Dr .

AYASKANTA SINGH
Introduction
NASH was described :
alcoholic- like liver disease that develops in people who
do not drink alcohol
Ludwig et al , J.mayo clin proc 1980

NAFLD : Defination
Presence of steatosis (accumulation of fat in excess of 5-10%
of liver weight ) detected by Imaging/Histopathological
study.
Exclusion of secondary causes of fatty liver.





NAFLD has reached epidemic proportions .
Prevalence in India- 24.5%
Singh SP et al Trop Gastroenterol 2004


Pathophysiology of NAFLD not well understood .
Multifactorial
Associated with obesity, diabetes, and insulin resistance,
Considered to be the liver manifestation of the metabolic
syndrome .
Several hypothesis

Spectrum
McCullough et al Clin. Liver Dis. 2004
The 2-hit hypothesis
Suggested by Day and James in 1998 .

1
st
hit
Hepatic triglyceride accumulation or Steatosis

2
nd
hit
Inflammatory cytokines/adipokines, mitochondrial
dysfunction and oxidative stress, which in turn lead to
steatohepatitis and/or fibrosis.
The traditional 2-hit hypothesis
The traditional 2-hit hypothesis: steatosis represents the first hit,
which then sensitises the liver to injury mediated by second hits
Pathogenesis of hepatic steatosis

Dysregulation of fatty acid metabolism leads to steatosis .

Disordered energy homoestasis .

Accumulation of triglycerides (TG) are formed from the
esterification of FFA and glycerol within the hepatocyte .



FFA accumulation :
1. Lipolysis (hydrolysis of FFA and glycerol from triglyceride)
within adipose tissue(64%)
2. De novo lipogenesis (DNL) (26%)
3. Dietary sources (15%).
Donnelly KL et al, J Clin Invest 2005

FFA utilization :
1. Beta oxidation of fatty acids
2. Re-esterification to TG and storage as lipid droplets,
3. Packaged and exported as very low density lipoprotein (VLDL)

VLDL particles are formed by the incorporation of TG into
apolipoprotein B (apoB) by microsomal transfer protein
(MTP).


Aberrant alterations of MTP/apoB synthesis and secretion
have been proposed as potential mechanisms.
Adams LA ,CMAJ 2005

Obesity
Obesity and hepatic steatosis strongly correlated .

Visceral fat more noxious than subcutaneous fat .
Elevated levels of FFAs in plasma which leads increased
hepatic uptake
Increases splanchnic lipolysis in portal vein .
Increasing visceral obesity results in increased production of
pro inflammatory cytokines [TNF , IL- 6, and CRP] and
decreased production of protective adiponectin .
G. Atzmon et al ,Hormone and Metabolic Research 2002

Lean NAFLD
The lean NAFLD approximately 13.2% of total NAFLD.

Dyslipidemia present in 90% of lean NAFLD patients .
IR present in 7.4% patients of lean NAFLD .

Compared to obese NAFLD, the severity of liver
histopathology was significantly lower in patients with
lean NAFLD .
Kumar et al , Ind J of Endocrinology and Metabolism 2013
Insulin Resistance
Insulin resistance is a universal phenomenon in NAFLD
G. C. Farrell et al., Hepatology 2002
Insulin resistance is intimately related to obesity

Proposed Mechanism :
1. Insulin suppress adipose tissue lipolysis. In situations of
IR impaired suppression leads to increased efflux of FFA
from adipose tissue .

2. Up -regulation of transcription factor Sterol Regulatory
Element Binding Protein-1c (SREBP-1c), a key gene
involved in DNL .

3. Down regulation mitochondrial beta oxidation of FFAs.

4. In the liver, IR increased intracellular degradation of
VLDL and apo B-100.


Forward vicious cycle

Many abnormalities reported in NAFLD :

FFAs ,
TNF- ,
Nuclear factor kappa (NF-k ),
Ceramide
SOCS(suppressors of cytokine signaling )
Cytochrome CYP2E1 .
Lipid metabolites like diacyl glycerol (DAG) .

Interfere with
insulin
signalling
cascade, and
contribute to
IR
NAFLD : Beyond I R
Hepatic De novo Lipogenesis
By product of hepatic gluconeogenesis and results in the
synthesis of FFA s in the liver .

Sensitive to insulin resistance

Normally responsible for <5% of hepatic FFAs ,but IR states
increases to about 20 % .

