Am J Physiol Endocrinol Metab 307: E115–E123, 2014.
First published May 20, 2014; doi:10.1152/ajpendo.00131.2014.
Consumption of a Western-style diet during pregnancy impairs offspring islet
vascularization in a Japanese macaque model
Lynley D. Pound,1 Sarah M. Comstock,1 and Kevin L. Grove1,2
1
2
Division of Diabetes, Obesity, and Metabolism, Oregon National Primate Research Center, Beaverton, Oregon; and
Division of Reproductive and Developmental Science, Oregon National Primate Research Center, Beaverton, Oregon
Submitted 14 March 2014; accepted in final form 15 May 2014
Pound LD, Comstock SM, Grove KL. Consumption of a
Western-style diet during pregnancy impairs offspring islet vascularization in a Japanese macaque model. Am J Physiol Endocrinol
Metab 307: E115–E123, 2014. First published May 20, 2014;
doi:10.1152/ajpendo.00131.2014.—Children exposed to a maternal
Western-style diet in utero have an increased risk of developing type
2 diabetes. Understanding the mechanisms and an investigation of
possible interventions are critical to reversing this phenomenon. We
examined the impact of maternal Western-style diet consumption on
the development of islet vascularization and innervation, both of
which are critical to normal islet function, in fetal and juvenile
offspring. Furthermore, we assessed whether improved dietary intake
or resveratrol supplementation could ameliorate the harmful consequences of Western-style diet consumption during pregnancy. Adult
female Japanese macaques were maintained on a control or Westernstyle diet for 4 –7 yr. One cohort of dams was switched back onto a
control diet, whereas another cohort received resveratrol supplementation throughout gestation. Pregnancies were terminated in the early
third trimester by C-section, or offspring were born naturally and sent
to necropsy at 1 yr of age. Western-style diet consumption resulted in
impaired fetal islet capillary density and sympathetic islet innervation.
Furthermore, this reduction in vascularization persisted in the juvenile
offspring. This effect is independent of changes in the expression of
key angiogenic markers. Diet reversal normalized islet vascularization
to control offspring levels, whereas resveratrol supplementation
caused a significant increase in capillary density above controls. These
data provide a novel mechanism by which maternal Western-style diet
consumption leads to increased susceptibility to type 2 diabetes in the
offspring. Importantly, an improved maternal diet may mitigate these
harmful effects. However, until the long-term consequences of increased vascularization can be determined, resveratrol use during
pregnancy is not advised.
high fat diet; nonhuman primate; pregnancy; resveratrol; vascularization
has risen dramatically in
the US, almost tripling in the past 30 years (48) and leading to
an increased risk for a range of metabolic complications,
including type 2 diabetes, cardiovascular disease, and stroke
(24, 58). Although diet and lack of physical activity play a key
role in this epidemic, a suboptimal in utero environment poses
significant risks to the developing fetus. Specifically, maternal
consumption of an obesogenic diet and increased maternal
adiposity prior to gestation contribute to an increased predisposition for the development of obesity and type 2 diabetes in
the offspring (7, 37, 57). The relationship between poor maternal nutrition and excessive weight gain with type 2 diabetes
predisposition in the offspring is poorly understood but likely
THE PREVALENCE OF CHILDHOOD OBESITY
Address for reprint requests and other correspondence: K. L. Grove, Oregon
National Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006
(e-mail: grovek@ohsu.edu).
http://www.ajpendo.org
involves a number of detrimental developmental changes in
key fetal metabolic organs, such as the pancreatic islet (reviewed in Ref. 17).
Our group has developed a model of maternal Western-style
diet (WSD) consumption during pregnancy in the Japanese
macaque (Macaca fuscata), a nonhuman primate (NHP) model
whose islet closely resembles the human islet in development,
structure, and function (12, 35). Previous data from our group
have indicated that maternal WSD consumption during pregnancy leads to a broad range of complications in the fetal and
juvenile offspring (21, 25–29, 43, 44, 48, 52–54). Specifically,
maternal exposure to a WSD is associated with placental
insufficiency (25), a reduction in islet ␣-cell mass, and an
increase in the ␤-cell/␣-cell ratio (21), which may have a
negative impact on islet paracrine interactions and thus function. Vascularization and innervation also play key roles in
intraislet communication as well as in the development of the
islet (reviewed in Ref. 18). The pancreatic islets are highly
vascularized micro-organs, and this dense capillary network
plays a critical role in its ability to detect changes in glycemia
and respond accordingly (10, 41, 47). Furthermore, intraislet
capillaries promote exposure to metabolic stimuli and critical
growth factors that stimulate normal islet development (20).
