Am J Physiol Gastrointest Liver Physiol 322: G295–G309, 2022.
First published January 5, 2022; doi:10.1152/ajpgi.00247.2021
RESEARCH ARTICLE
Inter-Organ Communication in Homeostasis and Disease
Maternal obesogenic diet regulates offspring bile acid homeostasis and hepatic
lipid metabolism via the gut microbiome in mice
Michael D. Thompson,1 Jisue Kang,1 Austin Faerber,1 Holly Hinrichs,1 Oğuz Özler,1 Jamie Cowen,1
Yan Xie,2 Phillip I. Tarr,3 and Nicholas O. Davidson2
1
Division of Endocrinology and Diabetes, Department of Pediatrics, Washington University School of Medicine, St. Louis,
Missouri; 2Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, St. Louis,
Missouri; and 3Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Washington University
School of Medicine, St. Louis, Missouri
Abstract
Mice exposed in gestation to maternal high-fat/high-sucrose (HF/HS) diet develop altered bile acid (BA) homeostasis. We hypothesized that these reflect an altered microbiome and asked if microbiota transplanted from HF/HS offspring change hepatic BA and lipid
metabolism to determine the directionality of effect. Female mice were fed HF/HS or chow (CON) for 6 wk and bred with lean males.
16S sequencing was performed to compare taxa in offspring. Cecal microbiome transplantation (CMT) was performed from HF/HS or
CON offspring into antibiotic-treated mice fed chow or high fructose. BA, lipid metabolic, and gene expression analyses were performed in recipient mice. Gut microbiomes from HF/HS offspring segregated from CON offspring, with increased Firmicutes to
Bacteriodetes ratios and Verrucomicrobial abundance. After CMT was performed, HF/HS-recipient mice had larger BA pools,
increased intrahepatic muricholic acid, and decreased deoxycholic acid species. HF/HS-recipient mice exhibited downregulated hepatic Mrp2, increased hepatic Oatp1b2, and decreased ileal Asbt mRNA expression. HF/HS-recipient mice exhibited decreased cecal
butyrate and increased hepatic expression of Il6. HF/HS-recipient mice had larger livers and increased intrahepatic triglyceride versus
CON-recipient mice after fructose feeding, with increased hepatic mRNA expression of lipogenic genes including Srebf1, Fabp1,
Mogat1, and Mogat2. CMT from HF/HS offspring increased BA pool and shifted the composition of the intrahepatic BA pool. CMT
from HF/HS donor offspring increased fructose-induced liver triglyceride accumulation. These findings support a causal role for vertical transfer of an altered microbiome in hepatic BA and lipid metabolism in HF/HS offspring.
NEW & NOTEWORTHY We utilized a mouse model of maternal obesogenic diet exposure to evaluate the effect on offspring
microbiome and bile acid homeostasis. We identified shifts in the offspring microbiome associated with changes in cecal bile
acid levels. Transfer of the microbiome from maternal obesogenic diet-exposed offspring to microbiome-depleted mice altered
bile acid homeostasis and increased fructose-induced hepatic steatosis.
bile acid metabolism; developmental programming; lipid metabolism; body mass index; microbiome
INTRODUCTION
As obesity rates increase, so do the prevalence and impact
of its complications, such as nonalcoholic fatty liver disease
(NAFLD). The global prevalence of NAFLD (nearly 25%)
makes this condition the most common chronic liver disease
at all ages (1, 2). Individual lifestyle is an important factor in
the development and progression of NAFLD, but genetic and
environmental factors play important roles. Exposures beginning in utero can initiate the development of metabolic
liver disease later in life. One exposure is maternal diet, a
well-established predisposing factor for development of
NAFLD in offspring (for review, see Ref. 3). Longitudinal
analysis of several birth cohorts has identified maternal prepregnancy BMI 30 as an independent predictor of offspring NAFLD (4, 5). Animal models corroborate these
findings and are being utilized to identify the mechanisms
for this developmental programming of liver disease (6–8).
We have found that maternal high-fat/high-sucrose (HF/
HS) diet changes bile acid (BA) homeostasis in mouse offspring (9), with increased BA pool size, increased expression
and activity of hepatic Cyp7a1, and a shifted intrahepatic BA
profile. However, the underlying mechanisms of transmission of altered BA homeostasis from dam to offspring is yet
to be defined. One potential mode for transmission is
through vertical transmission of an altered microbiome at
Correspondence: M. D. Thompson (thompsonmd@wustl.edu).
Submitted 23 July 2021 / Revised 22 November 2021 / Accepted 22 December 2021
http://www.ajpgi.org
0193-1857/22 Copyright © 2022 the American Physiological Society.
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DEVELOPMENTAL PROGRAMMING OF BILE ACID METABOLISM
birth. The role of resident gut bacteria in metabolizing bile
acids has been well described (for review, see Ref. 10). In
models of maternal obesogenic diet exposure (Fig. 1), the
microbiome of offspring is altered (11, 12). There are also
shifts in the microbiome of human offspring of mothers with
BMI 30 (13).
Although changes in bile acid metabolism and microbiome occur in models of maternal obesogenic diet exposure, a causal relationship for gut bacteria in regulating BA
pool size and intrahepatic BA profile remains unproven.
