Page 1 of 31
Diabetes
Insulin Receptor Substrate-2 (Irs2) in Endothelial Cells Plays a Crucial Role in
Insulin Secretion
Short running title: Role of Irs2 in endothelial cells in insulin secretion
Shinji Hashimoto1,*, Naoto Kubota1,2,3,4,*, Hiroyuki Sato1, Motohiro Sasaki1, Iseki
Takamoto1,2, Tetsuya Kubota1,3,4,5, Keizo Nakaya1, Mitsuhiko Noda6, Kohjiro Ueki1,2,
and Takashi Kadowaki1,2,3
1
Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The
University of Tokyo, Tokyo, Japan;
2
Translational Systems Biology and Medicine Initiative (TSBMI), The University of
Tokyo, Tokyo, Japan;
3
Clinical Nutrition Program, National Institute of Health and Nutrition, Tokyo, Japan;
4
Laboratory for Metabolic Homeostasis, RIKEN Center for Integrative Medical
Sciences, Kanagawa, Japan;
5
Division of Cardiovascular Medicine, Toho University Ohashi Medical Center, Tokyo,
Japan;
6
Department of Diabetes and Metabolic Medicine, International Medical Center of
Japan, Toyama Hospital, Tokyo, Japan
*These authors contributed equally to this work.
Corresponding author:
Takashi Kadowaki, M.D., Ph.D. and Naoto Kubota, M.D., Ph.D.,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan,
Tel: 81-3-5800-8815;
Fax: 81-3-5800-9797;
E-mail: [email protected] (T. Kadowaki); [email protected] (N.
Kubota)
Word Count: 3955
Tables: 2
Figures: 6
1
Diabetes Publish Ahead of Print, published online October 2, 2014
Diabetes
Page 2 of 31
Endothelial cells are considered to be essential for normal pancreatic β-cell
function. The present study attempted to demonstrate the role of Irs2 in
endothelial cells with regard to insulin secretion. Endothelial cell-specific insulin
receptor
substrate-2
knockout
(ETIrs2KO)
mice
exhibited
impaired
glucose-induced, arginine-induced, and glucagon-induced insulin secretion and
showed glucose intolerance. In batch incubation and perifusion experiments using
isolated islets, glucose-induced insulin secretion was not significantly different
between the control and the ETIrs2KO mice. In contrast, in perfusion experiments,
glucose-induced insulin secretion was significantly impaired in the ETIrs2KO mice.
The islet blood flow was significantly impaired in the ETIrs2KO mice. Following
the treatment of these knockout mice with enalapril maleate, which improved the
islet blood flow, glucose-stimulated insulin secretion was almost completely
restored to levels equal to those in the control mice. These data suggest that Irs2
deletion in endothelial cells leads to a decreased islet blood flow, which may cause
impaired glucose-induced insulin secretion. Thus, Irs2 in endothelial cells may
serve as a novel therapeutic target for preventing and ameliorating type 2 diabetes
and metabolic syndrome.
Type 2 diabetes is a heterogeneous disorder with varying degrees of insulin resistance
and insulin secretion (1, 2). The United Kingdom Prospective Diabetes Study clinical
trial revealed a progressive impairment in pancreatic β-cell function during the course
of the disease, implicating an important role of β-cell failure in the pathogenesis of type
2 diabetes (3). Actually, the progression of type 2 diabetes is associated with minimal
changes in the degree of insulin resistance; however, insulin secretion is progressively
2
Page 3 of 31
Diabetes
blunted with the transition from prediabetes to overt diabetes (4). β-cell failure or
dysfunction is inherently associated with type 2 diabetes and may precede the onset of
hyperglycemia. Thus, the amelioration of impaired insulin secretion might be a
reasonable therapeutic target.
Several studies have reported that the islet blood flow is involved in the regulation
of insulin secretion. Pancreatic islets are highly vascularized by a dense network of
capillaries, and various mediators, such as insulin, regulate the islet blood flow (5, 6).
Iwase et al. demonstrated that orally administered insulin secretagogues acutely
increased the islet blood flow (7). Moreover, renin-angiotensin system (RAS) inhibitors
that regulate the islet blood flow, such as angiotensin-converting enzyme (ACE)
inhibitors and angiotensin II receptor blockers (ARBs), increased insulin secretion in
response to glucose administration (8-10). In addition to RAS inhibitors, some
vasoactive drugs enhance pancreatic islet blood flow, augment insulin secretion and
improve glucose tolerance (11, 12). These findings suggest the involvement of the islet
blood flow in insulin secretion.
Irs2 is the major insulin receptor substrate isoform expressed in endothelial cells
(13). We previously reported that mice with the Irs2 deletion in endothelial cells
(ETIrs2KO mice) exhibited an attenuation of the insulin-induced capillary blood flow in
skeletal muscle (14). In the present study, we attempted to demonstrate the relationship
between insulin secretion and the islet blood flow using these mice. Although insulin
secretion from isolated islets was maintained, insulin secretion was significantly
impaired in the ETIrs2KO mice. These mice showed significant decreases in the islet
blood flow, similar to the results seen in skeletal muscle in a previous study (14). In
addition, following the administration of enalapril maleate, which enhances the islet
3
Diabetes
blood flow in these knockout mice, insulin secretion was almost completely restored to
levels equal to those in the control mice. These data suggest that the absence of Irs2 in
endothelial cells impairs the islet blood flow, which may be one of the mechanisms
responsible for the decrease in insulin secretion. We previously reported that
hyperinsulinaemia linked to obesity leads to the downregulation of Irs2 in endothelial
cells (14). Thus, Irs2 in endothelial cells may serve as a novel therapeutic target for
preventing and ameliorating type 2 diabetes and metabolic syndrome.
RESEARCH DESIGN AND METHODS
Animals
ETIrs2KO mice were generated as described previously (14). C57BL/6J mice were
obtained from CLEA Japan (Tokyo, Japan). Mice were housed under a 12-h light-dark
cycle and were given access ad libitum to normal chow MF consisting of 25% (w/w)
protein, 53% carbohydrates, 6% fat and 8% water (Oriental Yeast Co., Ltd., Osaka,
Japan). All the experiments in this study were performed on male mice. To evaluate the
effect of an ACE inhibitor on the islet blood flow and glucose-induced insulin secretion,
enalapril maleate (100 µg/kg body weight) or the corresponding volume of saline (0.1
mL) was injected intravenously. The animal care and experimental procedures were
approved by the Animal Care Committee of the University of Tokyo.
