Science 28Feb2020

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

-+- -

--+ -

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---

Differentiation --- +

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+

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+

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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