The Journal of Nutrition 153 (2023) 3382–3396

journal homepage: https://jn.nutrition.org/

Genomics, Proteomics, and Metabolomics

Maternal-Periconceptional Vitamin B12 Deficiency in Wistar Rats

Leads to Sex-Specific Programming for Cardiometabolic Disease Risk in
the Next Generation
Praveen Singh 1, 3, #, Lovejeet Kaur 2, 4, #, Subhoshree Ghose 1, 3, Swati Varshney 1, 3,
Vislavath Jyothi 2, Sourav Ghosh 1, 3, Pujitha Kommineni 2, Shamsudheen KV 1, Vinod Scaria 1, 3,
Sridhar Sivasubbu 1, 3, Giriraj Ratan Chandak 2, 3, **, Shantanu Sengupta 1, 3, *
1
CSIR-Institute of Genomics and Integrative Biology, New Delhi, India; 2 CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India;
3
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India; 4 Translational Health Science and Technology Institute, Faridabad,
India

A B S T R A C T

Background: Maternal vitamin B12 deficiency plays a vital role in fetal programming, as corroborated by previous studies on murine
models and longitudinal human cohorts.
Objectives: This study assessed the effects of diet-induced maternal vitamin B12 deficiency on F1 offspring in terms of cardiometabolic
health and normalization of these effects by maternal-periconceptional vitamin B12 supplementation.
Methods: A diet-induced maternal vitamin B12 deficient Wistar rat model was generated in which female rats were either fed a control AIN-
76A diet (with 0.01 g/kg vitamin B12) or the same diet with vitamin B12 removed. Females from the vitamin B12-deficient group were
mated with males on the control diet. A subset of vitamin B12-deficient females was repleted with vitamin B12 on day 1 of conception. The
offspring in the F1 generation were assessed for changes in body composition, plasma biochemistry, and molecular changes in the liver. A
multiomics approach was used to obtain a mechanistic insight into the changes in the offspring liver.
Results: We showed that a 36% reduction in plasma vitamin B12 levels during pregnancy in F0 females can lead to continued vitamin B12
deficiency (60%–70% compared with control) in the F1 offspring and program them for cardiometabolic adversities. These adversities, such
as high triglycerides and low high-density lipoprotein cholesterol, were seen only among F1 males but not females. DNA methylome analysis
of the liver of F1 3-mo-old offspring highlights sexual dimorphism in the alteration of methylation status of genes critical to signaling
processes. Proteomics and targeted metabolomics analysis confirm that sex-specific alterations occur through modulations in PPAR signaling
and steroid hormone biosynthesis pathway. Repletion of deficient mothers with vitamin B12 at conception normalizes most of the molecular
and biochemical changes.
Conclusions: Maternal vitamin B12 deficiency has a programming effect on the next generation and increases the risk for cardiometabolic
syndrome in a sex-specific manner. Normalization of the molecular risk markers on vitamin B12 supplementation indicates a causal role.

Keywords: maternal vitamin B12 deficiency, intergenerational programming, repletion, sex-specific effects, cardiometabolic abnormalities

Abbreviations: C, control; DMR, differentially methylated region; FFM, fat-free mass; iTRAQ, isobaric tags for relative and absolute quantitation; LBM, lean body
mass; LC, liquid chromatography; MetS, metabolic syndrome; MeDIP-Seq, methylated DNA immunoprecipitation sequencing; MS, mass spectrometry; OCM, one-
carbon metabolic; PPAR, peroxisome proliferator-activated receptors; RC, repletion at conception; TC, total cholesterol; TG, triglyceride; TSS, transcription start site;
UTR, untranslated region; VR, vitamin-restricted.
* Corresponding author.
** Corresponding author. E-mail addresses: chandakgrc@ccmb.res.in (G.R. Chandak), shantanus@igib.res.in (S. Sengupta).
#
PS and LK contributed equally to this work.

