Page 1 of 49 Diabetes
Beta cell-derived angiopoietin-1 regulates insulin secretion and glucose
homeostasis by stabilizing islet microenvironment
- Running title: Ang-1 contributes to glucose homeostasis -
Ho Seon Park,1,2,3 Hak Zoo Kim,2,3 Jong Suk Park,1,2,3 Junyeop Lee,4 Seung-Pyo Lee,5 Hail Kim,6
Chul Woo Ahn,1,2,3 Yoshikazu Nakaoka,7 Gou Young Koh,6,8 Shinae Kang1,2,3
1Department of Internal Medicine, 2Gangnam Severance Hospital, 3Severance Institute for
Vascular and Metabolic Research, Yonsei University College of Medicine, Yonsei University,
Seoul, South Korea; 4Department of Ophthalmology, Yeungnam University College of Medicine,
Daegu, South Korea; 5Department of Internal Medicine, Seoul National University Hospital, Seoul,
South Korea; 6Graduate School of Medical Science and Engineering, Korea Advanced Institute of
Science and Technology, Daejon, South Korea; 7Department of Vascular Physiology, National
Cerebral and Cardiovascular Center Research Institute, Osaka, Japan 8Center for Vascular
Research, Institute for Basic Science, Daejon, South Korea.
Corresponding author:
Gangnam Severance Hospital, 211 Eonju-ro, Gangnam-gu, Seoul, 135-710 Korea
Phone: +82-2-2019-3335
Fax: +82-2-3463-3882
E-mail: [email protected]
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Word count: 3999
Number of tables: 0
Number of figures: 7
Number of supplementary tables: 1
Number of supplementary figures: 9
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Abstract
Islets are highly vascularized for prompt insulin secretion. Although angiopoietin-1 (Ang1) is a
well-known angiogenic factor, its role in glucose homeostasis remains largely unknown. The
objective of this study was to investigate whether and how Ang1 contributes to glucose
homeostasis in response to metabolic challenge. We used inducible systemic Ang1-knockout
(Ang1sys-/-) and β-cell specific Ang1-knockout (Ang1β-cell-/-) mice fed a high-fat diet for 24 weeks.
Although the degree of insulin sensitivity did not differ between Ang1sys-/- versus Ang1sys+/+ mice,
serum insulin levels were lower in Ang1sys-/- mice, resulting in significant glucose intolerance.
Similar results were observed in Ang1β-cell-/- mice, suggesting a critical role of β-cell-derived Ang1
in glucose homeostasis. There were no differences in β-cell area nor vasculature density, but
glucose-stimulated insulin secretion was significantly decreased and PDX-1 expression and
GLUT2 localization were altered in Ang1β-cell-/- compared to Ang1β-cell+/+ mice. These effects were
associated with less pericyte-coverage, disorganized endothelial cell ultrastructure, and also,
enhanced infiltration of inflammatory cells and upregulation of adhesion molecules in the islets of
Ang1β-cell-/- mice. In conclusion, β-cell-derived Ang1 regulates insulin secretion and glucose
homeostasis by stabilizing the blood vessels in the islet and may be a novel therapeutic target for
diabetes treatment in the future.
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Introduction
The islet is composed of endocrine cells and its microenvironment which contains several types of cells such as endothelial and immune cells (1). During development, premature endocrine cells produce several angiogenic factors that attract endothelial cells into the premature islet cluster.
The recruited endothelial cells induce further β-cell differentiation and maturation (2, 3), suggesting the importance of close cross-talk between endocrine cells and their microenvironment
(1, 4).
Several studies have reported that vascular endothelial growth factor (VEGF)-A, a well- known angiogenic factor mainly produced from islet cells (3), acts as a crucial factor in islet development by orchestrating the communication between islet cells and nearby endothelial cells
(5-7). Another potent angiogenic factor, angiopoietin-1 (Ang1), is mainly produced from β-cells
(3). Ang1 exerts its signal via the Tie2 receptor on endothelial cells and mainly contributes to vascular maturity, stability, and integrity (8, 9). Systemic Ang1 (8) or Tie2 (10, 11) mutant/null mice are embryonically lethal because of defects in systemic vascular development. More specifically, the vessels display loose connective tissue and are barely covered with pericytes, indicating that Ang1 plays a role in connecting the blood vessels with the surrounding microenvironment (8, 9, 12).
With the development of time- and/or tissue-specific Ang1 deletion rodent models, Ang1 was predicted to be dispensable in the adult phase, at least under quiescent conditions, but the role of Ang1 is critical in adults when specific perturbations occur (13). Such context-dependent action of Ang1 makes it difficult to determine how Ang1 contributes to the function of a specific tissue/organ and when or under what circumstances Ang1 exerts its action. Although Ang1 has
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been demonstrated to enhance the efficacy of islet transplantation in diabetes research (14, 15), the
Tet-on inducible systemic Ang1 deletion or β-cell specific Ang1 overexpression showed no
dramatic metabolic changes under quiescent conditions (5).
However, because β-cells express relatively higher Ang1 compared to the surrounding
tissues in the adult stage (3) and precise communication between endocrine cells and vessels is
indispensable for β-cell function (4, 16), we hypothesized that Ang1 from the islets contributes
significantly to glucose homeostasis under metabolically stressed conditions such as high-fat diet
(HFD). The objective of the current study was to investigate the role of islet-derived Ang1 using
inducible systemic Ang1-knockout (Ang1sys-/-) and β-cell specific Ang1-knockout (Ang1β-cell-/-)
mice after 24 weeks of HFD, a rodent model that mimics obesity-induced type 2 diabetes mellitus
in humans.
