Metabolic effects following periconceptional alcohol exposure in rat adult offspring

273 Pages·2015·10.05 MB·English

Metabolic effects following periconceptional alcohol exposure in
rat adult offspring: modulations by a postnatal western diet
Emelie Marlene Gårdebjer
MS.c

A thesis submitted for the degree of Doctor of Philosophy at
The University of Queensland in 2015
School of Biomedical Science
Abstract
It is increasingly recognized that maternal health and nutritional status prior to and
during pregnancy are important determinants of fetal outcome. Fetuses exposed to a
suboptimal in utero environment are at higher risk of developing adult onset diseases
including: type 2 diabetes, insulin resistance and obesity. Maternal alcohol intake
has been extensively studied when consumed during pregnancy, at different doses,
and different exposure periods. Many of these – including studies investigating low
levels of prenatal alcohol consumption – have demonstrated abnormal development
of fetal organs and alterations in metabolic pathways that persists into adulthood and
commonly result in insulin resistance and glucose intolerance. Many women who
consume alcohol do however cease upon pregnancy recognition – but the effects of
alcohol consumption during the periconceptional period (defined as prior to
conception until preimplantation) on adult offspring metabolism are still to be
investigated. Therefore, the focus of this thesis was to ascertain whether
periconceptional alcohol exposure (PC:EtOH-exposure) results in persisting
metabolic effects in the adult offspring; and whether this was mediated via placental
dysfunction. In addition, as adult onset disease initially programmed in utero can be
potentiated or first revealed by a mismatch between the pre- and postnatal
environment, or a ‘second hit’, this thesis also investigated the interaction between
PC:EtOH-exposure and a postnatal consumption of a western diet (WD).

As a part of this thesis, a rat model of maternal alcohol consumption (moderate to
high) was developed. 12.5% alcohol (v/v) was administered via a liquid diet to dams
from 4 days pre-conception until 4 days after confirmation of pregnancy (PC:EtOH
group). Control dams were given a nutritional equal liquid diet but without alcohol
during the exposure period (untreated (U) group). Separate cohorts of rats were
generated to study the offspring at different ages: one subset of dams was assigned
for studies of the late gestation fetus and investigation of the placenta on embryonic
day (E)20 (Chapter 3); another subset of dams littered down naturally to allow for
examination of physiological parameters at 6-8 months of age in offspring consuming
a control diet (C) (U:C and PC:EtOH:C), and in combination with a WD (U:WD and
PC:EtOH:WD) (Chapter 4 & 5). Physiological parameters in the offspring included
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investigation of glucose- and insulin homeostasis via a glucose tolerance test (GTT)
and an insulin tolerance test (ITT); and assessment of the body composition via a
dual X-ray (DXA)-scan. Plasma at different ages throughout development was
assessed and tissue (predominantly the placenta, liver, adipose tissue and muscle)
were investigated with quantitative polymerase chain reaction (qPCR), western
blotting and histology.

PC:EtOH-exposure resulted in fetal growth restriction, increased the placenta:body
weight ratio, and increased placental length and width in both males and females.
Morphological analyses of the placenta revealed that the increase in placental size
primarily was accounted for by increases in the junctional zone and sex-specific
increases in the cross-sectional area of apparent glycogen cells. These changes
were associated with sex-specific alterations in a wide array of placental genes and
proteins involved in nutrient transportation and vasculogenesis. Importantly, the
protective enzyme 11-beta hydroxysteroid dehydrogenase (11βHsd-2) was
expressed at higher levels in the labyrinth zone of the placenta, which is suggestive
of an augmented stress-response, apparent well beyond the exposure window.
mRNA levels of DNA methyltransferases were also increased in the fetal liver,
suggesting a potential epigenetic involvement.