Glucose tolerance test
Mice were denied access to food for 16 h starting at 19:00 hours on the previous
evening and continuing until the end of the fasting period. Control and ETIrs2KO mice
were intraperitoneally injected with glucose (3.0 g/kg body weight) to evaluate insulin
secretion. Mice were also injected intraperitoneally with L-arginine monohydrochloride
4
Page 4 of 31
Page 5 of 31
Diabetes
(2.1 g/kg body weight) and glucagon (10 mg/kg body weight; Glucagon G Novo, Novo
Nordisk, Bagsvaerd, Denmark). Blood samples from tail snips were collected at the
indicated times, and the blood glucose level was immediately measured using an
automatic blood glucose meter (Glutest Pro, Sanwa Kagaku Kenkyusho, Nagoya,
Japan). Whole blood samples were collected and centrifuged in heparinized tubes, and
the plasma samples were stored at -30°C. The insulin levels were determined using an
insulin radioimmunoassay (RIA) kit (Institute of Isotopes, Budapest, Hungary) using rat
insulin as the standard.
Assay of insulin secretion from isolated islets and of the islet insulin content
Pancreatic islets were isolated from 12 week-old mice using collagenase digestion, as
described previously (15). Insulin secretion from the islets was measured under static
incubation with Krebs-Ringer-bicarbonate (KRB) buffer (129 mM NaCl, 4.8 mM KCl,
1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5 mM NaHCO3, and 10 mM
HEPES; pH7.4) containing 0.2% bovine serum albumin. In the static incubation
experiments, batches of 10 freshly isolated islets were preincubated at 37°C for 30 min
in 500 µL of KRB buffer containing 2.8 mM glucose. The preincubation solutions were
replaced with 500 µL of KRB buffer containing the test agents, and the batches of islets
were incubated at 37°C for 60 min. At the end of the incubation, aliquots of the buffer
were immediately sampled and stored at -30°C until assay. For measurement of the islet
insulin content, islets were solubilized in acid-ethanol solution overnight at -30°C. The
insulin concentration was measured using an insulin RIA kit, and the resulting
concentration was corrected according to the DNA content. The DNA content was
measured using a DNA assay kit (PicoGreen, Invitrogen, Carlsbad, CA, USA).
Islet perifusion
5
Diabetes
The kinetics of insulin secretion were studied in vitro using a perifusion system.
Isolated pancreatic islets were used immediately after isolation. Size-matched islets (n =
50) were placed in each column. Then, the columns were gently closed with the top
adaptors and immersed in a vertical position in a temperature-controlled water bath at
37°C. The perifusion medium was maintained at 37°C in a water bath. All the columns
were perifused in parallel at a flow rate of 0.6 mL/min. After 30 min of static incubation
with KRB buffer (2.8 mmol/L glucose), the islets were stimulated by the continuous
addition of 22.2 mmol/L of glucose. Samples were collected every 2 min and stored at
-30°C until further analysis.
Quantitative reverse-transcriptase PCR
Total RNA was extracted from the islets using an RNeasy kit (QIAGEN Sciences,
Maryland, USA), in accordance with the manufacturer’s instructions. After treatment
with RQ1 RNase-free DNase (Promega, Madison, WI) to remove genomic DNA,
cDNA was synthesized using MultiScribe reverse transcriptase (Applied Biosystems,
Foster City, CA), and TaqMan quantitative PCR (50°C for 2 min, 95°C for 10 min
followed by 40 cycles of 95°C for 15 s, 60°C for 1 min) was then performed using the
ABI Prism 7900 PCR system (Applied Biosystems) to amplify Insulin1, Insulin2 and
Cyclophilin cDNA. The primers that were used were purchased from Applied
Biosystems. The relative abundance of the transcripts was normalized to the constitutive
expression of cyclophilin mRNA.
Histological and immunohistochemical analysis of the islets
Isolated pancreata were fixed with 4% paraformaldehyde at 4°C overnight. Tissues
were routinely processed for paraffin embedding, and 4-µm sections were cut and
mounted on silanized slides. Pancreatic sections were stained with anti-rabbit insulin
6
Page 6 of 31
Page 7 of 31
Diabetes
antibodies (diluted 1:200; Santa Cruz Biotechnology, Santa Cruz, CA). Images of the
pancreatic tissue and islet β-cells were viewed on the monitor of a computer through a
microscope connected to a camera with a charged-coupled device (Olympus, Tokyo,
Japan). The areas of the pancreata and β-cells were traced manually and analyzed with
Win ROOF software (Mitani, Chiba, Japan), as previously described (15). At least 50
islets per mouse were analyzed. BrdU incorporation was analyzed as described
previously (16). In brief, BrdU (100 mg/kg in saline; Sigma-Aldrich, St. Louis, MO)
was injected intraperitoneally, and the pancreas was removed 6 hours later. The sections
were immunostained with anti-BrdU antibody (diluted 1:200; Dako, Glostrup,
Denmark). BrdU-positive β-cells were quantitatively assessed as a percentage of the
total number of β-cells by counting the cells in a minimum of 50 islets per mouse.
Immunohistochemical staining for pimonidazole were carried out as described
previously (17), with slight modifications. The oxygenation marker pimonidazole was
injected intravenously into the tail vein (60 mg/kg). Two hours later, the animals were
killed and their pancreata were removed and prepared for histological analysis. The
sections were immunostained with anti-pimonidazole antibody (diluted 1:100;
Hypoxyprobe, Burlington, MA). Pimonidazole-positive islets were quantitatively
assessed as a percentage of the total number of islets by counting in a minimum of 50
islets per mouse.
Perfusion experiments using mouse pancreata
Mice were used for the perfusion experiments after they had been denied access to food
overnight for 16 h, as previously reported (18) with slight modifications. Briefly, the
superior mesenteric and renal arteries were ligated, and the aorta was tied just below the
diaphragm. The perfusate was infused via a catheter placed in the aorta and collected
7
Diabetes
from the portal vein. The perfusate used was KRB HEPES buffer supplemented with
4.6% dextran and 0.25% BSA and gassed with 95% O2 / 5% CO2. The flow rate of the
perfusate was 1 mL/min. Pancreata were perfused with KRB HEPES buffer containing
2.8 or 22.2 mmol/L glucose. The perfusion protocols began with a 10-min equilibration
period with the same buffer used in the initial step (i.e., from 1 to 5 min), as shown in
the figures. The insulin levels in the perfusate were measured using an RIA kit.
