New K etone body receptor GPR43 regulates lipid metabolism under … · 2019. 10. 31. · the cells were cultured in DMEM containing 10 µg/mL blasticidin S (Funakoshi), 100 µg/mL

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Supplementary Information for Ketone body receptor GPR43 regulates lipid metabolism under ketogenic conditions Junki Miyamoto1, 2, #, Ryuji Ohue-Kitano1, 2, 3, #, Hiromi Mukouyama1, Akari Nishida1, Keita Watanabe1, Miki Igarashi1, Junichiro Irie2, 4, Gozoh Tsujimoto5, Noriko Satoh-Asahara3, Hiroshi Itoh2, 4, Ikuo Kimura1, 2, *

1Department of Applied Biological Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183-8509, Japan, 2AMED-CREST, Japan Agency for Medical Research and Development, Chiyoda-ku, Tokyo 100-0004, Japan, 3Department of Endocrinology, Metabolism, and Hypertension, Clinical Research Institute, National Hospital Organization Kyoto Medical Center, Kyoto 612-8555, Japan, 4Department of Endocrinology, Metabolism and Nephrology, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan, 5Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan. Corresponding author: Ikuo Kimura Email: [email protected]. This PDF file includes:

Supplementary text (Materials and Methods) Figures S1 to S9 Tables S1 to S2 SI References

www.pnas.org/cgi/doi/10.1073/pnas.1912573116

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Supplementary Information Text

Materials and Methods

Cell culture. The generation of GPR43-expressing HEK293 cells was described previously (1) and

the cells were cultured in DMEM containing 10 µg/mL blasticidin S (Funakoshi), 100 µg/mL

hygromycin B (Thermo Fisher Scientific), and 10% FBS. The cells were incubated at 37 °C in an

atmosphere of 5% CO2. The cells were further cultured under various conditions. For cAMP

determination, GPR43-expressing HEK293 cells were plated onto 24-well plates (1 × 105 cells/well)

and incubated for 24 hr. Each well was then treated with or without doxycycline (10 µg/mL) for 24 hr.

The cells were treated with forskolin (2 µM; Sigma; adenylate cyclase activator) and IBMX (500 µM;

Sigma; 3-Isobutyl 1-methylxanthine; phosphodiesterase inhibitor) to raise cAMP levels. Then, the

cells were stimulated with each monocarboxylic acid (sodium monocarboxylate, lithium α-

hydroxubutyrate, and lithium acetoacetate) for 10 min. cAMP concentration was determined by cAMP

EIA kit (Cayman Chemical) according to the manufacturer’s protocol. For [Ca2+]i response analysis,

cells were plated onto 96-well black plates (3 × 104 cells/well) and incubated for 24 hr. Each well was

treated with or without doxycycline (10 µg/mL) for 24 hr. After treatment, cells were further incubated

in HBSS (pH 7.4) containing calcium assay kit component A (Molecular Devices) for 1 hr at 37 °C.

Each of monocarboxylic acids used in the assay was dissolved in HBSS and prepared in another set of

96-well plates. These plates were set in the FlexStation 3 (Multi-Mode Microplate Reader), and the

mobilization of [Ca2+]i was monitored. Lithium acetoacetate and DL-Sodium β-hydroxybutyrate

(Sigma) were used as ketone bodies.

Animal study. C57BL/6J wild-type, C57BL/6J-background Gpr43−/−, adipose Gpr43TG, and

Gpr41−/− mice were housed under a 12-hr light–dark cycle and given regular chow (CE-2, CLEA). All

experimental procedures involving mice were performed according to protocols approved by the

Committee on the Ethics of Animal Experiments of the Tokyo University of Agriculture and

Technology (Permit Number: 28–87). The generation of Gpr43−/−, adipose Gpr43TG, and Gpr41−/−

mice was described previously (1, 2). To examine lipid metabolism by ketone bodies, each mouse (ca.

