0013-7227/91/1286-2739$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 6 Printed in U.S.A.
Hypersecretion of Islet Amyloid Polypeptide from Pancreatic Islets of Ventromedial HypothalamicLesioned Rats and Obese Zucker Rats* YOSHIHARU TOKUYAMA, AZUMA KANATSUKA, HARUHIKO OHSAWA, TAKAHIDE YAMAGUCHI, HIDEICHI MAKINO, SHO YOSHIDA, HAJIME NAGASE, AND SHUJI INOUE Second Department of Internal Medicine, Chiba University School of Medicine, Chiba, 2S0, and Third Department of Internal Medicine (H.N., S.I.) Yokohama City University School of Medicine, Yokohama, 236, Japan
ABSTRACT. To investigate the possible role of islet amyloid polypeptide (IAPP) in the development of type 2 diabetes mellitus, we examined the IAPP content and secretion in pancreatic islets isolated from ventromedial hypothalamic (VMH)-lesioned rats and genetically obese Zucker rats, using a specific RIA for IAPP. Obesity and hyperinsulinemia were observed in rats 21 days after VMH lesioning. IAPP content was increased in the islets of VMH-lesioned rats compared with findings in the shamoperated controls (100.9 ± 6.6 vs. 72.8 ± 3.85 fmol/islet; P < 0.01). Isolated islets of VMH-lesioned rats secreted larger amounts of IAPP in the presence of 2.8 mM and 16.7 mM glucose (2.99 ± 0.98 and 11.2 ± 1.29 fmol • islet"1 • 3 h"1) than was noted
I
SLET amyloid polypeptide (IAPP) also termed amylin is a major component of islet amyloid. This 37 amino acid peptide seems to be a natural product in pancreatic B cells, as determined in immunohistochemical studies (1-4) and with RIA (5). IAPP is secreted from the pancreatic islets in response to glucose (5, 6). An analysis of the complementary DNA encoding IAPP indicated that the IAPP precursor has a signal sequence and is likely to undergo amidation (7, 8). IAPP may inhibit the action of insulin on glycogenesis in isolated rat skeletal muscle (9, 10) and inhibit the glucose-stimulated insulin secretion from isolated rat pancreatic islets (11). In type 2 (noninsulin dependent) diabetes mellitus, the most significant pathological change in the pancreatic islets is amyloid deposits (12). In human type 2 diabetes mellitus, the extent of islet amyloid deposition increases with increase in the clinical severity (13). On the other hand, obesity and hyperinsulinemia are observed in the sub-
in sham-operated rats (ND and 6.65 ± 0.78 fmol islet"1 -3 h"1). In the obese Zucker rats, aged 14 weeks, IAPP concentrations in the islets were elevated compared with lean rats (133.3 ± 10.6 vs. 84.4 ± 8.5 fmol/islet; P < 0.01). The isolated islets secreted larger amounts of IAPP in response to 2.8 mM and 16.7 mM glucose (2.83 ± 0.88 and 15.81 ± 1.35 fmol • islet"1 • 3 h"1) than did those from lean control rats (0.36 ± 0.19 and 12.49 ± 1.20 fmol • islet"1 • 3 h"1). These results strongly suggest that overproduction and hypersecretion of IAPP occur in animals with obesity and hyperinsulinemia. {Endocrinology 128: 2739-2744, 1991)
jects with type 2 diabetes mellitus (14). To investigate changes in synthesis and secretion in subjects with this type of diabetes mellitus, we examined the amount of IAPP content and secretion in pancreatic islets isolated from ventromedial hypothalamic (VMH)-lesioned rats with obesity and hyperinsulinemia and from genetically obese Zucker rats that carry the recessive homozygous trait for obesity designated by fa/fa (15), and which are hyperinsulinemic.
Materials and Methods VMH-lesioned rats
Received October 2, 1990. Address all correspondence and requests for reprints to: Dr. Azuma Kanatsuka, Second Department of Internal Medicine, Chiba University School of Medicine, 1-8-1 Inohana, Chiba, 280, Japan. * This work was supported in part by Japan Ministry of Education Grant 01570625.