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Research Design and Methods
Animals
Ang1flox/flox mice (Acc.No.CDB0627K, RBRC09330,
www2.clst.riken.jp/arg/mutant%20mice%20list.html) were kindly provided by Dr. G.Y. Koh
which were originally developed and supplied by Dr. Y. Nakaoka and Riken BioResource Center
through the National Bio-Resource Project of the MEXT, Japan. The mice were generated by
insertion of the loxP allele into the two introns flanking exon1 of Ang1 (17, 18). Ang1flox/flox mice were crossed with Rosa26-CreERT2 and maintained as Rosa26-CreERT2;Ang1flox/wt in a C57BL/6N
genetic background to study inducible systemic Ang1 deletion. Rip-Cre (B6.Cg-Tg(Ins2-
cre)25Mgn/J, Jackson Laboratory, Bar Harbor, ME, USA) mice were purchased and crossed with
Ang1flox/flox mice to generate a Rip-Cre;Ang1flox/wt mice in a C57BL/6N genetic background. The
Rip-Cre;Ang1flox/wt mice were crossed to generate either the Rip-Cre;Ang1flox/flox (Ang1β-cell-/-) as
β-cell specific Ang1-knockout and Rip-Cre;Ang1wt/wt (Ang1β-cell+/+) as corresponding control mice
respectively throughout the entire experiments. To generate inducible systemic Ang1-knockout
mice (Ang1sys-/-) and their control comparator (Ang1sys+/+), Rosa26-CreERT2;Ang1flox/flox and their control Rosa26-CreERT2;Ang1wt/wt mice were intraperitoneally injected with 3mg of tamoxifen
(Sigma Aldrich, St. Louis, MO, USA) every other day for 6 days at 7 weeks of age. The mice were
fed with a standard normal diet (ND) or HFD with 60% of the total calories from fat (Research
Diets Inc., New Brunswick, NJ, USA), from 8 weeks of age for Ang1sys-/- and Ang1sys+/+ mice or
from 5 weeks of age for Ang1β-cell-/- and Ang1β-cell+/+ mice for a total 24 weeks. All animals were
maintained in a specific pathogen-free animal facility with 12-h light and dark cycles at the
Gangnam Biomedical Research Institute of Yonsei University College of Medicine with access to
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food and water ad libitum. All animal experiments were approved by the Yonsei University Health
System Institutional Animal Care and Use Committee (YUHS-IACUC).
Metabolic phenotyping
To evaluate glucose intolerance or insulin sensitivity, an intraperitoneal glucose tolerance
or insulin tolerance test was performed. Briefly, after 16 or 8-h of fasting, D-glucose (1.5g/kg for
Ang1sys+/+ and Ang1sys-/-, 0.5g/kg for Ang1β-cell+/+ and Ang1β-cell-/-) or insulin (1.5U/kg) was injected
into the abdominal cavity and then whole blood was collected from the tail vein. Blood glucose
level was measured using the ACCU-CHEK Performa glucometer (Roche Diagnostics Operations,
Inc., Basel, Switzerland) and serum insulin level with the Insulin ELISA kit (ALPCO, Salem, NH,
USA).
In vitro assay of glucose homeostasis
Mouse islets were isolated as previously described (19). For glucose-stimulated insulin
secretion (GSIS) analysis, islets were cultured overnight in RPMI-1640 (Thermo Fisher Scientific,
Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific) and 30 evenly sized
islets were picked per group. These islets were incubated for 1-h with no glucose in Krebs Ringer
Bicarbonate Buffer (KRBB) and then stimulated with 5.6, 16.7, or 20 mM of glucose each for 1-
h. The supernatant from each incubated buffer was collected and the amount of secreted insulin
was measured. The same set of islets was used to measure the amount of total insulin contents in
the islets. Briefly, the islets were incubated for 24-h in 75% of acidic-ethanol containing 0.2M HCl
at 4℃ and the supernatant processed for analysis. The degree of GSIS was presented as % release
calculated by dividing the amount of secreted insulin in the KRBB by the total insulin content in
the same batch of islets.
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To measure total insulin content per pancreas, the entire pancreas was dissected, weight measured, and then homogenized completely in 5mL of 0.2M acetic acid. The homogenate was transferred and boiled at 100℃ for 15-min. After cooling, the supernatant was taken and the insulin concentration was measured using ELISA. Total insulin content per pancreas was defined as the total amount of insulin per gram weight of the total pancreas.
For ex vivo islet culture experiment, high fat high sucrose diet (HFSD) with 58, 25, 17% calories from fat, carbohydrate, and protein (Research Diets) was fed for 4 weeks to 30 weeks-old
Ang1β-cell-/- and Ang1β-cell+/+ mice. For experiments using exogenous Ang1 supply, COMP-Ang1 protein (17, 20) was provided by Dr. G. Y. Koh.
Histologic analysis
Histological evaluation including functional vessel was performed according to previous report (19). Anti-insulin, anti-glucagon (Dako & Cell Signaling Technology Inc., Danvers, MA), anti-Ang1, anti-GLUT2 (Santa Cruz Biotechnology, Inc, Dallas, TX), anti-CD31, anti-CD45 (BD
Biosciences, San Diego, CA), anti-PDX-1 (Abcam, Cambridge, MA), anti-NG2, anti-collagen IV
(EMD Millipore Cooperation, Temecula, CA), anti-VEGFR2, anti-Tie2 (R&D Systems,
Minneapolis, MN), anti-PDGFRβ, anti-F4/80, anti-CD3e, and anti-B220 (eBioscience Life
Technologies , San Diego, CA) were used as primary Abs. 4',6-diamidino-2-phenylindole (DAPI)
(Thermo, Fisher Scientic) was used for nuclear staining. For whole pancreas imaging, the pancreas was scanned using a slide scanner (Leica SCN400F, Leica Microsystems GmbH, Wetzlar,
Germany) and the slide images were captured using the image viewer (SCN400image viewer version 2.2, Leica Microsystems CMS GmbH). The images were analyzed with the ImageJ program (version 1.49v, http://imagej.nih.gov/ij/, NIH, Bethesda, MD, USA). Electron microscopic image analysis was performed according to the previous report (21)
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Western blot
Western blot was performed as previously described (21) with anti-PDX-1, anti-GLUT2,
and anti-GAPDH (Sigma Aldrich) Abs.
Quantitative PCR
Total RNA was extracted using an RNA isolation kit (PicoPure, 12204-01, Thermo Fisher
Scientific). The cDNA was prepared (SuperScript III Reverse Transcriptase, 18080-044, Thermo
Fisher Scientific) and the amount of RNA analyzed by quantitative RT-PCR (StepOnePlus™ Real-
Time PCR system, Applied BiosystemsTM, Foster City, CA, USA) with the designated Taqman
primer and probes (Applied BiosystemsTM). See supplementary table for all primer information.
Statistical analysis
The statistical significance was tested with the independent t-test using SPSS 21.0 (SPSS
Inc., Chicago, IL, USA) and a p-value <0.05 was regarded as significant.
