# Human Islet Amyloid Polypeptide Transgenic Mice:  In Vivo and Ex Vivo Models for the Role of hIAPP in  Type 2 Diabetes Mellitus

Authors: J. W. M. Höppener; H. M. Jacobs; N. Wierup; G. Sotthewes; M. Sprong; P. de Vos; R. Berger; F. Sundler; B. Ahrén
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Human islet amyloid polypeptide (hIAPP), a pancreatic islet protein of 37 amino acids, is the main component of islet amyloid, seen at autopsy in patients with type 2 diabetes mellitus (DM2). To investigate the roles of hIAPP and islet amyloid in DM2, we generated transgenic mice expressing hIAPP in their islet beta cells. In this study, we found that after a long-term, high-fat diet challenge islet amyloid was observed in only 4 of 19 hIAPP transgenic mice. hIAPP transgenic females exhibited severe glucose intolerance, which was associated with a downregulation of GLUT-2 mRNA expression. In isolated islets from hIAPP males cultured for 3 weeks on high-glucose medium, the percentage of amyloid containing islets increased from 5.5% to 70%. This ex vivo system will allow a more rapid, convenient, and specific study of factors influencing islet amyloidosis as well as of therapeutic strategies to interfere with this pathological process.


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## Body

## 1. Introduction

Islet amyloid polypeptide (IAPP), also referred to as amylin, is a 37 amino acid  protein produced
in the pancreatic islet beta cells. Human IAPP
(hIAPP) is implicated in the pathophysiology of type 2 diabetes mellitus (DM2) since
it forms proteinaceous tissue deposits in the pancreatic islets (“islet amyloid”) [1–3].
Islet amyloid has been demonstrated in more than 80% of patients with DM2 [4, 5].
Islet amyloid formation is implicated in development of beta cell failure which,
in addition to insulin resistance, is a characteristic of DM2 [6]. Overproduction
of IAPP in insulin resistance may occur due to common transcription regulatory elements in
the promoter regions of the IAPP and insulin genes [7], and this might underly the
enhanced amyloid formation in DM2 [3]. This in turn
may induce impairment of beta cell function since aggregation of hIAPP
has been demonstrated to be cytotoxic [8–10]. However, involvement of islet
amyloid in development of DM2 is still not firmly established.
Recent data, both for hIAPP and for other amyloidogenic proteins (notably the
Alzheimer’s disease-related Abeta peptide), indicate that the degree of amyloid
formation does not correspond with the severity of disease [11, 12]. In
addition, prefibrillar aggregates of amyloidogenic proteins seem to be more
cytotoxic than mature amyloid fibrils [13, 14]. To explore the potential
diabetogenic effects of hIAPP and islet amyloid, we have generated transgenic
mice overproducing biologically active hIAPP in the
islet beta cells [15–17] (mouse IAPP
does not form islet amyloid). We previously showed that hIAPP overexpression in
itself does not induce hyperglycemia, hyperinsulinemia, or obesity in these
mice [15]. However, when the hIAPP transgenic mice were crossbred with leptin-deficient and insulin-resistant ob/ob mice, extensive islet amyloid formation with
worsening of the diabetes was observed [18]. In the present paper, we describe
two experimental studies. In an in vivo experiment, we examined
the influence of transgenic hIAPP expression on glucose tolerance of mice on a
high-fat diet for a long period of time. Previous studies had shown that
long-term, high-fat diet induces hyperglycemia, hyperinsulinemia, and obesity
in mice [19, 20]. Furthermore, high-fat diet may be involved in islet amyloid
formation in hIAPP transgenic mice [21]. We, thus, administered a high-fat diet
for 14 months to hIAPP transgenic and nontransgenic (control) mice and report
here the islet amyloid formation, glucose tolerance, and islet GLUT-2 mRNA
expression. In addition, we describe the development of an ex vivo  model system for islet amyloidosis, using pancreatic islets isolated
from the hIAPP transgenic mice. When such islets were cultured in high-glucose
medium, amyloid formation occurs more rapidly as compared to the in vivo situation. Thus, this ex
vivo model system will enable to study the process and effects of islet amyloid formation more specifically and
conveniently.


## 2. Materials and Methods

### 2.1. Animals

The generation of C57Bl/6J hIAPP transgenic mice with a rat insulin-2 gene promoter fragment
(position −695 to +8 relative to the transcription start site) linked to the
hIAPP gene has previously been described [15]. The hIAPP transgenic mice were
maintained by breeding heterozygous transgenic mice with mates of the C57BL/6J
strain. Transgenic mice were differentiated from nontransgenic (NT) littermates
by dot blot Southern hybridization, using a 588 bp hIAPP-specific DNA probe [15].
Mice were housed on hardwood bedding in polypropylene cages and maintained in
air-conditioned rooms at 20–22°C with a photoperiod of 12 hours light, 12 hours dark. Water was available
continuously and the mice received ad libitum a regular diet until 2.5 months
of age. This diet contained 4,500 kcal/kg and included 22.5% protein and 4.8%
fat (Hope Farms, Woerden, The Netherlands). At 2.5 months of age, the diet was
switched to a high-fat diet for 14 months containing 5,600 kcal/kg, 20.8%
protein, and 36.0% fat (30.0% cocoa oil, 6.0% corn oil; Hope Farms).


### 2.2. Glucose Tolerance Test

At 14 months after
the start of the high-fat diet, nonfasted mice were anaesthetized with an i.p.
injection of midazolam (0.4 mg/mouse) (Dormicum, Hoffman-La-Roche, Basel, Switzerland), and a combination of
fluanison (0.9 mg/mouse), and fentanyl (0.02 mg/mouse) (Hypnorm, Janssen, Beerse, Belgium).
D-glucose (British Drug Houses, Poole, UK) was
injected i.p. (1 g/kg) and blood was sampled from the retrobulbar, intraorbital,
capillary plexus before glucose administration and after 10, 30, 60, and
120 minutes. The samples were taken in heparinized tubes and stored on ice.
Following centrifugation, plasma was separated and stored at −20°C until
analysis. After the 120-minute blood sample, tissue was sampled (see below),
and trunk blood was obtained for measurement of IAPP levels. The blood was
collected in EDTA-tubes and kept on ice until centrifugation at 1500 g for 5
minutes at 4°C. Plasma was stored at −80°C.All animal experiments were approved by the Animal Welfare Committee of Utrecht University/University Medical Center
Utrecht, The Netherlands.


