Current Drug Targets, 2011, 12, 879-893 879

1389-4501/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.

Mitochondria in Chronic Liver Disease

Ignazio Grattagliano1, Stefan Russmann

2, Cátia Diogo

3, Leonilde Bonfrate

1, Paulo J. Oliveira

3,

David Q.-H. Wang4 and Piero Portincasa

*,1

1Department of Internal and Public Medicine, University Medical School, Bari, Italy

2Department of Clinical Pharmacology and Toxicology, University Hospital Zurich, Zurich, Switzerland

3Department of Life Sciences, Center for Neurosciences and Cell Biology, University of Coimbra, 3004-517 Coimbra,

Portugal

4Division of Gastroenterology and Hepatology, Department of Internal Medicine, Edward Doisy Research Center, Saint

Louis University School of Medicine, St. Louis, MO, USA

Abstract: Mitochondria are the main energy source in hepatocytes and play a major role in extensive oxidative

metabolism and normal function of the liver. This key role also assigns mitochondria a gateway function in the center of

signaling pathways that mediate hepatocyte injury, because impaired mitochondrial functions affect cell survival and

contribute to the onset and perpetuation of liver diseases. Altered mitochondrial functions have indeed been documented

in a variety of chronic liver diseases including alcohol-induced liver disease, nonalcoholic fatty liver disease, viral

hepatitis, primary and secondary cholestasis, hemochromatosis, and Wilson’s disease. Major changes include impairment

of the electron transport chain and/or oxidative phosphorylation leading to decreased oxidative metabolism of various

substrates, decreased ATP synthesis, and reduced hepatocyte tolerance towards stressing insults. Functional impairment of

mitochondria is often accompanied by structural changes, resulting in organelle swelling and formation of inclusions in

the mitochondrial matrix. Adequate mitochondrial functions in hepatocytes are maintained by mitochondrial proliferation

and/or increased activity of critical enzymes. The assessment of mitochondrial functions in vivo can be a useful tool in

liver diseases for diagnostic and prognostic purposes, and also for the evaluation of (novel) therapeutic interventions.

Keywords: Alcohol, cholestasis, fatty liver, hemochromatosis, hepatitis C virus, nitrosative stress, nonalcoholic fatty liver disease, oxidative stress, primary biliary cirrhosis, Wilson’s disease.

INTRODUCTION

Mitochondria are the main energy source in hepatocytes and play a major role in extensive oxidative metabolism and function of the liver in health. Impaired mitochondrial function may therefore play an important role in the onset and progression of chronic liver diseases [1]. Impaired functions of some subcellular organelles have been recently linked with mitochondrial dysfunction in liver diseases, in particular in fatty liver degeneration [2, 3]. Such alteration assigns a fundamental pathogenic role to mitochondrial impairment. Mitochondria are the common gateway where signals leading to cell injury converge [4, 5]. The study of mitochondrial functions is of major importance for the interpretation of several processes governing the energetic metabolism within the complex intracellular network of rela-tionships among organelles and other subcellular systems, including important connections with microsomes and the nucleus.

This review discusses the current knowledge on the general involvement of mitochondria during liver diseases as well as mitochondrial involvement during specific chronic liver diseases. Also, ways to assess mitochondrial function in

*Address correspondence to this author at the University of Bari Medical

School, Clinica Medica “A. Murri”, Department of Internal and Public

Medicine (DIMIMP), Hospital Policlinico - 70124 Bari, Italy; Tel: +39-080-

5478227; Fax: +39-080-5478232; E-mail: p.portincasa@semeiotica.uniba.it

vivo will be discussed, since this novel approach may be a useful tool for diagnostic and prognostic purposes, and for the evaluation of therapeutic interventions with mitochondria as potential drug targets. Although mitochondria also play a major role in acute liver diseases, this aspect is not the focus of this review.

ROLE OF MITOCHONDRIA IN THE DEVELOP-MENT AND PROGRESSION OF LIVER DISEASE

Mitochondria play an active role in several metabolic pathways by integrating signalling networks [6]. Signals, however, may damage mitochondria directly or indirectly by activating intracellular stress cascades or death receptor-mediated pathways. Reactive oxygen species (ROS) forma-tion, glutathione (GSH) depletion and protein alkylation are major events associated with mitochondrial dysfunction and represent critical initiating events in most forms of chronic liver diseases.

Although a number of new roles have been attributed to mitochondria, their main function remains the production of adenosine triphosphate (ATP). Finely tuned mechanisms involve an initial series of biochemical reactions in the Krebs cycle followed by electron transfer in the so-called mito-chondrial respiratory chain in the inner mitochondrial mem-brane (Fig. 1). Such processes are tightly dependent on the integrity of the mitochondrial membrane. The rate of ATP turnover, rather than its level, is critical for cell survival [7],

880 Current Drug Targets, 2011, Vol. 12, No. 6 Grattagliano et al.

and is a major determinant of necrotic or apoptotic cell death; indeed, apoptosis requires ATP for its initiation and completion. Several toxic compounds and some diseases determine structural alterations of mitochondrial membranes, resulting in functional impairment and decreased energy production.

A major injurious mechanism is mediated by ROS, which are responsible for oxidative modifications of lipid and protein components of the mitochondrial membrane as well as of mitochondrial DNA (mtDNA). Mitochondrial oxidative changes determine structural alterations, membrane fluidity change, appearance of hydrophilic moieties within hydro-phobic regions with loss of lipid-lipid and lipid-protein interactions, changes in membrane permeability, as well as inactivation of transport and receptor systems, among others. Oxidative damage is therefore the most likely causative process in a number of chronic liver diseases and may result in mtDNA alterations, stimulation of apoptotic pathways, and increased propensity to necrosis due to multi-level fail-ure to synthesize ATP. Overall mitochondrial health likely

depends on multiple factors including the mtDNA integrity, composition of membrane lipids, lipoprotein trafficking, balance between pro- and antioxidant factors, and metabolic demands placed on the liver [8].

Nitrosative stress is another important injurious mecha-nism, which relies on the local production of nitric oxide (NO) derivatives and their linkage with proteins and thiols, thus causing inactivation of enzymes, carriers and confor-mational changes. Indeed, at the mitochondrial level, NO is known to control both respiration and organelle biogenesis. Therefore, alterations in the local and global levels of NO may affect mitochondrial function as well [9].

