abc

Supplementary Figure 1

Relative expression

a 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

WT KO Spl

Organ mass (g)

b

Lun

0.2

0.1

0.05 0 TA

Gas

0.15 Organ mass (g)

Liv

Hrt

EDL

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

0.15

c

Kid

Hrt

Liv

Lun

Spl

Kid

Liv

Lun

Spl

Kid

1.2

1 0.1

0.8 0.6

0.05

0.4 0.2

0

0

TA

Gas

Hrt

Supplementary Figure 1. Adipose-specific knockout (KO) of Lkb1does not affect Lkb1 expression or mass of non-adipose tissues. (a) Relative levels of Lkb1 mRNA in several non-adipose tissues (n=4). (b, c) Masses of other organs in WT and KO mice. (b) Male mice (WT n=11, KO n=8, 10-wk-old); (c) Female mice (WT n=13, KO n=9, 9-wk-old). Error bars: s.e.m. TA, tibialis anterior muscle; Gas, gastrocnemius muscle; Hrt, heart; Liv, liver; Lun, lung; Spl, spleen; Kid, kidney.

b

Ki67


DAPI/Ki67

WT

a

DAPI

Supplementary Figure 2. Deletion of Lkb1 promotes BAT hyperplasia. (a-b) Representative BAT tissues (a) and Ki67 staining of BAT sections from P2 (postnatal day 2) WT and KO mice (b). The % Ki67+ cells was shown in the right panel (n=4). Error bars: s.e.m. ** P< 0.01, two-tailed Student’s t test. Scale bars: 100 µm .

0 2

5 1

0 1

5

0

KO

**

Ki67+/DAPI %


a

WT

b

KO

KO

OD490

WT

4 3

P=0.05

2 1 0 WT KO

kDa 55 35

100 90 40 35

d WT

KO

WT

KO Lkb1 Ucp1 PGC1a GAPDH

Relative expression

c

3.5 3 2.5 2 1.5 1 *** 0.5 0

*

WT

KO

*

**

Supplementary Figure 3. Ablation of Lkb1 increases expression of brown adipose specific genes in cultured adipocytes. (a) Oil red O staining of WT (Lkb1flox/flox) and KO (Adipoq-Lkb1) BAT SVF cells after differentiation. (b) Triglyceride (TG) levels in WT and Lkb1-KO cells after differentiation. n=5. (c) Protein levels of Lkb1, Ucp1 and PGC1a in WT and Lkb1-KO cells after differentiation. (d) mRNA levels of adipose tissue related genes in WT and Lkb1-KO cells after differentiation (n=9). Error bars: s.e.m. * P< 0.05, ** P< 0.01, *** P< 0.001, two-tailed Student’s t test. Scale bar: 100 µm.

a

Supplementary Figure 4 Control

Control

Lkb1-oe

b

c

OD 490

1.5

**

1 0.5 0 Control

7 Relative expression

2

Lkb1-oe

**

6 5 4 3 2 1 0 Control

Lkb1-oe

Lkb1-oe

d Control

Lkb1-oe

Relative expression

1.2 1.0 0.8

** **

0.6

**

***

0.4 0.2

***

0.0 Prdm16

Pgc1a

Ucp1

Pparg

C/ebpa

Fabp4

Supplementary Figure 4. Overexpression of Lkb1 inhibits lipid accumulation and expression of brown adipose specific genes in cultured adipocytes. (a) Oil red O staining of control and Lkb1 overexpression (Lkb1-oe) BAT SVF cells after differentiation. (b) Triglyceride (TG) levels were measured in control and Lkb1-oe cells based on OD490 (n=4). (c, d) Relative mRNA levels of Lkb1 (c) and BAT signature genes (d) in control and Lkb1-oe cells (n=4). Error bars: s.e.m. ** P< 0.01, *** P< 0.001, two-tailed Student’s t test. Scale bar: 100 µm.

a


b DNA Content per depot (µg)

250 200 150 100 50 0 WT WT KO

Relative expression

d

Ucp1

40

GAPDH

35

3.5 3 2.5 2 1.5 1 0.5 0

kDa

asWAT

kDa 35

KO

**

WT

KO Relative expression

iWAT

c

WT

*

**

WT

KO

KO

WT KO

35

Ucp1

40

GAPDH

35

70

*

50 30 6

*

3

*

*

**

0

f WT

5

KO Rectal Temperature(°C)

Relative expression

e

*

4 3 2 1 0

** Lkb1

WT KO WT KO WT KO Ucp1

40 35

GAPDH

Relative expression

h

12 10 8 6 4 2 0

WT

KO

37 36 35 34 33 0

Ucp1 Prdm16 Cidea Pgc1a Ppara Adipoq

g kDa 35

38

1

2 3 Hour

4

5

* * * **

*

Supplementary Figure 5. Ablation of Lkb1 promotes browning of subcutaneous WAT (a) Hematoxylin and eosin (H&E) staining of iWAT sections from WT and KO at 10-week-old. (b) Genomic DNA content per fat depot from 6-wkold male WT (n=5) and KO (n=4) mice. (c, d) Relative mRNA levels of brown fat selective genes in iWAT (c) and asWAT (d) from 4-6 week-old mice (n=8). Insets are Western blots showing Ucp1 protein levels in iWAT and asWAT. (e) Relative mRNA levels of brown fat related genes in eWAT WT and KO mice (n=4). (f) Rectal temperature of WT and KO male mice after cold stimulation (n=4). (g) Relative protein levels of Ucp1 in iWAT from WT and KO male mice (8-week-old) maintained at 4 ºC for 6 h. (h) Relative mRNA levels of browning marker genes in iWAT from WT and KO male mice (8-week-old) maintained at 4 ºC for 6 h (n=4). Error bars: s.e.m. * P< 0.05, ** P< 0.01, two-tailed Student’s t test. Scale bars: 100 µm.