https://doi.org/10.1016/j.tjnut.2023.08.032
Received 16 June 2023; Received in revised form 18 August 2023; Accepted 24 August 2023; Available online 1 September 2023
0022-3166/© 2023 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.
P. Singh et al.
Introduction
In utero maternal nutritional status is known to influence the
offspring’s health. Under and/or overnutrition can predispose
the fetus to the risk of developing noncommunicable diseases,
such as diabetes, hypertension, stroke, and coronary artery dis-
ease in adult life [1]. The Developmental Origins of Health and
Disease hypothesis has emerged based on epidemiologic obser-
vations that in utero malnutrition is associated with altered fetal
birth weight and future risk of the onset of metabolic syndrome
(MetS) [2–4]. Quantity and quality of macronutrients, such as
proteins, fat, and micronutrients, such as vitamins and minerals,
play a critical role in fetal development [5–7]. Deficiencies of
vitamin B12 and/or vitamin B9 (folate) are known to influence
fetal growth and differentiation through altered fetal program-
ming via regulating DNA methylation at several gene loci [8].
Both vitamin B12 and folate participate in the one-carbon
metabolic (OCM) pathway in which folate acts as a methyl
group carrier and participates in the biosynthesis of nucleotides
whereas vitamin B12 acts as a cofactor of methionine synthase,
which catalyzes the conversion of homocysteine to methionine.
Epidemiologic studies demonstrate that low serum vitamin B12
concentrations are common during pregnancy due to increased
demand throughout the gestational period [9]; severe deficiency
can be associated with intrauterine growth restriction, neural
tube defects, preterm birth and low-birth weight, adiposity, and
other adverse pregnancy outcomes [10–12].
Our previous studies have shown that a 90% plasma vitamin
B12 reduction in female Wistar rats is associated with insulin
resistance, adiposity, oxidative stress, and increased gluconeo-
genesis in male offspring [13]. Proteomics study performed on
the liver of offspring born to vitamin B12-deficient mothers
revealed alteration of carbohydrate, amino acid, fatty acid, and
lipid metabolism pathways [14]. Moreover, 90% reduction of
maternal plasma vitamin B12 has been shown to alter the
methylation status of candidate genes involved in the fatty acid
oxidation pathway and mitochondrial metabolism in the liver of
F1 generation male offspring [15]. Interestingly, repletion of
mothers with vitamin B12 at conception and parturition
normalized the methylation signatures as well as the plasma
lipid levels [15]. However, this extreme deficiency model does
not appropriately mimic the vitamin B12 deficiency that is
routinely observed in the human population. Importantly, it has
been shown previously that severe vitamin B12 deficiency could
modulate the anthropometric, biochemical parameters, and in-
flammatory cytokines to a greater extent than moderate defi-
ciency [12]. Therefore, it becomes pertinent to understand the
phenotypes and underlying mechanisms associated with this
range of vitamin B12 deficiency. Further, in our previous study,
we analyzed intergenerational effects of vitamin B12 deficiency
only in male offspring; however, several studies have shown
differential bioavailability of vitamin B12 in males and females
[16–19]. Some of these differences have been attributed to the
levels of sex hormones, such as testosterone and estrogen, which
also affect the OCM pathway at multiple levels [20,21]. Thus, it
is also important to independently understand the intergenera-
tional effects of vitamin B12 deficiency in males and females.
In the present study, we generated a female Wistar rat model
with 36% vitamin B12 deficiency in plasma (compared with
control) and assessed its molecular and biologic effects on the
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The Journal of Nutrition 153 (2023) 3382–3396
offspring in the next generation. To ascertain causality, we
looked for the reversal of these effects by maternal-
periconceptional vitamin B12 repletion. We observed that
vitamin B12 deficiency leads to increased adiposity and altered
plasma lipid profile in the F1 generation offspring. Multiomics
approaches, including DNA methylation and proteomics studies
on the liver of 3-mo-old F1 Wistar rats revealed altered metabolic
processes in male offspring but not in females, which were
supported by a targeted metabolomics analysis of the liver. Thus,
our study provides evidence that maternal vitamin B12 defi-
ciency dysregulates key metabolic pathways in a sex-specific
manner and consequent future cardiometabolic risk.
Methods
Animal experimentation and ethics statement
The female vitamin B12 deficient model was developed at
CSIR (Council of Scientific and Industrial Research) -Centre for
Cellular and Molecular Biology, Hyderabad, India on Wistar rats
in accordance with the Committee for the Purpose of Control and
Supervision of Experimental Animals guidelines of the Govern-
ment of India. The experimental procedures were approved by
the “Institute’s Ethical Committee on Animal Experiments”
(CCMB/IAEC/27/2013).
Generation of female Wistar rat model with vitamin
B12 deficiency
Female weanling Wistar (outbred) rats (Rattus norvegicus) (n ¼
18) were housed (n ¼ 2/cage) in propylene cages with wire mesh
bottom (to avoid coprophagy) and maintained at 22  2 C and
12-h light/dark cycle. The animals were divided into 2 groups and
consumed ad libitum for 3 mo either a casein-based control diet
(AIN-76A) (n ¼ 6) or the same diet with vitamin B12 restriction (0
g/kg vitamin B12 in the diet; n ¼ 12). The diets were purchased
from M/S Research Diets (diet composition: Supplementary
Table 1A–C) and all animals had free access to deionized water.
Feed intake and body weights were measured at 3 mo.
Biochemical estimations of vitamin B12, folate, homocysteine,
and lipids in plasma were performed at the end of 3 mo. All
control males were fed normal feed pellets from the time of
weaning, maintained under similar conditions, and housed in
pairs until mating. After confirmation of vitamin B12 deficiency,
the F0 females were mated with control males (at 2 females:1
male per cage). A subgroup of vitamin-restricted (VR) dams (n ¼
6) was repleted with control diet on day 1 of conception [repletion
at conception (RC)]. All 3 groups, Control (C), VR, and RC, were
allowed to deliver and were continued on their respective diets
during lactation (Figure 1). The pups of F0 dams, i.e., F1 gener-
ation, were continued on their mother’s respective diets until age
1 y, and biochemical and body composition analysis was con-
ducted at 3 and 12 mo. The rats were killed using standard pro-
cedure following ethical principles by inhalation of carbon
dioxide. Extracted tissues were washed twice with normal saline
(0.9% NaCl), aliquoted, and snap-frozen using liquid nitrogen
before storage in a 80 C freezer until further analysis.
Body composition analysis
Body composition was analyzed using total body electrical
conductivity at various time points as described previously [22].
P. Singh et al.
Figure 1. Schematic representation of the maternal vitamin B12 deficiency model of Wistar rat. RC, repleted with vitamin B12 at conception; VR,
vitamin B12 restricted.
Lean body mass (LBM) and fat-free mass (FFM) percentages were
measured in a small animal body composition analysis system
(model SA 3000 multidetector EMSCAN). The difference be-
tween LBM and FFM was used to calculate the percentage of
tissue-associated fat [13].
Measurement of plasma biochemical parameters
Plasma vitamin B12 and folate levels were measured using an
electrochemiluminescence immunoassay kit from Roche Di-
agnostics on a Cobas e411 automated platform using the man-
ufacturer’s guidelines. Plasma homocysteine and cysteine
concentrations were measured by HPLC equipped with a fluo-
rescence detector as described previously [23]. Lipid parameters
such as total cholesterol (TC), HDL cholesterol, LDL cholesterol,
and triglyceride (TG) levels were measured using enzymatic
colorimetric assay kits from Roche Diagnostics on the COBAS
INTEGRA 400 plus analyzer following the manufacturer’s
instructions.
DNA methylome study
Methylated DNA immunoprecipitation sequencing of F1 livers
To generate a genome-wide methylation profile, a methylated
DNA immunoprecipitation sequencing (MeDIP-Seq) based
approach was used. Genomic DNA was isolated from the liver of
3-mo-old Wistar rats from 3 groups (C, VR, and RC) of the F1
generation. MeDIP library construction and sequencing was
performed as mentioned previously [15]. Briefly, genomic DNA
was fragmented to an approximate size of 100–500 bp, purified
followed by end repair, 30 end adenylation, and adapter ligation
using NEBNext DNA library preparation kit (E6040L) following
the manufacturer’s instructions. Double-stranded DNA was de-
natured at 95 C to single-stranded DNA and then 5-methylcyto-
sine-specific antibody (BI-MECY-0100) was used to precipitate
and enrich for methylated DNA fragments. The efficiency of
enrichment was checked using PCR and qRT-PCR with primers
