Novel Treatment Targets for
Non Alcoholic Steatohepatitis
Natalie J. Török, MD
Professor
Gastroenterology and Hepatology
UC Davis, Northern California VA,
Mather
Cirrhosis Mortality
Cirrhosis Mortality
Mortality by underlying and multiple cause,
)
Economic Burden
• Direct cost (medications,
hospitalizations): $2.5 billion
• Indirect cost (loss of productivity):
$10.6 billion*
*Ruhl et al: The Burden of Digestive
Diseases in the United States.
Washington, DC: NIH publication; 2008.
pp. 137–143. US Dept. of Health and
Human Services, NIH, and NIDDK. 096443.
Obesity Epidemic in the U.S.
http://www.cdc.gov/diabetes/statistics.
NAFLD Worldwide
NAFLD/NASH
• The expected prevalence of NASH in
the USA, in 2025 could be > 25 million
people.
• This prevalence would exceed by 10fold the current US prevalence of HCV.
Non Alcoholic Fatty Liver
Disease (NAFLD)
Spectrum of disease
?
NAFLD
Steatosis
(macrovesicular)
Cirrhosis
Non Alcoholic
Steatohepatitis
(NASH):
Steatosis,
Inflammation,
Ballooning,
+ Mallory hyaline
+ Fibrosis
Inflammation,
Fibrosis StageIV
+ Steatosis
HCC
Predictors of fibrosis in NASH
Independent predictors:
– Age
– Obesity
– Diabetes
– AST/ALT>1
Angulo et al, Hepatology, 30, 1356, 1999
Treatment of NASH
• Lifestyle modifications
• Surgery (Roux-en Y gastrojejunostomy)
• Medications
– Protection of hepatocellular function
– Targeting the microbiome
– Anti fibrotics
Weight Loss
• In obese patients, weight loss remains
the main therapy.
• Loss of 5–10% of body weight
decreases liver fat by 40–80%.
• Weight loss pills: don’t work (no effect
on histology).
The Role of Bariatric Surgery
• Roux-en-Y gastric bypass
• Significant improvement in histology after
gastrojejunostomy
• Possible to perform in Child A cirrhotics (rule
out significant portal hypertension)
Medical Treatment of NASH
Lipid-lowering
Transam. Histology
Yes NP
Gemfibrozil
46
600 mg/day
4 weeks
Clofibrate
16
2 g/day
12 months
No
No
Insulin-sensitizing
* Troglitazone
400 mg/day
3 -6 months
Yes
Yes
Metformin
500 mg t.d.s.
4 months
Yes
NP
Hepatoprotective
*UDCA
24
13 -15 mg/kg/day
12 months
Yes
Yes
UDCA
24
250 mg t.d.s.
12 months
Yes
No
UDCA + diet
24
10 mg/kg/day
6 months
Yes
NP
UDCA + diet
31
10 mg/kg/day
6 months
Yes
NP
N-Acetylcysteine 11
1 g/day
3 months
Yes
NP
Betaine
8
20 g/day
12 months
Yes
Yes
Vitamin E
11*
400 -1200 IU/day
4 10 months
Yes
NP
22
300 mg/day
12 months
Yes
Yes
* Tocopherol
Obeticholic Acid
• Farnesoid X Receptor (FXR) agonism
(obeticholic acid): improvement of NASH
activity score (NAS) and ALT*
• NASH resolution in 22%
• Improvement in fibrosis in the diabetic
subpopulation
• Increase in LDL cholesterol
*Neuschwander-Tetri BA et al: Lancet.
2015;385:956–65.
