Nonalcoholic steatohepatitis: recent advances from experimental models to clinical management

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


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-


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


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


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


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.


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.


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.


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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Nonalcoholic steatohepatitis: recent advances from experimental models to clinical management

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