supplementary figure 1 - nature research · 2018-03-07 · tst a supplementary figure 1 nature...

††

h

Glu

cose

(mg/

dl)

Adi

poq/

Tbp

280 3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0LL FF

220

160

0 15 30min

60 120100

LLFF

i

f g

0.8 1.8

1.41.2

1.12.1

4.1

8.1

1.0

log 2

exp

ress

ion

ratio

(L:F

)

log 2 e

xpre

ssio

n ra

tio (L

:F)

Allele ratio (F:L)

0.6

0.4

0.2

-0.2

-0.6

-1.0

0.0

-0.4

-0.8

0.8 1.8

1.41.2

1.12.1

4.1

8.1

1.0

Allele ratio (F:L)

0.6

0.4

0.2

-0.2

-0.6

-1.0

0.0

-0.4

-0.8

b

37

FF FLGenotype

Bod

y w

eigh

t at 1

4 w

eeks

(g)

LL

36

35

34

33

32

d

50

FF FLGenotype

Bod

y w

eigh

t at 1

4 w

eeks

(g)

LL

49

48

47

46

c

4.6

FF FLGenotype

Adi

posi

ty in

dex

(g)

LL

4.4

4.2

4.0

3.8

3.6

3.4

3.2

e7.0

FF FLGenotype

Adi

posi

ty in

dex

(g)

LL

6.5

6.0

5.5

F L MG A GA G AC T CA T TC G GC T TG A AC G GA G GA C CG C C

SNPrs32402009rs32144501rs32292369rs32476725rs32512712

NES12083042NES12082613NES12076143NES12095289NES12093675NES12090622

Origin of ChrFat line

UnknownLean line

72Mbp

D15Mit93

D15Mit105

D15Mit28

Fob3b2

737475767778

80818283848586

79

M2 M F L

D15Mit68

D15Mit239

D15Mit238D15Mit71D15Mit261D15Mit2D15Mit95D15Mit32D15Mit70

D15Mit53

D15Mit189

D15Mit1

Tst

a

Supplementary Figure 1

Nature Medicine: doi:10.1038/nm.4115

Differential expression – M2 X Fat cross (qPCR)Differential expression – parental Fat and Lean (Microarray)

Expression metabolic tissuesTransgenic/Knockout phenotype

Function (Gene Ontology)Haplotypes-nonIBD

Pig QTLsChicken QTLs

Cattle QTLsHuman QTLs

Arhgap

39

Zfp251

Zfp7Mb Apo16Rbm

9=Rbfo

x2

Apo17

a

NO HIT NO DATAHIT

Apo19

a

Apo17

b

Apo11

0a

Apo17

c

Apo11

0b

Apo17

e

Myh9Pva

lbNcf4Csf2

rb2

Csf2rb

Tst

Mpst



Tst /

Tbp

Tst /

Tbp

3.5

2.53.0

2.01.51.0

6J 6JHF

0.50.0

***

1.4

1.01.2

0.80.60.4

6J Lepob

0.20.0

b c

†††


0.5

1.0

1.5

2.0

Exp

ress

ion

STD HF STD HF STD HF STD HF

C57BL/6

J

NZO/H1L

tJ

CAST/EiJ

WSB/EiJ

a


a b6

4

2

0

******

Tst/Tbp

1.2

0.8

0.4

1.0

0.6

0.20.0

Tst/Tbp

6N Ad-Tst 6N Ad-Tst

20

15

10

5

1 2 3weeks

4 5 60

c

d

Cum

mul

ativ

e w

eigh

tga

in (g

ram

s)

20

15

10

5

0

†††

†††

††††††

†††

25

20

15

10

5

2 3weeks

4 5 60

e

Wee

kly

food

inta

ke(g

ram

s)

Tota

l foo

d in

take

(gra

ms)

120

80100

604020

06N Ad-Tst

0.18

†

Gai

n (g

)/Int

ake

(g)

0.14

0.10

0.06

0.02

0.006N Ad-Tst

2.0

1.5

1.0

0.5

0.0

i

**

***

Ucp1/Tbp

† 4

3

2

1

0 ******

Ucp1/Tbp

j

6N Ad-Tst 6N Ad-Tst

1700

1600

1500

1400

1650

1550

1450

13501300

f

VO

2 m

l/hr/k

g

††

††

a.m. p.m.

