nonalcoholic steatohepatitis: recent advances from experimental models to clinical management
TRANSCRIPT
Clinical Biochemistry 3
Review
Nonalcoholic steatohepatitis: recent advances from experimental models
to clinical management
Piero Portincasa*, Ignazio Grattagliano, Vincenzo O. Palmieri, Giuseppe Palasciano
Department of Internal Medicine and Public Medicine, Clinica Medica bA. Murri,Q University Medical School of Bari, Piazza Giulio Cesare 11,
Policlinico, 70124 Bari, Italy
Received 20 July 2004; accepted 7 October 2004
Available online 10 December 2004
Abstract
A condition defined as nonalcoholic fatty liver disease (NAFLD) is frequently found in humans. Deemed as a benign condition until
recently, more emphasis is now put on the potential harmful evolution of the inflammatory form, that is, nonalcoholic steatohepatitis
(NASH), toward end-stage liver disease. This review highlights the major morphologic and pathophysiological features of NASH. The link
between experimental biochemical findings in animal models and clinical and therapeutic approaches in humans is discussed. Once all the
other causes of persistent elevation of serum transaminase levels have been excluded, the diagnosis of NASH can be only confirmed by liver
histology. Other noninvasive diagnostic tools, however, are being investigated to assess specific subcellular functions and to allow the follow-
up of patients at higher risk for major liver dysfunction. A better understanding of various pathogenic aspects of NASH will help in
identifying potential therapeutic approaches in these patients.
D 2004 The Canadian Society of Clinical Chemists. All rights reserved.
Keywords: Antioxidants; Breath test; Choline-deficient diet; Insulin resistance; Fatty liver; Microsomes; Mitochondria; Nonalcoholic steatohepatitis; Oxidative
stress; Peroxisomes
Contents
Experimental evidences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
From steatosis to steatohepatitis: the role of insulin resistance and free fatty acids (FFAs) . . . . . . . . . . . . . . . . . . . . 204
Role of microsomes and peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Redox balance in fatty livers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Mitochondrial abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
The intestinal–liver interaction hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Fasting and diet supplementation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Differences among models and susceptibility to necrotic cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Clinical approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
The natural course of disease and its relative complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Clinical presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
0009-9120/$ - s
doi:10.1016/j.cli
Abbreviation
hepatocellular ca
Alcoholism Scr
peroxisomal pro
ursodeoxycholic
* Correspon
E-mail addr
8 (2005) 203–217
ee front matter D 2004 The Canadian Society of Clinical Chemists. All rights reserved.
nbiochem.2004.10.014
s: ASH, Alcoholic steatohepatitis; BMI, body mass index; CAGE, Cut Annoyed Guilt Eye; FFAs, free fatty acids; GSH, glutathione; HCC,
rcinoma; HFE, hemochromatosis gene; IKK-h, inhibitor of kappa kinase beta; IL-8, interleukin-8; LPS, lipopolysaccharide; MAST, Michigan
eening Test; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OLT, orthotopic liver transplantation; PPAR,
liferation-activator receptor; ROS, reactive oxygen species; TNF-a, tumor necrosis factor-alpha; TGF-h, tumor growth factor-beta; UDCA,
acid.
ding author. Fax: +39 080 5478232.
ess: [email protected] (P. Portincasa).
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217204
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Since fatty infiltration of hepatocytes per se did not
appear to impair liver function, nonalcoholic fatty liver
disease (NAFLD) was traditionally considered to be a
benign condition with a low risk of progression toward more
serious illnesses. The finding of bfatty liverQ—most fre-
quently encountered during abdominal ultrasonography—
did not attract the attention of both investigators and
clinicians until very recently. However, two aspects must
be kept in mind when considering a bfatty liverQ. First,
among the NAFLD, the so-called nonalcoholic steatohepa-
titis (NASH) (the form resembling alcoholic steatohepatitis,
but occurring in patients without alcohol abuse [1]) is no
longer considered an invariably benign condition. Second,
the observation that grafted bfattyQ livers are indeed at
increased risk for primary nonfunction, which is propor-
tional to the degree of fatty degeneration and in turn leads to
discarding donated organs [2]. Due to the high prevalence in
the general population [3], fatty livers account for about
30% of the entire donor pool. Thus, the development of
therapeutic options able to preserve hepatic function after
transplantation of fatty livers will ultimately lead to the
expansion of the liver donor pool. A better understanding of
pathogenic mechanisms responsible for fatty accumulation
in hepatocytes and of mechanisms resulting in steatohepa-
titis will certainly help identify adequate measures for
decreasing the risk of evolution.
Here, we discuss the basic mechanisms responsible for
fatty degeneration of hepatocytes including current knowl-
edge on the behavior of hepatocyte subcellular organelles
occurring during metabolic derangements (i.e., insulin
resistance, oxidative stress, and excess free fatty acids
(FFAs)). The link between experimental and biochemical
findings in animal models and clinical and therapeutic
approaches in humans is also addressed.
Experimental evidences
From steatosis to steatohepatitis: the role of insulin
resistance and free fatty acids (FFAs)
Fatty infiltration of the liver implies accumulation of
triglycerides. This condition is classified as mild if the
amount of steatosis involves less than 30% hepatocytes,
moderate if it involves up to 60%, and severe if it is more than
60% [4]. Fatty liver degeneration occurs as a response of
hepatocytes to a variety of frequent conditions: namely
obesity [5], malnutrition [6], intestinal malabsorption [7],
metabolic and endocrine diseases including diabetes [8], and
thyroid diseases. A fatty liver can also be the consequence of
hepatotoxic drugs, accumulation of transition metals [9], and
hepatitis C infection [10]. In the presence of alcohol abuse,
the picture of fatty liver may occur as alcoholic steatohepatitis
(ASH). A fatty liver might also become a feature of the so-
called bmetabolic syndromeQ in which insulin resistance
plays a key role [8]. Notwithstanding all such causes of fatty
liver, no unique etiological factor has been identified in a
large number of cases. Fat accumulates in parenchymal liver
cells as a result of abnormal fatty acid metabolism [11], with
excessive delivery of free fatty acids to the liver compared to
the aliquot that can be metabolized, an increased mitochon-
drial synthesis of fatty acids, or a failure of the synthesis/
secretion of apolipoproteins or triglycerides [12].
