Originals Basic Effects of Exogenous Somatostatin and Insulin on Islet Amyloid Polypeptide (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
This study examined the effects of exogenous somatostatin and insulin on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas. Somatostatin inhibited the release of both amylin and insulin from the perfused pancreas to the same extent. The infusion of 10 nM somatostatin resulted in 40% inhibition of the secretion of both amylin and insulin induced by 11.1 mM glucose and 10 m M arginine, and this inhibition was significantly increased to 70% by the infusion of 100 nM somatostatin (p < 0.05). The amylin/insulin molar ratios remained constant at 0.8 % and were not changed by the infusion of somatostatin. On the other hand exogenous insulin at a concentration of 1.8 nM did not affect the release of amylin induced by 11.1 mM glucose and 10 mM arginine, whereas 180 nM insulin slightly, although not significantly, inhibited the release of amylin by 15 %. These findings suggest that the release of amylin may be negatively regulated by somatostatin and that circulating insulin may have no direct effect on the release of amylin at least at a physiological concentration. Key words Islet Amyloid Polypeptide — Amylin — Somatostatin — Exogenous Insulin — Pancreas Perfusion
amylin from the perfused rat pancreas in parallel with insulin release {Ogawa, Harris, McCorkle, linger and Luskey 1990; Inoue, Hisatomi, Umeda and Nawata 1991). In the present study, we evaluated the effects of exogenous somatostatin and insulin on the release of amylin from the perfused rat pancreas. Materials and Methods Isolated rat pancreas
Rat amylin Introduction Islet amyloid polypeptide (IAPP), or amylin, is a 37-amino acid peptide originally isolated from amyloid deposits in the pancreatic islets (Westermark, Wernstedt, Wilander, Hay den, O'Brien and Johnson 1987; Cooper, Willis, Clark, Turner, Sim and Reid 1987). Amylin has been reported to co-exist with insulin in the same secretory granules of the pancreatic p-cells {Johnson, O'Brien, Hay den, Jordan, Ghobrial, Mahoney and Westermark 1988) and to inhibit the synthesis of glycogen stimulated by insulin in isolated skeletal muscle {Leighton and Cooper 1988). These findings suggest that amylin may be a new secretory protein of the pancreatic (B-cells that may have endocrine actions. We and others reported that both glucose and arginine stimulated the release of
Horm. metab. Res. 24 (1992) 251 -253 © Georg Thieme Verlag Stuttgart • New York
perfusion
After an overnight fast, the pancreas glands were isolated from male Wistar-King albino rats weighing 350 g to 400 g under anesthesia induced by the intraperitoneal injection of 50 mg/kg sodium pentobarbital. The pancreas obtained was perfused according to the method oiGrodsky andFanska (1975) with minor modifications (Hisatomi, Maruyama, Orci, Vasko and Unger 1985) using a KrebsRinger 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; 11.1 mM glucose and 10 mM arginine. The celiac trunk and the portal vein were cannulated and then nonrecirculating perfusion was begun at a constant flow rate of 3.6 ml/min, maintained by a peristaltic pump. The perfusion medium was oxygenated with a mixture of 95% O2 and 5% CO2 and maintained at 37 °C. After an equilibration period of 20 min, somatostatin (Peptide Institute, Osaka, Japan) or semisynthetic human insulin (Novo Research Institute, Copenhagen, Denmark) dissolved in 0.2% BSA-saline was added to the circulating perfusate through a side-arm infusion pump. 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) and then stored until assay at - 20 °C. radioimmunoassay
Concentrations of amylin in the effluent were measured by radioimmunoassay (RIA) as described previously (Butler, Chou, Carter, Wang, Bu, Chang, Chang and Rizza 1990). Specific antiserum to rat amylin and 125I-rat amylin were purchased from Peninsula Laboratories (Belmont, CA). RIA was performed using the double antibody method and rat amylin standards diluted with perfusion medium. The serial dilution curve of the perfusate sample paralleled the standard curve of the assay. Insulin
radioimmunoassay
Concentrations of insulin in the effluent were measured by RIA using a kit purchased from Dainabot Company, Ltd. (Tokyo, Japan) (Inoue et al. 1991). Rat insulin standards (Novo Research Institute, Copenhagen, Denmark) diluted with perfusion medium were employed. The serial dilution curve of the perfusate sample paralleled the standard curve.
Received: 15 May 1991
Accepted: 19 Oct 1991
Downloaded by: University of British Columbia. Copyrighted material.
