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ORIGINAL PAPER
Presence of residual beta cells and co-existing islet autoimmunityin the NOD mouse during longstanding diabetes: a combinedhistochemical and immunohistochemical study
Shiva Reddy Æ Ryan Chau Chia Chai Æ Jessica Astrid Rodrigues ÆTzu-Hsuan Hsu Æ Elizabeth Robinson
Received: 15 May 2007 / Accepted: 6 July 2007 / Published online: 22 September 2007
� Springer Science+Business Media B.V. 2007
Abstract During type 1 diabetes, most beta cells die by
immune processes. However, the precise fate and charac-
teristics of beta cells and islet autoimmunity after onset are
unclear. Here, the extent of beta cell survival was deter-
mined in the non-obese diabetic (NOD) mouse during
increasing duration of disease and correlated with insulitis.
Pancreata from female NOD mice at diagnosis and at 1, 2,
3 and 4 weeks thereafter were analysed immunohisto-
chemically for insulin, glucagon and somatostatin cells and
glucose transporter-2 (glut2) and correlated with the degree
of insulitis and islet immune cell phenotypes. Insulitis,
although variable, persisted after diabetes and declined
with increasing duration of disease. During this period,
beta cells also declined sharply whereas glucagon and
somatostatin cells increased, with occasional islet cells co-
expressing insulin and glucagon. Glut2 was absent in
insulin-containing cells from 1 week onwards. CD4 and
CD8 T cells and macrophages persisted until 4 weeks, in
islets with residual beta cells or extensive insulitis. We
conclude that after diabetes onset, some beta cells survive
for extended periods, with continuing autoimmunity and
expansion of glucagon and somatostatin cells. The absence
of glut2 in several insulin-positive cells suggests that some
beta cells may be unresponsive to glucose.
Keywords Beta cells � NOD mouse � Diabetes �Insulitis � Glucose transporter-2
Introduction
The onset of human type 1 diabetes represents the culmi-
nation of a silent and prolonged pre-diabetic phase of
immune-mediated beta cell destruction (Eisenbarth et al.
1987). At clinical diagnosis, most beta cells are destroyed
and daily and life-long treatment of subjects with paren-
teral insulin becomes mandatory. Soon after diagnosis of
type 1 diabetes and commencement of insulin therapy,
many patients experience a ‘‘honeymoon’’ phase, which
can last for several months, when there is a marked
reduction in daily insulin dose (Assan et al. 1990; Palmer
et al. 2004). Thus, there may be some restoration of beta
cell function and/or mass during the early stages of clinical
diabetes and upon commencement of insulin therapy.
Earlier studies on pancreatic histology and immunohis-
tology showed that some beta cells remain in many subjects
with recent onset and for variable periods in longstanding
type 1 diabetes (Gepts 1965; Gepts and De Mey 1978; Foulis
and Stewart 1984; Foulis et al. 1986; Lohr and Kloppel
1987). A subsequent study involving 2,431 patients who
were greater than 18 years of age at onset of diabetes, showed
that *15 and 33% of patients had stimulated C-peptide
levels of >0.5 and 0.2–0.5 nmol/l, respectively, within the
first 5 years of diagnosis (Palmer et al. 2004). These data
confirm that some residual beta cell mass and function may
still exist in longstanding type 1 diabetic subjects.
Experimental studies with the ultimate aim of promoting
beta cell regeneration in human type 1 diabetes are being
pursued with renewed vigour. However, the limited avail-
ability of suitable human pancreatic material sampled at
defined time-points soon after onset of type 1 diabetes has
S. Reddy (&) � R. C. C. Chai � J. A. Rodrigues �T.-H. Hsu
School of Biological Sciences, University of Auckland,
Private Bag 92019, Auckland, New Zealand
e-mail: [email protected]
E. Robinson
Department of Epidemiology and Biostatistics, School of
Population Health, Faculty of Medical and Health Sciences,
University of Auckland, Private Bag 92019, Auckland,
New Zealand
123
J Mol Hist (2008) 39:25–36
DOI 10.1007/s10735-007-9122-5
hampered our understanding of the precise fate of residual
beta cells immediately after this phase and during increasing
duration of disease. They have also precluded direct studies
on the molecular, cellular and immune events mediating
further beta cell death and possible beta cell regeneration.
