Originals Basic
407
Effects of Glucagon-Like Peptide 1 (7—36) Amide and Glucagon on Amylin Release from Perfused Rat Pancreas K. Inoue, A. Hisatomi, F. Umeda and H. Nawata Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan
The effects of glucagon-like peptide 1 (7—36) amide [GLP-1 (7—36) amide ] and glucagon on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas were studied. In the presence of 5.6 mM glucose, GLP-1 ( 7 - 3 6 ) amide and glucagon stimulated the release of amylin from the perfused pancreas. The infusion of GLP-1 ( 7 - 3 6 ) amide at a concentration of 10~ 9 M elicited a biphasic release of amylin similar to that of insulin. The cumulative output of amylin induced by 1 0 - 9 M GLP-1 (7—36) amide was significantly higher than that by 10~ M glucagon (p < 0.01). The amylin/insulin molar ratios induced by GLP-1 ( 7 - 3 6 ) amide and glucagon were about 1 % and did not differ significantly. These findings suggest that GLP-1 (7—36) amide and glucagon stimulate the release of amylin from the pancreas and that the concomitant secretion of amylin and insulin might contribute to glucose homeostasis. Key words Amylin — Islet Amyloid Polypeptide — Glucagon-Like Peptide 1 — Glucagon — Pancreas Perfusion
which are insulinotropic peptides (Samols, Marri and Marks 1966; Weir, Mojsov, Hendrick and Habener 1989), on the secretion of amylin by the isolated perfused rat pancreas. Materials and Methods Isolated rat pancreas
perfusion
Male Wistar-King albino rats weighing 350 to 400 g were used in this study. The pancreas glands were isolated and perfused according to the method of Grodsky and Fanska (1975) with minor modifications (Hisatomi, Maruyama, Orci, Vasko and Unger 1985). Following an overnight fast, the rats were anesthetized by the intraperitoneal injection of sodium pentobarbital, 50 mg/kg. The pancreas 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 a peristaltic pump (Harvard Apparatus Company, Millis, Mass.) and using a Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 4.5% dextran T-70 (Pharmacia Fine Chemicals, Uppsala, Sweden), 1 % bovine serum albumin (BSA) (Sigma Chemical Company, St. Louis, MO), 5 mM each of pyruvate, fumarate and glutamate and 5.6 mM glucose. The perfusion medium was oxygenated with a mixture of 95 % O2 and 5 % CO2 and maintained at 37 °C. An equilibration period of 20 min preceded each experiment. GLP-1 (7—36) amide (Peninsula Laboratories, Belmont, CA) and glucagon (Peptide Institute, Osaka, Japan) were dissolved in 0.2% BSA-saline and added to the circulating perfusate through a side-arm infusion pump (Model 975; Harvard Apparatus Company). One-minute aliquots of the effluent were collected in chilled tubes containing 0.4 ml of an EDTA-benzamidine mixture (0.03 M/0.3 M). These samples were stored until assay at — 20 CC.