The term NASH currently indicates a steatohepatitis of
nonalcoholic origin that may progress to end-stage liver
disease, that is, liver cirrhosis and hepatocellular carcinoma
(HCC; Fig. 1). NASH is responsible for asymptomatic
elevation of serum aminotransferases in 40–90% of cases
and represents a frequent cause of abnormal liver tests in
blood donors. Most patients with NASH have no symptoms
or signs of liver disease at the time of diagnosis, which is
confirmed at liver histology [13].
The primary metabolic abnormality switching fatty livers
to NASH is still unknown. Insulin resistance plays a key role,
since it may influence several intracellular metabolic path-
ways [14]. Higher levels of fasting serum insulin have been
frequently noted in NASH patients [15], and diabetes is often
identified in the family of NASH patients [16]. Insulin
resistance is associated with hypertrophy of the microsomal
oxidant function due to increased activity of the cytochrome
P-450 system. In turn, this is caused by loss of the insulin
inhibitory effect and to upregulation mechanisms common
also to peroxisomal h-oxidation [17]. This condition may
lead to intracellular oxidative stress if there is an imbalance
between pro-oxidant and antioxidant molecules [16].
Peripheral insulin resistance, increased fatty acid h-oxidation, and hepatic oxidative stress are all present in both
liver with fatty degeneration and in NASH. Only in NASH,
however, have structural defects of mitochondria been
described [14]. In fact, hepatic injury in fatty livers is likely
associated with depletion of mitochondrial glutathione
(GSH) content, which precedes the decrease in the total
liver GSH levels and probably occurs because of a defect in
the mitochondrial GSH uptake mechanism [18]. Recent
experimental evidence suggests that the link between
obesity, insulin resistance, and NASH is the increased
release of FFAs from adipose tissue [19]. Such a condition is
likely to occur when patients with expanded adipose mass
undergo sudden weight loss [20]. Since central rather than
peripheral obesity is associated with NASH [21], the
Fig. 1. Current views on the onset and progression of fatty liver toward NASH (both entities seen within the NAFLD spectrum) and, ultimately, to liver
cirrhosis. The btwo hitsQ hypothesis is illustrated against a background of conditions including genetic defects, insulin resistance, hyperinsulinemia, obesity, and
lifestyle. Putative molecular mechanisms of damage are also shown and include fatty acids h-oxidation, cytokines, lipid peroxidation, and so forth (see also
text). Abbreviations: ROS, reactive oxygen species.
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217 205
increased presence of visceral fat in obese individuals
determines an enhanced delivery of FFAs from visceral
adipocytes into the portal system, and then to the liver. This
condition contributes to the reduced hepatic insulin clear-
ance with further increase of circulating insulin levels. FFAs
also stimulate hepatic gluconeogenesis and triglyceride
synthesis, impair the insulin ability to suppress hepatic
glucose output, affect other metabolic insulin actions [22],
and induce peripheral insulin resistance via inhibitor of
kappa kinase beta (IKK-h) activation [23]. FFAs compete
with glucose for its peripheral utilization [24], determining,
as a consequence, a reduced muscular glucose-6-phosphate
level, a diminished insulin-mediated GLUT4 translocation,
and glycogen synthesis [25]. One of the most deleterious
processes triggered by nonoxidative degradation of excess
FFAs in non-adipose cells seems to be the de novo synthesis
of ceramide [26]. Sphyngomielin-derived ceramide, in fact,
may trigger apoptotic mechanisms leading to cell death.
Accumulation of lipids and their further oxidation are
also under the influence of secretion and tissue sensitivity
by the hormone leptin, a protein of approximately 16 kDa
encoded by the obese (ob) gene and expressed predom-
inantly by adipocytes with important effects in regulating
body weight, metabolism, and reproductive function [27].
Deranged leptin secretion may contribute to the switch from
insulin sensitivity to insulin resistance. Hepatic insulin
resistance and high leptin concentrations are two factors
that favor the entry of FFAs into mitochondria and their
ligand action for the peroxisomal proliferation-activator
receptor-alpha (PPARa). PPARa is involved in lipid
metabolism in the liver by regulating the transcription of
some genes encoding enzymes involved in mitochondrial
and peroxisomal h-oxidation. Both hepatic insulin resist-
ance and the upregulation of PPARa-dependent genes by
FFAs can generate reactive oxygen species (ROS) by at least
three different mechanisms (see below). Also, increased
delivery of tumor necrosis factor-alpha (TNF-a) to liver
cells represents an additional mechanism of damage.
Role of microsomes and peroxisomes
Members of the microsomal cytochrome P-450 partic-
ipate in the generation of oxidative changes in fatty livers via
increased production of the free oxygen radical H2O2.
Increased oxidative stress in the liver is part of the damage
seen in NASH. Two enzymes, CYP2E1 and CYP4A, are
involved in the metabolism of long chain fatty acids (lipo-
oxygenation). Hepatic CYP2E1 increases with fasting,
diabetes, obesity, and insulin resistance, and initiates oxida-
tive stress in the fatty liver. In turn, this is associated with
hepatic microsomal lipid peroxidation [28,29]. Thus, several
compounds may induce liver toxicity following CYP2E1-
mediated bio-activation [30]. The enzyme CYP4A is
controlled by the transcription factor PPARa, governing
genes and is involved in intracellular fatty acid disposal
[31,32]. In particular, long chain and very long chain fatty
acids are also metabolized by CYP4A and the released
dicarboxylic acids serve as substrates for peroxisomal h-oxidation. Defective states of PPARa or of the peroxisomal
h-oxidation pathway may also play an important role in the
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217206
development of steatohepatitis [33]. It has been shown that
mice deficient in PPARa-inducible fatty acid oxidation
demonstrate an exaggerated steatotic response to fasting
[34]. Also, CYP2E1 has a great affinity for electrons and
easily forms reactive oxygen species (ROS) that react with
the unsaturated bonds of long chain fatty acids and initiate the
process of lipid peroxidation [35,36]. Therefore, in the
context of hepatic steatosis, both CYP2E1 and CYP4A could
generate the bsecond hitQ of cellular injury, particularly whenantioxidant reserves are depleted [17].
It has been recently demonstrated that CYP2E1 is
overexpressed in the liver of patients with NASH [37]; this
is similar to what has been observed in the animal model of
nutritional steato-hepatitis (i.e., lipid-rich, methionine-chol-
ine-deficient diet) [38,29]. The development of NASH in
these animals parallels the entity and lobular distribution of
CYP2E1 expression and is closely related to the distribution
of steatosis and inflammation [38]. It is likely that CYP2E1
has a role in the turnover of hepatic fatty acids, although this
is quantitatively less pronounced when compared with the
two major pathways of mitochondrial and peroxisomal h-oxidation [39]. Activated PPARa governs the expression of
both peroxisomal and microsomal lipid oxidation pathways
[31], and may become important especially when CYP2E1
levels are low and polyunsaturated fatty acids accumulate.