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Results
Inducible systemic deletion of Ang1 results in glucose intolerance with lower serum insulin level
after 24 weeks of HFD
To evaluate the effect of Ang1 on glucose homeostasis in adulthood, we deleted Ang1 from
7 weeks of age and fed Ang1sys-/- and the Ang1sys+/+ mice a ND or HFD for 24 weeks after 8 weeks
of age (Fig. 1A). Deletion of Ang1 from the genomic DNA, the corresponding transcripts and
protein after tamoxifen injection was confirmed (Fig. 1B and C, Supplementary Fig. 1). After 12 weeks of HFD, there was no difference in body weight nor blood glucose levels after glucose challenge between Ang1sys+/+ and Ang1sys-/- (Fig. 1D and E). However, after feeding HFD for 24 weeks, Ang1sys-/- mice developed glucose intolerance compared to Ang1sys+/+ (Fig. 1G), without significant differences in body weight and insulin sensitivity (Fig. 1F and H). Therefore, we measured serum insulin levels to investigate the cause of glucose intolerance by Ang1 deletion.
Although there was no change in the serum insulin level between Ang1sys-/- and Ang1sys+/+ mice fed with ND, HFD-fed Ang1sys-/- mice showed lower serum insulin levels after glucose challenge compared to HFD-fed Ang1sys+/+ mice (Fig. 1I). These findings suggest that the impaired glucose clearance from the blood in the Ang1sys-/- mice results from defective insulin secretion from β-cell.
β-cell-derived Ang1 is critical for glucose homeostasis after 24 weeks of HFD
Next, we generated β-cell specific Ang1-knockout mice (Ang1β-cell-/-) and fed them with
ND or HFD for 24 weeks starting from 5 weeks of age (Fig. 2A). Deletion of Ang1 from the genomic DNA was confirmed in the isolated islets (Fig. 2B) and depletion of Ang1 protein was also validated by immunostaining of β-cells (Fig. 2C). After 12 weeks of HFD, Ang1β-cell-/- showed
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a trend towards glucose intolerance without any difference in body weight (Fig. 2D and E). After
24 weeks of HFD, although there was no differences in both body weight and insulin sensitivity
in the Ang1β-cell-/- mice compared to the Ang1β-cell+/+ mice (Fig. 2F and G), the HFD-fed Ang1β-
cell-/- mice developed significant glucose intolerance compared to the HFD-fed Ang1β-cell+/+ mice
(Fig. 2H and I). Similar to the Ang1sys-/- mice, the serum insulin level of HFD-fed Ang1β-cell-/- mice
was lower compared to the HFD-fed control mice after glucose challenge (Fig. 2J). These findings
suggest that β-cell-derived Ang1 is crucial for maintaining glucose homeostasis under
metabolically challenged conditions, possibly by controlling the blood level of insulin.
No morphological and compositional difference in endocrine cells and vascular density in islets
by Ang1 deletion
Next, we investigated the mechanism underlying the impaired insulin secretion of Ang1β-
cell-/- mice fed with HFD. Because glucose intolerance was observed in Ang1β-cell-/- mice fed with
HFD without any difference in insulin sensitivity, we considered several possibilities such as
decreased β-cell mass resulting in decreased insulin production, hampered systemic insulin
circulation by problems in the intra-islet vasculature, or depressed insulin secretion from β-cells.
First, we evaluated whether changes occurred in endocrine cell composition or mass. However,
there was no difference in insulin-positive nor glucagon-positive area per whole pancreas or per
islets between Ang1β-cell+/+ and Ang1β-cell-/- mice (Fig. 3A–E). Neither the total insulin content per
pancreas weight nor the whole pancreas weight differed (Fig. 3F, Supplementary Fig. 2),
suggesting that the lower serum insulin concentration in the Ang1β-cell-/- mice compared to the
Ang1β-cell+/+ mice was not because of any difference in β-cell mass nor insulin production. Because
Ang1 is a potent angiogenic factor (8), we next evaluated whether decreased vascular density in
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the islets decreased the level of systemic insulin, even with appropriate production of insulin from
islets. However, there were no differences in the density of CD31-positive vessels (Fig. 3G and
H), the fluorescein isothiocyanate-lectin perfused functional vessel (Fig. 3G and I) nor the
VEGFR2-positive blood vessels (Supplementary Fig. 3), suggesting that the lower serum insulin
level after glucose challenge by Ang1 deletion is not because of the decrease of functional blood
vessels in the islets. These findings demonstrate that glucose intolerance in the Ang1β-cell-/- mice is
not a result of a defect in insulin production nor a change in intra-islet vascular density.
Impaired insulin secretion and defects in pancreatic and duodenal homeobox 1 (PDX-1) and
glucose transporter 2 (GLUT2) pathway by Ang1 deletion
Because there was no difference in endocrine mass nor vascular density in Ang1β-cell-/- mice
fed with HFD (Fig. 3), we next evaluated whether the decreased serum insulin level after glucose
challenge in vivo was because of a defect in insulin secretion from the islets. The islets were
isolated and the GSIS was tested under ex vivo culture. Although there was no difference in the
amount of insulin-2 transcript between the Ang1β-cell-/- and the Ang1β-cell+/+ mice (Fig. 4A), the
Ang1β-cell-/- islets in HFD showed significantly impaired insulin secretion for both low and high
glucose levels, suggesting that glucose intolerance in Ang1β-cell-/- mice occurred because of a defect
in insulin secretion (Fig. 4B). To further understand the mechanism related to impaired insulin
secretion in Ang1β-cell-/- islets, we evaluated various genes associated with β-cell maturation and function. The expression pattern of PDX-1, a major transcription factor involved in β-cell differentiation/maturation/GSIS (22, 23), was generally impaired in old Ang1β-cell+/+ mice
compared to young Ang1β-cell+/+ mice (Supplementary Fig 4). In Ang1β-cell-/- islets, the mRNA
(Supplementary Fig 5A) and protein (Fig. 4C and D) expression level of PDX-1, and the degree of
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nuclear localization (Fig. 4E) and the signal intensity of PDX-1 per islet (Fig. 4F) was significantly
decreased compared to Ang1β-cell+/+ with HFD. We next evaluated GLUT2, a molecule critical for
insulin secretion by sensing the change in serum glucose levels and known to be downregulated
under PDX-1-defective conditions (24-26). The degree of GLUT2 membrane localization (Fig. 4G)
and the signal intensity per islet (Fig. 4H) was significantly reduced in Ang1β-cell-/- mice, although
the mRNA and protein level did not differ (Supplementary Fig. 5B-E). Next, we tested the effect
of exogenous Ang1 (COMP-Ang1 protein, a well-known stable variant of Ang1 that mimics the
effect of Ang1 (20, 27) in the islets isolated from Ang1β-cell-/- and the Ang1β-cell+/+ mice fed with
HFSD. The decreased mRNA level of PDX-1 at day 0 recovered after 48-h even without COMP-
Ang1 treatment and the COMP-Ang1 treated islets showed higher PDX-1 level compared to the
COMP-Ang1 non-treated group (Supplementary Fig. 6A). The GLUT2 expression also increased
by COMP-Ang1 treatment (Supplementary Fig. 6B). Collectively, these results clearly
demonstrate that hyperglycemia in Ang1β-cell-/- mice occurred because of deterioration in the
insulin secretory function of the islets and that the PDX-1 and GLUT2 pathway is impaired when
β-cells are deprived of Ang1.