Introduction Islet amyloid polypeptide (IAPP), or amylin, is a 37-amino acid peptide originally isolated from amyloid deposits in human insulinomas (Westermark, Wernstedt, Wilander, Hayden, O'Brien and Johnson 1987) and from the pancreatic islets of patients with non-insulin-dependent diabetes mellitus (NIDDM) {Cooper, Willis, Clark, Turner, Sim and Reid 1987). The co-localization of amylin and insulin within the secretory granules of the pancreatic B-cells {Johnson, O'Brien, Hayden, Jordan, Ghobrial, Mahoney and Westermark 1988) suggests that amylin may be co-secreted with insulin from the P-cells. Recent studies have demonstrated that glucose stimulates the secretion of amylin from the isolated islets (Kanatsuka, Makino, Ohsawa, Tokuyama, Yamaguchi, Yoshida and Adachi 1989) and from the perfused pancreas (Inoue, Hisatomi, Umeda and Nawata 1990). This study was undertaken to evaluate the effects of glucagon-like peptide 1 ( 7 - 3 6 ) amide [GLP-1 ( 7 - 3 6 ) amide] and glucagon, both of
Horm.metab.Res.23(1991)407-409 © Georg Thieme Verlag Stuttgart • New York
Radioimmunoassay
of rat
amylin
Concentrations of amylin in the effluent were measured by radioimmunoassay (RIA) (Butler, Chou, Carter, Wang, Bu, Chang, Chang and Rizza 1990). Specific antiserum to rat amylin and I-rat amylin were purchased from Peninsula Laboratories. RIA was performed by a double-antibody technique. The specific antiserum to rat amylin was produced in rabbits by immunization with synthetic rat amylin. This antiserum exhibited a 13.3 % crossreactivity with human amylin, but less than 0.01 % cross-reactivity with human calcitonin-gene related peptides (CGRPs) and rat CGRPs. The minimum sensitivity of the assay was 2.6 pM. The serial dilution curve of perfusate samples paralleled the standard curve of the assay. Insulin
radioimmunoassay
Concentrations of insulin in the effluent were measured by RIA using a commercial kit purchased from Dainabot Company, Ltd. (Tokyo, Japan) (Hyodo, Nanba, Nakamura, Kaneko and Irie 1983). Rat insulin standards (Novo Research Institute, CoReceived: 13 July 1990
Accepted: 5 Febr. 1991
Downloaded by: University of Illinois. Copyrighted material.
Summary
Horm. metab. Res. 23 (1991)
K. Inoue, A. Hisatomi, F. Umeda and H. Nawata Table 1 Cumulative output of amylin and insulin induced by GLP-1 (7-36) amide and glucagon. Amylin (fmol/10 min)
Insulin (fmol/10 min)
Amylin/lnsulin Molar Ratio (%)
GLP-1 (7-36) amide (M)
10~9 10~8
1,542 ±278 552±125
143,300 ±15,000 45,100± 7,800
1.05±0.14 1.19±0.16
Glucagon (M)
10 ~9 10 " 8
279±121 429 ± 59
a2 a2
29,700±4,300 54,800 ±5,200
a3 a3
1.04±0.55 0.78 ±0.06
Values are given as mean ± SEM of six experiments. a2 p
407
Effects of Glucagon-Like Peptide 1 (7—36) Amide and Glucagon on Amylin Release from Perfused Rat Pancreas K. Inoue, A. Hisatomi, F. Umeda and H. Nawata Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan
The effects of glucagon-like peptide 1 (7—36) amide [GLP-1 (7—36) amide ] and glucagon on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas were studied. In the presence of 5.6 mM glucose, GLP-1 ( 7 - 3 6 ) amide and glucagon stimulated the release of amylin from the perfused pancreas. The infusion of GLP-1 ( 7 - 3 6 ) amide at a concentration of 10~ 9 M elicited a biphasic release of amylin similar to that of insulin. The cumulative output of amylin induced by 1 0 - 9 M GLP-1 (7—36) amide was significantly higher than that by 10~ M glucagon (p < 0.01). The amylin/insulin molar ratios induced by GLP-1 ( 7 - 3 6 ) amide and glucagon were about 1 % and did not differ significantly. These findings suggest that GLP-1 (7—36) amide and glucagon stimulate the release of amylin from the pancreas and that the concomitant secretion of amylin and insulin might contribute to glucose homeostasis. Key words Amylin — Islet Amyloid Polypeptide — Glucagon-Like Peptide 1 — Glucagon — Pancreas Perfusion
which are insulinotropic peptides (Samols, Marri and Marks 1966; Weir, Mojsov, Hendrick and Habener 1989), on the secretion of amylin by the isolated perfused rat pancreas. Materials and Methods Isolated rat pancreas
perfusion
Male Wistar-King albino rats weighing 350 to 400 g were used in this study. The pancreas glands were isolated and perfused according to the method of Grodsky and Fanska (1975) with minor modifications (Hisatomi, Maruyama, Orci, Vasko and Unger 1985). Following an overnight fast, the rats were anesthetized by the intraperitoneal injection of sodium pentobarbital, 50 mg/kg. The pancreas 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 a peristaltic pump (Harvard Apparatus Company, Millis, Mass.) and using a Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 4.5% dextran T-70 (Pharmacia Fine Chemicals, Uppsala, Sweden), 1 % bovine serum albumin (BSA) (Sigma Chemical Company, St. Louis, MO), 5 mM each of pyruvate, fumarate and glutamate and 5.6 mM glucose. The perfusion medium was oxygenated with a mixture of 95 % O2 and 5 % CO2 and maintained at 37 °C. An equilibration period of 20 min preceded each experiment. GLP-1 (7—36) amide (Peninsula Laboratories, Belmont, CA) and glucagon (Peptide Institute, Osaka, Japan) were dissolved in 0.2% BSA-saline and added to the circulating perfusate through a side-arm infusion pump (Model 975; Harvard Apparatus Company). One-minute aliquots of the effluent were collected in chilled tubes containing 0.4 ml of an EDTA-benzamidine mixture (0.03 M/0.3 M). These samples were stored until assay at — 20 CC.
