the pregnancy period (table S3). Offspring
were reared by CMF-fed conventional foster
mothers for 4 weeks. Over the course of the
experiments, newborns [litter size aligned to
10 ± 2 (ICR) or 6 ± 2 (C57BL/6J) mice] from
the GF and SPF mothers were reared by strain-
matched foster mothers under conventional
conditions for 4 weeks. Then, more than three
groups of littermates from each mother were
analyzed in individual experiments.
Heart rates were determined in conscious
mice using a tail-cuff system (Softron and
Muromachi Kikai), and body temperature was
determined in conscious mice using Lifechip
and a pocket reader (Destron Fearing), as de-
scribed elsewhere ( 16 ).
C57BL/6J-background WT,Gpr41−/−,and
Gpr43−/−mice andGpr41−/−Gpr43−/−double-
mutant pregnant mice were given CMF.Gpr41−/−
andGpr43−/−mice were generated as described
previously ( 15 , 16 );Gpr41−/−Gpr43−/−double-
mutant mice were generated by the CRISPR-
Cas9 system (fig. S17, A and B). Two guide RNAs
were identified using the publicly available
optimized CRISPR design tool (http://crispr.
mit.edu). Transgene insertion was established
by homology-directed DNA repair recombina-
tion, and mutations were analyzed using geno-
typing primers. Pregnant WT andGpr41−/−
mice were treated with propionate (150 mM)
and antibiotics (1 mg/ml neomycin) in drink-
ing water. After birth, pups were analyzed on P1.
Plasma samples were obtained from em-
bryos (6 to 12 per litter) delivered by WT and
mutant mice at E18.5 and subjected to meta-
bolome analysis. Plasma glucose concentra-
tions were measured in individual embryos
at E18.5. The SCG, heart, colon, and pancreas
were collected from WT or mutant mice at
E16.5, E18.5, P1, P7, P14, P28, or P49.
All experimental procedures involving mice
were performed according to protocols approved
by the Institutional Animal Care and Use Com-
mittee of Keio University School of Medicine
[permission no. 09036-(12)]; the Committee
on the Ethics of Animal Experiments of the
Tokyo University of Agriculture and Technol-
ogy (permit no. 28–87); and the Animal Care
and Use Committee, Okayama University (per-
mission no. OKU-2018346). Mice were treated
with lethal anesthesia with somnopentyl, and all
efforts were made to minimize animal suffering.
Histology
Livers, pancreas, and whole embryos were
embedded in OCT compound (Sakura Finetek),
and adipose tissues were embedded in paraffin.
These samples were sectioned into 7-mm-thick
slices that were stained with oil red O (Sigma-
Aldrich) or hematoxylin and eosin for micro-
scopic examination. Sections and cells were
fixed in 4% paraformaldehyde and immuno-
stained using primary antibodies raised against
tyrosine hydroxylase (TH) (Millipore) to detect
sympathetic neurons, nestin (BD Biosciences)
to detect undifferentiated neural cells, GLP-
1 (Abcam) to identify enteroendocrine cells,
and insulin (Sigma-Aldrich) to identify dif-
ferentiated pancreatic exocrine cells. This was
followed by signal development using second-
ary antibodies conjugated with a fluorescent
marker. Nuclei were counterstained with 4′,6-
diamidino-2-phenylindole (DAPI) (Roche).In situ hybridization
Mouseembryos(transverseandsagittalsec-
tions) and adult tissues were frozen in OCT
compound, after which 18-mm sections were
cutusingacryostatandstoredat–80°C until
hybridization. Mouse antisenseGpr41and
Gpr43RNA probes were transcribed using T7
RNA polymerase and uridine 5′-a-[^35 S]-thio-
triphosphate (Perkin Elmer). Tissue sections
were examined as described previously ( 15 , 16 )
and counterstained with hematoxylin and eosin
and TH antibodies (Millipore) to visualize SCGs.Biochemical analyses
Plasma samples were obtained from mice
fasted for 5 hours. Plasma glucose levels were
determined using OneTouch Ultra (LifeScan,
Inc.). Determinations of plasma TGs, free fatty
acids, and total cholesterol levels were per-
formed using commercial kits (TG, LabAssay
Triglyceride; free fatty acids, LabAssay NEFA;
and total cholesterol, LabAssay Cholesterol;
Wako Chemicals). Levels of plasma PYY [Mouse/
Rat PYY enzyme-linked immunosorbent assay
(ELISA) kit; Wako Chemicals], GLP-1 [GLP-
1 (Active) ELISA kit; Shibayagi], and insulin
[Insulin ELISA kit (RTU); Shibayagi] were
determined using ELISA kits, following the
respective manufacturer’s instructions. Levels
of total GLP-1 in colonic lysates were determined
using a commercial kit (Multi Species GLP-
1 Total ELISA; Millipore). For GLP-1 determi-
nations, plasma and tissue samples were treated
with dipeptidyl peptidase IV inhibitor (Merck
Millipore) to prevent the degradation of ac-
tive GLP-1.Glucose- and insulin-tolerance tests
For the glucose-tolerance test, mice fasted for
24 hours were intraperitoneally (i.p.) administered
1.5 mg of glucose (Wako Chemicals) per gram
of body weight. For the insulin-tolerance test,
mice fasted for 3 hours were administered
human insulin (3 mU/g, i.p.; Sigma-Aldrich).
