Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease


  • Potential conflict of interest: Nothing to report.

  • Supported by INSERM (Institut National de la Santé et de la Recherche Médicale).

Address reprint requests to: Bernard Fromenty, INSERM UMR991, Faculté de Pharmacie, 2, avenue du Professeur Léon Bernard, 35043 Rennes Cedex, France. E-mail: fax: +33 2 23 23 53 85


The worldwide epidemic of obesity and insulin resistance favors nonalcoholic fatty liver disease (NAFLD). Insulin resistance (IR) in the adipose tissue increases lipolysis and the entry of nonesterified fatty acids (NEFAs) in the liver, whereas IR-associated hyperinsulinemia promotes hepatic de novo lipogenesis. However, several hormonal and metabolic adaptations are set up in order to restrain hepatic fat accumulation, such as increased mitochondrial fatty acid oxidation (mtFAO). Unfortunately, these adaptations are usually not sufficient to reduce fat accumulation in liver. Furthermore, enhanced mtFAO without concomitant up-regulation of the mitochondrial respiratory chain (MRC) activity induces reactive oxygen species (ROS) overproduction within different MRC components upstream of cytochrome c oxidase. This event seems to play a significant role in the initiation of oxidative stress and subsequent development of nonalcoholic steatohepatitis (NASH) in some individuals. Experimental investigations also pointed to a progressive reduction of MRC activity during NAFLD, which could impair energy output and aggravate ROS overproduction by the damaged MRC. Hence, developing drugs that further increase mtFAO and restore MRC activity in a coordinated manner could ameliorate steatosis, but also necroinflammation and fibrosis by reducing oxidative stress. In contrast, physicians should be aware that numerous drugs in the current pharmacopoeia are able to induce mitochondrial dysfunction, which could aggravate NAFLD in some patients. (Hepatology 2013;58:1497–1507)




acetyl-CoA carboxylase


adiponectin receptor


adenosine diphosphate


adenine nucleotide translocator


adenosine triphosphate


carbohydrate responsive element-binding protein


coenzyme A


cytochrome c oxidase


carnitine palmitoyltransferase 1


cytochrome P450 2E1


de novo lipogenesis


endoplasmic reticulum


fatty acid


flavin adenine dinucleotide


fatty acid oxidation


fibroblast growth factor 21


forkhead box O1 transcription factor


reduced glutathione


high-fat diet


hypoxia inducible factor-1 alpha


interleukin 6


inducible nitric oxide synthase


insulin resistance


ketone bodies


ketoisocaproic acid


long-chain fatty acid


medium-chain acyl-CoA dehydrogenase


methionine/choline deficient


medium-chain fatty acid


manganese superoxide dismutase


mitochondrial respiratory chain


mitochondrial DNA


mitochondrial FAO


mitochondrial GSH


nicotinamide adenine dinucleotide


nonalcoholic fatty liver disease


nonalcoholic steatohepatitis


nonesterified fatty acid


nitric oxide


oxidative phosphorylation


PPARα coactivator 1alpha


peroxisome proliferator-activated receptor alpha


reactive nitrogen species


reactive oxygen species


short-chain fatty acid


sterol regulatory element-binding protein 1c




tricarboxylic acid


tumor necrosis factor alpha


uncoupling protein 2


very low density lipoprotein


white adipose tissue

Most obese people develop fatty liver, which is characterized by the presence of large vacuoles of lipids (mainly triacylglycerol) within the cytosol. Although fatty liver is a benign condition, it can progress in the long term to nonalcoholic steatohepatitis (NASH) in 10% to 20% of patients. In addition to macrovacuolar steatosis, NASH is characterized by microvesicular steatosis, inflammation, fibrosis, and the presence of hepatocyte injury in the forms of ballooning and apoptosis.[1, 2] Even though NASH is not by itself a severe hepatic lesion, it can progress to cirrhosis and liver cancer.[3] Collectively, the large spectrum of conditions ranging from fatty liver to NASH is referred to as nonalcoholic liver disease (NAFLD). In order to better assess the histological changes in NAFLD, in particular during therapeutic trials, the Pathology Committee of the NASH Clinical Research Network designed and validated the NAFLD activity score (NAS) system, derived from the sum of individual scores for steatosis, lobular inflammation, and hepatocellular ballooning.[2, 4]

During NAFLD, several metabolic adaptations are set up in order to curb fat accumulation. In particular, increased mitochondrial fatty acid oxidation (mtFAO) plays a significant role, but this adaptation secondarily induces oxidative stress.5,6 This could participate in the progressive reduction in mitochondrial respiratory chain (MRC) activity, which further aggravates oxidative stress and impairs energy output.5-7 Before considering mitochondrial adaptations and dysfunctions in NAFDL, we will recall key features of lipid and carbohydrate homeostasis and the role of mitochondria in FAO and energy production.