Islet blood flow measurements
The experiments were performed according to a protocol described in detail in a
previous report (19). Briefly, polyethylene catheters were inserted via the right carotid
artery into the ascending aorta and into the femoral artery. After the blood pressure
stabilized, nonradioactive microspheres (Dye-Trak; Triton Technology, Los Angeles,
CA) with a mean diameter of 10 µm were injected for 10 s via the catheter placed with
its tip in the ascending aorta. Starting 5 s before the microsphere injection and
continuing for a total of 60 s, an arterial blood sample was collected from the catheter in
the femoral artery at a rate of ~0.50 mL/min. The exact withdrawal rate was confirmed
in each animal by weighing the sample. After the reference sample was obtained,
another blood sample was drawn for the measurement of the blood glucose level. The
whole pancreata were removed, blotted, weighed, and treated using a freeze-thawing
technique to visualize the microspheres. The capillary blood flow values were
calculated according to the formula Qorg = Qref x Norg/Nref, where Qorg is the organ
capillary blood flow (mL/min), Qref is the withdrawal rate of the reference sample
(mL/min), Norg is the number of microspheres present in the organ, and Nref is the
number of microspheres in the reference sample. The microsphere contents of the
adrenal glands were used as a control to confirm that the microspheres had adequately
8
Page 8 of 31
Page 9 of 31
Diabetes
mixed in the arterial circulation (19). A <10% difference in the numbers of
microspheres between the right and left adrenal glands was taken to indicate sufficient
mixing. The islet blood flow was expressed per islet weight estimated by multiplying
the pancreatic weight with the islet volume fraction of the whole pancreas in each
animal. To evaluate the effect of insulin on the islet blood flow, insulin (0.75 U/kg body
weight) was injected intraperitoneally as described previously (20) with slight
modifications. The anaesthetised animals were left for 20 minutes. Before microsphere
injection, blood glucose levels were determined using arterial blood samples.
Lectin perfusion and vascular staining
Fluorescein-labeled lectin (Lycopersicon Esculentum; Vector, Buringame, CA), which
binds specifically to endothelial cells and epithelial cells, were injected into the tail vein
(1 mg/mL solution/0.1 mL/mouse) and were allowed to circulate for 3 min. The
pancreas was then excised and immersion-fixed in 4% paraformaldehyde for 16 h at
4°C. After fixation, the pancreata were immersed in 30% sucrose as a cryoprotectant,
after which the tissues were embedded in optimal cutting temperature compound
(SAKURA SEIKI, Tokyo, Japan). The block was sectioned into 15-µm-thick sections
and collected on microscope slides. Images of the vasculature were viewed on the
monitor of a computer through a CCD camera with Biozero (KEYENCE, Osaka, Japan).
The areas of the vasculature and β-cells were traced manually and analyzed with a
software program (Image J). At least 50 islets were analyzed per mouse.
Statistical analysis
Values were expressed as the mean ± SE. The statistical significance of differences
between groups was determined using a 2-tailed indirect Student t-test. Data involving
more than two groups were assessed using an analysis of variance (ANOVA).
9
Diabetes
RESULTS
Insulin secretion in response to glucose, glucagon, and arginine was impaired in
the ETIrs2KO mice
The blood glucose levels at 5 and 15 min after glucose loading were significantly higher
in the ETIrs2KO mice than in the control mice at 12 weeks (Fig. 1A). The plasma
insulin levels at 2 and 5 min were significantly lower in the ETIrs2KO mice than in the
control mice at 12 weeks (Fig. 1A). At 24 weeks, although the blood glucose level at 5
min was comparable to that in the control mice, the blood glucose level at 15 min was
significantly higher in the ETIrs2KO mice (Fig. 1B). The plasma insulin levels at 2 min
after glucose loading were significantly lower in the ETIrs2KO mice (Fig. 1B). In
addition to glucose, glucagon-induced and arginine-induced insulin secretion were also
significantly impaired in the ETIrs2KO mice (Fig. 1C, D). These findings suggest that
insulin secretion induced by various secretagogues is impaired in the ETIrs2KO mice.
Insulin secretion in isolated islets was not impaired in the ETIrs2KO mice
To clarify the molecular mechanism responsible for the impairment of insulin secretion
in the ETIrs2KO mice, we next measured insulin secretion from isolated islets using
static and perifusion analyses at 12 weeks. In contrast to the results of the in vivo study,
glucose-stimulated insulin secretion was comparable between the control and the
ETIrs2KO mice in a static incubation experiment (Fig. 2A). Moreover, the perifusion
experiments also demonstrated that insulin secretion under perifusion with a stimulating
glucose concentration was comparable between the control and the ETIrs2KO mice at
12 weeks (Fig. 2B). These data indicated that insulin secretion from isolated islets was
not impaired in the ETIrs2KO mice. The gene expression levels of Insulin1 and Insulin2
10
Page 10 of 31
Page 11 of 31
Diabetes
(Fig. 2C) and the insulin contents (Fig. 2D) in the isolated islets were not significantly
different between the control and the ETIrs2KO mice at 12 weeks. Consistent with our
previous study (14), the β-cell mass from the ETIrs2KO mice tended to be larger, but no
statistical difference was seen between the two groups (Fig. 2E, F) at 24 weeks. In
addition, the rate of BrdU incorporation into the pancreatic β-cell nuclei in the
ETIrs2KO mice tended to be increased but was not significantly different, compared
with that in the control mice at 24 weeks (Fig. 2G). These data suggest that the
impairment of β-cell function and/or mass often lead to impaired insulin secretion,
which was not observed in the ETIrs2KO mice.
Insulin secretion was significantly decreased in the ETIrs2KO mice during
pancreatic perfusion
The impairment of insulin secretion observed in vivo, but not in vitro, prompted us to
investigate the secretory responses of the pancreas in perfusion experiments that were
capable of assessing insulin secretion via blood vessels. The insulin responses to
glucose were significantly impaired in the ETIrs2KO mice pancreata at 12 weeks (Fig.
3A). In addition, the amount of secreted insulin (AUCinsulin) after glucose stimulation
(from 5 to 30 min) was also significantly impaired in the ETIrs2KO mice (Fig. 3B).