### 2.3. Plasma Measurements

IAPP levels were
measured in 25–100 ul plasma by RIA as described [15], using a rabbit,
polyclonal hIAPP antiserum (K1338) that shows full cross-reactivity with
synthetic amidated rat/mouse IAPP [22]. Free and bound radioactivity was
separated by use of double antibody immunoprecipitation. The sensitivity of the
assay is 3.5 pmol/l and the coefficiency of variation <10% at both low and
high levels. Insulin levels were measured in 20 ul plasma by RIA using guinea
pig anti-rat insulin antibody, 125I-labelled human insulin as tracer
and rat insulin as standard (Linco Research, St. Charles, Mo, USA). Free and bound radioactivity
was separated by use of an anti-IgG antibody (Linco). The sensitivity of the
assay is 12 pmol/l and the coefficiency of variation <3% at both low and
high levels. Glucose was determined in 10 ul plasma by the glucose oxidase
method.


### 2.4. Histological Analysis of Pancreatic Tissue

Pancreatic tissue samples were fixed in 3.7% phosphate-buffered formalin
(pH 7.4) for 24–48 hours and paraffin embedded. Sections of 5 μm were stained with Congo red for detection of
islet amyloid by polarized light microscopy (“apple-green” birefringence) and fluorescence
light microscopy (red-coloured autofluorescence). At least 10 islets per mouse
were examined. The percentage of individual islet areas occupied by amyloid, as
indicated by Congo red positive staining, was visually estimated and scored as
follows: 0% = score 0, between 0 and 26% = score 1, 26–50% = score 2, 51–75% =
score 3, and 76–100% = score 4. The Amyloid Index (range: 0–100) of an
individual mouse was calculated as(1×N1+2×N2+3×N3+4×N4)×25/n, where N1
is the number of islets with score 1, N2 the number with score 2, and so on,
and n is the total number of islets investigated. The degree of islet amyloid
formation was determined with the investigator being unaware of the genetic
status of the animals (i.e., “blind”).To examine the cellular expression of GLUT-2 mRNA, paraffin sections were
subjected to in situ hybridization using a previously described protocol [23] and a 35S-labelled
oligonucleotide probe covering the nucleotide sequence 247–276 of mouse GLUT-2 cDNA [24]. In order to
confirm beta cell expression of hIAPP mRNA in the transgenic mice, sections
were also hybridized with a 35S-labelled oligonucleotide probe specific for hIAPP mRNA [25].


### 2.5. Image Analysis and Morphometry

In situ hybridization radiolabelling was examined in a bright field microscope
(Olympus, BX60), and images were captured with a digital camera (Olympus, DP50). To quantify the density of labelling for
GLUT-2 mRNA within islets, areas of in
situ hybridization radiolabelling were calculated. Islets (n=5–8 per animal)
were randomly selected from different parts of the
sections from 4 mice, 2 males and 2 females in each group. The transgenic mice analyzed
were rated as negative for amyloid. The labelled area, that is, grain
density within an islet, and total islet area were measured, using NIH-image
software, and the density of labelling was expressed as percentage of the total
islet area [23, 26]. All sections used were hybridized simultaneously and under
identical conditions.


### 2.6. Isolation of Pancreatic Islets

For islet isolation, transgenic mice
were bred to homozygosity for the hIAPP transgenic locus. Homozygotes were
discriminated from heterozygous and nontransgenic littermates by dot blot
Southern hybridization of tail DNA using a human-specific IAPP probe [15] and
quantification of the hybridization signal using phosphor imaging and
Image-Quant software (Molecular Dynamics, Inc. Krefeld, Germany).Islets were isolated from the
pancreas of 6-month-old hIAPP transgenic male mice, essentially as previously
described [27]. Briefly, under halothane
anaesthesia, the abdomen was opened. The pancreas was excised starting from the
spleen site to the duodenum. Subsequently, the pancreas was brougth in 10 mL sterile Krebs-Ringer-buffer supplemented
with 25 mmol/L Hepes (KRH) and containing 10% Bovine Serum Albumin (BSA)
at 4°C. Next, the pancreas was chopped, digested using a two-stage
incubation of 20 minutes at 37°C with successively 1.0 and 0.7 mg/mL
collagenase (Sigma type XI, Sigma, St Louis, MO, USA). Islets were separated from exocrine
tissue by centrifugation over a discontinuous dextran gradient [28] and further
purified by handpicking into 9 cm petridishes with 12 mL KRH
buffer, pH = 7.4, supplemented with 10% BSA penicillin (100 units/mL)/streptomycin
(0.1 mg/mL) (KRH 10% BSA P/S) and glucose to a concentration of 11 mM. Two days
after isolation, the islets from 12 mice were pooled, mixed and split into portions.
Four portions of 75 islets each were fixed and embedded for amyloid
quantification. Eight portions of approximately 90 islets were transferred to culture
medium with 28 mM glucose. Medium was changed every 2-3 days, switching
between 11 mM and 28 mM of glucose (to prevent possible desensitization of the beta cells). Islets were counted, while being picked into the
dishes with fresh medium. At 3 weeks after islet isolation, the cultured
islets were fixed and embedded for amyloid quantification.


### 2.7. Fixation and Embedding of Pancreatic Islets

Islets were washed with phosphate buffered saline (PBS), and fixed in 0.5 mL islets
fixative (2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M Sörensen buffer) for
2 hours at room temperature. Fixative was removed and islets were washed with
0.5 mL  0.1 M Sörensen buffer. Sörensen buffer was removed and islets were resuspended
in 30μl 37°C heated 12% gelatin, cooled on ice and stored at −20°C.