Female Sprague-Dawley rats 10 weeks old and weighing 200250 g were housed in cages maintained at 24 C and given free access to laboratory chow (Oriental Yeast Co., Tokyo, Japan) and tap water. These rats were anesthetized with hexobarbital (50 mg/kg, ip), and VMH lesions or sham operations were carried out as described (16). Experiments were then performed 3 weeks after the lesioning. At termination of the experiments, the brains of all the rats were fixed in normal saline containing 10% formaldehyde, and it was histologically confirmed that the VMH had been destroyed.
2739
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 20:11 For personal use only. No other uses without permission. . All rights reserved.
2740
HYPERSECRETION OF IAPP IN VMH-LESIONED AND ZUCKER RATS
Endo • 1991 Vo! 128* No 6
TABLE 1. Characteristics of VMH-lesioned and control rats
Zucker rats Animals were obtained from the Zucker breeding colony maintained at Kiwa Laboratory Animals Co. (Wakayama, Japan). Groups of 16-week-old female, homozygous obese (fa/fa) and lean (Fa/?) Zucker rats were housed two to three per cage on a 12-h light-dark cycle at 24 C. All the animals were fed tap water and a laboratory chow diet. Islet preparation and incubation Rats were anesthetized with sodium pentobarbital, body wt was measured, and 2-4 ml of blood were withdrawn for determination of blood glucose and serum insulin levels. Blood glucose was immediately determined using a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA) based on the oxidase method. Serum was frozen at -20 C for the determination of insulin levels, using an enzyme immunoassay (EIA) kit (Enzyme-Test Insulin, Boehringer Mannheim Co., Mannheim, Germany). Intra- and interassay coefficients of variation were 4.5% and 5.6%, respectively. The pancreatic islets were isolated by a modification of reported methods (17). Collagenase digestion was carried out at 37 C for 25 min after collagenase (20 mg collagenase/10 ml Hanks' solution) injection into the common bile duct. The medium used for preincubation and incubation was KrebsRinger bicarbonate (KRB) buffer, pH 7.4, containing 1 g/liter BSA (RIA Grade Fraction V, Sigma Chemical, St. Louis, MO) and 500 kallikrein inhibiting units/ml aprotinin (Bayer Co., Osaka, Japan). Fifty islets per vial were preincubated for 60 min in 0.2 ml KRB buffer containing 2.8 mM glucose at 37 C under 95% O2-5% CO2, then were incubated for an additional 3 h in 0.1 ml KRB buffer containing 2.8 mM and 16.7 mM glucose. The incubation was terminated by cooling the vials in an ice bath. The medium of each vial was frozen at -80 C until insulin and IAPP assay. Measurement of IAPP and insulin release from the islets Aliquots of medium from each vial were taken for the RIA of IAPP (5). Intra- and interassay coefficients of variation were 4.2% and 5.5%, respectively. Another aliquot of the medium was used for the assay of insulin, using an EIA kit. Measurement of IAPP and insulin content in islets Isolated islets were homogenized in 70% formic acid by sonication. The formic acid was removed to near dryness with a vaporizer, and the extracts were lyophilized. Just before the assay, the extracts were dissolved in 0.1 ml 0.2 N acetic acid, diluted in the assay buffer, and adjusted to pH 7.4 with NaOH. The extracts were measured for IAPP (5), and insulin in the extracts was also determined by EIA using the insulin assay kit described above. The data in the text and figures are presented as means ± SEM. Student's t test was used for statistical analysis.
Results
Body wt (g) Serum insulin (pM) Plasma glucose (mM)
VMH-lesioned rats
Control rats
340 ± 17 (8)° 293.5 ± 26.5 (8)" 8.47 ± 0.49 (8) NS6
257 ± 8.5 (8) 127 ± 12.9 (8) 8.41 ± 0.35 (8)
Values are mean ± SEM with numbers of observations in parentheses. " P < 0.005 for comparison with controls. 6 NS, Not significant. TABLE 2. Characteristics of obese and lean Zucker rats Obese rats
Lean rats
502.8 ± 22 (10)° 223.3 ± 4.5 (10) Body wt (g) 119.1 ± 6.9 (10) Serum insulin (pM) 182.3 ± 12.9 (10)" 10.5 ± 0.38 (10) Plasma glucose (mM) 9.17 ± 0.71 (10) NS* Values are mean ± SEM with numbers of observations in parentheses. a P < 0.005 for comparison with controls. 6 NS, Not significant.