20–23 g, no significance) was kept under ketogenic condition. The mice were then sacrificed and

sampled for plasma, muscle, liver, intestine, and white adipose tissues. In some experiments, for

antibiotic treatment, mice were treated with ampicillin (Nacalai Tesque; 0.5 mg/ml), neomycin

(Nacalai Tesque; 0.5 mg/mL), metronidazole (Wako; 0.5 mg/mL), and vancomycin (Sigma; 0.2

mg/ml) in drinking water for 1 week before fasting. For the measurement of plasma TG and NEFA

concentrations, and LPL activity, the mice were administrated with lithium acetoacetate (Sigma; 500

mg/kg body weight), lithium α-hydroxybutyrate (TCI; 250 or 500 mg/kg body weight), or PBS by i.p.

injection at 20 min as previously described (2). For intermittent fasting, mice were fed with alternating

3

24 hr periods (15 cycles for 1 month) of free access to diet followed by 24 hr fasting (3). Body weight

change and food intake were monitored every time, and blood samples were collected on day 7, 14, 21

from tail, and last days. For eucaloric ketogenic diet trial, C57BL/6J and Gpr43−/− mice were fed

ketogenic diets (AIN-76A-Modified, Bio-Serv; SI Appendix, Table. S1) for 6 weeks (4). Body weight

change was monitored every week, and blood samples and tissues were collected.

Immunoblotting. GPR43-expressing HEK293 cells were seeded at a density of 1 × 105 cells/well in

24-well plates. After 24 hr, the cells were cultured in DMEM containing 10 µg/mL doxycycline and

10% FBS for 24 hr. Cells were then incubated in serum-free DMEM containing doxycycline

(10 µg/mL) for 24 hr. The cells were further cultured for 10 min in the presence of acetoacetate of

various concentrations, or propionate (1 mM). Cells and murine tissues were then lysed in TNE buffer

containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 50 mM NaF,

2 mM Na3VO4, 10 µg/mL aprotinin, and 1% Phosphatase inhibitor cocktail (Nacalai Tesque). Proteins

in the cell and murine tissue lysate were resolved by SDS gel electrophoresis and blotted onto a

nitrocellulose membrane. ERK1/2, its phosphorylated forms, LPL, and β-actin were detected by

immunoblotting using antibodies. The primary antibodies were used as follows: rabbit antibodies

against ERK1/2 (1:1000; Cell Signaling Technology), phosphorylated ERK1/2 (Thr202/Tyr204)

(1:1000; Cell Signaling Technology), goat antibody against LPL (1:1000; R&D Systems), mouse

antibody against β-actin (1:5000; Wako). The secondary antibody was a horseradish peroxidase-

conjugated donkey anti-rabbit, sheep anti-mouse antibodies (1:10000; GE Healthcare), and rabbit anti-

goat antibody (1:10000; Thermo Fisher Scientific). Immunoreactive bands were visualized using an

enhanced chemiluminescence detection system using ImageQuant LAS 500 (GE Healthcare). Image J

(National Institutes of Health) was used to quantify the integrated density of each band.

Ketone bodies measurement. Fresh plasma (20 µL) and tissues (50 mg) were immediately added

with cold acetonitrile (60 µL; plasma and 500 µL; tissues), and then tissue samples were homogenized.

The samples were vortex-mixed for 30 sec, and were centrifuged at 10,000 g for 5 min, and the

supernatant was subjected to ketone analysis. The ketone bodies were analyzed using an Acquity

UPLC system coupled to a Waters Xevo TQD mass spectrometry (Waters). The ketone bodies were

separated on an ACQUITY UPLC BEH C18 column (2.1 × 150 mm, 1.7 µm, Waters) using an

acetonitrile gradient in water containing 0.1% formic acid (from 20% to 25% acetonitrile in 3 min).

The flow rate was 0.15 ml/min, and the column temperature was maintained at 25 °C. MS detection

was in the negative ionization mode, and the source capillary voltage was set to 3000 V. The

desolvation and source temperatures were set at 500 °C and 150 °C, respectively. Individually

optimized multiple reaction monitoring (MRM) parameters were determined for target compounds

using standards. The following MRM transitions were monitored to quantify the ketone bodies: m/z

4

103.1→59.1 for β-hydroxybutyrate, m/z 101.1→101.1 for acetoacetate. The absolute levels of ketone

bodies were quantified using external standards.

