DOI: 10.5061/dryad.mpg4f4r31
title: Impact of maternal obesity on the gestational metabolome and infant metabolome, brain, and behavioral development in rhesus macaques

description: Maternal gestational obesity is associated with elevated risks for
 neurodevelopmental disorder, including autism spectrum disorder. However,
 the mechanisms by which maternal adiposity influences fetal developmental
 programming remain to be elucidated. We aimed to understand the impact of
 maternal obesity on the metabolism of both pregnant mothers and their
 offspring, as well as on metabolic, brain, and behavioral development of
 offspring by utilizing metabolomics, protein, and behavioral assays in a
 non-human primate model. We found that maternal obesity was associated
 with elevated inflammation and significant alterations in metabolites of
 energy metabolism and one-carbon metabolism in maternal plasma and urine,
 as well as in placenta. Infants born to obese mothers were significantly
 larger at birth compared to those born to lean mothers. Additionally, they
 exhibited significantly reduced novelty preference and significant
 alterations in their emotional response to stress situations. These
 changes coincided with differences in phosphorylation of enzymes in the
 brain mTOR signaling pathway between infants born to obese and lean
 mothers and correlated with the concentration of maternal plasma betaine
 during pregnancy. In summary, gestational obesity significantly impacted
 the infant systemic and brain metabolome and adaptive behaviors.
description: <em>2.1 Study population</em>
 Animal handling was approved by the UC Davis Institutional Animal
 Care and Use Committee (IACUC protocol#19299) and all experiments were
 performed in accordance with relevant guidelines and
 regulations. Pregnant female rhesus macaques
 (<em>Macaca mulatta</em>) with appropriate social behavior and
 a previous successful pregnancy were selected from an indoor breeding
 colony at the California National Primate Research Center (CNPRC). Fetal
 sex was determined by a qualitative real time PCR assay to detect the Y
 chromosome and only those with male fetuses were chosen for this study.
 Animals used in this study had maintained a consistent BCS for at least
 one year prior to the study. Obesity is defined when subjects have body
 fat above 30% for women (American Medical Association). A BCS of 3.5 was
 chosen as the cut off for inclusion in the obese group for this study as a
 BCS of 3.5 is correlated with 32 % body fat [14]. Mothers with a BCS of
 2.5 or lower comprised the Lean group. The sample size of the biological
 samples was not balanced due to fetal deaths for unknown reasons,
 misidentification of female offspring, technical issues in collecting
 enough sample volume for analysis, or recruitment of additional animals
 into the study in the middle of pregnancy to account for the sample loss
 (Supplementary Table S1). The final number of mothers and their offspring
 included six for the Lean and seven for the Obese groups (Supplementary
 Table S2). <em>2.2 Feeding and rearing of
 animals</em> Adult animals were fed
 seven biscuits (#5047; LabDiet, St. Louis, MO, USA) twice daily between
 6-9 am and 1-3 pm. All mothers were provided nine biscuits twice daily
 once pregnancy was determined, and twelve biscuits twice daily while
 nursing infants 4 months and older<em>.</em> Fresh produce was
 provided biweekly and water was available <em>ad libitum</em>.
 More detailed description is available in Appendix A.
 <em>2.3
 Sample collection and processing</em>
 All animals were coded by IDs, and therefore, the animal care
 takers and researchers who collected samples were blinded for group
 assignment. The collected biological samples were randomized using random
 numbers generated in R in conducting assessments, and the group assignment
 was blinded until the data was analyzed. On the day prior to sample
 collection, food was removed approximately 30 min after the feeding in the
 afternoon, and biological samples were collected before the morning
 feeding. Pregnancy in rhesus macaques lasts for 166.5 days on average.
 Plasma and urine samples were collected from mothers once during the
 1<sup>st</sup> and 2<sup>nd</sup> trimesters, and
 twice during the 3<sup>rd</sup> trimester on GD45, 90, 120,
 and 150 after anesthetizing animals with 5-30 mg/kg ketamine or 5-8 mg/kg
 telazol. Blood samples were collected in 5 mL lavender top (EDTA) or green
 (heparin) tubes and the supernatant was collected. Urine was collected
 from the bladder by ultrasound-guided transabdominal cystotomy using a
 22-gauge needle and subsequently centrifuged to collect supernatant.