These data suggest that the impaired insulin secretion in the ETIrs2KO mice is caused
by an impairment in the blood circulation, and not the dysfunction of the β- cells.
The islet blood flow was significantly decreased in the ETIrs2KO mice
The mean blood pressure in anesthetized mice was similar between the control and the
ETIrs2KO mice at 9-12 weeks (Fig. 4A). Although there was no significant difference
in the pancreatic blood flow (Fig. 4B), the number of microspheres in islets and islet
blood flow were significantly decreased in the ETIrs2KO mice, compared with that in
11
Diabetes
the control mice in the basal state (Fig. 4C, D). Moreover, in the insulin-treated state,
islet blood flow was significantly decreased in the ETIrs2KO mice at 9 weeks (Fig. 4E,
Table 1). These findings suggest that the absence of Irs2 in endothelial cells induce a
reduction in the islet blood flow, similar to previous observations in skeletal muscle
(14), leading to the decrease in insulin secretion in these mice.
The capillary area stained with lectin was significantly smaller in the islets from
the ETIrs2KO mice
The capillary area stained with lectin was significantly smaller in the islets from the
ETIrs2KO mice, compared with those from the control mice at 12 weeks (Fig. 5A, B).
However, the number of capillaries in the islets was comparable between the control
and the ETIrs2KO mice (Fig. 5C). Moreover, to assess islet oxygenation, we performed
immunohistchemical staining for pimonidazole. No difference in the frequency of
pimonidazole-positive islets was observed between the control and the ETIrs2KO mice
(Fig. 5D). These findings suggest that the capillary area, but not the number of
capillaries in the islets or islet oxygenation is reduced in the ETIrs2KO mice.
The islet blood flow was improved by treatment with an ACE-inhibitor, enalapril
maleate, resulting in the amelioration of insulin secretion in the ETIrs2KO mice
Recent studies have demonstrated that prevention of angiotensin II-formation, by ACE
inhibition with enalapril maleate, could preferentially increase the islet blood flow as a
result of a vasodilating action (21). It had been demonstrated that islet microvessels may
produce higher levels of angiotensin II than microvessels in the exocrine pancreas, and
therefore may be more sensitive to ACE-inhibition in islets. In this study, the
administration of enalapril maleate had no effect on the mean blood pressure (Fig. 6A),
blood glucose concentrations (Table 2), or pancreatic blood flow (Fig. 6B), as reported
12
Page 12 of 31
Page 13 of 31
Diabetes
previously (21). However, the islet blood flow in the ETIrs2KO mice was restored
significantly, almost to a level comparable to that in the control mice at 9-12 weeks (Fig.
6C). Furthermore, although enalapril maleate had no effect on the glucose-induced
insulin secretion from isolated islets (Fig. 6D), the glucose-induced insulin secretion
and glucose intolerance in the ETIrs2KO mice were significantly restored to levels
equal to those in the control mice (Fig. 6E). These results suggest that the impairment of
insulin secretion might have been caused by the impaired islet blood flow.
DISCUSSION
In this study, we demonstrated that the absence of Irs2 in endothelial cells impairs
insulin secretion (Fig. 1A-D) by reducing the islet blood flow (Fig. 4D). In fact,
following treatment with enalapril maleate, the glucose-stimulated insulin secretion was
restored to levels equal to those observed in the control mice (Fig. 6E), along with an
improvement in the islet blood flow (Fig. 6C). These data suggest that Irs2 in
endothelial cells regulates islet blood flow, mediating insulin secretion. Although
several studies have suggested that impaired insulin secretion was predominantly
caused by β-cell dysfunction (22, 23), the absence of Irs2 in the capillaries also might
cause insulin secretion in type 2 diabetes.
A recent study revealed that the correct integrity of the islet microvasculature is
essential for normal islet function, as it not only provides the means for the transport of
nutrients and oxygen but also ensures adequate paracrine interactions within the
individual islets. β-cell-specific vascular endothelial growth factor-A gene ablation
resulted in glucose intolerance and diabetes (24). These mice exhibited a decreased
density of the microvasculature, and the capillaries exhibited an abnormal
13
Diabetes
morphological appearance. Moreover, β-cell-specific FRK tyrosine kinase transgenic
mice exhibited an impaired glucose-stimulated insulin secretion in vivo (25). Insulin
secretion in isolated islets from these mice was similar to that of control mice. However,
the islet blood flow and capillary lumen diameter in the islets were decreased in these
mice. In these genetically modified animals, although insulin secretion from isolated
islets was maintained, insulin secretion was significantly impaired in vivo. These data
suggest that the disorganization of islet vascularization can impair glucose-stimulated
insulin secretion, even if the function of β-cells is not impaired. Moreover, recent
investigations have demonstrated that pancreatic islet adaptation to insulin resistance is
not limited to change within β-cells but also involves islet-specific neurovascular
remodeling. To accommodate the increased demand for insulin delivery into the
peripheral circulation, islet capillaries expand by dilation and not by angiogenesis (26).
Given these findings, both the correct integrity and function of the capillaries in the
islets might be essential for normal insulin secretion.
Why was the pancreatic blood flow similar but significantly decreased in the islets
from ETIrs2KO mice? In a previous study, although the capillary blood flow after
insulin treatment was impaired in the skeletal muscle of ETIrs2KO mice, this
phenomenon was not observed during fasting (14). These data suggest that endothelial
insulin signaling regulates capillary blood flow after insulin stimulation, but not in the
basal state. It is possible that the islet blood flow in the ETIrs2KO mice was decreased
because of the fact that the capillaries of the islets are constantly exposed to a high
concentration of insulin, whereas the pancreatic blood flow is not because of the lack of
insulin stimulation. In fact, the capillary blood flow was significantly higher in the islets
than in the whole pancreas in the control mice (Fig. 4B, D, 1.38 ± 0.16 vs. 0.73 ± 0.07,
14
Page 14 of 31
Page 15 of 31
Diabetes
P < 0.01).
In a present study, the islet blood flow was significantly decreased in the ETIrs2KO
mice. Although precise mechanisms remain unclear, it seems more likely that it was due
to microvascular dysfunction, but not anatomical abnormalities. In fact, following
treatment with enalapril maleate, the islet blood flow was restored to levels equal to
those observed in control mice via vasodilating action (Fig. 6C). In addition, we did not
detect anatomical abnormalities in islets of ETIrs2KO mice under the vascular staining
(Fig. 5A, C) or the electron microscope (date not shown). Further study is needed to
address this issue.