Similarly, angiogenic markers such as vascular endothelial
growth factor A (VEGF-A), angiopoietins, and ephrins are
produced by the islet and serve to enhance islet angiogenesis
(9, 49, 59). Islet innervation, like vascularization, plays a key
role in communication and function. Whereas sympathetic
fibers and their neurotransmitters, mediated primarily by norepinephrine, inhibit insulin secretion and stimulate glucagon
secretion, parasympathetic neurotransmitters stimulate insulin
secretion (reviewed in Ref. 3).
To date, however, the role of vascularization and innervation
in altered fetal islet development following maternal WSD
consumption has not been investigated. Extensive remodeling
of the islet and its vascularization occurs during the second half
of gestation, during which time islets become innervated (23).
Thus, we investigated the effect of WSD consumption during
pregnancy on islet vascularization and innervation in both the
fetus during the early third trimester and the 1-yr old juvenile
offspring. We hypothesized that exposure to WSD in utero
would result in altered vascularization and innervation, mirroring defects in islet development, that will persist into the
juvenile offspring.
Because the second half of gestation is a critical developmental period for the islet, therapeutic interventions during this
stage may mitigate some or all of the adverse effects of in utero
WSD exposure. Importantly, we have tested both lifestyle
modification as well as dietary supplementation with resveratrol to determine whether these might provide novel and
0193-1849/14 Copyright © 2014 the American Physiological Society
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clinically relevant methods to manage the detrimental developmental outcomes. In particular, we hypothesized that improved dietary intake during pregnancy by women who have
been chronically consuming a WSD could ameliorate the
harmful consequences on fetal development independently of
prevailing obesity and adiposity in the mother. Furthermore,
resveratrol, a small naturally occurring polyphenol, has garnered significant attention recently for its anti-aging, antiinflammatory, and provascular properties (5, 38) and thus is an
appealing option for women consuming a WSD. We hypothesized that the vasodilatory actions of resveratrol would improve WSD-induced impairments in the development of the
islet vasculature.
MATERIALS AND METHODS
Animal care. All animal procedures were conducted in accordance
with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Oregon National Primate Research Center
(ONPRC) and Oregon Health and Science University and were
approved by the ONPRC IACUC. The ONPRC abides by the Animal
Welfare Act and Regulations enforced by the USDA and the Public
Health Service Policy on Humane Care and Use of Laboratory
Animals in accordance with the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of Health.
Adult NHP model. Five adult male rhesus macaques (Macaca
mulatta) aged 9 –13 yr were fed ad libitum with a CTR diet (14%
calories from fat; Test Diet). Tissue was collected at necropsy by a
Veterinary Pathologist, as described previously (46). The pancreas
was isolated and divided into sections from head to tail to be fixed in
zinc formalin for immunohistochemical analysis.
Maternal WSD NHP model. This animal model has been described
previously (43). Briefly, Japanese macaques were provided ad libitum
with a control (CTR) diet (14% calories from fat) or WSD (32%
calories from fat, Test Diet; 5L0P, Purina Mills) supplemented with
calorically dense treats for 4 –7 yr. Metabolizable energy from CTR
and WSD diets was 2.87 and 3.80 kcal/g, respectively. The diet
reversal (REV) cohort was maintained on the WSD for 4 yr before
being switched onto the CTR diet during the breeding season of the
5th yr. The WSD/resveratrol (RESV) cohort received the WSD for 7
yr prior to being switched onto a WSD containing resveratrol (Highpurity trans-resveratrol, Resvida; DSM Nutritional Products) at a final
concentration of 0.37% 3 mo prior to the breeding season. Animals
were housed in indoor/outdoor pens with one to two males and three
to 11 females and were allowed to breed naturally. Gestational age
was estimated by ultrasound technology, as described previously (25),
and pregnancies were terminated by Cesarean section (C-section) at
GD130 in the Surgical Services Unit. Both male and female offspring
were used. Maternal body weight and composition and fetal body
weights can be found in Refs. 21, 43, and 52.
Juvenile studies were performed as described previously (21).
Briefly, male and female offspring were born naturally and maintained
on the dam’s diet until weaning. At ⬃8 mo, offspring were either
weaned onto their in utero diet or switched onto the opposing diet to
generate four treatment groups: CTR/CTR, CTR/WSD, WSD/CTR,
and WSD/WSD.
Figure 2, A and B, provides an overview of fetal and juvenile study
design, respectively.
Maternal intravenous glucose tolerance tests. Intravenous glucose
tolerance tests were performed on overnight-fasted dams at 1 wk prior
to C-section, as described previously (43). Briefly, animals were
sedated and administered a glucose bolus of 0.6 g/kg body wt via the
saphenous vein. Maternal insulin secretion was calculated as area
under the curve from fasting value at t ⫽ 0.
