Diabetes Research and Clinical Practice, 15 (1992) 0

1992 Elsevier

DIABET

Science Publishers

23

016%8227/92/$05.00

00576

Hypersecretion Yoshiharu ‘Second

23-30

B.V. All rights reserved

of IAPP from the islets of VMH-lesioned and obese Zucker rats

rats

Tokuyama ‘, Azuma Kanatsuka’, Haruhiko Ohsawa ‘, Takahide Yamaguchi Hideichi Makino ‘, Sho Yoshida ‘, Hajime Nagase2 and Shuji Inoue2

Department of Internal Medicine, Medicine,

Chiba University School of Medicine,

Chiba. Japan and ‘Third Department

Yokohama City Universitv School of Medicine.

‘,

of Internal

Yokohama, Japan

Summary

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 radioimmunoassay 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 sham-operated controls (100.9 + 6.6 vs 72.8 k 3.85 fmol/islet; P < 0.01). Isolated islets of VMH-lesioned rats secreted larger amounts of IAPP in the presence of 2.8 and 16.7 mM glucose (2.99 k 0.98 and 11.2 + 0.29 fmol islet ~~’ 3 h ‘) than was noted in sham-operated rats (ND and 6.65 k 0.78 fmol islet ’ 3 h- ‘). In the obese Zucker rats, aged 14 weeks, IAPP concentrations in the islets were elevated compared with lean rats (133.3 k 10.6 vs 84.4 k 8.5 fmol/islet; P < 0.01). The isolated islets secreted larger amounts of IAPP in response to 2.8 and 16.7 mM glucose (2.83 k 0.88 and 15.81 of- 1.35 fmol islet ’ 3 h- ‘) than did those from lean control rats (0.36 k 0.19 and 12.49 t 1.20 fmol islet ’ 3 h- ‘). These results strongly suggest that overproduction and hypersecretion of IAPP occur in animals with obesity and hyperinsulinemia. Key words:

Hyperinsulinemia; Zucker rat

Islet amyloid

polypeptide;

Introduction

Islet amyloid polypeptide (IAPP) also termed amylin is a major component of islet amyloid. This 37-amino acid peptide is a natural product in Correspondence to: A. Kanatsuka, Second Dept. of Internal Medicine, Chiba University School of Medicine, l-8-1 Inohana. Chiba, 280, Japan.

Ventromedial

hypothalamic

lesioned

rat;

pancreatic B cells, as determined in immunohistochemical studies [l-4] and with radioimmunoassay [ 51. IAPP is secreted from the pancreatic islets in response to glucose [ 5,6]. An analysis of the cDNA 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 +cletal muscle [ 9, lo] and inhibit the glucose-sti-

24

mulated insulin secretion from isolated rat pancreatic islets [II]. In type 2 (non-insulin-dependent) diabetes mellitus, the most significant pathological change in the pancreatic islets is amyloid deposition [ 121. In human type 2 diabetes mellitus, the extent of islet amyloid deposition increases with increase in the clinical severity [ 131. On the other hand, obesity and hyperinsulinemia are observed in subjects with type 2 diabetes mellitus [ 141. 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-lesioned rats with obesity and hyperinsulinemia [ 1.51 and from genetically obese Zucker rats that carry the recessive homozygous trait for obesity designated by fajfa [ 161, and which are hyperinsulinemic .

Materials and Methods VMH-lesioned rats Female Sprague-Dawley rats 10 weeks old and weighing 200-250 g 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 operation were carried out as described [ 151. Experiments were then performed 3 weeks after the lesioning. At the 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. 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 2-3 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 weight 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 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, F.R.G.). Intra- and interassay coefficients of variation were 4.5 and 5.6%, respectively. The pancreatic islets were isolated by a modification of reported methods [ 171. Collagenase digestion was carried out at 37 ‘C for 25 min after collagenase (20 mg collagenase/lO ml Hanks’ solution) injection into the common bile duct. The medium used for preincubation and incubation was Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4, containing 1 g/l bovine serum albumin (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% 0, - 5% CO,, 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 radioimmunoassay (RIA) of IAPP [ 51. Intraand 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

25 TABLE I Characteristics

of VMH-lesioned

and control rats

controls at the time of the 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.

