Bioscience Reports, Vol. 10, No. 4, 1990
The Effect of Islet Amyloid Polypeptide
(Amylin) and Calcitonin Gene-Related
Peptide on Glucose Removal in the
Anaesthetized Rat and on Insulin Secretion
from Rat Pancreatic Islets in vitro
Alison E. Tedstone, 1 Tania Nezzer, 2 Stephen J. Hughes, 3
A n n e Clark e and David R. Matthews 2
Received November 28th, 1989; revised version February 9, 1990
The effect of intravenous infusion of islet amyloid polypeptide (IAPP/amylin) and catcltonin
gene-related peptide (CGRP) on blood glucose and plasma insulin in the basal and glucose-stimulated
state was investigated in the anaesthetized rat. Both peptides had no effect on basal blood glucose or
plasma insulin but following an intravenous bolus of glucose, CGRP-treated rats were hyperglycaemic
and hyperinsulinaemic compared with control animals which were similar to IAPP-treated rats. IAPP
had no effect on glucose-stimulated islet insulin secretion. These results suggest that CGRP, but not
IAPP, alters glucose removal in vivo.
KEY WORDS: CGRP, IAPP/amylin, glucose removal, insulin release.
INTRODUCTION
Type II (non-insulin dependent) diabetes is characterized by abnormalities in
insulin release, insulin resistance [1] and by amyloid deposits in the pancreatic
islets of Langerhans [2]. Recently, the major peptide constituent of the islet
amyloid has been identified as a 37 amino acid peptide known as islet amyloid
polypeptide (lAPP) or amylin [3, 4]. This is a normal constituent of islet/3-cells in
both diabetic and non-diabetic humans [5] where it is localized in insulin granules
1 Metabolic Research Laboratory, Nutfield Department of Clinical Medicine and 2 Diabetes Research
Laboratories, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK. 3 Nufiield Department
of Clinical Biochemistry, John Radcliffe Hospital, Headley Way, Headmgton, Oxford OX3 9DU,
UK.
1To whom correspondence should be addressed.
339
0144-8463/90/0800-0339506 00/0 Q 1990 Plenum Pubhshmg Corporation
341t
Tedstone, Nezzer, Hughes, Clark and Matthews
and lysosomes [6]. The mechanisms underlying its formation into extracellular
amyloid deposits in Type II diabetes are uncertain.
cDNA analysis has shown that IAPP is derived from a larger propeptide and
that the predicted amino acid sequence is identical to the extracted peptide but
probably includes a c-terminal amidation [7, 8]. The physiological function of lAP
is unknown but it has 46% structural homology with calcitonin gene-related
peptide (CGRP), which has been shown to inhibit insulin secretion in rats and
mice, both in vivo and in vitro [9, 10, 11]. Leighton and Cooper [12, 13] have
demonstrated an inhibitory action of IAP and CGRP on insulin-mediated glucose
disposal to glycogen in the isolated stripped rat skeletal muscle; they have
postulated that a similar action of IAP could be associated with the development
of insulin resistance in Type II diabetes [14]. A prediction from the work of
Leighton and Cooper is that IAP and CGRP would cause decreased removal of a
glucose load in vivo. The aim of the present study was to test this hypothesis and
to examine the effect of IAPP on insulin secretion.
METHODS
Male Wistar rats (200 + 10 g) were maintained at 22~ in a 12 h light/dark
cycle (light starting at 07:00 h) on a diet of 52% carbohydrate, 21% protein, 4%
fat and the residue made up of non-digestible material (Special Diet Services,
Witham, Essex, UK).
Glucose Removal In Vivo
The experiments started at 09:00 h, the rats having been fasted for 18 h.
Under sodium pentobarbitone anaesthesia (60 mg/kg i.p.; Sigma Chemical Co.,
Poole, UK) saline-filled polyethylene cannulae were inserted into the right
carotid artery and left jugular vein for blood sampling and infusing respectively.