Tissue collection. Following C-section, fetuses were taken to the
Pathology Unit for full necropsy. Juvenile animals were sent to
necropsy at ⬃13 mo of age. Fetal and juvenile organs were dissected
by the veterinary pathologist, weights and measures were documented, and individual tissues were processed according to protocol
requirements. The pancreas was isolated and divided into sections
from head to tail to be fixed in zinc formalin for immunohistochemical
analysis or flash-frozen in liquid nitrogen for RNA isolation, as
described previously (21).
Pancreas RNA isolation and quantitative RT-PCR. Total RNA
isolation, reverse transcription, and quantitative PCR were performed
as described previously (21). RNA polymerase II was used to normalize real-time expression using the Pfaffl method (51).
Islet immunohistochemistry and quantitative analysis. Five-micrometer paraffin-embedded sections from the pancreatic tail were deparaffinized, and standard immunohistochemical methods were used for
subsequent analyses (21). Primary antibodies were applied for 48 h at
4°C. The following primary antibodies were used to detect pancreatic
antigens: guinea pig anti-insulin (1:2,500; Dako), rabbit anti-glucagon
(1:100; Cell Signaling Technology), sheep anti-platelet endothelial
cell adhesion molecule 1 (PECAM1; 1:250; R & D Systems), goat
anti-VEGF-A (1:100; R & D Systems), mouse anti-Ki67 (1:25; Dako)
and mouse anti-TH (1:100; Millipore). Secondary antibodies were
applied for 1 h at room temperature (1:1,500; Jackson ImmunoResearch Laboratories).
Images were acquired with the Marianas imaging workstation
(Intelligent Imaging Innovations, Denver, CO), as described previously (21). Cell vascularization and innervation were assessed using
the stereology module within the tail region of each pancreata. Vessels
and nerves were counted manually, where any fluorescently labeled
vessel or nerve fiber within the islet or making contact with an islet
was included. PECAM1⫹ area was calculated by intensity segmentation, using Slidebook 5.0 imaging software. Vascularization is
expressed as either number of capillaries or PECAM1⫹ area per islet
area. Area per capillary is calculated by dividing the total PECAM1⫹
area by the number of PECAM1⫹ fibers. Vascular proliferation was
measured by colocalization of PECAM1 with the proliferation marker
Ki67. Sympathetic innervation is expressed as number of TH⫹ fibers
per islet area. Representative images were acquired with the Leica
SP5 confocal microscope using the ⫻40 objective at 1,024 ⫻ 1,024
pixel resolution. Focal planes were 1 ␮m apart.
Data analysis. Fetal data were analyzed using a one-way ANOVA
with Tukey’s multiple comparison post hoc analysis (CTR vs. WSD
vs. REV vs. WSD/RESV). Juvenile data were analyzed using a
two-way ANOVA to test for maternal or postweaning diet effect.
Correlation analyses were performed using SPSS Statistics 19 software (Armonk, NY).
RESULTS
The adult NHP islet is highly vascularized and expresses
VEGF-A in the pancreatic ␤-cell. Although rodent and human
islet vasculature and its angiogenic signals have been relatively
well characterized, the vasculature in the NHP islet has not
been investigated previously. We sought to define and quantify
vascularization in the healthy NHP islet. Consistent with data
in both the rodent (10) and the human (reviewed in Ref. 31),
the NHP adult islet is highly vascularized compared with the
surrounding exocrine tissue, possessing an approximately sixfold greater capillary density (Fig. 1, A and B). In addition, we
investigated the expression pattern of VEGF-A, which in the
rodent is highly expressed and secreted by the pancreatic
␤-cell, where it recruits endothelial cells and promotes capillary growth and angiogenesis (6, 19, 32). Similarly, we demonstrated that the NHP ␤-cell expresses VEGF-A, indicating
overlapping angiogenic mechanisms between mammalian species (Fig. 1C).
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a
0.2
0.1
b
0.0
ENDO
EXO
a
3000
150
2000
1000
b
Area/capillary (µm2)
0.3
Capillary density (# mm-2)
B
Capillary area/endo or
exo area
A
Insulin/Glucagon/PECAM1
DAM DIET AND OFFSPRING ISLET VASCULATURE
100
50
0
0
ENDO
EXO
ENDO
EXO
Merge
VEGFA
Insulin
C
Fig. 1. Normal vascularization of the nonhuman primate (NHP) adult islet. A: representative immunohistochemical (IHC) image of endocrine and exocrine
vascularization in the adult control (CTR) pancreas. B: quantification of capillary area, capillary density, and area per capillary in endocrine (ENDO) and exocrine
(EXO) pancreatic tissue (n ⫽ 5 males). Data are expressed as means ⫾ SE. Different supercscripted letters indicate a significant difference (P ⬍ 0.05).
