Β-Klotho deficiency protects against obesity through a crosstalk between liver, microbiota, and brown adipose tissue

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30 Supplemental table 1. Primers list for quantitative real-time PCR.

Targeted gene Forward Reverse

Acaca 5’-AGGAAGATGGCGTCCGCTCTG-3’ 5’-GGTGAGATGTGCTGGGTCAT-3’

Adipoq 5’-GCACTGGCAAGTTCTACTGCAA-3’ 5’-GTAGGTGAAGAGAACGGCCTTGT-3’

Agrp 5’-AGCTTTGGCGGAGGTGCTA-3’ 5’-GCGACGCGGAGAACGA-3’

Atgl 5’-GTCCTTCACCATCCGCTTGTT-3’ 5’-CTCTTGGCCCTCATCACCAG-3’

Cd36 5’-AGATGACGTGGCAAAGAACAG-3’ 5’-CCTTGGCTAGATAACGAACTCTG-3’

Cidea 5’-TGCTCTTCTGTATCGCCCAGT-3’ 5’-GCCGTGTTAAGGAATCTGCTG-3’

Cpt1a 5’-AACCCAGTGCCTTAACGATG-3’ 5’-GAACTGGTGGCCAATGAGAT-3’

Crh 5’-CAACCTCAGCCGGTTCTGA-3’ 5’-CCCCAGGCGGAGGAAGTA-3’

Cyp7a1 5’-GCCTCTGAAGAAGTGAATGG-3’ 5’-CTAGTACTGGCAGGTTGTTTAG-3’

Cyp7b1 5’-CTGCAGTCAACAGGTCAAA-3’ 5’-GCCTCAGAACCTCAAGAATAG-3’

Cyp8b1 5’-CTGGCTTCCTGAGCTTATTC-3’ 5’-CCCAGTAGGGAGTAGACAAA-3’

Cyp27a1 5’-CAACCTCCTTTGGGACTTAC-3’ 5’-TGATCCATGTGGTCTCTTATTG-3’

Dgat2 5’-TGGGCCTTGGTGGTTTCTTAC-3’ 5’-GACTGCCCTTGCCCAGCTA-3’

Dio2 5’-CAGTGTGGTGCACGTCTCCAATC-3’ 5’-TGAACCAAAGTTGACCACCAG-3’

Elovl3 5’-TCCGCGTTCTCATGTAGGTCT-3’ 5’-GGACCTGATGCAACCCTATGA-3’

Fasn 5’-CATGACCTCGTGATGAACGTGT-3’ 5’-CGGGTGAGGACGTTTACAAAG-3’

Fgf21 5’-TCTATGGATCGCCTCACTTTGA-3’ 5’-CCTCCAGCAGCAGTTCTCTGA-3’

Fgfr1 5’-CCAAACCGTAGGCCTGTAGCT-3’ 5’-CATGCAGTTTCTTCTCCATTTTCTC-3’

Fgfr4 5’-CCAGAGTGACGTGTGGTCTTTC-3’ 5’-GCCCCCGAGGGTGAAG-3’

Hnf4a 5’-GGTAGGGGAGAATGCGACTC-3’ 5’-AAACTCCAGGGTGGTGTAGG-3’

Hmgcr 5’-CTTGTGGAATGCCTTGTGATTG-3’ 5’-AGCCGAAGCAGCACATGAT-3’

Hmgcs1 5’-GCCGTGAACTGGGTCGAA-3’ 5’-GCATATATAGCAATGTCTCCTGCAA-3’

Hsl 5’-TCTATGCGCAGGAGTGTGTC-3’ 5’-TTGACATCAGAGGGTGTGGA-3’

Il1b 5’-GATGAAGGGCTGCTTCCAAAC-3’ 5’-GTGCTGCTGCGAGATTTGAA-3’

Il6 5’-CCTTCAGAGAGATACAGAAACTCTAATTCA-3’ 5’-CTGTTAGGAGAGCATTGGAAATTG-3’

