The maternal environment programmes postnatal weight gain and glucose tolerance but placental and fetal growth are determined by fetal genotype in the Leprdb/+ model of gestational diabetes
1,2Raja Nadif, 1,2Mark R Dilworth, 1,2Colin P Sibley, 3Philip N Baker, 4Sandra T Davidge, 5J Martin Gibson, 1,2John D Aplin, 1,2Melissa Westwood
1Maternal and Fetal Health Research Centre, University of Manchester, Manchester Academic Health Sciences Centre, Manchester, UK2Maternal and Fetal Health Research Centre, St Mary’s Hospital Central Manchester Universities NHS Foundation Trust, Manchester Academic Health Sciences Centre, Manchester, UK3Gravida, University of Auckland, Auckland, New Zealand4Women and Children's Health Research Institute, University of Alberta, Edmonton, Alberta, Canada5Centre for Imaging Sciences, Institute of Population Health, University of Manchester, Manchester Academic Health Sciences Centre, Manchester, UK
Address for correspondence: Melissa Westwood, Maternal and Fetal Health Research Centre, University of Manchester, St Mary’s Hospital, Oxford road, Manchester, M13 9WL, UK. Tel: 44(0)161 2765461; Fax: 44(0)161 7016971; [email protected]
Word count: Text – 2770; Abstract – 250
1
Abstract
Mice heterozygous for a signalling-deficient leptin receptor (Leprdb/+(db/+)) are widely
used as a model of gestational diabetes that results in poor fetal outcomes. This study aimed
to investigate the importance of fetal genotype (db/+) relative to abnormal maternal
metabolism on placental function and therefore fetal growth and offspring health.
Wild type (wt) and db/+ females were mated to db/+ and wt males respectively to generate
litters of mixed genotype. Placentas and fetuses were weighed at E18.5; offspring weight,
hormone levels, glucose tolerance and blood pressure were assessed at 3 and 6 months.
Pregnant db/+, but not wt, dams had impaired glucose tolerance. db/+ placentas and fetuses
were heavier than wt but the maternal environment had no effect; wt placentas/fetuses from
db/+ mothers were no bigger than wt placentas/fetuses carried by wt mothers. Postnatal
growth, glucose metabolism and leptin levels were all influenced by offspring genotype.
However maternal environment affected aspects of offspring health as wt male offspring born
to db/+ dams were heavier and had worse glucose tolerance than the sex-matched wt
offspring of wt mothers. Blood pressure was not affected by maternal or fetal genotype.
These data reveal that studies using the db/+ mouse to model outcomes of pregnancy
complicated by gestational diabetes should be mindful of the genetically predisposed
fetal/post-natal overgrowth. Although inappropriate for dissecting the effect of maternal
hyperglycemia on the contribution of placental function to macrosomia, the db/+ mouse may
prove useful for investigating mechanisms underlying in utero programming of suboptimal
postnatal growth and glucose metabolism.
Keywords: placenta, birth weight, post-natal growth, GTT, leptin, blood pressure
2
Introduction
The incidence of obesity amongst women of child-bearing age has doubled over recent years
[1]. Pre-pregnancy weight is associated with the development of gestational diabetes (GDM)
and the frequency of this condition has also increased [2].
Pregnancy complicated by GDM is associated with increased fetal mortality and morbidity
[3]. Fetal overgrowth - macrosomia – occurs in a third of babies born to such mothers [4].
These infants are more likely to experience birth injuries, asphyxia and postnatal metabolic
disturbances [5]. Furthermore, in utero exposure to an adverse nutrient environment can
perpetuate disease; long-term studies have demonstrated that macrosomic offspring have
impaired glucose tolerance, increased adiposity and raised systolic blood pressure as children,
and an increased risk of developing diabetes, obesity and cardiovascular disease as adults
[6,7].
