SCFA was determined using external standard
calibration over an appropriate concentra-
tion range.
Commensal bacteria composition
Fecal, cecal, placental, colonic, and embryonic
DNA were extracted from frozen samples
using the FastDNA SPIN Kit for Feces (MP
Biomedicals) or Gentra Puregene Mouse Tail
Kit (Qiagen) according to the manufacturer’s
instructions. Bacteria (Bacteroides vulgatus
JCM5826T) were provided by the Japan Col-
lection of Microorganisms of RIKEN BRC and
used as standards specifically for the DNA-based
determination of bacterial counts. Bacterial
DNA was isolated using the MonoFas Bacte-
rial Genomic Kit IV (GLC Science) according
to manufacturer’s instructions. Quantitative
polymerase chain reaction (PCR) analysis was
performed using SYBR Premix Ex Taq II
(TaKaRa Bio) and a StepOne Real-Time PCR
System (Applied Biosystems). Standard curves
for quantification consisted of 10-fold serial
dilutions in the range of 10^8 to 10^0 copies of
the target 16Sribosomal RNA (rRNA) gene.
Universal bacterial primer sequences were
as follows: 5′-CRAACAGGATTAGAACCCT-3′
(forward) and 5′-GGTAAGGTTCCTCGCGTAT-
3 ′(reverse) ( 47 ).
For bacterial culture, amniotic fluids and
cecal contents from E16.5 pregnant ICR mice
were cultured in MRS (BD Biosciences) and
BL medium (Eiken Chemical) at 37°C for
24 hours under anaerobic conditions. The sam-
ples were then suspended at 0.1 mg/ml in ster-
ile PBS and diluted in 10-fold series.
For 16SrDNA amplicon sequencing, se-
quencing of the 16SrRNA variable regions 3
and 4 was performed as described previously
( 47 ). The libraries were sequenced using the
MiSeq system (Illumina) with 2 × 300 base
pair protocols. Microbial diversity and com-
position analyses were performed using QIIME
with the Greengenes reference database clustered
at 97% identity.
Indirect calorimetry
Energy expenditure was calculated as the pro-
duct of the calorific value of oxygen (3.815 +
1.232 × the respiratory exchange ratio) and
oxygen consumption (VO 2 ) as previously de-
scribed ( 15 ). Tyramine (100 mg/kg), a catechol-
amine releasing agent, was administrated at a
volume per i.p. injection as described ( 16 ).
RNA isolation and quantitative reverse
transcription PCR
Total RNA was extracted using the RNeasy
Mini Kit (Qiagen) and ISOGEN (Nippon Gene).
cDNA was reverse transcribed using isolated
RNA samples as templates and Moloney
murine leukemia virus reverse transcriptase
(Invitrogen). Expression analyses were per-
formed using SYBR Premix Ex Taq II and the
StepOnePlus Real-Time PCR System. Real-time
PCR cycling conditions were as follows: 95°C
for 30 s, followed by 40 cycles of 95°C for 5 s,
58°C for 30 s, and 72°C for 1 min. In addition,
dissociation was examined at 95°C for 15 s, fol-
lowed by 1 cycle of 60°C for 1 min and 95°C
for 15 s. The 18SrRNA gene was used as an
internal control. Each sample was tested in
duplicate for the average Ct value. Relative
mRNA expression was calculated after normal-
ization to the 18SrRNA reference gene using
the 2-DDCtmethod. Primer sequences are listed
in tables S4 and S5.Sympathetic neural cell culture
SCGs were dissected from P1 mice, trypsinized
in 0.05% trypsin in Hanks’balanced salt solu-
tion for 20 min at 37°C, and dissociated by
trituration. Dissociated cultures were plated
onto dishes coated with poly-L-lysine (20mg/ml;
Sigma-Aldrich) in DF medium (Gibco; Thermo
Fisher Scientific, Waltham, MA) containing
1% penicillin-streptomycin solution (Gibco).
Cells were cultured in conditioned medium
containing nerve growth factor (10 ng/ml;
Upstate Biotech) and 10% fetal bovine serum
(FBS) as described elsewhere ( 16 ). Neuronal
differentiation was quantified by counting TH-
positive cells; cells with outgrowths longer than
the cell body diameter were scored as positive
for neurites. ImageJ (NIH, Bethesda, MD) was
used to determine neurite outgrowth ( 48 ).Western blotting and cAMP determination
Hearts were homogenized in 0.1 M sodium
phosphate (pH 7.4) and centrifuged at 10,000g
for 20 min at 4°C. Supernatants were analyzed
by Western blotting as previously described
( 15 ). Proteins were detected by Western blot-
ting using anti-TH (Millipore) and anti-a-actin
antibodies (Sigma-Aldrich) for normalization.