In the non-obese diabetic (NOD) mouse model of human
type 1 diabetes, there is also a paucity of knowledge on the
precise fate of beta cell populations and islet immunopa-
thology at defined time-points after disease onset.
Information gained from such studies in the NOD mouse
would provide experimental platforms to assess the efficacy
of therapies aimed at beta cell regeneration and turnover and
thwarting recurrent autoimmunity. The present study has,
therefore, investigated the islet endocrine-inflammatory cell
pathology in diabetic NOD mice at specific time-points after
onset of disease. Immunohistochemical and histochemical
techniques were employed to examine changes in the num-
ber and distribution of the remaining beta cells and glucagon
and somatostatin cells. These results were correlated with the
degree of insulitis, the phenotypes of islet inflammatory cells
and expression of glucose transporter-2 (glut2).
Materials and methods
Animals
An inbred colony of NOD mice was established at the Ani-
mal Resources Unit of the School of Biological Sciences,
The University of Auckland. This colony originated from a
nucleus of three breeding pairs obtained from Dr. A. Mer-
riman at the University of Otago, Dunedin, New Zealand.
The University of Otago colony was established from
breeding pairs obtained from the Jackson Laboratories, Bar
Harbor, ME, USA. The Auckland colony is maintained on a
standard autoclaved diet (Harlan Teklad Global 18% Protein
Rodent Diet, code 2018S, UK) and sterile water ad libitum.
The current rate of spontaneous diabetes is *80% among
females usually between the ages of 80 and 250 days. Dia-
betes in NOD mice is defined as the presence of a non-fasting
hyperglycaemic value >12 mmol/l on three consecutive
days. In non-diabetic NOD mice, the non-fasting blood
glucose levels ranged from 4 to 6 mmol/l. All animal studies
were conducted in compliance with the guidelines of the
University of Auckland Animal Ethics Committee.
Study groups
Female NOD mice obtained from several breeding pairs
were monitored for the development of diabetes from day
70 onward. Following diagnosis of diabetes, mice were
sacrificed either immediately or following 1, 2, 3 and
4 weeks of diabetes (without insulin treatment; 5 NOD
mice per time-point). In addition, four newly diabetic NOD
mice were maintained on 0.5–1 U of Protophane insulin
(Novo Nordisk, Bagsvaerd, Denmark) administered once
daily by intra-peritoneal injection for 28 days to achieve a
daily blood glucose value <10 mmol/l. The ages of mice
enrolled in the various study groups are shown below:
Mouse
number
Newly
diabetic
(Days)
1 week
of
diabetes
(Days)
2 weeks
of
diabetes
(Days)
3 weeks
of
diabetes
(Days)
4 weeks
of
diabetes
(Days)
Newly
diabetic
treated
with
insulin
(Days)
1 124 127 182 173 190 149
2 117 111 139 183 164 154
3 95 144 148 93 156 154
4 117 117 121 136 211 246
5 95 104 136 150 142 –
Non-diabetic NOD mice between the ages of 100 and
200 days (n = 5) and NOD mice at various stages after
onset of diabetes were killed and the entire pancreas, with a
small piece of the adjoining spleen, was removed (see
below). Each pancreas was then divided immediately into
halves, one of which was snap-frozen (splenic half) in is-
opentane cooled in liquid nitrogen while the other fixed in
Bouin’s solution (duodenal portion). Bouin’s fixed pan-
creata were embedded in paraffin and sectioned (5 lm).
Frozen pancreata were cryosectioned (7 lm), air-dried
briefly, fixed in cold acetone and stored at �20�C until
required for immunohistochemistry.