C: representative IHC images of VEGF-A expression in adult CTR pancreas. Scale bars represent 25 ␮m.
Maternal WSD consumption impairs the development of the
islet vasculature in the fetal offspring. We have demonstrated
previously that WSD consumption during pregnancy results in
a significant reduction in ␣-cell mass and an increase in the
␤-cell/␣-cell ratio by the early third trimester (21). Because
islet vascularization is critical for normal islet development and
defects in vascularization often precede changes in islet morphology (30), we hypothesized that exposure to a WSD in
utero would result in impaired islet vascularity. We used
PECAM1 to visualize islet capillaries in the fetal pancreas at
gestational day (GD) 130, where term is ⬃170 days. WSD
offspring display a significant reduction in islet capillary area
(Fig. 3, A and B). This effect is due to both a decrease in the
number of islet-associated capillaries (Fig. 3C) and a smaller
average size of the remaining vessels (Fig. 3D), possibly
indicative of impaired capillary dilation. Interestingly, increased maternal insulin secretion during a glucose tolerance
test was weakly associated with reduced islet vascularization
(Fig. 3E), an effect that was influenced but not entirely dependent on three high-responding dams. These data indicate that
maternal insulin resistance may increase the risk of maladaptive islet development.
Improved maternal nutrition and maternal resveratrol supplementation increase islet vascularization. To investigate
whether the detrimental effects of maternal WSD consumption
on islet vascularization could be mitigated, we characterized
two models of dietary intervention. First, dams that had been
maintained on a WSD were switched onto a healthy CTR diet
prior to pregnancy (REV) (Fig. 2A). REV dams do not lose a
substantial amount of body weight, nor do they display significant metabolic improvements during this time (43). However,
diet reversal normalized islet vascular area (Fig. 3B), capillary
density (Fig. 3C), and average area per capillary (Fig. 3D) in
the fetal offspring at GD130 at a time when ␣-cell mass was
increased by 28% (6.525 vs. 5.086 mg in WSD) and the
␤-cell/␣-cell ratio decreased by 28% (1.889 vs. 2.611 in WSD)
with no change in islet mass compared with WSD offspring.
Similarly, islet size distribution did not differ between the
cohorts (data not shown).
In an additional cohort of WSD-fed dams, we investigated
whether resveratrol supplementation during pregnancy could
improve the WSD-induced impairment in islet vascularization.
Maternal resveratrol supplementation led to a significant increase in fetal islet capillary area (Fig. 3B), density (Fig. 3C),
and area per capillary (Fig. 3D) compared with WSD fetal
islets. However, supplementation also resulted in a 57% increase in capillary density over CTR offspring (Fig. 3C).
Alterations in islet vasculature are not mediated by changes
in angiogenesis. The development of the vasculature is tightly
regulated through a complex signaling network consisting of
both angiogenic growth factors and their respective receptors
(1, 4, 15, 50). To promote the dense capillary network within
the islet, islet cell types express several angiogenic factors to
attract and stimulate vessel growth and proliferation (Fig. 1) (1,
4, 10, 15, 39, 50). Thus, we investigated whether the alterations
in islet vascularization observed were associated with changes
in angiogenic factors and/or their receptors. Specifically, we
assessed changes in a number of key angiogenic families, i.e.,
VEGF, angiopoietins, fibroblast growth factor, and ephrins, all
of which play important roles in stimulating capillary ingrowth. WSD offspring did not exhibit reductions in the gene
expression levels of angiogenic factors within the pancreas
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A
4-7 Years
Conception
GD130
0
Fig. 2. Schematic overview of the fetal and
juvenile study design. A: fetal CTR and Western-style diet (WSD) exposure in utero with
diet reversal (REV) and WSD/resveratrol
(RESV) interventions. B: juvenile study design following maternal and postweaning
WSD consumption. GD130, gestational day
130.
CTR
Ad libitum CTR: 14% fat
WSD
Ad libitum WSD: 32% fat
REV
Ad libitum WSD
Ad libitum CTR
WSD/RESV
Ad libitum WSD
Ad libitum WSD/RESV
B
4-7 Years
CTR/CTR
Birth
Weaning
Necropsy
0
6-8 months
13 months
Ad libitum CTR
Ad libitum CTR
CTR/WSD
WSD/CTR
Ad libitum WSD
Ad libitum WSD
WSD/WSD
(Fig. 3F). Furthermore, compared with WSD offspring, fetal
expression levels were not altered significantly by diet reversal.