In adulthood – regardless of postnatal diet – both male and female offspring were
glucose intolerant and insensitive to exogenous insulin. This was associated with
increased hepatic gluconeogenesis and sex-specific alterations in peripheral insulin
signaling. Male, but not female offspring had an increased percentage of total- and
abdominal fat mass; and had developed a more advanced stage of hepatic steatosis.
The plasma lipid composition was abnormal; and the levels of gene expression of
inflammatory genes were increased in both sexes. While the interactive effects of
PC:EtOH and a postnatal WD were negligible in female offspring, a WD exacerbated
the degree of insulin insensitivity and hepatic steatosis in PC:EtOH-exposed male
offspring. Often, the phenotypes created by PC:EtOH and a WD were similar –
suggesting the effects of PC:EtOH on adult metabolism in some circumstances are
comparable to those seen after consuming a WD for the major part of adult life.

This thesis has provided compelling experimental evidence that maternal
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periconceptional alcohol consumption can program the offspring to adult onset
disease. This information should be taken into account when developing public
policy guidelines regarding alcohol intake prior to and during early pregnancy and
made available to women of childbearing age.
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Declaration by author
This thesis IS COMPOSED OF MY ORIGINAL WORK, AND CONTAINS no
material previously published or written by another person except where due
reference has been made in the text. I have clearly stated the contribution by others
to jointly-authored works that I have included in my thesis.
I have clearly stated the contribution of others to my thesis as a whole, including
statistical assistance, survey design, data analysis, significant technical procedures,
professional editorial advice, and any other original research work used or reported
in my thesis. The content of my thesis is the result of work I have carried out since
the commencement of my research higher degree candidature and does not include
a substantial part of work that has been submitted TO QUALIFY FOR THE AWARD
OF ANY other degree or diploma in any university or other tertiary institution. I have
clearly stated which parts of my thesis, if any, have been submitted to qualify for
another award.
I acknowledge that an electronic copy of my thesis must be lodged with the
University Library and, subject to the policy and procedures of The University of
Queensland, the thesis be made available for research and study in accordance with
the Copyright Act 1968 unless a period of embargo has been approved by the Dean
of the Graduate School.
I acknowledge that copyright of all material contained in my thesis resides with the
copyright holder(s) of that material. Where appropriate I have obtained copyright
permission from the copyright holder to reproduce material in this thesis.

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Publications during candidature
Peer-reviewed publications
GARDEBJER, E. M., CUFFE, J. S., PANTALEON, M., WLODEK, M. E., & MORITZ,
K. M. 2014. Periconceptional alcohol consumption causes fetal growth
restriction and increases glycogen accumulation in the late gestation rat
placenta. Placenta, 35, 50-7.

GARDEBJER, E. M., ANDERSON, S. T., PANTALEON, M., WLODEK, M. E., &
MORITZ, K. M. 2015. Maternal alcohol intake around the time of conception
causes glucose intolerance and insulin insensitivity in rat offspring which is
exacerbated by a postnatal high-fat diet. FASEB J, (fj.14-268979).

In preparation

GARDEBJER, E. M., WARD, L. C., BIELFELDT-OHMANN, H., WLODEK, M. E., &
MORITZ, K. M. The effects of maternal alcohol intake around conception and
a postnatal high-fat diet on adiposity in male and female rat offspring.

Other publications

PROBYN, M. E., PARSONSON, K. R., GARDEBJER, E. M., WARD, L. C.,
WLODEK, M. E., ANDERSON, S. T. & MORITZ, K. M. 2013. Impact of Low
Dose Prenatal Ethanol Exposure on Glucose Homeostasis in Sprague-
Dawley Rats Aged up to Eight Months. PLoS One, 8, e59718.

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International conference abstracts
DOREY, E*., GARDEBJER, E. M., CAMPBELL, F., PARAVICINI, T. M., WEIR, K.
A., WLODEK, M. E., & MORITZ, K. M. 2014. Periconceptional alcohol
exposure causes alterations to renal and cardiac function in aged female
offspring. Postgraduate Symposium in Biomedical Sciences, Brisbane,
Australia, November 3-5th.