Primary antibodies
Guinea pig anti-ovine insulin serum was available in this
laboratory and is specific for beta cells (Reddy et al. 1988,
2005). Rabbit anti-glucagon serum was purchased from
Dako, Glostrup, Denmark and sheep anti-somatostatin
serum was from Guildhay Limited, Guidford, Surrey, UK.
Rat monoclonal antibodies to mouse macrophages (clone
CD11b) and to CD8 T cells (clone KT15) were purchased
from Serotec, Oxford, UK. Rat monoclonal antibodies to
mouse CD4 T cells (clone GK1.5) and CD3 T cells (clone
KT3) were gifts from Dr. H Georgiou, Walter and Eliza Hall
Institute of Medical Research, Melbourne, VIC, Australia.
Highly specific rabbit anti-mouse glut2 was kindly provided
by Dr. B Thorens, University of Lausanne, Lausanne,
Switzerland, as affinity purified IgG. This antibody has been
used in immunocytochemical staining for mouse pancreatic
glut2 and specifically stains mouse beta cells (Thorens 1992;
Reddy et al. 1998). The immunohistochemical specificities
26 J Mol Hist (2008) 39:25–36
123
of the antibodies employed for detection of macrophages
and T cells in mouse pancreatic sections have been reported
previously (Reddy et al. 1995, 2003). In the immunohisto-
chemical procedure, all primary antibodies were titrated to
give maximum immunohistochemical reactivity. Normal
sera from a variety of mammalian species were available in
this laboratory for use as controls and blocking reagents for
immunohistochemistry.
Islet histopathology and insulitis
Pancreatic sections from different stages of diabetes and
from adult non-diabetic female NOD mice were stained by
H&E, studied for their histological characteristics and
graded for insulitis on a scale of 0–4 (Charlton et al. 1989;
Reddy et al. 1999). In this method, islets devoid of any
mononuclear cells = 0; minimum focal islet infiltrate = 1+;
peri-islet infiltrate of <25% of islet circumference = 2+;
peri-islet infiltration and <50% intra-islet area = 3+; intra-
islet infiltration >50% of islet area = 4+. All slides were
coded and at least ten separate islets from different levels
of each pancreas were scored. The insulitis score (%) for
each study group was calculated as follows:
Sum of (1 · number of islets with 1+, 2 · number of
islets with 2+, 3 · number of islets with 3+, 4 · number of
islets with 4+) divided by 4 · total number of islets scored.
The ratio obtained was expressed as a percentage. The
insulitis score (%) for each study group was expressed as
the mean ± SEM.
Immunolabelling of islet endocrine cells
Each of the three serial paraffin sections per slide was
immunolabelled for either insulin, glucagon or somato-
statin, with minor modifications (Reddy et al. 1988, 2005).
Briefly, sections were de-waxed with xylene, rehydrated in
increasing concentrations of ethanol, washed in water and
equilibrated in phosphate-buffered saline (PBS), pH 7.4. In
the immunohistochemical protocol, PBS acted as the wash
buffer and as a diluent, unless stated otherwise.
Sections were blocked with 5% normal donkey serum and
incubated with either guinea pig anti-ovine insulin (1:1,000
in 5% normal donkey serum), rabbit anti-glucagon (1:100) or
sheep anti-somatostatin (1:800 in 5% normal donkey serum)
for 1 h at 37�C. Following washing, sections were incubated
with either donkey anti-guinea pig IgG-FITC (1:50, Jackson
ImmunoResearch Laboratories, West Grove, PA, USA,
insulin section), donkey anti-rabbit IgG-Texas Red (1:100,
Jackson ImmunoResearch Laboratories, glucagon section)
or donkey anti-sheep IgG-FITC (1:100, Jackson Immuno-
Research Laboratories, somatostatin section) for 1 h at 37�C.