time of experiment. The serum insulin concentration in the rats with VMH lesions and obese Zucker rats was higher than that in the controls. No difference was observed in plasma glucose concentrations between obese rats and the controls. Release of insulin and IAPP from islets of VMHlesioned rats (Fig. 1, A and B) In both VMH-lesioned rats and the controls, more insulin was secreted from isolated islets during exposure to high glucose, 16.7 mM, compared with 2.8 mM glucose. Islets from the VMH-lesioned rats released a larger amount of insulin than did those of controls at each concentration of glucose. Concentrations of 2.8 mM glucose stimulated the secretion of insulin in the VMHlesioned rats 4-fold above that in controls (39.4 ± 6.35 vs. 9.83 ± 1.85 fmol-islet"1-3 h"1, P < 0.01). With 16.7 mM glucose, the secretion of insulin from the islets of VMH-lesioned rats was 2.2-fold over that of the controls (137.5 ± 17.4 vs. 62.4 ± 6.09 fmol• islet"1 • 3 h"1, P < 0.01). Figure IB compares the release of IAPP by stimulation of glucose in VMH-lesioned rats and controls. The islets from the VMH-lesioned rats released 1.68-fold the IAPP released from the controls, in response to 16.7 mM glucose (11.2 ± 1.29 vs. 6.65 ± 0.78 fmol • islet"1 • 3 h"1, P < 0.01). A small amount of IAPP was released from the islets of VMH-lesioned rats in case of exposure to 2.8 mM glucose (2.99 ± 0.98 fmol • islet"1 • 3 h"1). There was no detectable amount of IAPP released from islets of the controls.
Characteristics of animals
Release of insulin and IAPP from islets of Zucker rats (Fig. 2, A and B)
As shown in Tables 1 and 2, the VMH-lesioned and obese Zucker rats weighed more than the controls at the
In both the obese and lean rats, more insulin was secreted from the isolated islets exposed to high glucose,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 20:11 For personal use only. No other uses without permission. . All rights reserved.
HYPERSECRETION OF IAPP IN VMH-LESIONED AND ZUCKER RATS A
FIG. 1. Secretion of insulin and IAPP from isolated pancreatic islets obtained from VMH-lesioned rats (M) and from sham-operated rats (D) incubated for 3 h in the presence of 2.8 mM or 16.7 mM glucose. A, Secretion of insulin. B, Secretion of IAPP. Values are mean ± SEM with numbers of observations in parentheses. *, P < 0.01.
1
2741
B
200-
20-
150-
15-
p 10
100-
I
50-
(8)
ND
glucose
2.8
(mM)
glucose
16.7
(mM)
2.8
16.7
obese rats released 1.27-fold amounts of IAPP, compared with findings in the lean rats, in response to 16.7 mM glucose (15.81 ± 1.35 vs. 12.49 ± 1.20 fmol • islet"1 • 3 h"1, P< 0.01). Although small amounts of IAPP were released from the islets of obese rats in case of exposure to 2.8 mM glucose, the amounts were significantly greater than that from the islets of lean rats (2.83 ± 0.88 vs. 0.36 ± 0.19 fmol-islet"1-3 h"1, P < 0.05).
150
Insulin and IAPP content in islets of VMH-lesioned rats (Fig. 3)
(10) (10)
glucose (mM)
too)
(10)
2.8
16.7
glucose (mM)
2.8
(10)
(10)
Both insulin and IAPP content in islets of VMHlesioned rats were slightly but significantly greater than in the controls (insulin: 1074.0 ± 76.8 vs. 819.9 ± 62.6
16.7
FIG. 2. Secretion of insulin and IAPP from isolated pancreatic islets obtained from obese Zucker rats (H) and from lean rats (•) incubated for 3 h in the presence of 2.8 mM or 16.7 mM glucose. A, Secretion of insulin. B, Secretion of IAPP. Values are mean ± SEM with numbers of observations in parentheses. *, P < 0.01; **, P < 0.05.
16.7 mM, compared with 2.8 mM glucose. The islets from obese rats released larger amounts of insulin than did those of lean rats, at each concentration of glucose. Glucose, 2.8 mM, stimulated the secretion of insulin in obese rats 2.8-fold over that in lean rats (154.9 ± 17.9 vs. 55.5 ± 7.18 fmol-islet"1.3 h"1, P < 0.01). Secretion of insulin from the islets of obese rats was 1.5-fold greater than that of lean rats in the case of 16.7 mM glucose (221.8 ± 13.6 vs. 145.8 ± 5.31 fmol • islet"1 • 3 h"\ P < 0.01). Figure IB compares the release of IAPP by stimulation of glucose in obese and lean rats. The islets from the
A
B
U 1500 -
V 150H
o «£ 1000 c
rh
c
c
8 500 H _c "5 c 0
0) *->
o 50o
Q.