SCFAs measurement. SCFAs in cecum and plasma were determined following a modified protocol

as previously described (5, 6). In briefly, plasma and cecal contents were immediately added with 5-

sulfosalicylic acid, followed by the 1 min vortex-mixing. The samples were centrifuged at 15,000 g

for 15 min, and the supernatant was collected. The supernatant was added with 2-ethylbutyric acid (as

an internal control), hydrochloric acid, and diethyl ether, followed by the 1 min vortex-mixing. The

samples were centrifuged at 3,000 g for 5 min, and the SCFA-containing ether layers were collected

and pooled for GC-MS analysis using a GCMS-QP2010 Ultra (Shimadzu). The VF-WAXms (30 m ×

0.25 mm internal diameter × 1 µm; Agilent technologies) was used for chromatographic separation.

Helium (0.92 mL/min) was used as the carrier gas. The mass spectrometer was set to scan mode from

m/z 40–130 and in selected ion monitoring mode at m/z of 60 (retention time; 9.6) for acetate, m/z 74

(retention time; 10.7) for propionate, m/z 60 (retention time; 12.5) for n-butyrate, and m/z 88

(retention time; 13.6) for 2-ethylbutyric acid. The concentration of SCFAs in each sample was

determined using an external standard calibration over a specified concentration range.

Biochemical analyses. The blood glucose concentrations were measured using a One Touch Ultra

(LifeScan). The plasma triglyceride (LabAssay™ Triglyceride, Wako), NEFAs (LabAssay™ NEFA,

Wako), GLP-1 [GLP-1 (Active) ELISA KIT, Shibayagi], PYY (Mouse/Rat PYY ELISA Kit, Wako),

insulin [Insulin ELISA KIT (RTU), Shibayagi], total cholesterol (LabAssay™ Cholesterol, Wako),

active ghrelin (Active Ghrelin ELISA kit, SCETI KK, Tokyo, Japan) concentration was measured in

according with the manufacturer’s instructions. For plasma GLP-1 measurement, plasma sample was

treated with dipeptidyl peptidase IV inhibitor (Merck Millipore) to prevent the degradation of active

GLP-1. For plasma ghrelin measurement, plasma sample was treated with aprotinin (500 KIU/mL,

Sigma), EDTA (1.25 mg/mL, Nacalai Tesque), and HCl (0.1 M, Wako). The plasma and murine tissue

lysates were analyzed for LPL activity by using a LPL assay kit (Roar Biomedicals).

Indirect calorimetry. The oxygen consumption (VO2) and RER were determined with a CO2/O2

metabolic measuring system (Model MK-5000RQ6/04AD, Muromachi Kikai Co.) at 24 °C as

previously described (1). VO2 was expressed as the volume of O2 consumed per kilogram of lean body

mass weight per minute. The energy expenditure was calculated as the product of the calorific value of

oxygen (3.815+1.232 × RER) and VO2.

RNA isolation and real-time quantitative RT-PCR. Total RNA was extracted using the RNeasy

Mini Kit (Qiagen) and RNAiso Plus (TAKARA). Complementary DNA was transcribed using RNA

5

as templates with Moloney murine leukaemia virus reverse transcriptase (Invitrogen). Quantitative

reverse transcriptase PCR (qRT-PCR) analysis was performed with SYBR Premix Ex Taq II

(TAKARA) using the StepOneTM real time PCR system (Applied Biosystems). Primer sequences were

shown in SI Appendix, Table. S2.

Adipocyte culture. Murine embryonic fibroblasts (MEFs) was cultured at 37 °C in α-MEM (alpha

Modified Eagle Minimum Essential Medium, Invitrogen) containing 1% penicillin–streptomycin

solution (Gibco) with 10% FBS. Two days after confluence, the medium was replaced with α-MEM

containing 10% FBS and inducers [0.25 µM dexamethasone (Wako), 10 µg/ml insulin (Sigma), and

0.5 mM 3-isobutyl-1-methylxanthine] as well as pioglitazone (10 µM, Sigma) for 2 days. The medium

was then changed to DMEM supplemented with 10% FBS, 10 µg/ml insulin and 10 µM pioglitazone,

and then incubated for 2 days. After 5 days, the differentiated adipocytes were used for experiments

(1).