Further evidence indicates that ROS and NO species dis-rupt mitochondrial function through post-translational modi-fications of the mitochondrial proteome. This post-trans-lational modification of proteins may contribute to mito-chondrial dysfunction at least in some liver diseases. Recent proteomic techniques now allow the identification of defects in the assembly of multi-protein complexes in mitochondria

Fig. (1). Schematic representation of mitochondrial electron transport chain and its specific modulation at different steps. Electrons can enter

the mitochondrial respiratory chain at two specific sites. NADH is oxidized at Complex I, while succinate produced in the Krebs cycle is

oxidized to fumarate at Complex II. Electrons are delivered from Complex I and II to the mobile electron carrier ubiquinone (Q), which

shuttles them to complex III. Finally, electrons are channelled to cytochrome c, an extrinsic protein with a heme group, to cytochrome c

oxidase, a large complex with two copper and two heme centers. Electrons are delivered to oxygen and converted into water. The process of

electron transference is associated to ejection of protons at Complexes I, III and IV, which form an electrochemical potential composed of a

pH and electric components. The so called protonmotive force is used for several mitochondrial functions, including ATP synthesis by the

F0F1 ATP synthase, transport of electrolytes and metabolites (with one classical example being calcium) and to the processing of imported

proteins. Specific inhibitors exist that selectively inhibit points at the respiratory chain, including rotenone (Complex I),

thenoyltrifluoroacetone or malonate (Complex II), antimycin A or myxothiazol (Complex III) and azide or cyanide (Complex IV). The ATP

synthase is inhibited by oligomycin. The delicate and at the same time complex process of ATP generation in mitochondria by coupling

substrate oxidation to phosphorylation is called, logically, oxidative phosphorylation. The multitude of reaction steps implies that different

diseases, unbalance of metabolites/metal ions and different xenobiotics can perturb the process.

C II

FAD

Succinate

C I C III C IV

FMN b c1a a3

NADH+H+

Fumarate

Q c

½ O2

FMN 1cu cu

NAD+

Q c

H2O

Electrochemical

Potential Specific inhibitors:

C I - rotenone

ATP synthesis

Transport (electrolytes metabolites)

C II - thenoyltrifluoroacetone

C III - antimycin A

C IV - azide

Transport (electrolytes, metabolites)

Processing of imported proteins

Mitochondria in Chronic Liver Disease Current Drug Targets, 2011, Vol. 12, No. 6 881

and resolution of the highly hydrophobic proteins of the inner membrane and may therefore provide more insight into this mechanism in the future [10].

All mechanisms cited above are involved in the activa-tion of apoptotic and necrotic pathways, which are schema-tically depicted in Fig. (2). In the former case, structural and functional integrity of mitochondria is required for the maintenance of ATP supply. At the mitochondrial level, apoptosis can include alterations in the membrane distribu-tion of cardiolipin and phosphatidilcholine which can lead to increased mitochondrial permeability transition (MPT, see below), as well as to increased homing of pro-apoptotic proteins [11]. The MPT occurs for the opening of multiple pores (MPT pores) located in contact sites between the inner and outer membranes with a subsequent loss of proton gra-dient through the inner mitochondrial membrane and block of ATP synthesis. The resulting mitochondrial outer mem-brane permeabilization and rupture - due to mitochondrial

swelling - is then associated with several events: release of cytochrome C and other pro-apoptotic factors from the intermembrane space into the cytosol, disruption of nuclear chromatin, and activation of Ca

2+-depending proteins [12-

15]. Cytochrome C binds to a cytoplasmic scaffold (apaf-1), forming a complex in the presence of ATP called “apop-tosome”, which activates signalling pro-caspase 9. The apop-totic signalling is further amplified following activation of the executioner caspase 3 and interaction with pro-caspases 6, 7, and 2 in order to activate them and home them to their own target proteins [16]. Programmed apoptotic cell death is characterized by cytoplasmic and nuclear condensation and fragmentation without loss of membrane integrity. Apoptotic fragments are then removed by phagocytic cells with little accompanying inflammation.

In the presence of irreversible mitochondrial dysfunction, ATP production is heavily impaired, and this condition may favour necrotic over apoptotic cell death. When the initial

Fig. (2). Apoptotic and necrotic pathways highlighting the role of mitochondria during chronic liver diseases. Several factors involved in

chronic hepatic diseases lead to a stress of hepatic mitochondrial bioenergetics and failure. Mitochondrial stressors can include excess of

calcium/oxidative stress or activation and mitochondrial homing of pro-apoptotic proteins due to signals originated inside (for example due

to endoplasmic reticulum stress) or outside the cell (FasL or TNF� signalling). They can trigger alterations in the permeability of the outer

and inner membrane and lead to the formation of pro-apoptotic outer membrane channels, formed for example, by Bax multimerization and

antagonized by Bcl-2, or to the formation and induction of the MPT, which also leads to rupture of the outer membrane. Regardless of the

mechanism behind the loss of impermeability of the mitochondrial outer membrane, one of the consequences is the release of pro-apoptotic

proteins that are normally under enclosure in the inter-membrane space. Such proteins include cytochrome c (“a daily worker with a deadly

night job”), and the apoptosis-inducing and SMAC/Diablo, among others. Release of mitochondrial factors lead to the activation of the

caspase cascade, which will result into apoptosis if ATP levels are sufficient to sustain the programmed steps of the apoptosis program. In

case of a massive ATP depletion, which for example occurs during prolonged and extensive MPT induction, the process follows a necrotic

pathway, which is more prone to cause tissue inflammation.

Oxidative stress

Intact hepatocyte Dysfunctional hepatocyte

Oxidative stress

Calcium overload

Death signals

Mitochondrial Permeability Transition

Ca2+ depending protein activation

TNF� and FasL sensitization

Bax (Bcl-2), JNK, bim activationMassive ATP

depletion a ( c ), J , b act at o

Sustained ATP

production

depletion

production

Necrosis ApoptosisNecrosis p p

882 Current Drug Targets, 2011, Vol. 12, No. 6 Grattagliano et al.

injury is so severe that the MPT occurs irreversibly with rapid and severe mitochondrial ATP depletion in most of the mitochondrial population, the activation of the apoptotic pathway is inhibited. At this stage, necrosis occurs, charac-terized by cell swelling and lysis that follow severe dis-turbance of cell functions. Necrotic cell lysis induces inf-lammatory responses including release of cytokines and amplification of the initial injury through a sensitization of surrounding hepatocytes. This step can perpetuate further collateral damage. In spite of the conceptual differentiation between apoptotic and necrotic hepatocyte death, the dis-tinction between apoptosis and necrosis may not always be clear-cut in reality. Mixed phenomena have been described, and apoptosis and necrosis may in fact also be regarded as a continuous phenomenon [17, 18].

The following paragraphs will describe the role of mito-chondrial alterations in specific and most frequent chronic liver diseases in humans: alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), hepatitis C virus (HCV) infection, hemochromatosis, Wilson’s disease, and chronic cholestasis. Tests currently used for the assessment of mitochondrial function in vivo in the clinical setting will also be discussed, starting from some experimental animal models.

ROLE OF MITOCHONDRIA IN SPECIFIC LIVER DISEASES

Alcoholic Liver Disease (ALD)

In humans, excess ethanol consumption is a major cause of chronic liver injury with potential evolution towards fibrosis and cirrhosis. Animal models have clarified several issues, in this context. Chronic administration of ethanol in rats causes steatosis due to intracellular accumulation of tryglicerides, inflammation, necrosis and fibrosis, but not cirrhosis [19]. Liver cirrhosis often develops when ethanol administration is combined with other hepatotoxins [20]. Primates such as baboons, by contrast, develop liver cir-rhosis if chronically intoxicated with alcohol; this animal model can therefore be useful for the study of alcohol-induced liver disease in humans [21].

