lra.le.ac.uk Web viewWord count: Text – 2770; Abstract ... model does not mimic the placenta/fetal overgrowth often associated with gestational diabetes in women [4]

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

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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

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