corresponding to unmethylated and methylated regions reported
in humans previously [24]. PCR amplification was performed
using NEBNext Q5 HotStart HiFi PCR master mix (M0543S) for
12 cycles followed by agarose gel-based size selection of libraries
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The Journal of Nutrition 153 (2023) 3382–3396
with an approximate size of 300–350 bp fragments. The quality
of the libraries was checked using Agilent 2100 Bioanalyzer and
the concentrations were estimated using Qubit ds DNA high
sensitivity assay in Qubit 3.0 fluorometer (Q33216). The li-
braries were loaded onto the flow cell, and sequencing was
performed in the Illumina HiSeq2500 instrument using version 4
chemistry on single read mode for 50 cycles of amplification.
Proteomic profiling of liver tissue from F1 pups
Proteomic studies were performed on the liver of F1
offspring (both male and female) in 3 different groups (C, VR,
and RC) with 3 biologic replicates of each experimental con-
dition. Samples were homogenized in lysis buffer [7M urea,
2M thiourea, 4% 3-((3-cholamidopropyl) dimethylammonio)-
1-propanesulfonate, 25 mM ammonium bicarbonate, with
complete protease inhibitor cocktail] and centrifuged at
12,000  g for 30 min at 4 C. The supernatant was collected in
a fresh microcentrifuge tube. After quantification with Brad-
ford’s reagent, protein reduction, alkylation, tryptic digestion,
and peptide labeling with isobaric tags for relative and abso-
lute quantitation (iTRAQ) 4-plex reagent were performed as
described previously [25]. The labeled peptides were frac-
tionated into 8 fractions by strong cation exchange chroma-
tography (5 μm, 300 Å bead from SCIEX) with increasing
concentrations of ammonium formate (30–350 mM) buffer in
30% acetonitrile (vol:vol), pH 2.9.
The iTRAQ 4-plex labeled peptide fractions obtained after
strong cation exchange chromatography were separated on
reverse phase LC using the Eksigent NanoLC-Ultra 2D plus sys-
tem in trap-elute fashion. Peptides were desalted on an Eksigent
trap (ChromXP C18-3 μm, 120 Å, 350 μm  0.5 mm) column for
40 min at a flow rate of 3 μL/min. They were resolved on an
Eksigent C18 column (ChromXP C18, 75 μm  15 cm) in a 110-
min binary gradient with solvent A (0.1% formic acid) and sol-
vent B (99.9% acetonitrile and 0.1% formic acid) as the mobile
phase and a flow rate of 250 nL/min. The LC eluent was analyzed
using a NanoSpray III Source installed on the SCIEX TripleTOF
5600þ system. The instrument was operated in information-
dependent acquisition mode. The full mass spectrometry (MS)
P. Singh et al.
spectra were acquired in positive ion mode in m/z 350–1250 Da
with a 200 ms time-of-flight MS accumulation time, whereas the
MS/MS product ion scan was performed in the mass range of
100–1800 Da with a 70 ms accumulation time and a total cycle
time of 1.93 s. Parent ions with a charge state from þ2 to þ5
were considered for MS/MS acquisition. Optimized ion-source
parameters were used. Twenty-four candidate ions were moni-
tored per MS cycle, and the former target ion was excluded for 7
s. Information-dependent acquisition advanced “rolling collision
energy” was applied for subsequent MS and MS/MS scans.
Targeted metabolite profiling of liver in the F1
offspring
Metabolites were extracted from the liver of F1 generation
offspring (both male and female) from control, VR, and RC
groups with 3 biologic replicates in each experimental condition.
Ten milligrams of freeze-dried liver tissue was used for the
extraction of metabolites using the protocol defined previously
[26]. Metabolites were reconstituted in 125-μL methanol:
acetonitrile: water (2:1:1), and polar and nonpolar metabolites
were pooled in the ratio of 1:1 prior to LC-MS acquisition. Tar-
geted metabolic profiling was performed for amino acids and
intermediates of glycolysis, tricarboxylic acid (TCA) cycle, and
steroid hormone biosynthesis pathways (Supplementary Table 2
shows the metabolites and transitions information). Samples
were analyzed on a triple quadrupole hybrid ion trap mass
spectrometer (QTRAP 6500þ, SCIEX) coupled with a SCIEX
ExionLC UHPLC system, and data was acquired through Analyst
1.6.3 software in both negative and positive modes. Metabolites
were resolved on an Acquity UPLC BEH HILIC (1.7 μm, 2.1  100
mm) column using mobile phases of 10 mM ammonium acetate
and 0.1% formic acid in water (buffer A) and 95% acetonitrile
with 10 mM ammonium acetate and 0.1% formic acid (buffer B)
with a flow rate of 0.3 mL/min.
Data analysis
All biochemical variables are presented as mean  SEM.
Differences among the groups at specific ages were tested using
Student’s t test for 2 groups or analysis of variance (including a
test for the homogeneity of variance) for >2 groups, followed by
Bonferroni’s post hoc correction. Data sets were checked for any
outliers and if found deviated, Mann–Whitney test for 2 groups
and Kruskal Wallis for >2 groups were used. P values of 0.05
were considered significant. All heatmaps were generated using
Morpheus (https://software.broadinstitute.org/morpheus).
Venn diagrams were plotted using Venny 2.1.0 (https://
bioinfogp.cnb.csic.es/tools/venny/index.html).
MeDIP-Seq data analysis
The raw sequencing reads were processed through FastQC
(https://www.bioinformatics.babraham.ac.uk/projects/fastqc)
and Trimmomatic [27] to remove the adapters and low-quality
reads. The reads were mapped to the rat genome available in
the UCSC genome browser (rn6 assembly) using Bowtie 2
[28–30]. Alignment percentages were calculated using both
aligned and unaligned reads. Further, Samtools [31] was used to
filter the uniquely mapped reads, and they were used for meth-
ylated peak calling using MACS version 1.4.0 (Model-based
Analysis of ChIP-Seq) [32] with default parameters. We gener-
ated ~50 to 200 million reads in each sample, which was
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The Journal of Nutrition 153 (2023) 3382–3396
sufficient for whole genome coverage [33]. Depth of sequencing
was checked using a systematic data reduction approach with
MACS output [15,24]. The number of methylated peaks were
comparable to a previously published rat liver methylome study
[15]. Regions were considered to be differentially methylated
(DMR) if there was at least a 2-fold difference of methylation
with a hypergeometric P value  5  105. The number of peak
distributions was counted in different genomic regions (pro-
moter, 50 untranslated region [UTR], exon, introns, and 30 UTR)
using in-house PERL script. For gene annotation of the DMRs, we
downloaded the Ref-Seq (rn6) gene coordinates from the UCSC
genome browser, and regulatory region analysis was performed
in the 10-kb region surrounding the transcription start site (TSS).
KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway
analysis was performed using DAVID bioinformatics resources
[34,35]. Pathways were considered significant based on P value
of 0.05 with minimum 3 genes in each category.
Proteomics and metabolomics data analysis
All MS and MS/MS spectra generated were submitted for
database searching and quantitative analysis using ProteinPilot
version 4.0 (SCIEX). The Paragon algorithm was used in a
“Thorough ID” search mode against the Rattus norvegicus refer-
ence proteome from the UniProt database [36]. The following
parameters were included for search: trypsin as the digestion
enzyme with 2 missed cleavages, modifications by iodoaceta-
mide as cysteine blocking reagent, iTRAQ 4-plex modification of
the N-terminal of peptides and of the side chains of lysine. An
automatic decoy database search was also performed to calculate
the false discovery rate, and 1% global protein level false dis-
covery rate was considered for protein identification. The
resulting data set was normalized using auto-bias correction to
remove any experimental bias. A cutoff of 1.2-fold change was
set for identifying differentially expressed proteins, as used in
previous studies [37,38]. Pathway enrichment analysis was
performed in DAVID bioinformatics resources [34,35].
Metabolomics data was processed using MultiQuant software
v.3.0 (SCIEX), and data was exported to Microsoft Excel where
relative quantification and statistical analysis was performed.
Metabolites were considered significantly altered for a 1.5-fold
change and a P value of 0.05 in Student’s t test.
Results
Direct effects of plasma vitamin B12 deficiency on
F0 females
Plasma vitamin B12 levels were 36% lower in female rats fed
a vitamin B12-deficient diet for 3 mo than those fed with control
diet. There were no significant changes in plasma folate and
homocysteine levels (Table 1). Body weight of vitamin B12-
deficient female rats was lower than the control group
(Table 1). The vitamin B12-deficient dams (F0-VR) and those
repleted at conception (F0-RC) did not show any significant
difference in reproductive performance in terms of time to
conception and litter size (data not shown).
Effects of maternal plasma vitamin B12 deficiency
on F1 offspring
F1 generation offspring were followed at 3- and 12-mo,
equivalent to early and mid-adulthood in humans [39]. Body
P. Singh et al. The Journal of Nutrition 153 (2023) 3382–3396