Pathogenic mechanisms of ethanol toxicity include damage and functional alterations of subcellular organelles. Abnormalities of liver mitochondria are a common and early feature in humans and animals exposed to chronic ethanol toxicity. Morphological changes include megamitochondria (which can even exceed the diameter of the nucleus), red-uced number of cristae, and presence of crystalline inclu-sions. Mitochondria become pleomorphic, increase in size and show disorganization of cristae [22]; membranes are broken or disappear in different shapes under electron mic-roscopy or even show a U shape [23]. Taken together, the findings suggest that alcohol is a direct toxicant for mito-chondria, and that mitochondrial function is impaired at an early stage even at low ethanol concentrations [24].

An important step in ethanol-induced liver injury includes the generation of acetaldehyde and free radicals from ethanol metabolism, leading to mitochondrial altera-tions. Steps include destruction of essential components of mitochondrial membranes and consumption of antioxidant

molecules. Acetaldehyde can also react with proteins and form adducts [25], resulting in alteration of antigenic and functional properties [26]. The depletion of mitochondrial GSH appears to play a key role in the development of alcoholic liver disease [27]. The mitochondrial GSH pool decreases in ethanol-fed rats, and this is apparently due to suppression of cytosolic de novo synthesis of GSH [28], and to impaired influx of GSH from the cytosol into mitochon-dria [29]. Thus, the liver of chronic alcoholics becomes more vulnerable to the effect of hepatotoxic drugs which are normally detoxified by the GSH-dependent system (e.g. acetaminophen).

The most important functional alteration in liver mito-chondria isolated from ethanol-fed rats is a reduction of oxidative phosphorylation: this is shown by the existence of reduced activities of subunits of the electron transport chain, decreased mitochondrial ATP synthesis, decreased content of cytochromes, and altered phospholipid and fatty acid composition of membranes [1, 30, 31]. Mitochondrial res-piratory activity is impaired and superoxide radical genera-tion is enhanced in rats chronically fed with ethanol [32]. The reduced ATP synthase activity seems to depend mainly on an ethanol-induced defect of the F0 subunit of this enzyme [33], a likely consequence of decreased protein syn-thesis [34]. The same mechanism is invoked for the reduced activity of complexes I and IV of the respiratory chain. Such alterations induce a lower mitochondrial transmembrane potential, increased mitochondrial mass, and intracellular Ca

2+ with a broken mitochondrial MTP pore. Altogether,

data indicate that ethanol-induced mitochondrial injury is an important mechanism of alcoholic liver diseases [23]. Chan-ges are particularly evident if the tissue becomes hypoxic: the event might occur as a consequence of decreased mitochondrial and glycolytic activities between glucose and glyceraldehyde-3-phosphate [35], but also following inc-reased expression of inducible nitric oxide synthase (iNOs), and enhanced sensitivity of mitochondrial respiration to inhibition by NO [36]. Thus, hepatic response to chronic alcohol-dependent cytotoxicity involves a change in mito-chondrial function that also depends on iNOS induction.

Ethanol is first metabolized in the liver to acetaldehyde, a process occurring in the hepatocyte cytosol via a reaction catalyzed by the enzyme alcohol dehydrogenase (ADH). The generated acetaldehyde is subsequently metabolized to acetate: this process occurs in the mitochondria and is cata-lyzed by a different enzyme, acetaldehyde dehydrogenase (ALDH). Ethanol-intoxicated rats develop a damage of elec-tron transport chain and decreased ATP synthesis. Acetal-dehyde accumulating in mitochondria causes adduct forma-tion with structural and functional proteins and consequent oxidation [37, 38]. The use of alcohol dehydrogenase and aldehyde oxidase inhibitors has clarified that acetaldehyde accumulation and not its metabolites or ethanol itself are responsible for some of the effects related with alcohol-induced mitochondrial toxicity [37]. Toxicity is partly asso-ciated with changes in GSH compartmentalization and redox state [29].

Chronic ethanol administration also leads to alterations in membrane lipid composition and redox state. These also depend on dietary lipid content [39] and result into changes in membrane permeability and fluidity which impacts both

Mitochondria in Chronic Liver Disease Current Drug Targets, 2011, Vol. 12, No. 6 883

membrane and protein activity. Changes of fatty acid compositions in mitochondrial membranes and decreased antioxidant protection cause lipid peroxidation as the result of increased ROS production. These findings have been rep-eatedly observed as increased mitochondrial concentrations of conjugated dienes, malondialdehyde, and 4-hydroxynone-nal [37, 40].

Additional injurious mechanisms shown in chronically ethanol-fed rats include decreased mitochondrial translation, depressed respiratory complex levels and mitochondrial respiration rates, and decreased protein synthesis. Increased dissociation of mitoribosomes has also been documented by decreased sedimentation rates, larger hydrodynamic vol-umes, increased levels of unassociated subunits, and changes in the levels of specific ribosomal proteins [41]. Also, S-adenosyl-L-methionine (SAME) required for the assembly and subsequent stability of mitoribosomes, is depleted during chronic ethanol feeding [42]. Further studies revealed that ethanol also causes decreased state 3 respiration and res-piratory control ratio, mtDNA damage, and iNOS induction. Alcohol mediates losses in cytochrome C oxidase subunits, complex IV activity, and up-regulates the mitochondrial stress chaperone prohibitin. Exogenous SAME administra-tion preserves hepatic SAME levels and prevents several defects to mitochondrial genome and proteome that contri-bute to the bioenergetic defect in the liver after alcohol con-sumption [43]. However, a clear benefit of SAME substi-tution for the prevention of alcohol-induced liver disease in humans has not been observed.

Nonalcoholic Fatty Liver Disease (NAFLD)

A better understanding of mitochondrial dysfunction is of great pathogenic, prognostic, and therapeutic importance during metabolic alterations, and liver steatosis is a clear example.

NAFLD typically occurs in patients who do not drink alcohol or do not abuse alcoholic beverages, but develop metabolic abnormalities including visceral obesity, insulin resistance or frank type 2 diabetes mellitus, hypertension, and dyslipidemia [44]. Insulin resistance represents an early key factor which activates metabolic pathways promoting liver steatosis. Liver steatosis is also promoted by chronic intake of medications such as amiodarone or valproate [45], which accumulates in mitochondria and induces inhibition of fatty acid oxidation and electron transfer chain function [45]. NAFLD encompasses a wide spectrum of liver damage seen at histology, ranging from mild to massive steatosis, and from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis.

