Dr.dr.Siti Muchayat P.

, MS, Sp PK(K)
PPDS Patology Klinik & Laboratorium Kedokteran
FKMK Universitas Gadjah Mada
 BACKGROUND

NAFLD
A distinct clinicopathologic entity characterized
histologically by a spectrum ranging from simple
steatosis to steatohepatitis (NASH), cirrhosis and even
hepatocellular carcinoma (HCC).

The disease spectrum ranges from mild steatosis

to nonalcoholic steatohepatitis (NASH), advanced
stages of fibrosis, cirrhosis, and hepatocellular
carcinoma3,4
 BACKGROUND Prevalence
WHO 2014:
Globally obesity in 15% of women and 11% of Strong correlation between
men aged 18 and over. NAFLD prevalence & obesity
The prevalence of obesity in Asian countries indices

NASH in at least 20% of obese

adults or children 5% of those
who are overweight

NASH most common cause of

cryptogenic cirrhosis (India 6%)
and HCC worldwide

Japan, cirrhosis is now the

fourth most common cause of
death (4.7%) in DM T2,
and HCC is cause of cancer
death
(8.6%).1
 BACKGROUND Genetik

Proteins containing patatin 3

phospholipase (PNPLA3):
the first genes associated with
NAFLD in (GWA) study.

At risk of PNPLA3 rs738409 GG

genotype was found:
❖ 13-19% of the general
population in the Asian
❖ 4% in Caucasians
❖ 2% in African Americans and
25% in Hispanics.

This review provides an overview

of potential biomarkers for NASH
diagnosis and monitoring.
 PATHOGENESIS of HEPATIC STEATOSIS
A fatty liver is the result of the accumulation of various
lipids. Several mechanisms may lead to a fatty liver:
(1)  FFA supply due to  lipolysis from both
visceral/subcutan adipose tissue &/or
intake of dietary fat.
(2) FFA oxidation;
(3) de novo hepatic lipogenesis (DNL)
(4)  hepatic VLDL-TG secretion.

➢ FFA delivery to the liver accounts for almost two-

thirds of its lipid accumulation.
➢ Elevated peripheral fatty acids and DNL.
➢ Lipogenesis-controlling factors such as SREBP or
ChREBP, X-box binding protein 1 (XBP1), known as a
key regulator of the unfolded protein response UPR
secondary →ER stress
 BACKGROUND

NAFLD
The progression of fibrosis in 351 patients with NAFLD. Overall, fibrosis
progressed in ≥ 60 yo ≥ 50 yo , <50 correlation between NAFLD prevalence &
fibrosis & age
The prevalence of NAFLD differs from race and ethnicity. Hispanics had the
highest prevalence of NAFLD, hepatic steatosis, and elevated levels of
aminotransferas; followed by non- HispanicWhite; with the lowest rates reported in
Africa stages of fibrosis, cirrhosis, and hepatocellular carcinoma
Insulin resistance
 hepatic lipogenesis (DNL),
Inhibition of adipose tissue lipolysis
 fatty acid flux to the liver.
Promotes adipose tissue dysfunction→
changes in production and secretion adipokines and
inflammatory cytokines.
Fat accumulates in the liver:
Triglycerides
Increased lipotoxicity of; high levels of free fatty acids, free
cholesterol and other lipid metabolites
Autophagy
Inhibition of autophagy in hepatocytes increases the storage
of triglycerides.
ER stress and autophagy → inflammatory diseases.

Mitochondrial dysfunction, with oxidative stress

and the production of reactive oxygen species (ROS)
and stress-related endoplasmic reticulum
(ER)/Inflammation
 MITOCHONDRIAL DYSFUNCTIONS
Structural: encompass depletion of
mitochondrial DNA, morphological and Correlation between:
ultrastructural changes. Insulin resistance, obesity, TNF-
Functional alterations: αlevels and mitochondrial
dysfunction.
Include the respiratory chain and mitochondrial
β-oxidation. ROS, oxidized LDL activate Kupffer
& HSC, leading to inflammation and
Respiratory oxidation collapse: Impairment of fibrosis.
fat homeostasis, generation of lipid- derived
toxic metabolites & overproduction of ROS .
→hepatocytes necroinflammation & worsening
of mitochondrial damage.