Activation of peroxisomal h-oxidation finally occurs when
mitochondrial h-oxidation is impaired or saturated. This
results in peroxisomal proliferation and increased release of
H2O2, resulting in final activation of lipid peroxidation and
switch from steatosis to NASH [40].
Redox balance in fatty livers
Since not every fatty liver develops inflammation and
becomes fibrotic, the reason for the bsecond fibrogenic
hitQ—the oxidative stress—could involve dietary, environ-
mental, or genetic polymorphism. Abnormal oxidative stress
could also occur when hepatocyte radical-scavenging
systems are overwhelmed, that is, when the production of
ROS greatly exceeds the cellular defensive capacity.
Increased generation of ROS has been observed in several
models of fatty livers including alcohol [41] and caffeine
[42] intoxication, and lipotrope-deficient diets [43]. Exces-
sive production of ROS in NASH is suggested by several
reports and increased lipid peroxidation is a hallmark of
those fatty livers developing NASH. ROS originate in fatty
livers at three different intracellular sites, that is, (1) the
microsomal cytochrome P-450 system induced by free fatty
acids during endogenous metabolism of ketones and dietary
constituents [38,37]; (2) peroxisomal h-oxidation that
releases H2O2 when mitochondrial h-oxidation is saturated
by fatty acid excess or impaired [44]; and (3) mitochondria,
which physiologically generate ROS, but are damaged
themselves when the production of ROS is increased, such
as in the presence of altered respiration and oxidative
phosphorylation [45].
Impaired intracellular detoxification and ineffectiveness
of free radical scavenger systems are additional conditions
found in fatty livers. Both impairment of the transsulfuration
pathway [46] and decreased sulphydril content [47] have
been reported in livers with excessive fat accumulation.
We recently evaluated the occurrence of oxidative stress
in rats put on a steatogenic choline-deficient diet [48]. This
model resembles the human fatty liver due to excessive
intake of carbohydrates, with similar biochemical and
histological features [49]. Compared to control animals, rats
on a choline-deficient diet had significantly lower hepatic
concentrations of two important antioxidants, vitamin C and
a-tocopherol, and higher levels of lipid peroxides. Con-
sequently, the a-tocopherol/total lipid and a-tocopherol/lipid
peroxide ratios were found significantly lower in steatotic
livers, suggesting defective protection of unsaturated lipids
from oxidation and increased susceptibility to lipid perox-
idation. The imbalance between antioxidants and lipids has
also been observed in the plasma of obese children [50].
Taken together, all above-mentioned findings suggest that
the imbalance between oxidants and antioxidants predis-
poses fatty livers to greater injury when exposed to a second
hit involving generation of ROS. A proven model to validate
such hypotheses is the ischemia-reperfusion model that
invariably occurs during liver transplantation. A burst of
ROS generation occurs during reperfusion following either
warm or cold ischemia [51,52]. ROS generation causes cell
injury, either directly (i.e., by altering constitutive molecules
such as lipids, proteins, and nucleic acids) or indirectly (i.e.,
by promoting activation of transcription factors and adhesion
molecules) [53,16,54]. If compared to normal livers, fatty
infiltration is associated with a greater ROS-mediated lipid
peroxidation and liver injury during reperfusion of post-
ischemic organs [55]. A short course of vitamin E admin-
istration appears to prevent oxidative stress in fatty livers and
to improve survival following lethal ischemia [56]. By using
a chemiluminescence apparatus connected with an ultra-
sensitive camera, a recent study confirmed that ROS
production was greatly increased in steatotic livers during
post-ischemic reoxygenation [53]. Since the intracellular
mechanisms of oxidative stress cannot alone account for all
the inflammatory changes of NASH, other factors have been
invoked. TNF-a, a pro-inflammatory cytokine likely deriv-
ing from an endotoxemia-mediated activation of Kupffer
cells, is implicated in the pathogenesis of NASH [7]. Hepatic
macrophages from ob/ob mice, an animal model sponta-
neously developing NASH, express significantly greater
levels of TNF-a mRNA [57]. TNF-a may directly impair
mitochondrial respiration [58], causing the opening of the
mitochondrial permeability transition pore and depleting
mitochondrial cytochrome c [59]. Moreover, TNF-a pro-
moter polymorphism is higher in NASH patients with insulin
resistance compared to patients negative for TNF-a poly-
morphism [60]. Excessive lipid peroxidation might be
the ultimate trigger leading to the release of cytotoxic
cytokines (tumor growth factor (TGF-h), IL8), the expres-
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217 207
sion of FAS ligand, and the stimulation of fibrogenesis in
NASH livers [61,62]. Lastly, Bonkovsky et al. [63] found an
increased prevalence of hemochromatosis gene (HFE)
mutation among subjects with NASH. This finding may
have important pathogenic implications since it is known
that iron accumulation within hepatocytes represents a
stimulating factor for oxidative damages by activation of
Fenton’s reaction [64].
Mitochondrial abnormalities
As mentioned above, fatty infiltration in hepatocytes is
accompanied by a number of intracellular disorders as a
consequence or cause of excessive lipid infiltration [12].
Prominent abnormalities of subcellular organelles and
mitochondria have been described by electron microscopy
both in humans and in the experimental fatty degeneration
of hepatocytes [9,65–67]. Mitochondrial oxidative metabo-
lism represents the main energy source for the cell and
impairment of their specific functions may result in deficient
ATP production. Several commonly used drugs are potential
damaging factors for liver mitochondrial function [68,69]
due to their interference with fatty acid metabolism and
mitochondrial respiration. This condition promotes hepato-
cyte fatty degeneration and causes the mitochondrial
permeability transition pore opening. In addition, oxidative
mitochondrial damages, observed during reperfusion after
warm [45,55] or cold [70] ischemia, may strongly contribute
to the deterioration of hepatic energy metabolism observed
after transplantation of fatty livers [71]. Depending on a
major deterioration of energy metabolism, associated with
impairment of ketogenesis and glucose oxidation [70], the
recovery time after reperfusion is markedly prolonged in
steatotic livers [2]. Also, in the choline-deficient diet model,
the activity of mitochondrial complex I is altered in
association with an increased mitochondrial ROS formation
[72]. Thus, the closed link between oxidative stress and
impairment of ATP synthesis appears to be a major key
factor to explain the low tolerance of fatty livers to
ischemia-reperfusion injury and oxidative stress. The
oxidative balance and the capacity for ATP synthesis have
been recently investigated by our group in rat mitochondria
isolated from steatotic livers [73]. Fatty liver mitochondria
contained less glutathione, higher levels of lipid peroxida-
tion products, and lower intensity of the electrophoretic
protein band corresponding to the ATP synthase complex.
