was ameliorated by administration of propi-
onate during pregnancy (Fig. 3D). Heart rate
and body temperature after weaning, an index
of sympathetic nerve projection, exhibited a
similar trend, as did oxygen consumption (Fig.
3, E and F). Treatment with tyramine markedly
decreased oxygen consumption in offspring
from untreated or propionate- or antibiotic-
treated WT mothers, whereas these effects were
weakened in offspring from antibiotic-treated
WT mothers or untreatedGpr41−/−mothers
(Fig. 3F). On the basis of these observations, we
reasoned that propionate from the maternal
gut microbiota facilitates sympathetic nerve
development via GPR41. Deprivation of pro-
pionate resulted in sympathetic dysfunction,
including a reduction in body temperature
and heart rate fluctuations, as observed in the
GF offspring (Fig. 1C).
Embryonic insulin regulation via GPR43
GPR43 is expressed in adult enteroendocrine
Lcellsandpromotesthesecretionofguthor-
mones upon activation ( 28 ). BecauseGpr43
was also detected in the embryonic colon (fig.
S12C), we investigated the effect of GPR43 on
enteroendocrine cell differentiation at the em-
bryonic stage.Pax4andPax6were signifi-
cantly up-regulated in the colon ofGpr43−/−
C57BL/6J embryos in comparison with WT
embryos(Fig.4Aandfig.S15A).Suchchanges
were also observed in GF embryos with the
same background. By contrast,Gcgas well as
GLP-1 were down-regulated inGpr43−/−and
GF mice (Fig. 4A and fig. S15A). This result
suggests a retardation of enteroendocrine cell
differentiation inGpr43−/−and GF mice. To
directly assess the role of the SCFA-GPR43 axis
in enteroendocrine cell differentiation, we em-
ployed intestinal organoids, which recapitulate
cell differentiation in vivo by reducing canonical
Wnt signaling (fig. S15B). Propionate, which is
a more potent (EC 50 :~30mM) GPR43 ligand
than acetate or butyrate (EC 50 :~50or100mM,
respectively) ( 31 ), significantly promoted dif-
ferentiation of GLP-1+enteroendocrine cells
in intestinal organoids from WT embryos
(Fig. 4, B and C). In sharp contrast, this effect
was abrogated in organoids fromGpr43−/−
embryos (Fig. 4, B and C). Notably, the effect of
propionate on enteroendocrine cell differenti-
ation was not observed in organoids from
adult WT mice (fig. S15C), illustrating that
propionate-mediated GPR43 signaling is a
prerequisite for the development of enteroendo-
crine cells during the prenatal period.
Because GPR43 regulates insulin secretion
in pancreaticbcells ( 27 , 29 ) and this recep-
tor was expressed in the pancreas during the
perinatal-postnatal period (Fig. 2E), we sub-
sequently investigated the effect of GPR43 on
bcell differentiation during embryonic devel-
opment.Nkx6.1was significantly up-regulated
in the pancreas of embryos fromGpr43−/−and
GF mice compared with those from WT and
SPF mice, respectively, whereasIns2expres-
sion and insulin level were down-regulated
inGpr43−/−and GF mice (Fig. 4D and fig. S15D).
Moreover, during the induction of differen-
tiation of the rat pancreatic tumor cell line
AR42J intobcells, expression levels ofGpr43
mRNA, but notGpr41mRNA, were signifi-
cantly elevated in line with those ofIns2(fig.
S15E). Notably, propionate and the synthetic
GPR43 agonist phenylacetamide-1 (PA-1) pro-
moted differentiation into insulin+bcells;
however, this effect was compromised by RNA
interference–mediated knockdown ofGpr43
(Fig. 4E and fig. S15F). Thus, propionate-
mediated activation ofGPR43 facilitates dif-
ferentiation into pancreaticbcells as well as
GLP-1–positive enteroendocrine cells. GF ICR
as well as GF C57BL/6J embryos also demon-
strated abnormalities in these differentiation
and functional markers of sympathetic neuron,
enteroendocrine, and pancreaticbcells (fig. S15,
A, D, and G).
These findings raise the possibility that
SCFA-GPR43 signaling may play a vital role
in embryonic insulin secretion. Plasma insulin
levels inGpr43−/−embryos were markedly
lower than in WT embryos, although there
were no differences between WT andGpr43−/−
mothers (Fig. 4F). Likewise, reduced insulin
levels were also observed in GF embryos (fig.