Here, we tested the hypothesis that vertical transmission of
an altered microbiome after maternal HF/HS diet exposure
affects BA homeostasis in the offspring. We also sought to
define whether vertical transmission of an altered microbiome increases steatosis and alters expression of factors
involved in hepatic lipid metabolism in offspring exposed to
maternal obesogenic diet.
EXPERIMENTAL PROCEDURES
Mouse Breeding Scheme, Feeding Paradigm, and
Metabolic Analysis
All procedures were approved by the Animal Studies
Committee at Washington University School of Medicine
and conformed to National Institutes of Health guidelines.
Four-week-old female C57Bl/6J mice were fed either a highfat/high-sugar (HF/HS) [Test Diet 58R3; 59% fat, 26% carbohydrates (17% sucrose) and 15% protein] or standard (CON)
chow [Pico Lab Rodent diet 20; 13% fat, 62% carbohydrates
(3.2% sucrose) and 25% protein] for 6 wk as we have
reported previously (14). HF/HS- and CON-fed F0 female
mice were mated with chow-fed male mice to produce HF/
HS- and CON-exposed offspring. Tissues were collected at
necropsy at 6–8 wk of age. The numbers of offspring and
number of different litters represented are noted in each
figure legend.
To evaluate maternal obesogenic diet during pregnancy
and lactation only, we mated 10-wk-old lean female C57Bl/6J
mice with lean males. If a vaginal plug was identified, the
female mouse was separated and fed either HF/HS or standard chow (CON) throughout pregnancy and lactation.
To evaluate transmission across generations by male offspring, F1 male offspring from the CON or HF/HS were
mated with lean female mice. Offspring from this breeding
are denoted as PF2C if from the CON and PF2H if from the
HF/HS.
For all breeding, potential dams were staged to identify
the most likely period for successful mating. The sire was
placed in the cage for only 24 h to limit cohousing effects.
Figure 1. Diagram of mouse models. Schematic representation of the different mouse models used for the studies in this manuscript. HF/HS, high fat/
high sucrose. Created with BioRender.com.
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DEVELOPMENTAL PROGRAMMING OF BILE ACID METABOLISM
Bile Acid Analysis
BA pool size was measured in offspring as previously
reported (15). Briefly, after 4 h of fasting, gallbladder, liver,
and intestines including luminal contents were collected,
homogenized, and incubated in ethanol to extract BAs. Total
BA content was determined enzymatically (Crystal Chem,
Elk Grove Village, IL) and normalized to body weight.
Individual intrahepatic BA species were measured using
high-performance liquid chromatography (HPLC) and tandem mass spectrometry as we have previously described
(15). Individual BA concentrations were normalized to tissue
weight. The quantities of each BA type are presented as a
percent of the total intrahepatic BA pool. Primary and secondary bile acids were combined to calculate a ratio.
Quantitative PCR
Hepatic and ileal total RNA were extracted and cDNA was
prepared using an ABI high-capacity cDNA reverse transcription kit with 1 mg of total RNA. We applied real-time quantitative
PCR to cDNAs from six animals per group, performed in duplicate on an Applied Biosystems 7500 Real-Time PCR System
using SYBR Green PCR Master Mix (Applied Biosystems) and
primer pairs (provided on request) designed by Primer Express
software (Applied Biosystems). Relative mRNA abundance is
expressed as fold change to maternal chow-offspring chow
group after normalization to GAPDH.
Hepatic Triglyceride Measurement
Liver triglycerides (TG) were measured after 4 h of fasting
at necropsy using a colorimetric biochemical assay (Diabetes
Models Phenotyping Core, Diabetes Research Center at
Washington University).
Sequencing for Gut Microbiome Analysis
Stool DNA was isolated from cecal contents using the
PowerSoil DNA Isolation Kit (Qiagen) per the manufacturer’s
instructions. The samples were lysed with beads and spin filtered to elute purified DNA, which was stored at 20 C until
PCR amplification. A reengineered version of bTEFAP, a form
of amplicon sequencing utilizing next generation sequencing,
was used to evaluate the microbiota. This modern version of
bTEFAP has been adapted to current next generation sequencing technologies. We used 16S rRNA primer pairs 515F
GTGYCAGCMGCCGCGGTAA/806R GGACTACNVGGGTWTCTAAT, which were subjected to a single-step 30 cycle PCR was
used for the HotStarTaq Plus Master Mix Kit (95 C (5 min), 30
cycles at 95 C (30 s), 53 C (40 s), 72 C (1 min), and 72 C (10
min). The amplification products from the different samples
were mixed in equal concentrations, purified, and sequenced
(Illumina NovaSeq). The Mr. DNA ribosomal and functional
gene analysis pipeline was utilized to process the Q25 sequence
data. To note, sequences with point errors, shorter than 150 bp,
or containing ambiguous bases were removed. The quality of
the sequences was assessed by dereplicating sequences with
expected error >1.0. This process allows creation of a denoised
sequence or Zero-radius Operational Taxonomic Unit (zOTU).
The final sequence data were classified based on taxonomy
using BLASTn. Statistical analysis was conducted using XLstat,
NCSS 2007, “R,” and NCSS 2010. a and b diversity analyses
were viewed through Qiime 2.