Loss of pericytes from the vessels in Ang1-deleted islets
Because Ang1 coordinates vessel maturation and integrity, we investigated whether there
were changes in the coverage of pericytes, an important cell supporting the endothelium (28).
Generally, most islet endothelial cells were surrounded by NG2-positive pericytes, but the
proportion of NG2-positive cells per CD31-positive vessel area in the Ang1β-cell-/- islets was
significantly reduced compared to that of the control mice fed HFD (Fig. 5A and B). This
morphologic data was corroborated by the transcript level analysis (Fig. 5C). The trend was similar
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when pericytes were visualized by PDGFRβ immunostaining (Supplementary Fig. 7). Because pericytes are the key players in maintaining the vessel integrity, we examined whether the vascular ultrastructure was affected by Ang1 deletion, despite no change in the gross density of vessels. In electron microscopic examination, Ang1β-cell-/- mice fed with HFD showed a distinct pattern of disorganized fenestration and caveolae (Fig. 5D). These data demonstrate that β-cell-derived Ang1 is critical for maintaining normal vascular structures in the islets.
Higher degree of inflammation in Ang1-deleted islets
We also examined the inflammatory cells in islets, as these cells are important components of the islet microenvironment. The degree of inflammatory cell infiltration is generally increased by HFD (29, 30) and inflamed islets have defects in insulin secretion (29-32). Because Ang1 is known as an anti-inflammatory molecule under various conditions (9, 14, 15, 33), we evaluated the degree of CD45-positive cell infiltration into the islets in Ang1β-cell-/- mice with or without
HFD. Although 24 weeks of HFD itself did not increase the number of CD45-positive cells per islet in Ang1β-cell+/+ mice, Ang1 deletion together with 24 weeks of HFD induced significant infiltration of CD45-positive cells compared to their comparator mice (Fig. 6A and B). When we examined the subpopulation of the immune cells infiltrating the islets, the majority of the immune cells were F4/80+ suggesting that the macrophages are the major proportion of the CD45+ cells
(Supplementary Fig. 8A and B). Next, we investigated whether the impaired GSIS could be recovered when this insulitis phenotype was rescued. When the islets from 30 weeks old mice fed with HFSD were isolated, there were significantly higher number of CD45+ cells in the islets from the Ang1β-cell-/- compared to the Ang1β-cell+/+ at day 0, the difference of which almost disappeared after 7 days of culture (Supplementary Fig. 9A) (34). At day 0 of isolation, the Ang1β-cell-/- mice
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showed impaired insulin secretion compared to the Ang1β-cell+/+ mice at 20mM of glucose
stimulation. However, after 7 days of culture, there was no difference of GSIS between the Ang1β-
cell+/+ and the Ang1β-cell-/- both in the 5.6mM and the 20mM glucose stimulation. This means that
the impaired insulin secretory function in the Ang1β-cell-/- islets recover after 7 days of culture,
along with the disappearance of inflammatory cell infiltration (Supplementary Fig. 9B). The
inflamed islets were also associated with higher amounts of ICAM-1 and VCAM-1 production,
the main adhesion molecules that attract leukocytes from the blood vessel into the tissue (Fig. 6C
and D) (35). Furthermore, the expression of vitronectin, a matrix protein responsible for providing
a barrier to leukocyte infiltration and mediating Ang1 signaling via integrins (12, 36-38), was
significantly reduced in Ang1β-cell-/- mice fed a HFD, a pattern that differed from that in the Ang1β-
cell+/+ mice (Fig. 6E). This suggests that leukocytes transmigrate more easily into the inflamed islets.
Collectively, these data suggest that β-cell-derived Ang1, in response to chronic metabolic
challenges such as HFD, helps suppress inflammation in the islets and maintain insulin secretory
function.
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Discussion
In this study, we demonstrated that β-cell-derived Ang1 contributes to glucose homeostasis
by coordinating insulin secretion from islets. Mechanistically, Ang1 helps support the perivascular
ultrastructure of the intra-islet vessel and protect islets from inflammation, thereby enabling insulin
secretory machinery in the islets to function adequately after long-term HFD (Fig. 7). This is the
first study revealing that Ang1 can act as a crucial regulator of the interaction between endocrine
cells and its microenvironment such as blood vessels and inflammatory cells in the islets.
In our study, Ang1-deprived β-cells developed glucose intolerance and impaired GSIS after
24 weeks of HFD. In contrast to our initial hypothesis that the absence of Ang1 would cause severe
vascular/endocrine damage, Ang1-knockout mice did not present any gross abnormalities in intra-
islet vasculature nor islet endocrine cell morphology nor composition. This led us to investigate
the secretory function of insulin from the islets. The isolated islets of Ang1β-cell-/- mice showed
reduced insulin secretion with no change in the total amount of insulin production compared to
that of the control mice, indicating that Ang1 deletion compromises the secretory machinery. In
support of this, the degree of nuclear localization of PDX-1, a crucial factor for GSIS (22, 23), was
lower in Ang1β-cell-/- mice fed the HFD (Fig. 4E and F) and the cell surface translocation of GLUT2, another key factor in GSIS (24-26), was also decreased in Ang1β-cell-/- β-cells both under ND and
HFD (Fig. 4G and H). This clearly suggests that the lower insulin secretion in the Ang1β-cell-/- mice is associated with a defect in the glucose sensing/insulin secretory machinery in β-cells, albeit no effect on β-cell mass nor vasculature density.
