Molecular basis and mechanisms of progression of non-alcoholic steatohepatitis

Article (PDF Available)inTrends in Molecular Medicine 14(2):72-81 · March 2008with202 Reads
DOI: 10.1016/j.molmed.2007.12.003 · Source: PubMed
Non-alcoholic steatohepatitis (NASH), a cause of cirrhosis and hepatocellular carcinoma, is characterized by fatty infiltration of the liver, inflammation, hepatocellular damage and fibrosis. Progress has been made in understanding the molecular and cellular mechanisms implicated in the pathogenesis of this condition, therefore, we here review recent developments regarding the basic mechanisms of NASH development. Accumulation of triglycerides in the hepatocytes is the result of increased inflow of free fatty acids and de novo lipogenesis. Steatosis leads to lipotoxicity, which causes apoptosis, necrosis, generation of oxidative stress and inflammation. The resulting chronic injury activates a fibrogenic response that leads eventually to end-stage liver disease. A better understanding of these mechanisms is crucial for the design of novel diagnostic and therapeutic strategies.
This article was published in an Elsevier journal. The attached copy
is furnished to the author for non-commercial research and
education use, including for instruction at the author’s institution,
sharing with colleagues and providing to institution administration.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
Author's personal copy
Molecular basis and mechanisms of
progression of non-alcoholic
Fabio Marra
, Amalia Gastaldelli
, Gianluca Svegliati Baroni
, Gianluca Tell
and Claudio Tiribelli
Dipartimento di Medicina Interna and Centro di Ricerca, Trasferimento ed alta Formazione DenoTHE, University of Florence,
Viale Morgagni 85, I-50134, Florence, Italy
Istituto di Fisiologia Clinica, Consiglio Nazionale delle Ricerche, Via Moruzzi 1, I-56124, Pisa, Italy
Dipartimento di Gastroenterologia, University of Marche, Via Tronto 10, I-60020, Ancona, Italy
Dipartimento di Scienze e Tecnologie Biomediche, University of Udine, P. le Kolbe 4, I-33100, Udine, Italy
Centro Studi Fegato and Department BBCM, University of Trieste, Piazzale Europa 1, I-34127, Trieste, Italy
Non-alcoholic steatohepatitis (NASH), a cause of
cirrhosis and hepatocellular carcinoma, is characterized
by fatty infiltration of the liver, inflammation, hepato-
cellular damage and fibrosis. Progress has been made in
understanding the molecular and cellular mechanisms
implicated in the pathogenesis of this condition, there-
fore, we here review recent developments regarding the
basic mechanisms of NASH development. Accumulation
of triglycerides in the hepatocytes is the result of
increased inflow of free fatty acids and de novo lipogen-
esis. Steatosis leads to lipotoxicity, which causes apop-
tosis, necrosis, generation of oxidative stress and
inflammation. The resulting chronic injury activates a
fibrogenic response that leads eventually to end-stage
liver disease. A better understanding of these mechan-
isms is crucial for the design of novel diagnostic and
therapeutic strategies.
Introduction to the problem
The term non-alcoholic steatohepatitis (NASH) (see Glos-
sary; Box 1) was coined initially by Ludwig to describe
histopathological findings typical of alcoholic liver disease
in a group of patients without significant alcohol consump-
tion [1]. NASH is observed in a subset of patients with non-
alcoholic fatty liver disease (NAFLD), defined as fat
accumulation in the liver exceeding 5–10% by weight.
The clinical relevance of these conditions is related to
the high prevalence of NAFLD in the general population
and to the possible evolution of NASH towards end-stage
liver disease, including hepatocellular carcinoma, as well
as the need for liver transplantation [2].
NAFLD is considered the hepatic manifestation of the
metabolic syndrome, a cluster of closely related clinical
features linked to visceral obesity and characterized by
insulin resistance, dyslipidemia and hypertension [3].
With the rapidly increasing prevalence of the metabolic
syndrome in the general population, NAFLD has become
the most common cause of liver disease in Western
countries [2]. Hepatocellular injury, inflammation and
fibrosis are hallmarks of NASH, which are observed in
only a f raction of subjects with NAFLD, although the
exact mechanisms leading from NAFLD to NASH are still
largely unknown. In recent years, a large amount of
information on the mechanisms of fat infiltration,
damage, i nflammation and fibrosis in NASH has become
available. This review summarizes recent data that
have been gener ated on this topic. An understanding of
these aspects is crucial in this rapidly moving field, in
which developments in basic science are translated
quickly into diagnostic and/or therapeutic applications.
Data discussed herein are derived from studies on clinical
pathophysiology, cellular and molecular biology and
animal experimentation. The animal experimentation
is performed in several different models and the most
commonly used, or of greatest relevance to the present
review, are reported in Table 1.
Adipokines: cytokines produced predominantly at the level of adipose tissue.
Their action is both local (autocrine or paracrine) and distant (hormonal).
Adipokines produced by visceral fat target the liver primarily through the portal
De novo lipogenesis: endogenous synthesis of FFAs within the liver.
Hepatic stellate cells: these surround the sinusoids in the normal liver, where
they have a ‘quiescent’ state. After injury, these cells ‘activate’ and acquire
features that are relevant for the development of fibrogenesis.
Non-alcoholic fatty liver disease (NAFLD): fat accumulation in the liver of
subjects with absent or low (<20–30 g/day) alcohol consumption.
Non-alcoholic steatohepatitis (NASH): the histological picture observed in a
subset of patients with NAFLD. Besides fatty infiltration, variable degrees of
hepatocyte damage, inflammation and fibrosis are present.
Peroxisome proliferator-activated receptors (PPARs): molecules belonging to
the family of nuclear hormone receptors. Regulate transcription on binding
with ligands. Transcriptional activity is modulated by a wide number of co-
activators and co-repressors.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS): highly
reactive molecules, the levels of which can increase dramatically during
different types of stress, resulting in damage to cell structures. This cumulates
into a situation defined as oxidative stress.
Thiazolidinediones: a class of antidiabetic drugs that function as PPAR-g
Visceral adiposity: fat accumulation inside the abdominal cavity, localized
mostly in the omentum. Products of this type of adipose tissue are drained by
the portal circulation.
Corresponding author: Tiribelli, C. (
1471-4914/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2007.12.003 Available online 22 January 2008
Author's personal copy
Factors leading to fat accumulation in the liver
Accumulation of fat, mainly in the form of triglycerides, is
the sine qua non for the development of NAFLD and
NASH. The dynamics of lipid in the hepatocytes is both
under control of distantly produced hormones (primarily
insulin) and locally generated factors and represents the
result of complex interactions among multiple cell types
located in different tissues.
Insulin resistance
Insulin resistance (IR) is usually regarded as an impair-
ment in peripheral (i.e. skeletal muscle) glucose metab-
olism because the muscle is quantitatively the major site of
insulin-stimulated glucose uptake. To overcome IR and
promote glucose storage, insulin-resistant subjects
increase insulin secretion and reduce insulin clearance
[4], resulting in metabolic changes in other tissues
(Figure 1). Insulin is a potent inhibitor of hepatic endogen-
ous glucose production (HGP), which is impaired in the
presence of hepatic insulin resistance [5]. Another import-
ant target of IR is adipose tissue, in which the antilipolytic
effect of insulin is reduced, resulting in an increased
release of free fatty acids (FFAs) into the systemic circula-
Subjects with NAFLD are highly insulin resistant at the
level of: (i) muscle because they exhibit reduced glucose
uptake [6]; (ii) liver because they show impaired suppres-
sion of HGP [6,7] and (iii) adipose tissue because they
exhibit high lipolytic rates and increased circulating FFAs
[6,8]. IR in NAFLD is a primary defect independent of
obesity and/or diabetes, as shown recently in non-obese,
non-diabetic subjects with NAFLD [6]. The presence of
diabetes worsens the problem because these subjects tend
to accumulate more abdominal fat (both hepatic and vis-
ceral), which is correlated strictly to the degree of both
peripheral and hepatic IR [9].