### 2.8. Amyloid Quantification in Cultured Islets

From the gelatin-embedded islet blocks, 5μm frozen sections
were cut onto Superfrost Plus microscope slides (Menzel-Gläser) and stored at
−20°C until further use. Sections were fixed in acetone for 1′, rehydrated in PBS for 15′, stained with
heamatoxylin for 1′, washed in running tap water for 5′, and stained with Congo red (1 g/liter
saturated sodium chloride 80% ethanol, into which 10 mL/liter 1% sodium
hydroxide was added just before staining) for 30′. After dehydration in an
augmenting ethanol series (70%, 96%, 100%) and xylene (twice), sections were
enclosed with Depex. Amyloid-containing paraffin sections of hIAPP transgenic
mouse pancreatic tissue were  used as positive control for the Congo red staining.For the detection of amyloid, Congo red-stained islet sections were examined using
a fluorescence microscope. Amyloid deposits were visible as bright red
autofluorescent areas without cells, which showed a green birefringence upon
visualization with polarized light. An islet was scored as amyloid positive if at least 2 successive sections of that
islet contained Congo red-positive amyloid deposits. The scoring was performed
in a “blind” fashion, that is, with the investigator unaware of the source of
the islets.


### 2.9. Statistical Analysis

Values are means ±SEM, unless stated otherwise.P-values
indicate the probability level of random difference between groups, or of
random correlation, respectively. P-values <.05 were considered to represent statistical significance. Nonparametric
T-tests were used to compare 2 independent samples (Mann-Whitney-U test: hIAPP versus
NT, male versus female). Data of the mRNA in situ hybridizations were analyzed by Student’s
unpaired t-test. Differences in percentage of amyloid-positive islets between 2
days and 3 weeks of culture were analyzed by
use of one-way analysis of variance (ANOVA). Probability values of less than
.01 were considered significant.


## 2.1. Animals

The generation of C57Bl/6J hIAPP transgenic mice with a rat insulin-2 gene promoter fragment
(position −695 to +8 relative to the transcription start site) linked to the
hIAPP gene has previously been described [15]. The hIAPP transgenic mice were
maintained by breeding heterozygous transgenic mice with mates of the C57BL/6J
strain. Transgenic mice were differentiated from nontransgenic (NT) littermates
by dot blot Southern hybridization, using a 588 bp hIAPP-specific DNA probe [15].
Mice were housed on hardwood bedding in polypropylene cages and maintained in
air-conditioned rooms at 20–22°C with a photoperiod of 12 hours light, 12 hours dark. Water was available
continuously and the mice received ad libitum a regular diet until 2.5 months
of age. This diet contained 4,500 kcal/kg and included 22.5% protein and 4.8%
fat (Hope Farms, Woerden, The Netherlands). At 2.5 months of age, the diet was
switched to a high-fat diet for 14 months containing 5,600 kcal/kg, 20.8%
protein, and 36.0% fat (30.0% cocoa oil, 6.0% corn oil; Hope Farms).


## 2.2. Glucose Tolerance Test

At 14 months after
the start of the high-fat diet, nonfasted mice were anaesthetized with an i.p.
injection of midazolam (0.4 mg/mouse) (Dormicum, Hoffman-La-Roche, Basel, Switzerland), and a combination of
fluanison (0.9 mg/mouse), and fentanyl (0.02 mg/mouse) (Hypnorm, Janssen, Beerse, Belgium).
D-glucose (British Drug Houses, Poole, UK) was
injected i.p. (1 g/kg) and blood was sampled from the retrobulbar, intraorbital,
capillary plexus before glucose administration and after 10, 30, 60, and
120 minutes. The samples were taken in heparinized tubes and stored on ice.
Following centrifugation, plasma was separated and stored at −20°C until
analysis. After the 120-minute blood sample, tissue was sampled (see below),
and trunk blood was obtained for measurement of IAPP levels. The blood was
collected in EDTA-tubes and kept on ice until centrifugation at 1500 g for 5
minutes at 4°C. Plasma was stored at −80°C.All animal experiments were approved by the Animal Welfare Committee of Utrecht University/University Medical Center
Utrecht, The Netherlands.


## 2.3. Plasma Measurements

IAPP levels were
measured in 25–100 ul plasma by RIA as described [15], using a rabbit,
polyclonal hIAPP antiserum (K1338) that shows full cross-reactivity with
synthetic amidated rat/mouse IAPP [22]. Free and bound radioactivity was
separated by use of double antibody immunoprecipitation. The sensitivity of the
assay is 3.5 pmol/l and the coefficiency of variation <10% at both low and
high levels. Insulin levels were measured in 20 ul plasma by RIA using guinea
pig anti-rat insulin antibody, 125I-labelled human insulin as tracer
and rat insulin as standard (Linco Research, St. Charles, Mo, USA). Free and bound radioactivity
was separated by use of an anti-IgG antibody (Linco). The sensitivity of the
assay is 12 pmol/l and the coefficiency of variation <3% at both low and
high levels. Glucose was determined in 10 ul plasma by the glucose oxidase
method.


## 2.4. Histological Analysis of Pancreatic Tissue

Pancreatic tissue samples were fixed in 3.7% phosphate-buffered formalin
(pH 7.4) for 24–48 hours and paraffin embedded. Sections of 5 μm were stained with Congo red for detection of
islet amyloid by polarized light microscopy (“apple-green” birefringence) and fluorescence
light microscopy (red-coloured autofluorescence). At least 10 islets per mouse
were examined. The percentage of individual islet areas occupied by amyloid, as
indicated by Congo red positive staining, was visually estimated and scored as
follows: 0% = score 0, between 0 and 26% = score 1, 26–50% = score 2, 51–75% =
score 3, and 76–100% = score 4. The Amyloid Index (range: 0–100) of an
individual mouse was calculated as(1×N1+2×N2+3×N3+4×N4)×25/n, where N1
is the number of islets with score 1, N2 the number with score 2, and so on,
and n is the total number of islets investigated. The degree of islet amyloid
formation was determined with the investigator being unaware of the genetic
status of the animals (i.e., “blind”).To examine the cellular expression of GLUT-2 mRNA, paraffin sections were
subjected to in situ hybridization using a previously described protocol [23] and a 35S-labelled
oligonucleotide probe covering the nucleotide sequence 247–276 of mouse GLUT-2 cDNA [24]. In order to
confirm beta cell expression of hIAPP mRNA in the transgenic mice, sections
were also hybridized with a 35S-labelled oligonucleotide probe specific for hIAPP mRNA [25].