Pathogenesis of NAFLD is a complex phenomenon: with the increase of visceral adipose tissue, visceral adipocytes release substantial amounts of free fatty acids (FFAs) into the splanchnic circulation. FFAs influx in the liver leads to increased liver fat storage of triglycerides [46, 47]. The hepatic synthesis of FFAs is also increased as a consequence of increased plasma levels of glucose and insulin. One pathway that limits excess fat accumulation in the liver is the increased mitochondrial oxidation of FFAs, which becomes defective only when the respiration is severely impaired [48]. There might be a threshold above which fat infiltration

leads to hepatocyte injury. Fatty degeneration exposes the hepatocytes to a higher risk of oxidative damage, and a number of adaptive metabolic mechanisms have been described during the early phase of fatty infiltration [2, 49]. Such mechanisms include expression of intracellular sensors and signalling molecules for lipid metabolism and oxidative stress pathways [50, 51]. Alteration of these systems may have important pathogenic roles in NAFLD progression. In patients with more advanced forms of liver steatosis, the mitochondrial ability to synthesize ATP is decreased [52], and hepatic mitochondria exhibit ultrastructural changes [51]. mtDNA levels, protein expression and activity of res-piratory complexes also decrease in liver mitochondria [53, 54]. Mechanisms which deteriorate mitochondrial functions in patients with liver steatosis have not been completely elucidated. Oxidative stress plays a major role, in this res-pect. In fatty livers, ROS formation is increased at the mito-chondrial respiratory chain level. ROS determine oxidation of unsaturated lipids, directly related to the hepatic fat con-tent [2, 3, 49]. The activity of complex I of the respiratory chain is significantly reduced (-35%) and associated with changes in state 3 respiration [55]. In parallel, hydrogen peroxide (H2O2) generation is significantly increased. In a rodent model of fatty liver disease induced by a choline deficient diet, ROS and products derived from ROS-induced lipid oxidation damaged respiratory chain peptides and oxidized mitochondrial cardiolipin [3, 55]. Thus, ROS affect the mitochondrial complex I activity via oxidative damage of cardiolipin which is required for the functioning of this multisubunit enzyme complex [56].

Oxidation, glutathionylation and nitrosylation of mito-chondrial proteins occur as a response to oxidative stress and result in post-translational modification of proteins by car-bonyl and disulfide formation or thiol nitrogen exchange. All such alterations contribute to a further block of the electron flow in the respiratory chain resulting in further ROS for-mation. This vicious circle involves ROS-mediated antioxi-dant depletion, and the deficient capacity of mitochondria to inactivate ROS [2]. Ultimately, protein and lipid oxidation, and cytokine production are increased. Hepatocytes react to fat deposition with an early increase of GSH and thioredoxin stores both in the cytosol and in the mitochondria, likely to prevent lipid and protein oxidation [3]. However, in the chronic status, major redox changes occur particularly in mitochondria. Alterations including increased lipid peroxida-tion products with progressive decreases of GSH and thioredoxin, and increases of protein mixed disulfides (PSSG), nitrates and nitrosothiols are consistent with both pro-oxidant protein modifications and increased NO syn-thesis. A critical role for mitochondrial GSH in the develop-ment of the inflammatory form of nonalcoholic steato-hepatitis (NASH) has recently been proposed [57]. GSH depletion sensitizes hepatocytes to inflammatory cytokines and TNF-induced death pathways. We have recently shown that with ongoing steatosis, mitochondrial GSH content declines more rapidly than cytosolic GSH, suggesting that mitochondria are specific targets for oxidative changes in liver steatosis [3]. Likely, GSH is specifically consumed for maintenance of reduced protein sulfhydrils (PSH) and formation of nitrosothiols.

Other pathogenic factors including NO may be important for the progression of steatotic disease and appearance of

884 Current Drug Targets, 2011, Vol. 12, No. 6 Grattagliano et al.

fibrosis. Furthermore, a crucial role in disease progression has been recently assigned to the level and activity of mito-chondrial thioredoxin, a redox active protein with several biological activities, including regulation of PSH/PSSG ratio and other redox sensitive molecules. Thioredoxin is actively involved in NO activity regulation via cleavage of nitro-sothiols [58, 59]. Nitrosothiols, formed by conjugation of NO with free thiols, oppose dangerous reactions such as peroxynitrite formation and act as intracellular messengers that control cellular and mitochondrial functions [60, 61]. In fact, major alterations of thioredoxin levels have been observed with ongoing steatosis (3-14 days). With steatosis progression, thioredoxin changes have been associated with PSSG and nitrosothiols formation. iNOS expression and tyrosine nitrated proteins are markedly increased in liver mitochondria of ob/ob mice where the damage of complex I and cytochrome C is also evident [62]. Increased peroxy-nitrite formation is associated with a variety of interactions, including protein nitration to generate nitrotyrosine in NASH patients [51].

Additional mechanisms of mitochondrial damage in liver steatosis include increased production of angiotensin (ANG) II which is associated with impaired mitochondrial �-oxidation, and oxidative stress. Ren2 rats display elevated endogenous ANG II levels, and exhibit a number of mito-chondrial damages and reduced �-oxidation, as evidenced by ultrastructural abnormalities, decrease of mitochondrial con-tent, percentage of palmitate oxidation, enzymatic activities, and the expression levels of cytochrome C, cytochrome C oxidase subunit 1, and mitochondrial transcription factor A. These abnormalities are substantially improved with either ANG II receptor blocker valsartan or superoxide dismutase/ catalase mimetic tempol treatment (attenuation of mitochon-drial lipid peroxidation) [63].

All the above reported biochemical changes seem to play a fundamental role in the reduced tolerance of fatty livers to oxidative stress insults. Compared with normal livers under conditions of feeding a high fat diet, fatty livers show a greater mitochondrial content of oxidized lipids and proteins together with a low concentration of sulfhydryls and GSH. The mitochondrial catalytic �-F1 subunit of the F0F1-ATP synthase is about 35% lower in fatty livers. Starvation exa-cerbates mitochondrial oxidative injury to a greater extent in fatty livers. In the steatotic group, fasting induces a signi-ficant further decrease of the ATP levels, which is accom-panied by a 70% fall of the catalytic �-F1 subunit. These data indicate that mitochondrial oxidative alterations in fatty livers are associated with a reduction of F0F1-ATP synthase activity. These changes, which are greatly exacerbated after starvation, may account for the reduced synthesis of hepatic ATP observed in the presence of fatty infiltration. Indeed, the loss of reduced PSH is associated with a fall of GSH that likely reflects the oxidation of mitochondrial enzymes, such as the �-F1 subunit, which are known to contain functional sulfhydryl groups [64]. Therefore, mitochondrial dysfunction is a key factor exacerbating the reduced tolerance of fatty liver to an injurious insult. In fact, recent observations have shown that oxidative mitochondrial damages, observed during reperfusion after warm [65, 66] or cold [67] ischemia, may strongly contribute to the deterioration of hepatic energy metabolism observed after transplantation of fatty livers [68]. Depending on a major deterioration of energy

metabolism, associated with impairment of ketogenesis and glucose oxidation [67], the recovery time after reperfusion is markedly prolonged in steatotic livers [69]. Also, in the choline-deficient diet model of liver steatosis, the activity of mitochondrial complex I is altered in association with an increased mitochondrial ROS formation [70]. Thus, the close link between oxidative stress and impaired ATP synthesis appears to be a major key factor that would explain the low tolerance of fatty livers to ischemia-reperfusion injury and oxidative stress.