Sections previously immunolabelled for insulin and
somatostatin were dual-labelled for glucagon (Texas Red
fluorochrome). Selected sections previously immunola-
belled for glucagon (Texas Red fluorochrome) were dual-
labelled for somatostatin (FITC fluorochrome). All sections
were mounted and prepared for microscopy.
For triple-immunolabelling, sections were immuno-
stained for somatostatin as above (by sequential incubation
with sheep anti-somatostatin serum and donkey anti-sheep
IgG-FITC as secondary antibodies). Sections were then
blocked with 10% normal sheep serum to saturate any
remaining sites on donkey anti-sheep IgG conjugated to
FITC. Sections were washed and incubated with guinea pig
anti-insulin (1:1,000) followed by addition of goat anti-
guinea pig IgG-Alexa 568 (1:500, Invitrogen, Eugene, OR,
USA). After washing, they were incubated with rabbit anti-
glucagon (1:100) followed by addition of goat anti-rabbit
IgG-Alexa 350 (1:100, Invitrogen). Sections were washed
and prepared for microscopy.
Enumeration of insulin, glucagon and somatostatin
cells
Insulin, glucagon and somatostatin cells were enumerated
in each islet section either directly with the microscope
using the 20· objective or after photomicrography of im-
munolabelled islets. The corresponding cross-sectional area
of the islet in each pancreatic section was determined with
the Axiocam software provided with the Zeiss fluorescence
microscope. The mean number of immunoreactive insulin,
glucagon or somatostatin cells per millimeter square islet
area was calculated.
Immunolabelling of mouse glut2
Selected pancreatic sections were immunostained for
mouse glut2 and insulin as reported previously, with minor
modifications (Reddy et al. 1998). Briefly, paraffin sections
were blocked with 5% normal donkey serum and incubated
with a mixture of anti-glut2 (1:1,000 in 0.1% bovine serum
albumin) and guinea pig anti-insulin (1:1,000) at 4�C for
16 h, followed by reaction with donkey anti-rabbit IgG-
biotin and then with streptavidin Alexa 568 (1:500, Invi-
trogen). Following washing, sections were reacted with
donkey anti-guinea pig IgG-FITC (Jackson ImmunoRe-
search) and prepared for microscopy.
Immunolabelling of CD4 and CD8 T cells and
macrophages
Immunolabelling of CD4 and CD8 T cells and macrophages
was carried out as reported previously (Reddy et al. 1999,
J Mol Hist (2008) 39:25–36 27
123
2003). In this procedure, serial frozen sections of pancreas
were re-fixed in cold acetone for 10 min, equilibrated with
PBS and incubated with 5% normal donkey serum for 1 h at
37�C. Sections were washed and incubated with rat
monoclonal antibodies to CD4 T cells (undiluted), CD8 T
cells (1:50) and macrophages (1:10) and incubated for 16 h
at 4�C followed by washing. They were then incubated with
donkey anti-rat IgG-Alexa 488 (1:250, Invitrogen) for 1 h at
37�C, washed and prepared for microscopy.
Dual-immunolabelling of insulin and immune cells
Serial frozen pancreatic sections were immunolabelled for
CD3 T cells and macrophages as described above. Sections
Fig. 1 Photomicrographs of
pancreatic sections from non-
diabetic NOD mice and NOD
mice at various stages after
diabetes onset, stained by H&E,
showing the varying extent of
insulitis. Islets from 2 to 3 mice
are shown for each group. The
ages of mice and the
corresponding blood glucose
values prior to removal of
pancreata are indicated in each
photomicrograph. Scale bar in
(r) is 50 lm and applies to all
remaining photomicrographs
0
10
20
30
40
50
60
70
80
90
100
Non-diabetic Newly-diabetic 1 week 2 weeks 3 weeks 4 weeks
Stages of diabetes
Mea
n in
sulit
is s
core
(%
)
Fig. 2 The mean percent insulitis scores (%) ± SEM at various
stages of diabetes in NOD mice (five animals per time-point)
28 J Mol Hist (2008) 39:25–36
123
were then reacted with guinea pig anti-insulin (1:1,000) for
16 h at 4�C followed by incubation with goat anti-guinea
pig IgG-Alexa 568 (1:500, Invitrogen) for 1 h at 37�C,
washed and prepared for microscopy.
