Gosiset al.,Science 376 , eabf8271 (2022) 15 April 2022 6 of 12
ControlLiFKODKO010203040Body weight (g)Column min Column maxControlLiFKOControlLiFKODKOMlxiplAcacaAclyAcss2Srebf1Scd1FasnNormal
chowAMLN
dietABC(^13) C Fructose gavage
(^13) C Acetyl CoA
(^13) C Fatty acids
De novo
lipogenesis
AMLN diet
(7-11 days)
E
Incorporation of^13 C into
liver fatty acids
Body weights
Control LiFKO DKO
SREBP1 ro
sr
uc
er
P
D 16 week FPC diet
(^2) H
2 O i.p. injection
NADP^2 H
(^2) H Fatty acids
De novo
lipogenesis
ACLY
ACSS2
FASN
Protein levels
relative to HSP90 DKOLiFKO
Control
precursor
SREBP1
ACLY ACSS2 FASN
Arbitrary unit
0
10
0.0
2.5
- 1.5
**
- 0.0
2.5
**
FPC diet
(9 days)
F
Incorporation of^2 H into
liver fatty acids
Control
LiFKO
DKO
0.0
0.1
0.2
0.3
0.4
Enrichment (%)
(^) *
C14:0 C16
:0
C18:0
0
10
20
30
Body weight (g)
ControlLiFKO
DKO
Body weights
0.0
0.5
1.0
1.5
Relative expression
Srebp1a
0
1
2
3
4
5
Srebp1c
0.0
0.5
1.0
1.5
Acly
0.0
0.2
0.4
0.6
0.8
1.0
Acss2
0
1
2
3
4
5
Fasn
**
0
5
10
15
Scd1
0
1
2
3
Acaca
0.0
0.5
1.0
1.5
Chrebpa
0
1
2
3
4
5
Chrebpb
De novo lipogenesis genes (FPC diet) ControlLiFKO
Tfe3 KO
DKO
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
0
2
4
6
8
0
2
4
6
10
0
2
4
6
8
10
0.0
0.5
1.0
1.5
Relative expression
De novo lipogenesis genes (AMLN diet) ControlLiFKO
DKO
Srebp1a Srebp1c Acly Acss2 Fasn Chrebpa Chrebpb
0.0
0.2
0.4
0.6
C16:0 C18:0 C18:1 C20:0
Enrichment (%)
Control
LiFKO
DKO
**
Column min Column max
Fig. 4. Loss of FLCN in the liver suppresses de novo lipogenesis.(A) Heatmap
of normalized expression values of de novo lipogenesis genes from RNA-seq
described in Fig. 3. (BandC) mRNA expression of de novo lipogenesis genes
in livers of control, LiFKO,Tfe3KO, and DKO mice fed (B) an AMLN diet (n= 3 to 9)
for 17 to 18.5 weeks or (C) an FPC diet regimen (TD160785 with sugar water;n=5
to 11) for 16 weeks. (D) Protein expression of SREBP1, ACLY, ACSS2, and FASN
in livers from control, LiFKO, and DKO mice fed an FPC diet regimen (TD160785 with
sugar water) for 16 weeks. HSP90 from Fig. 3D was used as loading control. Mice
were euthanized after a 4- to 6-hour fast, and thus only the precursor form of SREBP1
was detected. (E) Control, LiFKO, and DKO mice (n= 6 or 7) were fed an AMLN
diet for 7 to 11 days, fasted from 9 a.m. to 7 p.m., refed for 2 hours, and force-fed
a bolus of^13 C-fructose and^12 C-glucose. The mice were fed overnight and killed
the next morning. LC-MS was performed to examine the amount of^13 C label
incorporation into hepatic fatty acids. (F) Control, LiFKO, and DKO mice (n= 9 to 11)
were fed an FPC diet regimen (TD190142 with sugar water) for 9 days and then
injected intraperitoneally (i.p.) with deuterium oxide (^2 H 2 O) at ~7 p.m. Five hours
later, the mice were killed, and their livers harvested. LC-MS was performed to
examine the amount of deuterium label incorporation into hepatic fatty acids.
P< 0.05, P< 0.01, P< 0.001, ****P< 0.0001; one-way ANOVA with TukeyÕs
multiple comparisons test. Data are depicted as mean ± SEM.
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