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26
Figure 1. 111In-exendin-4 accumulation study in isolated pancreatic islets.
Isolated islets (50, 100, 200, 300 and 400) from C57BL/6J mice were incubated with
111
exendin-4 for 30 min. A significant positive linear correlation was observed between
111
In-
In-
exendin-4 uptake values and the number of isolated islets (Pearson r = 0.97, p < 0.01).
Figure 2. Metabolic measurements after intraportal islet transplantation and ex-vivo liver
graft uptake of the 111In-exendin-4 probe.
(A) Nonfasting blood glucose and (B) body weight during the observation period. (C) Ex-vivo
liver graft uptake (%ID/g) using 111In-exendin-4 probe six weeks following islet transplantation.
All data are expressed as the mean ± SD. Control are indicated as open circles with a dotted line
and white bars with solid borders (n = 8), 150 IT are indicated as closed squares with a solid line
and checked bars with solid borders (n = 9), and 400 IT with successful engraftment are
indicated as closed triangles with a solid line, and gray bars with solid borders (n = 13) and 400*
IT with unsuccessful engraftment are indicated as open triangles with a dotted line and dotted
bars with solid borders (n = 3). 400 IT vs control: ††††p < 0.0001, 400 IT vs 150 IT: ***p < 0.001,
400 IT vs 400* IT: §§§p < 0.001, n.s., not significant. IT, islet transplantation.
Figure 3. Liver graft insulin content and ex-vivo liver graft and pancreas uptake of
111
In-
exendin-4 six weeks after islet transplantation.
(A) Liver graft insulin content, (B) ex-vivo liver uptake, and (C) ex-vivo pancreas uptake (%ID/g)
using the 111In-exendin-4 probe. All data are expressed as the mean ± SD. Control are indicated
as open circles with a dotted line and white bars with solid borders (n = 4), 150 IT are indicated
as closed squares with a solid line and checked bars with solid borders (n = 4), and 400 IT with
27
successful engraftment are indicated as closed triangles with a solid line and gray bars with solid
borders (n = 6) and 400* IT with unsuccessful engraftment are indicated as open triangles and
dotted bars with solid borders (n = 2). 400 IT vs. control: †††p < 0.001, ††††p < 0.0001; 400 IT vs.
150 IT: **p < 0.01, ***p < 0.001; 150 IT vs. control: ####p < 0.0001; n.s., not significant. IT, islet
transplantation.
Figure 4. Representative images of in-vivo
111
In-exendin-4 SPECT/CT and in-vivo liver
graft uptake after islet transplantation.
(A) In-vivo
111
In-exendin-4 SPECT/CT was performed six weeks after islet transplantation in
control, 150 IT, and 400 IT groups. Maximum to minimum intensity: red greater than orange,
yellow greater than green greater than blue greater than black. Signals from the graft in the liver,
yellow dashed circles; pancreas, white dashed circles; signals from the kidney, blue arrows. Lleft; R-right; V-ventral. (B) In-vivo liver graft
111
In-exendin-4 uptake (%ID/g) detected by
SPECT/CT. Data are expressed as the mean ± SD. 400 IT vs. control: ††††p < 0.0001; 400 IT vs.
150 IT: **p < 0.01; 150 IT vs. control: #p < 0.05. IT, islet transplantation.
Figure 5. Histological liver graft β-cell mass after islet transplantation and its correlation
with in-vivo 111In-exendin-4 SPECT/CT liver graft uptake.
(A)
Representative
images
of
hematoxylin
and
eosin
staining
(left
panel)
and
immunohistochemical anti-insulin staining (right panel) on the serial liver sections. Scale bars:
1000 μm in the overview images and 50 μm in the high magnification images. (B) Liver graft β
-cell mass (BCM) as calculated by histological analysis. Data are expressed as the mean ± SD.
Control are indicated as open circles with a dotted line and white bars with solid borders (n = 4),
28
150 IT are indicated as closed squares and checked bars with solid borders (n = 3), and 400 IT
with successful engraftment are indicated as closed triangles and gray bars with solid borders (n
= 3). 400 IT vs. control: †††p < 0.001; 400 IT vs. 150 IT: ***p < 0.001; 150 IT vs. control: n.s.,
not significant. (C) Significant correlation between liver graft uptake values on
111
In-exendin-4
SPECT/CT (percentage of injected dose/1 g, %ID/g) and histological quantification of BCM
(Pearson r = 0.95, p < 0.01). IT, islet transplantation.
Figure 6. Representative autoradiography and corresponding fluorescent images on the
liver islet grafts.
(A)Autoradiography (left panel) and fluorescence microscopy image with anti-insulin staining
(right panel) on the same liver graft section, which was harvested from the mice transplanted
with islets after incubation with
111
In exendin-4. (B) Autoradiography (left panel) and
fluorescence microscopy image with anti-insulin staining (right panel) on the same liver graft
section, harvested from the mice in which 111In exendin-4 was injected via the tail vein following
intraportal IT. Radioactive signals colocalized with the fluorescence signals of islet grafts
(dashed-line boxes). Box: high magnification image of the insulin-positive area. Scale bar: 200
µm in the overview images and 50 μm in the high magnification images.
