Vol. 170, No. 3, 1990 August

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

16, 1990

Pages

1223-1228

ISLET AMYLOID POLYPEPTIDE (IAPP) DOES NOT INHIBIT GLUCOSESTIMULATED INSULIN SECRETION FROM ISOLATED PERFUSED RAT PANCREAS Timothy

D. O'Brien*,

*Department

Per Westermark+,

of Veterinary

Medicine,

Pathobiology,

University

+Department

of

and Kenneth College

of Minnesota,

Pathology,

St.

Linksping

Ii.

Johnson*

of Veterinary

Paul,

Minnesota

University,

LinkGping,

Sweden Received

June

25,

1990

SUMMARY: Islet amyloid polypeptide (IAPP) is a recently discovered pancreatic islet hormone which is stored with insulin in the secretory vesicles of beta cells. Several lines of evidence suggested that IAPP might affect glucose-stimulated insulin secretion and, therefore, might play a role in the development of impaired insulin secretion which is typical of type 2 diabetes. In this study, the effects of human IAPP (amide) on glucose-stimulated insulin secretion was evaluated in the isolated perfused rat pancreas. IAPP in concetrations from 5 x lo-l2 to 1o-7 M had no significant effects on insulin secretion. IAPP, therefore, does not appear to be a significant modulator of glucose-stimulated insulin secretion at concentrations that are 0 1990Academic Press,Inc. physiologically relevant. Islet recently amyloid

amyloid

polypeptide

discovered deposits

diabetes, (theoretical

hormone which in the

diabetic

(IAPP)(also

pancreatic

cats,

molecular

is the

major

islets

of

and insulinomas mass of

known as amylin) constituent

of

humans with

type

2

IAPP

has

(1,2,3,4).

3850 daltons)

is a

is

IAPP carboxy-

terminally amidated and is comprised of 37 amino acids. approximately 45% homology with calcitonin gene-related

peptide

(CGRP) (1). IAPP has been shown by immunohistochemical

techniques

to

occur in the beta cells of normal pancreatic islets of humans, domestic cats, rats, and several other species (5,6). Immunoelectron

microscopic

immunoreactivity

occurs

studies

have

shown that

the

IAPP

in the secretory vesicles of feline and An immunohistochemical survey of many human beta cells (5,6,7). other feline tissues including central nervous system and other endocrine organs failed to detect IAPP immunoreactivity (5). 0006.291X&O$1.50 1223

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

170,

No.

3, 1990

BIOCHEMICAL

Similarly,

studies

probes for predominant

IAPP site

message

has

employing have for

been

insulin

role is

homology

(10,ll).

In

differing

results.

any

of

concentrations IAPP

MATERIALS

IAPP)

levels insulin

of 10e9 M but Three other humans,

into

inhibitory

The present

isolated

other

cDNA

the some

IAPP

tissues

(9).

in the beta cell secretory a role in the regulation

in inhibiting by the fact

has

been

IAPP

shown

was

on insulin (from

(h-IAPP), perfused

rats,

mice,

undertaken secretion to

IAPP

this

of

(r-IAPP)

inhibit study,

rabbits

further

by using 10.'

M) of

of

(10-l' injected

and

a

a range insulin

to

(14,15,16). evaluate

synthetic

using

effect

failed

secretion to

effect had

Another

and

has

studies rat islets

a stimulatory secretion at

on insulin

5 x lo-l2

have

lower concentrations in which IAPP was

on glucose-stimulated rat

to

in vitro isolated

detected insulin

not at studies

effects study

glucose-stimulated that CGRP (which

(10e6 M) of rat secretion (12).

perfused rat pancreas, on glucose-stimulated

peripherally

the

using

cells are although in

regard, two previous A study employing

high

concentration lo-lo Ml (13).

human

beta (8),

insulin have

may

COMMUNICATIONS

hybridization

be present

for IAPP suggested

to

this

indicated that glucose-stimulated

effect

situ

RESEARCH

metabolism.

extensive

detect

to

of IAPP with that IAPP

A possible secretion

isolated rat IAPP

in

BIOPHYSICAL

shown that the IAPP expression

reported

The localization vesicles suggests carbohydrate

AND

the

of amidated secretion

in

pancreas.