***1700

1600

1500

1400

1650

1550

1450

13501300

†† †

†† †

a.m. p.m.

***14001380

1340

1300

1260

1360

1320

1280

12401220

0.820.810.80

0.78

0.76

0.74

0.79

0.77

0.75

0.730.72

g

h

Act

ivity

(tot

al c

ount

s)

††

††

a.m. p.m.

***11001180

1040

1000

1060

1020

980960

*

*** *

a.m. p.m.

1.02

0.92

0.82

0.97

0.87

0.770.72

1.11

1.06

0.96

1.01

0.91

0.86

06:5

008

:20

09:5

011

:20

12:5

014

:20

15:5

017

:20

18:5

020

:20

21:5

023

:20

00:5

002

:20

03:5

005

:20

06:5

0R

ER

RE

R

Time

1800

1400

600

1000

1600

1300

400

800

2000

1800

1400

600

1000

1600

1300

400

800

2000

06:5

008

:35

10:2

012

:05

13:5

015

:35

17:2

019

:05

20:5

022

:35

00:2

002

:05

03:5

005

:35

Time

†† ††† †

a.m. p.m.

a.m. p.m.

06:5

708

:42

10:2

712

:12

13:5

715

:42

17:2

719

:12

20:5

722

:42

00:2

702

:12

03:5

705

:42

Time

3500030000

20000

10000

25000

15000

50000

3500030000

20000

10000

25000

15000

50000

a.m. p.m.

†† ††† †

a.m. p.m.


Tota

l wei

ght g

ain

(gra

ms)

VC

O2

ml/h

r/kg

VO

2 m

l/hr/k

g

RE

R

VC

O2

ml/h

r/kg

Act

ivity

(tot

al c

ount

s)

0.00.51.52.02.52.51.21.41.61.82.0

SC Epi Mes BAT Liver

†

Org

an w

eigh

t (gr

ams)

†††† ††


h

2500

2000

1500

1000

500

00 5 10 20

Time (min)60

TST

activ

ityC

onve

rsio

n K

CN

/FeC

N

0.04

0.03

0.02

0.01

0.00SC Epi Mes BAT Liv

i

orga

n/bo

dyw

eigh

tgr

am ti

ssue

/gra

m B

W

2.5

2.0

1.5

1.0

0.5

0.0

j

/Tbp

Pparg CebpD CebpB CebpA Dlk1 Klf5 Srebp1


a

†

25

15

20

10

5

0 1530 60Time (min)

1200

gluc

ose

(mm

ol/L

)

120

100

60

80

40

20

0 15 30 60Time (min)

0

b

% o

f bas

elin

e gl

ucos

e

††

Glu

cose

mm

ol/L

30

0 1530 60 120

25

20

15

10

0

††

Insu

lin (n

g/m

l)

9

0 1530 60 120

7

5

3

8

6

4

210

Time (min) Time (min)

c d

†1.5

1.0

0.5

0.0

e

Glut4/Tbp

6N Ad-Tst

120

100

80

60

0time (min)

150

f

NE

FA %

bas

al

††

3.0

perilipin

β-actin

2.0

1.0

2.5

1.5

0.5

0.0

g

Per

ilipi

n a.

u.

6N Ad-Tst

6N Ad-Tst

****


50 ***

**40

30

20

10

2weeks STS

4 6

Liv Sc Epi Mes

8 6NHF

6NHF

STS

0

a b(g

ram

s)bo

dy w

eigh

t

gram

gai

n/gr

am e

aten

feed

effi

cien

cy

0.140.12

0.080.10

0.060.040.020.00

6NHF

6NHF

STS

6NHF

6NHF

STS

d

(gra

m fa

t/gra

m d

ry fa

eces

)fa

ecal

fat

0.030

0.0200.025

0.0150.0100.0050.000

c

(gra

ms/

day/

mou

se)

food

inta

ke

18

12

6

0

e

(gra

ms)

final

bod

y w

eigh

t

60

40

20

50

30

100

(gra

ms)

orga

n w

eigh

ts

3.53.0

2.0

1.0

2.5

1.5

0.50.0

f



d

176

220

132

88

44

0

288

216

144

72

0

0:15 0:20 0:25 0:30 0:35 0:40Range [h:min]: 0:30

[O2]