Ikbkb 5’-GGCACCTTGGATGACCTAGA-3’ 5’-CCATATCCTGGCTGTCACCT-3’

Lep 5’-CACACACGCAGTCGGTATCC-3’ 5’-TTGGACTTCATTCCTGGGCT-3’

Lpl 5’-TTGCCCTAAGGACCCCTGAA-3’ 5’-TTGAAGTGGCAGTTAGACACAG-3’

Lrh1 5’-GATGTCCCTACTGTCGATTCAAAA-3’ 5’-TGGAAGCCGTAAGAGCCG-3’

Nfkbia 5’-TGCCTGGCCAGTGTAGCAGTCTT-3’ 5’-CAAAGTCACCAAGTGCTCCACGAT-3’

Npy 5’-TCTGCGACACTACATCAATCTCATC-3’ 5’-GTGTCTCAGGGCTGGATCTCTT-3’

Nr1h3 (Lxr) 5’-AGGAGTGTCGACTTCGCAAA-3’ 5’-CTCTTCTTGCCGCTTCAGTTT-3’

Nr1h4 (Fxr) 5’-CATCGGCAGGCAGAATAAA-3’ 5’-ACGGCGTTCTTGGTAATG-3’

Nrip (RIP140) 5’-TCAGCTTCCTTTCCCACATAGC-3’ 5’-TCATCTTTCGTTGCTCACCAAA-3’

Pgc1a 5’-GCACGAGAAGCGGGAGTCT-3’ 5’-CAGAAGCAGAAAGCAATTGAAGAG-3’

Pomc 5’-TGCTTCAGACCTCCATAGATGTGT-3’ 5’-GGATGCAAGCCAGCAGGTT-3’

Rps29 5’-GCCAGGGTTCTCGCTCTTG-3’ 5’-GGCACATGTTCAGCCCGTAT-3’

Shp 5’-CACTGTGTGAAGTCTTGGAG-3’ 5’-ATGATAGGGCGGAAGAAGA-3’

Shp2 5’-TCCATGGTCACTTGTCTGGA-3’ 5’-GACGTGGGTCACTTTGGACT-3’

Scd1 5’-AGATCTCCAGTTCTTACACGACCAC-3’ 5’-GACGGATGTCTTCTTCCAGGTG-3’

Socs3 5’-TGCGCCTCAAGACCTTCAG-3’ 5’-GCTCCAGTAGAATCCGCTCTC-3’

Srebp1c 5’-GGAGCCATGGATTGCACATT-3’ 5’-GGCCCGGGAAGTCACTGT-3’

Srebp2 5’-GGCGCCCCACTCAGAACACC-3’ 5’-CAGGAACGGCGGTCACCCAC-3’

Tnf 5’-CCACGCTCTTCTGTCTACTGAACTT-3’ 5’-GATGAGAGGGAGGCCATTTG-3’

Ucp1 5’-CCTCACTCAGGATTGGCCTCTA-3’ 5’-CTTCAGGGAGAGAAACACCTGC-3’

Ucp3 5’-GACCTACGACATCATCAAGGAGAAGT-3’ 5’-CTCCAAAGGCAGAGACAAAGTGA-3’

Pan-bacteria 5’-GCAGGCCTAACACATGCAAGTC-3’ 5’-CTGCTGCCTCCCGTAGGAGT-3’

Bacteroidetes 5’-CRAACAGGATTAGATACCCT-3’ 5’-GGTAAGGTTCCTCGCGTAT-3’

Firmicutes 5’-TGAAACTYAAAGGAATTGACG-3’ 5’-ACCATGCACCACCTGTC-3’

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31 SUPPLEMENTAL FIGURE 1

Supplemental Figure 1: Transcriptional changes in white adipose tissue (WAT) depots

in Klb

-/-

mice on high-fat diet (HFD).

A) Epididymal WAT (eWAT) mRNA levels of metabolic enzymes, transporters,

inflammatory markers and adipokines (A.U).

B) Perirenal WAT (prWAT) mRNA levels of metabolic enzymes, transporters, inflammatory

markers and adipokines (A.U).

