Ekihiro Seki, M.D.,Ph.D.

University of California San Diego

The Enhancement of Toxin-Induced Liver Fibrosis

in Fatty Liver Disease

Carbon tetrachloride (CCl4)

- A manufactured chemical.

- Does not exist naturally.

- Clear liquid with a sweet odor that smells like chloroform.

- Easily vaporizes.

- High levels or continuous low dose exposure influence to

liver, and cause hepatocyte injury, liver fibrosis and

hepatocarcinogenesis.

Trichloromethyl radical

(Free radical)

Lipid peroxidation

Hepatotoxicity

Carbon tetrachloride (CCl4)

- Has been used for dry-cleaning, solvent, reagent in

chemical synthesis, fire extinguisher fluid, and

refrigerants.

- The use and production of CCl4 was declined in the mid

1970s, but it has been diffusing into air, water and soil. It

takes for several years to break down

- In air of 0.1 ppb are common around the world.

- In water or soil, ranging from 50 to over 1,000 ppb at

22% of the Superfund sites.

- 47 th and 50 th RANK (2007 CERCLA and 2011

ATSDR priority lists)

• Nonalcoholic fatty liver disease (NAFLD) is one of

the leading causes of liver disease in the United

States

• The prevalence of obesity and diabetes increases

– 60-70% of obese adults have NAFLD

• 30-40% of adults in the western world have NAFLD

• 15-20% of NAFLD patients progress to NASH

• According to a 2008 estimation, NAFLD will be the leading cause for liver transplantation by 2020.

NAFLD

Question: Does Fatty liver disease affects

the sensitivity to toxin exposure?

High fat diet Chronic Industrial Toxin Exposure

Fatty Liver

?

Carbon tetrachloride, HBV, HCV, NASH, Alcohol

Activated Stellate cell

? TNF-

TGF-

Activation of Stellate cells in Liver Fibrosis

Cyp2E1

Hepatocyte Death

TGF-

HFD for 1M

LFD

HFD

8x CCl4 (0.25ul/g BW) or corn oil

8x CCl4 (0.25ul/g BW) or corn oil

The Effect of Fatty Liver in Toxin-induced Liver Fibrosis

HFD for 2M

LFD HFD

0

200

400

600

800

1000

1200

Se

rum

ALT

(U

/L)

Oil CCl4

* *

LFD + Oil HFD + Oil

LFD + CCl4 HFD + CCl4

HFD feeding enhances Toxin-induced Liver Injury

8 x CCl4 (0.25 ul/g BW)

LFD HFD

0

200

400

600

800

1000

1200

Se

rum

ALT

(U

/L)

Oil CCl4

* *

HFD feeding enhances Toxin-induced Liver Inflammation

8 x CCl4 (0.25 ul/g BW)

0

0.5

1

1.5

2

2.5

3

Oil CCl4 0

5

10

15

20

25

30

Oil CCl4

Tnf m

RN

A [

fold

]

Ccl2

mR

NA

[fo

ld]

* * *

LFD HFD

HFD feeding enhances Toxin-induced Liver Fibrosis

0

2

4

6

8

10

Oil CCl4

Siriu

s r

ed

sta

inin

g a

rea

(%

)

LFD

HFD

* *

Oil CCl4

LFD HFD

Col1

a1 m

RN

A [

fold

]

Acta

2 m

RN

A [

fold

]

Tim

p1 m

RN

A [

fold

]

Tg

fb1 m

RN

A [

fold

]

Oil CCl4 Oil CCl4 Oil CCl4

* * * * * * * *

HFD feeding enhances Toxin-induced Liver Fibrosis

0

5

10

15

20

25

0

50

100

150

0

5

10

15

20

25

0

1

2

3

4

5

6

What is the Underlying Mechanism of

Increased Sensitivity to CCl4?

High fat diet Chronic CCl4 Exposure

Fatty Liver

1. Increased sensitivity to

hepatocyte death.

2. Increased capacity to

produce cytokines.

3. Increased production of

extracellular matrix.

4. Increased production of

toxic metabolite from

CCl4.

1 30 306 579

TAB1 TAB2/3

184/187 412 192

Thr

P

Ser

P

Ser

P

Catalytic domain

158

Lys Lys

Ub

72 178

Thr

P

Serine Rich

region

Ub

TAK1 is a 78-80 kDa protein, which is encoded by the MAP3K7 gene.

TAK1 is a serine/ threonine kinase, and belong to MAP3K family.

Phosphorylation and ubiquitination of TAK1 are important for activation of TAK1

and its downstream molecules.

TAK1 interacts with TAB1, TAB2 and TAB3 that regulate TAK1 full activation.

TAK1 is activated in the signaling of TNF, IL-1, TLRs and TGF-.

TAK1 regulates activation of NF-B, JNK, p38, AMPK and NLK.