The Role of the Microbiome
Hepatocyte
Injury
TLRs
Macrophage
Activation
HSC Activation
Bacterial 16S DNA
LPS
Peptidoglycan
ROS
TNFα
IL-1β
IL-6
Permeability
Probiotics and synbiotics
• Improvement of transaminases
• ? Improvement in inflammation or
fibrosis*
* Ferolla et al: World J Hepatol. 2015 Mar 27;7(3)
Targeting Fibrosis-Pathogenesis
Normal
Liver
Quiescent HSC
SEC
Macrophage
Fibrosis
Active HSC
NADPH Oxidases as Core Inducers of
Stellate Cell Activation
Angiotensin II*
Phagocytosis
Leptin**
EtOH
NOXs
PDGF***
HCV
LPS
#
AGEs## Bile acid
*Bataller R et al: J Clin Invest. 2003 Nov;112(9):1383-94
**De Minicis et al: Hepatology, 2008 Dec;48(6):2016-26
***Adachi et al: Hepatology. 2005 Jun;41(6):1272-81
# Becker et al: Cell Physiol Biochem. 2007;19(1-4):89-98.
##Jiang et al: Gastro, 2010, Hepatology, 2013 Oct;58(4):133948, Bettaieb; Gastro, 2015.
NADPH Oxidases (NOXs)
Phagocytic NOX:
HSC
Kupffer cells
(liver
macrophages)
HSC
Hepatocytes
Hepatocytes
HSC,
SECs
Duox1
Duox2
NADPH + 2O2 ↔ NADP+ + 2O2- + H+
Altenhofer et al: Cellular and Molecular Life Sciences Vol. 69 Issue 14, 2012
NADPH oxidase 4
NADPH
NOX4
p22
NADPH+
H2O2
.-
O2 .-
• Expressed both in hepatocytes and activated
stellate cells
O .• Transcriptionally regulated
• Produces H2O2
NOX4
Coll.
*
10
8
6
4
2
0
Day1
NOX4/Arbp (fold)
?
NOX4/Arbp (fold)
NOX4 is induced by TGFβ in Stellate
Cells
Day 8
3
*
**
2
**
1
0
NT
TGF-β
Empty
vector
Wt
DN Smad3
Smad3
+TGF-β
*p<0.05, **p<0.01
Nox4/β-Actin αSMA/β-Actin Collagen1A1/β-actin
NOX4 induces Fibrogenic Transcripts
10
*
**
Collagen α I (2)
8
6
Scr+cata
4
MT
2
0
5
NOX4 si
Scr si
*
#
Non si
4
NOX4 si+cata
3
Scr si+cata
2
FL
1
0
*
NOX4 si
Scr si
*
6
No si
0
4
0.2
0.4
0.6
0.8
1
1.2
Luciferase Intensity (fold)
2
*P<0.005
0
1day
8days
WT
1day 8days
Nox4-/-
*p<0.05, #p<0.01,**p<0.001
Jiang et al: Free Radic Biol Med. 2012 Jul 15;53(2):289-96.
NOX4 in hepatocytes?
Biopsy samples from patients
Animal Models:
?
NOX4
Insulin
sensitivity
Stress
signaling
Hepatocyte-specific NOX4 deletion
Chow diet
Fast food diet
Studies on insulin sensitivity
In vitro Studies
*
*
Healthy Simple NASH
Control steatosis
*
3.0
2.0
NOX4
67kD
GAPDH
38kD
1.0
0.0
Chow
FFD
NOX4/GAPDH
(fold)
3
2.5
2
1.5
1
0.5
0
NOX4/Arbp
(fold)
NOX4/B2M (fold)
NOX4 Expression
is
Increased
in
NASH
control
NASH
Chow
3
FFD
*
2
1
0
Chow
FFD
Bettaieb et al: Gastroenterology. 2015 Apr 14,
NOX4 is Induced in Hepatocytes and
Stellate Cells in Patients with NASH
E-cadherin+NOX4
NL
NASH
αSMA+NOX4
CK19+NOX4
Oxidative Radicals and Lipid Peroxidation are
Decreased