Table 1 completely correct to the level of controls in the RC groups

Biochemical parameters and body weight of the F0 females after 3 mo (Supplementary Table 3).
of vitamin B12-restricted diet1 In an attempt to account for the effect of sex, we conducted a
Parameters Control (n ¼ 6) Vitamin-restricted (n ¼ 6)2 sex-specific analysis. Sex-specific analysis in 3 mo-old offspring
revealed that male offspring born to vitamin B12-restricted
Body weight (g) 230.72  3.81 216.91  3.983
Vitamin B12 (pg/mL) 823.17  27.07 529.2  78.783 mothers had significantly higher body fat, lower FFM, and
Folate (ng/mL) 8.62  1.03 8.96  1.26 lower LBM percentages whereas females did not show any sig-
Homocysteine (μmol/L) 3.47  0.3 3.1  0.04 nificant differences in the body composition with respect to
1
Values are presented as mean  SEM. controls. Interestingly, although a significant vitamin B12 defi-
2
Biochemical parameters could not be measured in one animal due ciency was present in both male and female offspring at 3 mo,
to low blood volume; hence, the values represent mean value of 5 plasma homocysteine levels were significantly higher only in
animals. males. There were no significant changes in plasma folate and
3
P < 0.05 by Student’s t test. cysteine levels. Repletion with vitamin B12 at conception cor-
rected (albeit partially) the plasma vitamin B12 levels in both
composition analysis using total body electrical conductivity in sexes and homocysteine levels only in males. We also observed
3-mo-old offspring showed no overall difference between significantly higher TG levels and a trend of increasing TC and
vitamin B12-restricted and control group (Supplementary LDL cholesterol in VR males with respect to control whereas VR
Table 3). We also noted a ~71% reduction in plasma vitamin females had significantly lower LDL cholesterol and a trend of
B12 with significant increase in homocysteine levels in the lower TG and TC levels. Males from the RC group showed
3-mo-old pups born to VR mothers. However, no significant reversal of TGs to the control levels whereas RC females
differences were noted in plasma lipid parameters in the VR continued to have low TC and LDL cholesterol levels (Table 2).
group except an increasing trend for TG levels compared to pups Both male and female offspring in the VR group showed
born to control rats. Offspring from the VR group at 12 mo significantly lower levels of plasma vitamin B12 (65% and 55%
continued the same trend as 3 mo offspring with no significant compared with respective controls) and elevated homocysteine
differences in body composition. However, along with a ~60% levels at 12 mo. However, only males showed elevated cysteine
plasma vitamin B12 deficiency and elevated plasma homocys- levels and altered body composition and plasma lipid parame-
teine, the 12-mo-old pups in the VR groups had significantly ters. Males from the VR group were obese with higher body
higher levels of cysteine and TGs. These changes did not weight and body fat percentages and decreased LBM and FFM

Table 2
Body composition and plasma biochemical parameters in female and male offspring at 3-mo time points1
Parameters Age (mo) Male Female
Control (n ¼ 6) VR (n ¼ 6) RC (n ¼ 6) Control (n ¼ 6) VR (n ¼ 6) RC (n ¼ 6)
Body weight (g) 3 361.4  10.2 361.7  5.5 373.7  6.5 219.1  6.3 214.6  6.4 221.2  6.0
12 440.2  21.4 492  15.6 583.3  9.3 b*&,c#$ 281.7  12.4 271.7  10.3 308.2  11.8a#$
Body fat (%) 3 14.2  0.5 18.4  1.1b*$ 18  0.8 b*$ 15.0  0.9 12.9  1 17.0  0.6b#$
12 17.4  0.5 19.5  0.9 14.8  1.8 18.0  1.4 16.8  1.3 19.4  0.4
Lean body mass (%) 3 85.8  0.5 81.6  1.1b*$ 82.0  0.8b*$ 85.0  0.9 87.1  1.0 83.0  0.6b#$
12 82.6  0.5 80.5  0.8 85.2  1.8 82.0  1.4 83.2  1.3 80.6  0.4
Fat-free mass (%) 3 38.8  0.1 37.1  0.4b*$ 37.2  0.3c*$ 41.5  0.5 42.4  0.6 40.6  0.3a#$
12 45.8  0.3 43.7  0.8a*$ 46.9  1.4 47.4  1.3 48.6  1.2 45.8  0.4
Vitamin B12 (pg/mL) 3 731.8  31.7 190.2  16.7b*& 673.0  23.2b#& 678.9  18.0 217.9  29.3c*$ 660.2  45.2c#$
12 902.1  37.6 320.0  10.0b*& 770.6  44.6a*&,c#$ 727.8  18.7 330.3  24.0c*$ 669.4  33.3c#$
Folate (ng/mL) 3 31.7  1.8 32.4  2.2 33.0  2.0 21.5  0.7 22.5  1.4 21.3  1.4
12 21.6  1.4 23.3  1.2 20.0  1.2 23.5  1.0 22.2  1.0 21.7  0.6
Homocysteine (μmol/L) 3 6.3  0.7 8.9  0.5a*$ 7.0  0.4a#$ 5.0  0.3 6.5  1.6 5.1  0.56
12 5.4  0.4 9.0  0.7b*$ 7.9  0.3c*$ 5.2  0.4 7.9  0.8a*$ 5.8  0.4a#$
Cysteine (μmol/L) 3 135.0  10.7 154.8  11.5 155.8  8.6 162.9  7.7 158.7  19.5 173.4  18.7
12 94.7  7.2 132.3  14.7a*$ 99.8  1.8 96.2  5.8 103.9  9.8 126.0  7.3a*&
TG (mg/dL) 3 77.2  7.9 170.8  28.1a*$ 56.5  2.8a*$,b#$ 94.6  11.8 80.2  10.2 112.4  14.5
12 57.9  5.1 95.2  9.3b*$ 82.3  8.1a*$ 64.2  12.7 72.4  6.3 77.8  7.9
HDL-C (mg/dL) 3 60.8  4.3 69.7  5.0 55.3  1.8a#& 85.7  3.9 77.1  8.1 73.8  5.9
12 91.7  2.1 69.9  2.7c*$ 76.3  3.9b*$ 67.0  6.2 75.4  4.1 70.5  4.9
LDL-C (mg/dL) 3 6.7  0.6 9.9  1.5 6.1  0.3 15.8  2.4 8.8  1.2a*$ 7.8  0.82a*$
12 21.7  1.8 24.6  2.7 20.6  2.8 9.1  1.9 8.3  2.5 9.0  1.7
TC (mg/dL) 3 70.1  4.6 88.0  7.4 63.7  2.3a#$ 109.2  6.4 89.9  7.9 84.5  5.6a*$
12 124.9  11.1 145.4  12.8 129.2  12.9 108.9  6.0 104.3  5.2 109.4  9.6
1
Wistar rat offspring fed different diets: Control (C), Vitamin B12 restricted (VR), Repleted at Conception (RC). Values are presented as the mean
 SEM. Values in a row with different superscripts (a/b/c) are significantly different from others at P value (a < 0.05, b < 0.01, c < 0.001).
*
Difference from control group.
#
Difference from the vitamin-restricted group. The normality test was done using the Shapiro test. For homogenous distribution, t test was used
(represented as $), and for non-homogenous distribution, non-parametric Mann–Whitney test was done (represented as &). Abbreviations: HDL-C,
high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride.

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P. Singh et al. The Journal of Nutrition 153 (2023) 3382–3396

percentages. These male rats also showed significantly elevated
plasma TG and low HDL cholesterol levels, indicating an
increased risk for developing cardiovascular diseases. However,
altered body composition and plasma lipid parameters in VR
males did not completely recover in the RC group and continued
to show earlier changes. However, females from the VR and RC
groups showed no difference in body composition and plasma
lipid parameters compared with control females (Table 2).
Methylome sequencing reveals sex-specific
differences in DNA methylation and associated
biologic pathways
RC rescued some of the phenotypic parameters, albeit
partially, hinting toward epigenetic alteration due to vitamin
B12 deficiency. We thus assessed the DNA methylation status in
the liver of 3-mo-old pups to ascertain DMRs in pups born to VR
mothers and also to check if these alterations normalized in the
repleted pups. The average methylation pattern in the 10-kb
region (5 kb upstream and downstream) around the TSS in a
100-bp sliding window showed a characteristic V-shaped curve
indicating low methylation density at the TSS, which increased
with increasing distance downstream of the TSS (Figure 2A).
This V-shaped pattern was consistent among all the groups of
offspring indicating that the global methylation pattern around
the TSS was conserved across different groups and is in agree-
ment with our previous study [15]. However, sex-specific anal-
ysis revealed that vitamin B12-restricted male offspring had an
increased methylation density in the gene body region compared
with controls, which was partially restored following RC
(Figure 2A). No significant differences in global methylation
density were observed between control, vitamin B12-restricted,
and vitamin B12-repleted female offspring suggesting
sex-specific differences in programming of methylation alter-
ations upon maternal vitamin B12 deficiency (Figure 2B).
Further, both hypomethylated and hypermethylated regions [in
all the genomic regions (promoter, 5’UTR, exon, 3’UTR and
intron)] were higher in males than in females in VR pups,
compared with control (Figure 2C, D). Interestingly, the majority

Figure 2. Average methylation pattern around the transcription start site (TSS) in control, vitamin B12 restricted, vitamin B12 repleted male
(A) and female (B) offspring in a 100-base pair (bp) sliding window. Distribution of hypermethylated (C) and hypomethylated (D) regions across
different genomic regions in the liver of F1 generation male and female offspring. Venn diagram showing the intersection of genes with
hypermethylated (E) and hypomethylated (F) promoter regions in vitamin-restricted male and female livers. UTR, untranslated region.