29
Figure 1
40000
Islet uptake
(CPM)
30000
20000
r=0.97
p<0.01
10000
50
100
200
300
Islet number
400
(A)
(B)Figure
Nonfasting blood glucose
Body weight
(C) Ex-vivo liver graft uptake
ns
(mg/dL)
(g)
1000
(%ID/g)
35
††††
ns
15
800
Control
✱✱✱ §§§
30
600
10
ns
150 IT
400 IT
400* IT
25
200
20
10
15
20
25
30
35
40
45
10
15
20
25
30
35
40
45
on
tr
15 ol
40 IT
40 IT
0*
IT
400
Days after IT
Days after IT
Liver graft insulin content
(ug/g)
†††
✱✱✱
1.0
(B)
Figure 3
Ex-vivo liver graft uptake
(%ID/g)
15
(C)
Ex-vivo pancreas uptake
(%ID/g)
†††
15
††††
####
Control
✱✱
0.8
150 IT
10
0.6
ns
10
0.4
0.0
ns
on
tr
15 l
IT
40
40 IT
0*
IT
on
tr
15 l
IT
40
40 T
0*
IT
ns
on
tr
15 l
IT
40
40 T
0*
IT
0.2
400 IT
400* IT
(A)
!"#$%"&
'()*+,-./(.*+,
722.34&5$4
Figure
'()*6,-./(.*6,
012.34&5$4
'()*6,-./(.*6,
(B) In-vivo liver graft uptake
on SPECT/CT
††††
!"#$%
(%ID/g)
✱✱
10
!"#$%
!"#$%
,"-)$.
Control
!"#$%
150 IT
400 IT
!"#$%&'()*#
IT
40
15
ol
'()*%$(+
on
tr
'()*%$(+
IT
'()*%$(+
&'()%$'*
liver graft BCM
†††
(mg/liver)
✱✱✱
0.8
Control
150 IT
0.4
400 IT
40
IT
ol
on
tr
-))*+,
0.0
IT
ns
0.2
15
'()*+,
0.6
In-vivo liver graft uptake &
histological liver graft BCM
(C)
(%ID/g)
In-vivo
liver graft uptake
!"#$%"&
Histological
(B) Figure
10
r=0.95
p<0.01
Control
400 IT
150 IT
0.0
0.2
0.4
0.6
0.8
Histological liver graft BCM (mg/liver)
Figure 6
Supplemental information for
Noninvasive quantitative evaluation of viable islet grafts using
111 In-exendin-4
SPECT/CT.
Ainur Botagarova,1,† Takaaki Murakami,1,† Hiroyuki Fujimoto,2 Muhammad Fauzi,1 Sakura Kiyobayashi,1 Daisuke Otani,1 Nanae Fujimoto,3
Nobuya Inagaki1,4*
1 Department
of Diabetes, Endocrinology, and Nutrition, Graduate School of Medicine, Kyoto University, Japan,
2 Radioisotope
3 Department
Research Center, Agency for Health, Safety and Environment, Kyoto University, Japan
of Regeneration Science and Engineering
Laboratory of Experimental Immunology, Institute for Life and Medical Sciences, Kyoto University, Japan
4 Medical
† A.B.
Research Institute KITANO HOSPITAL, PIIF Tazuke-kofukai, Osaka, Japan
and T.M. † have contributed equally to this work.
*Correspondence:
Nobuya Inagaki, MD, PhD
Department of Diabetes, Endocrinology, and Nutrition, Graduate School of Medicine, Kyoto University,
54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Medical Research Institute KITANO HOSPITAL
2-4-20 Ogimachi, Kita-ku, Osaka 530-8480, Japan
E-mail: inagaki@kuhp.kyoto-u.ac.jp
(A)
(B) AUC - glucose
Glucose Level
(mg/dL)
1000
800
(x103 mg/dL x min)
150
ns
###
600
100
400
200
50
(ng/mL)
15
30
60
Time(minutes)
120
Insulin Level
AUC - Insulin
0.6
(ng/mL x min)
60
0.4
####
40
0.2
0.0
✱✱
15
30
Time(minutes)
Control
150 IT
✱✱
400 IT
20
Control
150 IT
400 IT
Figure S1. Intraperitoneal glucose tolerance test (IPGTT). Blood glucose and insulin levels (A), AUC for glucose and insulin (B) during IPGTT
6 weeks after islet transplantation. Control are indicated as open circles with a dotted line and white bars with solid borders (n=4), 150 IT are
indicated as closed squares with a solid line and checked bars with solid borders (n=4), and 400 IT are indicated as closed triangles with a solid
line and grey bars with solid borders (n=4). Data are expressed as Mean ± SD. 400 IT vs 150 IT: *p<0.05, **p<0.01; 400 IT vs control; †p<0.05;
150 IT vs control: #p<0.05, ###p<0.001, ####p<0.0001 n.s., not significant.
...
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