Microbiome Depletion and Cecal Microbiome
Transplantation
Six-week-old male C57/Bl6 mice were purchased from
Jackson laboratories and acclimated to our mouse facility
for 1 wk before antibiotic treatment to deplete the resident
gut bacterial microbiome. The ABX mix contained 300 mL
of sterile water, to which vancomycin (150 mg), neomycin
(300 mg), and artificial sweetener (equal) (2 g) were added.
The antibiotic water was provided for 60 h followed by 12 h
of water containing polyethylene glycol.
Cecal contents were collected from 6-wk-old female HF/
HS and CON offspring and placed in reduced PBS with glycerol (adjusted to 16%) to create a slurry at 0.1 g/mL concentration, which was then stored ( 80 C) (Repeated using
male donor cecal contents in Supplemental Material;
see https://doi.org/10.6084/m9.figshare.15044439.v1). The
slurry mix was thawed on ice, homogenized with a glass
bulb, passed through a 100-mm nylon cell strainer, centrifuged for (3,000 rpm, 30 s), and vortexed to create a homogeneous mix 30 min before oral gavage of the recipient mice.
Microbiome-depleted mice were gavaged with stool slurry
mix from CON or HF/HS offspring five times over 11 days.
Statistical Analysis
Unpaired Student’s t test and ANOVA were used when
appropriate using GraphPad prism software. Data are presented as means (SE) with two-tailed P < 0.05 representing
significance and P > 0.05 and < 0.10 representing a trend.
RESULTS
Altered Microbiome in Offspring Exposed to Maternal
HF/HS Diet
Male and female offspring exhibited separation of cecal bacterial communities between offspring exposed to maternal
CON and HF/HS diet (Fig. 2A). This separation was also
observed in principal component analysis (PCoA) of excreted
stool in male offspring (Supplemental Fig. S1; all Supplemental
figures are available at https://doi.org/10.6084/m9.figshare.
15044439). Three measures of a-diversity (observed OTUs,
Faith phylogenetic diversity (PD), and Shannon diversity) in
male HF/HS offspring demonstrated decreased a-diversity by
observed OTUs and Faith PD (Fig. 2B). At the family-phylum
level, there was decreased abundance of Bacteroidetes and
Proteobacteria in male and female offspring after exposure to
maternal HF/HS diet (Fig. 2C). Conversely, Firmicutes and
Verrucomicrobia increased (Fig. 2C). The Firmicutes to
Bacteroidetes ratio was also increased in maternal HF/HS offspring (Fig. 2D).
Altered Cecal BA Profile and BA Metabolizing Bacteria
in Offspring Exposed to Maternal HF/HS Diet
We identified shifted abundance of numerous genera of
bacteria between HF/HS and CON offspring (Fig. 3A). We
specifically compared the abundance of five genera known
to metabolize BA’s between HF/HS and CON offspring(16).
No significant differences were observed in Lactobacillus,
Bacteroides, and Clostridium (Fig. 3B). There was a signifi-
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DEVELOPMENTAL PROGRAMMING OF BILE ACID METABOLISM
Figure 2. Shift in the cecal microbiome of high-fat/high-sucrose (HF/HS) male and female offspring. A: Bray–Curtis plots for b-diversity of cecal microbiome
from HF/HS and chow (CON) offspring. B: measures of a-diversity (observed OTUs, Faith PD, and Shannon diversity) in cecal microbiome of HF/HS and CON
offspring. C: relative abundance of each bacterial family in cecal microbiome of HF/HS and CON offspring. D: ratio of Firmicutes to Bacteroidetes in cecal microbiome of HF/HS and CON offspring. Quantitative data presented as means ± SE with n = 10 in each group and 5 separate litters represented in each group.
Male and female offspring included in each section. P values as indicated on graph for t tests. OTU, operational taxonomic unit; PD, phylogenetic diversity.
cant decrease in Bifidobacterium in males and Eubacterium
in females (Fig. 3B).
If the microbiome is playing a role in changing BA homeostasis, we would expect a shift in BA concentrations in stool.
Consistent with previous reports of intrahepatic BA profile
changes (9), cecal BA from HF/HS offspring showed
increased muricholic acid (MCA) and decreased deoxycholic
acid (DCA) (Fig. 4A). There was also decreased abundance of
urso-DCA (UDCA) species. These changes were similar in
male and female offspring. Consistent with these specific
shifts in BA abundance, the ratio of primary to secondary
bile acids was significantly increased in male HF/HS offspring (Fig. 4B). A trend toward this increase was observed
in female HF/HS offspring (Fig. 4B). Over 99% of cecal BAs
were unconjugated in each group. We measured expression
of bile acid transporters in the ileum of HF/HS and CON offspring. Male offspring did not show any differences in Asbt,
Mrp2, or Mrp3 (Fig. 4C). Female offspring exhibited a trend
toward a decrease in Asbt expression (Fig. 4D). Bile acidmediated activation of FXR represses Asbt expression via an
increase in Shp (17). Ileum expression of Shp was increased
in female HF/HS offspring, but not in males (Fig. 4, C and D).