Gut microbiota composition in cecum. Cecal DNA was extracted from frozen samples using the

FastDNA® SPIN Kit for Feces (MP Biomedicals) by following the manufacturer’s instructions.

Bacteria (Bacteroides vulgatus JCM5826T) was provided from Japan Collection of Microorganisms

of RIKEN BRC and used as standards specifically for the DNA-based determination of cecal bacterial

counts. Bacterial DNA was isolated using MonoFas Bacterial Genomic Kit IV (GL sciences)

following the manufacturer’s instructions. Quantitative PCR analysis was performed with using

SYBR Premix Ex Taq II (TAKARA) and StepOneTM real time PCR system (Applied Biosystems).

Standard curves for quantification consisted in ten-fold serial dilutions in the range of 108 to 100

copies of target 16S rRNA genes. Bacterial primer sequences are as follows; Universal, 5’-

CRAACAGGATTAGAACCCT-3’ (forward) and 5’-GGTAAGGTTCCTCGCGTAT-3’ (reverse) (1).

Partial 16S rRNA gene sequences were amplified targeting the hypervariable regions v4 using primers

515F (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA-

3’) and 806R (5’-

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3’) (7).

Amplicons generated from each sample were subsequently purified using AMPure XP (Beckman

Coulter). The 16S rRNA sequence data generated by the MiSeq sequencer (Illumina) using the MiSeq

Reagent kit v3 (600 cycles) were processed by the quantitative insights into microbial ecology

(QIIME 1.9.1) pipeline. Taxonomical assignments of operational taxonomic units (OTUs) were

performed using QIIME in accordance with the SILVA database (http://www.arb-silva.de) at the 97%

confidence level. Diversity analyses were used QIIME script core_diversity_analyses.py.

6

Statistical analysis. All values are presented as mean ± SEM. Differences between groups were

examined for statistical significance using two-tailed unpaired Student’s t-test (two groups) and two-

tailed one-way analysis of variance, followed by Tukey-Kramer’s post-hoc test and Dunnett’s post-

hoc test (≥ three groups).

7

Fig. S1. Acetoacetate did not induce nonspecific GPR43 responses in non-

doxycycline-treated HEK293 cells. (A) The mRNA expression of Gpr43 gene in Flp-In GPR43 T-Rex HEK293 cells treated with or without doxycycline (n = 4). ** P < 0.01 (Student’s t-test). (B, C)

cAMP levels (n = 4–5, B) and ERK1/2 phosphorylation (n = 6–7, C) in response to acetoacetate and

SCFAs (acetate and propionate) in a dose-dependent manner in Flp-In GPR43 T-Rex HEK293 cells

treated without doxycycline. The intracellular cAMP levels were determined using a cAMP assay kit,

and each data was presented as relative to the forskolin-induced cAMP levels. Dox; doxycycline. All

data are presented as means ± SEM.

A B

C

( - ) 0.1 0.3 1 3 10 300.5 propionate0

0.5

1

1.5

acetoacetate conc. (mM)

ER

K p

hosp

hory

latio

n(fo

ld s

timul

atio

n)

p-ERKERK

Supplementary Figure 1

( - ) 0.1 0.3 1 3 100.50

50

100

150

ligand conc. (mM)

Rel

ativ

e cA

MP

prod

uctio

n (%

)

forskolin

□ acetoacetate□ acetate

( - ) ( + )0

0.51

1.52

2.5m

RN

A e

xpre

ssio

n(Gpr43

/ 18S

)

× 10-3

Dox.

**

8

Fig. S2. Metabolic parameters in wild-type and Gpr43−/− mice under fasting

conditions. (A) Mice were fasted, and measured for ketone bodies in murine tissues (n = 6–8). (B) The same weight-matching was determined between C57BL/6J and Gpr43−/− mice (e.g. 20–23 g). (C)

The raw data of non-fasting mice. (D) Lean body mass at 0 and 48 hrs (n = 8). ** P < 0.01 (Tukey-

Kramer test). NS; not significant. All data are presented as means ± SEM.