Statistical analyses
Linear regression was used to investigate changes in in-
sulitis scores at various time-points after diabetes onset.
Poisson regression was used to investigate changes in the
number of beta, glucagon and somatostatin cells per unit
cross-sectional islet area.
Results
Insulitis and islet morphology
The degree of insulitis in NOD mice at various stages of
diabetes is shown in Fig. 1. Islets displayed considerable
variation in the extent of insulitis within each pancreas,
both before the onset of diabetes and subsequently.
However, at 4 weeks of diabetes, despite the persistence
of insulitis in some islets, a number were atrophied and
devoid of insulitis (Fig. 1p–r). The mean insulitis
scores ± SEM (%) at various stages of diabetes are
shown in Fig. 2. The insulitis scores showed a decrease
Fig. 3 Dual-label
immunohistochemistry of
pancreatic islets from non-
diabetic NOD mice, newly
diabetic and 1 week after
diabetes. Left panel: insulin
(green) + glucagon (red).
Middle panel: glucagon (red)
+ somatostatin (green). Right
panel: corresponding islets from
the left panel counterstained by
H&E. The stages of diabetes,
age of mice and blood glucose
levels immediately before
removal of pancreas are also
shown on the right. Note that in
(n) and (q) glucagon cells often
surround somatostatin cells.
Scale bar in (r) is 50 lm and
also refers to a–q
J Mol Hist (2008) 39:25–36 29
123
from the onset of diabetes until 4 weeks after onset
(p = 0.0031).
Islet endocrine cell populations
Representative photomicrographs of islets dual-labelled for
insulin and glucagon or glucagon and somatostatin are
shown in Figs. 3–5. The corresponding islets from adjacent
sections stained by H&E are also included. In non-diabetic
NOD mice, a central islet core of numerous beta cells
surrounded by glucagon and somatostatin cells with usually
peri-islet insulitis was observed, although islets with
advanced insulitis showed more extensive beta cell loss
(Fig. 3a–f). A similar pattern of immunostaining for the
three endocrine cell types was present in newly diabetic
NOD mice. However, several islets showed intra-islet in-
sulitis, a marked loss of beta cells and more centrally
located glucagon and somatostatin cells (Fig. 3g–l). After
1 week of diabetes, the pattern of immunostaining for the
three endocrine cell-types with concurrent insulitis was
similar to newly diabetic mice, although some islets
showed varying degrees of beta cell loss (Fig. 3m–r). Some
islets were beta cell negative with glucagon cells located in
a ring-like pattern often surrounding a cluster of somato-
statin cells (Fig. 3q). A similar immunohistochemical and
histochemical pattern was observed at 2, 3 and 4 weeks of
diabetes (Fig. 4).
Fig. 4 Dual-label
immunohistochemistry of
pancreatic islets at 2, 3 and
4 weeks of diabetes. Left panel:
insulin (green) + glucagon
(red). Middle panel: glucagon
(red) + somatostatin (green).
Right panel: corresponding
islets from the left panel
counterstained by H&E. The
stages of diabetes, ages of mice
and blood glucose levels
immediately before removal of
pancreas are also shown on the
right. Scale bar in (r) is 50 lm
and also applies to a–q
30 J Mol Hist (2008) 39:25–36
123
The relative distribution of insulin, glucagon and
somatostatin cells, determined by triple-immunolabelling, is
shown in Fig. 5a–f. Dual-labelling for insulin and glucagon
showed that occasional endocrine cells within some diabetic
islets were positive for both insulin and glucagon (Fig. 5g, h).
