Science - USA (2020-02-07)

LAKO had no effect on body weight, liver
weight, liver-to–body weight ratio, hepatic TG,
fasting glucose, glucose tolerance, or insulin
resistance (fig. S2, B to H). LAKO did not affect
the expression ofAdgre1, gluconeogenic genes
glucose-6-phosphatasecatalyticsubunit(G6pc)
and phosphoenolpyruvate carboxykinase 1 (Pck1),
lipogenic genes sterol regulatory element bind-
ing transcription factor 1 (Srebf1) and fatty

acid synthase (Fasn), or mitochondria and

lipid oxidation regulation genes peroxisome

proliferator activated receptorgcoactivator

1 a(Ppargc1a) and carnitine palmitoyltransfer-

ase 1a (Cpt1a) (fig. S2, I to L) in AMLN-fed mice.

However, LAKO significantly increased serum

ALT, AST, and ALP activities, suggesting en-

hanced liver damage (fig. S2, M to O). LAKO

mice had increased numbers of apoptotic liver

cells (fig. S2, P and Q). Furthermore, exaggerated

liver damage in LAKO mice led to increased

fibrosis (fig. S2, R to T). Similar to the results

from CD-HFD–fedmice,LAKOincreasedthe

expression of fibrosis markersActa2,Col1a1,

andCol3a1and fibrogenic growth factors

PdgfaandPdgfb, with no effect onTgfb,

Timp1,Pdgfra,orDdr2in AMLN-fed mice

(fig. S2U). Thus, although LAKO did not

Zhaoet al.,Science 367 , 652–660 (2020) 7 February 2020 3of9

Fig. 2. AMPK deficiency
increases caspase-6
cleavage to promote
liver damage in NASH.
(AtoE) Flox and LAKO
mice fed CD-HFD for
11 weeks. (A) Immunoblot
analysis of liver lysate;
n= 6 mice. (B) Casp6
activity in liver lysate;
n= 7 to 8 mice. (C) Liver
sections stained active
Caspase-6 (aCasp6, red)
and DAPI (blue). Scale
bar, 50mm. (D) Quantifi-
cation of aCasp6 staining
in (C);n= 7 mice.
(E) Liver sections stained
TUNEL (green), aCasp6
(red), and DAPI (blue).
Scale bar, 50mm. P<
0.05, Student’s unpaired
ttest. (FtoH) Flox
and LAKO mice fed with
CD-HFD for 3 weeks,
followed by intravenous
injection of 1.5 mg/kg
caspase-6 siRNA (KD) or
scrambled RNA (Sc) twice
per week for 3 weeks
while fed continuous
CD-HFD. (F) Liver sections
stained TUNEL (red) and
DAPI (blue). Scale bar,
50 mm. (G) Quantification
of TUNEL-positive nuclei
per field in (F);n=7mice.
(H) Serum ALT;n=7to
10 mice. Mean ± SEM;
P< 0.05, two-way anal-
ysis of variance (ANOVA).

A

D

BE

C

F

G

H

Flox LAKO

0

1

2

3

4

5

Casp6 activity

• 
  

LAKO

Flox

aCasp6 DAPI Merge

Flox-Sc

Flox-KD

LAKO-Sc

LAKO-KD

TUNEL DAPI Merge Zoom

Flox LAKO

0

1

2

3

4

5

aCasp6 Intensity

• 
  

Sc

KD

Flox LAKO

0

200

400

600

800

1000

1200

ALT (U/L)

• 
  

• 
  

n.s.

• 
  

Sc

KD

Flox LAKO

0

20

40

60

80

100

TUNEL

• cell/field *

• 
  

n.s.

• 
  

Flox LAKO

aCasp6 (18kDa)

Procaspase-6 (35kDa)

AMPKα

10μm

aCasp6

TUNEL

DAPI

Merge

Zoom

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