Kimuraet al.,Science 367 , eaaw8429 (2020) 28 February 2020 5of12
0
4
10
Relative mRNA expression
(Ins2
/18S
)
8
6
2
* **
**
Propionate
PA-1
Non-targeting siRNA
Gpr43 siRNA
-+- -
--+ -
Differentiation --- +
+
+
+
+
+
+
+
+
+
NS
NS
NS
D
E
F
**
0
0.5
2
2.5
Mo E18.5
Insulin (n g/mL)
1.5
1
0
1
2
3
4
Relative mRNA
expression(genes
/18S
)
0
1
2
** ** 3
Nkx6.1
** **
Ins2
0
2
4
8
Pancreatic insulin(ng/mg tissues)
6
* **
0
100
200
300
400
Insulin
+
fluorescence intensity / DAPI (%)
Differentiation
**
**
**
0
50
150
200
Mo E18.5
Plasma glucose (mg/dL)
100
B
A
C
0
1
2
3
5
Relative mRNA
expression(genes
/18S
)
** **
Pax4
4
0
1
2
3
4
** **
Pax6
0
0.5
1
1.5
** **
Gcg
0
1
2
3
5
Colonic total GLP-1(pmol/mg tissues)
4
** *
0
50
100
150
200
GLP -1+ cells
in organoids (%)
*
WT
Gpr43-/-
0
1
2
3
4
Relative mRNA
expression(Gcg
/18S
)
** ** ** NS
0
1
2
3
4
WT Gpr43-/-
Propionate
(+)(-) (+)(-)
Propionate
GLP-1
DAPI
Merge
50 μm
(-) Propionate
WT
(-)
Gpr43-/-
Propionate
(-)
Insulin
DAPI
Merge
Propionate PA-1 (-) Propionate PA-1
Differentiation
50 μm
Fig. 4. Propionate promotes enteroendocrine and pancreaticbcell differ-
entiation via embryonic GPR43.(A)Pax4,Pax6, andGcgmRNA expression
and GLP-1 protein levels in the colon (E18.5) (n= 7 or 8 tissues of embryos from
three litters per group). (BandC) Expression ofGcg(B) and distribution of GLP-1–
positive cells (C) after 24-hour treatment of embryonic organoids (E15.5) in the
presence (+) or absence (-) of Wnt with GPR43 ligands [1 and 10 mM (B); 100mM
propionate (C)]. GLP-1, green; DAPI, blue;n= 5 to 7 independent experiments from
two biological replicates per condition. (D) Expression ofNkx6.1andIns2mRNA
and insulin protein levels in the mouse pancreas (E18.5) (n= 7 or 8 tissues of
embryos from three litters per group). (E) Expression ofIns2(left) and localization
(middle; immunostaining) of insulin, and insulin positive cell count (right) in the
presence or absence of GPR43 ligands (10mM PA-1, 1 mM propionate) after
72-hour treatment (insulin, green; DAPI, blue;n= 4 to 10 independent experiments
from two or three biological replicates per condition). For induction ofbcell
differentiation, cells were cultured with betacellulin and activin A. (F) Plasma insulin
(left) and plasma glucose (right) levels in mothers and embryos of E18.5 (n=8plasma
samples andn= 8 plasma samples of embryos from eight litters per group). GF
and SPF mice with C57BL/6J background were analyzed [(A), (D), and (F)].
Student’sttest [(A), (D), and (F)] and Tukey–Kramer’s test [(B) and (E)]; **P<
0.01 and *P< 0.05. NS, not significant. All data are presented as means ± SEM.
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