TGF- activated kinase 1 (TAK1)

TAK1 Regulates NF-B and JNK Pathways,

and also Regulates AMPK-Autophagy Pathway

TAK1

mTORC1

Metabolic stress

(AMP/ATP, starvation)

AMPK

TAK1

TNF TGF

NEMO

IKK

NF-B

JNK

AP-1

Hepatocyte Death

autophagy

Hepatocyte Death

0

0.2

0.4

0.6

0.8

1

1.2

0

0.2

0.4

0.6

0.8

1

1.2

Ta

k1

mR

NA

[fo

ld]

Tak1 m

RN

A [

fold

]

LFD HFD

WT Ob/Ob

**

** WT Ob/Ob

TAK1

β-actin

0

0.2

0.4

0.6

0.8

1

1.2

WT Ob/Ob

*

LFD

TAK1

β-actin

8M-HFD

0

0.2

0.4

0.6

0.8

1

1.2

TA

K1

/-a

ctin [fo

ld]

LFD HFD

**

Hepatic TAK1 expression is decreased

in advanced fatty liver disease

Hepatic TAK1 expression is decreased in fatty liver

with toxin exposure

(2M)

(2M)

Does Decreased TAK1 Expression

Increase Sensitivity to CCl4?

High fat diet Chronic CCl4 Exposure

Fatty Liver

Does Decreased TAK1

Play a Role?

TAK1

Alb-Cre-Tg mice

Generation of hepatocyte-specific TAK1-deleted mice

Alb-promoter Cre-recombinase

X

Tak1 flox/flox mice

exon1 exon2 exon3 exon4

loxp loxp

exon1 exon3 exon4

loxp

TAK1

TAK1

Tak1HEP WT

TAK1

Tak1HEP WT

TAK1

Whole liver

Hepatocytes

CCl4 exposure augmentes liver fibrosis in Tak1HEP mice

WT-CCl4

Tak1HEP-oil Tak1HEP-CCl4

Siriu

s re

d pos

area

(%)

CCl4 - + - +

*

WT-oil

12 injections

(5-6 month old)

0

5

10

15

20

25 *

TAK1 deficiency enhanced liver injury and fibrogenic

response after chronic exposure to CCl4

0

200

400

600

800

1000

1200

1400

ALT

(U

/L)

CCl4 - + - +

*

*

CCl4 - + - + CCl4 - + - + CCl4 - + - +

0

10

20

30

40

50

60

Col1

a1 m

RN

A [

fold

]

0

10

20

30

Acta

2 m

RN

A [

fold

] 0

1

2

3

4

Tim

p1 m

RN

A [

fold

] * * *

Why Does Decreased TAK1 Expression

Increase Sensitivity to CCl4 Exposure?

1.Sensitivity to TNF-induced hepatocyte death

2.Sensitivity to TGF-induced hepatocyte death

3.Autophagy in hepatocytes.

(Inokuchi et al 2010, PNAS)

TNFR

TAK1

NF-B

JNK

Caspase-3

Apoptosis

Necrosis

CCl4 exposure

Hepatocytes

TGF

DAMPs

Kupffer cells

TNF

SMA

Hepatic stellate cells

TIMP1 Collagen

Liver fibrosis

TNF

IL-1

inflammation

Survival

TAK1-/- hepatocytes lack TNF-induced NF-B activation

and are susceptible to cell death

Tak1HEP WT

0 10 30 60 180 360 TNF-(min)

TAK1-/- hepatocytes lack TNF-induced NF-B activation

and are susceptible to cell death

num

ber

of c

ell d

eath

(%

) 0 5

10

15

20

25

30

necrosis apoptosis

Tak1HEP WT

**

TNF- - + - +

0 20

40

60

80

100

120

Ca

sp

ase

-3 a

ctivity

(R

FU

/min

)

**

Tak1HEP WT TNF- - + - +

0 10 30 60 180 360

Cleaved

caspase-3

caspase-3

NF-B

pJNK

JNK

(Inokuchi et al 2010, PNAS)

(Yang et al 2013, Gastroenterology)

TGFbR

TAK1

NF-B

JNK

Caspase-3

Apoptosis

Necrosis

CCl4 exposure

Hepatocytes

TGF

DAMPs

Kupffer cells

SMA

Hepatic stellate cells

TIMP1 Collagen

Liver fibrosis

TNF

IL-1

inflammation

Survival

TAK1-/- hepatocytes augment TGF-mediated Smad2/3

activation and are susceptible to cell death

TGF

Smad2/3/4

TU

NE

L-p

os c

ells

/HP

F

TGFβ - + - + - + - +

10ng/ml

0

5

10

15

20

25

30

*

*

** *

pSmad2

Smad2

pSmad3

Smad3

Cleaved

Caspase3

Caspase3

WT Tak1-/- Smad4-/- Tak1-/-

Smad4-/-

Tak1HEP WT

0 10 30 60 180 360 TGFβ(min) 0 10 30 60 180 360

TAK1-/- hepatocytes augment TGF-mediated Smad2/3

activation and are susceptible to cell death

(Yang et al 2013, Gastroenterology)

Autophagy is a process to degrade intracellular components (long-lived or

aggregated proteins, lipids, and damaged organelles, such as

mitochondria) in lysosomes to supply for energy generation for

maintaining cellular homeostasis.