In addition, despite the dramatic increase in islet vascularization following resveratrol supplementation, these offspring did
not display increased expression of key angiogenic factors
(Fig. 3F). It has been shown previously that resveratrol promotes vascularization through pathways involving endothelial
(eNOS) and inducible nitric oxide synthase (56). However,
WSD/RESV offspring displayed a small but significant decrease in the gene expression levels of eNOS compared with
CTR offspring (Fig. 3F), failing to explain the increased
capillary density. Furthermore, proliferation within the islet
capillaries was not impacted significantly across treatment
groups (Fig. 3G). Overall, these data suggest that the mechanism by which WSD and dietary interventions affect islet
vasculature is occurring independent of angiogenesis.
Maternal WSD consumption negatively impacts islet vascularization in the juvenile offspring. We have shown previously
that the early defect in ␣-cell mass observed at GD130 also
results in a reduction in ␣-cell mass in the juvenile offspring
(21). Similarly, we hypothesized that the early impairment in
the development of the islet vasculature would persist in the
juvenile offspring, paralleling the defects in islet morphology.
To address this, offspring from CTR or WSD offspring were
born naturally, maintained on their respective diets throughout
lactation, and weaned at ⬃8 mo of age. At this time, offspring
were either kept on their in utero diet or switched onto the
opposing diet (Fig. 2B), yielding four treatment groups. This
study design allows for the determination of the relative impact
of maternal vs. postweaning diet on islet vascularization and
innervation. Following postweaning WSD exposure (CTR/
WSD), juvenile animals tended to display an increase in islet
Ad libitum CTR
Ad libitum WSD
capillary area and area per capillary, consistent with an increase in metabolic demand (Fig. 4, A–D). However, there was
a significant reduction overall in islet vascularity in offspring
born to WSD dams (maternal diet effect: P ⬍ 0.01; Fig. 4B).
Furthermore, the reduction in capillary area was accompanied
by a reduction in the average size of the existing vessels (Fig.
4D). In WSD/CTR offspring, this results in islet vascularization similar to CTR/CTR offspring and is likely appropriate for
postweaning metabolic demand (Fig. 4B). In contrast, WSD/
WSD offspring fail to appropriately expand the islet vasculature in response to a postweaning WSD (Fig. 4B). The impact
of maternal WSD consumption on islet vascularization is
consistent with the significant impairment in islet capillary area
and size observed in the early third trimester (Fig. 3B).
Sympathetic islet innervation is impaired following in utero
WSD exposure but does not persist into the juvenile stage.
Because angiogenesis and neurogenesis are closely related
developmental processes, we hypothesized that maternal WSD
consumption would similarly impair the normal development
of sympathetic islet nerve fibers. To address this, we visualized
sympathetic fibers using tyrosine hydroxylase (TH). WSD
offspring displayed a 56% reduction in the density of sympathetic islet fibers at GD130 (Fig. 5, A and B). Neither maternal
diet reversal nor resveratrol supplementation during gestation
improved TH fiber density significantly.
Early developmental changes to both islet morphology (21)
and vascularization as a result of maternal WSD consumption
persist into childhood in the NHP model. However, significant
remodeling of islet nerve fibers occurs during the perinatal
period. To investigate whether the impaired innervation we
observe in the WSD fetus leads to altered sympathetic innervation in the juvenile offspring, we measured TH fiber density
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DAM DIET AND OFFSPRING ISLET VASCULATURE
CTR
WSD
REV
WSD/RESV
Insulin/Glucagon/PECAM1
A
b,c
0.2
b
0.0
CTR
WSD
c
a
a
100
2000
b
1000
CTR
WSD
1.0
†
**
*
3
S
O
F2
2
FG
A
1
A
H
P
E
FN
E
TE
K
G
P
P
A
N
G
TI
E1
T2
T1
R
D
N
A
K
FL
T1
E
G
FB
0.0
V
WSD
0
REV WSD/RESV
0
5000
10000 15000 20000
Insulin AUC
100
0.5
V
E
G
FA
Relative expression
**
‡
1000
G
WSD
REV
WSD/RESV
2.0
1.5
2000
b
CTR
REV WSD/RESV
F
**
a,b
0
0
REV WSD/RESV
3000
a
50
r2=0.15
p<0.05
5000
4000
a
150
3000
Ki67+/PECAM1+ area
(# mm-2)
a,c
E
200
N
Islet capillary
area/islet area
a
0.4
D
4000
Area/islet capillary
(µm2)
0.6
Islet capillary density
(# mm-2)
C
Islet capillary density
(# mm-2)
B
80
60
40