C) Inguinal WAT (iWAT) mRNA levels of metabolic enzymes, transporters, inflammatory

markers and adipokines (A.U).

Results are expressed as means ± SEM. N=5-6 Klb

-/-

and N=9-10 WT male mice on HFD per

**group. *p < 0.05 vs. WT determined by unpaired t test.**

A

C

B

Tnf

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.5 1.0 1.5 2.0 WT

Klb-/-*

Il1b Il6 Nfk bia _Lep

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.5 1.0 1.5 2.0 2.5 WT Klb-/-Adi poq

*

Hsl

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.5 1.0 1.5 2.0 WT Klb-/-Lpl Atg l Scd1

* *

Fasn_Cpt1a

*

Ucp 1

*

Tnf

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.5 1.0 1.5 2.0 WT Klb-/-Il1b Il6 Nfk bia

*

_*

*

Lep

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 WT Klb-/-Adi poq

*

Hsl

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

*

Fasn_Cpt1a_Ucp 1 p = 0 .0 9 Lep

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.5 1.0 1.5 2.0 2.5 WT Klb-/-Adi poq p=0.15

*

Tnf

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.5 1.0 1.5 2.0 WT

Klb-/-*

Il1b Il6 Nfk bia p=0.07 Hsl

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

*

Fasn_Cpt1a_Ucp 1

* *

p= 0 .1 0

eWAT

prWAT

iWAT

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32 SUPPLEMENTAL FIGURE 2

Supplemental Figure 2: Antibiotic-mediated (Vancomycin=VCM) blockade of

deoxycholic acid (DCA) production abrogates resistance to high-fat diet (HFD)-induced

obesity in Klb

-/-

mice.

A) Hepatic mRNA levels of BAs synthesis enzymes (A.U).

B) Lean and fat mass (g).

C) eWAT mRNA levels of inflammatory markers (A.U).

D) eWAT mRNA levels of metabolic enzymes and adipokines (A.U).

E) VCO

2

curves (ml/kg

0.75

/h) representing two consecutive days.

F) VCO

2

means (ml/kg

0.75

/h) during light and dark phases.

G) Hepatic mRNA levels of ligand and receptors of the FGF signaling pathway (A.U).

H) Hepatic mRNA levels of transcriptional regulators (A.U).

Results are expressed as means ± SEM. N=8 Klb

-/-

and N=11 WT male mice per group. *p <

0.05 vs. WT determined by unpaired t test. Individual values not represented (outside the

range of the Y-axis): Panel D (2.14, 2.34 for Ucp1 in WT group; 1.91 for Ucp1 in Klb-/-

Time

V

C

O

2 (

m

l/

k

g

0 .7

5 /h

)

0 200 400 600 800 1000 1200 1400 WT

Klb-/-Lean Fat

M

a

s

(

g

)

0 5 10 15 20 25 30 WT Klb-/-*

*

Light Dark

V

C

O

2 (

m

l/

k

g

0 .7

5 /h

)

200 400 600 800 1000 1200 WT Klb-/- Cyp 7a1

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0 1 2 3 4 WT

Klb-/-Liver

*

Cyp 8b1 Cyp 7b1 Cyp 27a1

*

Tnf

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.5 1.0 1.5 2.0 WT

Klb-/-WAT

Il1b Il6 Nfk bia Fgf2 1

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 WT

Klb-/-*

Fgfr 1 Fgfr 4

*

Liver

B

Hsl

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

WT

Klb-/-Lpl Atg l Scd 1 Fasn_Cpt1a

*

Ucp 1

WAT

Adi poq Lep

*

D

E

A

C

G

H

F

R

e

la

ti

v

e

g

e

n

e

x

p

re

s

io

n

(

A

.U

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 Shp

*

Sreb p1c WT

Klb-/-Liver

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33 group; 2.22, 2.01 for Lep in WT group); Panel G (5.41 for Fgf21 in Klb-/- group); Panel H

(1.59, 1.50 for Shp in WT group; 1.51 for Srebp1c in WT group).