(8)
(8) Sham
VMH
Sham
VMH
FIG. 3. Contents of insulin and IAPP in the pancreatic islets obtained from VMH-lesioned rats and sham-operated rats. A, Content of insulin. B, Content of IAPP. Mean ± SEM for IAPP and insulin contents are expressed in femtomolesi per islet. *, P < 0.01.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 20:11 For personal use only. No other uses without permission. . All rights reserved.
fmol/islet, P < 0.01; IAPP: 100.9 ± 6.6 vs. 72.8 ± 3.85 fmol/islet, P < 0.01).
TABLE 4. Secretion of rate of insulin and IAPP from the islets of obese and lean Zucker rats Obese rats
Insulin and IAPP content in islets of Zucker rats (Fig. 4)
Glucose
The insulin and IAPP content in islets of obese rats were increased, 1.31-fold and 1.57-fold, respectively, compared with findings in the lean rats (insulin: 2960.4 ± 119.9 vs. 2258.2 ± 80.4 fmol/islet, P < 0.01; IAPP: 133.3 ± 10.6 vs. 84.4 ± 8.5 fmol/islet, P < 0.01).
Insulin release {% of content) 5.2 ± 0.62 (10)° 2.8 mM 7.4 ± 0.47 (10) NS6 16.7 mM IAPP release (% of content) 2.1 ± 0.66 (10)c 2.8 mM 11.8 ± 1.14 (10) NS" 16.7 mM
Rate of secretion of insulin and IAPP expressed as a percent of content (Tables 3 and 4) Both insulin and IAPP secretion rates from the islets of VMH-lesioned rats and Zucker rats were expressed as percentage of content. In the VMH-lesioned rats, insulin release per content at each concentration of glucose and IAPP release per content at 2.8 mM glucose were significantly increased. However, the release of IAPP in 16.7 mM glucose showed no significant difference between the VMH-lesioned and A
U
B
3000-
V 150-
o I 2000
0)
c
•->
c o o
IOOO
50-
O-
(10)
v,0,
(10)
lean
obese
lean
obese
FIG. 4. Contents of insulin and IAPP in the pancreatic islets obtained from obese Zucker rats and lean rats. A, Content of insulin. B, Content of IAPP. Mean ± SEM for IAPP and insulin contents are expressed in femtomoles per islet. *, P < 0.01. TABLE 3. Secretion rate of insulin and IAPP from the islets of VMHlesioned rats and control rats Glucose
VMH-lesioned rats
Insulin release (% of content) 2.8 mM 3.6 ± 0.59 (8)° 16.7 mM 12.8 ± 1.6 (8)6 IAPP release (% of content) 2.8 mM 2.9 ± 0.97 (8)° 16.7 mM 11.1 ± 1.2 (8) NSC
Lean rats 2.4 ± 0.31 (10) 6.4 ± 0.23 (10) 0.42 ± 0.22 (10) 14.7 ± 1.42 (10)
Each value is calculated from the secretion (fmol • islet"1 • 3 h"1) and content (fmol/islet). Values are mean ± SEM with numbers of observations in parentheses. " P< 0.01 for comparison with controls. * NS, Not significant. c P < 0.05 for comparison with controls.
control rats. In obese Zucker rats, insulin release per content was increased with a low dose of glucose but was not significantly elevated with a high dose. IAPP release per content was slightly but significantly increased with 2.8 mM glucose. At 16.7 mM glucose there was no significant difference between the IAPP release in obese and lean rats. Discussion
E 100-
+-> 0)
8
Endo • 1991 Voll28-No6
HYPERSECRETION OF IAPP IN VMH-LESIONED AND ZUCKER RATS
2742
Control rats 1.1 ± 0.22 (8) 7.6 ± 0.74 (8) 0 (8) 9.1 ± 1.07 (8)
Each value is calculated from the secretion (fmol-islet"1^ h"1) and content (fmol/islet). Values are mean ± SEM with numbers of observations in parentheses. °P
Vol. 128, No. 6 Printed in U.S.A.