S16, A and B). Correspondingly, plasma glucose
levels inGpr43−/−(Fig. 4F) and GF embryos
(fig. S16, A and B) were significantly higher
than those in their control groups. Given that
dysregulation of fetal glucose levels renders
offspring susceptible to metabolic syndromes,
such as obesity and type 2 diabetes ( 32 ), we
speculate that the lack of SCFA-GPR43 signal-
ing during the prenatal period causes meta-
bolic syndrome in adulthood, most likely by
compromising energy homeostasis in the em-
bryos. Such retardation of the differentiation
of the sympathetic nerve, intestinal tract, and
pancreas was also evident inGpr41−/−Gpr43−/−
double-mutant C57BL/6J mice (fig. S17, A to E).Dietary fiber intake during pregnancy
To provide further evidence of the importance
of SCFAs in the developmental origin of obe-
sity resistance, we performed a dietary inter-
vention study in which pregnant ICR mice
were fed a high-fiber (HFi) or low-fiber (LFi)
diet under conventional conditions, after which
the susceptibility of their offspring to obesity
was examined (Fig. 5A). Although the body
weight of postpartum offspring from HFi-fed
mothers (HFi offspring) was significantly higher
than that of offspring from LFi-fed mothers
(LFi offspring) (fig. S18A), HFi intake sup-
pressed the HFD-induced body weight gain
from 13 weeks of age onward, in accordance
with reduced subcutaneous WAT and liver
weights (Fig. 5A). However, the effect of a HFidiet was abrogated when antibiotics were ad-
ministered to pregnant mice to eradicate the
gut microbiota (Fig. 5A and fig. S18A), indicat-
ing that microbial fermentation of dietary fiber
contributes to obesity suppression. Plasma me-
tabolic parameters were also improved in HFi
offspringcomparedwithLFioffspring(Fig.5B
and fig. S18, B and C). Likewise, HFi offspring
were resistant to HFD-induced glucose in-
tolerance and insulin resistance (fig. S18D), in
association with improved energy expenditure
(fig. S18E). Furthermore, sympathetic dysfunc-
tion, such as reduction in body temperature
and heart rate fluctuations, in LFi offspring
was ameliorated in HFi offspring (Fig. 5C).
Compositions of the gut microbiota were com-
parable between LFi offspring and HFi offspring
during adulthood but were significantly dif-
ferent between the two groups during infancy
(fig. S19, A and B). Metabolome profiling of
maternal and embryonic plasma samples re-
vealed that 11 and 4 metabolites were signif-
icantly altered between the LFi- and HFi-fed
groups (fig. S20), with 4 metabolites commonly
increased in the HFi-fed mothers and their
embryos (Fig. 5D). Among these 4 metabolites,
SCFAs were the only common factor in the
SPF versus GF and LFi versus HFi comparisons.
SCFA levels were significantly higher in the
embryos of HFi-fed mice (HFi embryos) than
in those of LFi-fed mice (LFi embryos) (Fig. 5E).
We also observed that plasma insulin levels
were significantly higher in HFi embryos than
in LFi embryos (Fig. 5F), and thereby plasma
glucose levels were significantly decreased in
HFi embryos (Fig. 5F). Thus, SCFAs generated
by the maternal gut microbiota through the
fermentation of dietary fiber are provided to
embryos via maternal circulation, improving
fetal glucose homeostasis and imparting resist-
ance to obesity in the offspring.SCFA supplementation during pregnancy
Plasma propionate levels in HFi embryos were
likely sufficient to activate the GPR41 and/or
GPR43 receptors, considering that the deter-
mined values were superior to their EC 50 values
(Fig. 5E). Positron emission tomography (PET)
imaging showed that [^11 C]-labeled propionate
in the colonic lumen reached the embryos
via the maternal liver and bloodstream within
40 min after infusion (fig. S21, A to D). Thus, to
rigorously examine the role of propionate in
obesity resistance of the offspring, we fed preg-
nant ICR mice a LFi diet supplemented with
propionate (Fig. 6A). The intake of this diet
raised plasma levels of propionate in both
mothers and embryos (fig. S22A). Treatment
with propionate suppressed the HFD-induced
increases in body weight, perirenal or subcu-
taneous WAT mass, and liver weight of adult
offspring (Fig. 6A). Plasma metabolic param-
eters were also improved in the offspring of
propionate-treated mothers (Pro offspring)Kimuraet al.,Science 367 , eaaw8429 (2020) 28 February 2020 7of12
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