Sources and dynamics of hepatic lipids
The mechanism(s) responsible for the increase in hepatic
fat accumulation is unclear and little information is avail-
able on the time course of the development of hepatic
steatosis. Animal models indicate that the liver might
accumulate lipids within a few weeks and even a few days
[10]. It has been suggested that fatty liver might result
from increased delivery of fatty acids, impaired hepatic
fatty acid oxidation and/or impaired synthesis or secretion
of very low-density lipoprotein (VLDL). Among these fac-
tors, the increased flow of FFAs to the liver is considered as
the most important. There is general agreement that
NAFLD subjects have increased lipolysis and high circu-
lating FFA levels; tracer studies have shown that neither
hepatic nor total lipid oxidation is reduced but rather
increased [6,8]. Elevated secretion of VLDL is also
observed in these subjects [11]. The increased availability
of FFAs to the liver might be multifactorial depending on
increased release of fatty acids from adipocytes, excess
lipid content in the diet or increased de novo lipogenesis
(DNL) (i.e. endogenous FFA synthesis in the liver) [12]
(Figure 1). Animal and human studies indicate that vis-
ceral adipose tissue is the major source of the increased
FFA flow to the liver. Increased visceral fat has been
associated with hepatic IR [7,9] because visceral fat is
highly lipolytic and FFAs are released directly into the
portal vein. Animals administered with high-fat feeding
show both hepatic steatosis and impaired suppression of
HGP, whereas, in humans, hypocaloric, low-fat diets
decrease hepatocellular lipid levels by 40–80% [13] . Thus,
it has been postulated that preferential influx of FFAs
through the portal circulation is a relevant determinant
of hepatic lipid accumulation. High splanchnic lipid flux is
observed not only after high fat meals but also during
fasting conditions in subjects with predominantly abdomi-
nal obesity [14]. Accordingly, visceral fat accumulation has
been associated with NAFLD because a strong correlation
exists between visceral and liver fat content [7,9]. DNL
might be another important source of hepatic FFAs.
Although the contribution of DNL during fasting is rather
small (5%) in normal subjects, in patients with NAFLD,
DNL is elevated, with rates of approximately 25% [15].
DNL requires transcription of lipogenic genes, which is
stimulated by insulin through sterol regulatory element
binding protein 1 (SREBP-1) and by glucose through carbo-
hydrate-response element-binding protein [16].
The enlargement of adipose tissue and in particular of
visceral fat has been associated with a decreased release
of insulin-sensitizing and anti-inflammatory cytokines and
increased expression of proinflammatory molecules. IR is
associated with adipose tissue inflammation, which
modifies adipokine secretion. Recent evidence indicates
Box 1. Non-alcoholic steatohepatitis (NASH) in a nutshell
It is observed in a subset of patients with NAFLD (daily alcohol
intake 20 g/day).
The metabolic syndrome (central obesity, hyperglycemia, dysli-
pidemia and hypertension) is a major risk factor for fatty liver and
Besides fatty infiltration of the liver, NASH is characterized by
histological evidence of inflammation, hepatocellular damage
(ballooning, Mallory bodies) and/or fibrosis.
More severe insulin resistance, susceptibility to oxidative stress,
cytokine imbalance favoring inflammation and a ‘profibrogenic’
phenotype have been proposed as factors involved in the
progression from bland steatosis to NASH.
In the absence of NASH, simple steatosis is considered benign,
although it might worsen the course of chronic viral hepatitis or of
alcoholic liver disease.
Most patients with NAFLD are referred to physicians for mild
elevations in liver enzymes (aminotransferases and/or gGT) and/
or for detection of a ‘bright liver’ on abdominal ultrasound.
Magnetic resonance spectroscopy enables a precise measure-
ment of the amount of fat in the liver.
No imaging technique has the capability to differentiate NASH
from simple steatosis; this is only achieved by liver biopsy.
In a fraction of patients, NASH progresses to severe fibrosis,
cirrhosis and liver decompensation. Evolution of NASH is now
considered the cause of the majority of cases formerly defined as
‘cryptogenic cirrhosis’.
As in other forms of cirrhosis, the appearance of hepatocellular
carcinoma might occur as a consequence of NASH evolution.
Diet (leading to reduction of body weight) and an increase in
physical activity have proven beneficial for patients with NAFLD.
No drugs are approved currently for the treatment of NASH.
Individual components of the metabolic syndrome should be
treated according to the existing guidelines.
Trends in Molecular Medicine Vol.14 No.2
Author's personal copy
that the chemokine monocyte chemoattractant protein-1
(MCP-1) is involved in the generation of adipose tissue
inflammation via an increased recruitment of macro-
phages, leading to fatty liver and insulin resistance [17].
Subjects with NAFLD exhibit decreased adiponectin
levels [18], which are correlated negatively with hepatic
TG content. The direct role of adiponectin in NAFLD is still
unclear, but this hormone is supposed to improve primarily
glucose and lipid metabolism. Adiponectin also inhibits the
expression of several proinflammatory cytokines [19], in-
cluding tumor necrosis factor (TNF)-a. Leptin is also
involved in the accumulation of hepatic TG through the
regulation of fat and its distribution [20] and the modu-
lation of hepatic oxidation [21]. A suggested mechanism for
the accumulation of hepatic TG could therefore be a resist-
ance to leptin action independently of IR [20]. Leptin also
has a multifunctional role in inflammation, acting as a
proinflammatory stimulus [22]. Among other factors
released within adipose tissue, TNF-a promotes lipolysis
and increases FFAs; both TNF-a and interleukin (IL)-6 are
related to mitochondrial dysfunction. The role of resistin is
still unclear. Resistin is produced by adipocytes and mono-
nuclear cells in rodents and mainly by stromal cells of
adipose tissue in humans [23]. Although plasma-resistin
levels correlate with hepatic TG and are decreased during
pioglitazone treatment, its circulating levels are not
increased with increased body-mass index or with insulin
resistance and are not elevated uniformly in Type 2 dia-
betes [23]. Intrahepatic resistin is increased in patients
with NASH and inflammatory cells contribute to its
expression [24]. Recently, the proinflammatory action of
visfatin, a protein produced preferentially by visceral adi-
pose tissue, has also been reported [25]. In spite of the
different molecules involved in the inflammatory cyto-
kines, the role of each of these factors and the interconnec-
tion among each other needs still to be elucidated.
Peroxisome proliferator-activated receptors
Three isoforms of peroxisome proliferator-activated recep-
tors [(PPARs); PPARa, PPARg and PPARb (or d)] have
been identified and are believed to have a central role in
the sensing of nutrient levels and in the regulation of lipid
and glucose metabolism [26]. Synthetic ligands for PPARg
(thiazolidinediones) and PPARa (fibrates) are used in the
treatment of diabetes and dyslipidemia. PPARg agonists
augment insulin-mediated adipose tissue uptake and sto-
rage of FFAs and also inhibit hepatic fatty acid synthesis
through the activation of adenosine monophosphate
(AMP)-activated protein kinase (AMPK) [27]. Thus, local
effects of PPARg agonists in adipose tissue can result in
reduced IR in distant sites, leading to a reduction of
circulating FFAs, glucose production, visceral fat accumu-
lation and liver steatosis [5,28]. Both PPARa and PPARg
have anti-inflammatory actions [27] and reduce adipose
Table 1. Animal models for the study of NAFLD and NASH
Description Limitations
Thrifty genotype
Psammomys obesus (sand rat) is a gerbil that shows normal metabolic
parameters when fed a low-calorie diet, whereas, on standard chow, it
develops all the parameters of the metabolic syndrome,
aminotransferase elevation and macrovesicular steatosis [78]
The possibility of a pathogenetic cascade leading
from the thrifty genotype to the metabolic
syndrome and to NAFLD or NASH remains to be
A contribution to obesity is provided by abnormalities in energy
expenditure, which depends in part on adaptive thermogenesis.
Adaptive thermogenesis involves sensing of dietary excesses, leading to
an increase in energy expenditure, that is, ‘diet-induced thermogenesis’.
The sympathetic nervous system is the efferent pathway by which the
brain regulates adaptive thermogenesis and mice lacking all three b-
adrenergic receptors (b-less mice) have been created [79]. When fed a
hypercaloric diet, b-less mice develop massive obesity, due entirely to a
defect in diet-induced thermogenesis
The target tissues of the sympathetic nervous
system in mediating diet-induced thermogenesis
are still unknown. No data on the development of
insulin resistance, NAFLD and NASH are available
on b-less mice
Models of
Mutations in the obese (ob )ordiabetes (db) genes prevent the synthesis
(ob/ob mice) or the effects (db/db mice and fa/fa rats) of leptin, a satiety
hormone that inhibits feeding and increases energy expenditure. These
strains share a common phenotype, being hyperphagic, obese, diabetic
and hyperlipidemic, with expanded white adipose tissue mass [22].