in the NOX4hepKO Mice
To address the specific role(s) of NOX4 in hepatocytes:
NOX4floxp X Alb-Cre → NOX4hepKO
ALT (U/L)
200
*
*
*
4
100
Chow
FFD
0
1.5
*
*
1
0.5
2
50
0
*
8
6
150
WT
NOX4hepKO
WT
NOX4hepKO
MDA (fold)
WT
NOX4hepKO
Lucigenin Intensity
(fold)
• Fast food diet
• Chow/CSAA/CDAA diet
0
Chow
FFD
Chow
FFD
*p<0.05
Inflammation and Apoptosis are Decreased in
the NOX4hepKO Mice
*
**
***
*
30
2
1.5
20
1
10
0.5
Chow
FFD
0
Chow
Active casp3
positive cells
(fold)
0
40
MCP1/ACT
(fold)
TNFα/ACT
(fold)
2.5
FFD
1.5
1
***
0.5
0
WT
wt
NOX4hepKO
NOX4hepKO
*P<0.05
**P<0.01
***p<0.001
Procollagen α1(I)/ACT
FFD
αSMA/ACT
(fold)
14
12
10
8
6
4
2
0
*
1
**
3
*
*
**
*
*
**
2
1
0
WT
NOX4hepKO
2
TGFβ/ACT
(fold)
OH-Proline
(mg/gm liver)
NOX4hepKO
WT
Chow
(fold)
Fibrosis is Decreased in the NOX4hepKO
Mice
1.5
0.5
1
0.5
0
Chow
FFD
*P<0.05
**P<0.01
0
Chow
FFD
WT
NOX4hepKO
Steatosis is Unchanged in the NOX4hepKO Mice on the
FFD
FFD
NOX4hepKO
WT
Chow
NOX4hepKO FFD
WT FFD
WT
NOX4hepKO
*
TG Content
(fold)
2
*
ns
1.5
1
*P<0.05
**P<0.01
0.5
*
2.5
2
1.5
1
0.5
0
1
5
0
Chow
FFD
0
Chow
FFD
*
Chow
Chow
*
FFD
CD36/ACT
(fold)
10
** *
2
PPARγ/ACT
(fold)
15
FAS/ACT
(fold)
SREBP1c
(fold)
WT
NOX4hepKO
0
FFD
5
4
3
2
1
0
*
Chow
*
FFD
The Role of NOX4 in Hepatocyte
Stress Signaling
?
NOX4
FA
O2
H2O2
?
p PKR
p JNK1
p
p
PERK
eIF2α
apoptosis
Insulin signaling
NOX4 is induced by Palmitate but not by Oleate
*
*
*
*
2
*
Luciferase activity
(fold)
Luciferase activity
(fold)
3
1.5
1
1
0.5
0
pGL-hNOX4-Luc
0
NT
BSA
OA
pGL-hNOX4-Luc
NOX4hepKO Mice on the FFD are Protected from ERstress,
JNK Activation and Apoptosis
NOX4hepKO
WT
Chow
pPKR
PKR
pPERK
PERK
pEiF2α
EiF2α
CHOP
pJNK1/2
JNK1/2
Active
Casp. 3
Tub
FFD
Chow
FFD
PP1c Phosphatase activity
The PKR/PERK phosphatase PP1c
activity is Decreased by NOX4/H2O2
*
*
*
WT
NOX4-/-
p<0.05
Insulin Sensitivity is Improved in the
NOX4hepKO Mice
WT
400
200
(min)
% of Basal Blood
Glucose
*
600
0
ITT
AUC (fold)
NOX4hepKO
0
150
30
60
90
WT
NOX4hepKO
*
100
*
**
50
0
(min) 0
30
60
90
1.2
**
0.8
0.4
0
120
WT
AUC (fold)
Blood Glucose
(mg/dL)
GTT
NOX4hepKO
1.2
*
0.8
0.4
0
WT
NOX4hepKO
*P<0.05
**p<0.01
NOX4 in NASH
NOX4
FA
IR
O2
Stress
signaling
p IRS-1
p Akt
Insulin
resistance
H2O2
PP1c
p PKR
p
p JNK1
p
eIF2α
PERK
ER stress
signaling
CHOP
Caspase-3
Apoptosis
NASH fibrosis
NOX inhibition
• Prolonged NOX2 inhibition may cause
increase in infections.
• NOX4 inhibition:
– Anti fibrogenic
– Anti apoptotic
– GKT137831, member of the
pyrazolopyridine dione family, inhibits
NOX1/4, orally available (GenKyoTex,
Geneva).