Cecal Microbiome Transplantation from HF/HS Donor
Alters BA Homeostasis in Recipient Mice
To determine if changes in the microbiome causally relate
to changes in BA homeostasis in the offspring, we performed
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cecal microbiome transplantation (CMT) from HF/HS and
CON offspring to mice whose microbiome was depleted by
antibiotic treatment. After CMT, HF/HS and CON recipients
exhibited similar body and liver weights (Fig. 5A). BA pool
size was significantly greater in HF/HS recipients (Fig. 5B).
Intrahepatic BA profiles were shifted in HF/HS recipient
mice with increased abundance of MCA and tauro-MCA
(TMCA), whereas tauro-DCA (TDCA) abundance decreased
(Fig. 5C). The cecal BA profile of HF/HS recipient mice
showed increased abundance of MCA and decreased abundance of DCA (Fig. 5D). We observed a trend toward an
increased primary to secondary BAs in livers of HF/HS
recipients but not in cecal BAs (Fig. 5E). Expression of
two primary BA synthetic genes, Cyp7a1 and Cyp8b1, was
not affected in HF/HS recipient livers (Fig. 5F). A trend
toward an increase in Nr1h4 expression was observed;
however, there was no associated difference in Shp
expression (Fig. 5G).
CMT from HF/HS Lineage Donor Altered Expression of
Bile Acid Transport Genes in Liver and Ileum
The expression of apical transporters Bsep and Mrp2 significantly decreased in livers of HF/HS recipient mice after
CMT, whereas the expression of the basolateral transporter
Oatp1b2 significantly increased (Fig. 6A). Hepatic expression of Ntcp, Mdr2, and Mrp3 remained constant. Ileal
Asbt, Mrp2, and Mrp3 expression decreased in HF/HS
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DEVELOPMENTAL PROGRAMMING OF BILE ACID METABOLISM
Figure 3. Abundance of bacterial genera involved in bile acid (BA) metabolism in offspring cecal contents. A: relative abundance of top 20 genera of
bacteria present in cecal contents of high-fat/high-sucrose (HF/HS) and chow (CON) offspring. B: percent abundance of five specific genera of bacteria
that can metabolize bile acids (Lactobacillus, Bacteroides, Bifidobacterium, Eubacterium, and Clostridium). Quantitative data presented as means ± SE
with n = 10 in each group and 4 separate litters represented in each group. Male and female offspring included in each section. P values as indicated
on graph for t tests.
recipients (Fig. 6B). Ileal Shp expression was increased
(Fig. 6B).
Gut Microbiome Altered in HF/HS CMT Recipients
Compared with CON
b-Diversity of gut microbial communities differed between
HF/HS and CON recipients. In contrast, three measures of
a-diversity remained unchanged between groups (Fig. 7B).
Analysis at the phylum level only showed minor differences in the abundance of specific bacterial phyla with more
differences present at the genus level (Fig. 7, C and D). Of
five different genera known to be involved in BA metabolism, only one (Lactobacillus) showed a trend toward
decreased abundance (Fig. 7E), whereas Prevotella,
Parabacteroides, and Allobaculum significantly increased
(Fig. 7F).
Changes in Short-Chain Fatty Acid Concentrations in
HF/HS Recipient Mice
Short-chain fatty acids (SCFAs) of bacterial origin can also
affect hepatic physiology. We measured concentrations of
SCFAs in cecal contents and serum from CMT recipient
mice. Cecal butyrate was significantly decreased in HF/HS
recipient mice whereas cecal acetate and propionate were
unchanged (Supplemental Fig. S2A), with no change in serum SCFA concentrations (Supplemental Fig. S2B). Hepatic
gene expression of Il6 was increased in HF/HS recipient
mice (Supplemental Fig. S2C).
Maternal HF/HS Exposures Restricted to Pregnancy and
Weaning Alters BA Homeostasis in Offspring
To determine whether exposure to maternal HF/HS diet
restricted to gestation affects BA homeostasis, F0 female
mice were placed on HF/HS diet the day after they were
paired with a male and continued on the diet to the day of
weaning. The weight of male offspring increased after
maternal HF/HS diet exposure during pregnancy and lactation (Fig. 8, A and C). Liver weights in male and female
offspring did not change (Fig. 8, A and C), but BA pool size
increased in both sexes after maternal HF/HS exposure
(Fig. 8, B and D).
A significant increase in Cyp7a1 expression was observed
in female offspring with a trend toward an increase (P =
0.066) in male offspring (Fig. 8, E and G). Neither male nor
female offspring exhibited differences in expression of
Cyp8b, and Cyp27a1 (Fig. 8, E and G), or of the BA nuclear receptor FXR or transcriptional repressor Shp (Fig. 8, F and H).
We observed a trend toward an increase in hepatic Bsep
expression and a decrease in Ntcp expression in male offspring exposed to maternal HF/HS diet between conception
and weaning (Supplemental Fig. S3A), but no difference in
female offspring was observed (Supplemental Fig. S3B).
Likewise, we found no differences in mRNA expression of
Oatp1b, Mrp2, and Mrp3 in offspring (Supplemental Fig. S3,
A and B).