Normal Role of Hepatic Mitochondria in Fat and Energy Homeostasis

Hepatic Fat Metabolism

Hepatic fatty acids (FAs) can be: (1) taken up from the pool of plasma nonesterified FAs (NEFAs) released by white adipose tissue (WAT); (2) generated from the hydrolysis of chylomicrons coming from the intestine; and (3) synthesized through de novo lipogenesis (DNL).8,9 Depending on the nutritional/hormonal status, hepatic FAs either enter mitochondria to undergo β-oxidation or are esterified into triacylglycerol (TAG), usually secreted in plasma as very low density lipoproteins (VLDL).10 Alternatively, the TAG molecules can accumulate as fat droplets surrounded by proteins belonging to the PAT family.11


Whereas short-chain and medium-chain FAs (SCFAs and MCFAs) freely enter mitochondria, the mitochondrial entry of long-chain FAs (LCFAs) depends on several enzymes, including carnitine palmitoyltransferase 1 (CPT1) (Fig. 1). Since CPT1 can be inhibited by malonyl-CoA (a metabolite generated during DNL), there is usually an inverse relationship between the rate of LCFA mitochondrial β-oxidation and that of DNL.[5]

Figure 1.

mtFAO and energy production by mitochondria. Whereas SCFAs and MCFAs freely enter mitochondria, the entry of LCFAs within these organelles requires different enzymes such as long-chain acyl-CoA synthetase (ACS), CPT1, and CPT2. SCFA-CoA, MCFA-CoA, and LCFA-CoA thioesters are then oxidized into acetyl-CoA moieties by way of the β-oxidation process. During fasting, acetyl-CoA moieties are mainly used to generate KB by way of ketogenesis. mtFAO generates NADH and FADH2, which transfer their electrons (e) to the MRC, thus regenerating NAD+ and FAD used for other β-oxidation cycles. Within the MRC, electrons are sequentially transferred to different polypeptide complexes (numbered I to IV) embedded within the inner membrane. Electron transfer from complex III to complex IV is mediated by cytochrome c (c). The final transfer of the electrons to oxygen takes place at the level of complex IV (also called COX), and generates water. The flow of electrons in the MRC is coupled with the extrusion of protons (H+) from the matrix, thus generating the mitochondrial membrane potential (ΔΨm). When energy is needed, these protons reenter the matrix through ATP synthase, thus leading to the partial dissipation of ΔΨm and phosphorylation of ADP into ATP. Adenine nucleotide translocator (ANT) then extrudes ATP from mitochondria, in exchange for cytosolic ADP. After a meal, pyruvate produced by anaerobic glycolysis enters the TCA cycle to be degraded into CO2, NADH, and FADH2, which are also oxidized by the MRC. Impairment of electron transfer within the MRC favors the leakage of electrons from complexes I and III, thus leading to ROS generation. Thirteen polypeptides of the MRC are encoded by mtDNA, and consequently any significant damage to this genome can impair MRC activity.

During fasting, low insulinemia and high glucagon levels favor TAG lipolysis in WAT, thus inducing NEFA release into the circulation and their oxidation in liver. Within mitochondria, every FA undergoes four sequential reactions, which generate one acetyl-CoA molecule and a shortened FA. The cycle is repeated to split FAs into several acetyl-CoA subunits, which produce acetoacetate and β-hydroxybutyrate. These ketone bodies (KBs) are then oxidized in extrahepatic tissues by the tricarboxylic acid (TCA) cycle to generate adenosine triphosphate (ATP).5,12

Besides decreased malonyl-CoA levels, higher FAO and ketogenesis during fasting also result from the increased expression of different enzymes through the activation of transcription factors, such as forkhead box A2 and peroxisome proliferator-activated receptor α (PPARα).5,13 For instance, LCFA-mediated PPARα activation increases the expression of the mitochondrial enzymes CPT1 and medium-chain acyl-CoA dehydrogenase (MCAD).

Fasting is also associated with the hepatic activation of sirtuins, which positively regulates different mitochondrial enzymes involved in FAO and MRC activity.14,15 Sirtuins also interact with PPARα coactivator 1α (PGC1α), thus favoring mitochondrial biogenesis.14,16 Finally, activation of adenosine monophosphate-activated protein kinase induces the inactivation of the lipogenic enzyme acetyl-CoA carboxylase (ACC), hence decreasing malonyl-CoA levels.[5]

In contrast, high insulin and glucose levels after a meal favor hepatic DNL by way of the synergic action of sterol regulatory element-binding protein 1c (SREBP1c) and carbohydrate responsive element-binding protein (ChREBP).5,8

Production of Energy and ROS by the MRC

mtFAO and other oxidative reactions (e.g., TCA cycle) produce NADH and FADH2, which are then reoxidized by the MRC, thus regenerating nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD) required for other cycles of oxidation.12,17 NADH and FADH2 oxidation is coupled to ATP synthesis through the oxidative phosphorylation (OXPHOS) process (Fig. 1).