20
0
CTR
WSD
REV WSD/RESV
Fig. 3. Vascularization of the fetal islet is reduced by maternal WSD consumption and restored by dietary intervention. A: representative IHC images of the
vascularization of fetal islets by platelet endothelial cell adhesion molecule 1 (PECAM1) staining. Scale bar represents 25 ␮m. B–D: quantification of islet
PECAM1⫹ area (B), vascular density (no. of PECAM1⫹ fibers/islet area; C), and area per vessel in CTR (n ⫽ 8; 4 males and 4 females), WSD (n ⫽ 9; 3 males
and 6 females), REV (n ⫽ 6; 2 males and 4 females), and WSD/RESV (n ⫽ 6; 4 males and 2 females) offspring (D). E: correlation of maternal insulin secretion
during a glucose tolerance test with islet capillary density. F: gene expression of key islet angiogenic factors and their receptors in CTR (n ⫽ 13; 6 males and
7 females), WSD (n ⫽ 11; 7 males and 4 females), REV (n ⫽ 6; 2 males and 4 females), and WSD/RESV (n ⫽ 6; 4 males and 2 females) pancreas. Dashed
line indicates relative CTR expression. G: quantification of proliferation (Ki67⫹ cells) within islet capillaries (PECAM1⫹ cells) in CTR (n ⫽ 7; 3 males and
4 females), WSD (n ⫽ 7; 2 males and 5 females), REV (n ⫽ 7; 2 males and 5 females), and WSD/RESV (n ⫽ 6; 4 males and 2 females) pancreas. Data are
expressed as means ⫾ SE. Different superscripted letters indicate a significant difference (P ⬍ 0.05). *P ⬍ 0.05, **P ⬍ 0.01 vs. CTR offspring; †P ⬍ 0.05,
‡P ⬍ 0.01 vs. WSD offspring.
in offspring at 1 yr of age (Fig. 6). Surprisingly, we observed
no change in sympathetic islet innervation in this age group,
suggesting that the offspring are capable of compensating
during the perinatal period.
DISCUSSION
In the current study, we demonstrate that maternal WSD
consumption during pregnancy results in alterations in the
development of islet vascularization and innervation in the
fetal offspring at the beginning of the third trimester. Specifically, WSD fetal offspring display significant reductions in
capillary area, density and size (Fig. 3) and fewer sympathetic
nerve fibers (Fig. 5). Importantly, the reduction in the size of
islet capillaries persists in juvenile offspring born to mothers
who consume a WSD (Fig. 4). The observation that vascularization and innervation are impaired is consistent with our
previously published data demonstrating altered development
of the islet in WSD offspring (21) and likely reflects the close
developmental relationship between these factors. Interestingly, recent data also suggest that impaired islet vascularization may in fact precede defects in islet development (30),
indicating that the impairments in ␣-cell mass observed previously in this model may be secondary to defects in the
development of the islet vasculature.
The longer-term impact of the observed reduction in vascularization and innervation is unknown but may provide a novel
mechanism by which these offspring have an increased predisposition to the development of type 2 diabetes later in life. In
rodent models of insulin resistance, intraislet capillaries dilate
in response to increased nutrient requirements and secretory
demand (22). Similarly, juvenile NHP offspring who receive
only a postnatal WSD exposure (CTR/WSD) tend to display an
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DAM DIET AND OFFSPRING ISLET VASCULATURE
A
CTR/WSD
WSD/CTR
WSD/WSD
Insulin/Glucagon/PECAM1
CTR/CTR
B
C
0.3
a
a,b
3000
b
0.2
2000
b
1000
0.1
0.0
Maternal: CTR
Post-weaning: CTR
150
4000
Islet capillary
area/islet area
0.4
D
Islet capillary density
(# mm-2)
Islet capillary
area/islet area
0.5
Diet Effect
* Maternal < 0.01
CTR
WSD
WSD
CTR
WSD
WSD
0
Maternal: CTR
Post-weaning: CTR
Diet Effect
* Maternal < 0.01
b
100
a,b
a,b
a
50
0
CTR
WSD
WSD
CTR
WSD
WSD
Maternal: CTR
Post-weaning: CTR
CTR
WSD
WSD
CTR
WSD
WSD
Fig. 4. Maternal WSD consumption impairs islet vascularization in the juvenile NHP. A: representative IHC images of the vascularization of juvenile islets by
PECAM1 staining. Scale bar represents 25 ␮m. B–D: quantification of islet PECAM1⫹ area (B), vascular density (no. of PECAM1⫹ fibers/islet area; C), and
area/vessel in CTR/CTR (n ⫽ 8; 4 males and 4 females), CTR/WSD (n ⫽ 6; 3 males and 3 females), WSD/CTR (n ⫽ 8; 3 males and 5 females), and WSD/WSD
(n ⫽ 11; 6 males and 5 females) juvenile offspring (D). Data are expressed as means ⫾ SE. Different superscripted letters indicate a significant difference (P ⬍
0.05). Significant effect of maternal diet by 2-way ANOVA is indicated.