 Within 15 days prior to delivery, a placental sample was collected
 transabdominally under ultrasound guidance using an 18-gauge needle
 attached to a sterile syringe and centrifuged to collect the
 pellet. Infant plasma was collected at PD30, 90, and
 110, and plasma, urine, and brain tissues were collected at PD180 when
 necropsy was conducted between 9:30 am-1:30 pm. Infants were anesthetized
 with ketamine and plasma and urine were collected, followed by euthanasia
 with 120 mg/kg pentobarbital. Heparin injection and clamping of the
 descending aorta was followed by flushing with saline at room temperature
 for 2 min and then by saline at 4 °C for 5 min at 250 mL/min until clear.
 The brain was extracted, and four regions (amygdala, hippocampus,
 hypothalamus, and prefrontal cortex) were dissected and immediately
 frozen. All collected samples were stored at -80 °C.
 <em>2.4
 Metabolite extraction and insulin, cytokine, and cortisol
 measurement</em> The plasma and
 urine samples were thawed on ice and filtered by Amicon Ultra Centrifugal
 Filter (3k molecular weight cutoff; Millipore, Billerica, MA, USA), and
 the filtrate was used for metabolomics analysis. Samples were stored at 4
 °C overnight and pH was adjusted to 6.8 ± 0.1. For the placental samples,
 polar metabolites were extracted as described with the following
 modification: after lyophilization of the polar metabolite layer, the
 dried sample was reconstituted in 270 µL of 10 mM phosphate buffer (pH
 6.85) prepared in deuterium oxide. Samples were transferred to 3 mm Bruker
 NMR tubes and kept at 4 °C until NMR data acquisition within 24 hours of
 sample preparation. A multiplex Bead-Based Kit
 (Millipore) was used to measure insulin as well as 17 cytokine and
 chemokine levels in heparin-treated plasma samples including hs-CRP,
 Granulocyte-macrophage colony-stimulating factor (GMCSF), interferon-γ
 (IFN-γ), tumor necrosis factor-α (TNF-α), transforming growth factor-α
 (TGF-α), monocyte chemoattractant protein-1 (MCP-1), macrophage
 inflammatory protein-1β (MIP-1β), interleukin (IL)-1β, IL-1 receptor
 antagonist (IL-1ra), IL-2, IL-6, IL-8, IL-10, IL-12/23 p40, IL-13, IL-15,
 and IL-17A. The assay was performed following the manufacturer’s protocol.
 Assessment of infant plasma cortisol was conducted as previously described
 [17,18]. Briefly, infants were separated from their mothers at 9 am and
 blood samples were collected at 11 am (Sample 1). Another blood collection
 was done at 4 pm (Sample 2), followed by intramuscular injection of 500
 μg/kg Dex. Blood was collected at 8:30 am of the following day (Sample 3)
 and 2.5 IU of ACTH was then injected intramuscularly. After 30 min, the
 last blood was collected (Sample 4). More detailed description is
 available in Appendix A. <em>2.5 <sup>1</sup>H
 nuclear magnetic resonance (NMR) spectroscopy data
 acquisition</em> We conducted an
 untargeted metabolomics analysis using <sup>1</sup>H NMR
 spectroscopy. All spectra were acquired at 25 °C using the noesypr1d pulse
 sequence on a Bruker Avance 600 MHz NMR spectrometer (Bruker, Billerica,
 MA, USA). Identification and quantification of metabolites were completed
 using Chenomx NMRSuite (version 8.1, Chenomx Inc., Edmonton,
 Canada). <em>2.6 Protein
 analysis</em> Protein was extracted
 from the cell layer collected after metabolite extraction of brain
 samples, and protein quantification and western blots were done as
 previously described. The following antibodies from Cell Signaling
 Technology (Danvers, MA, USA) were used for the western blots: rabbit
 anti-Akt (#9272), anti-phospho-Akt
 (#9275; Thr308), anti-AMPKα (#2603), anti-phospho-AMPKα
 (#2535; Thr172), anti-p70 S6K (#9202), anti-phospho-p70 S6K (#9234;
 Thr389), as well as goat anti-rabbit IgG antibody conjugated to
 horseradish peroxidase (#7074). Either Clarity Western ECL Blotting
 Substrates (Bio-Rad) or Radiance Plus (Azure biosystems, Dublin, CA, USA)
 were used depending on the strength of signal for chemiluminescent
 detection. Refer to Appendix A for more detailed description.