Maternal, and consequently fetal, hyperglycemia undoubtedly plays a role in fetal
overgrowth. However, good maternal glucose control does not abolish macrosomia [8]
suggesting that increased maternal-to-fetal transfer of other nutrients, for example lipids and
amino acids, may contribute to fetal overgrowth and importantly, that instead of merely
reflecting an increase in nutrient supply, macrosomia may be a consequence of abnormal
placental function. Indeed, numerous studies have shown that nutrient metabolism and
transport are altered in placentas from pregnancies complicated by GDM [9]. Furthermore,
placental mass is increased [10], exacerbating augmented nutrient transport by increasing the
surface area of the transporting epithelium (syncytiotrophoblast).
Interventions aimed at modulating placental function and thereby preventing fetal
macrosomia could be used to halt the transgenerational cycling of diabesity and reduce the
consequent global health burden. However, such advances are dependent upon the
availability of appropriate models to aid understanding of the role of the placenta in GDM
3
and to test potential therapies. Mice, like humans, have a haemochorial placenta and previous
studies have suggested that a strain that is heterozygous for a signalling-deficient leptin
receptor (C57BL/KSJ-Leprdb/+) is a good experimental model of GDM. Dams develop
diabetes (impaired glucose tolerance and elevated HbA1c) only during pregnancy [11,12] and
the offspring have significantly greater birth weights [11,13,14,15] and deranged metabolism
[15] compared to the offspring of wild-type (wt) mothers. These poor outcomes have been
attributed to the adverse maternal environment, however, the relative contribution and
importance of the fetal genotype (db/+) to placental function, and therefore fetal growth and
programming, have not been evaluated. This study aimed to determine the usefulness of the
db/+ mouse as a model for investigating placental function in pregnancies complicated by the
abnormalities in maternal metabolism that occur in gestational diabetes.
4
Methods
All experimental procedures were conducted in accordance with the Home Office Animals
(Scientific procedures) Act 1986 of the United Kingdom. All animals were maintained with
free access to food and water. Wild type and db/+ female were mated at twelve weeks of age
to db/+ and wt males respectively in order to generate litters of mixed genotype; day of plug
was counted as E0.5. Some dams (10 wt; 9 db/+) were euthanised on day E18.5 to enable
collection of placentas and fetuses (140 in total) which were weighed and then genotyped,
using DNA extracted from tail snips, by sequencing PCR products obtained using primers
flanking the Lepr mutation (F: 5′-CCCTCCCCTCTCCTAAGTGT-3′; R: 5′-
CAGCAACCGTCACACCATTA-3′) [16]. This analysis revealed that 58 of the
placenta/fetus pairs were wt (28 from wt dams; 30 from db/+ mothers) and 82 were db/+ (40
and 42 from wt and db/+ mothers respectively). Other dams were allowed to deliver and pup
genotype was determined by analysis of DNA extracted from ear punches obtained at
weaning (21 days of age). The F1 offspring were maintained for up to 6 months.
Dams (day 18.5 of pregnancy) and F1 offspring (3 and 6 months) were subjected to a glucose
tolerance test (fasted overnight, injected with 2g glucose/kg ip and tail vein blood samples
collected at 0, 20, 30, 60, 90 and 120 minutes) before sacrifice. Glucose concentrations were
measured using a glucometer (OneTouch Vita) and the 0 minutes sample was also used to
measure insulin and leptin levels using mouse-specific ELISAs (Millipore and R&D Systems
respectively).
The systolic and diastolic arterial pressure of the F1 offspring was measured at 6 months of
age by tail-cuff volume pressure recording (CODA system, Kent Scientific Corporation,
USA) as previously described [17], ensuring that mice were accustomed to the procedure
before collecting the blood pressure readings (average of 5/animal).
5
Data are presented as mean (±SEM). Within-litter comparisons were made using a paired t-
test. Data from different litters were analysed using an independent t-test; p<0.05 was
considered significant.
6
Results
db/+ dams have impaired glucose tolerance: db/+ dams had lower fasting insulin levels than
wt mothers (0.16(±0.04)ng/ml versus 0.31(±0.03)ng/ml; p<0.05) and analysis of glucose
levels confirmed impaired glucose tolerance during pregnancy (area under curve 1339(±85)
versus 987(±16); p<0.05). db/+ mothers had higher circulating leptin levels (760(±50)ng/ml)
than wild-type mothers (148(±15)ng/ml; p<0.05), and both had significantly higher levels
than their non-pregnant counterparts (33- and 19-fold respectively).