0 4805

Lean

bod

y m

ass

(g)

fasting time (hrs)

10152025

■□WT■□ Gpr43-/-

** NS

**

A

B

0 24 48fasting time (hrs)

0

0.4

0.8

1.2K

eton

e bo

dies

(mm

ol/m

g)in

WAT

acetoacetate


0

2

3

4β-hydroxybutyrate

1


0

0.4

0.8

1.2

Ket

one

bodi

es (m

mol

/mg)

in m

uscl

e

acetoacetate


0

2

3

4β-hydroxybutyrate

1


00.5

1.5

2.5

Ket

one

bodi

es (m

mol

/mg)

in li

ver

acetoacetate

1

2


0

4

6

8β-hydroxybutyrate

2


0

0.3

0.6

0.9

Ket

one

bodi

es (m

mol

/mg)

in in

test

ine

acetoacetate


00.5

1.5

2.5

1

2

β-hydroxybutyrate

used exclusion used exclusion

Gpr43-/-WT

0

16

18

20

22

24

26

28

30

Bod

y w

eigh

t (g)

at 7

wee

ks o

f age

C

D


Absolute baseline dataBody weight Blood glucose NEFAs TGs Total cholesterol LPL activity

(g) (mg/dL) (mEq/L) (mg/dL) (mg/dL) (μmol/L/min)Wild-type 21.38 ± 0.307 159.91 ± 3.132 0.223 ± 0.031 81.5 ± 12.51 51.95 ± 5.452 0.197 ± 0.006Gpr43-/- 21.71± 0.226 174.11 ± 6.447 0.233 ± 0.018 80.97 ± 8.99 58.85 ± 7.49 0.181 ± 0.004

9

Fig. S3. The expression of metabolic related factors under fasting condition. (A, B) Mice were fasted and measured plasma insulin (A), and active ghrelin (B) at 0 and 48 hrs (n = 8–10).

(C, D) Effects of fasting on the mRNA expression of Angptl4, Lpl, Gpr43 gene and LPL protein in

muscle (C) and liver (D) of wild-type and Gpr43−/− mice (n = 8). (E) The mRNA expression of Gpr41

gene in WAT, muscle, liver, and ileum of wild-type and Gpr43-/- mice in fasted condition (n = 8). (F)

Effects of acetoacetate-Li (AcAc-Li, 500 mg/kg i.p.), and α-hydroxybutyrate-Li (αHB-Li, 500 mg/kg

i.p.) on relative LPL activity in non-treated mice (n = 8). (G) The concentration of ketone bodies in

0 480

400

800

1200

Plas

ma

insu

lin (p

g/m

L)

fasting time (hrs)

D

E

0 48 0 480

1.5

fasting time (hrs)

1

Rel

ativ

e pr

otei

n ex

pres

sion

in m

uscl

e (L

PL /

β-ac

tin)

NS

0.5

NS

0 48 0 480

2

fasting time (hrs)

1.5

Rel

ativ

e pr

otei

n ex

pres

sion

in li

ver (

LPL

/ β-a

ctin

) NS

1

NS

0.5

0 48 0 480

1

2

3

Angptl4

fasting time (hrs)

Rel

ativ

e m

RN

A e

xpre

ssio

nin

mus

cle

(/ 18S

)

0 48 0 480

0.5

1.5

2

Lpl

fasting time (hrs)

1

0 48 0 48012

5

Gpr43

fasting time (hrs)

■□WT■□ Gpr43-/-

** ** NS

34

** **

F G

A

C

0 48 0 480

0.5

1

2

Angptl4

fasting time (hrs)

Rel

ativ

e m

RN

A e

xpre

ssio

nin

live

r (/ 18S

)

0 48 0 480

0.5

2

Lpl

fasting time (hrs)

1

1.5

0 48 0 480

5

Gpr43

fasting time (hrs)

2

** **

1

43

*NS NS

1.5

00

5

15

20

48 480fasting time (hrs)

Pla

sma

activ

e gh

relin

(fmol

/mL)

10

** **

NSB


00

0.5

1

1.5

2


** **

Ileum

00

0.5

1

1.5


** **

WAT

Rel

ativ

e m

RN

A e

xpre

ssio

n(Gpr41

/ 18S

)

00

0.5

1

1.5


** **

Muscle

00

0.5

1

1.5

2


** **

Liver

■□WT ■□ Gpr43-/-

0 10 20 30

1.2

0.8

0.4

0

Time (min)after acetoacetate i.p.