Increased expression of TNF-a and relatively low levels of adiponectin
promote lipolysis and FFA release. Activated sterol regulatory element
binding protein (SREBP)-1c promotes de novo lipogenesis
These models have been studied extensively,
although the ob mutation is not prevalent in the
human situation. Leptin levels correlate poorly
with the development of NASH. Little or no
fibrosis is observed in these animals
Models of
Feeding mice or rats with a methionine- and choline-deficient diet (MCD)
leads to the development of steatosis, inflammation and fibrosis. MCD
impairs VLDL assembly and secretion, reduces mitochondrial b-oxidation
and leads to induction of CYP2E1 expression, which induces ROS
production, mtDNA damage and apoptosis, with hepatic stellate
activation and extracellular matrix deposition [38]. Rats administered a
high-calorie diet containing 49% saturated fat develop visceral obesity,
increased levels of portal FFA, hepatic insulin resistance and increased
ROS production. Steatosis is associated with hepatocyte ballooning,
inflammation and apoptosis [80]
The MCD model does not replicate the phenotype
or the pathogenetic mechanisms of human NASH
because animals are cachectic, have low plasma
triglyceride levels and reduced liver weight/body
weight ratio and no insulin resistance. Collagen
deposition is evident after 6 months of the
high-fat diet
Several animal models have been proposed to investigate the different aspects that lead to NASH, although their ability to mirror the genetic or environmental abnormalities
observed in human beings might be limited. This table reports some of the aspects that might be investigated in rodent models, relevant for the exploration of mechanisms
leading to NASH. For detailed information on animal models of steatosis, please refer to [81].
Trends in Molecular Medicine Vol.14 No.2
Author's personal copy
tissue-derived circulating factors that could promote IR. In
particular, PPARg agonists increase adiponectin levels
and reduce resistin, TNF-a, IL-6 and C-reactive protein
levels in Type 2 diabetes patients and in patients with
NASH [29,30]. PPARg-induced increases in plasma
adiponectin could also mediate some of the insulin-sensi-
tizing effects of PPARg agonists. Recently, treatment with
thiazolidinediones was found to increase adiponectin levels
in NASH patients and this change was correlated with the
improvement in hepatic steatosis [30].
Mechanisms of damage in fat-laden hepatocytes
Fat accumulation in the cell might induce cytotoxicity
either directly or through sensitization to other agents.
Metabolic dysregulation, mitochondrial impairment and
oxidative stress have a significant role in determining
hepatocyte damage and result in profound changes in gene
expression, leading ultimately to apoptosis and contribut-
ing to the inflammatory process [31].
Mitochondrial dysfunction and oxidative stress
Mitochondria are involved in both FFA b-oxidation and
reactive oxygen species (ROS) generation. Patients with
NASH are characterized by abnormal mitochondria from
both a functional and a morphological point of view.
Accumulating evidence indicates that respiratory-chain
defects are a key determinant of mitochondrial dysfunc-
tion [32]. Mitochondrial dysfunction is mainly the
result of IR and the excess of FFAs (lipotoxicity). Mito-
chondrial impairment causes enhanced ROS production,
which, in turn, initiates a self-sustaining loop that leads
to chronic organelle damage. In fact, malondialdehyde
and 4-hydroxynonenal, resulting from cellular lipid
peroxidation, are able to inhibit cytochrome c oxidase
of mitochond rial complex IV and ROS per se might
damage both mitochondrial DNA (mtDNA) and iron–
sulphur cluster enzymes of the respiratory chain [33].
Inflammatory cy tokines, including TNF-a,alsocontrib-
ute to mitochondrial dy sfunctio n by inte rfering with the
mitochondrial respiratory chain and by forming super-
oxide anion [34]. An indirect effect of TNF-a in promot-
ing mitochondrial dysfunction is the increased
production of reactive nitrogen species (RNS) as a con-
sequence of nitric oxide synthase 2 (NOS2) induction.
RNS might inactivate proteins of the mitochondrial
respiratory chain functionally through nitration of tyro-
sine residues or the i ntermediate formation of S-nitroso-
cysteine or S-nitroso-glutathione [35] and might also
induce DNA damage [36]. The relevance of RNS f or
mitochondrial dysfunction is confirmed by data obtained
in ob/ob mice, in which administration of uric acid,
which reacts with peroxynitrite to form inactive
nitrogenous urates, reduced cytochrome c nitro-tyrosine
formation and lipoperoxide content [37].
The excess FFA oxidation in mitochondria, peroxysomes
and microsomes as well as the activation of Kupffer cells
Figure 1. Mechanisms of fat accumulation in non-alcoholic steatohepatitis. Insulin resistance causes an influx of FFAs to the liver, owing to increased lipolysis, especially in
the visceral adipose tissue. Increased de novo lipogenesis and fat from the diet also contribute to the fatty-acid pool. Both VLDL generation and FFA oxidation are increased
and are sufficient to prevent intrahepatic lipid accumulation. Abbreviations: DNL, de novo lipogenesis; GNG, gluconeogenesis; ROS, reactive oxygen species; TG,
triglycerides; VLDL, very low-density lipoproteins.
Trends in Molecular Medicine Vol.14 No.2
Author's personal copy
are also relevant sources of ROS, contributing to the
condition of oxidative stress typical of NASH. The
increased availability of FFAs determines the activation
of microsomal cytochrome P450 isoforms CYP2E1 and
CYP4A10/4A14, both involved primarily in FFA
v-oxidation, leading to an increased ROS production and
uncoupling mitochondrial respiration [32,38]. Accumu-
lation of cholesterol within mitochondria has been ident-
ified recently as an additional factor linking fatty
infiltration with the development of NASH [39].
Peroxisomal and microsomal b-oxidation cooperate in
limiting mitochondrial overload in the presence of excess
FFAs and it has been proposed that polymorphisms affect-
ing these pathways might contribute to the pathogenesis of
NASH [40]. Of notice is the finding that a non-mitochon-
drial source of ROS, such as the NADPH-oxidase system of
Kupffer cells, is also activated in NASH models, probably
as a consequence of lipoperoxide or endotoxin phagocytosis
[41]. This pathway is also present in activated hepatic
stellate cells (HSCs), possibly contributing to the general
oxidative stress condition. Nonetheless, inactivation of
NADPH oxidase by genetic deletion of gp91phox was
shown recently to be ineffective in blocking cytokine pro-
duction and fibrogenesis in dietary steatohepatitis [42].
Effects of oxidative stress
Chemical modification of essential biomolecules by ROS
and RNS causes their functional inactivation and leads to
either cell death or to an adaptive cellular response, for
example, activation of redox-sensitive transcription factors
[43,44] (nuclear factor (NF)-kB, Nrf-1 and Sp-1), which
contributes to the production of proinflammatory and
fibrogenic mediators by Kupffer cells and HSCs. Interest-
ingly, and potentially relevant for the pathogenesis of
NASH, Houstis showed a causal connection recently be-
tween disequilibrium in ROS production and IR [45].The
well established primary role of oxidative stress in the
pathogenesis of NASH is demonstrated by: (i) a significant
increase of markers of oxidative damage for lipids (i.e.
thiobarbituric acid reactive substances, malondialdehyde,
4-hydroxynonenal) [46,47], proteins (i.e. nitro-tyrosine
protein content) [8] and DNA (i.e. 8-OH-dG) [46],aswell
as an increase in thioredoxin, a plasmatic oxidative stress
marker [48]; and (ii) a concomitant reduction of antioxidant
factors, including coenzyme Q10, CuZn-superoxide dismu-
tase, catalase activity, glutathione and glutathione S-trans-
ferase [47]. Thus, as described earlier, consequences of
oxidative stress are associated with promotion and main-
tenance of cellular damage, involving the mitochondrial
‘vicious cycle’ both acting at local and systemic levels (Box 2).
Hepatocyte cell death in NASH
In spite of their powerful antioxidant resources, hepatocytes
suffer from the cytotoxic effect of oxidative stress, leading to
cell death by necrosis and apoptosis, as well as triggering
inflammation [49,50]. Both the intrinsic (triggered by cel-
lular stress) and the extrinsic (induced by death receptors)
pathways are involved in the pathogenesis of NASH [50].