nm

ol/m

l

Slo

pe p

mol

s-1 /

ml

m sA

cV C-II C-IIF1alpha Fe-S

SDHB/Aflavin

*** ***

†

†

3.5

2.5

1.5

3.0

2.0

0.5

0.0

a

/ β-a

ctin

3.0

2.5

1.5

2.0

0.5

0.0

b

AC

O2/

β-a

ctin

3.0

2.0

1.0

0.0

e

SO

D1/β-

actin

1.6

1.2

0.4

0.8

1.4

1.0

0.6

0.20.0

c

AC

O1/

β-a

ctin

6N Ad-Tst

6N Ad-Tst

6N Ad-Tst

2.0

1.6

1.2

1.8

1.4

1.00.80.60.40.20.0

Control ShTst

f

*

35

30

25

20

15

10

5

0Control

AU

den

sito

met

ry

Tst/T

bp

ShTst

TST

prot

ein

Lenti-viral shTst

0.35

0.25

0.30

0.20

0.15

0.10

0.05

0.000.1 1 10 0.1 1STS mM 2-PTS mM

10

gTS

T ac

tivity

FeC

N p

rodu

ctio

n (A

U)

**



0.12 r = 0.37p = 0.02n = 390.10

0.08

0.06

0.04

0.02

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.00

aPKACA

(R

.U.)

TST (R.U.)

3.0 r = 0.49p = 0.0001n = 492.5

2.0

1.5

1.0

0.5

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.0

b

FSP27

(R.U

.)


TST (R.U.)


Supplementary Table 1a ANALYSIS OF BODY WEIGHT TRAITS

Genotype BW3 (g) BW6 (g) BW14(g) FF 10.59 ± 0.62 22.31 ± 1.17 35.32 ± 1.07 FL 10.60 ± 0.62 21.38 ± 1.19 34.07 ± 1.08 LL 10.77 ± 0.62 21.73 ± 1.19 34.34 ± 1.10 Pr(FF > FL)1 0.51 0.92 0.96 Pr(FF > LL)1 0.81 0.82 0.92 a2 0.09 ± 0.10 -0.29 ± 0.31 -0.49 ± 0.36 d3 -0.08 ± 0.19 -0.64 ± 0.58 -0.76 ± 0.62 Pr(|a|>0)4 0.81 0.83 0.92 Pr(|d|>0)4 0.67 0.87 0.89

ANALYSIS OF FAT DEPOT TRAITS Genotype SC (g) EPI (g) ABD (g) MES (g) ADI (g) FF 0.608 ± 0.029 0.744 ± 0.052 0.309 ± 0.030 0.718 ± 0.049 4.071 ± 0.271 FL 0.570 ± 0.029 0.690 ± 0.053 0.299 ± 0.030 0.633 ± 0.049 3.763 ± 0.273 LL 0.576 ± 0.031 0.686 ± 0.054 0.282 ± 0.031 0.651 ± 0.050 3.743 ± 0.280 Pr(FF > FL)1 0.90 0.95 0.67 0.99 0.95 Pr(FF > LL)1 0.86 0.95 0.90 0.96 0.96 a2 -0.016 ± 0.014 -0.029 ± 0.017 -0.013 ± 0.010 -0.033 ± 0.019 -0.164 ± 0.095 d3 -0.021 ± 0.025 -0.025 ± 0.030 0.004 ± 0.018 -0.052 ± 0.031 -0.144 ± 0.167 Pr(|a|>0)4 0.86 0.95 0.91 0.96 0.96 Pr(|d|>0)4 0.81 0.80 0.59 0.95 0.81 Pr(X > Y)1 – posterior probability that a genotype X is heavier/fatter than genotype Y; 2 – average allele substitution effect – additive effect; 3 – dominance deviation; Pr(|X| > 0)4 – posterior probability that absolute value of X is greater than 0.