## 2.5. Image Analysis and Morphometry

In situ hybridization radiolabelling was examined in a bright field microscope
(Olympus, BX60), and images were captured with a digital camera (Olympus, DP50). To quantify the density of labelling for
GLUT-2 mRNA within islets, areas of in
situ hybridization radiolabelling were calculated. Islets (n=5–8 per animal)
were randomly selected from different parts of the
sections from 4 mice, 2 males and 2 females in each group. The transgenic mice analyzed
were rated as negative for amyloid. The labelled area, that is, grain
density within an islet, and total islet area were measured, using NIH-image
software, and the density of labelling was expressed as percentage of the total
islet area [23, 26]. All sections used were hybridized simultaneously and under
identical conditions.


## 2.6. Isolation of Pancreatic Islets

For islet isolation, transgenic mice
were bred to homozygosity for the hIAPP transgenic locus. Homozygotes were
discriminated from heterozygous and nontransgenic littermates by dot blot
Southern hybridization of tail DNA using a human-specific IAPP probe [15] and
quantification of the hybridization signal using phosphor imaging and
Image-Quant software (Molecular Dynamics, Inc. Krefeld, Germany).Islets were isolated from the
pancreas of 6-month-old hIAPP transgenic male mice, essentially as previously
described [27]. Briefly, under halothane
anaesthesia, the abdomen was opened. The pancreas was excised starting from the
spleen site to the duodenum. Subsequently, the pancreas was brougth in 10 mL sterile Krebs-Ringer-buffer supplemented
with 25 mmol/L Hepes (KRH) and containing 10% Bovine Serum Albumin (BSA)
at 4°C. Next, the pancreas was chopped, digested using a two-stage
incubation of 20 minutes at 37°C with successively 1.0 and 0.7 mg/mL
collagenase (Sigma type XI, Sigma, St Louis, MO, USA). Islets were separated from exocrine
tissue by centrifugation over a discontinuous dextran gradient [28] and further
purified by handpicking into 9 cm petridishes with 12 mL KRH
buffer, pH = 7.4, supplemented with 10% BSA penicillin (100 units/mL)/streptomycin
(0.1 mg/mL) (KRH 10% BSA P/S) and glucose to a concentration of 11 mM. Two days
after isolation, the islets from 12 mice were pooled, mixed and split into portions.
Four portions of 75 islets each were fixed and embedded for amyloid
quantification. Eight portions of approximately 90 islets were transferred to culture
medium with 28 mM glucose. Medium was changed every 2-3 days, switching
between 11 mM and 28 mM of glucose (to prevent possible desensitization of the beta cells). Islets were counted, while being picked into the
dishes with fresh medium. At 3 weeks after islet isolation, the cultured
islets were fixed and embedded for amyloid quantification.


## 2.7. Fixation and Embedding of Pancreatic Islets

Islets were washed with phosphate buffered saline (PBS), and fixed in 0.5 mL islets
fixative (2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M Sörensen buffer) for
2 hours at room temperature. Fixative was removed and islets were washed with
0.5 mL  0.1 M Sörensen buffer. Sörensen buffer was removed and islets were resuspended
in 30μl 37°C heated 12% gelatin, cooled on ice and stored at −20°C.


## 2.8. Amyloid Quantification in Cultured Islets

From the gelatin-embedded islet blocks, 5μm frozen sections
were cut onto Superfrost Plus microscope slides (Menzel-Gläser) and stored at
−20°C until further use. Sections were fixed in acetone for 1′, rehydrated in PBS for 15′, stained with
heamatoxylin for 1′, washed in running tap water for 5′, and stained with Congo red (1 g/liter
saturated sodium chloride 80% ethanol, into which 10 mL/liter 1% sodium
hydroxide was added just before staining) for 30′. After dehydration in an
augmenting ethanol series (70%, 96%, 100%) and xylene (twice), sections were
enclosed with Depex. Amyloid-containing paraffin sections of hIAPP transgenic
mouse pancreatic tissue were  used as positive control for the Congo red staining.For the detection of amyloid, Congo red-stained islet sections were examined using
a fluorescence microscope. Amyloid deposits were visible as bright red
autofluorescent areas without cells, which showed a green birefringence upon
visualization with polarized light. An islet was scored as amyloid positive if at least 2 successive sections of that
islet contained Congo red-positive amyloid deposits. The scoring was performed
in a “blind” fashion, that is, with the investigator unaware of the source of
the islets.


## 2.9. Statistical Analysis

Values are means ±SEM, unless stated otherwise.P-values
indicate the probability level of random difference between groups, or of
random correlation, respectively. P-values <.05 were considered to represent statistical significance. Nonparametric
T-tests were used to compare 2 independent samples (Mann-Whitney-U test: hIAPP versus
NT, male versus female). Data of the mRNA in situ hybridizations were analyzed by Student’s
unpaired t-test. Differences in percentage of amyloid-positive islets between 2
days and 3 weeks of culture were analyzed by
use of one-way analysis of variance (ANOVA). Probability values of less than
.01 were considered significant.


## 3. Results

### 3.1. Body Weight and Plasma IAPP Levels

Body weight after 14 months on the high-fat diet did not differ between the groups, being 57 ± 0.8 g versus 58 ± 0.9 g in male
hIAPP (n=8) and NT (n=5) mice, and 59 ± 1.1 g versus 64 ± 3 g in female hIAPP (n=11) and NT (n=6) mice.
Plasma IAPP levels were 462 ± 78 pmol/L in male hIAPP mice versus 195 ± 32 pmol/L in male NT mice,
and 346 ± 81 pmol/L in female hIAPP mice versus 130 ± 21 pmol/L in female NT mice, being
significantly higher in hIAPP mice  of
both genders (P < .01) without any
gender difference.