Most of the electrons provided to the MRC migrate along this chain, to finally reach cytochrome c oxidase (COX, or complex IV), where they safely combine with oxygen and protons to form water (Fig. 1). However, a fraction of these electrons leaks from complexes I and III to form the superoxide anion radical.5,17 This radical can then be dismutated by manganese superoxide dismutase (MnSOD) into hydrogen peroxide (H2O2), which is normally detoxified into water by glutathione peroxidase (GPx) and reduced glutathione (GSH).18 Thus, most mitochondrial reactive oxygen species (ROS) are usually detoxified and residual ROS serve as signaling molecules.[5] However, any significant reduction of MRC activity can induce ROS overproduction, thus triggering oxidative stress.5,7,17

The Mitochondrial Genome

Thirteen MRC polypeptides are encoded by mitochondrial DNA (mtDNA), a small circular DNA present in several copies within the matrix (Fig. 1),12,17 and sensitive to oxidative damage.5,19 The oxidative attack of mtDNA can generate 8-hydroxydeoxyguanosine, point mutations, and deletions. In addition to ROS, reactive nitrogen species (RNS) and lipid peroxidation products are able to damage mtDNA.20,21 Irreparable damages to mtDNA can induce its degradation by nucleases, thus leading to mtDNA depletion.19,22

Mitochondria also contain nuclear-encoded proteins required for mtDNA maintenance including mitochondrial transcription factor A (Tfam) involved in mtDNA transcription and mtDNA repair enzymes. Importantly, expression of Tfam and several MRC polypeptides is controlled by nuclear respiratory factors 1 and 2 (NRF1 and 2). Moreover, PGC1α interacts in the nucleus with NRF1, NRF2, and PPARα in order to coordinate the expression of nuclear genes governing mitochondrial function and biogenesis.16,23

Physiopathology of Fatty Liver and NASH

Role of Insulin Resistance and Hyperinsulinemia in Fatty Liver

Insulin resistance (IR) in muscle and WAT plays a central role in the pathogenesis of fatty liver (Fig. 2).8,24 In particular, IR in WAT favors TAG lipolysis, thus leading to uncontrolled NEFA release into the circulation.9,25 Because NEFA uptake by the hepatocytes is concentration-dependent, IR greatly increases the amount of NEFAs entering the liver.26 FAs are also synthesized more actively in liver during IR (Fig. 2), since hyperinsulinemia overactivates SREBP1c.5,26

Figure 2.

Metabolic impairment leading to fatty liver and adaptations. In the normal liver, lipid storage is minimal as there is a tight equilibrium between their accumulation (i.e., synthesis and uptake) and their removal (i.e., oxidation and export). In obese and diabetic individuals, this metabolic steady state is profoundly disturbed as insulin resistance and hyperinsulinemia favor both lipid uptake and synthesis (arrow 1). This leads to a progressive accumulation of lipids (mainly triglycerides). When type 2 diabetes develops, hyperglycemia can also promote lipid synthesis. The expansion of hepatic lipids can be limited by different hormonal and metabolic adaptations leading to increased peroxisomal and mitochondrial FAO, as well as enhanced VLDL secretion (arrow 2).

Excess of fat in liver can in turn cause IR in this organ.5,27 Intriguingly, IR in liver only affects some, but not all, insulin-sensitive metabolic pathways.28,29 For instance, whereas gluconeogenesis is less inhibited by insulin, DNL is overactivated by hyperinsulinemia.5,29 However, the mechanisms responsible for mixed hepatic insulin sensitivity and resistance are not fully understood, although different hypotheses have been put forward.29-34

Type 2 Diabetes and Liver

Hyperglycemia contributes to fatty liver during type 2 diabetes, in particular by overactivating ChREBP.5,35 Furthermore, high glucose levels promote ROS overproduction within hepatocytes, thus favoring oxidative stress and mitochondrial dysfunction.5,36 Type 2 diabetes can also be associated with high glucagonemia, which contributes to hyperglycemia and ketoacidosis.37 High glucagonemia could also impair hepatic function,38 possibly by increasing the expression of cytochrome P450 2E1 (CYP2E1).39