Effect of maternal diabetes on placental and fetal growth: There was no significant
difference in the average litter size of wt and db/+ dams (6.8±0.6 versus 8.0±0.6
respectively), nor in the number of wt and db/+ fetuses within each litter. Consequently the
total fetal and placental weight carried by wt dams (7375±583mg and 558±45mg,
respectively) was similar to that carried by db/+ mothers (fetal weight – 8534±610mg;
placental weight – 653±46mg). However, after accounting for the weight of the
fetal/placental unit, db/+ mothers were significantly heavier than their wt counterparts
(33.06±0.75g versus 30.37±0.72g; p<0.05). db/+ fetuses (n=40) carried by wt dams exhibited
a significantly higher (5%) birth weight than their wt littermates (n=28, p=0.05; see Figure 1
for individual pup data and Table I for mean litter weights). db/+ fetuses (n=42) from db/+
mothers were also bigger (3%) than their wt counterparts (n=30, p=0.05; Figure 1, Table I).
Surprisingly, maternal genotype had no effect on progeny birthweight. db/+ fetuses carried
by db/+ mothers were of similar size to those from wt dams (Figure 1,Table I). Moreover, wt
pups from db/+ mothers (offspring / dam combination that most closely models human
gestational diabetes) were no bigger than wt fetuses carried by wt mothers (Figure 1, Table I).
Similarly, placentas from db/+ fetuses (n=82) were larger (p<0.05) than those of wt fetuses
(n=58) irrespective of maternal genotype (Figure 1, Table I). Consequently, the fetal to
7
placental weight ratio, commonly used as an indicator of placental efficiency, was the same
in all animals (Figure 1).
Effect of maternal diabetes on offspring growth, metabolic parameters and blood pressure:
Initial analysis of F1 offspring weight suggested no difference between those from normal
pregnancy (23.72±0.97g and 27.86±1.05g at 3 and 6 months respectively) and those born to
dams with gestational diabetes (24.83±0.78g and 28.56±0.88g at 3 and 6 months). However,
analysis of data accounting for offspring genotype and sex revealed that wt males born to
db/+ mothers are heavier than wt males born to wt mothers (p<0.05) but the post-natal
growth of female offspring is not affected by the maternal environment (Figure 2A). In
keeping with our observations of the effect of the db/+ genotype on pre-natal growth, male
and female db/+ offspring, from both normal and complicated pregnancy, are heavier (Figure
2A) than their wt littermates at both 3 and 6 months of age.
A comparison of all offspring born to wt and db/+ mothers showed that at 6 months of age,
those from mothers with gestational diabetes had significantly lower fasting insulin levels
(0.16(±0.02)ng/ml versus 0.21(±0.02)ng/ml in wt; p<0.05) and worse glucose tolerance
(AUC 1603(±69) versus 1420(±41) in wt; p<0.05). Again there was an influence of sex and
genotype as the glucose tolerance of wt males born to db/+ mothers was significantly worse
than that of wt males from normal pregnancies at both 3 and 6 months (Figure 2B). db/+
offspring, both male and female, had impaired glucose tolerance, irrespective of the maternal
environment, in comparison to their wt littermates (Figure 2B).
Offspring leptin levels were affected by genotype (17.5(±2.2)ng/ml in six month old db/+
animals versus 5.4(0.81)ng/ml in wt mice; p<0.05) rather than sex or maternal environment
(Table II).
8
At six months of age, the systolic, diastolic and mean arterial (MAP) pressure of the male and
female wt offspring from db/+ mothers was similar to that of the sex-matched wt offspring
from uncomplicated pregnancies (MAP 132±7 versus 148±5mm Hg respectively); none of
the parameters measured were affected by offspring genotype (Table III).
9
Discussion
This study shows that the db/+ mouse is not ideal for investigating the effect of GDM on
placental function and therefore its contribution to fetal growth. However, the model may be
useful for dissecting mechanisms underlying in utero programming as the male, but not
female, offspring from db/+ mothers were heavier and had impaired glucose tolerance at six
months of age.