Intergenerational Transmission of Altered BA
Homeostasis after Maternal HF/HS Exposure
We previously reported that F2 offspring exhibited
increased BA pool sizes if bred from F1 female offspring from
an F0 female that had been fed HF/HS diet (9). To evaluate
whether an altered BA homeostasis phenotype is also passed
by male F1 offspring from an F0 female fed HF/HS diet, we
bred F1 generation offspring with lean females and evaluated
BA homeostasis in the subsequent F2 offspring (termed PF2).
No difference in body weight, liver weight, or liver weightto-body weight ratio was observed in male or female offspring (Fig. 9, A and B). PF2H offspring exhibited increased
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Figure 4. Shift in cecal bile acid concentrations in high-fat/high-sucrose (HF/HS) offspring. A: relative abundance of individual bile acids present in cecal
contents of HF/HS and CON offspring. B: ratio of primary to secondary bile acids present in cecal contents of HF/HS and chow (CON) offspring. C: relative expression of Asbt, Mrp2, Mrp3, and Shp in ileum of male HF/HS and CON offspring. D: relative expression of Asbt, Mrp2, Mrp3, and Shp in ileum of
female HF/HS and CON offspring. Quantitative data presented as means ± SE with n 5 in each group and 5 separate litters represented in each
group. Male and female offspring included in each section unless noted. P values as indicated on graph for t tests. CA, cholic acid; DCA, deoxycholic
acid; MCA, muricholic acid; TMCA, tauro-muricholic acid; UDCA, urso-deoxycholic acid. CDCA, chenodeoxycholic acid.
BA pool size (Fig. 9C), without a difference in intrahepatic
BA profile (Fig. 9D). Expression of Cyp7a1 mRNA exhibited a
trend toward an increase (P = 0.07) in PF2H offspring liver,
but other BA metabolism genes were unchanged (Fig. 9, E
and F).
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Microbiome from HF/HS Offspring is Sufficient to
Enhance Fructose-Induced Hepatic Steatosis
We next asked if the microbiome alteration due to maternal obesogenic diet exposure alters hepatic lipid metabolism,
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DEVELOPMENTAL PROGRAMMING OF BILE ACID METABOLISM
Figure 5. Recipients of cecal microbiome transplant from high-fat/high-sucrose (HF/HS) offspring develop altered bile acid (BA) homeostasis. A: body
weight, liver weight, and liver weight-to-body weight ratio of HF/HS and chow (CON) recipient mice. B: BA pool size in HF/HS and CON recipient mice. C:
abundance of individual BAs in liver tissue from HF/HS and CON recipient mice. D: abundance of individual BAs in cecal contents from HF/HS and CON
recipient mice. E: ratio of primary to secondary BAs in liver and cecal contents of HF/HS and CON recipient mice. F: relative expression of Cyp7a1 and
Cyp8b1 in liver of HF/HS and CON recipient mice. G: relative expression of Nr1h4 and Shp in liver of HF/HS and CON recipient mice. Quantitative data
presented as means ± SE with n 5 in each group with representation from 5 litters. All data from male mice receiving cecal microbiome transplantation (CMT) from female donors. P values as indicated on graph for t tests.
we performed CMT from HF/HS and CON offspring to antibiotic-treated mice followed by feeding a high-fructose diet for
7 days. No difference in body weight was observed (Fig. 10A),
but liver weight and liver weight-to-body weight ratio were
increased in HF/HS recipients (Fig. 10, B and C) along with
increased hepatic TG (Fig. 10D). Gene expression analysis
identified an increase in Srebf1 expression and a decrease in
Pdk4 expression (Fig. 10E). Analysis of genes involved in
lipid metabolism identified a significant decrease in Acaca
and significant increase in Acox1 (Fig. 10F), and trend toward
increased Mogat1 and Mogat2. No change in expression was
observed in several other lipid metabolism genes (Fig. 10G).
Genes involved in lipid transport were also affected with a
decrease in Slc27a1 and a trend toward an increase in Fabp1
(P = 0.0719) (Fig. 10H).
DISCUSSION
Developmental programming of NAFLD due to maternal
obesogenic diet has been described in multiple models with
an altered microbiome emerging as a candidate mode of
intergenerational transmission (6–9, 13, 18, 19). We and
others have reported changes in BA homeostasis after maternal obesogenic diet exposure. Here, we demonstrate 1)
increased bile acid pool size and a shift in the intrahepatic
BA profile after CMT from HF/HS offspring to antibiotictreated mice, 2) altered expression of several hepatic and
intestinal BA transporters following CMT for HF/HS recipient mice, 3) decreased fecal butyrate concentrations in
HF/HS recipient mice, 4) altered BA homeostasis in offspring exposed to time-limited maternal obesogenic diet
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Figure 6. Altered expression of bile acid (BA) transport genes in recipients of cecal microbiome from high-fat/high-sucrose (HF/HS) offspring. A: relative
expression of hepatic BA transporters in HF/HS and chow (CON) recipient mice. B: relative expression of ileal BA transporters and Shp in HF/HS and
CON recipient mice. Quantitative data presented as means ± SE with n 4 in each group with representation from 4 litters. All data from male mice
receiving cecal microbiome transplantation (CMT) from female donors. P values as indicated on graph for t tests.
(pregnancy and lactation only), and 5) enhanced fructoseinduced hepatic steatosis in HF/HS recipient mice. Several
of these findings warrant discussion.