3387
P. Singh et al.
of the DMRs in liver were sex-specific. We found a total of 4264
and 703 unique hypermethylated and 290 and 127 hypo-
methylated DMRs in the promoter region of male and female
liver, respectively. This corroborates with our previous study
that showed a significant association of DNA hypermethylation
with hyperhomocysteinemia due to vitamin B12 deficiency [15,
40]. Methylation levels at ~75% to 90% of DMRs were reversed
upon vitamin B12 RC, suggesting a causal inference (Supple-
mentary Table 4).
Global methylome study indicates perturbation of
important metabolic pathways
Biologic pathway enrichment analysis revealed that the DMR
genes in the VR male liver were majorly enriched in signaling
pathways like Hippo signaling, FoxO signaling, MAPK (Mitogen-
activated protein kinase) signaling, AGE-RAGE (Advanced
glycation end products (AGEs) and their receptor (Receptor for
Advanced Glycation Endproducts, RAGE)) signaling, Wnt
signaling, AMPK (AMP-activated protein kinase) signaling, and
PI3K-Akt signaling (Figure 3A). These signaling pathways are
complex, dynamic, and highly interconnected and regulate
glucose, lipid metabolism, and insulin sensitivity along with
processes, such as cell proliferation, differentiation, and stress
response [41–46]. DMR genes in VR female liver showed
enrichment in pathways like Rap1 signaling, phospholipase D
signaling, and Ras signaling (Figure 3B). Pathways in cancer
identified from DMR genes were enriched in both VR male and
female liver.
The list of DMR genes shows hypermethylation in the promoter
regions of 3 peroxisome proliferator-activated receptors (PPARs),
a family of nutrient sensing nuclear receptors (Ppara, Ppard,
Pparg). PPARα (Ppara) is the PPAR member that is majorly
expressed in liver and controls energy metabolism (glucose and
lipid metabolism) [47–49]. Promoter hypermethylation in PPARα
suggests lower expression under vitamin B12 restriction in liver,
Figure 3. KEGG pathway enrichment analysis of differentially methylated genes in vitamin B12-restricted male (A) and female (B) liver. X-axis
denotes -log10 (adjusted P value) and the Y-axis denotes pathway names.
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The Journal of Nutrition 153 (2023) 3382–3396
leading to metabolic changes. Therefore, we looked at the
methylation status of genes involved in energy metabolism. The
promoter regions of genes that code for enzymes in the glycolysis
pathway, glucose-6-phosphatase, catalytic subunit (G6pc), phos-
phofructokinase (Pfkl), 6-phosphofructo-2-kinase/fructose-2,
6-bisphosphatase (Pfklb3), glyceraldehyde 3-phosphate dehydro-
genase (Gapdh), phosphoglycerate mutase (Pgam1), enolase
(Eno3), and pyruvate kinase (Pk), were hypermethylated in VR
male liver. Similarly, genes coding for enzymes in the TCA cycle
such as citrate synthase (Cs), aconitase (Aco1 and Aco2), isocitrate
dehydrogenase (Idh1), fumarate hydratase (Fh), and malate de-
hydrogenase (Mdh2) were hypermethylated. Promoters of genes
coding for phosphoenolpyruvate carboxykinase (Pck1) and pyru-
vate carboxylase (Pc) also showed hypermethylation in the VR
male liver, indicating lower gene expression.
Genes related to fatty acid transport, desaturation, and elon-
gation were hypermethylated in the male liver, indicating that
β-oxidation in the mitochondria could be affected. The trans-
porter genes (Cpt1a, Cpt1b, Slc27a1, and Slc27a4) that import
fatty acids in the mitochondria showed hypermethylation. We
observed that the promoter of genes such as Elovl, Acsl4, Fads,
Fabp1, Hmgcs, Acat2, Acaa1b, and Acox2 were hypermethylated
in male liver, indicating downregulation. ACOX is the rate-
limiting enzyme in fatty acid β-oxidation. Srebf1 and Srebf2,
genes that code for sterol regulatory element binding proteins, a
family of transcription factors that regulate lipid homeostasis by
controlling the expression of a range of enzymes required for
endogenous cholesterol, fatty acid, triacylglycerol, and phos-
pholipid synthesis, also showed hypermethylation in their pro-
moter regions in VR male liver [50]. Interestingly, altered
methylation status in the promoters of genes from glucose and
lipid metabolism were only found in VR male liver and not in
female.
In the amino acid metabolism pathway, we observed that
genes involved in transamination (Gpt and Agxt) were hyper-
methylated in the male liver, suggesting altered levels of
P. Singh et al.
glutamate, glutamine, alanine, and serine. Moreover, aldehyde
dehydrogenases (Aldh4a1, Aldh5a1, and Aldh9a1) were also
hypermethylated in the male liver.
Rap1 signaling pathway was the most affected pathway in
female liver, and Rap1-deficient female liver is known to upre-
gulate genes involved in different metabolic pathways including
lipid, fatty acid, steroid, and cholesterol metabolism [51]. Thus,
alteration of multiple metabolic pathways, including branched
chain amino acid degradation, PPAR signaling pathway, glyc-
erolipids, and fatty acid metabolism, reflects alterations in lipid
homeostasis. Glycolysis and gluconeogenesis as well as
diabetes-related pathways have also been found to be deregu-
lated due to RAP1 deficiency [51]. We previously reported
altered methylation and protein expression of PPAR signaling in
the rat liver of male pups born to vitamin B12-deficient mothers
[14]. In the female liver, genes involved in cysteine and methi-
onine metabolism (Mthfr, Ahcyl, Sds, and Srm) were found to be
hypermethylated. Estrogen receptor (Esrra and Esrrg in male and
Esrrb in female), retinoid X receptor (Rxra in males and Rxrb in
both sexes), and glucocorticoid receptor (Nr3c1) genes in males
also showed hypermethylation in the promoter region in vitamin
B12-restricted offspring liver.
Proteomic profiling indicates sex-specific
differences of differential proteins and biologic
pathways
DNA methylation analysis in this study showed alterations in
methylation status of promoter and gene body region of a
number of genes in a sex-specific fashion. To confirm if these
changes were also reflected at the protein level, proteomic
profiling was performed in the liver tissue of F1 generation 3-mo-
old male and female offspring using iTRAQ 4-plex tag based
quantitative proteomics technique. A total of 1279 proteins in
males and 1134 proteins in females were quantified in the 3
biologic replicates, of which ~930 proteins were common be-
tween male and female liver. These proteins showed sex-specific
alterations in liver upon vitamin B12 restriction (Figure 4A).
Among the list of commonly quantified proteins, 239 (129
upregulated and 110 downregulated) and 305 (195 upregulated
Figure 4. (A) Heatmap represents distinct protein expression in vitamin-restricted male and female groups with respect to the control group.
(B) Venn diagram showing the common and unique proteins identified between vitamin B12 restricted male and female offspring liver as
compared to controls.
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The Journal of Nutrition 153 (2023) 3382–3396
and 110 downregulated) proteins were differentially expressed
in male and female offspring, respectively. Interestingly, only
~10% of the proteins that had altered expression were common
between male and female offspring, again suggesting sex-specific
proteome alterations (Figure 4B). The majority of the differen-
tially expressed proteins (62% in males and 65% in females)
showed reversal of expression in pups born to vitamin B12-
deficient mothers who were repleted at conception. KEGG
pathway analysis of differentially expressed proteins revealed
that metabolic pathways related to amino acid metabolism,
carbon metabolism, glutathione metabolism, and drug meta-
bolism involving cytochrome P450 were enriched in vitamin
B12-restricted males (Figure 5A) whereas females showed al-
terations in metabolic pathways such as oxidative phosphoryla-
tion and drug metabolism involving cytochrome P450 pathway
(Figure 5B).
Amino acid metabolism was the most significantly altered
metabolic process in males upon vitamin B12 restriction, with
differential expression of multiple proteins in the amino acid
biosynthesis process (Table 3).
Carbon metabolism showed significant alterations upon
maternal vitamin B12 deficiency as proteins majorly involved in
glycolysis, TCA cycle, and OCM were altered in both male and
female liver, which was in line with the methylome data.
Expression of glycolytic and TCA cycle enzymes PFKL, ENO3, CS,
and MDH in vitamin-restricted females showed elevated
expression with respect to controls whereas aldolase (ALDOA),
PC, and PCK1 showed diminished expression in at least 2 of the 3
replicates. In the male liver, pyruvate kinase enzyme level was
low in restricted group whereas level of isocitrate dehydrogenase
was high. Proteins involved in oxidative phosphorylation from
complex I (NADH dehydrogenase), complex III, complex IV, and
complex V (ATP synthase subunits) showed increased expression
upon vitamin restriction in female liver (Table 3).
Liver proteomic profiling of F1 tissue revealed that the
enzyme betaine-homocysteine methyltransferase (BHMT),
which is involved in remethylation of homocysteine to methio-
nine in liver, had elevated expression in the male offspring under
vitamin B12 restriction. Higher expression of enzymes involved
P. Singh et al. The Journal of Nutrition 153 (2023) 3382–3396