AND METHODS

Svnthetic neptides. C-amidated h-IAPP was synthesized using solid phase techniques on a Biosearch 960 peptide synthesizer with MBHA (p-methylbenzhydrylamine) resin and FMOC chemistry. The peptide was purified by reverse-phase high performance liquid chromatography. Amino acid composition was determined after acid hydrolysis and sequence analysis was performed using an Applied Biosystems 477A protein sequencer. Structure of the peptide was also confirmed by mass spectrometry. Pancreatic perfusion studies. Female Sprague-Dawley rats weighing 200-250 2 were maintained in a room with environmental temperature at 23 C and given free access to commercial rat chow and water. The rats were deeply anesthetized with sodium pentobarbital (60 mg/kg) by intraperitoneal injection. The pancreas was isolated and prepared for perfusion as previously reported using the celiac artery as the inlet and the portal vein as the outlet (17). The preparation was perfused with KrebsRinger bicarbonate solution (KRB)(pH 7.2) containing 15 mu HKPKS, 3.8% dextran, and 0.25% BSA at a rate of 2.5 ml/min. The 1224

Vol.

170,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

perfusion medium was continuously gassed with 95% O,-5% CO, and maintained at 39' C. Control pancreases were perfused for 20 minutes with KRB containing 2.75 mM glucose followed by 30 minutes with KRB containing 16.7 mM glucose. In experimental the pancreases were perfused during the initial 10 grows, minutes with KRB containing 2.75 mM glucose, followed by a 10 flinute perfusion with KRB containing 2.75 mM glucose and 5 x 10. 5 x lo-" 5 x 10e8 or 10e7 M h-IAPP (amide) and during the final 30 minutes with KRB containing 16.7 mM glucose and h-IAPP IAPP was first dissolved in 30% acetic concentration as above. acid before being added to the KRB (pH was then adjusted to pH 7.2). In each perfusion, samples were collected in plastic Eppendorf tubes at 1, 5, 10, 15, 20, 21, 22, 23, 24, 25, 27.5, and 50 minutes. 32.5, 35, 40, Samples were immediately 30, chilled on ice and frozen until assayed for insulin. Insulin was measured assay kit (ICN Biomedicals, standards (Novo Biolabs,

by radioimmunoassay Inc., Costa Mesa, Danbury, CT).

using an insulin CA) and rat insulin

RESULTS

at

Insulin concentration each sampling interval

Figure with IAPP

1 and Table 2.75 were

1.

mM glucose similar in

the minimal Statistical

Insulin

buffer for each group perfusion is shown in

concentrations

during

(minutes 0 to 20) either with all groups and were generally

detectable comparison

Insulin

in the perfusion during pancreatic

level for the insulin between the means of

perfusion or without at or below

radioimmunoassay. each group using

one

W/ml)

16OOy 1400

-

1200

-

1000

-

800

-

600 400 200 :

1 0

20

10

40

30

50

60

Time

Figure buffer perfusion perfusion mM and in the axis.

-

Controls

*

IAPP

0.5

+ nM

+

IAPP

100

IAPP

0.005

1. for

+

nM

IAPP

50

nM

nM

Insulin concentration (pU/ml) in effluent perfusion each experimental group at each sampling time during Glucose concentration in the of isolated rat pancreas. medium for the first 20 minutes of perfusion was 2.75 for the last 30 minutes was 16.7 mM. IAPP concentrations perfusion medium for each group are given below the x1225

Vol.

170,

No.

3, 1990

BIOCHEMICAL

AND

TABLE

Mean Insulin

BIOPHYSICAL

Concentration (fiU/ml)(l s.d.) in Perfusion Minutes 21 through 50 in Each Group

Controls

21

1156(312)

22

1207(563

23

100 nM

Medium

50 nM

0.5

nM

0.005

)

1017(163)

1003(615)

1550(608

847(357

1

708(287)

554(192)

962(367

24

658(222

1

551(137)

490 (247)

626(281

)

25

544(78)

466(113)

432(223)

807(303

)

27.5

720(193)

554(130)

600(305)

568(368)

289

132)

30

818(160)

579(113)

655(310)

713(456)

427

197)

32.5

872(262)

644(188)

745(254)

9661715)

474

156)

35

1104(329)

675(199)

854(293)

961(489)

