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Novel Treatment Targets for Novel Treatment Targets for Non Alcoholic SteatohepatitisNon Alcoholic Steatohepatitis
Natalie J. Török, MDProfessor
Gastroenterology and HepatologyUC Davis, Northern California VA,
Mather
Cirrhosis MortalityCirrhosis Mortality
Cirrhosis MortalityCirrhosis Mortality
Mortality by underlying and multiple cause, )
Economic BurdenEconomic 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. 09-6443.
Obesity Epidemic in the U.S.Obesity Epidemic in the U.S.
http://www.cdc.gov/diabetes/statistics.
NAFLD WorldwideNAFLD Worldwide
NAFLD/NASHNAFLD/NASH
• The expected prevalence of NASH in the USA, in 2025 could be > 25 million people.
• This prevalence would exceed by 10-fold the current US prevalence of HCV.
Non Alcoholic Fatty Liver Non Alcoholic Fatty Liver Disease (NAFLD)Disease (NAFLD)
NAFLDSteatosis(macrovesicular)
Non Alcoholic Steatohepatitis(NASH):
Steatosis,Inflammation, Ballooning,+ Mallory hyaline+ Fibrosis
Cirrhosis
Inflammation,Fibrosis StageIV+ Steatosis
Spectrum of disease
?
HCC
Predictors of fibrosis in NASHPredictors of fibrosis in NASH
Independent predictors:– Age– Obesity – Diabetes– AST/ALT>1
Angulo et al, Hepatology, 30, 1356, 1999
Treatment of NASH Treatment of NASH
• Lifestyle modifications
• Surgery (Roux-en Y gastrojejunostomy)
• Medications– Protection of hepatocellular function– Targeting the microbiome– Anti fibrotics
Weight LossWeight 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 SurgeryThe 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 NASHMedical Treatment of NASHLipid-lowering
Gemfibrozil 46 600 mg/day 4 weeks Yes NP
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
* Tocopherol 22 300 mg/day 12 months Yes Yes
Transam. Histology
Obeticholic AcidObeticholic 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 MicrobiomeThe Role of the Microbiome
Bacterial 16S DNABacterial 16S DNALPSLPSPeptidoglycanPeptidoglycan
TLRs
HepatocyteInjury
HSC Activation
Macrophage Activation
ROSROSTNFTNFααIL-1IL-1ββIL-6IL-6
Permeability
Probiotics and synbiotics Probiotics and synbiotics
• Improvement of transaminases
• ? Improvement in inflammation or fibrosis*
* Ferolla et al: World J Hepatol. 2015 Mar 27;7(3)
Targeting Fibrosis-PathogenesisTargeting Fibrosis-Pathogenesis
Quiescent HSCSEC
Active HSC
NormalLiver
Fibrosis
Macrophage
NADPH Oxidases as Core Inducers of NADPH Oxidases as Core Inducers of Stellate Cell ActivationStellate Cell Activation
Phagocytosis
Leptin** NOXs
Angiotensin II*
PDGF***
LPS
AGEs##
**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):1339-48, Bettaieb; Gastro, 2015.
*Bataller R et al: J Clin Invest. 2003 Nov;112(9):1383-94
EtOHBile acid#
HCV
Altenhofer et al: Cellular and Molecular Life Sciences Vol. 69 Issue 14, 2012
NADPH Oxidases (NOXs)NADPH Oxidases (NOXs)
Duox1Duox2
Phagocytic NOX:HSCKupffer cells (liver macrophages)
HSCHepatocytes
HepatocytesHSC,SECs
NADPH + 2O2 ↔ NADP+ + 2O2- + H+
NADPH oxidase 4NADPH oxidase 4
NOX4
p2
2
O2 .-
H2O2.-
O .-
NADPH NADPH+
• Expressed both in hepatocytes and activated stellate cells• Transcriptionally regulated• Produces H2O2
NOX4 is induced by TGFNOX4 is induced by TGFββ in Stellate in Stellate Cells Cells
NO
X4/
Arb
p (
fold
)N
OX
4/A
rbp
(fo
ld)
Day1Day1 Day 8Day 8
NTNT TGF-βTGF-β
NO
X4/
Arb
p (
fold
)N
OX
4/A
rbp
(fo
ld)
DN Smad3DN Smad3
****
****
**
Empty vectorEmpty vector
+TGF-β+TGF-β
Wt Smad3Wt Smad3
*p<0.05, **p<0.01
*
NOX4NOX4
?