GKT137831 Reduces ROS production
and activation of HSC in vitro
**
ROS Production
(fold)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Control
GKT137831
Target/Housekeeping
(fold)
1.2
1.0
Collagen
0.8
**
0.6
αSMA
TGFβ1
0.4
0.2
#
***
0.0
Control
GKT137831
*p=0.01, **p<0.01, ***p<0.001
Apoptosis Rate (%)
GKT137831 Reduces Hepatocyte
Apoptosis
60
*
50
40
30
20
10
0
Wt
control
FasL
GKT
137831
FasL
*p<0.05
Inhibition of NOX4 Improves
Steatohepatitis
NOX4 inhibitor 60 mg/kg, or vehicle:
daily gavage from week 6 of the 12 week FFD
*
*
60
40
20
Chow
veh
FFD
veh
4.0
***
3.0
FFD
inh
***
15.0
**
**
10.0
2.0
1.0
0.0
20.0
IL1β/Arbp
(fold)
TNFα/Arbp
(fold)
0
MCP1/Arbp
(fold)
ALT (U/L)
80
Chow
veh
FFD
veh
FFD
inh
5.0
0.0
Chow
veh
FFD
veh
FFD
inh
20
*
15
10
5
0
Chow
veh
FFD
veh
FFD
inh
Fibrosis improves after NOX4
inhibition
**
**
4.0
3.0
4.0
**
**
2.0
2.0
0.0
FFD
inh
1.0
0.0
Chow
veh
FFD
veh
FFD
inh
Chow
veh
FFD
veh
FFD
inh
TGFβ/Arbp
(fold)
6.0
FFD
veh
αSMA/Arbp
(fold)
Procollagenα1(I)/Arbp
(fold)
Chow
veh
2.5
2.0
1.5
1.0
0.5
0.0
*
Chow
veh
***
FFD
veh
FFD
inh
Insulin Sensitivity Improves after
NOX4 Inhibition
FFD
Vehicle
Glucose
(mg/dL)
Glucose
(mg/dL)
GTT
600
500
400
300
200
100
0
300
250
200
150
100
50
0
**
0
FFD
inhibitor
30
60
0
120 (Min)
FFD
Vehicle
ITT
*
90
FFD
inhibitor
*
**
30
60
90
120 (Min)
NOX1/4 Inhibition in NASH
•
•
•
•
Antifibrogenic
Hepatocyte protective
Improvement of insulin resistance
Phase 2 trial in diabetic nephropathy is
underway
Potential Future Agents for
NASH
• glucagon-like peptide-1 (GLP-1) activator (incretins,
Exenatide)-Phase II study-GI side effects.
• Selective cannabinoid receptor (CB) I antagonist-CP
945598
• CB II agonists
• 5HT-2B receptor antagonism, may inhibit
fibrosis, enhance regeneration
• 1,25-(OH)2 Vitamin D3
Summary
Novel Approaches in NASH the Future
• NASH-specific approaches
• Fibrosis stage-specific approaches
• Combined treatment strategies
• Individualized treatment
Acknowledgements
Joy X. Jiang, MD, PhD
Tzu-I Chao, PhD
Sophie Kiss, BSc
Xiangling Chen, MD, PhD
Yu Sasaki, MD, PhD
Jijing Tian, PhD
Hiroo Fukada, MD, PhD
Yannan Xi, BSc
Ahmed Bettaieb, PhD (UC Davis)
Fawaz Haj, PhD (UC Davis)
Kathrin Schröder (Goethe
Universitat, Frankfurt am Main)
Ralph Brandes, MD, PhD (Goethe
Universitat, Frankfurt am Main)
Dave Lambeth, PhD
(Emory University)
Masato Katsuyama, PhD (Kyoto
Prefectural University of Medicine,
Kyoto, Japan)
20
GIR
15
10
5
20
(mg/kg/min)
WT
NOX4hepKO
25
10
0
150 (min)
(mg/kg/min)
15
10
lucose uptake
(mg/kg/min)
HGP
WT
NOX4hepKO
5
0
Basal
Clamped
30
20
10
0
WT
NOX4hepKO
150
100
WT
NOX4hepKO
50
0
0
100
80
60
40
20
0
50
100
150 (min)
(uU/ml)
100
(mg/dL)
50
lamped Insulin
0
NOX4hepKO
WT
0
Blood Glucose
GIR [mg/(kg.min)]
Hyperinsulinemic-Euglycemic Clamp
WT
NOX4hepKO