microbial community in infants ( 19 )andthus
potentially influences the development of the
host metabolic system. However, 16Sribosomal
DNA (rDNA) amplicon sequencing showed
that the relative abundance of bacterial families
constituting the gut microbiota was similar in
offspring from SPF and GF ICR mothers during
adulthood, although GF offspring in infancy
showed significant decreases in Streptococcaceae
and Enterococcaceae compared with SPF off-
spring (fig. S3A). Principal coordinate analysis
based on weighted UniFrac distances con-
firmed that there were no differences between
the two groups during adulthood, but not during
infancy(fig.S3B).Toexcludethepossiblein-
fluence of the vaginal microbiota, newborns
from SPF and GF mothers were also delivered
by caesarean section. Consistent with vaginally
delivered offspring, caesarean GF offspring ex-
hibited severe obesity upon HFD feeding during
growth (fig. S4A). WAT and liver weight, plasma
metabolic parameters,and insulin levels were
also significantly higher in the caesarean GF
offspring at 16 weeks than in the caesarean SPF
offspring (fig. S4, B to D). Furthermore, the
former showed reduced energy expenditure
(fig. S4E). Additionally, compositions of the
gut microbiota were similar between caesarean-
delivered offspring from the SPF and GF mothers
during adulthood, whereas infant microbiota
may be affected slightly by the delivery modes
(fig. S5, A and B), consistent with the findings
of a recent study ( 19 ). Collectively, these data
show that the gut microbiota in adulthood is
not a primary factor in the obesity-prone phe-
notype of the GF offspring.
Interaction between the gut microbiota and
host genetics modulates the predisposition
to obesity, whereas environment factors such
as diet are indispensable for the regulation of
host-microbe interaction ( 20 – 22 ). Although gut
microbial compositions tended to be different
between offspring from ICR and C57BL/6J
mothers (fig. S6, A and B), they showed a sim-
ilar obesity phenotype (Fig. 1, A to F and figs.
Kimuraet al.,Science 367 , eaaw8429 (2020) 28 February 2020 2of12
Fig. 1. Offspring from GF mothers
exhibit severe obese phenotype when
fed a HFD.(A) Experimental scheme
(left). Body weight changes during the
HFD trial (middle) (n= 14 animals per
group) and tissue weights (right) (n=7to
9 tissues per group). epi, epididymal; peri,
perirenal; sub, subcutaneous. (B) Plasma
glucose, TGs, NEFAs, and total cholesterol
levels (n= 6 to 11 plasma samples per
group). mEq, milliequivalents. (C) Body
temperature (left) (n= 7 or 8 animals per
group) and heart rate (right) (n=7
animals per group). (D) Gut hormone PYY
(left) (n= 6 or 8 plasma samples per
group) and GLP-1 (right) (n= 6 plasma
samples per group) levels. (E) Energy
expenditure (n= 7 or 8 animals per
group). (F) Glucose tolerance test (left)
and insulin tolerance test (right) (n=8
animals per group). Male mice were
analyzed at 16 weeks of age. Student’s
ttest; **P< 0.01 and *P< 0.05. Data are
presented as means ± SEM. Offspring
from three or four litters per group were
used. SPF, conventional offspring derived
from ICR SPF mothers; GF, conventional
offspring derived from ICR GF mothers.
4 8 12 16
0
20
40
60
80
Body weight (g)
Weeks of age
SPF
GF
SPF GF
*
**
**
**
**
**
**
****
**
******
1cm
14
12
10
8
0
Energy expenditure
(kcal/kg/hr)
Time
*
Light Dark
**
**
SPF
GF
****
**
*
****
****
E
SPF
PYY (ng/mL)
2.5 *
1
0.5
0
1.5
2
GF SPF
GLP-1 (pg/mL)
18 *
6
0
12
GF
D
B
F
C
0
SPF
550
650
750
Heart rate (beats/min)
*
GF
0
SPF
37.5
38.5
39.5
Body temperature (
) *
GF
Plasma glucose (mg/dL )SPF GF
500 **
200
100
0
300
400
SPF GF
NEFAs (mEq /L)
0.8 **
0.2
0
0.4
0.6
Total cholesterol (mg/ dL )SPF GF
160 **
40
0
80
120
SPFGF
TGs (mg/dL)
80 **
20
0
40
60
0
Time (min) after glucose i.p.
0
200
400
500
Plasma glucose (%)
100
300
30 60 90 120 90
*
*
*
*
0
Time (min) after insulin i.p.
0
120
30 60 120
40
(^80) *
- SPF
GF
A
**
0
epi peri sub liver
1
2
3
4
5
Tissue weight (g)
WAT
SPF
GF
SPF GF
WAT (peri)
1cm
Pregnancy day 18.5
SPF Mother Germ-FreeMother
Conventional Foster MotherConventional Foster Mother
After weaning,
High-fat diet feeding
After weaning,
High-fat diet feeding
ConventionalFosterMotherConventionalFosterMo
RESEARCH | RESEARCH ARTICLE
- SPF