increase in islet capillary size, which is consistent with increased capillary dilation. However, WSD exposure in utero
significantly reduces islet capillary size compared with offspring who consume the WSD only postnatally (WSD/WSD
vs. CTR/WSD). The inability to appropriately expand the islet
vasculature likely results in a failure of the islet to respond to
metabolic demand and causes accelerated ␤-cell failure. In
fact, islet-specific deletion of VegfA in a murine model results
in hypovascularization of the islet and impaired glucose tolerance (10). Furthermore, the decrease in islet capillary size is
consistent with impaired dilation of the vessels. It has been
shown previously that rodent models of type 2 diabetes display
reduced vasodilation and impaired eNOS activity (22). Although we did not detect changes in eNOS expression (Fig.
3F), enzymatic activity may be altered without concomitant
changes in gene expression.
Interestingly, undernutrition and intrauterine growth restriction in a rodent model have also been associated with impaired
islet vascularity (8, 30). Maternal WSD consumption during
pregnancy has been linked to intrauterine growth restriction
and placental insufficiency (25, 33), suggesting shared attributes between these conditions. However, those authors attributed this deficiency to the significant reduction in expression of
VegfA (8, 30), unlike our NHP model of maternal WSD
consumption, indicative of distinct mechanisms of impaired
vascularization.
Previously, changes in islet innervation following maternal
WSD consumption had not been investigated in any species.
Although we demonstrate a significant reduction in sympathetic fiber density in WSD offspring, surprisingly, this effect
did not persist with juvenile offspring. In rodents, islet innervation undergoes significant remodeling in the postnatal period, resulting in a decrease in innervation after postnatal day
20 (13). This may explain the significant reduction in sympathetic innervation in the juvenile NHP islet compared with the
fetal islet. Interestingly, data from our group have indicated
that WSD consumption in adult NHPs results in an increase in
islet sympathetic fiber density, suggesting that this adaptation
is required for the response to increased metabolic demand
(Pound LD and Grove KL, unpublished observation). Similar
to impairments in vasculature, failure of the islet neural network development may also predispose the individual to the
development of type 2 diabetes later in life. Importantly,
animal models of insulin resistance or type 2 diabetes display
altered sympathetic islet innervation (36), likely leading to
enhanced ␣-cell function. Future studies will address whether
this observation is specific to sympathetic nerve fibers or is
indicative of an overall reduction in innervation.
Although compliance can be a challenge, maternal diet
reversal during pregnancy is a straightforward, cost-effective,
therapeutic option. Women consuming a WSD may be more
motivated to improve their dietary intake during pregnancy to
mitigate some of the potentially negative impacts on their fetus
than they may be otherwise. Despite no significant short-term
improvements in maternal metabolic health, a switch to a CTR
diet for the duration of pregnancy normalized islet vascularization in the fetal offspring in the early third trimester. The
observation that this occurred independent of improvements to
maternal weight, adiposity, and insulin sensitivity suggests that
the impairments in vasculature seen in the WSD fetus are
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DAM DIET AND OFFSPRING ISLET VASCULATURE
A
WSD
REV
WSD/RESV
Insulin/Glucagon/TH
CTR
# TH+ fibers/islet area
(# mm-2)
B
600
a
400
a,b
b
b
200
0
CTR
WSD
REV
WSD/RESV
Fig. 5. In utero WSD exposure results in impaired fetal islet innervation. A: representative IHC images of the innervation of fetal islets by tyrosine hydroxylase
(TH) staining. Scale bar represents 25 ␮m. B: quantification of islet innervation by TH staining in CTR (n ⫽ 9; 4 males and 5 females), WSD (n ⫽ 11; 3 males
and 8 females), REV (n ⫽ 7; 2 males and 5 females), and WSD/RESV (n ⫽ 5; 3 males and 2 females) offspring. Data are expressed as means ⫾ SE. Different
superscripted letters indicate a significant difference (P ⬍ 0.05).
# TH+ fibers/islet area
(# mm-2)
driven primarily by the diet itself and thus are easily reversible
with improved dietary intake. However, it was surprising that
there was not a similar normalization in ␣-cell mass or sympathetic innervation (Fig. 6). These factors may be more
sensitive to the phenotype of the mother than to the diet itself,
suggesting that the mechanisms are distinct from those driving
the impairment in islet vascularization.