Plasma glucose concentration was monitored
before injection and 15, 30, 60, 90, and 120 min
after injection using OneTouch Ultra.PET and CT imaging
Ten-week-old female Sprague-Dawley rats
(Charles River) were housed under a 12-hour
light-dark cycle and provided an AIN-93G diet
during pregnancy. For PET imaging, rats on
day16.5and17.5ofpregnancywereused.Syn-thesis of [^11 C]-propionate was performed by
modifying [^11 C]-acetate preparation as pre-
viously described ( 46 ). Briefly, [^11 C]-CO 2 was
bubbled into a mixture of 0.1 M ethyl mag-
nesium bromide and tetrahydrofuran (Wako
Chemicals) at−10°C, and then the mixture was
purified using solid-phase extraction columns
(OnGuard II Ag and OnGuard II H, DIONEX).
The resultant solution was converted to sodium
salt with NaHCO 3 aq (Maylon). [^11 C]-propionate
was confirmed by high-performance liquid
chromatography [Partisil-10-SAX (4.6 mm by
250mm)and20mMphosphatebuffer(pH4)
containing KH 2 PO 3 andphosphoricacid(for
adjusting pH) as an eluent at 0.7 ml/min and
at 40°C] at 96.5% radiochemical purity and
5.77% radiochemical yield. [^11 C]-Propionate
was diluted with physiological saline to 20
megabecquerels (250 to 300ml per rat) and
administered to the colonic lumen via the anus
using a feeding needle for rats under isoflurane
anesthesia. PET and computed tomography
(CT) scans were performed using ClairvivoPET
(Shimadzu Co., Ltd.) and Aquilion TSX-01A
(Tokyo Medical Systems) instruments, respec-
tively. After PET and CT imaging, rats were
laparotomized to collect blood from the in-
ferior vena cava under isoflurane anesthesia.
Subsequently, portions of the liver, kidney,
spleen, left hind paw muscle, and fetus with
placenta were dissected. Each tissue was washed
twice with physiological saline. After sufficiently
wiping off physiological saline, the radioactivity
of each organ was measured using ag-counter.
The distribution of [^11 C]-propionate in each
organ and in embryos was measured using an
AccuFLEXg7001 instrument (ARC-7001; Hitachi
Aloka Medical). Thereafter, the weight of each
tissue was measured. The obtained radio-
activity value was calculated using the follow-
ing equationValue of attenuation correction =
radioactivity value/(2{(T^1 −T2)/20.4})whereT1,T2, and 20.4 (in minutes) indicate
the time when each tissue was measured,
[^11 C] propionic acid administration time, and(^11) C half-life, respectively. After attenuation cor-
rection, each radioactivity value was divided
by the corresponding weight to calculate the
radioactivity value per fixed weight. The phar-
macokinetics of the tracer was evaluated as
counts per minute per gram of each tissue rel-
ative to that in the liver taken from the same
pregnant rat.
SCFA determinations
Plasma SCFAs were determined as previously
described ( 47 ). The SCFA-containing ether
layers were collected and pooled for gas
chromatography–mass spectrometry (GC-MS)
analysis using a GCMS-QP2010 Ultra instru-
ment (Shimadzu). The concentration of each
Kimuraet al.,Science 367 , eaaw8429 (2020) 28 February 2020 10 of 12
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