VMH-lesioned

rats

Control rats

340

+ 17

(8)*

257

f

293.5

+ 26.5

(8)*

127

+ 12.9 (8)

Release of insulin and MPP from islets of VMHlesioned rats (Table 3)

8.47 + 0.49 (8) ns

8.41 of: 0.35 (8)

Values are means f SEM with numbers parenthesis; *P < 0.005 for comparison not significant.

of observations in with controls; ns,

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 VMHlesioned rats released a larger amount of insulin than did those of controls at each concentration of glucose. 2.8 mM glucose stimulated the secretion of insulin in the VMH-lesioned rats fourfold above that in controls. 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. The islets from the VMH-lesioned rats released 1.68-fold the IAPP released from the controls, in response to 16.7 mM glucose. A small amount of IAPP was released from the islets of VMHlesioned rats in the case of exposure to 2.8 mM glucose. There was no detectable IAPP released from islets of the controls.

Body weight (8) Serum insulin (PM) Plasma glucose (mM)

8.5 (8)

were lyophilized. Just before the assay, the extracts were dissolved in 0.1 ml of 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 k SEM. Student’s t-test was used for statistical analysis.

Results Characteristics of animals (Tables 1 and 2)

As shown in Tables 1 and 2, the VMH-lesioned and obese Zucker rats weighed more than the

Secretion of insulin and IAPP from the islets of VMHlesioned rats and control rats

TABLE 2 Characteristics

TABLE 3

of obese and lean Zucker rats Obese rats

Glucose

VMH-lesioned

Lean rats

Insulin release (fmol islet _ ’ 3 h

Body weight 502.8

(lo)*

223.3 f 4.5 (IO)

2.8 mM 16.7 mM

(PM) Plasma glucose (mM)

182.3 f 12.9 (IO)*

119.1 + 6.9 (10)

IAPP release (fmol islet

9.17 f

0.71 (10) ns

10.5 f 0.38 (10)

Values are means + SEM with numbers of observations in parenthesis; *P < 0.005 for comparison with controls; ns, not significant.

2.8 mM 16.7 mM

Control rats

‘)

39.4 + 6.35 (8)* 137.5 k 17.4 (8)*

(g) Serum insulin

f 22

rats

9.83 f 1.85 (8) 62.4 f 6.09 (8)

’ 3 h ‘)

2.99 + 0.98 (8)* 11.2 + 1.29 (8)*

0 (8) 6.65 f 0.78 (8)

Values are means f SEM with numbers of observations parenthesis; *P < 0.01 for comparison with controls.

in

26 Release of insulin and IAPP from islets of Zucker rats (Table 4) In both the obese and lean rats, more insulin was secreted from the isolated islets exposed to high glucose, 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. Secretion of insulin from the islets of obese rats was 1.5fold greater than that of lean rats in the case of 16.7 mM glucose. The islets from the obese rats released 1.27-fold amounts of IAPP, compared with findings in the lean rats, in response to 16.7 mM glucose. Although small amounts of IAPP were released from the islets of obese rats in the case of exposure to 2.8 mM glucose, the amounts were significantly greater than that from the islets of lean rats.

TABLE 5

Insulin and IAPP content in islets of VMH-lesioned rats (Table 5) Both insulin and IAPP content in the islets of VMH-lesioned rats were slightly but significantly greater than in the controls.

Discussion

TABLE 4 Secretion of insulin and IAPP from the islets of obese and lean Zucker rats Glucose

Obese rats

Insulin release (fmol islet - ’ 3 h 2.8 mM 16.7 mM

Lean rats ‘)

154.9 f 0.62 (lo)* 221.8 f 13.6 (lo)*

2.83 + 0.88 (IO)* 15.81 5 1.35 (lo)**

VMH-lesioned Insulin (fmol islet IAPP (fmol islet

rats

1074.0 & 76.8 (8)*

islets of

Control rats 819.9 + 62.6 (8)

‘) 100.9 + 6.6 (8)*

72.8 k

3.85 (8)

‘)

Values are means f SEM with numbers of observations parenthesis; *P < 0.01 for comparison with controls.

in

Insulin and IAPP content in islets of Zucker rats (Table 6) 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.