In conclusion, NAFLD can be considered as a mitochon-drial disease [46], since mitochondrial dysfunction is involved in all pathogenic steps. Adipocytic transformation of hepatocytes is accompanied by major interrelated modi-fications of redox parameters and NO metabolism especially at a mitochondrial level, suggesting an early adaptive pro-tective response but also an increased predisposition towards pro-oxidant insults.

Hepatitis C Virus (HCV) Infection

Liver tissue from patients with HCV infection shows morphological mitochondrial changes and oxidative stress [71, 72]. Additional data suggest a clear relationship between HCV infection and mitochondrial dysfunction as a cause of hepatic oxidative stress and oxidative lipid and protein modifications [73]. This may, at least in part, depend on the primary location of HCV core protein in mitochondria other than in the cytoplasm and endoplasmic reticulum (ER) [74]. NS4A expression significantly alters the intracellular dis-tribution of mitochondria and causes mitochondrial damage [75]. In particular, the expression of HCV core proteins seems to directly interfere with mitochondrial function, which is affected by both direct interactions with viral pro-teins and by secondary effects of viral-activated signalling cascades [76].

HCV protein expression produces specific inhibition of complex I activity, depression of mitochondrial membrane potential and oxidative phosphorylation coupling efficiency, and increased production of reactive oxygen and nitrogen species. Effects are causally related to mitochondrial calcium overload; in fact, inhibition of mitochondrial calcium uptake completely reverses the observed bioenergetic alterations. This event occurs upstream of further mitochondrial dys-function, leading to alterations in the bioenergetic balance and nitro-oxidative stress [77]. Among the identified mito-chondrial proteins with consistently different expressions, prohibitin, a protein chaperon, appears to be up-regulated not only in core-expressing cells but also in full-genomic rep-licon cells and livers of core-gene transgenic mice. It has also been demonstrated that the interaction of prohibitin with mitochondrial DNA-encoded subunits of cytochrome C oxidase is disturbed by the core protein, resulting in a significant decrease in cytochrome C oxidase activity. This may ultimately lead to an impaired function of the mito-chondrial respiratory chain and subsequently to oxidative stress.

The relationship among subcellular organelles in HCV-infected hepatocytes is also supported by the observation that proteins from HCV are documented to traffic sequentially from the ER into mitochondria, probably through the mito-

Mitochondria in Chronic Liver Disease Current Drug Targets, 2011, Vol. 12, No. 6 885

chondria-associated membrane (MAM) compartment. The MAM are sites of ER-mitochondrial contact that enable the direct transfer of membrane bound lipids and the generation of high Ca

2+ microdomains for mitochondrial signalling and

responses to cellular stress. HCV core protein is associated with Ca

2+ regulation and apoptotic signals. HCV core

proteins directly increase mitochondrial Ca2+

uptake via a primary effect on the uniporter. This enhances the ability of mitochondria to sequester calcium in response to ER calcium release and increased mitochondrial ROS production and MPT [78]. In fact, it is generally accepted that trafficking of viral proteins to the MAM may allow viruses to manipulate a variety of fundamental cellular processes that converge at the MAM, including Ca

2+ signalling, lipid synthesis and transfer,

bioenergetics, metabolic flow, and apoptosis. Because of their distinct topologies and targeted MAM subdomains, mitochondrial trafficking of HCV proteins predictably involves alternative pathways and hence distinct targeting signals [79]. Because proper trafficking of viral proteins is necessary for their function, future studies on the require-ments for MAM to mitochondrial trafficking of essential viral proteins may provide novel targets for the rational design of anti-viral drugs.

Among the pathogenic mechanisms of damage, an inc-reased hepatic iron content in HCV-infected patients may be responsible for the increased ROS generation and lipid peroxidation [80]. These alterations may induce the appea-rance of megamitochondria in these patients, especially in those infected by genotype 1b [80]. One hypothesis to exp-lain the appearance of megamitochondria is that core protein alters the signal transduction pathways by binding to the cytoplasmic domains of some receptors that induce a signal-ling cascade promoting MPT [81, 82]. The inhibition of ROS production by the mitochondrial electron transport chain suggests that this is a possible explanation for the inhibitory effect exerted by HCV core protein on mitochondrial function [73]. Therefore, it is likely that excess ROS occur as a direct result of core protein expression with further impair-ment of mitochondrial electron transport chain and sensiti-zation of cells to oxidative insults. Accordingly, HCV-infec-ted hepatocytes die after activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of the DNA repair enzyme poly(ADP-ribose)-polymerase, finally resulting in apoptosis. Moreover, HCV infection activates Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes, resulting into mitochondrial outer membrane permeabilization and release of pro-apoptotic factors [83].

This state of chronic high susceptibility to oxidative stress determines a reduction in mitochondrial metabolic processes, which might contribute to the development of fatty degeneration of hepatocytes by an inhibition of mito-chondrial �-oxidation and oxidative damages to mitochon-drial DNA.

Hemochromatosis

Hemochromatosis is a hereditary iron overload disorder presenting with a common phenotype characterised by normal erythropoiesis, increased transferrin saturation and

serum ferritin levels, associated with excess parenchymal (especially liver) iron deposition [84]. Since the discovery of the hemochromatosis gene (HFE) and its main mutations (C282Y and H63D) in 1996, several novel gene defects have been detected, explaining the mechanism and diversity of iron overload diseases.

The main biochemical defect is related to innate low, but normally regulated, production of the hepatic peptide hormone hepcidin, a 25-amino-acid antimicrobial peptide,

which is the central regulator of iron homeostasis [85].

Iron is essential for life, as it works as a cofactor for enzymes involved in many metabolic processes. But, it can also be harmful especially for the liver, where iron overload causes many changes, including induction of oxidative stress, mitochondrial damage, altered oxidant defense systems and stimulation of hepatocyte proliferation [86]. Iron is transformed into its biologically available form in mitochondria by the iron-sulfur (Fe/S) cluster and heme synthesis pathways; therefore, mitochondria play a crucial role in maintaining cellular iron homeostasis [87]. Within mitochondria, iron is complexed in ferritin molecules which have a restricted tissue distribution and appear to protect mitochondria from iron toxicity and oxidative damage [88]. Iron physiologically works as an important bio-catalyst of oxidation-reduction reactions in the cell, and becomes dangerous when the fraction of redox-active metal ions exceeds that sequestered in specialized proteins or cellular

compartments [89].

Mitochondrial morphology and volume fraction are not altered neither in humans with hemochromatosis nor in rats with chronic iron overload [90]. Conversely, changes in mitochondrial membrane composition and function have been observed in rats with chronic iron overload [91, 92].