From Simple Fatty Liver to NASH

At least three major events are involved in the progression of fatty liver to NASH, including overproduction of ROS and RNS, lipotoxicity, and increased release of proinflammatory and profibrogenic cytokines.5,39-41

Different mechanisms could explain higher ROS generation during NAFLD: (1) Induction of CYP2E1,39,42 a ROS-producing enzyme located within the endoplasmic reticulum (ER) and mitochondria.43,44 (2) Enhanced peroxisomal FAO, the first enzymatic step of which produces H2O2.5,45 (3) Higher mtFAO, which is also able to generate ROS, possibly at the level of electron transfer flavoprotein-ubiquinone oxidoreductase,46,47 or downstream within the MRC.5 Reduced ROS detoxification could also favor oxidative stress. Indeed, several studies showed lower GSH levels in NAFLD, including within the mitochondria.48-51 Decreased expression and/or activity of antioxidant enzymes such as GPx and SODs could also occur, in particular at the mitochondrial level.48,52-55

NAFLD has been associated with higher hepatic expression of the inducible nitric oxide (NO) synthase (iNOS), mainly as a consequence of tumor necrosis factor-α (TNF-α) overproduction by the Kupffer cells.56,57 Increased expression of the neuronal NOS (nNOS) could be also a significant source of RNS.58 NO can readily react with the superoxide anion, thus generating the RNS peroxynitrite, which has deleterious effects on mitochondrial function and genome.20,58

Genetic Susceptibilities

Genetic susceptibilities could increase the risk of developing fatty liver, but also its progression to NASH in some individuals. Thus far, a polymorphism in the adiponutrin (PNPLA3) gene seems to be the most robust genetic determinant associated with fatty liver.59 Genetic polymorphisms affecting the mitochondrial ability to oxidize fat could also modulate the risk of NAFLD, in particular in genes encoding PPARα, leptin, adiponectin, or receptors of these adipokines.5,59,60 Polymorphisms in the TNF-α, transforming growth factor-β, and MnSOD genes have been shown to favor NASH.5,59,61

Mitochondrial Adaptations and Dysfunctions in Fatty Liver

During fatty liver, several metabolic adaptations can restrain fat accretion (Fig. 2). A major mechanism is the stimulation of mitochondrial and peroxisomal FAO.5,6,10,62,63 Increased mitochondrial oxidation of FAs and other substrates could also be an adaptation to produce more ATP needed for DNL and gluconeogenesis.64 However, higher FAO can be associated with different mitochondrial abnormalities, as discussed below (Table 1). Another important adaptation in fatty liver could be an increased release of VLDL.65-67

Table 1. Summary of the Main Mitochondrial Changes Reported in Fatty Liver, Mild to Moderate NASH, and Definite NASH and Comments Regarding Some Discrepancies Found in the Literature
Liver LesionMitochondrial Changes (and References)Comments
Simple fatty liver (humans and rodents)FAO and ketogenesis: 
 - Increased (53,64,68-70,73-80)Within each class of liver lesion, discrepancies between some studies could be due to differences in:
 - Reduced (71,82-86)1) Methodology, in particular for the assessment of FAO, which has been assessed in isolated mitochondria or in liver homogenates.
 Complex I activity:2) Degree of fatty liver, IR, oxidative stress and inflammation. For studies in rodents fed high-calorie diets, these parameters can greatly vary depending on the diet (see below) and the feeding duration.
 - Unchanged (86,88)3) Nutritional factors. Dietary lipids and fructose can have a significant impact on the expression of mitochondrial enzymes and FAO.
 - Reduced (132)4) Genetic factors. In humans, several polymorphisms (or less frequently mutations) are able to modulate mitochondrial function such as FAO and ketogenesis. Mitochondrial oxidative capacity can also vary between different strains of mice.
 Complex IV (COX) activity:5) Imperfect histological classification, in human and rodent investigations.
 - Unchanged (82,88,127,131)Reduced activity of the MRC complexes I and IV (i.e., electron transfer) is not necessarily associated with decreased mitochondrial respiration (i.e. oxygen consumption). Indeed, respiration is impaired only when MRC activity is significantly inhibited.
 - Increased (130)The variation of mtDNA levels could depend on the extent of lipid overload, oxidative stress and inflammation (e.g., TNFα). Classically, only marked mtDNA depletion (or mtDNA oxidative damage) can alter MRC activity.
 - Reduced (86,132) 
 Oxygen consumption (with succinate and ADP): 
 - Unchanged (82,88,131,133,134) 
 - Increased (53) 
 - Reduced (76,77,86,127,135) 
 mtDNA levels: 
 - Increased (73,79,126,127) 
 - Reduced (123,128,129) 
Mild to moderate NASH (ob/ob and db/db mice)FAO and ketogenesis: 
 - Increased (57,149,152,153,160) 
 - Reduced (161) 
 Complex I activity: 
 - Reduced (57,58,172-174) 
 Complex IV (COX) activity: 
 - Unchanged (58,174) 
 - Reduced (57) 
 Oxygen consumption (with succinate and ADP): 
 - Increased (152,169-171) 
 mtDNA levels: 
 - Increased (54,257) 
Definite NASH (humans and rodents)FAO and ketogenesis: 
 - Unchanged (179,180) 
 - Increased (42,71,72,97,181,186,187) 
 - Reduced (188) 
 Complex I activity: 
 - Reduced (208,211,212,213) 
 Complex IV (COX) activity: 
 - Unchanged (211) 
 - Increased (187) 
 - Reduced (208,210,212) 
 Oxygen consumption (with succinate and ADP): 
 - Unchanged (213) 
 - Increased (187) 
 - Reduced (7) 
 mtDNA damage: 
 - Increased (219-221) 