Autophagy

Cell death, Cancer, Fatty Liver, Type II Diabetes, Innate Immune

Systems, Aging, Infectious Disease, Toxin exposure, Fibrosis.

LC3B-I

-actin

WT

p62

Fed Fasted Fed Fasted

LC3B-II

Autophagy is suppressed in TAK1HEP mice

Faste

d

Fed

LC3B IHC

WT Tak1hep

Fe

d

Fa

ste

d

WT Tak1hep

Electron Microscope Tak1hep

Fed Fasted

N.D

.

% L

D w

ith

AV

0

10

20

30

40

50 * **

WT Tak1hep

Fed Fasted

LC

3B

aggre

ga

tes/ ce

ll

** **

**

WT Tak1hep

0

5

10

15

20

25

TAK1

mTORC1

Starvation

AMPK

Autophagy

Hepatocyte

Death

p-S6

(Inokuchi-Shimizu et al 2014, J Clin Invest)

WT-L

C3

B G

FP

TA

K1

-/- -

LC

3B

GF

P

starvation medium

medium

WT Tak1-/-

starvation

WT Tak1-/-

0

10

20

30

40

50

60

LC

3B

-GF

P (

do

ts/c

ell)

**

**

Autophagic LC3B aggregation is suppressed in TAK1-/- hepatocytes

WT

WT Tak1-/-

-actin

GFP-LC3B-I

GFP-LC3B-II

starvation

p62

starvation

Rat

io (

LC3B

-GF

P-I

I/I)

0

1

2

3 **

- + - +

Tak1-/-

starvation

Starvation-induced AMPK activation is inhibited in TAK1-/- hepatocytes

0 2h 6h

starvation

pAMPK

pULK1

WT Tak1-/-

0 2h 6h

starvation

AMPK

ULK1

pRaptor

Raptor

-actin

TAK1

pLKB1

LKB1

LC3B ↑

p62 ↓

P

TAK1

mTOR

Starvation

AMPK

Autophagy

LKB1

Raptor

ULK1 mTORC1

P

P

P

Rapamycin Restores Autophagy in TAK1-/- Hepatocytes

WT

TAK1-/-

- + rapa

WT TAK1-/-

-actin

GFP-LC3B-I

GFP-LC3B-II

- +

pS6

Tak1

p62

T-S6

P

TAK1

mTOR

Starvation

AMPK

Autophagy

LKB1

Raptor

P

P

Rapa

S6K

S6 P

(Inokuchi-Shimizu et al 2014, J Clin Invest)

Rapamycin Suppresses Liver Injury in Tak1HEP mice

0

10

20

30

40

50

60

TU

NE

L p

os c

ells

(/x

100 f

iled)

rapamycin

WT TAK1HEP

- - + +

0

500

1000

1500

2000

ALT

(U

/L)

rapamycin

WT TAK1HEP

- - + +

* *

(Inokuchi-Shimizu et al 2014, J Clin Invest)

Injection of 5mg/kgBW rapamycin 2 times/week from 28 to 36 weeks

Rapamycin Suppresses Liver Fibrosis in Tak1HEP mice

Vehicle rapa

*

0

2

4

6

8

Sir

ius R

ed

pos A

rea

(%

)

Vehicle rapamycin

Tak1HEP mice

(Inokuchi-Shimizu et al 2014, J Clin Invest)

The enhancement of toxin-induced liver

fibrosis in fatty liver disease

Carbon Tetrachloride Exposure High fat diet

Hepatocyte Death

Steatotic Hepatocytes

TAK1

NF-κB

Smad2/3

Autophagy

Stellate Cell Activation

Fatty liver disease changes the sensitivity to

toxin exposure that enhances liver fibrosis

High fat diet Chronic Industrial Toxin Exposure

Fatty Liver Liver Cirrhosis

Ekihiro Seki MD, PhD (Project5)

David A. Brenner MD (Co-leader)

Yoon Seok Roh DVM,PhD

Shuang Liang PhD

Hiroshi Matsushita MD, PhD

Sayaka Inokuchi-Shimuzu MD,PhD

Ling Yang MD,PhD

Bi Zhang

Jingyi Song

Robert Tukey PhD (Director)

Michael Karin PhD (Project 1)

Koji Taniguchi MD,PhD

XieFeng Wu PhD

Mark Ellisman PhD (Core)

Mason Mackey

Acknowledgement

UC San Diego

Superfund Research Center