Islets from insulin-treated NOD mice showed consid-
erable variation in the number and distribution of glucagon
cells and residual beta cells at the end of 28 days of
treatment, with several islets showing advanced insulitis
(results not shown).
The mean number of beta, glucagon and somatostatin
cells ± SEM per mm2 of cross-sectional islet area is shown
in Fig. 6. There was a decline in the mean number of beta
cells in diabetic NOD mice compared with non-diabetic
mice. There was no change in the number of beta cells from
onset until 4 weeks of diabetes (p = 0.21). However, there
was an increase in the number of glucagon cells during the
duration of the disease (p = 0.019). A similar increase was
also evident for somatostatin cells (p = 0.0009).
Glucose transporter-2 expression
Representative islets dual-immunolabelled for mouse glut2
and insulin from selected stages of diabetes are shown in
Fig. 7. Strong immunolabelling for glut2 was present in a
large proportion of beta cells in non-diabetic NOD mice
and to a lesser extent at onset of diabetes. Immunolabelling
for the transporter was absent in some insulin-positive cells
that were usually located adjacent to the region of insulitis
(Fig. 7d–f). From 1 week of diabetes and onward,
Fig. 5 Dual- and triple-label
immunohistochemistry of islets
after varying duration of
diabetes. a–c Insulin (red),
glucagon (blue) and
somatostatin (green); duration
of diabetes is indicated in each
micrograph. Islets
counterstained by H&E in d, e, fare the same as in a, b, c,
respectively. g Shows an islet
dual-labelled for insulin (green)
and glucagon (red). The arrowin g points to an islet cell co-
expressing insulin and
glucagon; also refer to inset in
g. h Shows an islet from an
NOD mouse with 3 weeks of
diabetes immunolabelled for
glucagon (red) and insulin
(green); arrows in h point to 2
cells which co-express glucagon
and insulin. Scale bar in f is
50 lm and also applies to a–e;
scale bar in g is 20 lm and in
inset = 10 lm; scale bar in his10 lm
0
1
2
3
4
5
Non-diabetic Newly-diabetic 1 week 2 weeks 3 weeks 4 weeksStages of diabetes
Mea
n n
um
ber
of
end
ocr
ine
cells
per
mm
2 isle
t ar
ea
Fig. 6 The mean number ± SEM of insulin cells (open bars),
glucagon cells (hatched bars) and somatostatin cells (solid bars)
per mm2 islet area at various stages of diabetes
J Mol Hist (2008) 39:25–36 31
123
immunolabelling for glut2 was largely absent, despite the
presence of cells which expressed insulin (Fig. 7g–o).
Intra-islet CD4 and CD8 T cells and macrophages
By immunohistochemistry, qualitative differences in the
number of CD4 and CD8 T cells and macrophages within
the islets of NOD mice were observed over the duration of
diabetes. In most islets with insulitis, CD4 and CD8 T
cells were more numerous than macrophages and were
distributed in the peri-islet and intra-islet areas (Fig. 8). At
4 weeks of diabetes, extensive insulitis consisting of
mostly CD4 and CD8 T cells and lesser numbers of
macrophages persisted in some islets (Fig. 8m–p). In non-
diabetic NOD mice and in mice with increasing duration
of diabetes, dual-labelling showed that some islets which
were positive for insulin had a mantle of extensive insu-
litis consisting of T lymphocytes and macrophages
(Fig. 9a–f, i, j). However, islets which were negative for
insulin showed minimal peri-islet T cells and macro-
phages (Fig. 9g, h).
Fig. 7 Dual-immunolabelling
of insulin (green; left panel) and
glut2 (red; middle panel at
various stages of diabetes. The
right panel shows the
corresponding islets
counterstained by H&E. In a–karrows point to cells which co-
express insulin and glut2, but
extremely weakly in k, while
arrowheads point to insulin
cells which do not express glut2.