Coll.
0
2
4
6
8
10
0
2
4
6
0
1
2
3
4
5
Collagen1A1/β-actin
αSMA/β-Actin
Nox4/β-Actin
*
*
*
8days 1day1day 8days
Nox4-/-WT
****
##
NOX4 induces Fibrogenic TranscriptsNOX4 induces Fibrogenic Transcripts
*p<0.05, #p<0.01,**p<0.001
Jiang et al: Free Radic Biol Med. 2012 Jul 15;53(2):289-96.
Collagen Collagen αα I (2) I (2)
Scr si
NOX4 si
Scr si+cata
NOX4 si+cata
Scr si
NOX4 si
FL
MT
Non si
Scr+cata
No si
*P<0.005*P<0.005
**
Luciferase Intensity (fold)
NOX4 in NOX4 in hepatocyteshepatocytes??
Biopsy samples from patients
Animal Models:Hepatocyte-specific NOX4 deletionChow dietFast food diet
Studies on insulin sensitivity
In vitro Studies
NOX4
?
Stress signaling
Insulin sensitivity
NOX4 Expression is Increased in NASHNOX4 Expression is Increased in NASH
HealthyControl
NASHNO
X4/
B2M
(fo
ld) *
control NASH
Simplesteatosis
*
Chow FFD
NOX4
GAPDH 38kD
67kD
**N
OX
4/G
AP
DH
(f
old)
Chow FFD
*
Chow FFD
NO
X4/
Arb
p(f
old)
NO
X4/
Arb
p(f
old)
Bettaieb et al: Gastroenterology. 2015 Apr 14,
E-cadherin+NOX4
NL
NASH
CK19+NOX4αSMA+NOX4
NOX4 is Induced in Hepatocytes and NOX4 is Induced in Hepatocytes and Stellate Cells in Patients with NASHStellate Cells in Patients with NASH
Oxidative Radicals and Lipid Peroxidation are Oxidative Radicals and Lipid Peroxidation are Decreased Decreased
in the in the NOX4NOX4hepKOhepKO Mice MiceTo address the specific role(s) of NOX4 in hepatocytes: NOX4floxp X Alb-Cre → NOX4hepKO
• Fast food diet• Chow/CSAA/CDAA diet
WTNOX4hepKO
WTNOX4hepKO
AL
T (
U/L
)A
LT
(U
/L)
Chow FFD
**
WTNOX4hepKO
Lu
cig
enin
In
ten
sit
y(f
old)
Chow FFD
**
MD
A (
fold
)
Chow FFD
**
*p<0.05*p<0.05
Inflammation and Apoptosis are Decreased in Inflammation and Apoptosis are Decreased in the NOX4the NOX4hepKOhepKO Mice Mice
***
MC
P1
/AC
T(f
old)
****
TN
Fα
/AC
T(f
old)
Chow FFD Chow FFD
***
WTWT NOX4hepKONOX4hepKO
Act
ive
casp
3 p
osi
tive
cel
ls(f
old)
Act
ive
casp
3 p
osi
tive
cel
ls(f
old)
NOX4hepKONOX4hepKO
wt
*P<0.05**P<0.01***p<0.001
Fibrosis is Decreased in the NOX4Fibrosis is Decreased in the NOX4hepKOhepKO
MiceMice O
H-P
rolin
e (m
g/gm
live
r)O
H-P
rolin
e (m
g/gm
live
r) ***
Chow FFD
WTNOX4hepKO
ChowChow FFDFFD
WT
WT
NO
X4h
epK
ON
OX
4hep
KO
**
**
***T
GF
β/A
CT
(fol
d)T
GF
β/A
CT
(fol
d)α
SM
A/A
CT
(fol
d)α
SM
A/A
CT
(fol
d)
Chow FFD
Pro
colla
gen
α1(
I)/A
CT
(fol
d)
WTNOX4hepKO
*
*P<0.05**P<0.01
Steatosis is Unchanged in the NOX4Steatosis is Unchanged in the NOX4hepKOhepKO Mice on the Mice on the FFDFFD
ChowChow FFDFFD
WT
WT
NO
X4h
epK
ON
OX
4hep
KO
Chow FFD
* nsns
TG
Co
nte
nt
(fol
d)
*
Chow FFD
* **
SR
EB
P1c
(fol
d)
Chow FFD
*
FA
S/A
CT
(fol
d)
PP
AR
γ/A
CT
(fol
d)
*
CD
36/A
CT
(fol
d)
Chow FFD Chow FFD
WTNOX4hepKO
***
WTNOX4hepKO
NOX4hepKO FFDWT FFD
*P<0.05**P<0.01
The Role of NOX4 in Hepatocyte The Role of NOX4 in Hepatocyte Stress SignalingStress Signaling
PKR
eIF2αJNK1
H2O2
NO
X4
O2
p
pp
FA
?PERKp
Insulin signaling
apoptosis
?