Introduction Islet amyloid polypeptide (IAPP), or amylin, is a 37-amino acid peptide originally isolated from amyloid deposits in human insulinomas (Westermark, Wernstedt, Wilander, Hayden, O'Brien and Johnson 1987) and from the pancreatic islets of patients with non-insulin-dependent diabetes mellitus (NIDDM) {Cooper, Willis, Clark, Turner, Sim and Reid 1987). The co-localization of amylin and insulin within the secretory granules of the pancreatic B-cells {Johnson, O'Brien, Hayden, Jordan, Ghobrial, Mahoney and Westermark 1988) suggests that amylin may be co-secreted with insulin from the P-cells. Recent studies have demonstrated that glucose stimulates the secretion of amylin from the isolated islets (Kanatsuka, Makino, Ohsawa, Tokuyama, Yamaguchi, Yoshida and Adachi 1989) and from the perfused pancreas (Inoue, Hisatomi, Umeda and Nawata 1990). This study was undertaken to evaluate the effects of glucagon-like peptide 1 ( 7 - 3 6 ) amide [GLP-1 ( 7 - 3 6 ) amide] and glucagon, both of
Horm.metab.Res.23(1991)407-409 © Georg Thieme Verlag Stuttgart • New York
Radioimmunoassay
of rat
amylin
Concentrations of amylin in the effluent were measured by radioimmunoassay (RIA) (Butler, Chou, Carter, Wang, Bu, Chang, Chang and Rizza 1990). Specific antiserum to rat amylin and I-rat amylin were purchased from Peninsula Laboratories. RIA was performed by a double-antibody technique. The specific antiserum to rat amylin was produced in rabbits by immunization with synthetic rat amylin. This antiserum exhibited a 13.3 % crossreactivity with human amylin, but less than 0.01 % cross-reactivity with human calcitonin-gene related peptides (CGRPs) and rat CGRPs. The minimum sensitivity of the assay was 2.6 pM. The serial dilution curve of perfusate samples paralleled the standard curve of the assay. Insulin
radioimmunoassay
Concentrations of insulin in the effluent were measured by RIA using a commercial kit purchased from Dainabot Company, Ltd. (Tokyo, Japan) (Hyodo, Nanba, Nakamura, Kaneko and Irie 1983). Rat insulin standards (Novo Research Institute, CoReceived: 13 July 1990
Accepted: 5 Febr. 1991
Downloaded by: University of Illinois. Copyrighted material.
Summary
Horm. metab. Res. 23 (1991)
K. Inoue, A. Hisatomi, F. Umeda and H. Nawata Table 1 Cumulative output of amylin and insulin induced by GLP-1 (7-36) amide and glucagon. Amylin (fmol/10 min)
Insulin (fmol/10 min)
Amylin/lnsulin Molar Ratio (%)
GLP-1 (7-36) amide (M)
10~9 10~8
1,542 ±278 552±125
143,300 ±15,000 45,100± 7,800
1.05±0.14 1.19±0.16
Glucagon (M)
10 ~9 10 " 8
279±121 429 ± 59
a2 a2
29,700±4,300 54,800 ±5,200
a3 a3
1.04±0.55 0.78 ±0.06
Values are given as mean ± SEM of six experiments. a2 p