Maternal Obesogenic Diet Induced Shifts in the
Offspring Microbiome Regulate BA Homeostasis
We initially hypothesized that an altered microbiome in
HF/HS offspring would be sufficient to induce the observed
changes in BA homeostasis. Indeed, we found that following
CMT from HF/HS offspring to microbiome-depleted mice
similarly increases BA pool size and shifts the intrahepatic
BA profile of the recipient mice. Although these changes are
consistent with our prior work (9), we did not observe a similar increase in Cyp7a1 following CMT from HF/HS offspring.
It is worth noting that Soderborg et al. (13) observed
increased Cyp7a1 expression in their human fecal microbiome transplantation transplant model. One possible explanation is that expression of some of these genes may be
under epigenetic regulation rather being specifically related
to microbiome changes or to concentrations of specific BAs
in our model. It is likely that the phenotypes observed after
maternal diet-driven developmental programming will combine microbiome and epigenetic drivers. Another possibility
is that differences in the type of maternal obesogenic diets
administered induces variability in offspring microbiomes
and BA metabolism. Changes in the offspring microbiomes
might also reflect sex (20). Although no difference in Cyp7a1
was observed after CMT, we did find differences in multiple
ileal and hepatic genes involved in BA transport. The
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decrease in all three ileal BA transporters could reflect a
compensatory response to the increased BA pool size in an
attempt to limit BA reuptake. We also observed increased hepatic Oatp1b2 (BA uptake into the hepatocyte) expression
and decreased Bsep and Mrp2 (BA export from hepatocytes)
expression. These findings would support overall BA retention within the hepatocyte. It is unclear if this reflects a
response to the increased BA pool size or if this represents
primary mediation of the changes in BA homeostasis secondary to microbiome changes. It is important to note that a
scenario of increased BA retention within the hepatocyte
could be cytotoxic or alter cell signaling (21, 22). Taken together, these studies implicate alterations in the microbiome
as one determinant of altered BA homeostasis in offspring
exposed to maternal high-fat diet (HFD).
Exposure during Pregnancy and Lactation Alone is
Sufficient to Change BA Homeostasis
We (9) and others (13, 20) have shown that maternal diet
affects bile acid metabolism when feeding starts before pregnancy. We now show that maternal HF/HS diet exposure
during and immediately after pregnancy is sufficient to alter
offspring BA homeostasis. We observed increased BA pool
sizes in male and female offspring with an associated
increase in Cyp7a1. These effects could be microbiome mediated as there is likely an effect on the microbiome after this
shorter-term HF/HS diet exposure. Indeed, HFD exposure
during gestation and lactation is sufficient to shift the microbiomes of Japanese macaques (23). Microbiome alterations
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DEVELOPMENTAL PROGRAMMING OF BILE ACID METABOLISM
Figure 7. Shifts in gut microbiome in high-fat/high-sucrose (HF/HS) recipient mice compared with chow (CON) recipient mice. A: Bray–Curtis plot for b-diversity of cecal contents from HF/HS and CON recipient mice. B: measures of a-diversity (observed OTUs, Faith PD, and Shannon diversity) in cecal
microbiome of HF/HS and CON recipient mice. C: relative abundance of each bacterial family in cecal microbiome of HF/HS and CON offspring. D: relative abundance of top 20 genera of bacteria present in cecal contents of HF/HS and CON offspring. E: percent abundance of five specific genera of bacteria that can metabolize bile acids (Lactobacillus, Bacteroides, Bifidobacterium, Eubacterium, and Clostridium). F: percent abundance of additional
genera of bacteria that are significantly increased in cecal microbiome of HF/HS recipient mice. Five separate litters represented in each group with triglyceride and cholesterol concentrations, 6 separate litters represented for all other quantitative data. All data from male mice receiving cecal microbiome transplantation (CMT) from female donors. P values as indicated on graph for t tests. OTU, operational taxonomic unit; PD, phylogenetic diversity.
are also present in mice offspring after maternal HFD exposure during gestation and lactation only with associated
impairments in the gut barrier (24). Exposure to HFD perinatally exacerbates HFD-induced hepatic stetatosis in the offspring (25). It is clear that exposure during this key period is
sufficient to drive changes in the offspring, so it will be important to differentiate and quantify the effect of prenatal
exposure versus exposure during gestation/lactation alone,
as transition to standard chow 1 wk before conception worsens steatosis (26).
Changes in the Microbiome Driven by Maternal HF/HS
Exposure Alter SCFA Metabolism in Offspring
Butyrate protects from hepatic steatosis and maintains
the intestinal barrier (27, 28). We did not observe changes in
circulating SCFAs leaving unanswered the impact on systemic SCFA metabolism, but the changes in SCFA concentrations in portal blood might be more informative of SCFA
delivery to the liver, and form a basis for future study.
Phenotypes May Be Affected by Timing of Microbiome
Changes
It is important to consider the timing of microbiome
changes and how that may affect phenotypes in the offspring. In the current study, we collected cecal contents
from 6-wk-old mice and transplanted into adult mice.