Mitochondria from HFE(-/-) mouse liver exhibit dec-reased respiratory capacity and increased lipid peroxidation. The decrease in state 3 respiration rate correlates well with the total liver iron content. Lipid peroxidation is the pro-posed major mechanism that explains mitochondrial iron toxicity [93], as a consequence of increased production but also of a reduced degradation of peroxidative products. The oxidative damage in HFE(-/-) mitochondria seems to be due to decreased MnSOD activity, as manganese supplemen-tation of HFE(-/-) mice leads to enhancement of MnSOD activity and suppresses lipid peroxidation [94]. Also, altered fatty acid composition of membrane phospholipids with increased amount of saturated fatty acids has been observed in rats with iron overload [95]. Exposure of isolated mito-chondrial fractions to iron results in a decrease of state 3 respiration which correlates with the level of lipid peroxi-dation products, thus further supporting the role for lipid peroxidation in mitochondrial impairment. Studies on iso-lated liver mitochondria from rats with chronic iron overload showed decreased activities of ferrocytochrome C reductase of the respiratory complex II, III and IV [96]. Therefore, mitochondrial impairment in experimental chronic iron over-load models results both from a disturbed lipid environment of the individual enzyme complexes of the electron transport chain and from direct oxidative damage to these enzymes [97].

886 Current Drug Targets, 2011, Vol. 12, No. 6 Grattagliano et al.

Wilson’s Disease

Like iron, copper is an essential transition metal ion. Its redox reactivity, whilst essential for the activity of mitochon-drial enzymes, can also be a source of harmful ROS if not chelated to biomolecules. Copper is an essential cofactor for over a dozen enzymes in which it is bound to specific amino acid residues in an active site [98]. Copper is sequestered by protein chaperones and moved across membranes by protein transporters with the excess held in storage proteins for future use. In mitochondria, these proteins are a distinct ceruloplasmin and metallothionein [99].

Structural changes in hepatocellular mitochondria are characteristic of Wilson’s disease (WD), an autosomal-recessive disorder, which is caused by mutations in a P-type ATPase and is associated with excess copper deposition in the liver. These alterations are more pronounced in the early stages and tend to disappear when the disease progresses to more advanced forms [100]. Different types of morpho-logical alterations have been described and include mega-mitochondria, matrix swelling and granular inclusion.

Mitochondrial dysfunction in copper disorders is asso-ciated with hepatocyte lipid accumulation, suggesting a de-fect in lipid metabolism related to mitochondrial impairment. The latter is related neither to the extent of steatosis nor to the hepatic concentration of copper, thus suggesting a genetic defect [101]. Mitochondrial DNA can be oxidatively damaged by copper accumulation with consequent multiple deletions and point mutations [102]. Animal studies with dietary copper overload have shown that increased lipid peroxidation products in mitochondria are partially pre-vented by vitamin E administration [103]. There is also evidence of severe mitochondrial dysfunction in the liver of patients with WD in which respiratory enzyme activities are decreased: complex I by 62%, complex II+III by 52%, com-plex IV by 33%, and aconitase by 71% [104]. Interestingly, copper ions have an inhibitory effect on cell respiration only at 500 �M concentration and after 48 h incubation but produce a significant uncoupling effect at lower concen-trations [105]. Copper induces an early and sharp increase of intracellular production of ROS which, in turn, inhibits pyruvate dehydrogenase (PDH) and �-ketoglutarate de-hydrogenase (KGDH). Liver PDH and KGDH activities are reduced in the Atp7b mouse model of WD prior to liver damage, and are partially restored by oral thiamine supple-mentation. These data support the hypothesis that copper-induced ROS may result in hepatocellular death after inhibition of PDH and KGDH [106].

The redox imbalance linked with copper accumulation catalyzes the oxidation of GSH to GSSG resulting in the generation of O2

-. Down-regulation of Cu-Zn superoxide dis-

mutase (SOD) consequent to the degradation of this enzyme causes decreased dismutation of O2

- that further contributes

to enhanced levels of O2- in the periportal region of the liver.

Decreased functioning of MnSOD activity, reduction in mitochondrial thiol/disulphide ratio and generation of O2

- are

much higher in mitochondria from the periportal region. This suggests involvement of mitochondria in the regional hepatotoxicity as evidenced by ATP depletion, collapse of mitochondrial membrane potential and induction of MPT [107]. In fact, incubation of hepatocytes with 10 �M copper results in a strong stimulation of ROS formation, and after

2 h of exposure a significant increase of both apoptotic and necrotic cells occurs. In a sub-population of cells, copper accumulation induces a decrease of mitochondrial membrane potential and occurrence of the MPT [108].

Because current medical treatment options are not effect-ive in all WD patients and adherence to therapy is a problem, at the light of the above reported observation, gene therapy might represent a future option. Meanwhile, thiamine or lipoic acid may constitute potential therapeutic agents.

Chronic Cholestasis

A number of biochemical abnormalities including toxic bile salt accumulation and oxidative mitochondrial altera-tions are likely to be implicated in the onset and evolution of cholestatic liver diseases of various causes [109].

In fact, the contribution of mitochondria to the genesis of liver injury in primary biliary cirrhosis (PBC) is documented by the presence of antimitochondrial antibodies (AMAs) in the serum of over 95% of patients with PBC. This antibody reacts with at least one of the five components of the M2 antigen identified as the 2-oxoacid dehydrogenase complex [110].

An important role in the progression of hepatic altera-tions appears to be linked to the exposure of hepatocytes to hydrophobic bile salts, which is in turn associated with time- and concentration-dependent increased generation of ROS and appearance of MPT [111]. In fact, it has been observed that increasing concentrations of different bile acids such as litocholic (LCA), deoxycholic (DCA), ursodeoxycholic (UDCA), chenodeoxycholic (CDCA), glycochenodeoxy-cholic (GCDCA), or taurochenodeoxycholic (TCDCA) acids decrease mitochondrial transmembrane potential. These compounds also decrease state 3 respiration and increase state 4 respiration. The concentration-dependent stimulation of state 4 by the above-mentioned bile salts is associated with enhanced permeability of mitochondria to H

+. Addition

of the same bile salts to succinct exposed mitochondria results in a dose-dependent membrane depolarization and stimulation of MPT [112]. This effect has also been reported with other experimental models of cholestasis [113].

Interestingly, in an acute experimental model of choles-tasis, liver mitochondria isolated from alpha-naphthyliso-thiocyanate-treated rats showed higher state 3 respiration, respiratory control ratio, ADP/O, and ATP/ADP ratio com-pared to controls. In this model, cholestatic mitochondria exhibit also an increased resistance to disruption of mito-chondrial Ca

2+ homeostasis due to MPT induction. These

mitochondria exhibit an improved efficiency, indicating that acute cholestatic events may lead to adaptive response to resist cell death that occurs during cholestasis [114]. The same research group demonstrated that as opposed to an acute insult, chronic exposure to alpha-naphthylisothio-cyanate results into enhanced MPT in the absence of mitochondrial respiratory and membrane potential alterations [115].