We demonstrate that fetal genotype influences both placental and fetal growth as the weight
of db/+ placentas and fetuses, carried by either wt or db/+ mothers, was significantly greater
than that of wt littermates. The leptin receptor is known to regulate leptin mRNA expression
in an autocrine manner [18] thus lepr heterozygosity likely affects placental leptin production
and consequently placental development and function, leading to increased fetal growth.
Indeed, others have noted increased leptin levels [19] and cellular hypertrophy [13] in db/+
placentas and in human placenta, leptin stimulates increased activity of the amino acid
transporter, system A [20].
Crucially however, our data suggest that in this model, fetal genotype is more important than
the maternal environment in determining placental and fetal growth as the placental and
birthweight of wt fetuses carried by db/+ and wt dams were similar. These data contrast with
that of other studies which report that db/+ mothers bear offspring with greater placental [13]
and birth [13,14,15,21,22] weights than wt mothers. It is possible that differences in
gestational age may have contributed to the discrepant findings as some studies [14,15]
assessed fetal weight at a later time point (E19 versus E18). However, changes in placental
growth usually precede changes in fetal growth [23] and, using the proxy measure of
placental:fetal weight ratio, we found no evidence of altered placental function in db/+
pregnancies. A more likely explanation lies in differences in experimental design. Previous
studies either set up matings such that db/+ pups were absent from wt pregnancies [21,22], or
10
compared all pups born from db/+ versus wt pregnancies without knowledge of pup genotype
[13], or commented, without detailing the results, that there were no significant differences in
placental and birth weight between wt and db/+ fetuses from the same litter, thus data from
each litter were grouped [14,15].
Interestingly, the weight of fetuses from db/+ mothers is reported to be greater than that of
pups from wt pregnancies even when maternal hyperglycemia was reduced by
overexpression of GLUT4 [14]. Increased placental growth, and therefore transfer of
nutrients, was mooted as an explanation of this finding, but the current study does not support
this hypothesis. Moreover, administration of leptin to db/+ mothers during late pregnancy
reduced their adiposity and circulating glucose levels, but fetal growth was not affected [21].
In that study [21], placental and fetal leptin levels were higher in db/+ compared to wt
pregnancies which, together with our own data, point towards fetal genotype as the dominant
regulator of placental and fetal growth in this model. Models of other pregnancy
complications have also noted that fetal genotype contributes to pregnancy outcome [24],
highlighting the need, where possible, to study mixed litters in order to truly appreciate the
influence of the maternal environment.
The fact that the db/+ model does not mimic the placenta/fetal overgrowth often associated
with gestational diabetes in women [4] is interesting and suggests that impaired maternal
glucose tolerance is not necessarily detrimental to placental function. A study of trophoblast
isolated from normal human placentas at term found that unlike elevated levels of non-
esterified fatty acids, raised glucose levels had little effect on placental structure, metabolism
and inflammation [25], leading the authors to postulate maternal dyslipidaemia as the key
determinant of placental dysfunction in pregnancies complicated by diabetes. In our study,
db/+ dams were heavier than wt dams at day18.5, which is in keeping with their reported
hyperphagia [15,21], though we did not assess maternal adiposity or profile circulating lipids.
11
Mice carrying other single gene mutations, such as those that are heterozygous for the
prolactin receptor or that lack the serotonin receptor also develop glucose intolerance during
pregnancy, as do animals with conditional deletion of genes for transcription factors such as
HNF-4α, FoxD3 and FoxM1 from pancreatic β cells [26]. However, these models have
mainly been explored in relation to understanding the mechanisms underlying maternal
pancreatic adaption to pregnancy and the pathogenesis of disease; further research is needed
to determine their suitability for studying the effect of GDM on placental function and fetal
growth. Alternative strategies include surgical removal or chemical (e.g. streptozotocin)
destruction of pancreatic β cells, though neither model accurately reflects the aetiology of
GDM and some clinical outcomes such as macrosomia are absent [26]. GDM induced by
nutritional manipulation, for example feeding mice a diet high in saturated fat, has been
reported to affect placental structure and function [27]; this supports the observations made in
human placenta discussed above, however GDM in women is a heterogeneous condition and
susceptibility is due to the combination of environmental and polygenic factors. It is unlikely
that any currently available model will be suitable for all studies [26] and researchers must
be careful to choose the most appropriate for their purpose.