Recent trials have suggested that inhibitors of RAS, such as ACE inhibitors and
ARBs, may reduce the incidence of new-onset type 2 diabetes in patients with or
without hypertension who have a higher risk of developing diabetes (27-29). In addition
to the amelioration of insulin secretion through the increased islet blood flow induced
by an ACE inhibitor, as seen in this study, the blockade of the RAS during the
development of diabetes has been attributed to improvements in peripheral insulin
sensitivity and β-cell dysfunction (30, 31). ACE inhibitors have been shown to improve
whole-body and skeletal muscle insulin resistance in hypertensive subjects with or
without type 2 diabetes (32-40). The reduction of angiotensin II-mediated vascular
resistance by ACE inhibition may improve insulin-stimulated glucose transport activity
in skeletal muscle (41). This improvement is associated with a favorable adaptive
response in glucose transporter-4 protein levels, glycogen storage, and the activities of
relevant intracellular enzymes of glucose catabolism (42). Moreover, locally generated
and physiologically active RAS components have functions that are distinct from the
classical vasoconstriction and fluid homeostasis actions of systemic RAS. Local RAS
15
Diabetes
can affect islet-cell function and structure in the adult pancreas as well as the
proliferation and differentiation of pancreatic stem/progenitor cells during development
(43, 44). In fact, the blockade of the RAS significantly attenuates islet damage and
restores the β-cell mass by reducing oxidative stress, apoptosis, and attenuating
profibrotic pathways (42, 45). Thus, RAS inhibition may decrease the development and
progression of diabetes through hemodynamic and non-hemodynamic effects or through
the protection of β-cells and non-β-cells.
In conclusion, we demonstrated that the absence of Irs2 in endothelial cells impairs
the islet blood flow, which may be one of the mechanisms responsible for the decrease
in insulin secretion. Thus, Irs2 in endothelial cells may serve as a novel therapeutic
target for preventing and ameliorating type 2 diabetes and metabolic syndrome.
Author Contributions. S.H., N.K., H.S., K.U. and T.K. designed this study and wrote
the manuscript. S.H., N.K., H.S., M.S., I.T., T.K. and K.N. conducted the experimental
research and analyzed the data. M.N. contributed to the data analysis and the
preparation of the manuscript. T.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 accuracy of the data analysis.
Acknowledgments. We wish to express our sincere appreciation to Dr. Wakako
Fujimoto and Dr. Susumu Seino (Division of Cellular and Molecular Medicine, Kobe
University Graduate School of Medicine, Kobe, Japan) for their advice and assistance in
the development of our perfusion experiments. In addition, we thank Emi
Hashimoto-Koga, Katsuko Takasawa, Norie Kowatari-Ohtsuka, Eri Yoshida-Nagata,
Ritsuko Hoshino, Eishin Hirata, Namiko Okajima-Kasuga and Hiroshi Chiyonobu for
16
Page 16 of 31
Page 17 of 31
Diabetes
their excellent technical assistance and animal care.
Funding. This work was supported by a grant for TSBMI from the Ministry of
Education, Culture, Sports, Science and Technology of Japan, and a Grant-in-aid for
Scientific Research in Priority Areas (A) (18209033) and (S) (20229008) from the
Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. K.).
Duality of Interest. No potential conflicts of interest relevant to this article were
reported.
17
Diabetes
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
REFERENCES
Kadowaki T, Miyake Y, Hagura R, Akanuma Y, Kajinuma H, Kuzuya N, Takaku F,
Kosaka K. Risk factors for worsening to diabetes in subjects with impaired glucose
tolerance. Diabetologia 1984;26:44-9
Kadowaki T. Insights into insulin resistance and type 2 diabetes from knockout
mouse models. J Clin Invest 2000;106:459-65
Reaven GM. HOMA-beta in the UKPDS and ADOPT. Is the natural history of type
2 diabetes characterised by a progressive and inexorable loss of insulin secretory
function? Maybe? Maybe not? Diab Vasc Dis Res 2009;6:133-8
Movassat J, Bailbé D, Lubrano-Berthelier C, Picarel-Blanchot F, Bertin E, Mourot
J, Portha B. Follow-up of GK rats during prediabetes highlights increased insulin
action and fat deposition despite low insulin secretion. Am J Physiol Endocrinol
Metab 2008;294:E168-75
Richards OC, Raines SM, Attie AD. The role of blood vessels, endothelial cells,
and vascular pericytes in insulin secretion and peripheral insulin action. Endocr Rev
2010;31:343-63
Jansson L. The regulation of pancreatic islet blood flow. Diabetes Metab Rev
1994;10:407-16.
Iwase M, Nakamura U, Uchizono Y, Nohara S, Sasaki N, Sonoki K, Iida M.
Nateglinide, a non-sulfonylurea rapid insulin secretagogue, increases pancreatic
capillary blood volume in the islet in rats. Eur J Pharmacol 2005;518:243-50
Pollare T, Lithell H, Berne C. A comparison of the effects of hydrochlorothiazide
and captopril on glucose and lipid metabolism in patients with hypertension. N
Engl J Med 1989;321:868-73
Haenni A, Andersson PE, Lind L, Berne C, Lithell H. Electrolyte changes and
metabolic effects of lisinopril/bendrofluazide treatment. Results from a randomized,
double-blind study with parallel groups. Am J Hypertens 1994;7: 615-22
Santoro D, Natali A, Palombo C, Brandi LS, Piatti M, Ghione S, Ferrannini E.