The immunoblot analysis of this band showed a 35% lower
detection of the catalytic h-F1 subunit of the F0-F1 ATP
synthase complex, which linearly correlated with a signifi-
cant decreased hepatic ATP content. Ultrastructural changes
of mitochondria, decreased mitochondrial respiration, and
impaired ATP generation capacity have also been described
in patients with NASH [74]. The activities of mitochondrial
respiratory complexes were decreased in the liver tissue of
patients with NASH, and this correlated with serum TNF-a,
insulin resistance, and body size [75]. In steatotic livers, the
excess of mitochondrial ROS generation may easily produce
fat deposit oxidation and the resulting lipid peroxides may
further impair the respiratory chain component and the
membrane transport capacity. This vicious circle involves
ROS-mediated antioxidant depletion and the deficient
capacity of mitochondria to inactivate ROS [76]. Thus,
irrespective of the cause responsible for steatohepatitis, this
condition itself seems either directly associated with an
increased generation of ROS or with an initial impairment of
electron transfer along the mitochondrial respiratory chain
that secondarily leads to mitochondrial ROS formation. This
mechanism finally increases both lipid peroxidation and
cytokine production.
The intestinal–liver interaction hypothesis
Evidence of severe damages caused to fatty livers
exposed to lipopolysaccharide (LPS) suggests that intestinal
bacteria may play a major causative role for NASH in
patients with prior benign liver steatosis. Some exogenous
and endogenous toxins (e.g., LPS and endogenously derived
ethanol derived by intestinal bacteria) are likely to amplify
hepatocyte oxidative imbalance by increasing the produc-
tion of pro-inflammatory cytokines. These molecules cause
organelle dysfunction, hepatocyte death, and accumulation
of inflammatory cells within the liver [77]. As observed in
animal models of ethanol-induced liver injury, intestinal
bacteria may dramatically enhance hepatic oxidative stress
by increasing the production of endogenous ethanol and by
activating nonparenchymal liver cells leading to inflamma-
tion. A similar pathogenic mechanism is also conceivable
for NASH. Studies in ob/ob mice [78] found increased
intestinal production of ethanol. Interestingly, administra-
tion of the luminal antibiotic neomycin decreased endoge-
nous ethanol production [79]. In the clinical setting, it has
been noted that severe forms of fatty liver injury occur after
jejunal–ileal bypass surgery and are associated with
intestinal bacterial overgrowth of the bblind loopQ [80].
Also, obesity and diabetes, which are major risk factors for
NASH, are often complicated by bacterial overgrowth [81]
and intestinal dysmotility [82].
It has also been hypothesized that the hepatic oxidative
stress in individuals genetically predisposed to NASH
development may be associated with an increased hepatic
toxicity of pro-inflammatory cytokines generated by intes-
tinal bacterial products, as observed in murine models of
fatty liver [83].
Fasting and diet supplementation
It is ascertained that obese patients with pure fatty change
of the liver have the advantage of weight reduction.
Progressive weight reduction also improves abdominal pain,
liver blood tests, and histology in patients with NASH [84].
However, fatal hepatic failure has been described following
sudden marked weight loss in morbidly obese patients with
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217208
severe NASH [5]. Therefore, careful clinical follow-up is
advised in patients undergoing weight-reduction surgery
[85], since fatty livers poorly tolerate excessive food
deprivation. In a recent report from our group [48], rats
with fatty livers starved for 18 h showed an 8-fold increase
in serum ALT. This finding was absent in control rats with
normal livers undergoing the same starvation period. Others
have noted that following normothermic ischemia reperfu-
sion, the survival rate during fasting was worse in rats with
fatty liver than in rats with a normal liver [86,87]. It seems
therefore that a well-preserved nutritional status protects the
liver against oxidative stress, while diet regimen may
increase the vulnerability of steatotic cells to oxidative
injury by depleting the cellular stores of antioxidants [76].
Prolonged fasting impairs the free radical scavenger
capacity of liver cells [88] by reducing the availability of
amino acid precursors of GSH synthesis. The hepatocyte
GSH content was reduced by 39% after 18 h of starvation in
normal livers [89], while prolonged food deprivation (36 h)
leads to more deleterious effects in steatotic than in control
hepatocytes [76]. In a recent study, [48] we showed that in
rats under a choline-deficiency diet, starvation determined a
significantly greater decrease of the hepatic concentrations
of glutathione, vitamin C, and vitamin E compared to
normal livers. This fall of antioxidant molecules was
accompanied by enhanced lipid and protein oxidation.
The protective role of intracellular GSH against pro-
oxidant conditions is stronger for fatty than for normal
livers. Rats with fatty liver depleted of GSH showed more
damage to fasting or pro-oxidant agents than rats with
normal livers [76]. The dangerous effects of starvation are
evident also in the mitochondrial compartment. Eighteen
hours of fasting significantly lowered the mitochondrial
GSH concentration only in fatty livers [73]. In these rats, the
most striking alteration regarded the ATP synthase complex,
whose band faded in normal livers, while almost completely
disappeared in steatotic livers. This latter finding was
accompanied by a 70% decrease of the immunodetected
h-F1 subunit and by a 25% reduction of the hepatic content
of ATP in steatotic livers.
Indeed, fatty acid oxidation represents the main cellular
source of energy between meals; subjects with impairment
of the mitochondrial h-oxidation do not tolerate fasting [90].Fasting may trigger hypoglycemia in these patients, thus
hampering energy production from glucose in extrahepatic
organs. Fasting also causes massive adipocyte lipolysis,
flooding the liver with fatty acids that are not oxidized by
the deficient mitochondria and therefore accumulate in the
liver [91]. FFAs and their derivatives inhibit and uncouple
mitochondrial respiration, and decrease energy production.