Previous studies have demonstrated that resveratrol supplementation improves vascular function and induces vasodilation
(16, 34, 56). Although human studies have not yet assessed the
300
200
100
0
Maternal: CTR
Post-weaning: CTR
CTR
WSD
WSD
CTR
WSD
WSD
Fig. 6. Islet innervation in the juvenile NHP is not affected significantly by
maternal WSD consumption. Quantification of islet innervation by TH staining
in CTR/CTR (n ⫽ 7; 3 males and 4 females), CTR/WSD (n ⫽ 6; 3 males and
3 females), WSD/CTR (n ⫽ 5; 1 male and 4 females), and WSD/WSD (n ⫽
8; 6 males and 2 females) juvenile offspring. Data are expressed as means ⫾
SE. Different letters indicate a significant difference (P ⬍ 0.05).
safety or efficacy of resveratrol treatment during pregnancy in
women consuming a WSD, our group demonstrated recently in
our NHP model of WSD consumption during pregnancy that
supplementation improved placental blood flow, consistent
with its role in enhancing vascular function (52). However, this
was accompanied by a concerning increase in total pancreas
mass and proliferation and an elevation in the ␤-cell/␣-cell
ratio (52). In this study, resveratrol supplementation during
pregnancy resulted in a significant increase in islet vascularization, consistent with its known vasodillatory effect. However, in particular, resveratrol consumption during pregnancy
resulted in hypervascularization in the offspring significantly
above CTR offspring. Although the long-term consequences of
this observation are unknown, hypervascularization has been
shown in rodent models to have a negative impact on islet
development, as the vasculature is critical for restricting pancreas growth and differentiation. Specifically, VegfA overexpression leads to reduced pancreatic branching and impaired
islet growth (2, 14). Furthermore, this effect may be of particular concern, as hypervascularization is a precursor to malignancy (11). Because resveratrol is a readily available dietary
supplement, the potentially harmful consequences in fetal pancreas warrant further investigation.
Interestingly, despite the enhanced islet vascularization observed following maternal WSD/RESV consumption, VegfA
expression was paradoxically impaired compared with CTR
offspring. Similarly, Kdr, the gene encoding the VEGF receptor-2, and Angpt2, the gene encoding angiopoietin-2, expres-
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DAM DIET AND OFFSPRING ISLET VASCULATURE
sion were impaired vs. WSD offspring. A number of studies
have reported reduced VegfA expression following resveratrol
supplementation (40, 42, 45, 55); however, this effect was
accompanied by a similar reduction in angiogenesis in one
study (55), in contrast to the data reported here. The mechanisms underlying this effect remain to be elucidated, but these
data indicate a complex role of resveratrol in islet angiogenesis. However, it is important to note that although the observed
increase in vascularization was specific to the islet, the alterations in the gene expression of key angiogenic markers were
taken from the whole pancreas and thus may at least partially
explain these paradoxical observations.
Overall, our data indicate that maternal WSD consumption
during pregnancy results in impaired islet vascularization and
innervation and, importantly, may present a novel mechanism
by which offspring are predisposed to developing type 2
diabetes later in life. Furthermore, an improvement in maternal
nutrition during pregnancy restored the loss of islet vascularity.
However, our islet data strongly indicate that women should
not consume resveratrol during pregnancy until followup studies are conducted.
ACKNOWLEDGMENTS
We thank Victoria Roberts for discussions and comments on the manuscript, Diana Takahashi, India Tindale, Peter Blundell, Leigh Ann Bauman,
and Rikley Buckingham (ONPRC) for technical assistance and guidance with
the animal studies, Anda Cornea for microscopy assistance, and Barbra Mason
for histology.
S. M. Comstock is presently at Corban University, Salem, OR.
GRANTS
This work was supported by National Institutes of Health funding: R24DK-090964 (K. L. Grove), P51-OD-011092 (partial salary support for K. L.
Grove and Marianas imaging), and S10-RR-024585 (Leica confocal imaging).
DISCLOSURES
The authors have no conflicts of interest, financial or otherwise, to disclose.
AUTHOR CONTRIBUTIONS
L.D.P. and K.L.G. conception and design of research; L.D.P. and S.M.C.
performed experiments; L.D.P. and S.M.C. analyzed data; L.D.P. and S.M.C.
interpreted results of experiments; L.D.P. prepared figures; L.D.P. drafted
manuscript; L.D.P., S.M.C., and K.L.G. edited and revised manuscript; L.D.P.,
S.M.C., and K.L.G. approved final version of manuscript.
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