### 3.2. Glucose Tolerance Test

After 14 months on the high-fat diet, nonfasted plasma glucose and insulin levels were not
different between hIAPP and NT mice of the same gender. However, both for the
hIAPP and NT mice, plasma insulin levels were higher in males as compared to
females (Figure1). When glucose was administered i.p. (1 g/kg), the insulin
response to glucose and the glucose elimination were similar in hIAPP and NT male
mice. In contrast, in female hIAPP mice, plasma glucose levels after the i.p.
glucose challenge were markedly higher at all time points as compared to female
NT mice (P < .05 or P < .01) in association with increased
insulin levels 30 minutes after glucose administration (P < .01). Hence, hIAPP overproduction was associated with severe
impairment of glucose elimination in female but not in male mice after high-fat diet.Plasma insulin and glucose levels immediately before and at different timepoints after an intraperitoneal injection of
glucose (1 g/kg body weight) in anaesthetized, nonfasted nontransgenic (NT,
dotted line), and hIAPP transgenic mice (solid line) on a high-fat diet for 14 months.
Mean values and SEM are shown;n=5–11 per group of mice; statistically significant changes between hIAPP and NT
mice are indicated by *(P < .05) and **(P < .01).
(a)(b)(c)(d)


### 3.3. Pancreatic Islet Amyloid Formation

Islet amyloid was detected in 4/19 hIAPP mice on high-fat diet but in none of the 11 NT mice. The
Amyloid Index for these 4 mice was11.0±6.2 (average
and SD). There was no gender difference in islet
amyloid formation in hIAPP transgenic mice (3/8 in males versus 1/11 in females).


### 3.4. Islet GLUT-2 mRNA Expression

As expected, a strong hIAPP mRNA labeling was observed in the islets of all
transgenic mice, while it was lacking in all NT mice (Figures2(a), 2(b)).
GLUT-2 mRNA labeling of weak to moderate density was observed in the islets of
NT mice (Figure 2(c)), with no overt difference between female
and male mice. In the transgenic mice, however, the GLUT-2 mRNA labeling was
generally weaker, and even barely detectable in some female mice (Figure 2(d)).
The GLUT-2 mRNA signal was reduced in all transgenic mice, regardless of the
presence or absence of islet amyloid. Image analysis revealed a significant
reduction of GLUT-2 mRNA labeling of islets in hIAPP transgenic versus NT mice
(P = .02, Figure 3).In situ mRNA hybridization (using radiolabeled oligoprobes) for hIAPP (a), (b), and GLUT-2 (c), (d) in islets of
nontransgenic (a), (c), and hIAPP transgenic (b), (d) female mice after 14 months
on high-fat diet. Note that hIAPP mRNA expression is absent in the nontransgenic
islet, and that GLUT-2 mRNA expression is reduced in
the transgenic islet. Scale bar = 30 μm.
(a)(b)(c)(d)Figure 3
Comparison of the average labeling density of GLUT-2 mRNA in situ hybridization in
pancreatic islets from nontransgenic (NT) and hIAPP transgenic (hIAPP) mice (P = .02). For both groups 4 mice were analysed, 2 males, and 2 females. The 4
transgenic mice did not have amyloid.


### 3.5. Ex Vivo Survival and Amyloid Formation in Cultured hIAPP Transgenic Pancreatic Islets

The percentage of 3-week survival of hIAPP transgenic islets was 83.8 ± 1.0% (n=8). Of all islet cultures, 22–30 islets were scored for the presence of amyloid. The percentage of amyloid-positive
islets significantly increased (P < .001) from 5.5±3.4% (n=4) after 2 days
of culture to 70 ± 3.1% (n=8) at the end of the culture period. Thus, the percentage
of amyloid-positive islets increased more than 10 times in three weeks of
culture at high glucose conditions in this ex vivo islet amyloidosis system. An example of a cultured hIAPP
transgenic islet containing amyloid is shown in Figure 4.Detection of islet amyloid in islet of Langerhans isolated from an hIAPP transgenic mouse,
and cultured in medium with a high glucose concentration. Frozen section of a gelatine-embedded
islet was stained with the amyloid-specific dye Congo red and visualized with
light microscopy (a) and  fluorescence microscopy (b), respectively.
(a)(b)


## 3.1. Body Weight and Plasma IAPP Levels

Body weight after 14 months on the high-fat diet did not differ between the groups, being 57 ± 0.8 g versus 58 ± 0.9 g in male
hIAPP (n=8) and NT (n=5) mice, and 59 ± 1.1 g versus 64 ± 3 g in female hIAPP (n=11) and NT (n=6) mice.
Plasma IAPP levels were 462 ± 78 pmol/L in male hIAPP mice versus 195 ± 32 pmol/L in male NT mice,
and 346 ± 81 pmol/L in female hIAPP mice versus 130 ± 21 pmol/L in female NT mice, being
significantly higher in hIAPP mice  of
both genders (P < .01) without any
gender difference.


## 3.2. Glucose Tolerance Test

After 14 months on the high-fat diet, nonfasted plasma glucose and insulin levels were not
different between hIAPP and NT mice of the same gender. However, both for the
hIAPP and NT mice, plasma insulin levels were higher in males as compared to
females (Figure1). When glucose was administered i.p. (1 g/kg), the insulin
response to glucose and the glucose elimination were similar in hIAPP and NT male
mice. In contrast, in female hIAPP mice, plasma glucose levels after the i.p.
glucose challenge were markedly higher at all time points as compared to female
NT mice (P < .05 or P < .01) in association with increased
insulin levels 30 minutes after glucose administration (P < .01). Hence, hIAPP overproduction was associated with severe
impairment of glucose elimination in female but not in male mice after high-fat diet.Plasma insulin and glucose levels immediately before and at different timepoints after an intraperitoneal injection of
glucose (1 g/kg body weight) in anaesthetized, nonfasted nontransgenic (NT,
dotted line), and hIAPP transgenic mice (solid line) on a high-fat diet for 14 months.
Mean values and SEM are shown;n=5–11 per group of mice; statistically significant changes between hIAPP and NT
mice are indicated by *(P < .05) and **(P < .01).
(a)(b)(c)(d)


## 3.3. Pancreatic Islet Amyloid Formation

Islet amyloid was detected in 4/19 hIAPP mice on high-fat diet but in none of the 11 NT mice. The
Amyloid Index for these 4 mice was11.0±6.2 (average
and SD). There was no gender difference in islet
amyloid formation in hIAPP transgenic mice (3/8 in males versus 1/11 in females).