Mitochondrial β-Oxidation and Ketogenesis

Thanks to different noninvasive methods, increased hepatic mtFAO was found in patients with fatty liver, or in obese individuals with prodromal features of the metabolic syndrome.68-70 Importantly, higher fat oxidation seems to persist in patients with NASH,42,71,72 as discussed later on (Table 1).

In rodents, enhanced mtFAO was found in fatty liver induced by overfeeding,64,73-79 or by monosodium L-glutamate.53 Hepatic ketogenesis was also augmented in mice fed a high-fat diet (HFD),80 thus suggesting that higher acetyl-CoA generation by way of mtFAO is followed by efficient KB production. However, during severe IR, excess acetyl-CoA could also enter the TCA cycle, in particular to serve as a carbon source for gluconeogenesis.79,81 This could explain why increased mtFAO is not always associated with higher levels of plasma KB.79

Increased hepatic FAO and/or higher expression of FAO genes were not always found in patients and rodents with fatty liver (Table 1).71,82-86 Different factors could explain this discrepancy:

  1. First, different methods were used to assess FAO. For instance, some studies reported increased mtFAO in liver homogenates but normal (or reduced) mtFAO in isolated mitochondria, which could be explained by higher mitochondrial mass.64,74,76
  2. Second, mitochondrial adaptations could vary during the development of NAFLD and IR79,87,88 and could also depend on nutritional factors such as dietary lipids89-92 and fructose.83,93
  3. Finally, the capacity of liver mitochondria to oxidize substrates and to adapt to nutrient excess is under complex genetic control in mice94,95 and in human, as mentioned previously.

Possible Mechanisms Leading to Higher mtFAO

The precise mechanisms responsible for higher mtFAO in NAFLD are poorly understood but several hypotheses can be put forward (Fig. 3):

  1. Increased levels of NEFAs. When IR occurs in WAT, the mere expansion of the pool of NEFAs in plasma can augment the global rate of mtFAO in liver.96,97 Higher FA levels in liver can also activate PPARα, as discussed later on.
  2. Higher production of hormones and cytokines. mtFAO in liver could be favored by increased levels of hormones and other circulating factors such as leptin,5,98-100 FGF21,101-104 and interleukin 6 (IL6).105,106
  3. Hepatic IR. Little is known regarding the impact of IR on mtFAO. However, some investigations suggest that FAO and ketogenesis are overall up-regulated during IR,68,71 although insulin-induced down-regulation of lipid oxidation can still occur in the insulin-resistant fatty liver.13,68,71,107 The paradoxical coexistence of increased FAO and higher DNL in fatty liver will be discussed below.
  4. Activation of hepatic PPARα. Numerous studies showed increased expression of PPARα in fatty liver.73,86,108-113 Different cues such as FAs, leptin, and IL6 could activate PPARα and its target genes involved in mtFAO (CPT1, MCAD), peroxisomal FAO (acyl-CoA oxidase), and VLDL production (microsomal triglyceride transfer protein).114-116 In contrast, hepatic PPARα expression was either unchanged, or even reduced, in some investigations.88,101,117,118
  5. Increased hepatic CPT1 expression and activity. Hepatic CPT1 expression and/or activity is often enhanced in rodents and patients during NAFLD and obesity.56,75,77,86,101,108,110,119-121 Increased CPT1 expression can be due to PPARα activation, but some studies also suggest a PPARα-independent mechanism.5,56 Expression and/or activity of other mtFAO enzymes can be increased during fatty liver, such as different dehydrogenases.108,111,120,122,123
Figure 3.