Pla

sma

keto

ne b

odie

s (m

M)

〇 acetoacetate〇 β-hydroxybutyrate

H

( - ) AcAc0

0.6

0.8

1

1.2

Rel

ativ

e LP

L ac

tivity

in p

lasm

a *

i.p.

〇WT〇 Gpr43-/-

Rel

ativ

e LP

L ac

tivity

in p

lasm

a

1.4

1.2

1

0.8

0.60

( - ) i.p.

○ AcAc-Li○ αHB-Li

**

10

plasma following the injection of acetoacetate (250 mg/kg i.p.) (n = 8). (H) Plasma LPL activity was

measured in wild-type and Gpr43−/− mice at 20 min after treatment with AcAc (250 mg/kg i.p., n = 8).

LPL activity was expressed as relative to the LPL levels of non-treated mice. ** P < 0.01; * P < 0.05

(Tukey-Kramer test). NS; not significant. All data are presented as means ± SEM.

11

Fig. S4. The expression of LPL in muscle and liver of Gpr43TG mice under fasting

condition. (A, B) Effects of fasting on the Lpl gene, and LPL protein expression in muscle (A) and liver (B) of wild-type and adipose Gpr43TG mice (n = 7–8). NS; not significant. All data are presented

as means ± SEM.

480

fasting time (hrs)

0

3R

elat

ive

mR

NA

exp

ress

ion

in m

uscl

e (Lpl

/ 18S

)

2

1

480

NS

480

fasting time (hrs)

0

3

Rel

ativ

e m

RN

A e

xpre

ssio

nin

live

r (Lpl/

18S

)

2

1

480

NSA B

480fasting time (hrs)

0

0.6

1.2

1.8

Rel

ativ

e pr

otei

n ex

pres

sion

in m

uscl

e (L

PL /

β-ac

tin)

480

NS

480fasting time (hrs)

0

0.6

1.2

1.8

Rel

ativ

e pr

otei

n ex

pres

sion

in li

ver (

LPL

/ β-a

ctin

)

480

NS

■□WT■□ Gpr43TG


12

Fig. S5. Changes in gut microbial composition and intestinal metabolic parameters

in fasted mice. (A–C) The gut microbiota composition was determined for total bacterial numbers (A), relative abundance of microbial taxa in phylum level (B), and diversity (C) in fasted-mice (n = 7–

9). (D) The mRNA expression of Angptl4, Lpl, and Gpr43 gene in colon of wild-type and Gpr43−/−

E F

D

A B

16

8

4

0Plas

ma

GLP

-1 (p

g/m

L)

12

0 48

fasting time (hrs)

1.5

1

0.5

0Plas

ma

PYY

(ng/

mL)

*

○WT○ Gpr43-/-

0 48

fasting time (hrs)

G H

Tota

l bac

teria

(×10

6co

pies

/ ng

DN

A)

0 48 0 480

3

fasting time (hrs)

2

1

** **

■□WT■□ Gpr43-/-

C


-5 0 5

-10

-5

PC

2 [2

2.6%

]

PC1 [26.6%]

0

5●WT_0 hr〇WT_48 hr● Gpr43-/-_0 hr〇 Gpr43-/-_48 hr

00

6

12

18


** **

Angptl4

Rel

ativ

e m

RN

A e

xpre

ssio

nin

col

on (/

18S

)

00

0.5

1

2


** **

Lpl

1.5

00

0.5

1

1.5


**

Gpr43

■□WT ■□ Gpr43-/-

0

3

1

Tota

l bac

teria

(×10

6co

pies

/ ng

DN

A)

Normal

2

WT Gpr43-/-7 9 7 9

time (days)after Abx. treatment

0

80

40

Cec

alS

CFA

s (m

M)

20

Normal

acetate

0

12

Normal

propionate

6

9

0

16

12

8

Normal

n-butyrate

60

3 4

WT Gpr43-/-7 9 7 9


WT Gpr43-/-7 9 7 9


WT Gpr43-/-7 9 7 9


13

mice (n = 8). (E, F) Plasma GLP-1 (E) and plasma PYY (F) were measured at 0 and 48 hrs (n = 6). (G,

H) Wild-type and Gpr43−/− mice were treated with antibiotics in drinking water for 1 week, followed

by fasting for 48 hr. (G) The gut microbiota composition was determined by qRT-PCR for total

bacterial numbers in the fasted, antibiotic-treated mice (n = 7–8). (H) Cecal short-chain fatty acids

were measured by GC/MS in fasted-mice with or without antibiotics treatment (n = 7–8). ** P < 0.01;

* P < 0.05 (Tukey-Kramer test). Abx; antibiotics. All data are presented as means ± SEM.