## 3.4. Islet GLUT-2 mRNA Expression

As expected, a strong hIAPP mRNA labeling was observed in the islets of all
transgenic mice, while it was lacking in all NT mice (Figures2(a), 2(b)).
GLUT-2 mRNA labeling of weak to moderate density was observed in the islets of
NT mice (Figure 2(c)), with no overt difference between female
and male mice. In the transgenic mice, however, the GLUT-2 mRNA labeling was
generally weaker, and even barely detectable in some female mice (Figure 2(d)).
The GLUT-2 mRNA signal was reduced in all transgenic mice, regardless of the
presence or absence of islet amyloid. Image analysis revealed a significant
reduction of GLUT-2 mRNA labeling of islets in hIAPP transgenic versus NT mice
(P = .02, Figure 3).In situ mRNA hybridization (using radiolabeled oligoprobes) for hIAPP (a), (b), and GLUT-2 (c), (d) in islets of
nontransgenic (a), (c), and hIAPP transgenic (b), (d) female mice after 14 months
on high-fat diet. Note that hIAPP mRNA expression is absent in the nontransgenic
islet, and that GLUT-2 mRNA expression is reduced in
the transgenic islet. Scale bar = 30 μm.
(a)(b)(c)(d)Figure 3
Comparison of the average labeling density of GLUT-2 mRNA in situ hybridization in
pancreatic islets from nontransgenic (NT) and hIAPP transgenic (hIAPP) mice (P = .02). For both groups 4 mice were analysed, 2 males, and 2 females. The 4
transgenic mice did not have amyloid.


## 3.5. Ex Vivo Survival and Amyloid Formation in Cultured hIAPP Transgenic Pancreatic Islets

The percentage of 3-week survival of hIAPP transgenic islets was 83.8 ± 1.0% (n=8). Of all islet cultures, 22–30 islets were scored for the presence of amyloid. The percentage of amyloid-positive
islets significantly increased (P < .001) from 5.5±3.4% (n=4) after 2 days
of culture to 70 ± 3.1% (n=8) at the end of the culture period. Thus, the percentage
of amyloid-positive islets increased more than 10 times in three weeks of
culture at high glucose conditions in this ex vivo islet amyloidosis system. An example of a cultured hIAPP
transgenic islet containing amyloid is shown in Figure 4.Detection of islet amyloid in islet of Langerhans isolated from an hIAPP transgenic mouse,
and cultured in medium with a high glucose concentration. Frozen section of a gelatine-embedded
islet was stained with the amyloid-specific dye Congo red and visualized with
light microscopy (a) and  fluorescence microscopy (b), respectively.
(a)(b)


## 4. Discussion

### 4.1. High-Fat Diet and Amyloid Formation

In this study, transgenic mice overproducing the amyloidogenic hIAPP  in
their pancreatic islet beta cells, as well as NT control mice, were fed a
high-fat diet for 14 months, in order to evaluate the impact on islet amyloid
formation and glucose homeostasis when combining these two potentially
diabetogenic factors. We anticipated a marked islet amyloid formation in the
hIAPP transgenic mice on the high-fat diet because we previously observed that
crossbreeding the hIAPP transgenic mice with the Obese mouse (being severly
insulin resistant) resulted in extensive islet amyloid formation [18]. Also,
when insulin resistance was induced in hIAPP mice by crossbreeding with the
obese Agouti viable yellow mice [29] or by exogenous growth hormone and
glucocorticoids [30], islet amyloid formation was promoted. In addition,
high-fat feeding induced islet amyloid formation in approximately 80% of male
mice in another hIAPP transgenic colony [21]. However, we found that only four out
of the 19 hIAPP mice (approx. 40% of the males) that were followed for 14
months on the high-fat diet did develop islet amyloid. This lower frequency
might be due to differences in the genetic background and/or the composition of
the diet, influencing insulin resistance and IAPP expression. The amyloid index
in those four high-fat fed mice was higher than in six of 33 similarly aged
hIAPP mice (approx. 30% of the males) which developed amyloid on a  regular diet (11.0±6.2 versus 4.2±2.9,
P=.024) [18]. This indicates that although long-term, high-fat diet indeed has
the capacity to promote islet amyloid formation in these hIAPP transgenic mice,
the efficiency is not high. Crossbreeding the hIAPP mice with leptin-deficient
Obese mice introduced more severe obesity and insulin resistance [18] as
compared to the high-fat diet. Consequently, these other factors seem of
importance for the promotion of islet amyloid formation. Another factor might
be hyperglycemia, which is more severe in the hIAPP ob/ob mice as compared to
the hIAPP mice on high-fat diet. Such a hypothesis is supported by the finding
that in isolated pancreatic islets of our hIAPP mice, islet amyloid was detected
by electron microscopy after culture in high-glucose medium but not
in low-glucose medium [31]. Other mechanisms may, however, also be of
importance.


### 4.2. High Fat Diet and Glucose Tolerance

In this study, we also observed severe glucose intolerance in female but not in male hIAPP transgenic
mice. The finding that in all groups of mice plasma insulin levels failed to
return to basal within 2 hours after the glucose load is in accordance with
high-fat diet inducing insulin resistance [32]. Also,
the higher insulin levels in male mice versus female mice is well known
from previous studies [33]. Thus, our data indicate that the overproduction of
insulin in response to insulin resistance after high-fat diet was adequate in
hIAPP males but not in hIAPP females. Since there was no gender difference in
the amyloid formation in high-fat fed hIAPP mice, these results suggest that a metabolic impact of high levels of circulating IAPP
underlies the gender difference in glucose tolerance of hIAPP transgenic
mice  after high-fat diet. IAPP has, thus,
been shown to inhibit insulin secretion [34, 35] as well as to inhibit glycogen
synthesis in rat muscle tissue [36] through inhibition of glycogen synthase and
stimulation of glycogen phosphorylase [37]. In addition, it has been observed
that IAPP administration induces insulin resistance in rats [38], although no
such effect was evident in humans [39]. IAPP has also in some studies [40] but
not in others [41] been shown to increase liver glucose production. Whether
these actions show gender differences, and thus may explain the remarkable
glucose intolerance observed in female but not in male hIAPP transgenic mice on
the high-fat diet, is not known. Indeed, it is striking that although male
rodents generally are more prone to insulin resistance than females, the hIAPP
transgenic females on high-fat diet are more glucose intolerant than their male
littermates. Since insulin levels are not lower in the hIAPP females compared
to the NT females, these data suggest that insulin sensitivity is impaired in
the female hIAPP mice.