Possible mechanisms leading to increased mitochondrial β-oxidation during NAFLD. During NAFLD, mtFAO can be up-regulated in order to counteract enhanced lipid synthesis and deposition in liver. Different mechanisms could be responsible for this metabolic adaptation such as increased circulating levels of hormones and cytokines including leptin, FGF21, and IL6, as well as higher levels of circulating NEFAs. Indeed, the mere expansion of the pool of NEFAs in plasma augments the global rate of mtFAO in liver. FAs, leptin, and IL6 can activate PPARα and its target genes involved in mtFAO such as CPT1 and MCAD (not shown). CPT1 could also be less sensitive to malonyl-CoA, possibly as the consequence of insulin resistance.

Since CPT1 activity is inhibited by the lipogenic precursor malonyl-CoA, there are at least two hypotheses that can explain how CPT1 could still be active with high malonyl-CoA levels. First, hepatic CPT1 could be less inhibitable by this endogenous metabolite.124 Second, malonyl-CoA could exist as two different pools. Whereas malonyl-CoA produced by ACC1 could be used for DNL, malonyl-CoA generated by ACC2 could specifically serve as a CPT1 inhibitor.125

Other Mitochondrial Components and Metabolic Pathways

Experimental and clinical investigations showed increased hepatic mtDNA levels during fatty liver,73,79,126,127 although other studies showed reduced mtDNA content (Table 1).123,128,129 Hepatic mtDNA levels could depend on the severity of lipid overload123 and/or oxidative stress.128,129Discrepancies also exist regarding MRC activity (i.e., electron transfer), mitochondrial respiration (i.e., oxygen consumption), and OXPHOS efficiency (Table 1). For instance, COX activity was either increased,130 normal,82,88,127,131 or moderately reduced.86,132 Oxygen consumption in isolated liver mitochondria with adenosine diphosphate (ADP) (i.e., state 3 respiration) and different substrates was either augmented,53 unchanged,82,88,131,133,134 or significantly reduced (but with mild to moderate decreased respiration).73,76,77,86,127,135 OXPHOS efficiency and the mitochondrial membrane potential ΔΨm were either increased,64,76,86,88 unchanged,131,133 or decreased,136 which could reflect OXPHOS uncoupling. Increased OXPHOS efficiency could be an adaptive mechanism to produce more ATP that is needed to promote DNL and gluconeogenesis.64 Finally, hepatic ATP levels were either unchanged,130,137 or moderately decreased.132,138,139

The noninvasive functional test with ketoisocaproic acid (KICA) has been used to assess mitochondrial function in patients with NAFLD. KICA is a leucine catabolite which enters mitochondria to be fully degraded into H2O and CO2.140 In one study, 13C-KICA decarboxylation was unchanged in patients with simple steatosis but it was significantly impaired in NASH.141 In another study, 13C-KICA decarboxylation was unaffected in patients with nonalcoholic fatty liver, while it was significantly reduced in patients with alcoholic steatosis.142

Altogether, these data suggest that some, but not all, of the mitochondrial metabolic pathways are able to adapt to fat overload in liver. The discrepancies between the above-mentioned studies regarding mtDNA levels, MRC activity, OXPHOS efficiency, and ATP levels could be due to differences in the severity of fatty liver and oxidative stress. Accordingly, reduced mtDNA levels and impairment of MRC and OXPHOS seem to be more consistently observed in fatty liver induced by a choline-deficient (CD) diet, which is associated with overt oxidative stress.128,134,143-146

Mitochondrial Adaptations and Dysfunctions in Ob/ob and Db/db Livers

Since numerous investigations have been performed in genetic leptin-deficient ob/ob mice and leptin-resistant db/db mice, a specific section is dedicated to these murine models of mild to moderate steatohepatitis. Indeed, liver lesions in these mice are characterized by moderate hepatic necroinflammation and mild fibrosis,147-150 in addition to steatosis, which is more severe in the ob/ob genotype.147,151 However, hepatocellular ballooning degeneration is not a typical feature in ob/ob and db/db mice.147

Mitochondrial β-Oxidation and PPARα Expression

Higher LCFA oxidation was found in liver mitochondria and peroxisomes isolated from ob/ob mice (Table 1).57,149,152,153 Increased mtFAO capacity in ob/ob liver was associated with enhanced CPT activity and/or CPT1 expression,109,152,154 and higher expression of other mtFAO enzymes.119,154-157 Moreover, PPARα expression is augmented in ob/ob liver,109,154,158 although some studies found normal or reduced PPARα expression.157,159 In db/db mice, mtFAO was enhanced in one study,160 whereas total hepatic FAO was decreased in another report (Table 1).161 PPARα expression in db/db liver was either increased,109,162,163 unchanged,164-166 or decreased.167,168