NUMBER OF MICE ANALYSED PER F2 GENOTYPE AND TRAIT Genotype BW3 BW6 BW14 SC EPI ABD MES ADI FF 35 34 36 36 36 36 34 34 FL 49 49 53 53 53 53 50 50 LL 35 35 35 35 35 35 34 34

Supplementary Table 1b ANALYSIS OF BODY WEIGHT TRAITS

Genotype BW3 (g) BW6 (g) BW14(g) FF 12.70 ± 0.82 33.13 ± 0.52 49.12 ± 0.75 FL 12.45 ± 0.81 32.23 ± 0.45 47.73 ± 0.65 LL 12.03 ± 0.82 31.77 ± 0.49 46.77 ± 0.71 Pr(FF > FL)1 0.85 0.96 0.97 Pr(FF > LL)1 0.99 0.99 1.00 a2 -0.33 ± 0.13 -0.68 ± 0.28 -1.17 ± 0.40 d3 0.09 ± 0.19 -0.22 ± 0.41 -0.21 ± 0.59 Pr(|a|>0)4 0.99 0.99 1.00 Pr(|d|>0)4 0.68 0.70 0.64

ANALYSIS OF FAT DEPOT TRAITS Genotype SC (g) EPI (g) ABD (g) MES (g) ADI (g) FF 1.170 ± 0.037 0.890 ± 0.066 0.631 ± 0.022 1.123 ± 0.057 6.495 ± 0.282 FL 1.108 ± 0.033 0.777 ± 0.055 0.602 ± 0.019 1.053 ± 0.047 6.025 ± 0.240 LL 1.076 ± 0.036 0.768 ± 0.062 0.596 ± 0.021 1.053 ± 0.053 5.949 ± 0.266 Pr(FF > FL)1 0.95 0.94 0.90 0.86 0.94 Pr(FF > LL)1 0.99 0.94 0.92 0.83 0.95 a2 -0.047± 0.020 -0.061 ± 0.039 -0.017 ± 0.012 -0.035 ± 0.036 -0.273 ± 0.162 d3 -0.015 ± 0.030 -0.052 ± 0.058 -0.011 ± 0.018 -0.035 ± 0.054 -0.197 ± 0.240 Pr(|a|>0)4 0.99 0.94 0.92 0.84 0.95 Pr(|d|>0)4 0.69 0.82 0.74 0.74 0.80 Pr(X > Y)1 – posterior probability that a genotype X is heavier/fatter than genotype Y; 2 – average allele substitution effect – additive effect; 3 – dominance deviation; Pr(|X| > 0)4 – posterior probability that absolute value of X is greater than 0.

NUMBER OF MICE ANALYSED PER F2 GENOTYPE AND TRAIT Genotype BW3 BW6 BW14 SC EPI ABD MES ADI FF 40 40 40 40 40 40 40 40 FL 62 62 62 62 62 62 62 62 LL 42 42 42 42 42 42 42 42


Supplementary Table 2.

a Line Diet Body

weight Sc Epi Mes BAT Liver

L C 38.89 0.6347 0.8355 0.6209 0.1456 2.2382 L C 35.98 0.6716 0.6832 0.5482 0.166 1.9244 L C 44.01 1.1701 1.2253 0.9099 0.2672 2.2959 L C 38.55 0.7285 0.9078 0.7715 0.1571 2.308 L C 37.88 0.5487 0.5944 0.4505 0.1109 1.4063 L C 39.34 0.8561 0.8515 0.8309 0.2329 2.2051 L C 33.06 0.5465 0.5326 0.5106 0.1071 1.5835 L HF 27.48 0.2485 0.1791 0.2153 0.0818 1.4893 L HF 31.78 0.3553 0.3891 0.2685 0.0904 1.7095 L HF 38.09 0.6796 0.6775 0.4431 0.1333 1.7926 L HF 36.38 0.4635 0.5985 0.4006 0.0964 1.5368 L HF 31.45 0.3551 0.3451 0.2896 0.1209 1.6336 L HF 30.96 0.3354 0.251 0.3246 0.0884 1.3888 L HF 29.63 0.4813 0.339 0.299 0.1175 1.4803 F C 45.82 2.2637 1.6313 1.2175 0.3381 2.5542 F C 48.86 1.9608 1.8702 1.0955 0.4009 2.5106 F C 45.07 1.766 1.171 0.7921 0.3091 1.9753 F C 51.18 1.5382 1.0669 0.8493 0.3714 2.6535 F C 51.97 1.9533 1.3479 0.9482 0.3349 2.78 F C 50.7 1.7834 1.4621 1.0432 0.5273 2.6411 F C 44.23 1.4928 1.3369 1.0615 0.3059 2.3615 F HF 55.26 2.791 1.2906 0.9475 0.3191 2.4268 F HF 50.16 2.5404 1.4631 1.009 0.3594 2.8345 F HF 50.84 2.7991 1.4769 1.0932 0.3248 2.5642 F HF 55.15 3.3862 1.3312 1.0002 0.4043 2.5881 F HF 49.2 2.5198 1.3259 0.8793 0.3564 2.459 F HF 48.66 2.1636 2.3034 1.4775 0.3273 2.2931 F HF 39.94 1.2334 1.4211 1.084 0.2223 2.3164 b