Figure 5. KEGG pathway enrichment analysis of differentially expressed proteins in the vitamin B12-restricted male (A) and female liver (B).

Table 3
Differentially expressed proteins in different metabolic pathways in the liver upon vitamin B12 restriction1
Metabolic pathways VR male liver VR female liver
Up Down Up Down
(>1.2-fold change) (<0.8-fold change) (>1.2-fold change) (<0.8-fold change)
Amino acid metabolism ASL1, ASS1, CPS1, CTH, PKM, SDSL ENO3, OTC CPS
ENO3, GLUL, GPT, GOT1,
IDH1, IDH2, OTC, PAH, SDS
Energy metabolism IDH PKM PFKL, ENO3, CS, MDH, ALDOA, PC, PCK1
(glycolysis, TCA cycle, NDUFA6, NDUFA4, NDUFS3,
oxidative phosphorylation) NDUFS8, NDUFA9,
NDUFA13, NDUFB6,
UQCRC2, COX5A, COX6B1,
COX6A1, COX7A2, ATP5A1,
ATP5H, ATP5O, ATP5I,
ATP5B, ATP5I
One-carbon metabolism BHMT, CTH
Glutathione metabolism GST, GPX, GPT2, GOT1, GCLC, GST, GLS2
GLUL, GLS2
Arginine biosynthesis and ASS1, ASL, OTC, CPS1, GLUL, OTC CPS1, GLS2
urea cycle GPT2, GOT1
Cytochrome P450-dependent UGT2B, FMO3, GSTA3, GSTA1, AKR7A3 UGT2B37, GSTA3, GSTK1, GSTM2, ADH1
xenobiotics and drug GSTK1, GSTM3, GSTT2 GSTP1, SULT2A1, FMO1,
metabolism CYP1A2, F1LTB8,
RGD1559459
Steroid hormone biosynthesis UGT2B, AKR1D1, CYP2D5, CYP1A2, CYP2C12, CYP2B3, HSD11B1
SULT1E1 UGT2B37, RGD1559459
1
Differentially expressed proteins in the liver under vitamin B12 restriction in males (VR male) and females (VR female), revealed by iTRAQ
analysis. Abbreviations: iTRAQ, isobaric tags for relative and absolute quantitation; TCA, tricarboxylic acid.

in the transsulfuration pathway, such as cystathionine gamma-
lyase indicated an increased flux of homocysteine toward
glutathione biosynthesis and metabolism through increase in
cysteine levels (as reflected in plasma). Proteins, such as gluta-
thione S-transferase (GST) and glutathione peroxidases (GPX)
showed elevated levels under vitamin restriction in male liver.
Glutamate pyruvate transaminase 2 (GPT2), glutamate-
oxaloacetate transaminase 1 (GOT1), GLUL (glutamine synthe-
tase), and glutaminase (GLS2) were other proteins involved in
glutamate biosynthesis and glutamine/glutamate metabolism
that showed elevated expression upon vitamin restriction in
male liver. Females also showed alterations in expression of
GSTs, glutamate cysteine ligase, and GLS2. Proteins involved
directly in arginine biosynthesis, and the urea cycle in general,
showed increased expression upon vitamin restriction in males
(Table 3).
Cytochrome P450-dependent metabolic processes were
among the top dysregulated pathways in liver of the vitamin-
restricted male and female offspring. Several proteins
involved in cytochrome P450-dependent xenobiotics and drug
metabolism pathways show differential levels (Table 3).
Interestingly, some of these cytochrome p450 monooxygenases