0
1
2
3
BSA Pal
pGL-hNOX4-Luc
Lu
cife
rase
ac
tivi
ty
(fol
d)
Ctrl A
d
+BSACtrl
Ad
+Pal
dnSmad3
+Pal
*
* *
Ctrl A
d
Ctrl A
d
+TGFβ
dnSmad3
+TGFβ
* *
NOX4 is induced by Palmitate but not by OleateNOX4 is induced by Palmitate but not by Oleate
Lu
cife
rase
ac
tivi
ty
(fol
d)
NT BSA OA
pGL-hNOX4-Luc
WT NOX4hepKO
Chow
pPKR
pPERK
PERK
PKR
pEiF2α
EiF2α
CHOP
Tub
FFD Chow FFD
pJNK1/2
JNK1/2
Active Casp. 3
NOX4NOX4hepKOhepKO Mice on the FFD are Protected from ER- Mice on the FFD are Protected from ER-stress, stress,
JNK Activation and ApoptosisJNK Activation and Apoptosis
NOX4NOX4hepKOhepKO Mice on the FFD are Protected from ER- Mice on the FFD are Protected from ER-stress, stress,
JNK Activation and ApoptosisJNK Activation and Apoptosis
The PKR/PERK phosphatase PP1c The PKR/PERK phosphatase PP1c activity is Decreased by NOX4/Hactivity is Decreased by NOX4/H22OO22
PP
1c
Ph
osp
hat
ase
act
ivit
y
NTBSAH 2
O 2Pal
GSH+P
al NTBSAPal
**
* WTNOX4-/-
p<0.05
Insulin Sensitivity is Improved in the Insulin Sensitivity is Improved in the
NOX4NOX4hepKOhepKO Mice Mice
0
200
400
600
3030 6060 9090 12012000
Blo
od
Glu
cose
(m
g/dL
) *
0
50
100
150
3030 6060 909000% o
f B
asal
Blo
od
G
luco
se
WTWT
NOX4hepKONOX4hepKO*
* ** *A
UC
(fo
ld)
WT NOX4hepKO
*
WT NOX4hepKO
AU
C (
fold
)
GTT
ITT
WTWT
NOX4hepKONOX4hepKO
(min)(min)
*
(min)(min)
*P<0.05**p<0.01*P<0.05**p<0.01
NOX4 in NASHNOX4 in NASH
PKR
eIF2αJNK1
Caspase-3
H2O2
NO
X4
O2
p
pp
CHOP
Apoptosis
FA
NASH fibrosis
PP1c
p
p
IR
IRS-1
Akt
PERKp
ER stress ER stress signalingsignaling
Stress Stress signalingsignaling
Insulin Insulin resistanceresistance
NOX inhibitionNOX 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).