Sc/BW Epi/BW Mes/BW BAT/BW Liv/BW LC mean 0.019 0.0208 0.0172 0.0044 0.052

SEM 0.0015 0.0016 0.0013 0.0005 0.0029 LHF mean 0.0127** 0.0119** 0.0098** 0.0032** 0.0492

SEM 0.0012 0.0015 0.0005 0.0002 0.0017 FC mean 0.0379††† 0.0294††† 0.0209††† 0.0076††† 0.0517

SEM 0.0023 0.0023 0.0014 0.0005 0.0014 FHF mean 0.0492** 0.0308 0.0218 0.0066 0.0504

SEM 0.0036 0.0031 0.0019 0.0003 0.002



High Fat Diet (16hr fast) C57BL/6N Ad-Tst

Fasting glucose (mmol/L) 7.8±0.4 6.7±0.5 Fasting insulin (ng/ml) 0.59±0.19 0.38±0.06

Basal EndoRa (mmol/kg/min) 0.32±0.07 0.24±0.02

Clamp insulin (ng/ml) 0.99±0.17* 1.05±0.08* Average clamp GIR (mmol/kg/min) 0.12±0.03 0.30±0.02†††

Rd Clamp (mmol/kg/min) 0.38±0.05 0.44±0.05* vs Basal EndoRa Clamp EndoRa (mmol/kg/min) 0.25±0.06 0.14±0.02*, P=0.07 vs 6N

Glycolysis (mmol/kg/min)

0.22±0.09 0.14±0.04



Non-Obese Obese Obese-T2D ANOVA p value

p value

n=28 n=45 n=23 P Age (years) 51.9 ± 13.2 45.2 ± 9.7* 45.04 ± 11.2 0.01 BMI (kg/m2) 25.57 ± 2.72 44.26 ± 8.25* 44.30 ± 3.75* <0.0001 Fasting Glucose

(mg/dl)

93.2 ± 11.16 96.04 ± 14.5 137.3 ± 56.9*# <0.0001 VAT gene expression

(n)

28 45 23 - PPARγ (A.U.) 0.031 ±

0.019

0.0063 ±

0.004*

0.0052 ±

0.0029*

<0.0001 GLUT4 (A.U.) 0.057 ±

0.030

0.026 ± 0.021* 0.015 ± 0.007*# <0.0001 IRS1 (A.U.) 0.015 ±

0.007

0.010 ± 0.004* 0.009 ± 0.005* 0.01 TST (A.U.) 0.047 ±

0.021

0.039 ± 0.023 0.034 ± 0.015* 0.02 SAT gene expression

(n)