The finding that the anti-apoptotic Bcl-2 is significantly
elevated in NASH patients compared with controls [51] is
suggestive of a major involvement of the extrinsic pathway,
in which Bcl-2 action is secondary. Inhibition of apoptosis
induced by death receptors and evaluation of plasmatic
content of caspase-activity products (i.e. cytokeratin-18
fragments, as in [50]) are potentially promising targets
for non-invasive diagnosis and therapy of NASH.
Gene expression control and NASH
The complex changes in hepatocyte biology imply a
relevant alteration in the gene expression profile. In
patients with NASH, several cellular responses coexist,
involving apoptotic genes, lipid metabolism, chaperone
activity, extracellular matrix remodeling, mitochondrial
activity and nucleic acid metabolism, as well as genes
involved in the adaptive response to oxidative stress
[52]. The transcription factors Sp1 and Sp3 have been
identified as potential new factors in the development of
NASH, being responsible for the early activation of genes,
such as type I procollagen, which is involved in active
fibrogenesis [31,53],orCCAAT-enhancer-binding protein
b (C/EBP b), which is involved in lipid synthesis, the
inflammatory response and endoplasmic reticulum stress
[54]. Of interest is the recent finding of a role for the
transcription factor Nrf-1 [44], a pivotal player in controlling
gene expression of antioxidant genes through the binding to
AU-rich element sequences, and the upregulation in NASH
biopsies [31] of apurinic/apyrimidinic endonuclease-1/redox
factor-1 (APE/Ref-1), a central coordinator of the adaptive
cellular response to oxidative stress. Interestingly, both Nrf-
1 and APE/Ref-1 are overexpressed in liver cancer [44].A
new system emerging from gene-deletion studies is
represented by the phosphatidylinositol 3-kinase/Akt/phos-
phatase and tensin homolog (PTEN) pathway [55] because
PTEN-deficient mice display features characteristic of
NASH as well as hepatocellular carcinoma. Despite the
enormous amount of data provided by platforms for gene-
expression analysis, little information on proteins, both in
terms of expression levels and post-translational modifi-
cations, or on potential circulating biomarkers for the diag-
nosis of NASH is available currently. Future work in this
Box 2. Mechanisms leading to hepatocyte damage in NASH
Excessive FFA oxidation can cause oxidative stress through
mitochondria, peroxisomes and microsomes.
Plasmatic markers of oxidative stress are increased in NASH
Increase in FFA (i) determines hyperactivation of microsomal
CYP450 enzyme isoforms CYP2E1 and CYP4A leading to an
increase in ROS production and (ii) acts in uncoupling mitochon-
drial respiration.
NASH is characterized by functional and morphological mito-
chondrial abnormalities.
Impairment of oxidative phosphorylation inhibits the respiratory
chain and b-oxidation, ultimately causing a marked impairment of
mitochondrial fatty acid oxidation and leading to ROS production.
TNF-a secreted by adipose tissue, hepatocytes and Kuppfer cells
contributes significantly to mitochondrial dysfunction by promot-
ing ROS and RNS production by iNOS.
Hepatocyte apoptosis (both by the intrinsic and the extrinsic
pathway) is involved in the pathogenesis of NASH.
A deep alteration in gene expression profiles occurs during NASH
pathogenesis. The transcription factors Sp1 and Sp3, C/EBPb, Nrf-1
and NF-kB are actively involved in controlling inflammation and
Trends in Molecular Medicine Vol.14 No.2
Author's personal copy
direction is needed to fully understand whether some kind of
specificity can be ascribed to the onset of NASH with respect
to other etiologies and to provide the reliable availability
of serum biomarkers for non-invasive diagnosis for this
It should be underscored that recent data indicate that
other factors, besides the overall amount of fat, might be
crucial for the induction of metabolic abnormalities, cell
damage and fibrogenic progression of NASH. Disruption of
the gene encoding the elongase Elovl6 in mice prevents the
formation of very-long-chain fatty acids [56]. When admi-
nistered a high-fat, high-sugar diet, the Elovl6-deficient
mice had a comparable degree of steatosis when compared
with wild-type mice but their sensitivity to insulin was
much higher. Recent reports indicate that the lipid
accumulation in the form of triglyceride might even be
protective for the development of liver fibrosis. Inhibition
of diacylglycerol acyltransferase 2 in mice by the use of
antisense oligonucleotides was effective in limiting trigly-
ceride accumulation, although oxidative stress, lobular
necroinflammation and fibrosis were more severe [57].
These changes were associated with higher intrahepatic
levels of FFAs, which are likely to have a major detrimen-
tal role in the process of lipotoxicity.
NASH: inflammation and fibrosis
Together with hepatocyte damage, inflammation and fibro-
sis are the key features denoting the progression from
simple steatosis to steatohepatitis. The molecular mechan-
isms of inflammation are largely cross-talking with those
responsible for hepatocellular damage and fibrosis. In a
large series of patients, hepatic inflammation or alanine
aminotransferase (ALT) levels, as a proxy of necroinflam-
matory activity, are independent predictors of the fibrogenic
progression of the disease. Finally, liver inflammation con-
tributes to insulin resistance and possibly to cardiovascular
risk through systemic inflammation [58].
Signaling pathways leading to hepatic inflammation
Among different components of the inflammatory infil-
trate, neutrophils are an important source of oxidative
stress-related molecules [59], although different types of
mononuclear cells are also present. Inflammation in NASH
is the result of a cross-talk between parenchymal and non-
parenchymal cells through biologically active soluble
mediators. Liver cells are also the target of factors gener-
ated by the visceral adipose tissue, especially when infil-
trated by inflammatory cells, as in obesity. Activated
Kupffer cells and hepatic stellate cells also contribute
significantly to cytokine expression during steatohepatitis
[60]. Finally, sinusoidal endothelial cells are important for
the interaction with circulating leukocytes, through the
expression of adhesion molecules (Figure 2). Hepatocyte
damage and oxidative stress are initial triggers to inflam-
mation, although additional factors, including endoplas-
mic reticulum stress, contribute to the generation of
inflammatory signals and IR [61]. These upstream events
converge on the activation of proinflammatory transcrip-
tion factors that ultimately cause the recruitment of
leukocytes. NF-kB activation is crucial for inflammation,
Figure 2. Mechanisms of inflammation in NASH. Damage to hepatocytes caused by fat loading and resulting lipotoxicity leads to the activation of intracellularsignaling
pathways, which leads to the expression of several cytokines that are responsible for the recruitment of inflammatory cells. Hepatic damage also affects the biology of other liver
cells, such as Kupffer cells, which become activated and contribute to cytokine secretion. Recruitment of inflammatory cells is also conditioned by factors produced by activated
hepatic stellate cells and sinusoidal endothelial cells. Most liver-resident cells are targeted by adipokines secreted by visceral adipose tissue These events lead to a vicious circle
that causes worsening of liver damage, further inflammation, maintenance of steatosis, disease progression and insulin resistance. Abbreviations: CAM, cell adhesion
molecule; ECM, extracellular matrix; ER, endoplasmic reticulum; JNK, c-Jun N-terminal kinase, PDGF, platelet-derived growth factor; TGF-b, transforming growth factor-b.
Trends in Molecular Medicine Vol.14 No.2
Author's personal copy
although it is also involved in the maintenance of cell
survival [43]. The NF-kB pathway is upregulated in rodent
models and in patients with NASH [62] (Figure 2). Con-
ditional activation of NF-kB within hepatocytes is suffi-
cient to trigger low-grade liver inflammation, steatosis and
IR [60]; deletion of the upstream kinase IkB kinase b
(IKKb) preserves the sensitivity to insulin when mice
are placed on a high-fat diet [63]. Recent data indicate a
much more complex role of this transcription factor
because neutralization in liver parenchymal cells of NF-
kB essential modulator (NEMO or IKKg), which is
required for NF-kB activation, causes steatohepatitis,
fibrosis and the development of liver cancer in mice [64].
This apparent discrepancy might be explained by the fact
that severe deficiency of NF-kB activation, such as in
NEMO conditional-knockout mice, leads to enhanced apop-
tosis, triggering compensatory hepatocyte proliferation,
oxidative stress, inflammation and fibrosis. It could be
speculated that strategies that are extremely effective in
neutralizing NF-kB activation might be detrimental ulti-
mately for the evolution of steatohepatitis and might even
favor the development of cancer.