The mechanism by which Ang-1 is related to the GSIS of β-cells was intriguing. Because
Ang1 signaling is known to contribute to vessel maturation, stability, and integrity mainly by
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orchestrating the interaction of endothelial cells with nearby cells or connective tissue (9, 12, 39),
we evaluated whether Ang-1 exerts its effect by changing the coverage of the vessels by pericytes,
the major vascular components supporting the vascular integrity (28). Although additional studies
are needed to examine the role of pericytes, most previous studies found that pericytes are crucial
players in guiding the proper differentiation and function of adjacent cells by communicating with
endothelial cells (28, 40, 41). In line with previous studies showing that Ang1 deletion mice
develop poor perivascular coverage of the vessels by pericytes (8, 42), the NG2-positive pericyte
covered area per CD31-positive area decreased significantly in Ang1β-cell-/- mice (Fig. 5A and B).
Considering the known role of pericytes in tissue fibrosis, inflammation (28, 41), and maintenance
of vascular ultrastrucure (40, 43), it appears that the depletion of pericytes would lead to impaired
β-cell maturation or function (43-46). Indeed, our findings in Ang1β-cell-/- mice demonstrated the
role of pericytes in maintaining glucose homeostasis, which stresses that a ‘healthy’ interaction
between the endothelial cells and the pericytes is an important cornerstone for maintenance of a
‘healthy’ islet function. Mechanistically, pericyte defect may hamper GSIS because of the defect
in Tcf7L2/BMP4 through PDX-1/GLUT2 pathway (46) or may lead to insufficient production of
the basement membrane protein, ultimately leading to β-cell dysfunction (47).
In addition to defects in pericytes and the ultrastructural breakdown of the intra-islet
vasculature, we observed significant inflammatory cell infiltration in the islets of Ang1β-cell-/- mice
(Fig. 6A and B). Ang1 was originally found to be a pro-survival and anti-inflammatory factor (33,
39), and overexpression of Ang1 in rodent islets showed anti-apoptotic and anti-inflammatory
effects (15). We also observed similar actions of Ang1, as Ang1 knockout developed more
infiltration of CD45-positive cells into the islets. Upregulation of adhesion molecules, such as
ICAM-1 and VCAM-1, in Ang1-knockout islet may attract leukocytes into the inflamed area, a
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finding supported by previous studies of Ang1 deletion (33, 48). Vitronectin is a candidate molecule that mediates Ang1 signaling between cells and connective tissue and thus, maintains vascular integrity (36, 38). Thus, the increase in vitronectin may be a defense mechanism of the islets to overcome metabolic stress under the HFD (37, 48). In contrast, the impaired vitronectin pathway in Ang1β-cell-/- mice fed the HFD may accelerate the breakdown of the islet microenvironment by inflammation. A HFD itself or inflammation also appears to inhibit PDX-1 signaling (29, 30), GLUT2 membrane localization (24, 49), which was supported by our series of experiments as well (Fig. 4), finally leading to impaired GSIS in Ang1β-cell-/- mice.
Although it has been consistently shown that Ang1 governs vascular development resulting in embryonic lethality when defective from the developmental stage (8, 10, 42), Ang1 does not appear to play a crucial role in maintaining life, at least under the quiescent conditions of the postnatal stage (13). In our study, Ang1 depletion impaired GSIS after long-term HFD challenge in both β-cell-specific knockout from the developmental stage or systemic knockout from the adult stage. Thus, the action of Ang1 may be context-dependent, such as development or severe defect by systemic deletion, together with destabilized/inflamed/injured conditions by long-term HFD.
This may explain why VEGF-A-defective mice developed glucose intolerance only when VEGF-
A was deleted in the developmental stage (50). The effect of Ang1 on vasculature formation in the islets may not be as strong as VEGF-A. β-cell-specific Ang1-knockout adult mice in our study showed no significant changes in vascular density nor endocrine mass. However, Ang1 is a very delicate and elaborate factor that orchestrates the vascular maturity, integrity, and stability in terms of vascular health, particularly under stressed conditions. Under conditions of balance breakdown by chronic metabolic stress, Ang1 may help maintain intra-islet vascular health, a role that was not evident in quiescent state of metabolism.
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In conclusion, a defect in β-cell-derived Ang1, inducing islet inflammation and disrupting
the normal intra-islet vascular ultrastructure, impairs the insulin secretory mechanism in response
to metabolic challenge. This is the first report demonstrating that Ang1 governs the
microenvironment of the islets under chronic stress conditions, which may contribute to adequate
glucose control. More detailed studies of the downstream target of Ang1 in the islets is warranted
and may lead to the discovery of novel therapeutic targets for diabetes in the future.
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Acknowledgments
We appreciate the wonderful help from Professor Bum Jin Lim on analyzing the electron microscopy image.
Author Contributions
H.S.P., H.Z.K., S.K., designed the study. H.S.P, H.Z.K. S.K. conducted the research. H.S.P.,
H.Z.K., S.K. analyzed data. H.S.P., S.K. wrote the manuscript. J.L., S.-P.L., H. K., G.Y.K. gave constructive comments regarding the study concept. J.S.P., C.W.A., S.-P.L., H. K., Y.N., G.Y.K. reviewed and edited the manuscript.
Guarantor Statement
S.K. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Conflict of Interest
No potential conflicts of interest relevant to this article were reported
Funding
This research was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
Technology (A1061486) and by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2013M3A9D5072550).
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Prior Presentation
Parts of this study were presented in an abstract form at the 77th Scientific Sessions of the
American Diabetes Association, San Diego, CA, 9–13 June 2017 and at the 2015 International
Conference on Diabetes and Metabolism of the Korean Diabetes Association, Seoul, Korea, 15–
17 October 2015.
Data and Resource Availability
The data and resource generated during and/or analyzed during the current study are available from
the corresponding author on reasonable request.