When mitochondrial respiration is impaired, not enough
NAD+ is regenerated to sustain h-oxidation, thus a worsen-ing of hepatic steatosis may easily occur [92]. Fasting also
predisposes mitochondria to a greater oxidative injury
during ischemia reperfusion. Shorter periods of food
deprivation in rats with fatty liver are in fact sufficient to
obtain mitochondrial GSH depletion and produce oxidative
damages during warm ischemia reperfusion of the same
entity as those observed in rats with normal livers under-
going longer periods of fasting [93]. In a recent study, the
role of diet was accurately investigated in humans; again,
findings suggested that dietary habits may strongly promote
steatohepatitis both directly (by modulating hepatic trigly-
ceride accumulation and antioxidant activity) and indirectly
(by affecting insulin sensitivity and postprandial triglyceride
metabolism). This study provides further rationale for more
specific alimentary intervention in patients with NASH [94].
Thus, a good nutritional status renders hepatocytes more
resistant to ischemic insults and oxidative stress because of
larger glycogen stores [95,96]. With glycogen depletion (as
it occurs in steatosis), hepatocytes become more susceptible
to damage [97]. It appears, in fact, that liver parenchymal
cell function deranges only when endogenous glucose
reserves are lacking. Thus, supplementation of nutrients
rather than a simple diet restriction would ameliorate the
response of fatty liver to damaging insults [98].
Differences among models and susceptibility to necrotic cell
death
Several metabolic pathways may be deranged in livers
with fatty infiltration, including the activity of subcellular
organelles [17,66,99]. However, differences emerge among
experimental models of liver steatosis. Livers with fatty
degeneration show disturbances in the regulation of intra-
cellular GSH compartmentation and homeostasis
[48,53,56]. Obese Zucker rats [56], a genetic model of
obesity in animals, had a hepatic content of GSH, vitamin
E, and catalase lower than nonobese controls. In rats on a
choline-deficient diet [48], the hepatic concentration of
GSH was not decreased under basal conditions, whereas
vitamin E and the ascorbate levels were significantly lower.
Also, in ob/ob mice, a transgenic model developing insulin
resistance, the mitochondrial GSH concentration was found
to be higher compared to nonobese mice [79], and the
hepatic mitochondrial GSH content was found to be lower
in rats under a choline-deficient diet compared to their
control littermates [73]. Similar changes were found in rats
with fatty liver due to chronic overload by copper [9] or
iron [100] and in ethanol intoxicated rats [101]. While the
first finding has been interpreted as an adaptive mechanism
to an increased release of free radicals, in the other fatty
liver models the lower mitochondrial GSH level has been
explained as a defective import of GSH from the cytosol,
resulting in an increased susceptibility of these organelles
to oxidative insults. Moreover, while the mitochondrial
ATP synthase activity was unaffected in obesity-associated
fatty liver [79], the same enzymatic activity was signifi-
cantly decreased both in choline-deficient diet and in
chronic ethanol-induced rat fatty liver [73]. In ob/ob mice,
the cellular response to an acute regenerative stimulus has
been shown to be inhibited as a result of adapted
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217 209
hepatocyte signaling mechanisms to survive chronic oxi-
dative stress [102]. Similarly, the liver regenerative capacity
was declined in obese Zucker rats [99,103], which are
defective in leptin receptor signaling. Liver regeneration
has been shown to be impaired also in other models of fatty
infiltration, such as the alcoholic [104] and the hypothyroid
[105,106] ones. By contrast, liver regeneration does not
appear to be impaired in methionine-choline-deficient rats
[107], in orotic acid-induced liver steatosis [103], and in a
choline-deficient diet model [108]. It is conceivable that
different proliferative responses in these models may be in
relation to the extent of chronic hepatic oxidative stress that
might also modify the response to acute oxidative stress
following partial hepatectomy. In spite of different mito-
chondrial antioxidant status and energy production, all
these experimental models of fatty liver elicited an
increased vulnerability to hepatocyte necrosis when chal-
lenged by insults that produce minor damages in normal
livers. In obese mice, this vulnerability is interpreted as an
adaptive promotion of anti-apoptotic molecules by mito-
chondria that effectively protect hepatocytes from apoptotic
death [79]. By contrast, in choline-deficient rats, an
inefficient mitochondrial ATP synthesis and a deep loss
of mitochondrial GSH under stressing conditions add up to
an early cytosolic loss of antioxidants. In fact, these organs
are further disadvantaged because they are extremely poor
in ascorbate [48] and, consequently, do not allow antiox-
idant sparing activity in the presence of low GSH levels.
All these factors are associated with a high susceptibility to
necrosis. In fact, it is ascertained that in the presence of
poor ATP availability, necrosis ensues before the activation
of the energy requiring apoptotic pathway. Based on this
consideration, it is not surprising that necrosis rather than
apoptosis is the predominant process of cell death in fatty
livers, especially when challenged with injuring insults
[99]. Contrasting results emerge from a recent human
study. In this report, an increased number of TUNEL-
positive cells and higher expression of FAS receptors,
which are features of apoptosis, have been identified in
liver specimens obtained from patients with obesity-related
NASH [109]. In these patients, hepatocyte apoptosis was
greater in liver samples of patients with simple steatosis
and controls, and correlated with the disease severity.
Altogether, these observations clearly indicate the existence
of major differences among experimental models and
suggest the need to investigate the metabolic response to
stress conditions in each experimental model and in the
human fatty liver.
Clinical approach
The natural course of disease and its relative complications
The relatively recent identification of NAFLD–NASH
and the notion that the evolution toward end-stage liver
disease is possible have prompted researches to better screen
for subgroups of patients at a higher risk of disease
evolution. The step involves better knowledge of the natural
history of the disease. Whereas the presence of elevated
liver enzymes is insensitive and cannot be used to reliably
confirm the diagnosis or stage the extent of fibrosis in fatty
livers [110], the presence of older age, diabetes, and obesity
may be predictors of fibrosis [111]. Also, the coexistence of
metabolic disorders is associated with more severe and
potentially progressive forms of liver disease [112]. To date,
the lack of specific and sensitive noninvasive tests has
greatly limited the chance for detection of NASH [113]; this
explains why identification of better noninvasive predictors
of disease evolution is currently a major priority.
With this in mind, subgroups of patients are considered
with attention, that is, children with morbid obesity, adults
with associated conditions and especially with the metabolic
syndrome, and familial forms of NASH associated with
hereditary predisposition such as lipodystrophy. Such
conditions will be discussed in the following paragraphs.