A tracheotomy was performed. Anaesthesia and body temperature were maintained throughout the 90 min of the experiment. The animals were divided into
four experimental groups, all of which were infused with 0.9% NaCI (1 ml/h) for
the first 30min. Animals then received (on a random basis) either IAPP
(83nmol/kg/h; n = 7 ; Peninsula, Liverpool, UK), hCGRP-1 (83nmol/kg/h;
n = 5; Peninsula, Liverpool, UK) or continuous infusion of saline (n = 10) for a
further 30min. After 60min, glucose (0.5g/kg in 0.2ml saline) was given
intravenously as bolus over 2 min to all peptide-treated and 5 saline-treated rats.
Peptide and saline infusions were continued for a further 30 min.
At intervals throughout the experimental period carotid arterial blood
samples (0.15 ml) were taken into heparinized syringes and replaced with an
equal volume of saline. Samples were kept on ice for measurement of blood
glucose (Beckman autoanalyser), then centrifuged and the plasma frozen for
subsequent radioimmunoassay of plasma insulin (sensitive to 2 +0.2/~U/ml)
against rat insulin standard [15].
Effect of Peptides on Blood Glucose
341
In Vitro Insulin Secretion
Islets of Langerhans were prepared by collagenase (Sigma Chemical Co.,
Poole, UK) digestion of pancreatic tissue from non-starved rats [16] and cultured
for 20 h in RPMI 1640 supplemented with antibiotics and 10% foetal calf serum
(Gibco, Paisley, UK). Islets were collected under a dissecting microscope with a
wire loop and incubated in batches of 5 for 2 h at 37~ in Hepes-buffered Krebs
medium containing 2 mg BSA/ml (Boehringer, W. Germany) with 2 mM glucose,
10mM glucose + l A P P (0.03-300mM)~ Aliquots of the medium were then
collected and assayed for insulin by radioimmunoassay [17].
Results are expressed as mean values+S.E.M, and differences were
compared by unpaired Student's t tests.
RESULTS
Glucose Removal In Vivo
Intravenous infusion of lAPP or CGRP had no significant effect on blood
gl!ucose levels (Fig. 1) or basal plasma insulin (Fig. 2). Following an intravenous
16 84
GLUCOSE
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gs
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0
m
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PEPTIDE
00
1'0
2'0
3'0
4'0
5'0
6'0
7()
8()
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90 ......
TREATMENT TIME (rnins)
]Fig. 1. The effect of IAPP and C G R P on blood glucose before and after an intravenous glucose
challenge. Results are mean values • S.E.M. (S.E.M. time 0 - 6 0 m i n = 0 . 1 - 0 . 4 m M ) for rats
infused with saline (O), IAPP ( 0 ) or C G R P (I-q) before and after an i.v. glucose challenge. O n e
group of rats received only saline (11). Statistical differences between glucose challenged salineand CGRP-treated rats are shown by **P < 0.01 (unpaired Student's t-test).
342
Tedstone, Nezzer, Hughes, Clark and Matthews
glucose bolus, blood glucose increased rapidly, reaching a peak at 5 min and then
slowly returned towards the basal level of glycaemia throughout the remaining
30 min of the experiment in all treatment groups (Fig. 1). The hyperglycaemia
was significantly greater at time 70 to 90 min in the group treated with CGRP. No
significant differences in blood glucose were seen between the saline- and
IAP-treated rats throughout the experimental period. The rate of glucose
removal (decline in glycaemia) was similar for all three treatment groups. The
glucose load induced an increase in plasma insulin in all experimental groups
(Fig. 2). Although the increase was higher in the CGRP-treated group it was only
significantly elevated above saline controls at 90 min and no significant differences
were found between IAPP and saline-treated controls.
To estimate the effect of the peptides on the relative sensitivity of islet cells
for basal and elevated blood glucose the insulin: glucose ratio was calculated for
each animal at each time point (data not shown). No differences were found
between any treatment group.
In Vitro Insulin Secretion
Elevation of glucose from 2 to 10 mM induced a 5-fold increase in insulin
secretion (from 3 1 + 3uU/islet/h to 169 + 16uU/islet/h mean + S . E . M . ,
100
GLUCOSE
90
80
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q
q
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PEPTIDE
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TREATMENT TIME (mins)
Fig. 2. The effect of IAPP and CGRP infusion on plasma glucose before and after an intravenous
glucose challenge. Results are mean values + S.E.M. (S.E.M. time 0-60 min = 0.7-2.5/~U/ml) for
rats infused with saline (O), IAPP (Q) or CGRP (D) before and after an i.v. glucose challenge.