We obtained evidence that the pancreatic islets of obese rats secrete and contain larger amounts of IAPP than do those of lean control rats, determined using a specific radioimmunoassay for IAPP which we developed [ 51. This is the first evidence that IAPP content and secretion in pancreatic islets are increased in the presence of obesity and hyperinsulinemia. The rats used here are typical models of ani-

TABLE 6 Contents ofinsulin and IAPP in the pancreatic islets of obese Zucker rats and lean rats

55.5 f 0.31 (10) 145.8 f 5.31 (10)

IAPP release (fmol islet _ ’ 3 h - ‘) 2.8 mM 16.7 mM

Contents of insulin and IAPP in the pancreatic VMH-lesioned rats and control rats

0.36 f 0.19 (10) 12.49 k 1.20 (10)

Lean rats

Obese rats Insulin (fmol islet IAPP (fmol islet

2960.4 f 119.9 (IO)* ‘) 133.3 f ‘)

2258.2 k 80.4 (10)

10.6 (lo)* 84.4 T 8.5 (10)

Values are means + SEM with numbers of observations in parenthesis; *P < 0.01, **P < 0.05 for comparison with controls.

Values are means k SEM with numbers of observations parenthesis; *P < 0.01 for comparison with controls.

in

27

mals with obesity and hyperinsulinemia [ 1516,181. The isolated islets of both VMHlesioned rats and genetically obese Zucker rats contained larger amounts of insulin, consistent with previous reports [ 19,201. It has been suggested that VMH-lesioning alters autonomic neural activities, resulting in hyperinsulinemia and obesity [ 19,21-241. Thus, one possible mechanism is that the increased content of IAPP is caused by an augmented production of IAPP in /? cells as a result of the altered autonomic neural activity after VMH-lesioning. In genetically obese rats, the hyperplastic/hypertrophic nature of pancreatic islets has been noted and the pancreatic insulin content was increased [25,26]. It has been suggested that disruption in the hypothalamus induces an abnormal pituitary function, resulting in hypersecretion of adrenal steroids and insulin [ 27,281. It was reported that chronic dexamethasone treatment leads to elevation of IAPP mRNA in adult rats [29]. Thus, hypersecretion of glucocorticoids seems to enhance the production of IAPP in obese rats. Since increased content of IAPP was observed in obese experimental rodents in which obesity and hyperinsulinemia were produced by different mechanisms, we propose that IAPP production is enhanced, in part, through mechanisms which are involved in overproduction and hypersecretion of insulin in the pancreas. This notion is supported by our recent finding that IAPP concentration in the pancreas increases in C57BL/6J ob/ob mice which inherit obesity and diabetes as an autosoma1 recessive mutation on chromosome 6 and which have hyperinsulinemia [30] (data not shown). Recent analyses of the human IAPP gene indicated that there were no obvious conserved sequences in the promoter regions of the human IAPP and insulin genes [31]. This may suggest that for the two genes, the regulation of transcription differs. However, dexamethasone treatment of adult rats caused an increase both in IAPP and insulin mRNA in the pancreas [ 291. In the streptozotocin-treated rats, both IAPP and insulin mRNA levels were reduced [29]. Obesity is commonly associated with hyper-

insulinemia and resistance to insulin [32]. It has been reported that IAPP potentially inhibits the rate of insulin-stimulated glycogen synthesis in the rat soleus muscle in vitro [ 9, lo] and causes peripheral insulin resistance in vivo [ 33,341. Islet IAPP content and secretion seem to be augmented in association with the development of obesity. It is possible that peripheral insulin resistance of obese animals is related, in part, to hypersecretion of islet IAPP. The prevalence of type 2 diabetes associated with obesity is high in North American Pima Indians. Longitudinal population studies showed that subjects who later developed type 2 diabetes had higher fasting and post-load insulin concentrations than did the controls [ 141. Recently, it was reported that 77% of diabetic Pima Indians had islet amyloid identified by immunoreactivity for IAPP, compared with only 7% of nondiabetic subjects [35]. In human subjects, enhanced IAPP production and secretion accompanied by obesity and hypersecretion of insulin is perhaps related to the progression of islet amyloid deposition, the result being impairment of pancreatic @-cell function.

Acknowledgements We thank Mrs. K. Amano for expert technical assistance and M. Ohara for editorial comments. This work was supported in part by Grant 0 1570625 from the Ministry of Education, Japan.

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34 Sowa, R., Sanke, T., Hirayama, J. et al. (1990) Islet amyloid polypeptide amide causes peripheral insulin resistance in vivo in dogs. Diabetologia 33, 118-120. 35 Clark, A., Saad, M.F., Nezzer, T. et al. (1990) Islet amyloid polypeptide in diabetic and non-diabetic Pima Indians. Diabetologia 33, 285-289.

Hypersecretion of IAPP from the islets of VMH-lesioned rats and obese Zucker rats.

To investigate the possible role of islet amyloid polypeptide (IAPP) in the development of type 2 diabetes mellitus, we examined the IAPP content and ...
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