Hypersecretion of Islet Amyloid Polypeptide from Pancreatic Islets of Ventromedial HypothalamicLesioned Rats and Obese Zucker Rats* YOSHIHARU TOKUYAMA, AZUMA KANATSUKA, HARUHIKO OHSAWA, TAKAHIDE YAMAGUCHI, HIDEICHI MAKINO, SHO YOSHIDA, HAJIME NAGASE, AND SHUJI INOUE Second Department of Internal Medicine, Chiba University School of Medicine, Chiba, 2S0, and Third Department of Internal Medicine (H.N., S.I.) Yokohama City University School of Medicine, Yokohama, 236, Japan
ABSTRACT. To investigate the possible role of islet amyloid polypeptide (IAPP) in the development of type 2 diabetes mellitus, we examined the IAPP content and secretion in pancreatic islets isolated from ventromedial hypothalamic (VMH)-lesioned rats and genetically obese Zucker rats, using a specific RIA for IAPP. Obesity and hyperinsulinemia were observed in rats 21 days after VMH lesioning. IAPP content was increased in the islets of VMH-lesioned rats compared with findings in the shamoperated controls (100.9 ± 6.6 vs. 72.8 ± 3.85 fmol/islet; P < 0.01). Isolated islets of VMH-lesioned rats secreted larger amounts of IAPP in the presence of 2.8 mM and 16.7 mM glucose (2.99 ± 0.98 and 11.2 ± 1.29 fmol • islet"1 • 3 h"1) than was noted
I
SLET amyloid polypeptide (IAPP) also termed amylin is a major component of islet amyloid. This 37 amino acid peptide seems to be a natural product in pancreatic B cells, as determined in immunohistochemical studies (1-4) and with RIA (5). IAPP is secreted from the pancreatic islets in response to glucose (5, 6). An analysis of the complementary DNA encoding IAPP indicated that the IAPP precursor has a signal sequence and is likely to undergo amidation (7, 8). IAPP may inhibit the action of insulin on glycogenesis in isolated rat skeletal muscle (9, 10) and inhibit the glucose-stimulated insulin secretion from isolated rat pancreatic islets (11). In type 2 (noninsulin dependent) diabetes mellitus, the most significant pathological change in the pancreatic islets is amyloid deposits (12). In human type 2 diabetes mellitus, the extent of islet amyloid deposition increases with increase in the clinical severity (13). On the other hand, obesity and hyperinsulinemia are observed in the sub-
in sham-operated rats (ND and 6.65 ± 0.78 fmol islet"1 -3 h"1). In the obese Zucker rats, aged 14 weeks, IAPP concentrations in the islets were elevated compared with lean rats (133.3 ± 10.6 vs. 84.4 ± 8.5 fmol/islet; P < 0.01). The isolated islets secreted larger amounts of IAPP in response to 2.8 mM and 16.7 mM glucose (2.83 ± 0.88 and 15.81 ± 1.35 fmol • islet"1 • 3 h"1) than did those from lean control rats (0.36 ± 0.19 and 12.49 ± 1.20 fmol • islet"1 • 3 h"1). These results strongly suggest that overproduction and hypersecretion of IAPP occur in animals with obesity and hyperinsulinemia. {Endocrinology 128: 2739-2744, 1991)
jects with type 2 diabetes mellitus (14). To investigate changes in synthesis and secretion in subjects with this type of diabetes mellitus, we examined the amount of IAPP content and secretion in pancreatic islets isolated from ventromedial hypothalamic (VMH)-lesioned rats with obesity and hyperinsulinemia and from genetically obese Zucker rats that carry the recessive homozygous trait for obesity designated by fa/fa (15), and which are hyperinsulinemic.
Materials and Methods VMH-lesioned rats
Received October 2, 1990. Address all correspondence and requests for reprints to: Dr. Azuma Kanatsuka, Second Department of Internal Medicine, Chiba University School of Medicine, 1-8-1 Inohana, Chiba, 280, Japan. * This work was supported in part by Japan Ministry of Education Grant 01570625.