The problem of NAFLD is being increasingly recognized
in pediatric patients. Sixty percent of adolescents with
elevated ALT levels are obese or overweight [114]. Overall,
this is an emerging problem as childhood obesity becomes
increasingly prevalent [115,116]. Whereas cirrhosis has
been reported rarely, fibrosis is common in pediatric NASH
[117]. Thus, once drug hepatotoxicity and genetic or
inherited metabolic disorders have been excluded, liver
biopsy remains the gold standard for diagnosis and
prognosis [118].
Wanless and Lentz [20] found steatosis in 70% of obese
and 35% of lean patients and NASH in 18.5% of obese and
2.7% of lean patients at autopsy. Among type II diabetic
patients, it is estimated that 75% have some forms of fatty
liver [119,120]. The association of NASH with the other
main features of the metabolic syndrome (low HDL-
cholesterol, high triglycerides, arterial hypertension, fasting
hyperglycemia, central obesity) is going to be confirmed by
recent studies also investigating the prevalence of fatty liver
among hypertensive patients [121,122].
The fact that NASH is observed in only a subset of
patients with type II diabetes and that it is uncommon in
patients with other manifestations of insulin resistance
syndrome, such as the polycystic ovarian disease, indicates
that other factors, that is, genetic predisposition, might be
involved. Similarly, the finding of familial clustering of
NASH and cryptogenic cirrhosis supports a role for genetic
polymorphisms in the factors that predispose one to NASH
[123,124]. Therefore, a number of gene abnormalities,
including the group of adipocyte-derived cytokines (leptin,
resistin, adiponectin, a-TNF, and IL-6), have been consid-
ered. Studies in this direction may provide information also
on liver disease progression among patients with NASH
[125]. Of particular interest are studies conducted in people
with lipodystrophy and in murine models of diabetes
associated with lipoatrophy. Lipodystrophies, a disorder of
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217210
peripheral fat deposition, is characterized by nearly absent
peripheral fat, severe hepatic steatosis, and diabetes—
conditions that almost completely disappear with leptin
administration [126,127]. Leptin and adiponectin, in fact,
exert their action by modulating insulin sensitivity in
insulin-sensitive tissues such as adipose, muscle, and liver.
In the latter, leptin may also play an important role in
regulating the partitioning of fat between mitochondrial h-oxidation and triglyceride synthesis [128].
It is likely that the evolution rate of liver disease in
NASH is influenced by factors acting within and outside the
hepatocyte. For example, the progression of NASH toward
cirrhosis varies in association with the features of the
metabolic syndrome [129,130]. Ratziu et al. [131] noted that
obesity-related cirrhosis is often as aggressive as hepatitis
C-related cirrhosis. Once liver cirrhosis has been established
from NASH, however, the typical richness of steatosis can
be lost. A likely explanation is the formation of porto-
systemic shunts and diminished exposure to the fat-storing
signal generated by insulin. Also, the sinusoidal capillariza-
tion may alter the passage of lipoproteins from portal
circulation to hepatocytes.
Already in 1986, Lawson et al. [132] observed a 4-fold
greater incidence of diabetes among patients with HCC
compared with controls. More recently, it has been noted
that the risk of developing NASH and HCC is doubled
among people with diabetes [133]. Such findings suggest
that a strong relationship may exist between diabetes,
obesity, and insulin resistance in the pathogenesis of
HCC. A major question therefore is if HCC incidence is
rising in developed countries as a consequence of the
increasing obesity and diabetes rates. The development of
HCC in a patient with NASH and excess body weight but
without prior cirrhosis has already been reported [134].
Also, incidental HCC is not uncommonly observed among
patients with bcryptogenicQ cirrhosis of probable NASH
origin [135]. Several animal models, mimicking human
conditions, have provided useful information with respect to
the complexity of NASH. Among these experimental
models of HCC, dietary choline deficiency is known to
produce both fatty liver and nongenotoxic liver cancer,
supporting the relation between fat accumulation and cancer
development [136]. Also, hepatocyte hyperplasia and
decreased apoptosis have been implicated in the develop-
ment of HCC in leptin-deficient mice [137].
In spite of a potential link between NASH, metabolic
conditions, and HCC, the impact of screening NASH
conditions to prevent HCC is still less defined than
screening of patients with HBV and HCV chronic infection
[138,139].
Clinical presentation
With the exception of a scant number of patients
suffering from postprandial abdominal pain and fatigue,
most of the NASH cases show asymptomatic elevation of
aminotransferases (40–90%). Usually, there are no symp-
toms or signs of liver disease at the time of diagnosis,
although an enlarged liver can be found at physical exam.
NASH is suspected once other liver diseases have been
ruled out.
NAFLD often occurs in people living in Western
countries reporting absent or very low alcohol consumption.
Individuals are frequently overweight and can show a mild
increase of serum lipids. Some of them are diabetics or have
a family history for diabetes. In some cases, drugs such as
amiodarone or anti-epileptic medications may have some
implication. Geographical differences may exist: NASH was
common among nonobese males in a large multicenter study
in Italy [140,141] and among females with morbid obesity in
the USA [142]. The presence of the bmetabolic syndromeQshould be actively searched for in these patients: this would
imply a screening for elevated blood pressure, obesity,
elevated triglyceridemia, low HDL cholesterol, and insulin
resistance. In fact, a coexisting metabolic syndrome carries a
high risk of NASH among subjects with fatty liver [112].
Once other liver disease or extrahepatic causes of trans-
aminase elevation have been excluded, the definitive
diagnosis of NASH relies on liver biopsy and histology [13].
A standard set of liver function tests is used in the
workup of patients with suspected NASH. None of these
tests is really specific for NASH. The AST/ALT ratio is
often less than 1 in NASH, whereas a ratio above 1 would
suggest an alcoholic steatohepatitis or evolution toward liver
cirrhosis. In this respect, the higher AST levels would reflect
more extensive mitochondrial damages. g-Glutamyltrans-
peptidase is of limited use, as it can increase in both
nonalcoholic and alcoholic steatohepatitis. Red cell mean
corpuscular volume (MCV) is frequently elevated in
alcoholic patients. The use of CAGE or Michigan Alcohol-
ism Screening Test (MAST) questionnaires, however, can
help in disclosing some alcoholic habits.