Mitochondrial Respiration, MRC Activity, and Ultrastructure

In ob/ob mice, hepatic mitochondrial oxidation of glutamate (providing electrons to complex I) was either unchanged or increased, whereas that of succinate (providing electrons to complex II) was consistently enhanced (Table 1).152,169-171 In db/db liver, glutamate and succinate-driven mitochondrial respiration was increased.170 However, the activity of different hepatic MRC complexes was significantly reduced in ob/ob57,58,172,173 and db/db mice (Table 1).172,174,175 These data, reporting higher (or normal) rates of oxygen consumption and reduced activity of different MRC complexes, are not necessarily discordant. Indeed, mitochondrial respiration is significantly impaired only when the activity of MRC complexes is severely inhibited.176 An important ATP depletion was observed in ob/ob liver,171,177 which could be due to OXPHOS uncoupling.171,178 Finally, electron microscopic analysis of ob/ob liver showed enlarged mitochondria with abnormal cristae organization and granular matrix, but without crystalline inclusions.153

Taken together, these data in ob/ob and db/db indicated higher oxidative capacity of liver mitochondria with different respiratory substrates including FAs, but impaired activity of different MRC complexes. These mitochondrial alterations are leading to ROS overproduction since more substrate-derived electrons are entering the MRC and leak from complexes I and III.5,7,17,63,171 Increased hepatic mtFAO in ob/ob and db/db mice was associated with higher, normal, or even reduced PPARα expression. The exact reasons of this discrepancy are not known, but differences in age and diet could be involved.

Mitochondrial Dysfunctions in Definite NASH

Mitochondrial β-Oxidation and PPARα Expression

Three studies assessed whole-body 13C-octanoate oxidation in patients with NASH. In one study, patients with NASH had higher whole-body 13C-octanoate oxidation when compared to the controls,72 whereas the other studies showed no difference (Table 1).179,180 Using indirect calorimetry and KB production as surrogate markers of mtFAO, other investigations found higher fat oxidation in patients with NASH.42,71,97,181 In contrast, reduced PPARα mRNA expression was found in patients with NASH compared to patients with simple fatty liver,111,113,182 thus suggesting that PPARα induction progressively declines when fatty liver progresses to NASH.

Hepatic PPARα expression has been investigated in rodents fed a high-calorie diet and presenting NASH. Interestingly, PPARα expression was reduced in these models.117,118,183-185 In two of these studies, PPARα expression progressively declined with the duration of the high-calorie feeding and this was accompanied with a worsening of liver injury. Moreover, during these longitudinal experiments performed over several weeks, serum TNF-α and adiponectin levels progressively increased and decreased, respectively.118,185

Rodents fed a methionine/choline deficient (MCD) diet develop severe steatohepatitis, but this is associated with a strong reduction in body weight and lower glycemia.186-188 In two studies, respectively carried out in mice and rats fed an MCD diet, hepatic mtFAO with palmitate and octanoate was increased (Table 1).186,187 However, other investigations in rats revealed reduced mitochondrial oxidation of palmitate.188 Hepatic PPARα expression was unchanged, or reduced, in several studies carried out with this dietary model of steatohepatitis.189-193

Rodents fed a choline-deficient, ethionine-supplemented (CDE) diet can also develop steatohepatitis.194,195 A recent study in mice fed a CDE diet showed a significant decrease in hepatic expression of PGC1α, NRF1, Tfam, and two MRC proteins.128 Hence, these data suggest impaired mitochondrial biogenesis in NASH, beyond blunted PPARα expression. Interestingly, oxidative stress and inflammation are able to reduce PGC1α expression.196-198

Taken together, these data suggest that mtFAO could be preserved in NASH, and could even be enhanced in some situations (Table 1). On the contrary, mtFAO could be reduced in severe forms of NASH characterized by extensive oxidative stress and lipid peroxidation,188 or at the cirrhotic stage.199,200 However, in contrast to what happens in simple fatty liver, hepatic PPARα expression in NASH is often unchanged, or even reduced. Impaired PPARα induction could be related to higher TNF-α expression and/or lower adiponectin levels, which are frequently observed in NASH.113,201,202 Indeed, TNF-α is able to down-regulate the expression of PPARα and its targets genes,203,204 whereas inhibition of adiponectin-induced signaling through the adiponectin receptor-2 (AdipoR2) reduces PPARα activity in liver.205,206

Other Mitochondrial Functions, MRC Activity, and ATP Levels

Noninvasive breath tests carried out with 13C-KICA reported an inverse relationship between KICA metabolism and the severity of NASH.141 In addition, investigations in obese women with NAFLD found an inverse relationship between KICA oxidation and serum levels of ALT and γ-glutamyltransferase.207

Thus far, only one study reported a severe reduction in the hepatic activity of the five MRC complexes in patients with NASH, and this was correlated with serum levels of TNF-α.208 Recent clinical investigations also suggested that mitochondria in NASH had lower membrane potential compared to fatty liver.209 This could be due to reduced MRC activity, although OXPHOS uncoupling could play a role in the early stage of NAFLD, as discussed later on.