Female Sprague-Dawley rats 10 weeks old and weighing 200250 g were housed in cages maintained at 24 C and given free access to laboratory chow (Oriental Yeast Co., Tokyo, Japan) and tap water. These rats were anesthetized with hexobarbital (50 mg/kg, ip), and VMH lesions or sham operations were carried out as described (16). Experiments were then performed 3 weeks after the lesioning. At termination of the experiments, the brains of all the rats were fixed in normal saline containing 10% formaldehyde, and it was histologically confirmed that the VMH had been destroyed.
2739
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 20:11 For personal use only. No other uses without permission. . All rights reserved.
2740
HYPERSECRETION OF IAPP IN VMH-LESIONED AND ZUCKER RATS
Endo • 1991 Vo! 128* No 6
TABLE 1. Characteristics of VMH-lesioned and control rats
Zucker rats Animals were obtained from the Zucker breeding colony maintained at Kiwa Laboratory Animals Co. (Wakayama, Japan). Groups of 16-week-old female, homozygous obese (fa/fa) and lean (Fa/?) Zucker rats were housed two to three per cage on a 12-h light-dark cycle at 24 C. All the animals were fed tap water and a laboratory chow diet. Islet preparation and incubation Rats were anesthetized with sodium pentobarbital, body wt was measured, and 2-4 ml of blood were withdrawn for determination of blood glucose and serum insulin levels. Blood glucose was immediately determined using a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA) based on the oxidase method. Serum was frozen at -20 C for the determination of insulin levels, using an enzyme immunoassay (EIA) kit (Enzyme-Test Insulin, Boehringer Mannheim Co., Mannheim, Germany). Intra- and interassay coefficients of variation were 4.5% and 5.6%, respectively. The pancreatic islets were isolated by a modification of reported methods (17). Collagenase digestion was carried out at 37 C for 25 min after collagenase (20 mg collagenase/10 ml Hanks' solution) injection into the common bile duct. The medium used for preincubation and incubation was KrebsRinger bicarbonate (KRB) buffer, pH 7.4, containing 1 g/liter BSA (RIA Grade Fraction V, Sigma Chemical, St. Louis, MO) and 500 kallikrein inhibiting units/ml aprotinin (Bayer Co., Osaka, Japan). Fifty islets per vial were preincubated for 60 min in 0.2 ml KRB buffer containing 2.8 mM glucose at 37 C under 95% O2-5% CO2, then were incubated for an additional 3 h in 0.1 ml KRB buffer containing 2.8 mM and 16.7 mM glucose. The incubation was terminated by cooling the vials in an ice bath. The medium of each vial was frozen at -80 C until insulin and IAPP assay. Measurement of IAPP and insulin release from the islets Aliquots of medium from each vial were taken for the RIA of IAPP (5). Intra- and interassay coefficients of variation were 4.2% and 5.5%, respectively. Another aliquot of the medium was used for the assay of insulin, using an EIA kit. Measurement of IAPP and insulin content in islets Isolated islets were homogenized in 70% formic acid by sonication. The formic acid was removed to near dryness with a vaporizer, and the extracts were lyophilized. Just before the assay, the extracts were dissolved in 0.1 ml 0.2 N acetic acid, diluted in the assay buffer, and adjusted to pH 7.4 with NaOH. The extracts were measured for IAPP (5), and insulin in the extracts was also determined by EIA using the insulin assay kit described above. The data in the text and figures are presented as means ± SEM. Student's t test was used for statistical analysis.
Results
Body wt (g) Serum insulin (pM) Plasma glucose (mM)
VMH-lesioned rats
Control rats
340 ± 17 (8)° 293.5 ± 26.5 (8)" 8.47 ± 0.49 (8) NS6
257 ± 8.5 (8) 127 ± 12.9 (8) 8.41 ± 0.35 (8)
Values are mean ± SEM with numbers of observations in parentheses. " P < 0.005 for comparison with controls. 6 NS, Not significant. TABLE 2. Characteristics of obese and lean Zucker rats Obese rats
Lean rats
502.8 ± 22 (10)° 223.3 ± 4.5 (10) Body wt (g) 119.1 ± 6.9 (10) Serum insulin (pM) 182.3 ± 12.9 (10)" 10.5 ± 0.38 (10) Plasma glucose (mM) 9.17 ± 0.71 (10) NS* Values are mean ± SEM with numbers of observations in parentheses. a P < 0.005 for comparison with controls. 6 NS, Not significant.
