DOI: 10.5061/dryad.6hdr7sr43
title: Calorie restriction and pravastatin administration during pregnancy in obese rhesus macaques modulates maternal and infant metabolism and infant brain and behavioral development

description: Maternal obesity has been associated with a higher risk of
 pregnancy-related complications in mothers and offspring; however,
 effective interventions have not yet been developed. We tested two common
 interventions, calorie restriction and pravastatin administration, during
 pregnancy in a rhesus macaque model with the hypothesis that these
 interventions would normalize metabolic dysregulation in pregnant mothers
 leading to an improvement in infant metabolic and cognitive/social
 development. A total of 19 obese mothers were assigned to either one of
 the two intervention groups (n=5 for calorie restriction; n=7 for
 pravastatin) or an obese control group (n=7) with no intervention, and
 maternal gestational samples and postnatal infant samples were compared
 with lean control mothers (n=6). Gestational calorie restriction
 normalized one-carbon metabolism dysregulation in obese mothers but
 altered energy metabolism in their offspring. Although administration of
 pravastatin during pregnancy tended to normalize blood cholesterol in the
 mothers, it potentially impacted the gut microbiome and kidney function of
 their offspring. In the offspring, both calorie restriction and
 pravastatin administration during pregnancy tended to normalize the
 activity of AMPK in the brain at 6 months, and while results of the Visual
 Paired-Comparison test, which measures infant recognition memory, were not
 significantly impacted by either of the interventions, gestational
 pravastatin administration, but not calorie restriction, tended to
 normalize anxiety assessed by the Human Intruder test. Although the two
 interventions tested in a non-human primate model led to some improvements
 in metabolism and/or infant brain development, negative impacts were also
 found in both mothers and infants. Our study emphasizes the importance of
 assessing gestational interventions for maternal obesity on both maternal
 and offspring long-term outcomes.
description: <strong>Study
 population</strong> Pregnant female
 rhesus macaques (<em>Macaca mulatta</em>) from an indoor
 breeding colony at the California National Primate Research Center with
 appropriate social behavior and previous successful pregnancies were
 enrolled. Animal handling was approved by the UC Davis Institutional
 Animal Care and Use Committee (IACUC) (#19299). A qualitative real-time
 PCR assay (Jimenez & Tarantal, 2003) was used to identify mothers
 with male fetuses to include in this study. Since obesity is defined as
 subjects with body fat above 30% for women, according to guidelines from
 the American Society of Bariatric Physicians, American Medical
 Association, and in some publications (Okorodudu et al., 2010; Shah
 & Braverman, 2012), a Body Condition Score (BCS) of 3.5 (32.8 %
 body fat on average (Summers et al., 2012)) was used as the cutoff.
 Therefore, mothers with BCS of 3.5 and above were categorized as obese.
 Obese mothers were randomly assigned to the Obese Control (OC) group, OR
 group (received calorie Restriction), or OP group (received Pravastatin).
 Mothers with BCS of 2.5 and below were assigned to the Lean Control (LC)
 group. The unbalanced sample size was because some mothers were removed
 from the analyses due to fetal deaths for unknown reasons,
 misidentification of a female fetus, different timing for study
 enrollment, or technical issues upon collecting samples. The number of
 animals was six for the LC, seven for the OC, five for the OR, and seven
 for the OP groups. <strong>Feeding, rearing, and
 interventions</strong> Adult female
 animals were provided monkey diet (High Protein Primate Diet Jumbo #5047;
 LabDiet, St. Louis, MO, USA) twice a day between 6–9 am and 1–3 pm. The
 calories were provided as 56% from carbohydrates, 30% from protein, and
 13% from. Mothers in the LC, OC, and OP groups were fed nine biscuits
 twice a day once pregnancy was confirmed. Mothers in the OR group received
 a restricted supply of food once the pregnancy was detected and was
 maintained throughout pregnancy. The food restriction was set such that
 the average total weight increase would be 8% body weight from the last
 day before conception because the recommended total weight gain in the
 2<sup>nd</sup> and 3<sup>rd</sup> trimesters is
 5-9 kg for the average US woman with obesity who weighs 80 kg and is 1.6 m
 in height (Body Mass Index of 30), according to the Institute of Medicine
 2009 guidelines
 (Institute of Medicine and National Research Council, 2009).