For cyclic adenosine monophosphate (cAMP)
determinations, cellswere lysed in 0.1 N HCl.
After acetylation, cAMP levels were determined
in duplicate using enzyme immunoassay kits
(Cayman) as previously described ( 16 ).Pancreatic cell line culture
AR42J cells (rat pancreatic exocrine cells) were
purchased from the American Type Culture
Collection (ATCC) and cultured in F-12K (Thermo
Fisher Scientific) containing 20% FBS and
maintained at 37°C under 5% CO 2. For cell
differentiation, the cells were plated in 24-well
plates (1 × 10^5 cells per well) and cultured with
betacellulin (2 nM; Wako Chemicals) and ac-
tivinA(1nM;WakoChemicals),inthepresence
or absence of propionate (1 mM; Sigma-Aldrich)
or PA-1 (10mM) for 72 hours. Then, total RNA
was isolated using ISOGEN. For small inter-
fering RNA (siRNA) knockdown experiments,
AR42J cells were transfected with 30 nM siRNA
(Gpr43; target sequence: GGCCAUUGCACCA-
UCGUCA; GE Healthcare) using Lipofectamine2000 transfection reagent (Invitrogen) according
to the manufacturers’instructions.Organoid culture
Colonic crypts were isolated from 7-week-old
mice or embryos on day 15.5 and cultured on
Matrigel, as previously described ( 49 ). Culture
medium contained advanced DMEM/F12 (Life
Technologies) supplemented with gentamicin/
amphotericin B solution (Life Technologies),
10 mM HEPES (Nacalai Tesque), 1% (v/v) N2
(Life Technologies), 1% (v/v) B27 (Life Tech-
nologies), 1mMN-acetylcysteine (Sigma-Aldrich),
2mML-alanyl-L-glutamine (Nacalai Tesque),
500 nM A-83-01 (Wako Chemicals), 10 nM
[Leu^15 ]-gastrin 1 (Sigma-Aldrich), 1 mM nico-
tinamide (Wako Chemicals), 10mM SB202190
(Wako Chemicals), 2.5mMCHIR99021(StemRD),
and 2.5mM thiazovivin (StemRD) supplemented
with the growth factors EGF (epidermal growth
factor; 50 ng/ml; Peprotech), noggin (100 ng/ml;
Sigma-Aldrich), and R-spondin (1mg/ml; Sigma-
Aldrich). The medium was incubated in the
presence or absence of Wnt3a for 24 hours.
Organoid cultures were then treated with pro-
pionate(1or10mM)for24hours,andtotal
RNA was isolated using the RNeasy Mini Kit.Metabolite analysis
Liquid chromatography–tandem MS (LC-MS/
MS)–based lipidomics analysis was performed
as described elsewhere ( 50 ). Briefly, samples
were subjected to solid-phase extraction on a
Sep-Pak C18 cartridge (Waters) with deuterium-
labeled internal standards (arachidonic acid-d 8 ,
leukotriene B4-d 4 , 15-hydroxyeicosatetraenoic
acid-d 8 , and prostaglandin E2-d 4 ). Lipidomics
analyses were performed using a Waters UPLC
system with a linear ion-trap quadrupole mass
spectrometer (QTRAP 5500; AB SCIEX) equip-
ped with an Acquity UPLC BEH C18 column
(1.0 mm by 150 mm by 1.7mm; Waters). MS/MS
analyses were conducted in negative-ion mode,
and lipophilic metabolites were identified and
quantified by multiple-reaction monitoring.
Hydrophilic metabolites from biological samples
were extracted and derivatized as described
previously ( 51 ). GC-MS analysis was per-
formed using a GCMS-QP2010 Ultra instru-
ment (Shimadzu) with a fused silica capillary
column (CP-SIL 8 CB low bleed/MS; 0.25 mm
by 30 m by 0.25mm; Agilent Technologies) as
described previously ( 51 ). Acquired data were
exported in the CSV file format and analyzed
using in-house analytical software (AI output).Statistical analysis
All values are presented as means ± SEM.
Statistical significance of differences between
groups was determined using two-tailed un-
paired Student’sttest (two groups) or two-
tailed one-way analysis of variance followed by
Tukey-Kramer’s post hoc test and Dunnett’s
post hoc test (three or more groups).PvaluesKimuraet al.,Science 367 , eaaw8429 (2020) 28 February 2020 11 of 12
RESEARCH | RESEARCH ARTICLE