ControlControl GKT137831 GKT137831
****R
OS
Pro
du
cti
on
(f
old
)T
arg
et/H
ou
seke
ep
ing
(fo
ld)
##
****
******
CollagenCollagen
αSMAαSMA
TGFβ1TGFβ1
ControlControl GKT137831 GKT137831
GKT137831 Reduces ROS production GKT137831 Reduces ROS production and activation of HSC and activation of HSC in vitroin vitro
*p=0.01, **p<0.01, ***p<0.001
Ap
op
tosi
s R
ate
(%)
Wt control
FasL GKT137831
FasL
**
GKT137831 Reduces Hepatocyte GKT137831 Reduces Hepatocyte ApoptosisApoptosis
*p<0.05
Inhibition of NOX4 Improves Inhibition of NOX4 Improves SteatohepatitisSteatohepatitis
AL
T (
U/L
)A
LT
(U
/L)
Chowveh
FFDveh
* *
Chowveh
FFDveh
FFDinh
TN
Fα
/Arb
p(f
old)
TN
Fα
/Arb
p(f
old)
*** *** ** **
Chowveh
FFDveh
FFDinh
*
Chowveh
FFDveh
FFDinh
IL1β
/Arb
p(f
old)
IL1β
/Arb
p(f
old)
FFDinh
MC
P1/
Arb
p(f
old)
MC
P1/
Arb
p(f
old)
NOX4 inhibitor 60 mg/kg, or vehicle:daily gavage from week 6 of the 12 week FFD
Fibrosis improves after NOX4 Fibrosis improves after NOX4 inhibitioninhibition
Pro
coll
age
nα
1(I)
/Arb
p(f
old)
Chowveh
FFDveh
FFDinh
Chowveh
FFDveh
FFDinh
αS
MA
/Arb
p(f
old)
αS
MA
/Arb
p(f
old)
Chowveh
FFDveh
FFDinh
TG
Fβ
/Arb
p(f
old)
TG
Fβ
/Arb
p(f
old)
** **** ** * ***
Chowveh
FFDveh
FFDinh
0100200300400500600
Glu
cose
(m
g/dL
)G
luco
se (
mg/
dL)
3030 6060 909000 120120 (Min)
050
100150200250300
3030 6060 909000 120120
FFDVehicle
FFDVehicle
FFDinhibitor
FFDinhibitor
* * **
Glu
cose
(m
g/dL
)G
luco
se (
mg/
dL)
GTT
ITT
**
FFDVehicle
FFDVehicle
FFDinhibitor
FFDinhibitor
(Min)
Insulin Sensitivity Improves after Insulin Sensitivity Improves after NOX4 InhibitionNOX4 Inhibition
NOX1/4 Inhibition in NASHNOX1/4 Inhibition in NASH
• Antifibrogenic
• Hepatocyte protective
• Improvement of insulin resistance
• Phase 2 trial in diabetic nephropathy is underway
Potential Future Agents for Potential Future Agents for NASHNASH
• 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
SummarySummaryNovel Novel Approaches in NASH the FutureApproaches in NASH the Future
• NASH-specific approaches
• Fibrosis stage-specific approaches
• Combined treatment strategies
• Individualized treatment
Ahmed Bettaieb, PhD (UC Davis) Ahmed Bettaieb, PhD (UC Davis)
Fawaz Haj, PhD (UC Davis)Fawaz Haj, PhD (UC Davis)
Kathrin SchröderKathrin Schröder (Goethe (Goethe Universitat, Frankfurt am Main)Universitat, Frankfurt am Main)
Ralph Brandes, MD, PhD (Goethe Ralph Brandes, MD, PhD (Goethe Universitat, Frankfurt am Main)Universitat, Frankfurt am Main)
Dave Lambeth, PhDDave Lambeth, PhD(Emory University)(Emory University)
Masato Katsuyama, PhD (Kyoto Masato Katsuyama, PhD (Kyoto Prefectural University of Medicine, Prefectural University of Medicine, Kyoto, Japan)Kyoto, Japan)
Joy X. Jiang, MD, PhDJoy X. Jiang, MD, PhD
Tzu-I Chao, PhDTzu-I Chao, PhD
Sophie Kiss, BScSophie Kiss, BSc
Xiangling Chen, MD, PhDXiangling Chen, MD, PhD
Yu Sasaki, MD, PhDYu Sasaki, MD, PhD
Jijing Tian, PhDJijing Tian, PhD
Hiroo Fukada, MD, PhDHiroo Fukada, MD, PhD
Yannan Xi, BScYannan Xi, BSc
AcknowledgementsAcknowledgements
Blo
od
Glu
cose
(m
g/dL
)(min)(min)
Glu
cose
up
take
(mg/
kg/m
in)
WT NOX4hepKOWT NOX4hepKOC
lam
ped
Insu
lin(u
U/m
l)
GIR
[mg/
(kg.
min
)]
(min)(min)
NOX4hepKONOX4hepKO
WT
WT NOX4hepKO
GIR
(m
g/kg
/min
)
HG
P(m
g/kg
/min
)
WTNOX4hepKO
Basal Clamped
NOX4hepKONOX4hepKO
WT
Hyperinsulinemic-Euglycemic ClampHyperinsulinemic-Euglycemic Clamp