Effects of chronic angiotensin converting enzyme inhibition on glucose tolerance
and insulin sensitivity in essential hypertension. Hypertension 1992;20:181-91
Huang Z1, Jansson L, Sjöholm A. Vasoactive drugs enhance pancreatic islet blood
flow, augment insulin secretion and improve glucose tolerance in female rats. Clin
Sci (Lond) 2007;112:69-76
Nyström T1, Ortsäter H, Huang Z, Zhang F, Larsen FJ, Weitzberg E, Lundberg JO,
Sjöholm Å.Inorganic nitrite stimulates pancreatic islet blood flow and insulin
secretion. Free Radic Biol Med 2012;53:1017-23
Kubota T, Kubota N, Moroi M, Terauchi Y, Kobayashi T, Kamata K, Suzuki R,
Tobe K, Namiki A, Aizawa S, Nagai R, Kadowaki T, Yamaguchi T. Lack of insulin
receptor substrate-2 causes progressive neointima formation in response to vessel
injury. Circulation 2003;107:3073-80
Kubota T, Kubota N, Kumagai H, Yamaguchi S, Kozono H, Takahashi T, Inoue M,
Itoh S, Takamoto I, Sasako T, Kumagai K, Kawai T, Hashimoto S, Kobayashi T,
Sato M, Tokuyama K, Nishimura S, Tsunoda M, Ide T, Murakami K, Yamazaki T,
Ezaki O, Kawamura K, Masuda H, Moroi M, Sugi K, Oike Y, Shimokawa H,
Yanagihara N, Tsutsui M, Terauchi Y, Tobe K, Nagai R, Kamata K, Inoue K,
Kodama T, Ueki K, Kadowaki T. Impaired insulin signaling in endothelial cells
reduces insulin-induced glucose uptake by skeletal muscle. Cell Metab.
18
Page 18 of 31
Page 19 of 31
Diabetes
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
2011;13:294-307
Kubota N, Terauchi Y, Tobe K, Yano W, Suzuki R, Ueki K, Takamoto I, Satoh H,
Maki T, Kubota T, Moroi M, Okada-Iwabu M, Ezaki O, Nagai R, Ueta Y,
Kadowaki T, Noda T. Insulin receptor substrate 2 plays a crucial role in beta cells
and the hypothalamus. J Clin Invest 2004;114:917-27
Terauchi Y, Takamoto I, Kubota N, Matsui J, Suzuki R, Komeda K, Hara A,
Toyoda Y, Miwa I, Aizawa S, Tsutsumi S, Tsubamoto Y, Hashimoto S, Eto K,
Nakamura A, Noda M, Tobe K, Aburatani H, Nagai R, Kadowaki T. Glucokinase
and IRS-2 are required for compensatory beta cell hyperplasia in response to
high-fat diet-induced insulin resistance. J Clin Invest 2007;117: 246-57
Olsson R1, Carlsson PO. A low-oxygenated subpopulation of pancreatic islets
constitutes a functional reserve of endocrine cells. Diabetes 2011;60:2068-75
Miki T, Minami K, Shinozaki H, Matsumura K, Saraya A, Ikeda H, Yamada Y,
Holst JJ, Seino S. Distinct effects of glucose-dependent insulinotropic polypeptide
and glucagon-like peptide-1 on insulin secretion and gut motility. Diabetes
2005;54:1056-63
Jansson L, Hellerström C. A rapid method of visualizing the pancreatic islets for
studies of islet capillary blood flow using non-radioactive microspheres. Acta
Physiol Scand 1981;113:371-4
Sparrow RA1, Beckingham IJ. Islet blood flow following insulin administration. J
Anat 1989;163:75-81
Carlsson PO, Berne C, Jansson L. Angiotensin II and the endocrine pancreas:
effects on islet blood flow and insulin secretion in rats. Diabetologia
1998;41:127-33
Ueki K, Okada T, Hu J, Liew CW, Assmann A, Dahlgren GM, Peters JL,
Shackman JG, Zhang M, Artner I, Satin LS, Stein R, Holzenberger M, Kennedy RT,
Kahn CR, Kulkarni RN. Total insulin and IGF-I resistance in pancreatic beta cells
causes overt diabetes. Nat Genet 2006;38:583-8
Goren HJ. Role of insulin in glucose-stimulated insulin secretion in beta cells. Curr
Diabetes Rev 2005;1:309-30
Lammert E, Gu G, McLaughlin M, Brown D, Brekken R, Murtaugh LC, Gerber HP,
Ferrara N, Melton DA. Role of VEGF-A in vascularization of pancreatic islets.
Curr Biol 2003;13:1070-4
Annerén C, Welsh M, Jansson L. Glucose intolerance and reduced islet blood flow
in transgenic mice expressing the FRK tyrosine kinase under the control of the rat
insulin promoter. Am J Physiol Endocrinol Metab 2007;292:E1183-90
Dai C1, Brissova M, Reinert RB, Nyman L, Liu EH, Thompson C, Shostak A,
Shiota M, Takahashi T, Powers AC. Pancreatic islet vasculature adapts to insulin
resistance through dilation and not angiogenesis. Diabetes 2013;62:4144-53
Zanchetti A, Ruilope LM. Antihypertensive treatment in patients with type-2
diabetes mellitus: what guidance from recent controlled randomized trials? J
Hypertens 2002;20:2099-110
Suzuki K, Nakagawa O, Aizawa Y. Improved early-phase insulin response after
candesartan treatment in hypertensive patients with impaired glucose tolerance.
Clin Exp Hypertens 2008;30:309-14
Jandeleit-Dahm KA, Tikellis C, Reid CM, Johnston CI, Cooper ME. Why blockade
of the renin-angiotensin system reduces the incidence of new-onset diabetes. J
19
Diabetes
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
Hypertens 2005;23:463-73
Kahn SE, Andrikopoulos S, Verchere CB. Islet amyloid: a long-recognized but
underappreciated pathological feature of type 2 diabetes. Diabetes. 1999;48:241-53
McFarlane SI, Kumar A, Sowers JR. Mechanisms by which angiotensin-converting
enzyme inhibitors prevent diabetes and cardiovascular disease. Am J Cardiol
2003;91:30H-37H
Jauch KW, Hartl W, Guenther B, Wicklmayr M, Rett K, Dietze G. Captopril
enhances insulin responsiveness of forearm muscle tissue in non-insulin-dependent
diabetes mellitus. Eur J Clin Invest 1987;17:448-54
Gans RO, Bilo HJ, Nauta JJ, Popp-Snijders C, Heine RJ, Donker AJ. The effect of
angiotensin-I converting enzyme inhibition on insulin action in healthy volunteers.
Eur J Clin Invest 1991;21:527-33
Torlone E, Rambotti AM, Perriello G, Botta G, Santeusanio F, Brunetti P, Bolli GB.
ACE-inhibition increases hepatic and extrahepatic sensitivity to insulin in patients
with type 2 (non-insulin-dependent) diabetes mellitus and arterial hypertension.