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References
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37. Korpos E, Kadri N, Kappelhoff R, Wegner J, Overall CM, Weber E, Holmberg D, Cardell S, Sorokin L: The peri-islet basement membrane, a barrier to infiltrating leukocytes in type 1 diabetes in mouse and human. Diabetes 2013;62:531-542 38. Leavesley DI, Kashyap AS, Croll T, Sivaramakrishnan M, Shokoohmand A, Hollier BG, Upton Z: Vitronectin--master controller or micromanager? IUBMB Life 2013;65:807-818 39. Brindle NP, Saharinen P, Alitalo K: Signaling and functions of angiopoietin-1 in vascular protection. Circ Res 2006;98:1014-1023 40. Hellstrom M, Gerhardt H, Kalen M, Li X, Eriksson U, Wolburg H, Betsholtz C: Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol 2001;153:543-553 41. Chatterjee S, Naik UP: Pericyte-endothelial cell interaction: a survival mechanism for the tumor vasculature. Cell Adh Migr 2012;6:157-159 42. Vikkula M, Boon LM, Carraway KL, 3rd, Calvert JT, Diamonti AJ, Goumnerov B, Pasyk KA, Marchuk DA, Warman ML, Cantley LC, Mulliken JB, Olsen BR: Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2. Cell 1996;87:1181-1190 43. Hayden MR, Karuparthi PR, Habibi J, Lastra G, Patel K, Wasekar C, Manrique CM, Ozerdem U, Stas S, Sowers JR: Ultrastructure of islet microcirculation, pericytes and the islet exocrine interface in the HIP rat model of diabetes. Exp Biol Med (Maywood) 2008;233:1109-1123 44. Nakamura M, Kitamura H, Konishi S, Nishimura M, Ono J, Ina K, Shimada T, Takaki R: The endocrine pancreas of spontaneously diabetic db/db mice: microangiopathy as revealed by transmission electron microscopy. Diabetes Res Clin Pract 1995;30:89-100 45. Sasson A, Rachi E, Sakhneny L, Baer D, Lisnyansky M, Epshtein A, Landsman L: Islet Pericytes Are Required for beta-Cell Maturity. Diabetes 2016;65:3008-3014 46. Sakhneny L, Rachi E, Epshtein A, Guez HC, Wald-Altman S, Lisnyansky M, Khalifa-Malka L, Hazan A, Baer D, Priel A, Weil M, Landsman L: Pancreatic Pericytes Support beta-Cell Function in a Tcf7l2- Dependent Manner. Diabetes 2018;67:437-447 47. Nikolova G, Jabs N, Konstantinova I, Domogatskaya A, Tryggvason K, Sorokin L, Fassler R, Gu G, Gerber HP, Ferrara N, Melton DA, Lammert E: The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation. Dev Cell 2006;10:397-405 48. Imai Y, Patel HR, Doliba NM, Matschinsky FM, Tobias JW, Ahima RS: Analysis of gene expression in pancreatic islets from diet-induced obese mice. Physiol Genomics 2008;36:43-51 49. Wu Y, Wu T, Wu J, Zhao L, Li Q, Varghese Z, Moorhead JF, Powis SH, Chen Y, Ruan XZ: Chronic inflammation exacerbates glucose metabolism disorders in C57BL/6J mice fed with high-fat diet. J Endocrinol 2013;219:195-204 50. Reinert RB, Brissova M, Shostak A, Pan FC, Poffenberger G, Cai Q, Hundemer GL, Kantz J, Thompson CS, Dai C, McGuinness OP, Powers AC: Vascular endothelial growth factor-a and islet vascularization are necessary in developing, but not adult, pancreatic islets. Diabetes 2013;62:4154-4164
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Figure Legends
Figure 1. Inducible systemic deletion of Ang1 results in glucose intolerance with low serum
insulin level after high fat diet.
A: Schematic diagram of the animal experiment for inducible systemic Ang1 deletion. Conditional
knockout of Ang1 was induced with tamoxifen at 7 weeks of age and the mice were fed with
normal diet (ND) or high fat diet (HFD) starting at 8 weeks of age for 24 weeks. B: Ang1 deletion
was confirmed at 1 or 6 weeks after tamoxifen administration. Genomic DNA extracted from the
tail was used for PCR with primers detecting Cre, Ang1flox, Ang1wt or the deleted Ang1. C: Ang1
deletion was confirmed by qRT-PCR of the mRNA transcripts from the indicated tissues after
tamoxifen administration. The amount of Ang1 transcript was presented as a relative value after
normalization to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript
and compared to the value of Ang1sys+/+ set at 1 in each tissue (n=4~6 per group). D: Body weight
after 12 weeks of the indicated diet (n=6~7 per group). E: Intraperitoneal glucose tolerance test
(IPGTT) after 12 weeks of the indicated diet (n=5~7 per group). F: Body weight after 24 weeks of
the indicated diet (n=5~7 per group). G: IPGTT after 24 weeks of the indicated diet (n=8~10 per
group). H: Intraperitoneal insulin tolerance test after 24 weeks of the indicated diet (n=6~7 per
group). I: Serum insulin concentration was measured at baseline and at 15 min after IPGTT
(n=8~12 per group). All data are presented as mean±SE. E, G and H: Closed circle, Ang1sys+/+ with
ND; open circle, Ang1sys-/- with ND; closed triangle, Ang1sys+/+ with HFD; open triangle, Ang1sys-/-
with HFD. *, p<0.05 Ang1sys-/- versus the Ang1sys+/+ in each diet group at each time point. N.S.,
Not significant. Rosa26-CreERT2;Ang1sys+/+, Ang1sys+/+. Rosa26-CreERT2;Ang1sys-/-, Ang1sys-/-.
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Figure 2. β-cell derived Ang1 is critical for glucose homeostasis after high fat diet.
A: Schematic diagram of the animal experiment for β-cell specific deletion of Ang1. Ang1β-cell+/+
and Ang1β-cell-/- mice were fed with normal diet (ND) or high fat diet (HFD) starting at 5 weeks of
age for 24 weeks. B: The genomic DNA extracted from the islets was used for PCR with primers detecting Cre, Ang1flox, Ang1wt or the deleted Ang1 to confirm the β-cell specific deletion of Ang1.
C: β-cell specific deletion of Ang1 was confirmed using the immunofluorescent staining for Ang1.