663

252)

40

1263(315)

967(276)

1199(464)

1176(578)

813(340)

50

1474(438)

1104(477)

1156(210)

1151(664)

1043(468)

with

minute

(PC 0.01) following group

21 (16.7

was done at each time

mmol/l

glucose).

between groups was only the first phase insulin

receiving

5 x 10“'

593 (795)

nM

1062(774)

of variance

831(579)

1

410(253) 225(102) 65)

interval

A significant

M IAPP had insulin

differences

948 (642)

beginninc difference

seen at minutes 23, 24, and 25 secretion at which times the

than the other groups. The group receiving responsible for the statistical difference was excluded, the group significant (P> 0.01).

at

of IAPP)

1323(1026)

way analysis

the the

COMMUNICATIONS

1

Group (concentration Minute

RESEARCH

were not

concentrations 5 x lo-" (i.e., if

lower

M IAPP was that group

statistically

However, a two sample T test comparing means at minutes 23, 24, and 25 of the control group versus group receiving 5 x lo-" M IAPP showed no significant

differences

(P> 0.05).

DISCUSSION Impairment

of glucose-stimulated

insulin

secretion

is

one of

the major metabolic defects that occurs in type 2 diabetes mellitus. The possibility that IAPP may participate in the pathogenesis of type 2 diabetes through this mechanism was suggested by reports showing an inhibitory effect on glucosestimulated insulin secretion (10,ll) by CGRP (which has extensive homology to IAPP)(l). This hypothesis was further supported by 1226

Vol.

the

170,

No.

fact

islets

3, 1990

that of

IAPP

most

formation

BIOCHEMICAL

AND

accumulates

type

as

2 diabetics,

may be a reflection

disease

(18).

states by the

Increased

of impaired demonstration

pancreatic

of

fact that vesicles

response

to

that

IAPP

islet

cell

highest

with

IAPP (5,6) and

of

COMMUNICATIONS

in

the

that islet IAPP secretion

IAPP

have

impaired

in

(20)

paracrine

with supported in

tolerance

(19).

the beta cell with insulin

provides

effects

amyloid in this

association

glucose

is located within and is co-released arginine

pancreatic

the

on beta

in

possibility

cell

or

other

types.

In this study, on glucose-stimulated employed from the

amyloid

suggesting abnormal

secretion

cats

glucose

might

islet

RESEARCH

glucose tolerance was subsequently of increased IAPP immunoreactivity

islets

Also, the secretory

of

BIOPHYSICAL

ranged lowest

we found insulin

that

may be achieved thus probably (23)],

solutions

physiologically relevant range report demonstrated inhibition rat islets above those human

or

human

IAPP

using rat reported

rabbit that

Findings in pharmacologic

the

effect of Concentrations

from 5 x lo-l2 to 10e7 M [which spans reported plasma IAPP levels (21,22)

concentration

physiologic

no significant secretion.

IAPP at to-date

plasma can

for this of insulin

be achieved

previous effect of

the

hormone. secretion

in

the range to near the

A previous from

concentration in normal

highest

or

isolated is far diabetic

concentration

physiologic

study therefore, IAPP and may not

IAPP IAPP

for h-IAPP in spanning the entire

lo-' M. This as occurring

and exceed

human of

of

solution.

appear to be a be physiologically

relevant. Although 5 x lo-" was not physiologic findings, indicating

the

peak

M was higher statistically relevance therefore, a stimulatory

insulin than that significant

secretion of

the controls, and appeared

because no dose do not agree with effect

in

of

phase insulin secretion from perfused response was proven in the previous possibility that it was a spurrious

our

perfusions this unlikely

difference to be of Our seen.

response was a previous report

r-IAPP

using

on first

(13)

and second No dose

rat pancreas. paper leaving open the finding. Another possible

explanation for the differences these studies is that the previous study employed r-IAPP whereas we used h-IAPP which allows the possibility that the differences were due to differing Additional credence is given to the actions of the peptides. 1227

Vol.

170,

No.

BIOCHEMICAL

3, 1990

results of the present IAPP injected peripherally several

different

species

study

AND

BlOPHYSlCAL

by previous had no effect

RESEARCH

COMMUNICATIONS

in vivo studies in on insulin secretion

which in

(14,15,16).

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

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