 During nursing of infants older than 4 months, all mothers were provided
 twelve biscuits. Fresh produce was provided biweekly, and water was
 provided <em>ad libitum</em> for all mothers. Mothers in the
 OP group were given pravastatin sodium (ApexBio Technology, Houston, TX,
 USA) at 20 mg/kg body weight prepared in a neutralized syrup (20 mg/mL
 sodium bicarbonate dissolved in a fruit-flavored syrup (Torani, San
 Leandro, CA, USA)) once a day from the time pregnancy was confirmed until
 delivery. The caloric value of the administration was made so as not to
 influence body weight or skew nutritional value of the diet among all
 treatment groups. Both interventions were applied only during
 gestation.<em> </em>Although most mothers were allowed to
 deliver naturally, cesarean delivery was performed for fetal indications
 when recommended by veterinarians (2 for each of the LC and OC groups, and
 1 for the OP group). These mothers did not accept their infant following
 birth, so foster mothers were provided. <strong>Sample Collection and
 pre-processing prior to sample storage</strong> The animal caretakers and researchers who
 collected samples were blinded for group assignment by coding all animals
 by IDs. The collected biological samples were randomized by using random
 numbers and the group assignment was blinded during the data
 collection. Both mothers
 (during pregnancy) and infants were weighed every week. One day before
 sample collection, food was removed 30 min after the afternoon feeding,
 and biological samples were collected prior to the morning feeding. To
 collect biological samples, animals were anesthetized using 5–30 mg/kg
 ketamine or 5–8 mg/kg telazol. Both maternal and infant blood was
 collected using 5 mL lavender top (EDTA) tubes (Monoject, Cardinal Health,
 Dublin, OH, USA) and urine was collected from the bladder by
 ultrasound-guided transabdominal cystotomy using a 22-gauge needle and
 stored in a 15 mL Falcon tube. A placental sample was collected at GD150
 transabdominally under ultrasound guidance using an 18-gauge needle
 attached to a sterile syringe. Sample processing was as previously
 described in (Hasegawa et al., 2022). Necropsy was conducted between 9:30
 am–1:30 pm. First, infants at the age of PD180 were fasted and
 anesthetized with ketamine, and
 plasma and urine were collected. Then, euthanasia was performed with 120
 mg/kg pentobarbital, followed by heparin injection, clamping of the
 descending aorta, and flushing with saline until clear. The kidney and
 brain (amygdala, hippocampus, hypothalamus, and prefrontal cortex) were
 collected, weighed, and immediately frozen on dry ice or
 liquid nitrogen to store at -80 °C until further
 analyses<em>.</em> <strong>Metabolite extraction and
 analysis by <sup>1</sup>H NMR, and measurement of insulin,
 cholesterol, cytokine, and cortisol</strong> Detailed procedures were previously
 described (Hasegawa et al., 2022). Briefly, plasma and urine samples were
 filtered using Amicon Ultra Centrifugal Filter (3k molecular weight
 cutoff; Millipore, Billerica, MA, USA), and the supernatant was used for
 analysis. For both the placental and brain tissue samples, polar
 metabolites were extracted using our previously reported method (Hasegawa
 et al., 2020). A total of 180 μL of sample (tissue extract or filtered
 urine or serum) was transferred to 3 mm Bruker NMR tubes (Bruker,
 Billerica, MA, USA). Within 24 h of sample preparation, all
 <sup>1</sup>H NMR spectra were acquired using the noesypr1d
 pulse sequence on a Bruker Avance 600 MHz NMR spectrometer (Bruker,
 Billerica, MA, USA) (O’Sullivan et al., 2013). Chenomx NMRSuite (version
 8.1, Chenomx Inc., Edmonton, Canada) (Weljie et al., 2006) was used to
 identify and quantify metabolites. Heparin-treated plasma
 samples were used to measure insulin and 17 cytokines and chemokines
 (hs-CRP, Granulocyte-macrophage colony-stimulating factor, IFN-γ, TNF-α,
 transforming growth factor-α, monocyte chemoattractant protein-1,