(Musso et al., Progress in Lipid Research, 2013)

Juanjuan et al. J Clin and Transl Hepatol, 2016
ENDOPLASMIC RETICULUM STRESS

NAFLD, factors that induce UPR:

hyperglycemia, mitochondrial injury
that depletes ATP,
hypercholesterolemia, depletion of
phosphatidylcholine & oxidative
stress.
UPR to the activation of c-jun
terminal kinase (JNK), an activator of
inflammation and apoptosis.
X-Box binding protein-1 (XBP-1)
is the main regulators of UPR
and interacts with the PI3K insulin
signaling pathway, with increased
nuclear translocation induced by
insulin .
The interaction of PI3K and XBP-
1 is both modulated by and
modulates the cellular response to
ER stress.
 INFLAMMATORY STATE IN NAFLD :

Main inflammatory pathways, JNK-AP-1

and IKK-NF-κB, are critically involved in
the development of the chronic
inflammatory state in NAFLD.

Nuclear factor-jB kinase-b (NF-kB) is a

transcription factor and a primary
regulator of inflammatory activation, and
its IKK2 subunit is the major component
required for its activation during the acute
inflammatory response

Persistent NF-κB pathway activation has

been shown in animal models of
NAFLD/NASH

(Liu et al. Lipids in Health and Disease 2010)

 Serum and hepatic levels of TNF-α, IL-6
: increased in NASH & correlate with
histological severity of liver damage

Inflammation and NF-kB activation can

promote chronic inflammatory state of
NAFLD, and carcinogenesis,

(Zhan YT, and An W, World J Gastroenterol 2010)

 They regulate effector proinflammatory
INFLAMMASOMES : cytoplasmic multi-
cytokines by activation of families of
protein complexes, consisting of
germ-line encoded PRRs as the TLRs,
caspases and molecules derived from
NOD-like receptors (NLRs) and C type
pathogen microorganisms or liberated by
lectin receptors (CLRs).
injured cells, which can trigger
Release of DAMPs can be induced by
inflammation. They act as sensors of
cell necrosis, apoptotic cells that are
endogenous or exogenous PAMPs or
not removed by phagocytes and
DAMPs.
oxidative stress
 APOPTOSIS MARKERS

Increased hepatocyte cell death

(apoptosis) occurs in NASH, suggests the
role of extrinsic (death receptor-
mediated) and intrinsic (organelle-
initiated) pathways.

Expression of Fas, activation of the

effector caspases (especially caspase 3)
including cytokeratin 18 (CK-18),

Plasma CK-18 fragments independently

predict NASH in multivariate analysis.

(Wieckowska et al., Hepatology, 2007)

 ADIPOSE TISSUE DYSFUNCTION

Adipose tissue :
Lowgrade inflammatory states .
producing pro-inflammatory cytokines
(IL-6 , TNF-α)

Increased expression of inflammatory

genes and macrophages activation in the
visceral and subcutaneous adipose tissue
of patients with NAFLD correlates with
progression from simple steatosis to
NASH and fibrosis
16-kDa anorexigenic hormone
proinflammatory.

Leptin increases in obese subjects, leptin

resistance and profibrogenic .

Leptin activates HSC through the hedgehog

and mTOR pathways.

Kupffer cells are targeted by leptin

Low-dose gut-derived endotoxin induces

hyper-responsiveness to leptin-mediated
signaling and subsequent upregulation of
CD14 and accelerated fibrosis in NASH mice.

(Buzzetti et al. Metabolism, 2016)

 ADIPONECTIN
Enhancing the deacylation of the
sphingolipid ceramide independently of
AMPK, especially in hepatocytes,
cardiomyocytes and β- cells .
Finelli et al.World J Gastroenterol. 2013
Anti-inflammatory effects:
blocking the activation of NF-kB,
secreting anti-inflammatory cytokines
and inhibiting the release of pro-
inflammatory cytokines such as TNF-α
and IL-6 .
Direct antifibrotic effect, which could
be mediated by the activation of
AMPK. World J Gastroenterol. 2013
Major source of resistin →from the
peripheral blood macrophages and adipocytes
and correlate with IL-6, BMI
Resistin promotes systemic IR by AMPK
activation and decreasing upregulation of
GLUT-4 in adipocytes whereas anti-resistin
antibody administration improves IR.
Acylation-stimulating protein (ASP) :
derived from the interaction of complement
C3, factor B and adipsin. Production of ASP
may also be increased by IL-6 and its levels
in NAFLD are high correlating with
HOMAIR
score.
Angiotensinogen: Angiotensinogen is also
found in adipocytes and its levels are
increased in obesity.
i
 Adipose Tissue Cytokines
Qureshi, and Abrams, World J Gastroenterol 2007