However, recent work has identified a key role for the
early microbiome in programming the responsiveness of
the immune system to future inflammatory insults (29),
specifically a weaning reaction that occurs between 2 and
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Figure 8. Altered bile acid (BA) homeostasis in offspring exposed to maternal obesogenic diet during pregnancy and lactation only. A: body weight, liver
weight, and liver weight-to-body weight ratio of male offspring from chow (CON) or high fat/high sucrose (HF/HS) (P&L only). B: BA pool size in male offspring from CON or HF/HS (P&L only). C: body weight, liver weight, and liver weight-to-body weight ratio of female offspring from CON or HF/HS (P&L
only). D: BA pool size in female offspring from CON or HF/HS (P&L only). E: relative expression of bile acid metabolism genes in male offspring from CON
or HF/HS (P&L only). F: relative expression of Nr1h4 and Shp in male offspring from CON or HF/HS (P&L only). G: relative expression of bile acid metabolism genes in female offspring from CON or HF/HS (P&L only). H: Relative expression of Nr1h4 and Shp in female offspring from CON or HF/HS (P&L
only). Quantitative data presented as means ± SE with n 5 in each group with 5 separate litters represented in each group. Male and female offspring
in each section. P values as noted on each graph for t tests. P&L, pregnancy and lactation.
4 wk and is dependent on the early microbiome. In the absence of the microbiome, the weaning reaction, defined
by temporary increases in colonic TNF-a and IFN-c, did
not occur and subsequently led to worse inflammatory
response in the adult. It is not yet defined how maternal
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HFD may impact this weaning reaction, but there is
increased intestinal inflammation and gut permeability in
weaning age offspring after such exposure (30). The consequences of maternal diet-induced changes in the offspring will need to be more closely studied in the
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DEVELOPMENTAL PROGRAMMING OF BILE ACID METABOLISM
Figure 9. Altered bile acid (BA) homeostasis in second generation offspring of male exposed to maternal obesogenic diet. A: body weight, liver weight,
and liver weight/body weight in male offspring. B: body weight, liver weight, and liver weight-to-body weight ratio in female offspring. C: BA pool size of
PF2 high-fat/high-sucrose (HF/HS) and chow (CON) male offspring. D: abundance of individual BAs in liver of PF2 HF/HS and CON male and female offspring. E: relative expression of bile acid metabolism genes in PF2 HF/HS and CON male offspring liver. F: relative expression of Nr1h4 and Shp in PF2
HF/HS and CON male offspring liver. Quantitative data presented as means ± SE with n 6 in each group and 6 separate litters represented in each
group. P values as noted on each graph for t tests.
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Figure 10. Transfer of microbiome from high-fat/high-sucrose (HF/HS) offspring increases fructose-induced hepatic triglyceride (TG) accumulation and
alters hepatic lipid metabolism. A: body weight of HF/HS and chow (CON) recipient mice after fructose diet feeding. B: liver weight of HF/HS and CON recipient mice after fructose diet feeding. C: liver weight-to-body weight ratio of HF/HS and CON recipient mice after fructose diet feeding. D: hepatic triglyceride concentration in HF/HS and CON recipient mice after fructose diet feeding. E: relative expression of transcription factors that control lipid
metabolism in liver of HF/HS and CON recipient mice after fructose diet feeding. F: relative expression of lipid metabolism genes with significant or trend
toward a change in liver of HF/HS and CON recipient mice after fructose diet feeding. G: relative expression of lipid metabolism genes that are
unchanged in liver of HF/HS and CON recipient mice after fructose diet feeding. H: relative expression of lipid transport genes in liver of HF/HS and
CON recipient mice after fructose diet feeding. Quantitative data presented as means ± SE with n 6 in each group and 6 separate litters represented
in each group. All data from male mice that received cecal microbiome transplantation (CMT) from female donors. P values as noted on each graph for t
tests.
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DEVELOPMENTAL PROGRAMMING OF BILE ACID METABOLISM
perinatal period given the programming impact of the
early microbiome.
Intergenerational Transmission of Altered Bile Acid
Homeostasis
Developmental programming studies can evaluate whether
phenotypes are passed in an intergenerational (to F2) or transgenerational (to F3) manner. We previously reported that F2
offspring after F0 maternal HF/HS exposure have increased
BA pool size but no difference in intrahepatic BA profile was
observed (9). F2 offspring in these studies came from F1
females exposed to maternal HF/HS. In this report, we show
that F2 offspring from F1 males exposed to maternal HF/HS
also have increased BA pool size. This shows that this phenotype can be passed through male offspring and also that
changes in BA pool size may also be driven by factors other
than the microbiome as the F1 males are not contributing
directly to the offsprings microbiome. In future, more mechanistic studies will be necessary to evalaute the factors that are
driving the nonmicrobiome-mediated changes in offspring
BA homeostasis after maternal HF/HS diet exposure.
Potential Sex Differences in Response to Maternal
Obesogenic Diet
Previous studies have identified sex differences in response
to maternal obesogenic diet and the development of offspring
NAFLD (31–33). Although some commonalities exist beteween
male and female offspring in BA homeostasis in our studies,
there are also differences that merit consideration. Male offspring exposed to maternal obesogenic diet exhibited an
increase in the ratio of primary to secondary BAs in cecal contents; however, only a trend was observed in females. This could
suggest variability in how the microbiome is modifying BAs
between male and female offspring, which could be a direct
result of differences in the microbiome makeup. We observed
similarites in the microbiome between sexes at the phylum
level; however, analysis at the genus level revealed important
differences, most notably a decrease in Bifidobacterium in
males and a decrese in Eubacterium in females. Further studies
will be necessary to decipher how these specific genus level differences affect offspring bile acid homeostasis through more
reductionist approaches. Another sex difference we observed is
that the expression of the ileal bile acid transporter Asbt is
decreased in female HF/HS offspring as the BA pool increases,
but those changes were not observed in male HF/HS offspring.
