compared with control LFi offspring (Fig. 6B
and fig. S22, B and C). HFD-induced glucose
intolerance and insulin resistance were also
significantly ameliorated in Pro offspring (fig.
S22D), and energy expenditure was improved
in Pro offspring (fig. S22E). Furthermore, sym-
pathetic dysfunction in the LFi offspring was
rescued in Pro offspring (Fig. 6C). Composi-
tions of the gut microbiota were similar be-
tween LFi offspring and Pro offspring during
both infancy and adulthood (fig. S23, A and B).
Additionally, maternal intervention with pro-
pionate reversed the retardation of sympa-
thetic nerve projections to the heart as well as
the retardation of GLP-1+enteroendocrine cell
and pancreaticbcell differentiation in embryos
of LFi-fed mothers (Fig. 6, D and E); it also
enhanced plasma insulin levels in the embryos,
restoring them to levels comparable to those
of HFi embryos (Fig. 6F). Consequently, an
increase in plasma glucose levels in control
embryos was efficiently suppressed in embryos
of mothers given propionate (Fig. 6F). Consist-
ent with the offspring from HFi-fed mothers
treated with antibiotics, offspring from HFi-fed
GF ICR mothers recapitulated the obesity-prone
phenotype, which was rescued by administra-
tion of propionate during pregnancy (fig. S24,
A to H). Together, these observations define
the importance of maternal propionate, which
renders offspring resistant to obesity.
Discussion
In this study, we determined that maternal
gut microbiota during pregnancy confers resist-
ance to obesity in offspring via the SCFA-GPR41
and SCFA-GPR43 axes. During pregnancy,
SCFAs from the maternal gut microbiota are
sensed by GPR41 and GPR43 in the sympa-
thetic nerve, intestinal tract, and pancreas of
the embryo. SCFAs are known to exert pleio-
tropic effects through several mechanisms
( 10 , 11 , 13 , 15 , 16 , 24 , 33 – 36 )suchashistone
deacetylase (HDAC) inhibition by butyrate
[median inhibitory concentration (IC 50 ): ~90
to 170mM] ( 37 , 38 ) and activation of GPR109A
by butyrate (EC 50 : ~700mM) ( 39 ) and Olfr78
by acetate (EC 50 : ~2300mM) and propionate
(EC 50 : ~1000mM) ( 33 ), in addition to GPR41
and GPR43. Considering the low concentra-
tions of SCFAs (acetate: ~400mM, propionate:
~50mM, and butyrate: ~10mM) in the em-
bryonic circulating plasma, SCFAs were unlikely
to interact with Olfr78 and GPR109A. Mean-
while, the concentrations of SCFAs were suf-
ficient to activate GPR41 and GPR43 ( 30 , 31 ).
Activation of embryonic GPR41 and GPR43 by
SCFAs promoted sympathetic neuronal, enter-
oendocrine, and pancreaticbcell differenti-
ation, which were essential for maintaining
energy homeostasis (e.g., thermogenesis and
heart rate) via the sympathetic nervous system
and fetal glucose homeostasis. Furthermore,
given that the expression ofGpr43tended to
be down-regulated in the pancreas and colon
of GF offspring, SCFAs such as butyrate and
propionate [which also serves as an HDAC
inhibitor ( 40 )]mayregulateembryonicdevel-
opment by regulatingGpr41andGpr43gene
expression through epigenetic modifications.
Several metabolites other than SCFAs showed
similar changes in the plasma of mothers and
embryos between SPF and GF conditions. How-
ever, in HFi-fed mothers and their embryos, only
SCFAs were commonly elevated compared with
those in the LFi-fed counterparts. Treatment
of LFi-fed mothers with propionate repaired
the defects in the differentiation of intestinal
enteroendocrine cells and sympathetic neurons
in the embryos, and the effects of propionate
were abolished underGpr41andGpr43defi-
ciencies. These observations underscore the con-
tribution of the SCFA-GPR41 and SCFA-GPR43
axes in prenatal development of the metabolic
and neural systems. Additionally, although off-
spring from GF-ICR and GF-C57BL/6J mothers
developed obesity, the severity of the obese
phenotype was slightly different between the
two groups. Such variation may be attributed
to gut microbial composition, which is subs-
tantially affected by host genetic backgrounds
( 20 , 22 ). Consistent with this notion, we
found that plasma SCFA levels differed be-
tween ICR and C57BL/6J mothers (Fig. 2B and
fig. S13C).