Human adipose tissue

Source of inflammatory cytokines
Adipocytes secrete PAI-1 (plasminogen
activator inhibitor-1), MCP-1 (macrophage
chemotaxis protein), IL-8 and IL-6.
Steatosis, macrophage infiltration and
macrophage chemokine expression of
MCP-1 attractant is significantly increased.
NASH develops with CD4 (+) and CD8 (+)
Tcell infiltration increases, and levels of
inflammatory cytokines, such as IL-6 or IL-
8, also increase

Takaki A, Research on Immunology,2016

GENETIC DETERMINANTS
EPIGENETIC FACTORS
EXTRACELLULAR VESICLE MARKERS
EXTRA VESICLES NAFLD

Ban LA et al.Int. J. Mol. Sci. 2016

 DIETARY FACTORS
Fructose is a lipogenic, pro-
inflammatory dietary factor that
results in oxidative stress and
upregulation of TNF-α.
It is cleared by the liver within first-pass
metabolism by 90% and is
metabolized by specific hepatic kinases,
independently of
insulin action, into fructose-1-
phosphate.
This metabolite is converted into triose
phosphate, which enters the glycolytic
pathway generating substrates for DNL
NAFLD is associated with bacterial
overgrowth and increased intestinal
permeability, induce copper deficiency
with increased fibrosis
Sweetened beverages that contain
fructose-containing sugars such as
sucrose and high fructose corn syrup are
associated with increased risk of
developing steatosis and NASH,
especially in overweight/obese individuals
.
Influence of The Microbiota: The Gut-Liver Axis
 PROTEOMIC MARKERS
Bell et al utilised an ion-intensity based, label-free quantitative proteomics approach
(LFQP) and discovered 55 proteins that changed significantly between NAFLD and
NASH with advanced fibrosis.
Bell et al further revealed 15 proteins that changed significantly between early NASH
and NASH with advanced fibrosis.

6 proteins diagnostic method (fibrinogen β chain, retinol binding protein, Serum

amyloid P component, lumican, transgelin 2 and CD5 antigen-like) and a 3 proteins
diagnostic method (component C7, insulin-like growth factor acid labile subunit and
transgelin 2) were developed to diagnose the different stages of NAFLD

Rodríguez-Suárez et al with DIGE in combination with MALDI TOF/TOF. Ten

of the proteins were further validated by Western blotting to confirm.
demonstrated that both serum concentration of carbamoyl phosphate synthase 1
(CPS1) and 78 kDa glucose-regulated protein (GRP78) decreased gradually to
steatosis and NASH
 Biomarker NAFLD

The association between serum hemoglobin and NAFLD may be partially

modulated by haptoglobin (Hpt), an acute α2-glycoprotein phase protein produced
in response to inflammatory in fl ammation.

Pentraxin 3 (PTX-3), acute-phase protein like CRP in function complementary used

as a marker for disease severity in inflammatory and infectious diseases.

Li et al found that HO-1 may inhibit the proliferation of hepatic myofibrobblasts, is

the metabolic end product of heme catabolism such as haem oxygenase-1 (HO-1),
protein-responsive stress that degrades the pro-oxidant heme to biliverdin.
 MATRIX EXTRACELLULAR MARKERS

Activated HSC produces crucial cytokines, growth factors and

inflammatory mediators, adhesion molecules (I-CAM-1, V-CAM-1, N-
CAM), MMP (MMP-1, -2, -3, -14) , and protease inhibitors (TIMP-1, -
2, plasminogen activator inhibitor-1), among other proteins.

Increase in TIMP-1 expression and simultaneously reduce the activity

of MMPs with subsequent protein accumulation, especially type I, III,
and IV collagen in the extracellular space.115 In the murine model of
cirrhosis and NASH,
PANEL BIOMARKER
PANEL BIOMARKER
 FOXO (Forkhead box O) MARKERS

FOXOs have pleiotropic functions, with effects on cell survival, anti-oxidative

stress, autophagy, and metabolism.
Forkhead box O (FOXO) transcription factors are key downstream regulators
in range of cellular functions including the regulation of glucose, triglyceride,
and cholesterol homeostasis.
 SUMMARY

The pathogenesis of NAFLD and its development is a

complex process ('multi hit' hypothesis).
A number of diverse parallel processes contribute to
the development of NASH liver steatosis and
inflammation. Intestinal microbiomas have a key role
through the intestine axis, along with insulin
resistance, hormones secreted from adipose tissue
and obesity, ER stress, mitochondrial dysfunction
and activation of inflammatory responses.
Furthermore, genetic factors may explain progres-
sivity (monitoring) in NAFLD.
5/8/2018 ThankYou 33