Note that at 3 weeks after
diabetes onset (m–o), the
remaining insulin cells are
negative for glut2. Scale bar in
q is 50 lm and also applies to
a–n
32 J Mol Hist (2008) 39:25–36
123
Discussion
The sequential changes in the cellular and molecular
pathology of the islet endocrine-immune cell axis, includ-
ing the expression of locally produced proinflammatory
molecules, during human type 1 diabetes remain incom-
plete. The relative unavailability and practical and ethical
difficulties in obtaining rapidly cryopreserved pancreatic
samples from diabetic subjects have limited such studies.
To address some of these issues, the present studies were
undertaken in the NOD mouse and pancreata analysed at
defined time-points at and after diabetes onset. Surpris-
ingly, our studies showed that insulitis persisted in most
islets of NOD mice even after onset of disease and until
4 weeks. Daily treatment of diabetic NOD mice with
insulin did not result in the retraction of insulitis or influ-
ence beta cell number during protracted disease. However,
therapeutically administered insulin may not only control
hyperglycaemia but also act as an autoantigen and invoke
an autoimmune response. Pancreata from diabetic NOD
mice without insulin treatment demonstrated islet atrophy
accompanied by minimum peri-insulitis, particularly with
increasing disease duration. The islet infiltrate consisted of
mostly CD4 and CD8 T cells with lower numbers of
macrophages, a pattern also seen in adult prediabetic NOD
mice (Reddy et al. 1995). Retention of this characteristic
immunophenotype during an increasing duration after
diabetes onset suggests that the remaining beta cells may
continue to act as immune targets after diabetes onset,
although it is possible that some of the surviving beta cells
may be refractory to immune recognition and injury. In this
study, although detailed enumeration of immune cells and
the possible presence of islet-located dendritic cells were
not conducted, there were qualitative variations in the
number of intra-islet CD4 and CD8 T cells and macro-
phages. In earlier studies by others, pancreatic samples
from children with new onset type 1 diabetes also showed
insulitis that was often patchy, in a majority of cases and in
a majority of islets (Foulis and Stewart 1984; Foulis et al.
1986, 1991). In children with recent-onset disease, the islet
inflammatory cells consisted of mainly lymphocytes and a
smaller number of macrophages (Foulis et al. 1991). By
immunohistochemistry, pancreatic sections from a recent-
onset diabetic child showed predominance of CD8 T cells
and a lesser number of B cells, macrophages and IgG
deposits, a pathology that is different from islets of newly
Fig. 8 Immunohistochemical
staining of CD4 (first row) and
CD8 T cells (second row) and
macrophages (third row) at
various stages of diabetes in
three serial sections. Islets in d,h, l, p are adjacent sections
stained by H&E and show the
same islet as in a–c, e–g, i–kand m–o, respectively. Scalebar in p is 50 lm and applies to
all photomicrographs. The
stages of diabetes, ages of mice
and blood glucose levels before
removal of pancreas are
indicated above the
photomicrographs
J Mol Hist (2008) 39:25–36 33
123
diabetic NOD mice where CD4 T cells predominate
(Bottazzo et al. 1985; Reddy et al. 1995). More recently,
pancreatic biopsies obtained from recent-onset diabetic
subjects showed that the remaining beta cells were usually
associated with the presence of T cells within the islets and
that macrophages and dendritic cells sometimes persisted
even in islets that were devoid of beta cells (Uno et al.
2007). However, studies carried out in biopsies offer his-
tolopathological patterns seen in only a restricted number
of islets.
At diabetes onset in the NOD mouse, there was a
marked decline in the number of beta cells which persisted
until 4 weeks after diabetes but this decline did not reach
statistical significance. It is unclear if beta cells from NOD
mice have the capacity to regenerate spontaneously after
diabetes onset or even in the prediabetic period. Any net
increase in beta cell turnover may be thwarted by ongoing
autoimmune destruction and glucose toxicity during clini-
cal disease and thus, could escape detection. In humans
with long-standing type 1 diabetes, recent evidence sug-
gests that there may be limited beta cell regeneration and it
has been proposed that concurrent activation of the apop-
totic pathway may result in net beta cell loss (Meier et al.