Indeed male wt offspring from db/+ mothers were heavier and had poorer glucose tolerance
than those from normal pregnancies in agreement with a previous study which reported
differences in the weight of 8-week old wt male, but not female, offspring from db/+ and wt
pregnancies [22]. However, differences observed in the leptin levels of such animals are not
replicated herein; in our study, the levels of circulating leptin in 6 month old animals are
related to genotype rather than the maternal environment. It is possible that the adverse
maternal influence resolves with increasing age. Others have reported that at 6 months, the
weight of wt offspring born to db/+ and wt mothers is similar in both sexes but that female
offspring have increased body fat and insulin resistance [15]. Offspring adiposity was not
12
assessed in our study but we did not detect sex differences in fasting insulin levels. A sex
difference in offspring outcomes, with males often faring worse, is a common observation in
studies of in utero programming, especially in relation to glucose intolerance [28], and has
been ascribed to differences in maternal investment of energy depending on fetal sex [29].
Data from this and other published studies suggest that future investigations of the db/+
model should consider offspring sex when assessing outcomes.
db/db mice are known to have raised systolic, diastolic and mean arterial blood pressures in
comparison to their db/+ littermates [30] but a comparison of db/+ and wt offspring, born to
either wt or db/+ mothers, has not been reported. In this study, all measures of blood pressure
were similar between offspring. However, it is possible that more sensitive assessment, for
example using radiotelemetry might reveal subtle effects of the maternal environment and /
or offspring genotype or that a secondary stressor may not be as well tolerated.
It will be interesting to uncover the mechanisms that can programme the postnatal health of
male offspring from db/+ dams in the absence of placental / fetal overgrowth. In vitro studies
suggest that GDM could influence epigenetic programming [31,32] and more recently, genes
involved in appetite control and energy metabolism have been shown to be epigenetically
modified in placentas and cord blood of infants from pregnancies complicated by GDM
[33,34]. Furthermore, a mouse model of GDM induced by administration of streptozotocin
found that although the birthweight of F1 offspring was not affected, the male offspring had
impaired glucose tolerance as adults and altered methylation of the imprinted genes Igf2/H19
that are important for pancreatic islet development [35]. Altered nutrition in the perinatal
period can also cause epigenetic changes that affect adult health [36,37]. Nothing is known
about the quantity and quality of milk from db/+ dams, thus cross-fostering experiments will
be important to determine how maternal nutrient supply during this critical period of
development contributes to the long-term health of the wt offspring born to db/+ dams.
13
In summary, our study highlights the need to genotype offspring when interpreting the effect
of the maternal environment on placental and fetal weight in genetic models of GDM. The
db/+ mouse may be most useful for investigating mechanisms underlying GDM
programming of postnatal growth and glucose metabolism.
14
Figure Legends
Figure 1. Heterozygosity in a leptin receptor gene mutation predisposes to placental and
fetal overgrowth. Ten wild type (wt) females at twelve weeks of age were mated to db/+
males and nine age-matched db/+ females were mated to wild type males in order to generate
litters of mixed genotype. Day of plug was counted as E0.5 and fetuses and their
corresponding placentas were collected for assessment of weight and genotype at E18.5.
Offspring carrying the db/+ genotype (n=82) have increased body and placental weights in
comparison to wt offspring (n=58), regardless of maternal genotype. The weight of each pup
was divided by the weight of its corresponding placenta to give the fetal: placental ratio,
commonly used as an indicator of placental efficiency, which was not affected by maternal or
fetal genotype. Data points represent individual pups / placentas; bar represents mean. * -
p<0.05.
Figure 2. Effect of maternal diabetes on postnatal weight gain and glucose tolerance.