 macrophage inflammatory protein-1β (MIP-1β), and 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) using a multiplex Bead-Based Kit (Millipore) on
 a Bio-Plex 100 (Bio-rad, Hercules, CA) following the manufacturer’s
 protocol. For each sample, a minimum of fifty beads per region were
 collected and analyzed with Bio-Plex Manager software using a 5-point
 standard curve with immune marker quantities extrapolated based on the
 standard curve. Two samples were removed for analysis of TNF-α and IL-1ra
 as technical errors (both from Animal ID 1132103: 895.2 and 1115.1 pg/mL
 at gestational days (GD) 90; 510.8 and 617.2 pg/mL at GD120,
 respectively). Plasma cholesterol level was measured by Clinical
 Laboratory Diagnostic Product (OSR6116) on Beckman Coulter AU480 (Beckman
 Coulter, Brea, CA). Infant plasma cortisol level at PD110 was
 assessed as previously described (Vandeleest et al., 2019; Walker et al.,
 2018). In short, infants were transferred to a test room at 9 am and blood
 was drawn at 11 am (Sample 1), followed by another blood collection at 4
 pm (Sample 2) and intramuscular injection of 500 μg/kg dexamethasone (Dex)
 (American Regent Laboratories, Inc., Shirley, NY). On the next day, a
 blood sample was collected at 8:30 am (Sample 3), and then 2.5 IU of
 adrenocorticotropic hormone (Amphastar Pharmaceuticals, Inc., Rancho
 Cucamonga, CA) was injected intramuscularly. The last blood was collected
 (Sample 4) 30 min after adrenocorticotropic hormone injection. The
 collected blood samples were processed and stored, and cortisol
 concentration was assessed by a chemiluminescent assay on the ADVIA
 Centaur CP platform (Siemens Healthcare Diagnostics, Tarrytown, NY,
 USA). <strong>Protein quantification
 and western blot analysis</strong> Protein quantification and western blot
 analysis were conducted as previously described (Hasegawa et al.,
 2020)<em>.</em> Briefly, a DC Protein Assay kit (Bio-Rad) was
 used for total protein quantification. For the western blot, the following
 antibodies were used: rabbit anti-Akt
 (#9272), anti-phospho-Akt (#9275; Thr308), anti-Adenosine
 Monophosphate-activated Protein Kinase (AMPK)α (#2603),
 anti-phospho-AMPKα (#2535; Thr172), anti-p70 S6 Protein Kinase (S6K)
 (#9202), anti-phospho-p70 S6K (#9234; Thr389), and goat anti-rabbit IgG antibody
 conjugated to horseradish
 peroxidase (#7074) (Cell Signaling
 Technology, Danvers, MA, USA). For chemiluminescent
 detection, Clarity Western ECL Blotting Substrates (Bio-Rad) or Radiance
 Plus (Azure biosystems, Dublin, CA USA) were used. <strong>Visual Paired Comparison Test
 at ~1 month of age</strong> On post-conception day 200 ± 3 days,
 recognition
 memory development of infants was assessed using Visual Paired Comparison (VPC) conducted between 8:30–10:30 am (Burbacher & Grant, 2012; Golub et al., 2006, 2014). In brief, two identical black and white contrast abstract pictures (Fagan Test of Infant Intelligence; Infantest Corporation, Cleveland, OH) were placed right and left of center, and infants were allowed to observe for a total of 20 sec (familiarization trial). Then, the pictures were switched to the familiar and novel ones placed either right or left of center based on a predetermined random order, and the frequency and duration of looking were video recorded using The Observer software (Noldus, Inc., Wageningen, The Netherlands) for a 10 sec from the time of the first fixation (preference trial 1). The side of the pictures was switched, and another 10 sec test period was recorded (preference trial 2). In total, four problems were conducted to each infant. Novelty preference was calculated by dividing the number of fixations at the novel stimulus by number of fixations at both the novel and familiar stimulus. <strong>Human Intruder (HI) test at ~4 months of age</strong> HI test was conducted as described before (Walker et al., 2018). Briefly, we measured the frequency of scratch (as an indicator of anxiety (Troisi, 2002)) for 1 min in four graded