Diabetologia 1991;34:119-25
Shieh SM, Sheu WH, Shen DD, Fuh MM, Jeng CY, Jeng JR, Chen YD, Reaven
GM. Improvement in metabolic risk factors for coronary heart disease associated
with cilazapril treatment. Am J Hypertens 1992;5:506-10
Paolisso G, Gambardella A, Verza M, D'Amore A, Sgambato S, Varricchio M.
ACE inhibition improves insulin-sensitivity in aged insulin-resistant hypertensive
patients. J Hum Hypertens 1992;6:175-9
Morel Y, Gadient A, Keller U, Vadas L, Golay A. Insulin sensitivity in obese
hypertensive dyslipidemic patients treated with enalapril or atenolol. J Cardiovasc
Pharmacol 1995;26:306-11
Shamiss A, Carroll J, Peleg E, Grossman E, Rosenthal T. The effect of enalapril
with and without hydrochlorothiazide on insulin sensitivity and other metabolic
abnormalities of hypertensive patients with NIDDM. Am J Hypertens
1995;8:276-81
Suzuki M, Ikebuchi M, Yokota C, Shinozaki K, Harano Y. (1995) Normalization of
insulin resistance in non-obese essential hypertension by cilazapril treatment. Clin
Exp Hypertens 1995;17:1257-68
Lender D, Arauz-Pacheco C, Breen L, Mora-Mora P, Ramirez LC, Raskin P. A
double blind comparison of the effects of amlodipine and enalapril on insulin
sensitivity in hypertensive patients. Am J Hypertens 1999;12:298-303
Jacob S, Henriksen EJ, Fogt DL, Dietze GJ. Effects of trandolapril and verapamil
on glucose transport in insulin-resistant rat skeletal muscle. Metabolism.
1996;45:535-41
Bergman RN, Finegood DT, Kahn SE. The evolution of beta-cell dysfunction and
insulin resistance in type 2 diabetes. Eur J Clin Invest 2002;32: 35-45
Leung PS. The physiology of a local renin-angiotensin system in the pancreas. J
Physiol 2007;580:31-7
Wang L1, Leung PS. The role of renin-angiotensin system in cellular
differentiation: implications in pancreatic islet cell development and islet
transplantation. Mol Cell Endocrinol 2013;381:261-71
Tikellis C, Wookey PJ, Candido R, Andrikopoulos S, Thomas MC, Cooper ME.
Improved islet morphology after blockade of the renin- angiotensin system in the
20
Page 20 of 31
Page 21 of 31
Diabetes
ZDF rat. Diabetes 2004;53:989-97
21
Diabetes
Page 22 of 31
Table 1. Effect of insulin on blood glucose level.
Strain
No. of animals
The change of blood glucose, mmol/L
Control
ETIrs2KO
10
10
-2.69 ± 0.35
-2.58 ± 0.47
Values are the mean ± SE
Table 2. Effect of enalapril maleate on blood glucose and plasma insulin levels.
Strain
Control
ETIrs2KO
ETIrs2KO
Substance given
Saline
Saline
Enalapril
maleate
No. of animals
10
10
10
Blood glucose, mmol/L
6.23 ± 0.19
6.47 ± 0.20
6.21 ± 0.24
Plasma insulin, nmol/L
0.23 ± 0.03
0.26 ± 0.05
0.22 ± 0.04
Values are the mean ± SE
22
Page 23 of 31
Diabetes
FIGURE LEGENDS
Figure 1- Insulin secretion was impaired in the ETIrs2KO mice. Blood glucose levels
(left) and plasma insulin levels (right) during an intraperitoneal glucose tolerance test in
the control and the ETIrs2KO mice at 12 (A) and 24 (B) weeks (n = 9-10). (C) Plasma
insulin levels during an intraperitoneal glucagon tolerance test in the control and the
ETIrs2KO mice at 14 weeks (n = 9-10). (D) Plasma insulin levels during an
intraperitoneal arginine tolerance test in the control and the ETIrs2KO mice at 13 weeks
(n = 9-10). Values are the mean ± SE. Statistical significance is depicted as * (P < 0.05).
Figure 2- Insulin secretion in the isolated islets was not impaired in the ETIrs2KO mice.
(A) Glucose-induced insulin secretion using batch-incubated islets at 12 weeks (n = 9).
(B) Glucose-induced insulin secretion as assessed in an islet perifusion experiment at 12
weeks (n = 8). (C) mRNA expression levels expressed as the values in the islets of the
ETIrs2KO mice relative to those of the control mice at 12 weeks (n = 5). (D) Insulin
content per DNA concentration in islets isolated from the control and the ETIrs2KO
mice at 12 weeks (n = 8). (E) Insulin staining of pancreatic sections from the control
and the ETIrs2KO mice at 24 weeks (scale bar = 100 µm). (F) The β-cell mass was
calculated as estimated islet weight (n = 5). (G) Replication rate of β-cells assayed on
the basis of BrdU incorporation in the control and the ETIrs2KO mice at 24 weeks (n =
7). Results are shown as the percentage of BrdU-positive cells relative to the total
number of β-cells. Values are the mean ± SE.
Figure 3- Insulin secretion was significantly decreased in the ETIrs2KO mice during
pancreatic perfusion. (A) Insulin secretion from perfused pancreata of the control and
23
Diabetes
the ETIrs2KO mice at 12 weeks (n = 8). (B) Amounts of insulin secreted in the control
and the ETIrs2KO mice after glucose stimulation expressed as the AUCinsulin values
from 5 to 30 min in A. Values are the mean ± SE. Statistical significance is depicted as *
(P < 0.05) and ** (P < 0.01).
Figure 4- The islet blood flow was significantly decreased in the ETIrs2KO mice. The
mean blood pressure (A), pancreatic blood flow (B), the number of microspheres in islet
tissue (C) and islet blood flow (D) in anaesthetized 9-12-week-old control and
ETIrs2KO mice in the basal state (n = 10). The islet blood flow (E) in anaesthetized
9-week-old control and ETIrs2KO mice in insulin-treated state (n = 10) Values are the
mean ± SE. Statistical significance is depicted as * (P < 0.05) and *** (P < 0.001).