n=28 n=34 n=22 - PPARγ (A.U.) 0.043 ±

0.022

0.010 ± 0.005* 0.0075 ± 0.003* <0.0001 GLUT4 (A.U.) 0.065 ±

0.032

0.048 ± 0.032 0.024 ± 0.011* 0.001 IRS1 (A.U.) 0.016 ±

0.008

0.012 ± 0.006 0.0092 ± 0.002* 0.01 TST (A.U.) 0.055 ±

0.024

0.056 ± 0.025 0.041 ± 0.012*# 0.01


Supplementary Figure 1. Chromosomal composition, fat mass lowering effect, allele effect on Tst mRNA and metabolic protection of congenic lines M and M2. a. A portion of mouse Chr15 between 72-86 Mbp is shown, as defined by microsatellite markers (D15MitX). SNP and genotypes were used to fine-map the breakpoints between the chromosomal segments derived from the Fat line and Lean line. The red line (right) shows the 95% confidence interval of the previously mapped fatness QTL Fob3b26. b – e. Plots for body weight (b, d) at 14wk and Adiposity Index (c, e) by genotype in the F2 population from crosses between the Fat (F) line and congenic lines M (b, c) or M2 (d, e). Statistical analysis to determine mean values and confidence intervals are detailed in the supplementary online information and related data are shown in Supplementary Table 1a, b. f. A calibration curve was first determined using genomic DNA from FF or LL homozygotes mixed in various molar ratios of F allele/L allele: 8:1, 4:1, 2:1, 1:1, 1:2, 1:4 and 1:8. g. Tst allele-specific expression was determined in WAT RNAs from heterozygotes, n = 8. Through intercepting measured fluorescence intensities on the standard curve, the allele ratio in heterozygous individuals was extrapolated and the average relative Lean: Fat line allele expression was determined (red line). h. Mice carrying 2 Lean line Fob3b2-M2 intervals (LL; broken line) had improved oral glucose tolerance compared to FF mice (solid line) and i. Significantly elevated WAT adiponectin mRNA levels compared to mice carrying 2 F-line intervals (FF). †† = P < 0.01 by t-test. Data are mean ± SEM, n = 6 mice per line. Supplementary Figure 2. Tst is the top candidate lean gene positioned within the minimized Fob3b2-M2 sub-sub congenic region. The red color of the heat map indicates a positive outcome for an association of the 20 non-identical by descent genes (arranged in chromosome order along the top) found within the minimal sub-sub congenic M2 region with: known QTLs for adiposity across species; bioinformatic predictions of function; expression within relevant metabolic tissues; meaningful and differential adipose tissue expression between M2 x F line F2 congenic mice and between the adipose tissues of the parental Fat and Lean lines (left column). Supplementary Figure 3. Tst mRNA levels are high in lean founder strains and low in dietary and genetic obesity. a. RNA-Seq expression data from female mice of 4 selected founder strains (C57BL/6J, NZO/HiLtJ, CAST/EiJ and WSB/EiJ) on control (STD) or HFD (n = 3 per group). B. Tst mRNA level in subcutaneous adipose tissue from C57BL/6J (6J) mice fed control diet (black bar) or high fat diet (HF, grey bar) for 18 weeks corrected to TATA binding protein (Tbp) mRNA level. c. Tst mRNA level in subcutaneous adipose tissue from 10 week old control (black bar, n = 6) or age-matched genetically obese (white bar) leptin-deficient mice (Lepob: C57BL/6J-Lepob/ob, n = 5), corrected to Tbp mRNA. *** = P < 0.001, ††† = P <0.01 by t-test. Supplementary Figure 4. Adiponectin promoter-driven adipose Tst overexpression is selectively for white fat and causes reduced fat mass on high fat diet due to increased energy expenditure that is not related to canonical brown or white fat activation. Real-Time PCR of Tst mRNA in a. brown adipose tissue or b. homogenized whole bone of C67BL/6N fed control


