocrine hormone which contributes to glucose homeostasis through its opposing action against insulin. Amylin was originally purified and characterized from insular amyloid deposits in patients with NIDDM and diabetic cats. Overproduction of this peptide by feline pancreatic fi cells has recently been proven to antedate the onset of overt diabetes mellitus [ 161. From these data, this novel peptide might be a significant factor in the pathogenesis of NIDDM. Further study is needed to clarify the pathophysiological significance of amylin in diabetes. References Westermark, P. and Wilander, E. (1978) The influence of amyloid deposits on the islet volume in maturity onset diabetes mellitus. Diabetologia 15, 4 17-42 1. Westermark, P., Wernstedt, C., Wilander, E. and Sletten, K. (1986) A novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochem. Biophys. Res. Commun. 140, 827-831. Westermark, P., Wernstedt, C., Wilander, E., Hayden, D.W., O’Brien, T.D. and Johnson, K.H. (1987) Amyloid fibrils in human insuhnoma 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, 3881-3885. Cooper, G.J.S., Willis, A.C., Clark, A., Turner, R.C., Sim, R.B. and Reid, K.B.M. (1987) Purification and characterization of a peptide from amyloid-rich pancreases of
10
11 12
13
14
15
16
type 2 diabetic patients. Proc. Natl. Acad. Sci. USA. 84, 8628-8632. Asai, J., Nakazato, M., Kangawa, K., Matsukura, S. and Matsuo, H. (1989) Isolation and sequence determination of rat islet amyloid polypeptide. Biochem. Biophys. Res. Commun. 164, 400-405. Leffert, J.D., Newgard, C.B., Okamoto, H., Milburn, J.L. and Luskey, K.L. (1989) Rat amylin: cloning and tissuespecific expression in pancreatic islets. Proc. Natl. Acad. Sci. USA. 86, 3127-3130. Clark, A., Cooper, G.J.S., Lewis, C.E. et al. (1987) Islet amyloid formed from diabetes-associated peptide may be pathogenic in type-2 diabetes. Lancet ii, 231-234. Leighton, B. and Cooper, G.J.S. (1988) Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature 335, 632-635. Johnson, K.H., O’Brien,T.D., Hayden, D.W. et al. (1988) Immunolocalization of islet amyloid polypeptide (IAPP) ain pancreatic fi-cells by means of peroxidase-antiperoxidase (PAP) and protein A-gold techniques. Am. J. Pathol. 130, l-8. Lukinius, A., Wilander, E., Westermark, G.T., Engstriim, U. and Westermark, P. (1989) Co-localization of islet amyloid polypeptide and insulin in the fi-cell secretory granules of the human pancreatic islets. Diabetologia 32, 240-244. Grodsky, G.M. and Fanska, R.E. (1975) The in vitro perfused pancreas. Methods Enzymol. 39, 364-372. Hisatomi, A., Maruyama, H., Orci, L., Vasko, M. and Unger, R.H. (1985) Adrenergically mediated intrapancreatic control of the glucagon response to glucopenia in the isolated rat pancreas. J. Clin. Invest. 75, 420-426. Chang, J.K., Chou, J., Butler, P. et al. (1990) Determination of amylin concentration in rat and human plasma. FASEB J. 4, Part II, A 1226, No. 5575 (abstract). Hyodo, J., Nanba, O., Nakamura, K., Kaneko, H. and Irie, M. (1983) Clinical application of plasma IRI determination using solid phase bead method-with the special reference to the comparison with a previous double antibody method. Clin. Endocrinol. 3 1, 1129-l 138. Sorenson, R.L. and Elde, R.P. (1983) Dissociation of glucose stimulation of somatostatin and insulin release from glucose inhibition of glucagon release in the isolated perfused rat pancreas. Diabetes 32, 561-567. Johnson, K.H., O’Brien, T.D., Jordan, K. and Westermark, P. (1989) Impaired glucose tolerance is associated with increased islet amyloid polypeptide (IAPP) immunoreactivity in pancreatic /?-cells. Am. J. Pathol. 135,245-250.