Whereas compositions of the gut microbiota
during adulthood were comparable in the SPF
and GF offspring, compositions in infancy were
different between the two groups. A similar
trend was also observed in infant HFi off-
spring compared with LFi offspring, raising
the possibility that altered infant microbiota
partially contributes to development of the
obesity phenotype of GF and LFi offspring.
Nevertheless, propionate administration to LFi
mothers during pregnancy ameliorated the
obesity-prone phenotype of the offspring with-
out affecting the infant microbiota (fig. S23).
Furthermore, propionate treatment also im-
proved hyperglycemia in GF embryos and lower
energy expenditure in LFi and GF offspring.
Although GPR41 deficiency also caused lower
energy expenditure, propionate administration
during pregnancy failed to prevent this pheno-
type. On the basis of these observations, we
reason that maternal microbiota–derived SCFAs,
particularly propionate, play a vital role in pre-
venting the development of metabolic disorder
in offspring. Meanwhile, it should be noted
that GF offspring exhibited the abnormality
in energy expenditure in both light and dark
cycles, whereas LFi offspring displayed the
abnormality only in the dark cycle. Therefore,
we cannot formally exclude the possibility that
the presence of intestinal microbiota and/or
theirproducts,otherthanSCFAs,maycontrib-
ute to the enhancement of energy expenditure
in the light cycle.Embryonic insulin regulation was impaired
in embryos from GF mothers, and insulin levels
were significantly elevated in the adult stage.
Excessive insulin levels in adult GF offspring
aremostlikelyattributedtometabolicadap-
tations in response to low birth weight and the
retardation of pancreaticbcell differentiation,
eventually increasing susceptibility to obesity
upon HFD feeding. This abnormality is rem-
iniscent of catch-up growth, whereby children
born small for gestational age (SGA) face a risk
ofexcessivebodyweightgainandmetabolic
syndrome later in life ( 18 , 41 ), as evidenced by
multiple birth cohort studies ( 42 – 44 ). Although
the etiology of SGA births remains to be cla-
rified, maternal factors, including malnutrition,
smoking, and alcohol consumption, have been
implicated ( 45 ). We further propose that de-
privation of SCFAs as a result of disturbances
in intestinal microbiota may be another caus-
ative factor for SGA births, leading to catch-up
growth and susceptibility to obesity. Our study
provides evidence for the crucial contribution
of the maternal gut environment during preg-
nancy to the metabolic programming of off-
spring to prevent metabolic syndrome. This
finding opens new research avenues into pre-
emptive therapies for metabolic disorders by
targeting the maternal gut microbiota.Materials and methods
Animal study
Allanimaldietsunlessotherwiseindicatedwere
provided by Research Diets. GF ICR (Sankyo
Labo Service) and C57BL/6J (Clea Japan) mice
were housed in vinyl isolators under a 12-hour
light-dark cycle and given regular chow (CMF,
Oriental Yeast). For fecal microbiota transplan-
tation, pregnant GF mice (day 18.5) received
oral gavage of fecal suspension in phosphate-
buffered saline (PBS) from strain-matched
pregnant SPF mice. In caesarean section exper-
iments, progesterone (2 mg per mouse) was
subcutaneously injected into pregnant GF or
SPF IQI mice (Clea, Japan) on days 17.5 and
18.5 to avoid preterm delivery. On day 19.5,
newborns were delivered by caesarean section
and reared by foster IQI mothers under con-
ventional conditions for 4 weeks. Then, 4-week-
old male mice were fed a HFD (D12492) for
12 weeks and housed individually (table S1). In
separate experiments, conventional ICR mice
(SLC, Inc.) were fed AIN-93G formula–based
LFi or 10% inulin–containing HFi diets during
pregnancy (table S2). To eliminate commensal
bacteria, HFi-fed ICR mice were treated with
1 mg/ml neomycin (Nacalai Tesque) in drink-
ing water during pregnancy. Offspring were
reared by CMF-fed conventional foster mothers
for 4 weeks; weaned mice were fed a HFD for
12 weeks and housed individually. In SCFA-
supplementation experiments, conventional
ICR mice were given an AIN-93G–based diet
supplemented with 5% (w/w) propionate forKimuraet al.,Science 367 , eaaw8429 (2020) 28 February 2020 9of12
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