Offspring (male and female) from wild type (wt) ♀ / db/+ ♂ or db/+ ♀ / wild type ♂ crosses
were weighed (A) or subjected to a glucose tolerance test (B) at 3 and 6 months of age. Data
are shown as mean±SEM; AUC – area under curve; n – number of offspring analysed; * -
p<0.05.
15
wt mothers (n=10) db/+ mothers (n=9)wt offspring db/+ offspring wt offspring db/+ offspring
Placental weight(mg) 78.61 (±1.30) 83.80 (±1.91)a 79.50 (±1.63) 83.40 (±1.23)b
Fetal weight(mg) 1037 (±39.6) 1122 (±35.8)a 1041 (±15.4) 1087 (±10.7)b
Table I. Placental and fetal weights of day E18.5 litters. The mean placental and fetal weight
of all the wt or db/+ offspring within each litter was calculated from ten wild type (wt) and
nine db/+ mothers and are presented as mean±SEM. a – p<0.05 versus wt pups from wt dam;
b – p<0.05 versus wt pups from db/+ dam.
16
wt mothers db/+ mothers
Offspring
wt (n=8) db/+ (n=12) wt (n=13) db/+ (n=13)
♂ (n=4)
♀ (n=8)
♂ (n=7)
♀ (n=9)
♂ (n=4)
♀ (n=4)
♂ (n=6)
♀ (n=4)
leptin (ng/ml) 4.5 (±1.8)
7.9 (±3.0)
16.6a
(±2.5)23.3a
(±6.5)
3.1 (±0.7
)
7.6 (±0.8)
16.3b
(±4.8)17.8b
(±3.8)
Table II. Serum leptin levels (mean (±SEM)) of offspring from wild type (wt) and db/+
mothers measured at 6months of age. a - p<0.05 versus sex-matched wt littermates from wt
dam; b – p<0.05 versus sex-matched wt littermates from db/+ dam.
17
wt mothers db/+ mothers
Offspring
wt (n=9) db/+ (n=12) wt (n=10) db/+ (n=11)
♂ (n=4)
♀ (n=5)
♂ (n=8)
♀ (n=4)
♂ (n=4)
♀ (n=6)
♂ (n=7)
♀ (n=4)
Systolic pressure (mmHg)
186 (±3)
157 (±4)
179 (±8)
169 (±14)
166 (±11)
149 (±8)
163 (±8)
151 (±8)
Diastolic pressure(mmHg)
152 (±3)
125 (±4)
141 (±6)
130 (±11)
129 (±12)
115 (±7)
131 (±7)
118 (±8)
Table III. Systolic and diastolic pressure (mean (±SEM)) of offspring born to wild type (wt)
and db/+ mothers measured at 6 months of age. Neither parameter was significantly affected
by offspring sex, genotype or the maternal environment.
18
Acknowledgements
This work was supported by Diabetes UK (08/0003816).
Duality of Interests
The authors have nothing to declare.
Author Contributions
R.N. - researched data, reviewed/edited manuscript. M.R.D. - researched data,
reviewed/edited manuscript. C.P.S. - contributed to experimental design and discussion,
reviewed/edited manuscript. P.N.B. - contributed to experimental design, reviewed/edited
manuscript. S.T.D. - contributed to experimental design, reviewed/edited manuscript. J.M.G.
- contributed to experimental design, reviewed/edited manuscript. J.D.A. - contributed to
experimental design and discussion, reviewed/edited manuscript. M.W. - contributed to
experimental design and discussion, researched data, performed data and statistical analyses,
wrote manuscript.
19
References
1. James WP (2008) The epidemiology of obesity: the size of the problem. J Intern Med 263:336-352.
2. Simmons D (2011) Diabetes and obesity in pregnancy. Best Pract Res Clin Obstet Gynaecol 25:25-36.
3. Wendland EM, Torloni MR, Falavigna M, Trujillo J, Dode MA, Campos MA, Duncan BB, Schmidt MI (2012) Gestational diabetes and pregnancy outcomes--a systematic review of the World Health Organization (WHO) and the International Association of Diabetes in Pregnancy Study Groups (IADPSG) diagnostic criteria. BMC Pregnancy Childbirth 12:23
4. Moore TR (1997) Fetal growth in diabetic pregnancy. Clin Obstet Gynecol 40:771-786.
5. Grassi AE, Giuliano MA (2000) The neonate with macrosomia. Clinical Obstetrics and Gynecology 43:340-348.
6. Vasudevan C, Renfrew M, McGuire W (2011) Fetal and perinatal consequences of maternal obesity. Arch Dis Child Fetal Neonatal Ed 96:F378-F382.