(6N, black bars, n = 4) or high fat diet (HFD, grey bars, n = 5) or Ad-Tst mice (n = 6) fed control (purple bars) or HFD (pink bars). *** = P < 0.001 by 2-way ANOVA. c. Absolute tissue weights after 6 weeks of HFD and feeding efficiency (right-hand panel: ratio of grams gained for grams of food consumed) and d. cumulative weight gain of C57BL/6N (broken black lines, grey bars) compared to Ad-Tst mice (broken purple lines, pink bars) on HFD. Total average weight gain is shown in the right hand panel. n = 6 mice per group. †, ††, ††† = P < 0.05, P < 0.01 and P < 0.001 by RM-ANOVA (d) and t-test (c). e. Weekly food intake for C57BL/6N and Ad-Tst mice on high fat diet. Total average food intake is shown in the right hand panel. f. Quantification of light (a.m.) and dark phase (p.m.) mean oxygen consumption (VO2), CO2 production (VCO2) and respiratory exchange ratio (RER) shown longitudinally and as mean a.m. and p.m. RER values (rightmost panel) in control C57BL/6N littermate (black lines and bars) or Ad-Tst mice (purple lines and bars) on chow diet. g. VO2, VCO2 and RER as above in C57BL/6N littermate (grey bars) or Ad-Tst mice (pink bars) after 6 weeks HFD. n = 4 for both diets, †, ††, ††† = P < 0.05, P < 0.01 and P < 0.001, *, **, *** = P < 0.05, 0.01 and 0.001 by 2-way ANOVA. h. Longitudinal (left panels) and total activity as the sum of central region beam breaking (right panels) in C57BL/6N and Ad-Tst mice on control (f) or high fat diet (g). i. Ucp1 mRNA in brown fat or j. white fat of C57BL/6N mice on control (black bars, n = 5) or high fat diet (grey bars, n = 5) and Ad-Tst mice on control (purple bars, n = 6) or high fat diet (pink bars, n = 6). *, **, *** = P < 0.05, 0.01 and 0.001, † = P < 0.05 by 2 way-ANOVA. All values were corrected to Tbp mRNA internal control. Supplementary Figure 5. Adiponectin promoter-driven adipose Tst overexpressing mice exhibit improved glucose homeostasis and adipose insulin-sensitization whereas Tst gene knockout mice have similar fat mass and unaltered adipogenic gene expression profiles. a. Glucose tolerance and b. insulin tolerance in C57BL/6N (black line) and Ad-Tst mice (purple line) on control diet. c. Glucose tolerance and d. insulin excursion during the glucose tolerance test in C57BL/6N (broken black line) and Ad-Tst mice (broken purple line) after 6 weeks of HF diet. n = 6. Data are mean ± SEM. † = P < 0.05, †† = P < 0.01 by RM ANOVA. e. Adipose tissue glucose transporter 4 (Glut4) mRNA levels in white fat of C57BL/6N mice on control (black bars, n = 5) or HFD (grey bars, n = 6) and Ad-Tst mice on control (purple bars) or HFD (pink bars). n = 6, † = P < 0.05 by 2 way ANOVA. Values are corrected to Tbp mRNA internal control. f. Plasma non-esterified free fatty acid levels (NEFA) after a 5 hour fast and 15 minutes after an oral glucose bolus was administered to C57BL/6N (black line) or Ad-Tst mice (purple line). n = 6, †† = P < 0.01 by paired t-test . g. PERILIPIN1 protein levels in adipose tissue from C57BL/6N on control (black bars, n = 6) or HFD (grey bars, n = 4) or Ad-Tst (purple bars, n = 6) fed control or HFD (pink bars, n = 4). The upper panel shows a representative western blot of protein probed with antibodies to perilipin (upper panel) and β-actin (lower panel). * = P < 0.05, ** = P < 0.01 by 2 way-ANOVA. h. TST activity (conversion of KCN to FeCN, see online methods) was assayed in liver of Tst–/–(open circles, blue broken line), Tst+/– (grey circles, blue solid line) and C57BL/6N littermate (Tst+/+, black circles, black line) male adults over time. A representative plot is


shown from individual mice of each genotype. i. Tissue to bodyweight ratios of C57BL/6N (grey bars) and Tst–/–mice (light blue bars) after 6 weeks HFD. j. Adipose stromal vascular fraction mRNA levels of the adipogenic transcription factors or markers Pparg, Cebpd, Cebpb, Cebpa, Dlk1, Klf5 and Srebp1 in C57BL/6N (black bars) or Tst–/–mice (blue bars). mRNA levels were determined by quantitative real-time PCR, n = 6 per genotype. Supplementary Figure 6. The effects of thiosulfate administration on body weight in HF-fed C57BL/6J mice and C57BL/KsJLeprdb/db mice. C57BL/6N mice were exposed to a high fat diet and either normal drinking water (black broken lines and white bars, n = 6) or water supplemented with 3mg/ml thiosulfate (STS, grey broken lines and orange bars, n = 6). a. Weight gain b. feeding efficiency (weight gain per gram of food eaten) c. food intake and d. fecal fat. e. Final bodyweight in C57BL/KsJLeprdb/db mice given access to normal chow and drinking water (white bars, n = 5) or drinking water supplemented with 3mg/ml STS (orange bars, n = 6). f. final organ weights between water or STS-treated C57BL/KsJLeprdb/db mice. All data are mean ± SEM. ** = P < 0.01 by t-test. *** = P < 0.001 by RM ANOVA. Supplementary Figure 7. Effects of Tst overexpression, TST substrate activation and TST inhibition on mitochondrial proteins and metabolism. a. Protein levels of the complex 5 ATP synthase F1 alpha subunit (cV F1alpha; mitochondrial protein loading control) and the Fe-S (SDHB) or flavin (SDHA) containing subunits of succinate dehydrogenase (SDH) in C57BL/6N (black bars) or Ad-Tst (purple bars) mice on control diet. Protein levels of iron-sulfur cluster (Fe-S)-containing mitochondrial b., or c. cytosolic, aconitase (ACO2, 1, respectively) in adipose tissue of C57BL/6N on control (black bars) or HFD (grey bars) or Ad-Tst mice fed control (purple bars) or high fat diet (pink bars). n = 5, Data are mean ± SEM. † = P < 0.05, *** = P < 0.001 by 2 way ANOVA. d. The effects of adding thiosulfate (arrow s, 1 mM) to isolated mitochondria (added at arrow m) in the presence of 2 mM ADP (arrow A). The blue upper line denotes the oxygen concentration and the red line denotes the change in the rate of oxygen concentration with time. Traces are representative of 3 independent experiments in isolated mouse liver mitochondria. e. Protein levels of cytosolic superoxide dismutase (SOD1) in adipose tissue of C57BL/6N fed control (black bars) or HFD (grey bars) or Ad-Tst mice fed control (purple bars) or HFD (pink bars). n = 5. f. Tst mRNA and TST protein levels in mature 3T3-L1 adipocytes exposed to a control lenti-mir (white bars) or a lenti-shRNAmir (black bars) directed against Tst (shTst). * = P < 0.05, ** = P < 0.01 by t-test. All data are mean ± SEM and n = 6 replicates. g. The effects of sodium thiosulfate (STS) or the TST-inhibitor 2-propenyl thiosulfate (2-PTS) on TST activity in 3T3-L1 adipocyte homogenates. Supplementary Figure 8. Correlation of adipose TST mRNA with adipose mRNA of genes involved in lipolytic and lipid droplet functions. a. Correlation with the protein kinase A Alpha subunit (PKACA) mRNA and b. correlation with mRNA for the lipid droplet associated protein FSP27 mRNA in human adipose tissues (n = 39–49). R.U., relative units. SAT, subcutaneous adipose tissue. Significance of correlational data for TST and other adipose genes was assessed with Scheffé post hoc tests.