2005).
Our demonstration of an increase in the number of
glucagon and somatostatin cells after diabetes onset is
noteworthy. In a previous study, an increase in glucagon
cells was shown in the NOD mouse at onset of diabetes
Fig. 9 Dual-immunolabelling
of insulin (red) + CD3 T cells
(green), left panel and insulin
(red) + macrophages (green),
right panel, at various stages of
diabetes. The ages of mice and
the blood glucose levels
immediately before removal of
pancreas are also shown. Note
that most islets show insulin
staining surrounded by T cells
and macrophages. In g and h a
few peri-islet immune cells
surround an islet that is negative
for insulin. Scale bar in j is
50 lm and applies to c–i; scale
bar in b is 100 lm and applies
to a
34 J Mol Hist (2008) 39:25–36
123
(O’Reilley et al. 1997). However, in our study, this
increase as well as an increase in somatostatin cells were
more pronounced at 3 and 4 weeks of diabetes and
restricted to diabetic islets but not to pancreatic ductal cells
as reported previously (O’Reilley et al. 1997). The mech-
anisms which underlie the increase in glucagon and
somatostatin cell number are unclear. In the low dose
streptozotocin mouse model, there is an expansion of
glucagon cells at onset of diabetes also (Li et al. 2000). In
humans, there are conflicting reports regarding changes in
glucagon cell number after onset of type 1 diabetes (Gepts
and De Mey 1978; Stefan et al. 1982; Rahier et al. 1983;
Somoza et al. 1994).
The significance of some islet cells double-positive for
insulin and glucagon after onset of diabetes is unclear. We
have previously shown a similar co-expression in islets
from NOD mice rendered diabetic with cyclophosphamide
(Reddy et al. 2005). It appears that an extreme diabetic
environment may recapitulate some of the embryonic
developmental processes governing islet hormones (Bon-
ner-Weir 2000). During pancreatic development in the fetal
rat, transient co-expression of insulin and glucagon has
been observed in a proportion of pancreatic cells from day
12.5 of gestation (Hashimoto et al. 1988).
The absence of glut2 expression in insulin-positive cells
in the later stages of clinical diabetes implies that most
surviving beta cells may fail to release insulin in response
to glucose and thus, may not be fully functional. Several
studies have suggested that hyperglycaemia can impair
beta cell function due to glucotoxicity and it is likely that
this dysfunction may be present in NOD mice with long-
standing hyperglycaemia. Indeed, adult NOD mice exhibit
defects in insulin release in response to glucose but not to
arginine even before diabetes onset (Kano et al. 1986).
In conclusion, the present studies have clearly estab-
lished that some beta cells are present in the NOD mouse
for increasing periods following onset of the disease.
Whether they represent immune-resistant or newly formed
cells is not clear. A net increase in beta cell mass may be
limited due to ongoing autoimmunity which can invoke
beta cell death by apoptosis. Co-administration of specific
peptides to diabetic NOD mice has been shown to reverse
diabetes (Suarez-Pinzon et al. 2005). Short-term experi-
mental therapies aimed at protecting the limited number of
beta cells present immediately after diagnosis, promoting
their regeneration and blocking beta cell autoimmunity
may lead to the development of promising interventions
aimed at reversing established disease.
Acknowledgements We thank Beryl Davy and Lorraine Rolston for
expert histological support and Jacqueline Ross for valuable advice
on image preparation and Vernon Tintinger for assistance with the
careful maintenance of the NOD mouse colony. Financial assistance
from the Child Health Research Foundation, the Maurice & Phyllis
Paykel Trust, University of Auckland School of Biological Sciences
Summer Studentship Programme and the Auckland Medical Research
Foundation is gratefully acknowledged.
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