JNK (c-Jun N-terminal kinase), a mediator of TNF-
induced cell death, is another signaling pathway associated
with both inflammation and IR. Genetic deletion of both
JNK-1 and JNK-2 in hepatocytes is accompanied by a
reduction of inflammation and IR in mice [65]. However,
although JNK-1-deficient mice are protected markedly from
the development of inflammation and liver injury, the phe-
notype of JNK-2-knockout mice is similar to that of wild-
type animals [66]. These data point to JNK, and particularly
the JNK-1 isoform, as an attractive target to limiting the
development of inflammation, injury and apoptosis associ-
ated with lipid accumulation in the hepatocyte.
Cytokines as effectors of proinflammatory signals
Intrahepatic gene expression and/or plasma levels of TNF-
a and IL-6 are increased in fatty liver and in NASH in
humans [67,68]. These factors are produced under the
control of NF-kB, JNK or p38
. Modulation of TNF-a
expression by genetic deletion or other means results in
amelioration of steatosis, inflammation and hepatocyte
damage in ob/ob mice and in dietary models of steatohe-
patitis [69], suggesting a pivotal role for this cytokine in
NASH. Contrary to this conclusion, it was reported
recently that, in dietary steatohepatitis, lipid peroxidation
and hepatocellular damage were similar in wild-type mice
or in animals with targeted deletion of TNF-a or its re-
ceptor, TNFR1 [62]. By contrast, interference with NF-kB
activation protected significantly from the development of
steatohepatitis and reduced the expression of TNF-a and
intercellular adhesion molecule-1 [62]. These observations
Table 2. Molecular mechanisms of hepatic fibrosis in NASH
Mechanism Evidence Potential clinical relevance
Inflammatory cells release fibrogenic factors, such as platelet-derived
growth factor and TGF-b1, which stimulate the profibrogenic actions of
HSCs. Depletion of natural killer cells worsens matrix accumulation,
inducing programmed cell death of activated HSCs
Anti-inflammatory drugs or cytokines might block
fibrosis. Chemokine antagonists are in development
Oxidative stress
Oxidative stress-related molecules mediate progression of liver fibrosis
and increase procollagen expression in human HSCs. Reactive
aldehydes induce nuclear translocation of JNK in HSCs and induce
expression of TGF-b and MCP-1 [82]
Potential usefulness of antioxidant approaches,
although their efficacy has not been proven
convincingly in humans
Generation of apoptotic hepatocytes stimulates fibrogenesis in vivo
and in vitro [83]. Phagocytosis of apoptotic bodies by HSCs amplifies
the process, causing NADPH-mediated production of ROS
IDN-6556, a general caspase inhibitor, is being
tested currently in clinical trials
Altered glucose
Diabetes is a potent predictor of fibrosis. Elevated glucose levels or
insulin upregulate TGF-b and connective tissue growth factor and also
affect HSC biology [84]. Receptors for advanced glycation end-products
are expressed by HSCs and have a role in migration of activated HSCs
Aggressive management of hyperglycemia might
slow fibrosis progression
Nuclear hormone
PPARg is expressed in quiescent HSCs and decreases with the
activation process. PPARg ligands revert most features of the activated
phenotype of HSCs and inhibit fibrosis in vivo [86]
Experimental work indicates that the antifibrotic
response occurs when animals are treated early.
Clinical trials suggest a beneficial action of
thiazolidinediones on fibrosis during NASH
Cannabinoids act through CB1 and CB2 receptors. CB2 mediates
antifibrogenic actions whereas CB1 modulates fibrosis positively.
Inactivation of CB1 receptors decreases fibrogenesis by lowering
hepatic TGF-b and reducing accumulation of fibrogenic cells in the
liver [87]
Availability of rimonabant, a CB1 receptor
antagonist, which reduces obesity and ameliorates
parameters of the metabolic syndrome
Angiotensin II induces profibrogenic actions in HSCs, which express
all components of the renin–angiotensin system. Interference with the
renin–angiotensin system attenuate fibrosis development in different
experimental models of chronic liver injury [88,89]
Patients with the metabolic syndrome and NASH
often require pharmacological treatment of
hypertension. Clinical trials are underway
Leptin is a potent profibrogenic factor, acting directly on HSCs and
potentially involved in the development of cancer [22,90,91].
Adiponectin ameliorates liver damage in models of steatohepatitis and
blocks fibrogenesis after toxic liver damage [92,93]
Adiponectin receptors have been characterized and
are probably a target for future studies in NASH
Information is derived from studies in animal models of steatohepatitis and in cultures of fibrogenic cells, primarily human or rodent stellate cells.
Trends in Molecular Medicine Vol.14 No.2
Author's personal copy
suggest that TNF-a is only one of the multiple effectors of
steatohepatitis, under the control of NF-kB, especially in
the dietary model.
Serum levels of IL-6, another NF-kB target, correlate
with parameters of chronic low-grade inflammation [68].
The role of IL-6 is somewhat controversial because this
cytokine has stimulatory effects on liver regeneration and
a possible beneficial effect on fatty liver in mice has also
been reported [70]. Nonetheless, chronic exposure to IL-6 is
actually characterized by sensitization to liver damage and
several lines of evidence identify IL-6 as a mediator of
inflammation and IR [71]. IL-6 signals through activation
of the Jak–STAT pathway, which is regulated negatively
by the suppressors of cytokine signaling (SOCS) family
[72]. SOCS1 and 3 mediate insulin resistance in response
to cytokines, blocking insulin-receptor signaling at differ-
ent levels [72]. SOCS also interfere with leptin-receptor
signaling, suggesting their contribution to the induction of
leptin resistance.
Other proinflammatory mechanisms
The pattern-recognition receptor (PRR) family has also been
suggested as contributing to the proinflammatory responses
in fatty liver, especially in the later stages of the disease,
when bacterial-derived products are increasingly absorbed
from the gut and target non-parenchymal liver cells, in-
cluding Kupffer cells. PRRs comprise Toll-like receptors
(TLRs) and other receptors that recognize endotoxin and
other bacterial products [73,74]. TLR activation triggers
multiple intracellular signaling pathways, including NF-
kB, and could be important for the amplification and main-
tenance of inflammatory signals and fibrosis [74,75].Che-
mokines are a subfamily of cytokines that regulates
inflammation through receptor-mediated recruitment of
different leukocyte subclasses. IL-8 and MCP-1, which
recruit neutrophils and mononuclear cells, respectively,
are under the control of NF-kB and their expression is
increased in patients with steatohepatitis and in animals
models thereof [68,76].
Molecular mechanisms of hepatic fibrosis in NASH
Fibrosis and cirrhosis are the final outcomes of all chronic
liver disease, however, some morphological and biological
differences distinguish fibrosis due to NASH from the one
secondary to other causes of liver damage. NASH-related
fibrosis develops primarily in the pericentral areas, where
thin bundles of fibrotic tissue surround groups of hepato-
cytes and thicken the space of Disse, in a ‘chicken wire’
fashion. The main cell type responsible for extracellular
matrix deposition is represented by HSCs, which undergo
activation in conditions of liver injury, acquiring a pheno-
type that enables them to participate in the liver wound-
healing process (reviewed in [77]). The profibrogenic mech-
anisms operating in NASH are partly in common with
those observed in other chronic liver diseases. However,
the increase in circulating adipokines, oxidative stress
generated by accumulation of fat in hepatocytes and the
hormonal profile associated with the metabolic syndrome
might have a specific role for the induction of fibrogenesis
in this condition. The molecular mechanisms of fibrogen-
esis in NASH are summarized in Table 2.
Concluding remarks
An impressive amount of information has been accumulated
in the past 5 years on all aspects of pathophysiology, mol-
ecular and cellular biology of NASH. Fatty infiltration of the
liver is closely linked to IR and is considered a component of
the metabolic syndrome. Ectopic fat infiltration might also
lead to hepatocyte damage, a process defined ‘lipotoxicity’.
Lipotoxicity and cell death are associated with activation of
proinflammatory pathways, leading to leukocyte infiltra-
tion, damage amplification and fibrosis. Despite these
advances, several unanswered questions remain (Box 3).