One group of rats received only saline (B). Statistical differences between glucose challenged
saline- and CGRP-treated rats are shown by *P < 0.05 (unpaired Student's t-test).
Effect of Peptides on Blood Glucose
343
30OHM"
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!
100riM"
10ram glucose
+IAPP
30aM"
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3.0nM"
0.3nM"
0.03nM"
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10mM glucose
2mM glucose
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40
60
80
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120
Fig. 3. The effect of IAPP on glucose stimulated insulin release from isolated
islets. Results are mean values + S.E.M. for 10-20 observation concentration
points. For 2 mM glucose insulin release = 31 • 3/tU/islet/h and for 10 mM
glucose insulin release = 169 5:16/*U/islet/h. Statistical differences in insulin
release compared with the 10 mM glucose value are shown by *P < 0.05 and
**P < 0.01 (unpaired Student's t-test).
P < 0 . 0 5 ; Fig. 3). IAP (0.03 nM to 300 nM) had no significant effect on glucose
stimulated insulin secretion with the exception of the observations made at 30 nM
IAPP when a small but significant decrease was noted.
DISCUSSION
Rats treated with CGRP were both hyperglycaemic and hyperinsulinaemic
compared with saline-treated control animals following an intravenous glucose
load. This suggests that CGRP impaired the removal of glucose from the
circulation. This may be due to a peptide-induced defect in peripheral glucose
removal and this could result from a reduction in peripheral insulin sensitivity
rather than an action of CGRP on the pancreatic islets. /g-Cell sensitivity to
glucose (as judged by the plasma insulin concentrations) was similar in both
peptide- and saline-treated groups of rats. This suggests that the observed
hyperinsulinaemia in the presence of CGRP was a fl-cell response to the elevated
glucose stimulus. Therefore, the data presented here supports the in vitro
observation of Leighton and Cooper [12] who showed that CGRP inhibited
insulin stimulated glucose disposal to glycogen in isolated skeletal muscle, without
altering lactate production. However, in the present study, although the
CGRP-treated rats were relatively hyperglycaemic throughout the post-glucose
period, the rate of decline in glucose was similar to that of saline-treated control
rals; this suggests that the elevation of blood glucose and increased plasma insulin
allowed a similar rate of clearance of glucose from the circulation in the presence
of CGRP to that of the control rats.
CGRP treatment did not affect basal levels of plasma insulin or blood
glucose in this study. Earlier studies with CGRP have produced conflicting
results: increased basal insulin levels in the rat [10] and decreased in the mouse
344
Tedstone, Nezzer, Hughes, Clark and Matthews
[9] and the pig [18], as well as a decreased ability of glucose to stimulate insulin
secretion both in vivo [10] and in vitro [11]. These differences may reflect
differences in experimental protocol, for example, the animals used in this study
were fasted for 18 h prior to the experiment compared with non-fasted animals
used by earlier investigators.
There was no evidence in the present study that IAPP had any effect on
either insulin secretion in vitro or glucose removal in vivo. This is in contrast with
the findings of Leighton and Cooper [12] who describe lAPP and CGRP as
having similar inhibitory effects on insulin-mediated glycogen formation in
skeletal muscle, in vitro. Small structural differences between the lAP used in
these experiments (IAPPl_37) and that used by Leighton and Cooper [12] might
explain this discrepancy. However, it should be noted that both the amidated and
non-amidated forms of lAPP have similar effects on Ca 2§ metabolism in the rat
[191.
We conclude that although CGRP may have an initial inhibitory action on
peripheral glucose removal in vivo which is rapidly overcome by an increase in
plasma insulin, no such action can be demonstrated for lAPP which also has little
effect on insulin secretion. Alternatively, it may be that the previously described
in vitro effects are not representative of the whole-body situation and that lAPP
is not a causative agent of insulin resistance.
ACKNOWLEDGEMENTS
We thank Dr D. H. Williamson for advice, Mrs Vera Ilic for measurement of
plasma insulin and Mrs M. Barber for preparation of the manuscript. This work
was supported in part by the Medical Research Council (UK).
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