Scale bars, 100µm. D: Body weight after 12 weeks of the indicated diet (n=10 per group). E:
Intraperitoneal glucose tolerance test (IPGTT) after 12 weeks of the indicated diet (n=16~23 per
group). F: Body weight after 24 weeks of the indicated diet (n=10 per group). G: Intraperitoneal
insulin tolerance test after 24 weeks of the indicated diet (n=5~8 per group). H: IPGTT after 24
weeks of the indicated diet (n=13~23 per group). I: Area under the curve (AUC) of blood glucose level during IPGTT (n=13~23 per group). J: Serum insulin concentration was measured at baseline and at 15 min after IPGTT (n=5~7 per group). All data are presented as mean±SE. *, p<0.05
Ang1β-cell-/- versus Ang1β-cell+/+ in each diet group at each time point. E, G and H: Closed circle,
Ang1β-cell+/+ with ND; open circle, Ang1β-cell-/- with ND; closed triangle, Ang1β-cell+/+ with HFD;
open triangle, Ang1β-cell-/- with HFD. N.S., Not Significant. Rip-Cre;Ang1β-cell+/+, Ang1β-cell+/+.
Rip-Cre;Ang1β-cell-/-, Ang1β-cell-/-.
Figure 3. No morphological and compositional difference of endocrine cells and vascular
density by Ang1 deletion.
Total pancreas was harvested after 24 weeks of HFD. A: Representative immunofluorescent
staining of insulin, glucagon and the blood vessels in the Ang1β-cell-/- or the Ang1β-cell+/+ mice fed either normal diet (ND) or high fat diet (HFD). B, C: Insulin-positive (B) or glucagon-positive (C)
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area per entire area of the pancreas (n=5~6 per group). D, E: Insulin-positive (D) or glucagon-
positive (E) area per entire area of the islet (n=5~6 per group). F: Total insulin amount per
pancreatic weight (n=4 per group). G: Representative immunofluorescent staining of the blood
vessels by CD31 and the perfused vessels by lectin in the Ang1β-cell-/- or the Ang1β-cell+/+ fed either
ND or HFD. H, I: Area of the blood vessels (CD31-positive area) (H) or perfused blood vessels
(lectin-positive area) (I) per insulin-positive area in the islet (n=4~6 per group). All data are
presented as mean±SE. N.S., Not Significant. Scale bars, 100µm. Rip-Cre;Ang1β-cell+/+, Ang1β-
cell+/+. Rip-Cre;Ang1β-cell-/-, Ang1β-cell-/-.
Figure 4. Impairment of insulin secretion together with the defect in PDX-1 and GLUT2
pathway by Ang1 deletion.
A: Islets were isolated after the indicated diet and the amount of Ins2 transcript analyzed. The
relative amount of Ins2 transcript are presented as a relative value after normalization to GAPDH
compared to the value of Ang1β-cell+/+ with normal diet (ND) set at 1 (n=5~6 per group). B: Cultured
islets from the indicated mice and diet were incubated in either 5.6 or 16.7 mM glucose for 1-hr.
The secreted amount of insulin from each islet was measured from the supernatant and the
percentage of insulin release was calculated by dividing the concentration of insulin in the
supernatant with the total amount of insulin per islet. Insulin content per group was
2314.9±121.13ng (Ang1β-cell+/+) and 2108.3±119.12ng (Ang1β-cell-/-) under ND, and
5163.6±701.96ng (Ang1β-cell+/+) and 4494.4±636.09ng (Ang1β-cell-/-) under HFD (n=4~7 per group).
C: Representative image of PDX-1 Western blot. D: The signal intensity of PDX-1 in Western blot
was divided by that of GAPDH, and then presented as a relative value of Ang1β-cell+/+ with ND set
at 1 (n=4 per group). E: Representative immunostaining of PDX-1. F: The signal intensity of PDX-
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1 per insulin-positive area presented as a relative value of Ang1β-cell+/+ with ND set at 100 (n=4 per
group). G: Representative immunostaining of GLUT-2. H: The signal intensity of GLUT-2 per
insulin-positive area presented as a relative value of Ang1β-cell+/+ with ND set at 100. (n=4 per
group). All data are presented as mean±SE. †, p<0.05 Ang1β-cell-/- versus Ang1β-cell+/+ in each diet
group at each glucose concentration. #, p<0.05 16.7mM versus 5.6mM in the respective diet group
in Ang1β-cell+/+. *, p<0.05 Ang1β-cell-/- versus Ang1β-cell+/+ in each diet group. N.S., Not Significant.
Scale bars, 100µm. Rip-Cre;Ang1β-cell+/+, Ang1β-cell+/+. Rip-Cre;Ang1β-cell-/-, Ang1β-cell-/-.
Figure 5. Loss of pericytes from the vessels in the Ang1 deleted islets.
A: Representative immunostaining of the pericytes (NG2) and the blood vessels (CD31) in the islet
of the Ang1β-cell-/- or the Ang1β-cell+/+ fed either normal diet (ND) or high fat diet (HFD). Scale bar,
100µm. B: The area of blood vessels covered by the pericytes were presented as a percentage by
analyzing the CD31-positive area costained with NG2 (n=4~6 per group). C: Islets were isolated
and the amount of NG2 transcript analyzed. The relative amount of NG2 transcript are presented
as a relative value after normalization to GAPDH compared to the value of Ang1β-cell+/+ with ND
set at 1 (n=4~6 per group). D: The section of pancreas was visualized in the vicinity of the β-cell
and the blood vessel with transmission emission microscopy. The inlet of the image of the lower
panel is a high magnification view. Arrow, disorganized fenestration; Arrow head, caveolae; Lm,
blood vessel lumen; Scale bars, 1µm. All data are presented as mean±SE. *, p<0.05 Ang1β-cell-/-
versus Ang1β-cell+/+ in each diet group. N.S., Not Significant. Rip-Cre;Ang1β-cell+/+, Ang1β-cell+/+.
Rip-Cre;Ang1β-cell-/-, Ang1β-cell-/-.
Figure 6. Higher degree of inflammation in the Ang1 deleted islets.
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A: Representative immunostaining to visualize the CD45-positive cells, together with collagen IV
in the islet of the Ang1β-cell-/- or the Ang1β-cell+/+ fed either normal diet (ND) or high fat diet (HFD).
Scale bars, 100µm. B: Number of CD45-positive cells per islets (n=4~5 per group). C, D and E:
Islets were isolated and the amount of ICAM-1 (C), VCAM-1 (D) and vitronectin (E) transcript
analyzed. The relative amount of each transcript are presented as a relative value after
normalization to GAPDH compared to the value of Ang1β-cell+/+ with ND set at 1 (n=4~6 per group).
All data was presented as mean±SE. *, p<0.05 Ang1β-cell-/- versus Ang1β-cell+/+ in each diet group.