Figure 5- The capillary area stained by lectin was significantly decreased in the islets of
the ETIrs2KO mice. Lectin staining of pancreatic section from the control and the
ETIrs2KO mice at 12 weeks (scale bar = 50 µm). The approximate islet boundary is
marked by a dotted line (A). Vessel area expressed as a relative value to the total islet
volume % (B) and the number of capillaries per square millimeters of islet (C).
Pimonidazole-positive islets were quantitatively assessed as a percentage of the total
number of islets (D). Values are the mean ± SE for 5 animals. Statistical significance is
depicted as *** (P < 0.001).
Figure 6- The islet blood flow was improved by treatment with an ACE-inhibitor,
enalapril maleate, resulting in the amelioration of insulin secretion in the ETIrs2KO
mice. The mean blood pressure (A), the pancreatic blood flow (B), and the islet blood
24
Page 24 of 31
Page 25 of 31
Diabetes
flow (C) in anaesthetized 9-12-week-old control and ETIrs2KO mice at 10 min after the
intravenous injection of saline or enalapril maleate (n = 10). (D) Effect of enalapril
maleate on glucose-induced insulin secretion using batch-incubated islets of C57BL/6J
mice at 12 weeks (n = 5). (E) Blood glucose levels (left) and plasma insulin levels
(right) during an intraperitoneal glucose tolerance test in 12-week-old control and
ETIrs2KO mice at 10 min after the intravenous injection of saline or enalapril maleate
(n = 11-13). Values are the mean ± SE. Statistical significance is depicted as * (P < 0.05,
control + saline vs. ETIrs2KO mice + saline) and # (P < 0.05, ETIrs2KO mice + saline
vs. ETIrs2KO mice + enalapril).
25
Diabetes
Figure 1- Insulin secretion was impaired in the ETIrs2KO mice. Blood glucose levels (left) and plasma insulin
levels (right) during an intraperitoneal glucose tolerance test in the control and the ETIrs2KO mice at 12 (A)
and 24 (B) weeks (n = 9-10). (C) Plasma insulin levels during an intraperitoneal glucagon tolerance test in
the control and the ETIrs2KO mice at 14 weeks (n = 9-10). (D) Plasma insulin levels during an
intraperitoneal arginine tolerance test in the control and the ETIrs2KO mice at 13 weeks (n = 9-10). Values
are the mean ± SE. Statistical significance is depicted as * (P < 0.05).
190x274mm (284 x 284 DPI)
Page 26 of 31
Page 27 of 31
Diabetes
Figure 2- Insulin secretion in the isolated islets was not impaired in the ETIrs2KO mice. (A) Glucose-induced
insulin secretion using batch-incubated islets at 12 weeks (n = 9). (B) Glucose-induced insulin secretion as
assessed in an islet perifusion experiment at 12 weeks (n = 8). (C) mRNA expression levels expressed as
the values in the islets of the ETIrs2KO mice relative to those of the control mice at 12 weeks (n = 5). (D)
Insulin content per DNA concentration in islets isolated from the control and the ETIrs2KO mice at 12 weeks
(n = 8). (E) Insulin staining of pancreatic sections from the control and the ETIrs2KO mice at 24 weeks
(scale bar = 100 µm). (F) The β-cell mass was calculated as estimated islet weight (n = 5). (G) Replication
rate of β-cells assayed on the basis of BrdU incorporation in the control and the ETIrs2KO mice at 24 weeks
(n = 7). Results are shown as the percentage of BrdU-positive cells relative to the total number of β-cells.
Values are the mean ± SE.
190x274mm (284 x 284 DPI)
Diabetes
Figure 3- Insulin secretion was significantly decreased in the ETIrs2KO mice during pancreatic perfusion. (A)
Insulin secretion from perfused pancreata of the control and the ETIrs2KO mice at 12 weeks (n = 8). (B)
Amounts of insulin secreted in the control and the ETIrs2KO mice after glucose stimulation expressed as the
AUCinsulin values from 5 to 30 min in A. Values are the mean ± SE. Statistical significance is depicted as *
(P < 0.05) and ** (P < 0.01).
190x274mm (284 x 284 DPI)
Page 28 of 31
Page 29 of 31
Diabetes
Figure 4- The islet blood flow was significantly decreased in the ETIrs2KO mice. The mean blood pressure
(A), pancreatic blood flow (B), the number of microspheres in islet tissue (C) and islet blood flow (D) in
anaesthetized 9-12-week-old control and ETIrs2KO mice in the basal state (n = 10). The islet blood flow (E)
in anaesthetized 9-week-old control and ETIrs2KO mice in insulin-treated state (n = 10) Values are the
mean ± SE. Statistical significance is depicted as * (P < 0.05) and *** (P < 0.001).
190x274mm (284 x 284 DPI)
Diabetes
Figure 5- The capillary area stained by lectin was significantly decreased in the islets of the ETIrs2KO mice.
Lectin staining of pancreatic section from the control and the ETIrs2KO mice at 12 weeks (scale bar = 50
µm). The approximate islet boundary is marked by a dotted line (A). Vessel area expressed as a relative
value to the total islet volume % (B) and the number of capillaries per square millimeters of islet (C).
Pimonidazole-positive islets were quantitatively assessed as a percentage of the total number of islets (D).
Values are the mean ± SE for 5 animals. Statistical significance is depicted as *** (P < 0.001).
190x274mm (284 x 284 DPI)
Page 30 of 31
Page 31 of 31
Diabetes
Figure 6- The islet blood flow was improved by treatment with an ACE-inhibitor, enalapril maleate, resulting
in the amelioration of insulin secretion in the ETIrs2KO mice. The mean blood pressure (A), the pancreatic
blood flow (B), and the islet blood flow (C) in anaesthetized 9-12-week-old control and ETIrs2KO mice at 10
min after the intravenous injection of saline or enalapril maleate (n = 10). (D) Effect of enalapril maleate on
glucose-induced insulin secretion using batch-incubated islets of C57BL/6J mice at 12 weeks (n = 5). (E)
Blood glucose levels (left) and plasma insulin levels (right) during an intraperitoneal glucose tolerance test
in 12-week-old control and ETIrs2KO mice at 10 min after the intravenous injection of saline or enalapril
maleate (n = 11-13). Values are the mean ± SE. Statistical significance is depicted as * (P < 0.05, control +
saline vs. ETIrs2KO mice + saline) and # (P < 0.05, ETIrs2KO mice + saline vs. ETIrs2KO mice + enalapril).
190x274mm (284 x 284 DPI)