3390
P. Singh et al.
(CYPs) are also involved in the process of steroid hormone
biosynthesis. Steroid hormone biosynthesis involves enzymes
from 2 classes—CYPs and hydroxysteroid dehydrogenases
(HSDs) along with some accessory enzymes. Proteins like
UGT2B, AKR1D1, CYP2D5, and SULT1E1 were dysregulated in
males whereas females showed dysregulated expression of
proteins encoded by CYP1A2, CYP2B3, CYP2C12, HSD11B1,
UGT2B37, and RGD1559459. Changes in levels of these pro-
teins from the steroid hormone biosynthesis pathway might
result in sex-specific outcomes.
Targeted metabolite profiling to understand the
effects of maternal vitamin B12 restriction
DNA methylation and proteomics studies in the liver of F1
offspring born to VR females hinted toward the alteration of
amino acid, glycolysis, energy metabolism, and steroid hormone
biosynthesis pathways, which could be due to PPARα dysregu-
lation. To check if the metabolites in these pathways show al-
terations, we performed a targeted metabolomics study, majorly
focusing on these pathways.
Glycolysis intermediates, fructose bisphosphate (FBP), 3-
phosphoglycerate/2-phosphoglycerate (3-PG/2-PG), and phos-
phoenolpyruvate were lower only in VR male liver. FBP is an
important regulator of glycolytic flux, and low FBP, 3-PG/2-PG,
Figure 6. Status of dysregulated proteins and biologic pathways in the vitamin B12-restricted group with respect to controls with their metabolite
status involved in (A) glucose metabolism, (B) one-carbon metabolism and glutathione biosynthesis, (C) arginine metabolism along with some
intermediates of steroid hormone biosynthesis, and (D) amino acid profile among vitamin-restricted and vitamin-repleted rat livers with respect to
control liver.
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The Journal of Nutrition 153 (2023) 3382–3396
and phosphoenolpyruvate indicate lower glycolytic flux in VR
males. Altered TCA intermediates (α-ketoglutarate and oxaloac-
etate in VR males and succinate and oxaloacetate in VR females)
may also result in alteration of amino acid metabolism and
transamination reactions [as highlighted in pathway enrichment
analysis using differentially expressed proteins and methylated
genes (Figure 6A)] as they utilize TCA cycle intermediates like
α-ketoglutarate and oxaloacetate [52,53].
Alteration in amino acid levels was distinctly visible in VR
male liver as all the measured amino acids levels were altered in
males, whereas in females the extent of alteration was lower
(Figure 6D). Glutamine and glutamate, involved in glutathione
biosynthesis, particularly showed sex-specific patterns as they
were only altered in VR males and were unaffected in VR fe-
males, as also indicated in proteomics data. Glutathione
biosynthesis was also impacted through an increased trans-
sulfuration process from the OCM pathway through different
metabolic intermediates. Metabolites like S-adenosylmethionine
(SAM), 5,10-methylenetetrahydrofolate, 5-methyltetrahy-
drofolate, cystathionine, and cysteine sulfinate are all elevated in
VR males (>1.5-fold change, P < 0.05) whereas only VR females
showed elevated SAM while other intermediates were largely
unaltered. Proteomics data showed increased BHMT expression
only in VR males. Betaine levels were low specifically in VR male
P. Singh et al.
liver indicating increased utilization in males for the generation
of SAM through methionine (Figure 6B).
An altered arginine biosynthesis and urea cycle flux was also
found in VR males at the metabolite level, as high arginine,
argininosuccinate, ornithine, citrulline, and aspartate were
found only in VR males. This is consistent with high ASS1, ASL,
OTC, and CPS1 found at the protein level. In females, this
pathway remained unimpacted at the metabolite level
(Figure 6C).
Differential steroid hormone regulation as indicated from
methylation and proteomic data was reflected at the metabolite
level, as several intermediates from steroid hormone biogenesis
like pregnenolone, hydroxypregnenolone, aldosterone, deoxy-
corticosterone, and hydroxycorticosterone, were all elevated in
VR males and relatively unaltered in VR females. Cortisol, a
glucocorticoid associated with stress, was also significantly
increased only in VR male liver whereas VR female showed
levels comparable with control female.
Discussion
Vitamin B12 is a key micronutrient essential for fetal growth,
development, and various physiologic functions [54–57]. Defi-
ciency of vitamin B12 at critical stages of development is asso-
ciated with poor gestational outcome and adult onset of
cardiometabolic diseases [56,58–60]. The present study high-
lights the effects of dietary vitamin B12 deficiency in pregnant
Wistar rats leading to distinct effects on the OCM pathway, body
composition, and lipid parameters in the offspring in a
sex-specific manner. Multiomics (DNA methylome, proteome,
and metabolome) profiling of liver tissue of F1 generation Wistar
rat offspring combined with analysis of anthropometric and
biochemical parameters provide us an insight into the molecular
mechanisms of early developmental programming for risk of
cardiometabolic diseases in the future due to perturbation of in
utero vitamin B12 levels. Our present study shows that offspring
in the F1 generation continued on the same vitamin
B12-restricted diet as their mothers show a further reduction in
plasma vitamin B12 with elevated plasma homocysteine levels.
Our study further provides evidence that repletion of pregnant
mothers with vitamin B12 at conception could partially restore
plasma vitamin B12 levels and also attenuate the metabolic ab-
normalities in the repleted offspring through altered epigenetic
programming. Sex-specific analysis of body composition and
plasma biochemical parameters are in line with an earlier report
[14]. We show here that F1 generation male offspring developed
higher adiposity and had lower HDL cholesterol and higher TG
levels under maternal vitamin B12 deficiency at 3 mo, whereas
females did not show these abnormalities. These differences in
biochemical parameters and body composition indicate an
increased cardiometabolic disease risk in adulthood in vitamin
B12-restricted male offspring but not in female offspring, similar
to the observations in human longitudinal studies. A recent study
in the Pune Maternal Nutrition Study cohort from India shows
similar findings; in children born to mothers with nutritional
deficiency (vitamin B12 deficiency among ~70% of mothers),
28% of children were glucose intolerant at the age of 18 y. The
glucose intolerance was more common in men (37%) compared
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The Journal of Nutrition 153 (2023) 3382–3396
with women (20%), and there was a decrease in insulin sensi-
tivity with age. Those with glucose intolerance showed lower
compensatory insulin secretion from childhood [61]. Another
study in the Mysore Parthenon Cohort showed an association of
low maternal vitamin B12 status with greater cortisol response to
stress in the offspring, and higher plasma total homocysteine
concentration was significantly associated with a higher heart
rate response to stress at 13.5 y [62]. Both studies provide evi-
dence that vitamin B12-deficient status during pregnancy leads
to fetal programming for future risk of cardiometabolic
disorders.
Global DNA methylome analysis of liver tissues from 3-mo-
old vitamin B12-restricted F1 male offspring show alteration of
signaling pathways like Hippo, FoxO, MAPK, Wnt, and AMPK,
which regulate complex metabolic functions and maintain en-
ergy homeostasis by regulating glucose, lipid metabolism, and
insulin sensitivity [41–46]. These signaling pathways are also
involved in processes like cell proliferation, differentiation, and
stress response. VR F1 female offspring show altered Rap1,
phospholipase D, and Ras signaling in liver. Rap1 and Ras
signaling pathways affect processes like cell growth, prolifera-
tion, differentiation, and cell survival [63]. Rap1 is a crucial
player in the process of tumor cell migration, invasion, and
metastasis [64,65]. Rap1-deficient mice show deregulation of
metabolic programs, including fatty acid, glucose metabolism,
and PPARα signaling [51]. Phospholipase D signaling has been
shown to be positive regulator of glucose metabolism and insulin
sensitivity [66]. Dysregulation of these signaling pathways
highlights impaired energy homeostasis (glucose and lipid
metabolism) and suggests progression toward a MetS phenotype
with insulin resistance and fatty liver in males. Major hepatic
transcription factors involved in nutrient sensing and regulating
metabolism that are reported to be dysregulated in MetS-related
perturbations in both clinical and preclinical studies are perox-
isome proliferator-activated receptor α (Ppra), cAMP responsive
element binding protein 3 like 3 (Creb3l3), and forkhead box O1
(Foxo1) [67]. Interestingly, these genes show promoter hyper-
methylation in VR male liver but not in VR female. Targets of
these transcription factors also show perturbed methylation