Cholestatic rats with bile duct ligation (BDL) exhibit a decreased oxygen consumption per mitochondrial mass, indicating a reduced oxidative metabolism [116] and in particular decreased complex I, II and III activity [117]. Since complex I and III are limiting steps in hepatic fatty

Mitochondria in Chronic Liver Disease Current Drug Targets, 2011, Vol. 12, No. 6 887

acid metabolism, these alterations likely depend on oxidative lipid and protein changes and may have important metabolic significance [118]. Increased concentration of lipid peroxi-dation products and accumulation of oxidized proteins have been observed in mitochondria isolated from BDL rats [9, 119], confirming the hypothesis of oxidative damages to functional structures as a major cause of mitochondrial impairment in chronic cholestasis. In particular, the concen-tration of thiobarbiturate reacting substances, a lipid peroxi-dation product, increases in mitochondria from BDL rats as a function of ongoing cholestasis. This oxidative process is associated with a decrease of mitochondrial GSH content and of ubiquinones 9 and 10, other antioxidant molecules involved in the electron transport chain. The activity of complex II and III of the electron transport chain is also decreased in BDL rats as a consequence of oxidative damages to mitochondrial lipids and proteins [109]. This has also been observed in rats where ongoing cholestasis was associated with early oxidative changes in mitochondria. PSH is decreased and negatively correlated with PSSG [120]. In this rat model of cholestasis, the mtDNA copy number is altered and apoptotic signaling is stimulated. In addition, peroxisome proliferator-activated receptor-coacti-vator-1� and transcriptional factor A are impaired, thus indicating that transcriptional regulation of mitochondrial biogenesis is imbalanced already after few hours of complete bile duct obstruction, resulting in later mitochondrial dys-function and consequent cholestatic liver injury via activa-tion of the intrinsic apoptotic pathway [121].

Morphometric and biochemical analyses have shown that the mitochondrial content per hepatocyte is increased in BDL rats, suggesting that mitochondrial proliferation is a mechanism to maintain liver mitochondrial function during cholestatic conditions. It is known that NO is a determining factor for mitochondrial proliferation [122, 123] and com-pensatory mitochondria proliferation appears to be a specific characteristic of chronic cholestasis [1]. In fact, high hepatic levels of nitrosothiols during cholestasis may provide bene-ficial effects by supporting cell survival and mitochon-driogenesis, although at this point it is not known if ampli-fication of an already damaged mitochondrial population is beneficial for the cell. However, with the progression of cholestasis, the decreased availability of thioredoxin together with increased production of NO and with GSH depletion [124] may result in enhanced protein nitrosation [125] and PSH oxidation.

Patients with extra-hepatic cholestasis due to bile duct obstruction display oxidative alterations of liver proteins but bile duct re-canalization improves oxidative changes [126]. Thus, changes seen in hepatic cholestatsis also depend on the retention of toxic products, which contribute to oxidative alterations. Antioxidants may effectively reduce liver injury and MPT stimulation caused by toxic bile salts [111].

The mechanism by which hydrophilic bile salts provide protection on mitochondrial electron transport chain, dam-aged by hydrophobic bile salts accumulation, appears to be related to a decreased incorporation of toxic bile salts into mitochondrial membranes [127]. Also, interventions directed to sustain mitochondrial antioxidant activity, such as that exerted by thioredoxin and to support NO metabolism in the early phase of cholestasis may yield protection by favoring

mitochondrial proliferation and allowing protein redox status maintenance.

mtDNA DEPLETION SYNDROME AND CITRULLI-NEMIA

mtDNA depletion syndromes (MDS) are a group of severe mitochondrial disorders usually occurring during infancy or childhood, resulting from tissue-specific reduction of mtDNA copy number [128]. All these syndromes result from defects in nucleus-encoded factors and are mostly inherited as an autosomal recessive trait [129]. The hepato-cerebral form is probably the most common variant of MDS and it is characterized by an infantile onset of acute liver failure and is associated with lethargy, hypotonia, vomiting, seizures and hypoglycaemia [130]. Three genes have been identified as being involved in this form of disease: DGUOK, MPV17 and POLG1 [131-133]. DGUOK encodes the deoxyguanosine kinase that, together with thymidine kinase 2 (TK2), maintains the supply of dNTPs for mtDNA synthesis [131]; MPV17 seems to be involved in the mtDNA maintenance and regulation of mitochondrial proteins invol-ved in oxidative phosphorylation [133] and POLG1 encodes the catalytic subunit of mitochondrial DNA polymerase [132]. POLG1 is the major disease gene in mitochondrial disorders [134]. Mutations in this gene can be associated with depletion but also with multiple deletions or point mutations of mitochondrial DNA. Bortot and coworkers described the case of an 18-month-old patient with recurrent hypoketotic hypoglycaemia and fatal liver dysfunction with tissue-specific mtDNA depletion due to mutations in the POLG1 gene [135].

Citrin is a calcium-binding mitochondrial solute-carrier protein primarily expressed in the liver, heart and kidney [136]. The mitochondrial aspartate-glutamate carrier (AGC) is important in conjugation with the malate/aspartate shuttle, in urea synthesis and in gluconeogenesis [137]. Citrin is acti-vated by calcium on the external side of the inner mito-chondrial membrane [138]. Citrin deficiency induces two clinical features: neonatal intrahepatic cholestasis caused by citrin deficiency and adult-onset type II citrullinemia, CTLN2 [139, 140]. Mutations in the human gene coding for citrin are responsible for CTLN2, which is an autosomal recessive disease caused by a liver-specific deficiency in arginino-succinate synthase [137, 141]. Sawada and co-workers found evidences that citrin downregulation induces apoptosis of hepatocytes through the mitochondrial death pathway, highlighting the importance of citrin in survival of hepatocytes and maintenance of liver function [142].

ASSESSMENT OF MITOCHONDRIAL FUNCTION IN VIVO

A number of tests have been suggested for assessing mitochondrial function in the living subject (Table 1). Considering the important role of mitochondria in chronic liver disease and the difficulty to assess liver function with current routine tests, these may indeed serve several pur-poses; they could support diagnosis and prognosis of chronic liver disease, and they may also be used to assess the effect of new therapies.

888 Current Drug Targets, 2011, Vol. 12, No. 6 Grattagliano et al.

Respiratory Chain Activity

The mitochondrial respiratory chain activity can be assessed in vivo by measuring the ratio acetoacetate/�-hydroxybutyrate or pyruvate/lactate, which is related to the NAD/NADH ratio in the arterial blood. A reduced activity of the respiratory chain is associated with a decrease in NAD/NADH ratio, which consequently results in decreased acetoacetate/�-hydroxybutyrate (mitochondrial redox state) and pyruvate/lactate (cytosolic redox state) ratios. Patients with chronic liver disease may have a decreased ratio of the above reported parameters in arterial blood [143, 144].