Nuclear transcription factors regulates Hepatic DNL

The principal nuclear receptors are
1. SREBP1 (sterol regulatory element binding protein1c)
2. ChREBP (carbohydrate response element-binding protien)
3. LXR Alpha
4. FXR (Farnesoid X Receptor )
5. PPAR Gamma
6. PPAR Alpha

Mutations leads to hepatic steatosis ,observed in mice
models .
McPherson R,et al , Biochem Cell Biol 2004


Dietary factors
Dietary Fatty Acids
Contribute to increased fatty acid influx into the liver .
Trans fatty acid, derived from partial dehydrogenation of
vegetable oils ( found in processed food) have a causative
role in hepatic steatosis.
Kim HJ et al ,J Lipid Res 2002
Fructose
Increased consumption of high fructose corn syrup (e.g soft
drinks) is linked with insulin resistance syndrome.
The hepatic metabolism of fructose favors de novo lipogenesis
and ATP depletion .
Xiaosen Ouyang et al , Journal of Hepatology 2008
Pathogenesis of steatohepatitis

1. Inflammatory cytokines
2. FFA and lipotoxicity
3. Adipokines
4. Oxidative stress and mitochondrial dysfunction ,ER stress
5. Bacterial overgrowth
Inflammatory cytokines

Steatosis associated with chronic hepatic inflammation .

This effect is mediated by activation of the IKK-B/NF-KB
signalling pathway.
Activated directly by FFA .

Increases pro-inflammatory mediators - TNF , IL-1 , IL-6 .
Interferes with insulin signalling IR .


Serum and hepatic levels of TNF- elevated NASH patients, levels
correlate with histological severity.
Crespo J et al ,Hepatology 2001
TNF- promotes IR.
Inhibition of TNF- signalling improves IR and NASH.

Serum IL-6 levels elevated in IR and NAFLD, which correlate with
inflammation and fibrosis .
Klover PJ, Endocrinology 2005

Cytokines can replicate all of the histological features of NASH, i.e
neutrophil chemotaxis, hepatocyte apoptosis /necrosis , Mallory
body formation and stellate cell activation.
Day CP. Et al Gastroenterology 2006
FFA and lipotoxicity
Increased levels of FFA - directly toxic to hepatocytes .
Mechanisms :
1. Leads to stimulation of TNF-.
2. Up-regulates cytochrome P450 isoenzymes , generation of
ROS and lipid peroxidation.

3. Up-regulates PPAR-, which promotes FA oxidation but
increases oxidative stress through dicarboxylic acid
derivatives .

4. Directly toxic to cellular membranes, forms toxic FA ethyl
ethers and disruption of mitochondrial function .

Modified 2-hit hypothesis
The accumulation of FFA alone is sufficient to induce liver
damage, without recourse for a second hit.

Rather than being harmful, triglyceride accumulation in the
form of steatosis may actually be protective by preventing FFA-
induced inflammation and oxidative stress.

Yamaguchi K, Hepatology 2007
Adipokines

Adipose tissue - actively secreting endocrine organ.

Adipokines (adipocyte-derived peptides) :- leptin and adiponectin

Adiponectin
Anti-inflammatory (antagonises TNF-)
Increases insulin sensitivity
Increases fatty acid -oxidation .

Serum adiponectin levels reduced in obesity, IR , diabetes mellitus,
and metabolic syndrome .
The administration of recombinant adiponectin improves
biochemical and histological parameters of NAFLD in a murine
model.
Tomita K et al Hepatology 2008

Leptin
Role in pathogenesis is unclear .

Satiety hormone , controls food intake and energy regulation .
Involved with insulin signaling and regulation of glucose
metabolism in peripheral tissues .
Role in regulating the partitioning of fat between mitochondrial -
oxidation and triglyceride synthesis in the liver ????


Severe steatohepatitis develop in leptin-deficient (ob/ob) mice .
Higher levels observed in obese and NAFLD patients which are
regarded as states of leptin resistance .
Huang XD et al , World J Gastroenterol 2008









Oxidative stress and mitochondrial
dysfunction

Plays important role in progression .

Increased FFA load oxidation overwhelmed
reactive oxygen species (ROS).

ROS induce oxidative stress , activates inflammatory pathways
causes mitochondrial damage .

Elevated expression of hepatic microsomal FA oxidizing enzyme
Cytochrome P450 (CYP2E1) -- observed in human and animal
models of NASH and represents a potent source of ROS.
Farrell GC et al ,Hepatology 1998
Mitochondrial Damage
Structurally abnormal; they become ballooned and lose
cristae ,inclusion bodies .

Decrease in the activity of mitochondrial respiratory
chain complexes , with a concomitant increase in
mitochondrial ROS formation .

Altered ATP homeostasis
Bacterial overgrowth and endotoxin

Endotoxins are suspected in the pathogenesis of alcoholic
steatohepatitis .