time of experiment. The serum insulin concentration in the rats with VMH lesions and obese Zucker rats was higher than that in the controls. No difference was observed in plasma glucose concentrations between obese rats and the controls. Release of insulin and IAPP from islets of VMHlesioned rats (Fig. 1, A and B) In both VMH-lesioned rats and the controls, more insulin was secreted from isolated islets during exposure to high glucose, 16.7 mM, compared with 2.8 mM glucose. Islets from the VMH-lesioned rats released a larger amount of insulin than did those of controls at each concentration of glucose. Concentrations of 2.8 mM glucose stimulated the secretion of insulin in the VMHlesioned rats 4-fold above that in controls (39.4 ± 6.35 vs. 9.83 ± 1.85 fmol-islet"1-3 h"1, P < 0.01). With 16.7 mM glucose, the secretion of insulin from the islets of VMH-lesioned rats was 2.2-fold over that of the controls (137.5 ± 17.4 vs. 62.4 ± 6.09 fmol• islet"1 • 3 h"1, P < 0.01). Figure IB compares the release of IAPP by stimulation of glucose in VMH-lesioned rats and controls. The islets from the VMH-lesioned rats released 1.68-fold the IAPP released from the controls, in response to 16.7 mM glucose (11.2 ± 1.29 vs. 6.65 ± 0.78 fmol • islet"1 • 3 h"1, P < 0.01). A small amount of IAPP was released from the islets of VMH-lesioned rats in case of exposure to 2.8 mM glucose (2.99 ± 0.98 fmol • islet"1 • 3 h"1). There was no detectable amount of IAPP released from islets of the controls.
Characteristics of animals
Release of insulin and IAPP from islets of Zucker rats (Fig. 2, A and B)
As shown in Tables 1 and 2, the VMH-lesioned and obese Zucker rats weighed more than the controls at the
In both the obese and lean rats, more insulin was secreted from the isolated islets exposed to high glucose,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 20:11 For personal use only. No other uses without permission. . All rights reserved.
HYPERSECRETION OF IAPP IN VMH-LESIONED AND ZUCKER RATS A
FIG. 1. Secretion of insulin and IAPP from isolated pancreatic islets obtained from VMH-lesioned rats (M) and from sham-operated rats (D) incubated for 3 h in the presence of 2.8 mM or 16.7 mM glucose. A, Secretion of insulin. B, Secretion of IAPP. Values are mean ± SEM with numbers of observations in parentheses. *, P < 0.01.
1
2741
B
200-
20-
150-
15-
p 10
100-
I
50-
(8)
ND
glucose
2.8
(mM)
glucose
16.7
(mM)
2.8
16.7
obese rats released 1.27-fold amounts of IAPP, compared with findings in the lean rats, in response to 16.7 mM glucose (15.81 ± 1.35 vs. 12.49 ± 1.20 fmol • islet"1 • 3 h"1, P< 0.01). Although small amounts of IAPP were released from the islets of obese rats in case of exposure to 2.8 mM glucose, the amounts were significantly greater than that from the islets of lean rats (2.83 ± 0.88 vs. 0.36 ± 0.19 fmol-islet"1-3 h"1, P < 0.05).
150
Insulin and IAPP content in islets of VMH-lesioned rats (Fig. 3)
(10) (10)
glucose (mM)
too)
(10)
2.8
16.7
glucose (mM)
2.8
(10)
(10)
Both insulin and IAPP content in islets of VMHlesioned rats were slightly but significantly greater than in the controls (insulin: 1074.0 ± 76.8 vs. 819.9 ± 62.6
16.7
FIG. 2. Secretion of insulin and IAPP from isolated pancreatic islets obtained from obese Zucker rats (H) and from lean rats (•) incubated for 3 h in the presence of 2.8 mM or 16.7 mM glucose. A, Secretion of insulin. B, Secretion of IAPP. Values are mean ± SEM with numbers of observations in parentheses. *, P < 0.01; **, P < 0.05.
16.7 mM, compared with 2.8 mM glucose. The islets from obese rats released larger amounts of insulin than did those of lean rats, at each concentration of glucose. Glucose, 2.8 mM, stimulated the secretion of insulin in obese rats 2.8-fold over that in lean rats (154.9 ± 17.9 vs. 55.5 ± 7.18 fmol-islet"1.3 h"1, P < 0.01). Secretion of insulin from the islets of obese rats was 1.5-fold greater than that of lean rats in the case of 16.7 mM glucose (221.8 ± 13.6 vs. 145.8 ± 5.31 fmol • islet"1 • 3 h"\ P < 0.01). Figure IB compares the release of IAPP by stimulation of glucose in obese and lean rats. The islets from the
A
B
U 1500 -
V 150H
o «£ 1000 c
rh
c
c
8 500 H _c "5 c 0
0) *->
o 50o
Q.