 <em>2.7
 VPC Test</em> Recognition memory was
 tested with infants on post-conception day 200 ± 3 days using a VPC test
 conducted between 8:30-10:30 am. Briefly, infants were hand-held in front
 of a testing booth to look at two identical black and white high contrast
 abstract pictures (Fagan Test of Infant Intelligence; Infantest
 Corporation, Cleveland, OH, USA) placed to the right and left of center
 for a total of 20 sec of cumulative looking time (familiarization trial).
 Then, the familiar and novel pictures were placed either right or left of
 center according to a pre-decided random order for a 10 sec test period
 from the time of the first fixation (preference trial 1). The side of the
 pictures was switched and a second 10 sec test period was conducted
 (preference trial 2). Four problems were presented to each infant. The
 trials were video recorded for later scoring of frequency and duration of
 looking patterns using The Observer software (Noldus, Inc., Wageningen,
 The Netherlands). Novelty preference was calculated as: number of
 fixations at the novel stimulus/number of fixations at both the novel and
 familiar stimulus. <em>2.8 HI
 test</em> The HI test was conducted
 as described previously. In short, we examined the frequency of scratch
 (as an indicator of anxiety) in response to the following four graded
 levels of stress (1 min each): Profile-Far (technician presented the left
 profile from ~1 m away from an infant in a cage), Profile-Near (presented
 left profile from ~0.3 m), Stare-Far (made direct eye contact with the
 animal from far), and Stare-Near (direct eye contact from near
 position). <em>2.9
 Statistics</em> The overall
 gestational weight gain (GWG) rate was obtained using the following
 equation: 1000 * ln(W2/W1)/(D2-D1), where D1 is the date the last weight
 (W1) was obtained before pregnancy and D2 is the date the last weight (W2)
 was obtained before birth. Homeostatic model assessment for insulin
 resistance (HOMA-IR) was calculated using fasting glucose (mg/dL) *
 fasting insulin (μU/mL)]/405.
 Estimated placental volume (EPV) was calculated as follows: (πT/6) *
 [4H(W−T)+W(W−4T)+4T<sup>2</sup>, where T is thickness at
 maximal height, H is height at maximal width, and W is maximal width
 measured by ultrasound. Metabolite concentrations were
 log-transformed prior to application of the following statistics. A linear
 mixed-effects model was fitted followed by ANOVA to test the group
 difference of samples with multiple time points using lme4 package
 (version 1.1.21). Estimated marginal means were obtained using emmeans
 (version 1.4.4), and pairwise comparison was applied as a post-hoc test.
 R<sup>2</sup> values were obtained as the effect size using
 r2glmm (version 0.1.2) (medium effect when
 0.15&lt;R<sup>2</sup>&lt;0.35; large when 0.35
 R<sup>2</sup>). T-tests were used to test for
 group differences of samples collected at a single time point. The effect
 size was calculated using Cohen’s D (d) measurement (medium effect when
 0.5&lt;d&lt;0.8; large when 0.8
 d&lt;1.3; very large when 1.3
 d). For metabolomics analysis, Benjamini-Hochberg false
 discovery rate correction was applied to p-values. For post-hoc tests, a
 p-value of 0.05 was used as the cut-off. Correlation between HOMA-IR and
 EPV was assessed by repeated measures correlation using rmcorr package
 (version 0.4.1) and other correlations were tested by the Pearson or
 Spearman methods and visualized using ggscatter (version 0.2.5). The
 correlation coefficient (R) was used as the effect size (medium effect
 when 0.3&lt;R&lt;0.5; large when 0.5
 R 1). In order to address the small sample size and the inherent large heterogeneity and variability of rhesus macaque data, we utilized a combination of p-values &lt; 0.1 and medium-large effect size to define statistical significance. More detailed description is available in Appendix A.