Insulin resistance is defined by a suboptimal response to
the biological action of insulin to endogenous insulin with
resulting hyperinsulinemia [143]. Although the gold stand-
ard for measuring insulin resistance is the euglycemic
insulin clamp, the so-called homeostasis model assessment
(HOMA) formula is deemed a reliable surrogate measure of
in vivo insulin sensitivity in humans ([fasting serum insulin
(AIU/ml) � fasting serum glucose (mmol/L)] divided by
22.5). Patients are classified as insulin resistant if HOMA is
over 1.64 [21,144]. Insulin resistance and systemic hyper-
tension are both independent factors associated with
advanced forms of NASH [145]. Hyperinsulinemia and
insulin resistance in patients with NASH are likely derived
from an enhanced pancreatic insulin secretion that compen-
sates for the reduced insulin sensitivity [146].
Liver imaging is essential in patients who are likely to
have NAFLD. Liver ultrasonography detects the presence of
fat in the liver as bbright liverQ [141,147] but is unable to
predict fibrosis. Although CT scan and NMR can provide
information on fat accumulation in the liver [148], their use
Table 1
Grading and staging for NASH
Grading Steatosis Ballooning Inflammation
Grade 1,
Mild
1–2 (b33%) Minimal Lobular: 1–2;
Portal:
none–mild
Grade 2,
Moderate
2–3 (33–66%) Present Lobular: 2;
Portal:
mild–moderate
Grade 3,
Severe
3 (z66%) Marked Lobular: 3;
Portal:
mild–moderate
Staging Perisinusoidal
fibrosis
Portal-based
fibrosis
Bridging
fibrosis
Cirrhosis
Stage 1 Focal or
extensive
0 0 0
Stage 2 As above Focal or
extensive
0 0
Stage 3 Bridging
septa
Bridging
septa
+ 0
Stage 4 F; zone 3
incorporated
into septa
Portal tract
replaced or
incorporated
into septa
Extensive +
Readapted from Brunt et al. [185].
Fig. 2. General principles of the 13C-stable-isotope breath test for the
dynamic study of liver function. (Upper panel) The use of ketoisocaproic
acid (KICA) is shown: (1) oral administration of 13C-KICA; (2) rapid
absorption of the substrate at the proximal intestine and portal delivery to
the liver; (3) liver metabolism of the substrate with ultimate production of
labeled 13CO2; (4)13CO2 is promptly diffused in the lung and expired in
breath; (5) breath collection in appropriate test tubes and 13CO2 measured
by mass spectrometry. (Lower panel) The decarboxylation of KICA
uniquely occurs at the mitochondrial level and depends on NAD+
availability. The asterisk (*) indicates the labeled carbon as 13C.
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217 211
is discouraged because they are not able to differentiate
between NASH and nonprogressive NAFLD. Therefore,
liver biopsy in patients with fatty liver and elevated serum
transaminases should be considered to establish the diag-
nosis of NASH, staging the disease (e.g., extent of fibrosis)
and assessing treatment effectiveness [149] (Table 1). A
study from the Mayo Clinic concluded that age was the
most significant predictor of the degree of fibrosis; severe
fibrosis is rare in nonobese nondiabetic patients younger
than 45 years [150]. Ratziu et al. [151] found that age
greater than 50 years correlated with septal fibrosis in obese
patients and that no patients showed septal fibrosis or
cirrhosis when age was under 50 years, body mass index
(BMI) lower than 30 kg/m2, and ALT elevation less than 2-
fold. Thus, if a patient present with elevated transaminases
plus fatty liver at ultrasonography and other chronic liver
diseases have been accurately excluded, liver biopsy would
only be necessary when age is over 40 years. Much attention
should therefore be put on predicting advanced or pro-
gressive disease to select patients suitable for liver biopsy. A
recent report suggests an algorithm including serum
hyaluronate and carbohydrate-deficient transferrin/transfer-
rin ratio as a noninvasive method to predict liver fibrosis in
patients with metabolic syndrome [152]. Despite the fact
that this approach may predict the presence of fibrosis, it
cannot predict which patients are developing more aggres-
sive forms of NASH. Fargion et al. [153] found that patients
with fatty liver and persistent high serum ferritin level may
be at high risk of developing NASH; this is particularly true
if data are simultaneously associated to glucose or lipid
metabolism disorders.
In order to develop more accurate noninvasive tests for
the study of NASH patients, recent investigations focused
on the use of breath tests as diagnostic tools to investigate
liver function. By using substrates marked with the non-
radioactive and naturally occurring stable isotope 13C,
specific enzyme function can be investigated in the liver.
Thus, breath tests provide accurate information on meta-
bolic processes occurring in patients with various degrees of
liver disease (Fig. 2). Mion et al. [154] used ketoisocaproic
acid to explore mitochondrial function in vivo [155] in
patients with alcoholic and nonalcoholic fatty livers. The
test was altered only in patients with alcoholic fatty liver. A
potential pitfall, however, was that patients with non-
alcoholic fatty liver had normal levels of serum trans-
aminases. Therefore, the possibility that NASH was absent
in this group could not be ruled out, since liver biopsy was
not performed in this study. In another preliminary study,13C-methionine could better distinguish between the two
groups [156]. Since microsomal enzymes have been found
to be hypertrophic in NASH, the breath test may be adapted
to investigate microsomal functional mass. The 13C-meth-
acetin breath test has potential interest: it is easy to perform,
not burdened by side effects, and has low cost [157]. The
usefulness of such breath tests for studying bdynamicQ liverfunction is being tested also in our laboratory and compared
with other bstaticQ liver function tests.
Fig. 3. Schematic representation of the ligands and relative effects of
thiazolidinediones. The dimension of the arrows is proportional to the
receptor selectivity for thiazolidinediones.
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217212
Treatment
Current therapeutic approaches of NASH are largely
conservative, and a summary is depicted in Table 2. Patients
should avoid alcohol and other hepatotoxins [158]. A
program including progressive weight reduction, metabolic
control, and gradual physical exercise may contribute to
improve liver abnormalities. A gradual weight loss is
recommended if obesity is present and a fatty liver is found
[5]. Conversely, a sudden weight reduction, as following
weight-reduction surgery, must be avoided since it may
result in fatal hepatic failure. This has been shown in
severely obese patients with NASH [159], and a potential
mechanism includes great transport of FFAs to the liver.