GARDEBJER, E. M*., MCMAHON, K., MORITZ, K. M., & PANTALEON, M. 2014.
Periconceptional alcohol exposure programs sex specific hyperinsulinemia
possibly through dysregulation of placental O-linked glycosylation. World
Congress of Reproductive Biology, Edinburgh, Scotland, September 2-4th.
Abstract P288. (Poster presentation).

GARDEBJER, E. M*., WARD, L. C., ANDERSON, S., & MORITZ, K. M. 2014.
Periconceptional alcohol consumption or a postnatal western diet causes
insulin insensitivity in a sex-specific manner in rat. Scandinavian
Physiological Society, Stockholm, Sweden, August 22-24th. (Oral
presentation).

GARDEBJER, E. M*., CUFFE, J. S & MORITZ, K. M. 2013. Periconceptional alcohol
consumption induces placental stress and causes fetal growth restriction.
Australiasian Fetal Alcohol Spectrum Disorders Conference, Brisbane,
Australia, November 19-20th, 2013. (Oral Presentation).

DOREY, E*., KALISCH-SMITH, J., GARDEBJER, E. M., & MORITZ, K. M. 2013.
Alterations in male kidney structure and function following periconceptional
ethanol exposure. Australiasian Fetal Alcohol Spectrum Disorders
Conference, Brisbane, Australia, November 19-20th.

GARDEBJER, E. M*., WARD, L. C., PANTALEON, M., WLODEK, M. E., & MORITZ,
K. M. 2013. Periconceptional alcohol exposure contributes to insulin
resistance in rat adult offspring. 2013 International Postgraduate Symposium
in Biomedical Sciences, Brisbane, Australia, October 28-30th. (Poster
presentation).

DOREY, E*., KALISCH-SMITH, J., GARDEBJER, E. M., WLODEK, M, E., WEIR, K.,
PANTALEON, M., & MORITZ, K. M. 2013. The effect of periconceptional
ethanol on kidney development and function. 2013 International
Postgraduate Symposium in Biomedical Sciences, Brisbane, Australia,
October 28-30th.

GARDEBJER, E. M*., WARD, L. C., WLODEK, M. E., PANTALEON, M., & MORITZ,
K. M. 2013. Periconceptional alcohol exposure contributes to insulin
resistance. 35th European Society for Clinical Nutrition and Metabolism,
Leipzig, Germany, August 31st – September 3rd. Clinical Nutrition, 32 (Suppl
1):S104. Abstract PP216-SUN. (Poster presentation).

GARDEBJER, E. M*., CUFFE, J. S & MORITZ, K. M. 2013. Periconceptional alcohol
exposure impacts on placental structure and gene expression. 37th Congress
of the International Union of Physiological Sciences, Birmingham, United
Kingdom, July 21-26th. Wiley online. Abstract PBC343. (Poster presentation).

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GARDEBJER, E. M*., & MORITZ, K. M. 2012. Periconceptional alcohol exposure
alters fetal and placental development. 2012 International Postgraduate
Symposium in Biomedical Sciences, Brisbane, Australia, September 24-26th.
(Poster presentation).

GARDEBJER, E. M*., & MORITZ, K. M. 2012. Periconceptional alcohol
consumption alters placental and fetal development. Society for Gynecologic
Investigation 2012 International Summit – Prematurity and Stillbirth, August
3-5th. Brisbane, Australia. (Poster presentation).

National conference abstracts
DOREY, E*., GARDEBJER, E. M., CAMPBELL, F., PARAVICINI, T. M., WEIR, K.
A., WLODEK, M. E., & MORITZ, K. M. 2014. Periconceptional alcohol alters
adult renal and cardiac function in a sex dependent manner. Australian
Physiological Society, Brisbane, Australia, November 30th-December 3rd.