N.S., Not Significant. Rip-Cre;Ang1β-cell+/+, Ang1β-cell+/+. Rip-Cre;Ang1β-cell-/-, Ang1β-cell-/-.
Figure 7. Schematic diagram of the current investigation.
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Supplementary Table
Table 1. Primer sequence
F: Forword Gene name Sequence (5’ to 3’) R: Reverse
F CCGGATTCAACATGGGCAATGTGCC Ang1 R CAGTCAAAATGCCTAAGATAAAC
Ang1 F GCAGCCATAGCAATGCCAGAGGT Genotyping (deleted allele) R TCCCATGGCAACTCACAAAACTCC
F AGGTTCGTTCACTCATGGA Cre R TCGACCAGTTTAGTTACCC
F ATACCGGAGATCATGCAAGC RipCre R TGTGCCAAAGGGATTTTAGG
Gene name Taqman assay ID
Ang1 Mm00456503_m1
Ins2 Mm00731595_gH
Gene NG2 Mm00507257_m1
expression ICAM-1 Mm00516023_m1
VCAM-1 Mm01320970_m1
Vitronectin Mm00495976_m1
GAPDH Mm99999915_g1
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Supplementary Figure Legends
Supplementary Figure 1. Deletion of Ang1 in the islets of the Ang1sys-/- mice.
Total pancreas was harvested and immunostained with Ang1 and insulin to demonstrate the effective deletion of Ang1 in the Ang1sys-/- mouse. Representative images are shown. Scale bars,
100µm.
Supplementary Figure 2. No difference in the total pancreas weight between the Ang1β-cell+/+ and the Ang1β-cell-/- mice.
Experimental groups were composed of the Ang1β-cell+/+ and Ang1β-cell-/- mice, each fed with either the ND or the HFD. The entire pancreas was harvested and the weight measured (n=4 per group).
Note that there was no statistical difference in the total pancreas weight between the Ang1β-cell+/+ and the Ang1β-cell-/- mice. All data was presented as mean±SE. N.S., Not Significant.
Supplementary Figure 3. No difference in the VEGFR2-positive blood vessels between the
Ang1β-cell+/+ and Ang1β-cell-/- mice.
Experimental groups are composed of the Ang1β-cell+/+ and Ang1β-cell-/- mice, each fed with either the ND or the HFD. VEGFR2 were visualized together with CD31 and insulin to demonstrate that there was no difference in the VEGFR2-positive blood vessels between the Ang1β-cell+/+ and Ang1β- cell-/- mice. Representative images of the islets are shown for each experimental group. Scale bars,
100µm. Ins, insulin.
Supplementary Figure 4. Impaired PDX-1 expression in old mice compared to young mice
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PDX-1 was immunostained in 7 and 30 weeks of control mice (Rip-Cre;Ang1β-cell+/+) and a
representative image was presented. Scale bars, 100µm.
Supplementary Figure 5. Difference in the expression level of the PDX-1 and the GLUT2
between the Ang1β-cell+/+ and Ang1β-cell-/- mice.
Experimental groups are composed of the Ang1β-cell+/+ and Ang1β-cell-/- mice, each fed with either
the ND or the HFD (n=4 per group). A: Comparison of the PDX-1 mRNA. B: Comparison of the
GLUT2 mRNA level C: Western blot of the GLUT2 expression. D: Comparison of the GLUT2
protein level. All data was presented as mean±SE. *, p<0.05 Ang1β-cell-/- versus Ang1β-cell+/+ in each
diet group. N.S., Not significant.
Supplementary Figure 6. Recovery of the PDX-1 and GLUT2 mRNA level with exogenous
Ang1 supplementation.
Experimental groups are composed of 0 weeks old Ang1β-cell+/+ and Ang1β-cell-/- mice, each fed with
high fat, high sucrose diet for 4 weeks. The isolated islets were treated with 200ng/ml of COMP-
Ang1 or vehicle for 48hr (n=4 per group). A: PDX-1 transcription by exogenous Ang1 treatment.
Note the significant enhancement of the PDX-1 transcription when Ang1 was supplied
exogenously. B: GLUT2 transcription by exogenous Ang1 treatment. Note the significant
enhancement of the GLUT2 transcription when Ang1 was supplied exogenously. All data was
presented as mean±SE. *, p<0.05 Ang1β-cell-/- versus Ang1β-cell+/+ at 0 hr. *, p<0.05 COMP-Ang1-
treated-group versus vehicle-treated-group in each genotype mice at 48 hr after COMP-Ang1
treatment. N.S., Not significant.
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Supplementary Figure 7. Loss of pericyte coverage from the vessels in the HFD fed Ang1β-
cell-/- mice.
Experimental groups are composed of the Ang1β-cell+/+ and Ang1β-cell-/- mice, each fed with either
the ND or the HFD. PDGFRβ was visualized together with CD31 and insulin to demonstrate the
degree of pericyte coverage in the endothelial cells. Note the loss of PDGFRβ+ pericytes in the
HFD fed Ang1β-cell-/- mice. Scale bars, 100µm.
Supplementary Figure 8. Predominance of F4/80+ immune cell subpopulation in the HFD
induced insulitis.
Experimental groups are composed of the Ang1β-cell+/+ and Ang1β-cell-/- mice, each fed with either
the ND or the HFD. Lymph node was stained as positive control for CD3e and B220. A: Abundant
F4/80+ cells in the islets in the HFD fed Ang1β-cell-/- mice. B: Scarce CD3e+ or B220+ cells in the
islets. Red arrow head, CD3e; Green arrow head, B220. Scale bars, 100µm.
Supplementary Figure 9. Rescue of impaired glucose-stimulated insulin secretion with the
improvement of insulitis in the ex vivo cultured islets.
Experimental groups are composed of 30 weeks old Ang1β-cell+/+ and Ang1β-cell-/- mice, each fed with high fat high sucrose diet for 4 weeks (n=4 per group). A: Recovery of the insulitis phenotype with 7 days of ex vivo culture of the islets in the Ang1β-cell-/- mice. Scale bars, 100µm. B: A significant recovery of the impaired glucose-stimulated insulin secretion was noted along with the improvement of the insulitis. All data was presented as mean±SE. *, p<0.05 Ang1β-cell-/- versus
Ang1β-cell+/+. N.S., Not significant.
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