Summary
Horm. metab. Res. 24 (1992)
K. Inoue, A. Hisatomi, F. Vmeda and H. Nawata Table 1 Effects of exogenous somatostatin and insulin on the secretion of amylin and insulin induced by 11.1 mM glucose and 10 mM arginine in the perfused rat pancreas. Period (min)
Amylin (fmol/min)
Somatostatin (nM) 0 -10- 0 1-10 10 0 16-25 100 26-35
710 + 136 381 ± 40 811 + 140 226 ± 28
Insulin (nM) 0 -10- 0 1.8 1-10 0 16-25 180 26-35
775 + 76 793 + 80 823 + 85 700 + 70
Insulin (pmol/min)
a1
b2 c1
'
85.8 + 8.1 49.3 + 2.7 a2 100.7 + 9.5 27.9+1.0 b1'c 1
Amylin/lnsulin Molar Ratio (%)
0.83 + 0.06 0.79 + 0.03 0.79 + 0.07 0.80 + 0.07
Values are given as mean ± SEM of four experiments. Statistical analysis was determined by the paired Student's t-test. a1 p < 0.05,a2p< 0.01 vs period (-10-0). b1 p < 0.05, b2 p < 0.01 vs period (1-10). c1 p
This study examined the effects of exogenous somatostatin and insulin on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas. Somatostatin inhibited the release of both amylin and insulin from the perfused pancreas to the same extent. The infusion of 10 nM somatostatin resulted in 40% inhibition of the secretion of both amylin and insulin induced by 11.1 mM glucose and 10 m M arginine, and this inhibition was significantly increased to 70% by the infusion of 100 nM somatostatin (p < 0.05). The amylin/insulin molar ratios remained constant at 0.8 % and were not changed by the infusion of somatostatin. On the other hand exogenous insulin at a concentration of 1.8 nM did not affect the release of amylin induced by 11.1 mM glucose and 10 mM arginine, whereas 180 nM insulin slightly, although not significantly, inhibited the release of amylin by 15 %. These findings suggest that the release of amylin may be negatively regulated by somatostatin and that circulating insulin may have no direct effect on the release of amylin at least at a physiological concentration. Key words Islet Amyloid Polypeptide — Amylin — Somatostatin — Exogenous Insulin — Pancreas Perfusion
amylin from the perfused rat pancreas in parallel with insulin release {Ogawa, Harris, McCorkle, linger and Luskey 1990; Inoue, Hisatomi, Umeda and Nawata 1991). In the present study, we evaluated the effects of exogenous somatostatin and insulin on the release of amylin from the perfused rat pancreas. Materials and Methods Isolated rat pancreas
Rat amylin Introduction Islet amyloid polypeptide (IAPP), or amylin, is a 37-amino acid peptide originally isolated from amyloid deposits in the pancreatic islets (Westermark, Wernstedt, Wilander, Hay den, O'Brien and Johnson 1987; Cooper, Willis, Clark, Turner, Sim and Reid 1987). Amylin has been reported to co-exist with insulin in the same secretory granules of the pancreatic p-cells {Johnson, O'Brien, Hay den, Jordan, Ghobrial, Mahoney and Westermark 1988) and to inhibit the synthesis of glycogen stimulated by insulin in isolated skeletal muscle {Leighton and Cooper 1988). These findings suggest that amylin may be a new secretory protein of the pancreatic (B-cells that may have endocrine actions. We and others reported that both glucose and arginine stimulated the release of
Horm. metab. Res. 24 (1992) 251 -253 © Georg Thieme Verlag Stuttgart • New York
perfusion
After an overnight fast, the pancreas glands were isolated from male Wistar-King albino rats weighing 350 g to 400 g under anesthesia induced by the intraperitoneal injection of 50 mg/kg sodium pentobarbital. The pancreas obtained was perfused according to the method oiGrodsky andFanska (1975) with minor modifications (Hisatomi, Maruyama, Orci, Vasko and Unger 1985) using a KrebsRinger 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; 11.1 mM glucose and 10 mM arginine. The celiac trunk and the portal vein were cannulated and then nonrecirculating perfusion was begun at a constant flow rate of 3.6 ml/min, maintained by a peristaltic pump. The perfusion medium was oxygenated with a mixture of 95% O2 and 5% CO2 and maintained at 37 °C. After an equilibration period of 20 min, somatostatin (Peptide Institute, Osaka, Japan) or semisynthetic human insulin (Novo Research Institute, Copenhagen, Denmark) dissolved in 0.2% BSA-saline was added to the circulating perfusate through a side-arm infusion pump. 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) and then stored until assay at - 20 °C. radioimmunoassay
Concentrations of amylin in the effluent were measured by radioimmunoassay (RIA) as described previously (Butler, Chou, Carter, Wang, Bu, Chang, Chang and Rizza 1990). Specific antiserum to rat amylin and 125I-rat amylin were purchased from Peninsula Laboratories (Belmont, CA). RIA was performed using the double antibody method and rat amylin standards diluted with perfusion medium. The serial dilution curve of the perfusate sample paralleled the standard curve of the assay. Insulin
radioimmunoassay
Concentrations of insulin in the effluent were measured by RIA using a kit purchased from Dainabot Company, Ltd. (Tokyo, Japan) (Inoue et al. 1991). Rat insulin standards (Novo Research Institute, Copenhagen, Denmark) diluted with perfusion medium were employed. The serial dilution curve of the perfusate sample paralleled the standard curve.
Received: 15 May 1991
Accepted: 19 Oct 1991
Downloaded by: University of British Columbia. Copyrighted material.
Summary
Horm. metab. Res. 24 (1992)
K. Inoue, A. Hisatomi, F. Vmeda and H. Nawata Table 1 Effects of exogenous somatostatin and insulin on the secretion of amylin and insulin induced by 11.1 mM glucose and 10 mM arginine in the perfused rat pancreas. Period (min)
Amylin (fmol/min)
Somatostatin (nM) 0 -10- 0 1-10 10 0 16-25 100 26-35
710 + 136 381 ± 40 811 + 140 226 ± 28
Insulin (nM) 0 -10- 0 1.8 1-10 0 16-25 180 26-35
775 + 76 793 + 80 823 + 85 700 + 70
Insulin (pmol/min)
a1
b2 c1
'
85.8 + 8.1 49.3 + 2.7 a2 100.7 + 9.5 27.9+1.0 b1'c 1
Amylin/lnsulin Molar Ratio (%)
0.83 + 0.06 0.79 + 0.03 0.79 + 0.07 0.80 + 0.07
Values are given as mean ± SEM of four experiments. Statistical analysis was determined by the paired Student's t-test. a1 p < 0.05,a2p< 0.01 vs period (-10-0). b1 p < 0.05, b2 p < 0.01 vs period (1-10). c1 p