Serum levels of bacterial endotoxin and LPS stimulate
hepatic production of TNF-, IL-6, and IL-8 and activate an
inflammatory response that leads to hepatic necrosis .


Portal endotoxemia was believed to contribute to NASH
associated with surgical jejunoileal bypass (performed in the
past to treat obesity), the risk of which was reduced with
antibiotics .
Hocking MP, et al ,Dig Dis Sci 1998


Small intestinal bacterial overgrowth and increased gut
permeability have been found more frequently in patients with
NASH when compared with controls .
Miele L et al , Hepatology 2009
Revealed over-representation of Lactobacillus species and selected
members of phylum Firmicutes

A significant increase in fecal ester VOC in obese NAFLD patients
Fibrosis /cirrhosis
Fibrosis frequent histologic finding in advanced NAFLD.
Exact pathogenesis unknown .

Fibrosis :
activation and proliferation of hepatic stellate cells in the
subendothelial space of Disse
secretion of extracellular matrix(ECM) components,i.e collagen
types I and III

Factors involved in fibrogenic process :
inflammatory cytokines, angiotensin, alterations in the ECM,
growth factors, and oxidative stress
Enhanced hepatic production of transforming growth factor-
(TGF- ) --- activates stellate cells .


Hyperinsulinemia associated with NAFLD may stimulate
release of
Platelet-derived growth factor (PDGF),
Connective tissue growth factor (CTGF ) ,
Vascular endothelial growth factor (VEGF),
Fibroblast growth factor and other cytokines .

Genetic predisposition
Play an important role in hepatic steatosis progressing into NASH
and cirrhosis .

Polymorphisms in genes related to lipid metabolism, IR, oxidative
stress, cytokines/ adipokines and fibrogenesis may all increase
susceptibility to NASH development .

SNP in the angiotensinogen and TGF-1 genes to be associated
with advanced hepatic fibrosis in obese patients.
Yoneda M,et al Liver Int 2009
Environmental factors
The third hit hypothesis

Third-hit has been added to reflect inadequate hepatocyte
proliferation.

In the healthy liver, cell death stimulates replication of mature
hepatocytes which replace the dead cells and reconstitute normal
tissue function.

However oxidative stress, lipotoxicity inhibits the replication of
mature hepatocytes which results in expansion of the hepatic
progenitor cell (oval cell) population.
Richardson MM,et al Gastroenterology 2007;

These HPCs are strongly correlated with fibrosis stage .


Activation of these cells has also been implicated in
hepatocellular carcinogenesis .


Inappropriate proliferation of hepatocyte progenitors
represents the proposed third hit in NAFLD pathogenesis .
Jou j et al ,Semin Liver Dis 2008
Conclusion
The pathogenesis of NAFLD and NASH is a complex multi step process.

Genetic factors
obesity
hyperinsulinemia and metabolic syndrome and
environmental and dietary factors.

Clinical profile varies .
Recent advances in the understanding of pathogenesis of NAFLD can help in
the development of potential therapeutic strategies .



Impact steatosis
and the
subsequent
progression to
steatohepatitis
Thank you
Proposed pathogenesis of NASH. The likelihood of progression to advanced
NASH/cirrhosis results from a complex interplay between genetic predisposition and the
mechanisms described earlier.
Oxidation of FFas
Mitochondrial beta oxidation

Peroxisomal Beta oxidation

PPAR Gamma provides key enzymes for these
processes which leads to ATP production.
Impaired FFA elimination
Intrahepatic FFAs are metabolized by two pathways .
a) Esterified to VLDL particles ,excreted into systemic
circulation .

b) Mitochondrial Beta oxidation .
Transmembrane Carrier families and
entry of FFAs into liver
Influx of FFAs into hepatocytes is mediated by four
transmembrane carrier families .
1. Fatty acid transport proteins
2. Fatty acid translocase
3. Fatty acid binding protein and
4. Caveolins

The overexpression of these in mice models have shown to
encourage hepatic steatosis .
Ref..

LIPOGENIC ENZYMES


1. Diacylglycerol Acyltransferase1 and 2 (DGAT1 and
DGAT 2 )

2. Acetyl Co-A Carboxylase 1 and 2 (ACC1 and 2)
DGAT 1 and DGAT 2 enzymes catalyze the final step in
triglyceride synthesis .

DGAT 1 facilitates VLDL secretion while DGAT2 promotes
hepatic lipid accumulation .

Mice who over expresses DGAT2 develop increased
hepatic steatosis .

Inhibition of DGAT2 causes a marked reduction in
hepatic TG content via downregulation of SREBP1 and
upregulation of Beta oxidation of FFAs .