7. Yessoufou A, Moutairou K (2011) Maternal diabetes in pregnancy: early and long-term outcomes on the offspring and the concept of "metabolic memory". Exp Diabetes Res 2011:218598-
8. Langer O, Rodriguez DA, Xenakis EM, McFarland MB, Berkus MD, Arrendondo F (1994) Intensified versus conventional management of gestational diabetes. Am J Obstet Gynecol 170:1036-1046.
9. Jansson T, Cetin I, Powell TL, Desoye G, Radaelli T, Ericsson A, Sibley CP (2006) Placental transport and metabolism in fetal overgrowth -- a workshop report. Placenta 27 Suppl A:S109-S113.
10. Lao TT, Lee CP, Wong WM (1997) Placental weight to birthweight ratio is increased in mild gestational glucose intolerance. Placenta 18:227-230.
11. Kaufmann RC, Amankwah KS, Dunaway G, Maroun L, Arbuthnot J, Roddick JW, Jr. (1981) An animal model of gestational diabetes. Am J Obstet Gynecol 141:479-482.
12. Stanley JL, Cheung CC, Rueda-Clausen CF, Sankaralingam S, Baker PN, Davidge ST (2011) Effect of gestational diabetes on maternal artery function. Reprod Sci 18:342-352.
13. Lawrence S, Warshaw J, Nielsen HC (1989) Delayed lung maturation in the macrosomic offspring of genetically determined diabetic (db/+) mice. Pediatr Res 25:173-179.
14. Ishizuka T, Klepcyk P, Liu S, Panko L, Liu S, Gibbs EM, Friedman JE (1999) Effects of overexpression of human GLUT4 gene on maternal diabetes and fetal growth in spontaneous gestational diabetic C57BLKS/J Lepr(db/+) mice. Diabetes 48:1061-1069.
20
15. Yamashita H, Shao J, Qiao L, Pagliassotti M, Friedman JE (2003) Effect of spontaneous gestational diabetes on fetal and postnatal hepatic insulin resistance in Lepr(db/+) mice. Pediatr Res 53:411-418.
16. Bannon P, Wood S, Restivo T, Campbell L, Hardman MJ, Mace KA (2013) Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice. Dis Model Mech 6:1434-1447.
17. Ahmed A, Singh J, Khan Y, Seshan SV, Girardi G (2010) A new mouse model to explore therapies for preeclampsia. PLoS One 5:e13663-
18. Zhang Y, Olbort M, Schwarzer K, Nuesslein-Hildesheim B, Nicolson M, Murphy E, Kowalski TJ, Schmidt I, Leibel RL (1997) The leptin receptor mediates apparent autocrine regulation of leptin gene expression. Biochem Biophys Res Commun 240:492-495.
19. Hoggard N, Crabtree J, Allstaff S, Abramovich DR, Haggarty P (2001) Leptin secretion to both the maternal and fetal circulation in the ex vivo perfused human term placenta. Placenta 22:347-352.
20. Jansson N, Greenwood SL, Johansson BR, Powell TL, Jansson T (2003) Leptin stimulates the activity of the system A amino acid transporter in human placental villous fragments. J Clin Endocrinol Metab 88:1205-1211.
21. Yamashita H, Shao J, Ishizuka T, Klepcyk PJ, Muhlenkamp P, Qiao L, Hoggard N, Friedman JE (2001) Leptin administration prevents spontaneous gestational diabetes in heterozygous Lepr(db/+) mice: effects on placental leptin and fetal growth. Endocrinology 142:2888-2897.
22. Lambin S, van BR, Caluwaerts S, Vercruysse L, Vergote I, Verhaeghe J (2007) Adipose tissue in offspring of Lepr(db/+) mice: early-life environment vs. genotype. Am J Physiol Endocrinol Metab 292:E262-E271.