14

Fig. S6. Eucaloric ketogenic diet fed wild-type and Gpr43−/− mice and the gut

microbial composition. (A, B) After eucaloric ketogenic diet (EKD) feeding for 6 weeks, plasma insulin (A) and blood glucose (B) were measured (n = 8–12). (C, D) The gut microbiota composition

was determined for relative abundance of microbial taxa in phylum level (C) and diversity (D) in

normal diet (ND)-fed and EKD-fed mice (n = 8–11). All data are presented as means ± SEM.

A B

WTGpr43-/-0

80

120

160

Blo

od g

luco

se (m

g/dL

)

40

C

WTGpr43-/-0

200

400

600

Pla

sma

insu

lin (p

g/m

L)

WT Gpr43-/-

0

20

40

60

80

100

Rel

ativ

e ab

unda

nce

(%)

ND EKD ND EKD

VerrucomicrobiaTenericutesSaccharibacteriaProteobacteriaFirmicutesDeferribacteresBacteroidetesActinobacteria

D


-6 -4 -2 0 2

-4

-2

2

PC

2 [1

5.3%

]PC1 [29.6%]

●WT_ND〇WT_EKD● Gpr43-/-_ND〇 Gpr43-/-_EKD

0

4

4

15

Fig. S7. Circulating levels of insulin, SCFAs, and ketone bodies in wild-type and

Gpr43−/− mice under ketogenic conditions. (A) Plasma insulin was measured in intermittent fasted condition (n = 8–9). (B–D) Wild-type and Gpr43−/− mice were treated with antibiotics in

drinking water for 1 week, followed by fasting for 48 hrs. Plasma short-chain fatty acids (B), plasma

insulin (C), plasma acetoacetate (D, Left), and plasma β-hydroxybutyrate (D, Right) were measured in

the fasted, antibiotic-treated mice (n = 7–10). ** P < 0.01 (Student’s t-test). ## P < 0.01; # P < 0.05,

compared with wild-type mice (Student’s t-test). Abx; antibiotics. All data are presented as means ±

SEM.

ad libitum intermittentfasting

0

1000

1500

2500

Plas

ma

insu

lin (p

g/m

L)

2000

500

○WT○ Gpr43-/-

A

C

B

D


Pla

sma

SC

FAs

(μM

)

0

900

600

300

Normal

acetate

0

20

10

5

Normal

propionate

15

0

16

8

4

Normal

n-butyrate

12

WT Gpr43-/-7 9 7 9


WT Gpr43-/-7 9 7 9


WT Gpr43-/-7 9 7 9


7 90

500

1000

Plas

ma

insu

lin (p

g/m

L)


○WT○ Gpr43-/-

WT Gpr43-/-

0

0.6

1.2

1.8

Pla

sma

keto

ne b

odie

s (m

M)

7 9 7 9


acetoacetate

WT Gpr43-/-

0

1

2

3

7 9 7 9


β-hydroxybutyrate

16

Fig. S8. Schematic drawing showing the lipid metabolism via GPR43 under

ketogenic conditions.


Ketogenic condition(Fasting, Ketogenic diet, etc.)Normal condition

Shift in Nutrition

Plasma SCFAs

GPR43

Systemic effectsPlasma SCFAs

GPR43

Systemic effectsAcetoacetate

Intestinal function ↓Systemic energy utilization ↑

Ketone Body Receptor GPR43 Regulates Lipid Metabolismunder Ketogenic Condition

Lipase → TG lipolysis Lipase → TG lipolysis

Intestinal SCFAs

GPR43

Gut

LPL → TG lipolysis

Intestinal SCFAs

GPR43

Gut

LPL → TG lipolysis

OH

O

Angptl4

OH

O

OH

OO

17

Fig. S9. The expression of ketone body receptor GPR43 in brain under ketogenic

condition. The mRNA expression of Gpr43 gene in brain under fasting condition, ketogenic diet feeding, or intermitting fasting condition (n = 8). ** P < 0.01; * P < 0.05 (Student’s t-test). IF;

intermittent fasting. All data are presented as means ± SEM.