(8)
(8) Sham
VMH
Sham
VMH
FIG. 3. Contents of insulin and IAPP in the pancreatic islets obtained from VMH-lesioned rats and sham-operated rats. A, Content of insulin. B, Content of IAPP. Mean ± SEM for IAPP and insulin contents are expressed in femtomolesi per islet. *, P < 0.01.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 20:11 For personal use only. No other uses without permission. . All rights reserved.
fmol/islet, P < 0.01; IAPP: 100.9 ± 6.6 vs. 72.8 ± 3.85 fmol/islet, P < 0.01).
TABLE 4. Secretion of rate of insulin and IAPP from the islets of obese and lean Zucker rats Obese rats
Insulin and IAPP content in islets of Zucker rats (Fig. 4)
Glucose
The insulin and IAPP content in islets of obese rats were increased, 1.31-fold and 1.57-fold, respectively, compared with findings in the lean rats (insulin: 2960.4 ± 119.9 vs. 2258.2 ± 80.4 fmol/islet, P < 0.01; IAPP: 133.3 ± 10.6 vs. 84.4 ± 8.5 fmol/islet, P < 0.01).
Insulin release {% of content) 5.2 ± 0.62 (10)° 2.8 mM 7.4 ± 0.47 (10) NS6 16.7 mM IAPP release (% of content) 2.1 ± 0.66 (10)c 2.8 mM 11.8 ± 1.14 (10) NS" 16.7 mM
Rate of secretion of insulin and IAPP expressed as a percent of content (Tables 3 and 4) Both insulin and IAPP secretion rates from the islets of VMH-lesioned rats and Zucker rats were expressed as percentage of content. In the VMH-lesioned rats, insulin release per content at each concentration of glucose and IAPP release per content at 2.8 mM glucose were significantly increased. However, the release of IAPP in 16.7 mM glucose showed no significant difference between the VMH-lesioned and A
U
B
3000-
V 150-
o I 2000
0)
c
•->
c o o
IOOO
50-
O-
(10)
v,0,
(10)
lean
obese
lean
obese
FIG. 4. Contents of insulin and IAPP in the pancreatic islets obtained from obese Zucker rats and lean rats. A, Content of insulin. B, Content of IAPP. Mean ± SEM for IAPP and insulin contents are expressed in femtomoles per islet. *, P < 0.01. TABLE 3. Secretion rate of insulin and IAPP from the islets of VMHlesioned rats and control rats Glucose
VMH-lesioned rats
Insulin release (% of content) 2.8 mM 3.6 ± 0.59 (8)° 16.7 mM 12.8 ± 1.6 (8)6 IAPP release (% of content) 2.8 mM 2.9 ± 0.97 (8)° 16.7 mM 11.1 ± 1.2 (8) NSC
Lean rats 2.4 ± 0.31 (10) 6.4 ± 0.23 (10) 0.42 ± 0.22 (10) 14.7 ± 1.42 (10)
Each value is calculated from the secretion (fmol • islet"1 • 3 h"1) and content (fmol/islet). Values are mean ± SEM with numbers of observations in parentheses. " P< 0.01 for comparison with controls. * NS, Not significant. c P < 0.05 for comparison with controls.
control rats. In obese Zucker rats, insulin release per content was increased with a low dose of glucose but was not significantly elevated with a high dose. IAPP release per content was slightly but significantly increased with 2.8 mM glucose. At 16.7 mM glucose there was no significant difference between the IAPP release in obese and lean rats. Discussion
E 100-
+-> 0)
8
Endo • 1991 Voll28-No6
HYPERSECRETION OF IAPP IN VMH-LESIONED AND ZUCKER RATS
2742
Control rats 1.1 ± 0.22 (8) 7.6 ± 0.74 (8) 0 (8) 9.1 ± 1.07 (8)
Each value is calculated from the secretion (fmol-islet"1^ h"1) and content (fmol/islet). Values are mean ± SEM with numbers of observations in parentheses. °P