### 4.3. High-Fat Diet and GLUT-2 Expression

Islet GLUT-2 mRNA expression was reduced in hIAPP transgenic versus NT mice, and this
reduction appeared more severe in female than in male mice. Beta cell GLUT-2
expression is known to correlate with glucose responsiveness of the cells [42].
However, insulin levels were not reduced in the male or female hIAPP mice.
Therefore, it is presently not known if and how the reduced GLUT-2 mRNA expression
among the transgenic mice might be related to the glucose intolerance in the
female hIAPP mice. Also, the mechanism of hIAPP mediated downregulation of islet GLUT-2
mRNA expression is unknown, but our data indicate that in addition to
inhibition of insulin action in muscle [33, 34] IAPP can (in) directly inhibit
glucose responsiveness of islet beta cells by affecting GLUT-2 expression.In conclusion, this in vivo  study shows that promoting insulin
resistance over a long period of time by giving a high-fat diet for 14 months
promotes islet amyloid formation in hIAPP transgenic mice, although less
extensively than in the severe insulin-resistant Obese, leptin-deficient  hIAPP mice. This suggests that the degree of
insulin resistance is important for extensive development of islet amyloid. In
addition, we observed a remarkable gender difference in that severe glucose
intolerance
was observed only in female hIAPP transgenic mice given high-fat diet and not
in males. We suggest that this gender difference is due to the high level of circulating
IAPP rather than to islet amyloid formation. If and how this glucose
intolerance might be mediated by downregulation of beta cell GLUT-2 gene
expression, as observed in the hIAPP mice, is presently unknown.


### 4.4. Ex Vivo Islet Amyloidosis Model

The rationale for the ex vivo study was to
examine if amyloid would be formed in isolated and cultured pancreatic islets
from hIAPP mice, to such a degree that it would be detectable with light
microscopy. Since both the present and previous [18]  in
vivo data indicated  the development of islet amyloid notably in
male hIAPP mice, we decided to investigate amyloid formation in such islets
specifically from male mice. To increase the potential for amyloid formation,
we bred the mice to homozygosity for the hIAPP transgene. At an age of 6 months,
homozygous transgenic hIAPP males had islet amyloid in about 5% of their
pancreatic islets, at 2 days after islet isolation. Previously, we detected
amyloid fibrils by electron microscopy in islets from 4–10 months old heterozygous
hIAPP transgenic mice, cultured for 1 week in medium with 11 or 28 mM glucose [31].
With the present model, using islets from 6-month-old homozygous hIAPP transgenic
males, cultured in medium with high glucose (switching between 11 and 28 mM), we
can detect amyloid deposits with Congo red staining and light microscopy, thus enabling
quantification of the degree of islet amyloidosis. The number of
amyloid-positive islets increased more than 10 fold (from 5.5 to 70%) after 3 weeks of culture
in medium containing a high glucose concentration. Although an accurate
comparison between the degrees of islet amyloid formation in
vivo  and ex vivo was not made, our data certainly indicate stronger islet amyloid formation in hIAPP
islets cultured ex vivo as compared to in vivo. This might be
explained by the higher glucose concentrations in the ex vivo system. hIAPP
transgenic mice in vivo have normal plasma glucose concentrations [15, 18],
whereas ex vivo the glucose concentration in the medium switched between  11 and 28 mM. It is known that a high glucose
concentration triggers both insulin and IAPP secretion, and the hIAPP transgene
is under control of an insulin promoter. In addition, macrophages have been
implicated in in vivo removal of (beginning) amyloid deposits
[43] and such macrophages are absent in the ex vivo system,
potentially allowing increased amyloid formation. When combined with a more
accurate amyloid quantification procedure involving image analysis, this ex vivo system may present a fast and convenient model to study the
process (and factors involved) of islet amyloidosis, as well as the detrimental
consequences for individual beta cells (apoptosis) and islet function (insulin
producing capacity). In addition, such a model system might be used as an
amyloidosis assay to assess the potency of known and novel therapeutic
strategies, aimed at reducing, or even preventing, islet amyloid formation, and its effects on beta cell and
islet  function.


## 4.1. High-Fat Diet and Amyloid Formation

In this study, transgenic mice overproducing the amyloidogenic hIAPP  in
their pancreatic islet beta cells, as well as NT control mice, were fed a
high-fat diet for 14 months, in order to evaluate the impact on islet amyloid
formation and glucose homeostasis when combining these two potentially
diabetogenic factors. We anticipated a marked islet amyloid formation in the
hIAPP transgenic mice on the high-fat diet because we previously observed that
crossbreeding the hIAPP transgenic mice with the Obese mouse (being severly
insulin resistant) resulted in extensive islet amyloid formation [18]. Also,
when insulin resistance was induced in hIAPP mice by crossbreeding with the
obese Agouti viable yellow mice [29] or by exogenous growth hormone and
glucocorticoids [30], islet amyloid formation was promoted. In addition,
high-fat feeding induced islet amyloid formation in approximately 80% of male
mice in another hIAPP transgenic colony [21]. However, we found that only four out
of the 19 hIAPP mice (approx. 40% of the males) that were followed for 14
months on the high-fat diet did develop islet amyloid. This lower frequency
might be due to differences in the genetic background and/or the composition of
the diet, influencing insulin resistance and IAPP expression. The amyloid index
in those four high-fat fed mice was higher than in six of 33 similarly aged
hIAPP mice (approx. 30% of the males) which developed amyloid on a  regular diet (11.0±6.2 versus 4.2±2.9,
P=.024) [18]. This indicates that although long-term, high-fat diet indeed has
the capacity to promote islet amyloid formation in these hIAPP transgenic mice,
the efficiency is not high. Crossbreeding the hIAPP mice with leptin-deficient
Obese mice introduced more severe obesity and insulin resistance [18] as
compared to the high-fat diet. Consequently, these other factors seem of
importance for the promotion of islet amyloid formation. Another factor might
be hyperglycemia, which is more severe in the hIAPP ob/ob mice as compared to
the hIAPP mice on high-fat diet. Such a hypothesis is supported by the finding
that in isolated pancreatic islets of our hIAPP mice, islet amyloid was detected
by electron microscopy after culture in high-glucose medium but not
in low-glucose medium [31]. Other mechanisms may, however, also be of
importance.