0 480

0.5

1

1.5

Rel

ativ

e m

RN

A e

xpre

ssio

n(Gpr43

/ 18S

)

Fasting

fasting time(hrs)

NC EKD0

0.5

1

1.5

Ketogenic diet

2

0

0.5

1

1.5

Intermittent fasting

** ***

18

Table S1. Composition of diets.

Table S1. Composition of dietsNormal chow (NC) Ketogenic diet (KD)

Proximate Profile % %Protein 8.65 8.6

Fat 4.48 75.1Fiber 5.15 4.8Ash 6.95 3

Moisture 8.65 < 10Carbohydrate 49.68 3.2

Caloric Profile kcal/gm kcal/gmProtein 0.35 0.34

Fat 0.4 6.76Carbohydrate 2 0.13

Total 2.75 7.24

Fatty acids gm% gm%C18:2, Linoleic acid 44.44 115C18:3, Linolenic acid 3.26 6.7

Total saturated fatty acids 20.84 303Total monounsaturated fatty acids 25.32 288Total polyunsaturated fatty acids 51.26 122

19

Table S2. Primer sequences.

TableS2. Primersequences .

Gene Sequence

18SForward 5’-CTCAACACGGGAAACCTCAC -3’

Reverse 5’-AGACAAATCGCTCCACCAAC -3’

Angptl4Forward 5’-GTGGTAACGCTTGACAGGGG -3’

Reverse 5’-AAAGTCCACTGTGCCGCTCC-3’

LplForward 5’-CTGCTGGCGTAGCAGGAAGT-3’

Reverse 5’-GCTGGAAAGTGCCTCCATTG-3’

Gpr43Forward 5’-GGCTTCTACAGCAGCATCTA-3’

Reverse 5’-AAGCACACCAGGAAATTAAG -3’

Gpr41Forward 5’-GTGACCATGGGGACAAGCTTC-3’

Reverse 5’-CCCTGGCTGTAGGTTGCATT-3’

20

References

1. I. Kimura et al., The gut microbiota suppresses insulin-mediated fat accumulation via the short-

chain fatty acid receptor GPR43. Nat. Commun. 4, 1829 (2013).

2. I. Kimura et al., Short-chain fatty acids and ketones directly regulate sympathetic nervous system

via G protein-coupled receptor 41 (GPR41). Proc. Natl. Acad. Sci. U S A. 108, 8030–8035 (2011).

3. R. M. Anson et al., Intermittent fasting dissociates beneficial effects of dietary restriction on

glucose metabolism and neuronal resistance to injury from calorie intake. Proc. Natl. Acad. Sci.

U S A. 100, 6216–6220 (2003).

4. F. R. Jornayvaz et al., A high-fat, ketogenic diet causes hepatic insulin resistance in mice, despite

increasing energy expenditure and preventing weight gain. Am. J. Physiol. Endocrinol. Metab.

299, E808–815 (2010).

5. A. Nakajima et al., The short chain fatty acid receptor GPR43 regulates inflammatory signals in

adipose tissue M2-type macrophages. PLoS One 12, e0179696 (2017).

6. J. Miyamoto et al., Barley β-glucan improves metabolic condition via short-chain fatty acids

produced by gut microbial fermentation in high fat diet fed mice. PLoS One 13, e0196579 (2018).

7. J. Miyamoto et al., Gut microbiota confers host resistance to obesity by metabolizing dietary

polyunsaturated fatty acids. Nat. Commun. 10, 4007 (2019).

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New K etone body receptor GPR43 regulates lipid metabolism under … · 2019. 10. 31. · the cells were cultured in DMEM containing 10 µg/mL blasticidin S (Funakoshi), 100 µg/mL