23. Sibley CP, Brownbill P, Dilworth M, Glazier JD (2010) Review: Adaptation in placental nutrient supply to meet fetal growth demand: implications for programming. Placenta 31 Suppl:S70-S74.
24. Kulandavelu S, Whiteley KJ, Qu D, Mu J, Bainbridge SA, Adamson SL (2012) Endothelial nitric oxide synthase deficiency reduces uterine blood flow, spiral artery elongation, and placental oxygenation in pregnant mice. Hypertension 60:231-238.
25. Pathmaperuma AN, Mana P, Cheung SN, Kugathas K, Josiah A, Koina ME, Broomfield A, ghingaro-Augusto V, Ellwood DA, Dahlstrom JE, Nolan CJ (2010) Fatty acids alter glycerolipid metabolism and induce lipid droplet formation, syncytialisation and cytokine production in human trophoblasts with minimal glucose effect or interaction. Placenta 31:230-239.
26. Pasek RCGannon M (2013) Advancements and challenges in generating accurate animal models of gestational diabetes mellitus. Am J Physiol Endocrinol Metab 305:E1327-E1338.
21
27. Liang C, DeCourcy K, Prater MR (2010) High-saturated-fat diet induces gestational diabetes and placental vasculopathy in C57BL/6 mice. Metabolism 59:943-950.
28. Rees DA, Alcolado JC (2005) Animal models of diabetes mellitus. Diabet Med 22:359-370.
29. Aiken CE, Ozanne SE (2013) Sex differences in developmental programming models. Reproduction 145:R1-13.
30. Su W, Guo Z, Randall DC, Cassis L, Brown DR, Gong MC (2008) Hypertension and disrupted blood pressure circadian rhythm in Type 2 diabetic db/db mice. Am J Physiol Heart Circ Physiol 295:H1634-H1641.
31. Chiang EP, Wang YC, Chen WW, Tang FY (2009) Effects of insulin and glucose on cellular metabolic fluxes in homocysteine transsulfuration, remethylation, S-adenosylmethionine synthesis, and global deoxyribonucleic acid methylation. J Clin Endocrinol Metab 94:1017-1025.
32. Wellen KE, Hatzivassiliou G, Sachdeva UM, Bui TV, Cross JR, Thompson CB (2009) ATP-citrate lyase links cellular metabolism to histone acetylation. Science 324:1076-1080.
33. Ruchat SM, Houde AA, Voisin G, St-Pierre J, Perron P, Baillargeon JP, Gaudet D, Hivert MF, Brisson D, Bouchard L (2013) Gestational diabetes mellitus epigenetically affects genes predominantly involved in metabolic diseases. Epigenetics 8:935-943.
34. El HN, Pliushch G, Schneider E, Dittrich M, Muller T, Korenkov M, Aretz M, Zechner U, Lehnen H, Haaf T (2013) Metabolic programming of MEST DNA methylation by intrauterine exposure to gestational diabetes mellitus. Diabetes 62:1320-1328.
35. Ding GL, Wang FF, Shu J, Tian S, Jiang Y, Zhang D, Wang N, Luo Q, Zhang Y, Jin F, Leung PC, Sheng JZ, Huang HF (2012) Transgenerational glucose intolerance with Igf2/H19 epigenetic alterations in mouse islet induced by intrauterine hyperglycemia. Diabetes 61:1133-1142.
36. Pentinat T, Ramon-Krauel M, Cebria J, Diaz R, Jimenez-Chillaron JC (2010) Transgenerational inheritance of glucose intolerance in a mouse model of neonatal overnutrition. Endocrinology 151:5617-5623.
37. Zhang X, Yang R, Jia Y, Cai D, Zhou B, Qu X, Han H, Xu L, Wang L, Yao Y, Yang G (2014) Hypermethylation of Sp1 binding site suppresses hypothalamic POMC in neonates and may contribute to metabolic disorders in adults: impact of maternal dietary CLAs. Diabetes 63:1475-1487.
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