## 4.2. High Fat Diet and Glucose Tolerance

In this study, we also observed severe glucose intolerance in female but not in male hIAPP transgenic
mice. The finding that in all groups of mice plasma insulin levels failed to
return to basal within 2 hours after the glucose load is in accordance with
high-fat diet inducing insulin resistance [32]. Also,
the higher insulin levels in male mice versus female mice is well known
from previous studies [33]. Thus, our data indicate that the overproduction of
insulin in response to insulin resistance after high-fat diet was adequate in
hIAPP males but not in hIAPP females. Since there was no gender difference in
the amyloid formation in high-fat fed hIAPP mice, these results suggest that a metabolic impact of high levels of circulating IAPP
underlies the gender difference in glucose tolerance of hIAPP transgenic
mice  after high-fat diet. IAPP has, thus,
been shown to inhibit insulin secretion [34, 35] as well as to inhibit glycogen
synthesis in rat muscle tissue [36] through inhibition of glycogen synthase and
stimulation of glycogen phosphorylase [37]. In addition, it has been observed
that IAPP administration induces insulin resistance in rats [38], although no
such effect was evident in humans [39]. IAPP has also in some studies [40] but
not in others [41] been shown to increase liver glucose production. Whether
these actions show gender differences, and thus may explain the remarkable
glucose intolerance observed in female but not in male hIAPP transgenic mice on
the high-fat diet, is not known. Indeed, it is striking that although male
rodents generally are more prone to insulin resistance than females, the hIAPP
transgenic females on high-fat diet are more glucose intolerant than their male
littermates. Since insulin levels are not lower in the hIAPP females compared
to the NT females, these data suggest that insulin sensitivity is impaired in
the female hIAPP mice.


## 4.3. High-Fat Diet and GLUT-2 Expression

Islet GLUT-2 mRNA expression was reduced in hIAPP transgenic versus NT mice, and this
reduction appeared more severe in female than in male mice. Beta cell GLUT-2
expression is known to correlate with glucose responsiveness of the cells [42].
However, insulin levels were not reduced in the male or female hIAPP mice.
Therefore, it is presently not known if and how the reduced GLUT-2 mRNA expression
among the transgenic mice might be related to the glucose intolerance in the
female hIAPP mice. Also, the mechanism of hIAPP mediated downregulation of islet GLUT-2
mRNA expression is unknown, but our data indicate that in addition to
inhibition of insulin action in muscle [33, 34] IAPP can (in) directly inhibit
glucose responsiveness of islet beta cells by affecting GLUT-2 expression.In conclusion, this in vivo  study shows that promoting insulin
resistance over a long period of time by giving a high-fat diet for 14 months
promotes islet amyloid formation in hIAPP transgenic mice, although less
extensively than in the severe insulin-resistant Obese, leptin-deficient  hIAPP mice. This suggests that the degree of
insulin resistance is important for extensive development of islet amyloid. In
addition, we observed a remarkable gender difference in that severe glucose
intolerance
was observed only in female hIAPP transgenic mice given high-fat diet and not
in males. We suggest that this gender difference is due to the high level of circulating
IAPP rather than to islet amyloid formation. If and how this glucose
intolerance might be mediated by downregulation of beta cell GLUT-2 gene
expression, as observed in the hIAPP mice, is presently unknown.


## 4.4. Ex Vivo Islet Amyloidosis Model

The rationale for the ex vivo study was to
examine if amyloid would be formed in isolated and cultured pancreatic islets
from hIAPP mice, to such a degree that it would be detectable with light
microscopy. Since both the present and previous [18]  in
vivo data indicated  the development of islet amyloid notably in
male hIAPP mice, we decided to investigate amyloid formation in such islets
specifically from male mice. To increase the potential for amyloid formation,
we bred the mice to homozygosity for the hIAPP transgene. At an age of 6 months,
homozygous transgenic hIAPP males had islet amyloid in about 5% of their
pancreatic islets, at 2 days after islet isolation. Previously, we detected
amyloid fibrils by electron microscopy in islets from 4–10 months old heterozygous
hIAPP transgenic mice, cultured for 1 week in medium with 11 or 28 mM glucose [31].
With the present model, using islets from 6-month-old homozygous hIAPP transgenic
males, cultured in medium with high glucose (switching between 11 and 28 mM), we
can detect amyloid deposits with Congo red staining and light microscopy, thus enabling
quantification of the degree of islet amyloidosis. The number of
amyloid-positive islets increased more than 10 fold (from 5.5 to 70%) after 3 weeks of culture
in medium containing a high glucose concentration. Although an accurate
comparison between the degrees of islet amyloid formation in
vivo  and ex vivo was not made, our data certainly indicate stronger islet amyloid formation in hIAPP
islets cultured ex vivo as compared to in vivo. This might be
explained by the higher glucose concentrations in the ex vivo system. hIAPP
transgenic mice in vivo have normal plasma glucose concentrations [15, 18],
whereas ex vivo the glucose concentration in the medium switched between  11 and 28 mM. It is known that a high glucose
concentration triggers both insulin and IAPP secretion, and the hIAPP transgene
is under control of an insulin promoter. In addition, macrophages have been
implicated in in vivo removal of (beginning) amyloid deposits
[43] and such macrophages are absent in the ex vivo system,
potentially allowing increased amyloid formation. When combined with a more
accurate amyloid quantification procedure involving image analysis, this ex vivo system may present a fast and convenient model to study the
process (and factors involved) of islet amyloidosis, as well as the detrimental
consequences for individual beta cells (apoptosis) and islet function (insulin
producing capacity). In addition, such a model system might be used as an
amyloidosis assay to assess the potency of known and novel therapeutic
strategies, aimed at reducing, or even preventing, islet amyloid formation, and its effects on beta cell and
islet  function.



---

*Source: 697035-2008-05-12.xml*