Particular attention should be directed to the factors that
cause progressive liver damage, interplay among IR, inflam-
mation and fibrosis, and the relationships between fatty
liver, NASH and cardiovascular risk. In addition, the mol-
ecular factors of lipotoxicity need to be identified more
precisely, unraveling the role of different FFAs in this
context. It will be crucial to generate animal models that
more accurately reflect the metabolic changes characteristic
of NASH, to reliably test novel therapeutic approaches
because effective treatments for this condition are still
Conflicts of interest
The authors have no conflicts of interest to disclose in
relation to the present paper.
Work in F.M.’s laboratory was supported by grants from the Italian
Ministry of University and Research and from the University of Florence.
C.T. was suppo rted in part by a grant from Fondo Ricerca Stategica (FIST
2004), Fo ndo Studi Fegato-Onlus and Fonda zione CRT. G.T. was
supported in part by a grant from Progetto Ricerca Interesse Nazionale
(PRIN) and from FIST (04). The authors gratefully acknowledge the
contribution of the other members of the Fatty Liver Italian Network
(FLIN): Stefano Bellentani, Giorgio Bedogni, Elisabetta Bugianesi, Lory
S. Croce
, Giulio Marchesini and Gianluca Perseghin.
1 Ludwig, J. et al. (1980) Nonalcoholic steatohepatitis: Mayo Clinic
experiences with a hitherto unnamed disease. Mayo Clin. Proc. 55,
Box 3. Outstanding questions
Is the recruitment of different subsets of leukocytes relevant to the
pathogenesis of NASH?
What are the molecular effectors of lipotoxicity? Could triglyceride
accumulation be protective, at least in some instances? What is
the role of different FFAs?
What are the basic mechanisms underlying the lack of effects of
antioxidants in clinical trials?
Can we identify the molecular and genetic mechanisms of
transition from simple steatosis to NASH?
Is leptin resistance relevant to NASH? Are all liver cells leptin
Reversibility of NASH: is the return to steatosis without inflam-
mation possible?
Is the role of locally generated, intrahepatic molecules more
important than that of fat-derived or circulating factors?
Can we dissociate the molecular factors associated with increased
cardiovascular risk from those leading to advanced liver disease?
Are we getting closer to obtaining animal models that resemble
human NASH thoroughly?
Is the development of fibrosis more linked to inflammation or to
hepatocellular damage?
Trends in Molecular Medicine Vol.14 No.2
Author's personal copy
2 Angulo, P. (2002) Nonalcoholic fatty liver disease. N. Engl. J. Med. 346,
3 Marchesini, G. et al. (2003) Nonalcoholic fatty liver, steatohepatitis,
and the metabolic syndrome. Hepatology 37, 917–923
4 Utzschneider, K.M. and Kahn, S.E. (2006) Review: The role of insulin
resistance in nonalcoholic fatty liver disease. J. Clin. Endocrinol.
Metab. 91, 4753–4761
5 Gastaldelli, A. et al. (2006) The effect of rosiglitazone on the liver:
decreased gluconeogenesis in patients with type 2 diabetes. J. Clin.
Endocrinol. Metab. 91, 806–812
6 Bugianesi, E. et al. (2005) Insulin resistance in non-diabetic patients
with non-alcoholic fatty liver disease: sites and mechanisms.
Diabetologia 48, 634–642
7 Seppala-Lindroos, A. et al. (2002) Fat accumulation in the liver is
associated with defects in insulin suppression of glucose production
and serum free fatty acids independent of obesity in normal men. J.
Clin. Endocrinol. Metab. 87, 3023–3028
8 Sanyal, A.J. et al. (2001) Nonalcoholic steatohepatitis: association of
insulin resistance and mitochondrial abnormalities. Gastroenterology
120, 1183–1192
9 Gastaldelli, A. et al. (2007) Relationship between hepatic/visceral fat
and hepatic insulin resistance in nondiabetic and Type 2 diabetic
subjects. Gastroenterology 133, 496–506
10 Samuel, V.T. et al. (2004) Mechanism of hepatic insulin resistance in
non-alcoholic fatty liver disease. J. Biol. Chem. 279, 32345–32353
11 Musso, G. et al. (2005) Adipokines in NASH: postprandial lipid
metabolism as a link between adiponectin and liver disease.
Hepatology 42, 1175–1183
12 Carpentier, A. et al. (2002) Sensitivity to acute insulin-mediated
suppression of plasma free fatty acids is not a determinant of
fasting VLDL triglyceride secretion in healthy humans. Diabetes 51,
13 Petersen, K.F. et al. (2005) Reversal of nonalcoholic hepatic steatosis,
hepatic insulin resistance, and hyperglycemia by moderate weight
reduction in patients with Type 2 diabetes. Diabetes 54, 603–608
14 Nielsen, S. et al. (2004) Splanchnic lipolysis in human obesity. J. Clin.
Invest. 113, 1582–1588
15 Donnelly, K.L. et al. (2005) Sources of fatty acids stored in liver and
secreted via lipoproteins in patients with nonalcoholic fatty liver
disease. J. Clin. Invest. 115, 1343–1351
16 Tamura, S. and Shimomura, I. (2005) Contribution of adipose tissue
and de novo lipogenesis to nonalcoholic fatty liver disease. J. Clin.
Invest. 115, 1139–1142
17 Kanda, H. et al. (2006) MCP-1 contributes to macrophage infiltration
into adipose tissue, insulin resistance, and hepatic steatosis in obesity.
J. Clin. Invest. 116, 1494–1505
18 Bugianesi, E. et al. (2005) Plasma adiponectin in nonalcoholic
fatty liver is related to hepatic insulin resistance and hepatic fat
content, not to liver disease severity. J. Clin. Endocrinol. Metab.
19 Ouchi, N. et al. (1999) Novel modulator for endothelial adhesion
molecules: adipocyte-derived plasma protein adiponectin. Circulation
100, 2473–2476
20 Fishman, S. et al. (2007) Resistance to leptin action is the major
determinant of hepatic triglyceride accumulation in vivo. FASEB J.
21, 53–60
21 Huang, W. et al. (2007) Hepatic steatosis and plasma dyslipidemia
induced by a high-sucrose diet are corrected by an acute leptin infusion.
J. Appl. Physiol. 102, 2260–2265
22 Marra, F. (2007) Leptin and liver tissue repair: do rodent models
provide the answers? J. Hepatol. 46, 12–18
23 Arner, P. (2005) Resistin: yet another adipokine tells us that men are
not mice. Diabetologia 48, 2203–2205
24 Bertolani, C. et al. (2006) Resistin as an intrahepatic cytokine:
overexpression during chronic injury and induction of
proinflammatory actions in hepatic stellate cells. Am. J. Pathol. 169,
25 Moschen, A.R. et al. (2007) Visfatin, an adipocytokine with
proinflammatory and immunomodulating properties. J. Immunol.
178, 1748–1758
26 Gervois, P. et al. (2007) Drug insight: mechanisms of action and
therapeutic applications for agonists of peroxisome proliferator-
activated receptors. Nat. Clin. Pract. Endocrinol. Metab. 3, 145–156
27 Browning, J.D. and Horton, J.D. (2004) Molecular mediators of hepatic
steatosis and liver injury. J. Clin. Invest. 114, 147–152
28 Gastaldelli, A. et al. (2006) The effect of pioglitazone on the liver: role of
adiponectin. Diabetes Care 29, 2275–2281
29 Bajaj, M. et al. (2004) Plasma resistin concentration, hepatic fat
content, and hepatic and peripheral insulin resistance in
pioglitazone-treated type II diabetic patients. Int. J. Obes. Relat.
Metab. Disord. 28, 783–789
30 Belfort, R. et al. (2006) A placebo-controlled trial of pioglitazone in
subjects with nonalcoholic steatohepatitis. N. Engl. J. Med. 355,
31 Rubio, A. et al. (2007) Identification of a gene-pathway associated with
non-alcoholic steatohepatitis. J. Hepatol. 46, 708–718
32 Begriche, K. et al. (2006) Mitochondrial dysfunction in NASH:
causes, consequences and possible means to prevent it.