Supplementary Table 1. Phenotype effect of the Tst-containing Fob3b2 QTL region in two independent F2 populations from crosses between congenic lines M and parental F line (Table 1a) or M2 and parental F line (Table 1b). See Fig. 1b for chromosomal composition of M lines. Table 1a. The top panel shows analyses of body weight traits, the middle panel shows adipose traits and the bottom panel shows the number of mice examined with FF, FL or LL genotypes at the relevant Fob3b2 subcongenic locus. Genotype designations are: F = Fat-line alleles and L = Lean-line Fob3b2 QTL alleles present in the M and M2 sub-congenics. Traits: BW = body weight at 3, 6 and 14 weeks of age, and fat depots : SC: subcutaneous, EPI: epididymal, ABD: abdominal and MES: mesenteric. ADI = adiposity index = 2 (ABD + EPI + SC) + MES. Table 1b. shows an experiment where the sub-subcongenic M2 line F2 progeny were exposed to high fat diet for 11 weeks. Values in bold are highly significant. Pr(X > Y)1 – posterior probability that a genotype X is heavier/fatter than genotype Y; 2 – average allele substitution effect – additive effect; 3 – dominance deviation; Pr(|X| > 0)4 – posterior probability that absolute value of X is greater than 0. Supplementary Table 2. Body and organ weights of Lean and Fat mice on HFD. (a) Body weight and wet organ weights of Lean (L) and Fat (F) mice fed control (C) or high fat (HF) diets for 20 weeks. (b) Wet weight of the organ at necropsy was divided by the final bodyweight (BW) to give a ratio. Sc; subcutaneous, Epi; epididymal, Mes; visceral mesenteric, BAT; interscapular brown adipose tissue, Liv; liver. ** = P < 0.01 effect of diet within the line. ††† = P < 0.001 difference between the F and L line by 2-way ANOVA. n = 7. Supplementary Table 3. Summary data for hyperinsulinemic euglycemic clamps in C57BL/6N (6N) or Ad-Tst littermates after 2 weeks of high fat diet exposure (HFD). n = 6. EndoRa; endogenous glucose production, Rd; rate of glucose disposal. ††† = P < 0.001, indicates a significant difference between genotype. * = P <0.05 indicates a significant difference between the clamp value and the appropriate basal value within a genotype. Supplementary Table 4. Anthropometric characteristics and gene expression in visceral (VAT) and subcutaneous (SAT) adipose tissue in a Spanish case-control obesity/obesity T2D study31. * = P < 0.05 in obese-T2D or obese compared with non-obese subjects; # = P < 0.05 in obese-T2D compared with obese subjects (Scheffé post hoc test). T2D; type 2 diabetes.


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