levels of stress: Profile-Far (the left profile of a technician was presented from ~1 m away from an infant), Profile-Near (left profile presented from ~0.3 m), Stare-Far (direct eye contact was made with the infant from far position), and Stare-Near (direct eye contact made from near position). <strong>Statistical analyses</strong> All statistical analyses were done in R (version 3.6.1). GWG was obtained using the following equation: [(delivery weight) - (last weight before conception)] / (last weight before conception) * 100, where the delivery weight is the last weekly weight before delivery. Homeostasis model assessment for insulin resistance (HOMA-IR) was obtained using the following equation: [fasting glucose (mg/dL) * fasting insulin (μU/mL)] / 405. kidney tissue weight was divided by the body weight at PD7 to obtain the normalized kidney mass. One animal with ID1215218 (OC group) was removed due to a technical error in measurement of insulin, as the insulin level was higher than biologically reasonable (Zhang et al., 2011). The following equation was used to calculate estimated placental volume (EPV): (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 (Sonek et al., 2018). Either the OR or the OP group was separately compared with both control groups (OC and LC). For data that were collected at a single time point, one-way ANOVA was performed and Tukey honestly significant difference as a post-hoc test was applied. Eta squared was calculated as the effect size measurement using sjstats package (version 0.18.0), with a medium effect defined as 0.06&lt; eta squared &lt;0.14, and a large effect defined as eta squared ³ 0.14. For data collected at multiple time points, a linear mixed-effects model was fit followed by ANOVA to test for group differences using the lme4 package (version 1.1.21). Estimated marginal means were calculated by using emmeans (version 1.4.4) and pairwise comparison was applied as a post-hoc test. Effect size for multiple time points was calculated as coefficient of determination (R<sup>2</sup>) using r2glmm (version 0.1.2), with a medium effect defined as 0.15&lt;R<sup>2</sup>&lt;0.35, and a large effect defined R<sup>2</sup> &gt;=0.35. For metabolomics data, metabolite concentrations were log-transformed prior to statistical analysis. Benjamini-Hochberg false discovery rate for multiple testing correction was applied. After testing for the model fit, the statistical model was adjusted by maternal age for maternal plasma samples, by the centered GWG values for maternal urine samples, and by maternal age for infant plasma samples. Infant weights were corrected for gestational length. In order to avoid false negative results due to the small sample size, statistical significance was defined as a combination of uncorrected p-value of less than 0.1 and medium-large effect (Sullivan & Feinn, 2012). Unless otherwise stated, data are presented as mean ± standard error. We used duration of looking from eye tracking test measured at months 0.25, 1, 3, and 6 as the primary outcome to calculate the power. Based on the previous study (Bauman et al., 2013), we assumed a correlation among the four repeated observations ranges between 0.3 and 0.7, for a type one error α = 0.05, the intended sample size of eight monkeys per group can provide a power of 80-93% to detect the rates of change per month between the two groups as small as 1.1 SD (Diggle et al., 2002). For cytokine/chemokine data, Partial Least Squares Discriminant Analysis (PLS-DA) was performed as well to examine whether different combinations of multiple cytokines could be used to differentiate between treatment groups. The PLS-DA model was used to cluster the groups and the Variable Importance in Projection (VIP) scores were obtained measure the importance of cytokine/chemokine in the PLS-DA model. Cytokine/chemokines with VIP scores above 1.0 were defined to have meaningful impact on the group separation. PLS-DA model was performed via MetaboAnalyst 5.0 (Xia et al., 2009).