status in the gene promoter region. Proteomics analysis in the VR
male liver tissue also shows enrichment of metabolic pathways
from dysregulated proteins. Pathways related to biosynthesis
and metabolism of amino acids, such as arginine metabolism,
glutathione metabolism, carbon metabolism, cytochrome
P450-related metabolic processes, pentose and glucuronate
metabolism, and cysteine and methionine metabolism, are
enriched in VR males. VR females show alterations in metabolic
pathways related to carcinogenesis, oxidative phosphorylation,
and cytochrome P450-related processes. Metabolomics study
targeting energy metabolism (glycolysis and TCA cycle), amino
acids, arginine metabolism, and steroid hormone biosynthesis
shows some sex-specific alterations. Dysregulated glucose
metabolism is suggestive of lower glycolytic flux in liver of male
offspring born to vitamin B12-restricted females than controls.
Alteration in amino acids levels in liver is more prominent in VR
male compared with VR female liver. OCM and arginine
biosynthesis intermediates also show alteration only in VR male
liver. Elevated BHMT enzyme expression in the VR male
P. Singh et al.
offspring from proteomics data and lower levels of betaine in
metabolomics experiments suggest possible utilization of betaine
through BHMT pathway to maintain the flux of SAM/SAH under
vitamin B12 deficient conditions in the male offspring. Inter-
estingly, metabolic profiling also shows increased SAM levels in
both male and female offspring. Increased homocysteine levels
in male offspring can activate the transsulfuration pathway,
resulting in increased cysteine levels. Earlier reports have linked
higher homocysteine levels with DNA hypermethylation, which
supports our present findings of increased hypermethylation
signatures in the liver of VR male offspring [68]. Steroid hor-
mones show elevated levels in VR male whereas they are unal-
tered in VR female. Cortisol, a glucocorticoid associated with
stress is also increased in VR male liver. Cortisol regulates blood
glucose level by regulating the release of insulin and glucagon
[69,70]. In the liver, increased cortisol levels can increase
gluconeogenesis from nonhexose precursors like pyruvate and
glycogenic amino acids like α-ketoglutarate, decrease glycogen
synthesis, and induce insulin resistance and hepatic steatosis
[71,72]. Evidence from the literature indicates an association of
vitamin B12 deficiency with increased stress levels and secretion
of corticosteroids [62]. In the present study, we show that
upregulation of intermediates of the corticosteroid biosynthesis
pathway and cortisol levels in the liver of male offspring, which
could also be due to impaired glucose metabolism in the males,
whereas, in females cortisol levels are unchanged. All these ob-
servations from body composition, plasma biochemical param-
eter analysis, and omics analysis (DNA methylomics, proteomics,
and metabolomics) demonstrate a sexually dimorphic outcome
in which VR male offspring in the F1 generation show an obese
phenotype with metabolic dysregulation, indicative of insulin
resistance and MetS, whereas VR females present with relatively
less prominent outcomes.
Energy homeostasis in liver is tightly controlled by the family
of transcription factors that perform the task of nutrient sensing.
PPARs are ligand-activated nuclear transcription factors that
play a key role in nutrient homeostasis [73]. In the current study,
we find gene promoter hypermethylation in all the 3 PPAR iso-
types [PPARα, PPARδ (also known as PPARβ), and PPARγ] in the
VR male liver, which indicates their lower protein expression.
PPARs control expression of genes involved in carbohydrate,
lipid, lipoprotein, and energy metabolism [74]. PPARα is the
predominant PPAR isotype in liver that controls mitochondrial
and peroxisomal β-oxidation pathways, TG metabolism, and
fatty acid uptake, especially during fasting [75]. This observa-
tion is in line with our previous study showing PPARα hyper-
methylation in F1 generation 12-mo male offspring under
maternal vitamin B12 deficiency and PPAR signaling pathway to
be a key modulator of liver proteome in pups born to vitamin
B12 deficient rats [14,15].
The sexually dimorphic perturbation in metabolic processes
and phenotypic outcomes as observed in this study can result from
sex hormones and/or sex-specific developmental programming.
Genes involved in steroid hormone metabolism like androgen and
estrogen metabolism have been shown to exhibit PPARα- depen-
dent sexual dimorphism [76]. Estrogen, a sex-steroid hormone,
has been shown to inhibit the action of PPARα on obesity and lipid
metabolism by regulating PPARα-dependent target genes [77].
Impaired estrogen synthesis in aromatase-null mice causes inac-
tivation of estrogen receptors, which induces hepatic steatosis,
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The Journal of Nutrition 153 (2023) 3382–3396
with elevated hepatic TGs only in males, which is reversed by
estrogen replacement [78]. Etomoxir treatment in PPARα null
mice with steatosis phenotype causes 100% mortality in males but
only 25% in females. However, 17β-estradiol treatment reduces
the mortality in males to 20% [79]. We speculate that steroid
hormone biosynthesis alteration in VR offspring liver could
change the concentration of estrogen, which may lead to
sex-specific outcomes.
In summary, our study shows that vitamin B12 deficiency
causes a sexually dimorphic dysregulation of the OCM pathway,
altering DNA methylation pattern, which leads to differential
proteomic and metabolic alterations in key metabolic pathways.
These signatures correct to near-normal on vitamin B12 RC, and
thus are likely to be causally associated.
This study has several strengths and a few limitations. The
major strength is its comprehensive nature, both in terms of in-
clusion of a group of pregnant animals, repleted with vitamin
B12 at conception and a multiomic data analysis approach from
liver, the single most important tissue associated with metabolic
abnormalities. Overall, these approaches allow identification of
correlated differential signatures highlighting strong corrobora-
tion of findings from one to another and propose a causal
inference based on reversion of changes upon vitamin B12
repletion. Due to limited availability of blood samples, some
limitations such as unavailability of data on plasma glucose,
glucose tolerance tests, and estrogen levels have crept in, which
could help emphasize the hormonal regulation of sexually
dimorphic phenotypes with MetS-like outcomes only in male
offspring born to vitamin B12-restricted mothers.
Further, investigations utilizing metabolic tracer techniques
would be required to understand the detailed mechanisms of
metabolic dysregulation in offspring born to vitamin B12-
restricted mothers. Studies with vitamin B12 restriction in
PPARα null rodents and application of steroid hormones like
estrogen could specifically ascertain the role of sex-ster-
oid–mediated PPAR regulations and consequent sexually
dimorphic outcomes as observed in this study. Identifying
detailed signaling mechanisms playing a causal role in the
metabolic abnormalities due to in utero vitamin B12 deficiency
is relevant to future studies.
Author contributions
The authors’ responsibilities were as follows – PS: per-
formed the metabolomics experiment; SV: performed the
biochemical analysis and proteomics work; SG (Subhoshree):
performed the MeDIP experiments and was involved in data
analysis; SG (Sourav): was involved in establishing the
MeDIP-Seq analysis pipeline on Wistar rat tissues; VS: pro-
vided valuable inputs in MeDIP-Seq data analysis; SKV: hel-
ped in troubleshooting and running the sequencing workflow;
SSB: supervised design of the sequencing experiments and
provided critical inputs to the study; LK, VJ, PK: were
involved in all the animal experiments; PS, SG, SV, LK: per-
formed the integrated multiomics data analysis; PS: wrote the
initial draft of the manuscript; SG, LK: provided critical input
in improving the manuscript; SS, GRC: conceptualized the
study and designed the experiments and were also involved
in data analysis and writing the manuscript; and all authors:
read and approved the final manuscript.
P. Singh et al.
Conflicts of interest
The authors report no conflicts of interest.
Funding
The study was supported by grant no. MLP1804 and
MLP2014 from Council of Scientific and Industrial Research
(CSIR), India, to SS and GRC. PS and SG (Subhoshree) were
supported by research fellowships from Council of Scientific and
Industrial Research (CSIR), India, and SV was supported by
research fellowships from University Grants Commission, India.
Data availability
All the data supporting the finding is available within the
article and its supplementary materials. Raw data that supports
the findings of this study are available from the corresponding
author, upon reasonable request.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tjnut.2023.08.032.
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