a


b WT

3

KO

*

OD490

WT

2.5 2 1.5 1

KO

0.5 0 WT

d kDa 55

WT

KO

WT

KO Lkb1

35

Ucp1

40 35

GAPDH

Relative expression

c

70 65 60 55 50 10 8 6 4 2 0

KO

***

*** ** ***

*** **

**

**

*

Supplementary Figure 6. Deletion of Lkb1 promotes browning of cultured white adipocytes. (a) Oil red O staining of WT and KO iWAT SVF cells after differentiation. (b) Triglyceride (TG) levels based on OD490 (WT n=4, KO n=5). (c) Relative protein levels of Lkb1 and Ucp1 in WT and Lkb1-KO cells after differentiation. (d) Relative mRNA levels of adipogenesis related genes in WT and Lkb1-KO cells after differentiation (n=9). The Error bars: s.e.m. * P< 0.05, ** P< 0.01, *** P< 0.001, two-tailed Student’s t test. Scale bars: 100 µm.

a

Control

Control

Lkb1-oe

c 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

Relative expression

d

**

Lkb1-oe

6

Relative expression

OD 490

b


*

5 4 3

2 1

0 Control

Lkb1-oe

Control

Control

3.0 2.5 2.0 1.5 1.0 0.5 0.0

Lkb1-oe

Lkb1-oe

***

**

***

Prdm16

Pgc1a

Ucp1

Pparg

C/ebpa

***

Fabp4

Supplementary Figure 7. Overexpression of Lkb1 inhibits white adipocyte differentiation and expression of adipogenic genes. (a) Oil red O staining of control and Lkb1 overexpression (Lkb1-oe) iWAT SVF cells after differentiation. (b) Triglyceride (TG) levels based on OD490 (n=4). (c, d) Relative mRNA levels of Lkb1(c), adipogenic and BAT signature genes (d), n=4. Error bars: s.e.m. ** P< 0.01, *** P< 0.001, two-tailed Student’s t test. Scale bar: 100 µm.


VO2 (ml kg-1 h-1)

a

WT

KO

6000 5000 4000 3000 6 11 16 21 2 7 12 17 22 3 8

VCO2 (ml kg-1 h-1)

b

WT 5500 5000 4500 4000 3500 3000

6 11 16 21 2 7 12 17 22 3 8

c

Heat (Kcal kg-1 h-1)

KO

WT

KO

0.65 0.55 0.45 0.35 6 11 16 21 2 7 12 17 22 3 8

Supplementary Figure 8. Adipose specific deletion of Lkb1 in mice increases systemic energy expenditure. Oxygen consumption (VO2, a), carbon dioxide production (VCO2, b), and heat production (c) were measured using indirect calorimetry (WT n=3, KO n=4). Error bars: s.e.m.


a

b 0.2

***

30 20 10

2.5 Organ mass (g)

Body weight (g)

40

KO Muscle mass (g)

WT

50

c

0.15 0.1 0.05 0

0

TA

Blood glucose (mg/dL)

200

Chow diet

e

0.5

*

f

50

50

50

0

0

0 WT

Fasting 16h

i

KO

WT

Spl

300 250 200 150 100 50 0 WT

Kin

Fasting 4h

HFD

100

KO

Lun

g

150

100

h

Liv

200

150

WT

* Hea

Fasting 4h

200

100

1

Gas

250

150

**

1.5

0

WT KO

d

2

KO

**

WT

KO

KO

200

**

150

HE

Blood glucose (mg/dL)

250

100 50 0 WT

KO

Supplementary Figure 9. Adipose specific deletion of Lkb1 improves glucose metabolism and protects high fat diet (HFD)-induced obesity. (a) Body weight of the WT (n=6) and KO (Adipoq-Lkb1, n=7) mice after 12 weeks on HFD. (b, c) Muscle (b) and organ (c) mass of the WT (n=6) and KO (n=7) mice after 12 weeks HFD. (d, e) Blood glucose levels of the 10-week old WT and KO mice fed on chow diet (d, WT n=6, KO n=4) or after fasting 4 h (e, n=8). (f-h) Fed (f, WT n=5, KO n=7) and fasted (g, 4 h, WT n=9, KO n=10; h, 16h, WT n=8, KO n=9) blood glucose levels of the WT and KO mice after 10 weeks HFD. (i) H&E staining of liver sections of WT and KO mice after 12 weeks HFD. Error bars: s.e.m. * P< 0.05, ** P< 0.01, *** P