Alpha-Ketoacid Dehydrogenase

Specific mitochondrial metabolic pathways can be assessed in vivo by performing breath test analysis using substrates delivering labeled CO2 during mitochondrial metabolism. The most interesting tools include �-ketoiso-caproic acid (KICA), benzoic acid, octanoic acid, and methionine. KICA breath test assesses the activity of branched-chain �-ketoacid dehydrogenase, an enzyme located in the mitochondrial matrix [145]. This method is particularly reliable to assess mitochondrial function in patients with alcoholic liver disease or after administration of mitochondrial toxic drugs [146, 147]. Chronic alcoholics have a decreased KICA decarboxylation to a greater extent than conventional quantitative tests of hepatic function such as galactose elimination capacity or the aminopyrine clear-ance. This specific enzymatic alteration is likely related to the ethanol-induced redox shift (NADH/NAD ratio). More recently, KICA breath test helps to distinguish patients with different histological stages of NAFLD [148]: the more advanced the disease is, the greater is the impairment of KICA decarboxylation. In addition, mitochondrial impair-ment as reflected by KICA analysis appears to be also proportional to the BMI of the patient.

Octanoic Acid

Mitochondrial �-oxidation can be assessed by the use of fatty acids breath test [149]. There are a few reports with oral [

13C]-octanoate that have shown how the rate of mito-

chondrial metabolism of octanoate is increased in patients with NAFLD without advanced disease [150]. This would indicate that the mitochondrial fatty acids degradation in vivo is increased in patients with early stages of fatty liver disease.

Benzoic Acid

Benzoic acid undergoes hepatic conversion to hippuric acid after mitochondrial activation to form a coA-derivative [151]. The final product is excreted in the urine. Decreased availability of ATP and/or coA results in a decreased renal excretion of hippurate after administration of benzoate. This test has a good reproducibility in patients with organic acidurias [152] and in experimental liver cirrhosis [153].

Urea Production

Quantification of urea production is a method to assess liver function in patients with liver cirrhosis [154, 155]. The principle is based on the assessment of urea synthesis after infusion of amino acids as substrates, resulting in plasma accumulation of �-amino nitrogen. Urea synthesis increases in parallel to plasma �-amino nitrogen, thus representing the “functional hepatic nitrogen clearance” [155] which correlates with the galactose elimination capacity, a liver function test reflecting hepatocellular mass.

[31

P] Nuclear Magnetic Resonance Spectroscopy

This technique allows to assess changes in hepatic phos-phate metabolism of cirrhotic patients after the adminis-tration of fructose [156]. This method offers the possibility to monitor oxidative phosphorylation under different meta-bolic conditions [157].

THERAPEUTIC PERSPECTIVES

Maintenance of adequate mitochondrial function is not only important for the survival of hepatocytes but also for providing functionally valid mitochondria for new cells. The improvement of mitochondrial function is of great import-ance in patients with chronic liver disease and an intriguing challenge for research.

There is increasing evidence that the integrity of mito-chondrial antioxidant defense is of vital importance parti-cularly with regard to the functioning of the whole liver. In particular, the improvement of mitochondrial function following UDCA exposure in rats with chronic cholestasis [127] demonstrates that mitochondrial alterations are susceptible to therapeutic interventions and that by acting on pathogenic mechanisms, the damage could be partially reverted. Of particular interest are results concerning the

Table 1. Assessment of Mitochondrial Function in vivo

Method Measured Reaction Clinical Conditions where the

Reaction can be Impaired [Refs.]

Acetoacetate/�-hydroxybutyrate ratio Respiratory chain activity Chronic liver diseases [126, 127]

�-ketoisocaproic acid (KICA) 13C-breath test

Activity of branched-chain �-ketoacid dehydrogenase

ALD; NAFLD; toxic liver injury [143-145]

Octanoic acid 13C-breath test Fatty acid �-oxidation NAFLD [148]

Benzoic acid Hippuric acid formation after mitochondrial

activation to form coA-derivatives Liver cirrhosis [149]

Urea production Urea synthesis after infusion of amino acids Liver cirrhosis [152, 153]

31P-NMR spectroscopy Oxidative phosphorylation Liver cirrhosis [154]

Legend: ALD, alcoholic liver disease; NAFLD, nonalcoholic liver disease.

Mitochondria in Chronic Liver Disease Current Drug Targets, 2011, Vol. 12, No. 6 889

effect of rosiglitazone on mitochondrial morphology in patients with NAFLD [158]. The available data show that administration of a PPAR modulator such as rosiglitazone results in mitochondrial changes, and therefore confirm the hypothesis of a functional network between mitochondria and other subcellular organelles, such as peroxisomes.

New data showing maintenance of adequate mitochon-drial function during the onset and development several other diseases are eagerly awaited and should be widely explored in the near future.

ACKNOWLEDGEMENTS

Work was supported in part by research grants from the Italian Ministry of University and Research (FIRB 2003 RBAU01RANB002), the Italian National Research Council (CNR) (short-term mobility grant 2005), the University of Bari (grants ORBA09XZZT, ORBA08YHKX) (P.P.), and from the National Institutes of Health (US Public Health Service) (research grants DK54012 and DK73917) (D.Q.-H.W.). C.D. is sponsored by the Portuguese Foundation for Science and Technology (FCT, Ph.D. fellowship # SFRH/BD/48133/2008).

AUTHORS’ INVOLVEMENT WITH THE MANU-SCRIPT

I. Grattagliano and P. Portincasa have contributed by drafting of the manuscript. All authors have equally contri-buted to manuscript concept, literature search, critical revi-sion of the manuscript for important intellectual content, including tables and figures.

ABBREVIATIONS

ALD = Alcoholic liver disease

AMA = Antimitochondrial antibodies

ANG = Angiotensin

ATP = Adenosine triphosphate

BDL = Bile duct ligation

ER = Endoplasmic reticulum

Fe/S = Iron-sulfur

FFA = Free fatty acid

GSH = Glutathione

HFE = Hemochromatosis gene

iNOs = Inducible nitric oxide synthase

KGDH = �-ketoglutarate dehydrogenase

KICA = �-ketoisocaproic acid

MAM = Mitochondria-associated membrane

mtDNA = Mitochondrial DNA

MPT = Mitochondrial permeability transition

NAFLD = Nonalcoholic fatty liver disease

NASH = Nonalcoholic steatohepatitis

NO = Nitric oxide

PBC = Primary biliary cirrhosis

PDH = Pyruvate dehydrogenase

PSSG = Protein mixed disulfides

PSH = Protein sulfhydrils

ROS = Oxygen species

SAME = S-adenosyl-L-methionine

SOD = Superoxide dismutase

WD = Wilson’s disease

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Received: October 29, 2009 Revised: May 28, 2010 Accepted: June 10, 2010

PMID: 21269263