DOREY, E*., GARDEBJER, E. M., CAMPBELL, F., PARAVICINI, T. M., WEIR, K.
A., WLODEK, M. E., & MORITZ, K. M. 2014. Periconceptional alcohol
exposure alters renal and cardiac function in aged female offspring. The
Annual Scientific Meeting of the ANZSN & Renal Society of Australasia
Annual Conference. August 25-27th. . . . .

GARDEBJER, E. M*., & MORITZ, K. M. 2012. The effect of periconceptional alcohol
consumption on the late gestation fetus. The Australian Early Origin of
Hypertension Workshop, Adelaide, Australia, September 27-29th. (Poster
presentation).

GARDEBJER, E. M*., & MORITZ, K. M. 2012. Periconceptional ethanol
consumption increase glucose concentrations in pregnant dams and alters
fetal and placental growth. The Annual Scientific Meeting of the Endocrine
Society of Australia and the Society for Reproductive Biology, August 26-
29th. Gold Coast, Australia. (Poster presentation).

GARDEBJER, E. M*., PANTALEON, M., & MORITZ, K. M. 2012. Does
periconceptional ethanol intake predispose the embryo to adult metabolic
syndrome? 26th Annual Meeting, The Fetal and Neonatal Workshop of
Australia and New Zealand, Port Stephens, Australia, March 16-17th. (Oral
Presentation).

*, presenting author
Other presentations

GARDEBJER, E. M. 2014. Don’t drink and get pregnant! Lund University seminar,
Clinical research center, Malmö, Sweden, October 13th. (Oral presentation).

GARDEBJER, E. M. 2014. Early and late effects of periconceptional alcohol
exposure in rat. Nestlé Research Center, Lausanne, Switzerland, December
17th. (Oral presentation).
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Publications included in this thesis
GARDEBJER, E. M., CUFFE, J. S., PANTALEON, M., WLODEK, M. E., & MORITZ,
K. M. 2014. Periconceptional alcohol consumption causes fetal growth
restriction and increases glycogen accumulation in the late gestation rat
placenta. Placenta, 35, 50-7.

Incorporated as Chapter 3
Contributor Statement of contribution
Gårdebjer, E. M. Study design (45%)
Animal treatment (95%)
Tissue collection (50%)
Gene and protein studies (100%)
Histological studies (95%)
Interpretation of results (70%)
Writing manuscript (70%)
Cuffe, J. S. Histological studies (5%)
Reviewing and editing manuscript (5%)
Interpretation of results (15%)
Pantaleon, M. Interpretation of results (5%)
Reviewing and editing manuscript (5%)
Wlodek, M. E. Reviewing and editing manuscript (10%)
Moritz, K. M. Study design (55%)
Animal treatment (5%)
Tissue collection (50%)
Interpretation of results (10%)
Reviewing and editing manuscript (10%)

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GARDEBJER, E. M., ANDERSON, S., PANTALEON, M., WLODEK, M. E., &
MORITZ, K. M. 2014. Maternal alcohol intake around the time of
conception causes glucose intolerance and insulin insensitivity in rat
offspring which is exacerbated by a postnatal high-fat diet. FASEB. J,
(fj.14-268979).

Incorporated as Chapter 4
Contributor Statement of contribution
Gårdebjer, E. M. Study design (50%)
Animal treatment* (80%)
Tissue collection† (60%)
Glucose/Insulin tolerance testing (100%)
Plasma assays (100%)
Gene and protein studies (100%)
Interpreting results (65%)
Writing manuscript (75%)
Anderson, S. Interpretation of results (15%)
Reviewing and editing manuscript (10%)
Pantaleon, M. Tissue collection† (5%)
Interpretation of results (10%)
Reviewing and editing manuscript (5%)
Wlodek, M. E. Reviewing and editing manuscript (5%)
Moritz, K. M. Study design (50%)
Animal treatment* (5%)
Tissue collection† (20%)
Interpretation of results (10%)
Reviewing and editing manuscript (5%)

*, Remaining 15% performed by Kalisch-Smith, J; †, remaining 15% contributed to by
other members of Moritz’s lab
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