Mitochondrion 6, 1–28
33 Paradies, G. et al. (1992) The effect of aging and acetyl-L-carnitine on the
activity of the phosphate carrier and on the phospholipid composition in
rat heart mitochondria. Biochim. Biophys. Acta 1103, 324–326
34 Sanchez-Alcazar, J.A. et al. (2000) Tumor necrosis factor-alpha
increases the steady-state reduction of Cytochrome b of the
mitochondrial respiratory chain in metabolically inhibited L929
cells. J. Biol. Chem. 275, 13353–13361
35 Gow, A.J. and Stamler, J.S. (1998) Reactions between nitric oxide
and haemoglobin under physiological conditions. Nature
391, 169–
36 Szabo, C. et al. (1996) DNA strand breakage, activation of poly (ADP-
ribose) synthetase, and cellular energy depletion are involved in the
cytotoxicity of macrophages and smooth muscle cells exposed to
peroxynitrite. Proc. Natl. Acad. Sci. U. S. A. 93, 1753–1758
37 Garcia-Ruiz, I. et al. (2006) Uric acid and anti-TNF antibody improve
mitochondrial dysfunction in ob/ob mice. Hepatology 44, 581–591
38 Leclercq, I.A. et al. (2000) CYP2E1 and CYP4A as microsomal catalysts
of lipid peroxides in murine nonalcoholic steatohepatitis. J. Clin.
Invest. 105, 1067–1075
39 Mari, M. et al. (2006) Mitochondrial free cholesterol loading sensitizes
to TNF- and Fas-mediated steatohepatitis. Cell Metab. 4, 185–198
40 Fan, C.Y. et al. (1998) Steatohepatitis, spontaneous peroxisome
proliferation and liver tumors in mice lacking peroxisomal fatty
acyl-CoA oxidase. Implications for peroxisome proliferator-activated
receptor alpha natural ligand metabolism. J. Biol. Chem. 273, 15639–
41 De Minicis, S. et al. (2006) NADPH oxidase in the liver: defensive,
offensive, or fibrogenic? Gastroenterology 131, 272–275
42 dela Pena, A. et al. (2007) NADPH oxidase is not an essential mediator
of oxidative stress or liver injury in murine MCD diet-induced
steatohepatitis. J. Hepatol. 46, 304–313
43 Schwabe, R.F. and Brenner, D.A. (2007) Nuclear factor-kB in the liver:
friend or foe? Gastroenterology 132, 2601–2604
44 Xu, Z. et al. (2005) Liver-specific inactivation of the Nrf1 gene in adult
mouse leads to nonalcoholic steatohepatitis and hepatic neoplasia.
Proc. Natl. Acad. Sci. U. S. A. 102, 4120–4125
45 Houstis, N. et al. (2006) Reactive oxygen species have a causal role in
multiple forms of insulin resistance. Nature 440, 944–948
46 Seki, S. et al. (2002) In situ detection of lipid peroxidation and oxidative
DNA damage in non-alcoholic fatty liver diseases. J. Hepatol. 37, 56–62
47 Yesilova, Z. et al. (2005) Systemic markers of lipid peroxidation and
antioxidants in patients with nonalcoholic fatty liver disease. Am. J.
Gastroenterol. 100, 850–855
48 Sumida, Y. et al. (2003) Serum thioredoxin levels as a predictor of
steatohepatitis in patients with nonalcoholic fatty liver disease. J.
Hepatol. 38, 32–38
49 Feldstein, A.E. et al. (2003) Hepatocyte apoptosis and Fas expression
are prominent features of human nonalcoholic steatohepatitis.
Gastroenterology 125, 437–443
50 Wieckowska, A. et al. (2006) In vivo assessment of liver cell apoptosis as
a novel biomarker of disease severity in nonalcoholic fatty liver disease.
Hepatology 44, 27–33
51 Ramalho, R.M.
et al. (2006) Apoptosis and Bcl-2 expression in the
livers of patients with steatohepatitis. Eur. J. Gastroenterol. Hepatol.
18, 21–29
52 Younossi, Z.M. et al. (2005) A genomic and proteomic study of the
spectrum of nonalcoholic fatty liver disease. Hepatology 42, 665–674
Trends in Molecular Medicine Vol.14 No.2
Author's personal copy
53 Garcia-Ruiz, I. et al. (2002) Sp1 and Sp3 transcription factors mediate
malondialdehyde-induced collagen alpha 1(I) gene expression in
cultured hepatic stellate cells. J. Biol. Chem. 277, 30551–30558
54 Rahman, S.M. et al. (2007) CCAAT/enhancing binding protein beta
deletion in mice attenuates inflammation, endoplasmic reticulum
stress, and lipid accumulation in diet-induced nonalcoholic
steatohepatitis. Hepatology 45, 1108–1117
55 Watanabe, S. et al. (2005) Hepatocyte-specific Pten-deficient mice as a
novel model for nonalcoholic steatohepatitis and hepatocellular
carcinoma. Hepatol. Res. 33, 161–166
56 Matsuzaka, T. et al. (2007) Crucial role of a long-chain fatty acid
elongase, Elovl6, in obesity-induced insulin resistance. Nat. Med. 13,
57 Yamaguchi, K. et al. (2007) Inhibiting triglyceride synthesis improves
hepatic steatosis but exacerbates liver damage and fibrosis in obese
mice with nonalcoholic steatohepatitis. Hepatology 45, 1366–1374
58 Van Gaal, L.F. et al. (2006) Mechanisms linking obesity with
cardiovascular disease. Nature 444, 875–880
59 Ikura, Y. et al. (2006) Localization of oxidized phosphatidylcholine in
nonalcoholic fatty liver disease: impact on disease progression.
Hepatology 43, 506–514
60 Cai, D. et al. (2005) Local and systemic insulin resistance resulting
from hepatic activation of IKK-b and NF-kB. Nat. Med. 11, 183–190
61 Marciniak, S.J. and Ron, D. (2006) Endoplasmic reticulum stress
signaling in disease. Physiol. Rev. 86, 1133–1149
62 Dela Pena, A. et al. (2005) NF-kB activation, rather than TNF,
mediates hepatic inflammation in a murine dietary model of
steatohepatitis. Gastroenterology 129, 1663–1674
63 Arkan, M.C. et al. (200 5) IKK-b links inflammation to obesity-induced
insulin resistance. Nat. Med. 11, 191–198
64 Luedde, T. et al. (2007) Deletion of NEMO/IKKg in liver parenchymal
cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell
11, 119–132
65 Hirosumi, J. et al. (2002) A central role for JNK in obesity and insulin
resistance. Nature 420, 333–336
66 Schattenberg, J.M. et al. (2006) JNK1 but not JNK2 promotes the
development of steatohepatitis in mice. Hepatology 43, 163–172
67 Crespo, J. et al. (2001) Gene expression of tumor necrosis factor alpha
and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis
patients. Hepatology
34, 1158–1163
68 Haukeland, J.W. et al. (2006) Systemic inflammation in nonalcoholic
fatty liver disease is characterized by elevated levels of CCL2. J.
Hepatol. 44, 1167–1174
69 Li, Z. et al. (2003) Probiotics and antibodies to TNF inhibit
inflammatory activity and improve nonalcoholic fatty liver disease.
Hepatology 37, 343–350
70 Hong, F. et al. (2004) Interleukin 6 al leviates hepatic steatosis and
ischemia/reperfusion injury in mice with fatty liver disease. Hepatology
40, 933–941
71 Klover, P.J. et al. (2005) Interleukin-6 depletion selectively improves
hepatic insulin action in obesity. Endocrinology 146, 3417–3427
72 Howard, J.K. and Flier, J.S. (2006) Attenuation of leptin and insulin
signaling by SOCS proteins. Trends Endocrinol. Metab. 17, 365–371
73 Brun, P. et al. (2005) Exposure to bacterial cell wall products triggers
an inflammatory phenotype in hepatic stellate cells. Am. J. Physiol.
Gastrointest. Liver Physiol. 289, G571–G578
74 Schwabe, R.F. et al. (2006) Toll-like receptor signaling in the liver.
Gastroenterology 130, 1886–1900
75 Seki, E. et al. (2007) TLR4 enhances TGF-b signaling and hepatic
fibrosis. Nat. Med. 13, 1324–1332
76 Leclercq, I.A. et al. (2004) Curcumin inhibits NF-kB activation and
reduces the severity of experimental steatohepatitis in mice. J.