More recently, however, it was shown that weight loss after
surgery in severely obese individuals results in major
improvement of obesity, obesity-related metabolic syn-
drome, and liver histology [160]. As NASH patients with
diabetes mellitus are at higher risk to develop more
aggressive outcomes [161], the potential role for antidiabetic
drugs in NASH patients is being explored. Drugs that
decrease insulin resistance and increase hepatic insulin
sensitivity are of interest [162–164]. Recently, metformin
(850 mg b.i.d. for 24 weeks) improved the hepatic necro-
inflammatory activity in NASH [165]. In a pilot study, the
use of rosiglitazone (a PPARg ligand) belonging to the
thiazolidinediones family was associated with decreased
insulin resistance and improved liver histology in NASH
patients [166]. Thiazolidinediones exert their function
through activation of PPARg nuclear receptors. This
activation ameliorates insulin sensitivity by promoting
glucose utilization at the muscular level and by decreasing
hepatic glucose production (Fig. 3).
Although diet restriction remains the mainstay of treat-
ment in patients with liver steatosis, encouraging results
have been reported in pilot studies testing other drugs of
different categories, including gemfibrozil, metformin,
vitamin E, N-acetylcysteine, and S-adenosyl-l-methionine
[56,57,167–170]. One study showed that oral supplementa-
tion of vitamin E (as antioxidant agent) at high doses (600
IU/day) normalized aminotransferase levels in children with
Table 2
Therapeutic approaches showing the beneficial effects in NASH patients or
in animal models
Strategy Treatment
Gradual weight reduction Caloric restriction [111]
Exercise [5]
Weight-reduction surgery [160]
Insulin sensitization Metformin [165]
PPARs ligand (Rosiglitazone, Pioglitazone)
[166,186]
Lipid-lowering drugs Fibrates (Gemfibrozil) [187]
Fish oil [178]
Antioxidants Vitamin E [171,172]
N-Acetyl-cysteine [173]
Betaine [179]
NASH [171], while in another study transaminases dropped
and liver histology significantly improved after vitamin E
and weight reduction in patients with NASH [172]. Other
authors have described amelioration of liver parameters after
a combination of probiotics with prebiotics and vitamins
[173] or after alternative medication products [174].
Results with the more hydrophilic-less cytotoxic dihy-
droxy bile salt ursodeoxycholate (a well-known oral litholitic
agent in patients with cholestrol gallstones) yielded con-
troversial results. Okan et al. [175] reported that ursodeox-
ycholic acid (UDCA) prevented the appearance of liver
steatosis in rats on a choline-deficient diet but was ineffective
to prevent steatosis when added to the diet at a later stage. In
a recent study from the Mayo Clinic, however, 2 years of
UDCA therapy at high doses (13–15 mg/kg/d), although safe
and well tolerated, was not better than placebo for patients
with NASH, assessed by liver histology at baseline and after
treatment [176]. Results available so far would therefore not
warrant the use of UDCA in patients with NAFLD.
Carnitine and coenzyme-A, essential co-factors in the
transport of fatty acids into the mitochondria, might
contribute to increase the subsequent oxidation of the same
fatty acids. However, the need for high dose parenteral
administration to reach appreciable hepatic concentrations is
so far against their routine clinical use. Finally, ciprofibrate,
a PPARa ligand and inducer of fatty acid oxidation,
decreased the severity of choline-deficient diet induced
fatty change and hepatitis [177].
Very recently, dietary omega-3 fatty acids have been
shown to decrease hepatic triglycerides in Fisher 344 rats
[178]. Betaine, an antioxidant with potential hepatoprotec-
tive effects, was effective (20 mg/day) as other medica-
tions in reducing serum transaminase levels with
associated amelioration of liver histology [179]. It must
be underscored, however, that all the above-mentioned
medications need to be evaluated in carefully controlled
long-term studies before a clear recommendation is
formulated.
P. Portincasa et al. / Clinical Biochemistry 38 (2005) 203–217 213
Finally, invasive procedures may have indications in
severely obese patients refractory to diet and exercise. The
positioning of a gastric balloon can result in a decreased
stimulus to eat, but its application is limited to 6–10 months.
The external gastric banding is an alternative choice [180].
The most widely performed bariatric surgical procedure,
Roux-en-Y gastric bypass, achieves permanent and signifi-
cant weight loss but can be followed by severe complications
in patients with NASH.
Orthotopic liver transplantation (OLT) is an option for
end-stage liver disease patient, including those with
NASH-related cirrhosis. However, the de novo occurrence
of NASH with progression to cirrhosis has been reported
also following liver transplantation [181]. The high
recurrence rate following transplantation and the clinical
outcomes similar to those of other group of patients
undergoing OLT support the assertion that NASH repre-
sents per se a cause of liver cirrhosis and end-stage liver
disease [182,183].
Conclusions and perspectives
The histological features of what we now call NASH
were described since 1962 by Thaler [184] and better
characterized by Ludwig in 1980 et al. [1]. Morphological
findings range from fatty degeneration to inflammation and
fibrosis, and may end in liver cirrhosis. NAFLD and NASH,
however, are likely to represent the tip of the iceberg
including several complex biochemical, metabolic, and
clinical conditions. Despite the well-defined morphological
features, our knowledge on the pathogenic mechanisms is
mostly lacking as well as an appropriate therapeutic
approach. Therefore, a better understanding of the mecha-
nisms leading to fat accumulation and oxidative balance
impairment in steatotic livers is greatly expected to improve
the therapeutic approach against the risk to develop NASH,
as well as to increase the tolerance of these organs toward
oxidative stress conditions. In this view, biochemical
investigations may drive the identification of new diagnostic
tools that may allow the diagnosis and follow-up of these
patients without the need for liver biopsy. Treatment of co-
morbidities is essential to exclude additional factors of liver
injury in patients with NASH as well as it is of fundamental
prognostic importance to identify and treat underlying liver
steatosis or steatohepatitis in patients also carrying other
causes of liver disease. Attractive pharmacological
approaches include new molecules that modulate the
activation of PPARs, which regulate both microsomal and
peroxisomal lipid oxidation pathways [37] and improve
insulin sensitivity. Drugs that increase the efflux of
triglycerides from the liver or that augment their utilization
might be another option. Therefore, based on the evidence
reported so far, any strategy enabling fatty livers to increase
resistance against stressing conditions will protect them
from inflammation and will necessarily improve the basal
function of these organs. Thus, beyond diet education, lipid
and glucose metabolism control, and improved physical
exercise, a rationale approach aiming to increase the
tolerance of steatotic livers to stress-induced injury should
simultaneously enhance the hepatocellular content in anti-
oxidant molecules, ameliorate their distribution within
subcellular organelles, enlarge the glycogen reserves, and
improve their utilization.
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