We speculate that Asbt is decreased in response to elevated BA
levels, possibly as a compensatory adaptation to decrease reabsorption and normalize the total BA pool. Since we do not
observe this same adaptation in male offspring, that difference
may in part explain why we observe more sigificant alterations
in BA homeostasis in male HF/HS offspring. This speculation,
however, will be a focus of future work to delineate sex differences in phenotypes driven by maternal obesogenic diet exposure.
There are additional limitations we want to highlight in relation to the current studies. The molecular analysis presented
here centers around changes in gene expression by quantitative
PCR. These changes may not reflect what is happening at the
level of protein function or localization within the cell. In particular, FXR and Shp control gene expression in the nucleus
and their cell-specific localization and function will be
important to evaluate in future studies. Another limitation is
that some but not all analyses were completed in both sexes in
the offpsring. Although there is clear overlap in some of the
changes in BA homeostasis and microbiome shifts, there
appear to be sex differences that warrant further evaluation.
Most of our analysis in the CMT studies utilized female donor
cecal contents, and it is possible that there may be sex differences reflecting the source of donor microbiome. Nevertheless,
we observed a similar increase in BA pool size when utilizing
donor cecal contents from male offspring (Supplemental Fig.
S4). Likewise, CMT studies were only performed in male recipients and it is possible the response to CMT in female recipients
may be different. There are also inherent limitations in relation
to performance of CMT studies, including the observation that
antibiotic treatment followed by microbiome transplantation
does not result in an exact engraftment of the same bacterial
abundances (34). Although we observed similarities in the general directionality of abundances in donor and recipient abundances at the genus level, none of the differences achieve
statistical significance in both groups. An alternative approach
to increase efficiency of engraftment is to perform similar studies in gnotobiotic mice. However, gnotobiotic mice exhibit developmental defects due to the importance of the early
microbiome, which could affect the outcomes of analyses performed here (for review, see Ref. 35). Given these limitations,
the current data do not definitively show that certain specific
changes in the offspring microbiome cause the observed
changes in BA homeostasis and thus can only be attributed to
global changes in the microbiome following maternal obesogenic diet exposure.
In summary, alteration of the offspring microbiome by
maternal HF/HS diet exposure is sufficient to change BA homeostasis in offspring, increasing the bile acid pool size and
shifting the intrahepatic BA pool. Furthermore, transferring
the microbiome after maternal obesogenic diet exposure
increases fructose-induced steatosis. Given the clear effect of
maternal diet-driven microbiome alteration on offspring, interventions that reverse these changes could provide a pathway
for disease prevention. Future experiments will need to further
define the role of specific bacteria that are affected in the offspring microbiome. We anticipate that that future studies,
including epigenetic effects of the microbiome, will guide the
path for reversing developmental programming of NAFLD.
SUPPLEMENTAL DATA
Supplemental Figs. S1–S4: https://doi.org/10.6084/m9.figshare.
15044439;
Supplemental Material: https://doi.org/10.6084/m9.figshare.
15044439.v1.
ACKNOWLEDGMENTS
The authors acknowledge the expert assistance of David
Scherrer and Xuntian Jiang in the Metabolomics Core for performance of BA composition and SCFA analysis and Sangeeta Adak
for completing triglyceride measurements.
GRANTS
This study was supported by National Institutes of Health (NIH)
Grants DK-122018 (to M. D. Thompson); AGA Research Scholar
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DEVELOPMENTAL PROGRAMMING OF BILE ACID METABOLISM
Award (to M. D. Thompson); NIH Washington University
Digestive Diseases Research Core Center Grant No. NIDDK P30
DK052574 (to M. D. Thompson, P. I. Tarr and N. O. Davidson);
NIH Grant P30 DK056341 (Nutrition Obesity Research Center)
(to M. D. Thompson); and by the Washington University Diabetes
Research Center, Grant No. P30 DK020579 (to M. D. Thompson).
N. O. Davidson was also supported by NIH Grants DK-119437, HL151328, and DK-128169.
11.
12.
13.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by
the authors.
AUTHOR CONTRIBUTIONS
M.D.T., P.I.T., and N.O.D. conceived and designed research;
M.D.T., J.K., A.F., H.H., O.Ö., J.C., and Y.X. performed experiments;
M.D.T., J.K., A.F., H.H., O.Ö., and J.C. analyzed data; M.D.T., J.K.,
and N.O.D. interpreted results of experiments; M.D.T., J.K., A.F.,
and O.Ö. prepared figures; M.D.T. drafted manuscript; M.D.T., J.K.,
H.H., O.Ö., P.I.T., and N.O.D. edited and revised manuscript;
M.D.T., J.K., H.H., O.Ö., P.I.T., and N.O.D. approved final version
of manuscript.
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