Hepatol. 41, 926–934
77 Bataller, R. and Brenner, D. (2005) Liver fibrosis. J. Clin. Invest. 115,
78 Zoltowska, M. et al. (2001) Circulating lipoproteins and hepatic sterol
metabolism in Psammomys obesus prone to obesity, hyperglycemia and
hyperinsulinemia. Atherosclerosis 157, 85–96
79 Bachman, E.S. et al. (2002) bAR signaling required for diet-induced
thermogenesis and obesity resistance. Science 297, 843–845
80 Svegliati-Baroni, G. et al. (2006) A model of insulin resistance and
nonalcoholic steatohepatitis in rats: role of peroxisome proliferator-
activated receptor-alpha and n-3 polyunsaturated fatty acid treatment
on liver injury. Am. J. Pathol. 169, 846–860
81 Koteish, A. and Diehl, A.M. (2001) Animal models of steatosis. Semin.
Liver Dis. 21, 89–104
82 Parola, M. and Robino, G. (2001) Oxidative stress-related molecules
and liver fibrosis. J. Hepatol. 35, 297–306
83 Canbay, A. et al. (2004) Apoptosis: the nexus of liver injury and fibrosis.
Hepatology 39, 273–278
84 Svegliati-Baroni, G. et al.
(1999) Insulin and insulin-like growth factor-
1 stimulate proliferation and type I collagen accumulation by human
hepatic stellate cells: differential effects on signal transduction
pathways. Hepatology 29, 1743–1751
85 Fehrenbach, H. et al. (2001) Up-regulated expression of the receptor
for advanced glycation end products in cultured rat hepatic stellate
cells during transdifferentiation to myofibroblasts. Hepatology 34,
86 Marra, F. (2006) Thiazolidinediones and hepatic fibrosis: don’t wait too
long. Gut 55, 917–919
87 Teixeira-Clerc, F. et al. (2006) CB1 cannabinoid receptor antagonism:
a new strategy for the treatment of liver fibrosis. Nat. Med. 12,
88 Bataller, R. et al. (2003) NADPH oxidase signal transduces angiotensin
II in hepatic stellate cells and is critical in hepatic fibrosis. J. Clin.
Invest. 112, 1383–1394
89 Caligiuri, A. et al. (2003) Antifibrogenic effects of canrenone, an
antialdosteronic drug, on human hepatic stellate cells.
Gastroenterology 124, 504–520
90 Aleffi, S. et al. (2005) Upregulation of proinflammatory and
proangiogenic cytokines by leptin in human hepatic stellate cells.
Hepatology 42, 1339–1348
91 Saxena, N.K. et al. (2007) Concomitant activation of the JAK/STAT,
PI3K/AKT, and ERK signaling is involved in leptin-mediated
promotion of invasion and migration of hepatocellular carcinoma
cells. Cancer Res. 67, 2497–2507
92 Kamada, Y. et al. (2003) Enhanced carbon tetrachloride-induced
liver fibrosis in mice lacking adiponectin. Gastroenterology 125,
93 Xu, A. et al. (2003) The fat-derived hormone adiponectin alleviates
alcoholic and nonalcoholic fatty liver diseases in mice. J. Clin. Invest.
112, 91–100
Coming soon in the quarterly magazine for the history and philosophy of science:
Earthquake theories in the early modern period by F. Willmoth
Science in fiction - attempts to make a science out of literary criticism by J. Adams
The birth of botanical Drosophila by S. Leonelli
Endeavour is available on ScienceDirect,
Trends in Molecular Medicine Vol.14 No.2
    • "HCC ranks as the third highest cause of cancerrelated death globally, requiring an early diagnosis of NAFLD as a potential risk factor [25,30]. Steatosis is characterized by enhanced fatty infiltration within the liver in the absence of alcohol consumption, which may promote the progression to the more severe NASH, featured by mixed inflammatory-cell infiltration, hepatocyte ballooning and necrosis, portal hypertension and fibrosis [30,31] . However, the exact molecular mechanisms underlying NAFLD pathogenesis and progression are far from clear, and need to be further elucidated. "
    [Show abstract] [Hide abstract] ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease worldwide, ranging from simple steatosis to nonalcoholic steatohepatitis, which may progress to cirrhosis, eventually leading to hepatocellular carcinoma (HCC). HCC ranks as the third highest cause of cancer-related death globally, requiring an early diagnosis of NAFLD as a potential risk factor. However, the molecular mechanisms underlying NAFLD are still under investigation. So far, many in vitro studies on NAFLD have been hampered by the limitations of 2D culture systems, in which cells rapidly lose tissue-specific functions. The present liver-on-a-chip approach aims at filling the gap between conventional in vitro models, often scarcely predictive of in vivo conditions, and animal models, potentially biased by their xenogeneic nature.
    Full-text · Article · Jul 2016
    • "Inflammatory process in NASH and atherosclerosis may share common mechanisms, involving the local presence of activated macrophages [19]. An increase in M1 cytokines is also associated with the development of NASH in both experimental animals and humans [20][21][22]. Treatment of ob/ob mice with IL-33 strongly enhanced the mRNA expression of M2 markers in liver, including L-arginase (Arg1) and Chi313 [12]. In this work, treatment of NASH mice with IL-33 enhanced expression of Th2 cytokines (IL-4, IL-5 and IL-13) and M2 markers (Arg-1 and CD206), and reduced Th1 cytokines (IFN-γ) and M1 markers (IL-12p70 and TLR2), indicating that IL-33 promoted Th2 cytokine synthesis leading to the polarization of liver macrophages/Kupffer cells toward an M2 phenotype, and ultimately shifted the cytokine imbalance towards anti-inflammation, which might be beneficial for reversing hepatic steatosis. "
    [Show abstract] [Hide abstract] ABSTRACT: The aim of our work was to investigate the role of interleukin-33 (IL-33) and its receptor ST2 in the progression of diet-induced nonalcoholic steatohepatitis (NASH) in mice, and the characteristic expression in livers of patients with NASH. Mice were fed with high-fat diet (HFD) or methionine-choline 4-deficient diet (MCD) and injected intraperitoneally with IL-33. Both mRNA and protein expression levels of IL-33 and ST2 were up-regulated in the livers of mice fed with HFD or MCD. Treatment with IL-33 attenuated diet-induced hepatic steatosis and reduced activities of ALT in serum, as well as ameliorated HFD-induced systemic insulin resistance and glucose intolerance, while aggravated hepatic fibrosis in diet-induced NASH. Furthermore, treatment with IL-33 can also promote Th2 response and M2 macrophage activation and beneficial modulation on expression profiles of fatty acid metabolism genes in livers. ST2 deficiency did not affect hepatic steatosis and fibrosis when fed with controlling diet. IL-33 did not affect diet-induced hepatic steatosis and fibrosis in ST2 knockout mice. Meanwhile, in the livers of patients with NASH, IL-33 was mainly located in hepatic sinusoid, endothelial cells, and hepatic stellate cells. The mRNA expression level of IL-33 and ST2 was elevated with the progression of NASH. In conclusion, treatment with IL-33 attenuated diet-induced hepatic steatosis, but aggravated hepatic fibrosis, in a ST2-dependent manner.
    Article · May 2016
    Yinjie GaoYinjie GaoYuan LiuYuan LiuMei YangMei Yang+1more author...[...]
    • "Previous data examined the role of cytokine-and enzyme-induced inflammation and their link with IR and fatty liver [39][40]. Metabolic dysregulation, mitochondrial impairment and oxidative stress have a crucial role in determining hepatocyte damage, contributing to inflammatory process and NAFLD progression [41]. In particular, mitochondrial dysfunction is mainly related to the IR and lipotoxicity due to FFA excess [42]. "
    [Show abstract] [Hide abstract] ABSTRACT: The potential mechanisms of action of polyphenols in non alcoholic fatty liver disease (NAFLD) are overlooked. Here we evaluate the beneficial therapeutic effects of hydroxytyrosol (HT), the major metabolite of the oleuropein, in a nutritional model of insulin-resistance and NAFLD by high fat diet. Young male rats were divided into 3 groups receiving: 1. standard diet (STD; 10.5% fat); 2. high fat diet (HFD; 58.0% fat); 3. HFD + HT (10 mg/kg/die by gavage). After 5 weeks the oral glucose tolerance test was performed and at 6th week blood sample and tissues (liver and duodenum) were collected for following determinations.
    Article · Jan 2016 · Oncotarget
Show more