Experimental studies investigated mitochondrial respiration and MRC activity in HFD models of NASH. In one study, mitochondrial respiration with glutamate/malate and succinate was significantly reduced, albeit moderately (Table 1).7 Investigations on MRC activity showed a strong reduction of COX activity in one study,210 whereas this MRC complex was unaltered in another one.211 In the latter study, however, activity of complex I was significantly reduced (Table 1).211 Finally, longitudinal investigations in mice showed that decreased complex I and COX activities in NASH after 15 weeks were alleviated after 30 weeks.212 This may suggest that some compensatory mechanisms at the MRC level could still be activated in NASH.

In the MCD diet model of steatohepatitis, one study showed increased mitochondrial respiration with glutamate/malate and succinate after 6 weeks of the diet and this was associated with higher activity of COX (Table 1).187 In a longitudinal study in rats, higher mitochondrial respiration with glutamate/malate and succinate was observed after 3 weeks of MCD diet feeding, but oxygen consumption returned to normal values after 7 and 11 weeks.213 Moreover, activity of complexes I and II progressively decreased over time in these investigations.213

Reduced liver ATP content has consistently been observed in patients and rodents with NASH, although the extent of this reduction greatly varied between these studies.137,139,213,214 Interestingly, longitudinal investigations in rats showed a progressive reduction of ATP content during the development of NASH, with lower ATP levels compared to simple fatty liver.139,185 Hence, the hepatic energy status worsens during NAFLD progression.

A possible mechanism responsible for impaired ATP synthesis in the early stage of NAFLD could be OXPHOS uncoupling by way of uncoupling protein 2 (UCP2) up-regulation.5,137,184,215 However, uncoupling activity and localization of endogenous UCP2 are still debated,216,217 and its pathophysiological role in NAFLD has been questioned.148 Alternatively, other OXPHOS uncoupling proteins could be involved.218 During NASH, on the other hand, lower ATP production could be due to reduced activity of different MRC complexes.208,210-212

mtDNA and Mitochondrial Ultrastructure

During NASH, different types of mtDNA damage have been detected including deletions, point mutations, and increased 8-hydroxydeoxyguanosine levels (Table 1).219-221 Moreover, the last two mtDNA lesions were more frequently observed in NASH compared with simple fatty liver.220,221 A significant depletion of mtDNA was also reported in patients with NAFLD, although cases of fatty liver and NASH were not distinguished in this study.222

Investigations in patients with NASH showed that liver mitochondria were often swollen and presented ultrastructural abnormalities, including para-crystalline inclusions.223,224 However, a recent study found no statistical difference between simple fatty liver and NASH regarding the presence of megamitochondria and intramitochondrial crystalline inclusions.225 In contrast, the mean mitochondrial diameter was higher in NASH compared with fatty liver.225 Actually, some ultrastructural abnormalities of liver mitochondria could appear well before NASH.225,226

Possible Mechanisms Leading to Reduced MRC Activity in NASH

At the moment, there is no definite explanation for the progressive decline of MRC activity during NASH, although some hypotheses can be put forward (Fig. 4).

Figure 4.

Possible mechanisms of progressive decrease in MRC activity during NAFLD. During NAFLD, MRC activity progressively declines and this could participate in the progression of fatty liver to NASH by further enhancing ROS generation. A first mechanism which could lead to reduced MRC activity is ROS overproduction resulting from increased mtFAO (arrow 1). A second mechanism is ROS generation due to CYP2E1 overexpression and reduced levels of mtGSH. Whereas CYP2E1 induction could be secondary to higher levels of glucagon, FAs and KBs, reduced mtGSH could result from higher mitochondrial levels of cholesterol. A third mechanism could be RNS overproduction due to TNF-α-induced iNOS overexpression, whereas TNF-α could also reduce MRC activity by increasing HIF-1α activity. Other mechanisms leading to impaired MRC activity could include higher levels of different lipid intermediates including free FAs, increased generation of lipid peroxidation products such as 4-HNE, and reduced adiponectin action. Finally, higher FOXO1 activity and oxidative stress could be involved, by way of impaired mitochondrial biogenesis. Although lower MRC activity during NASH does not hamper the ability of mitochondria to oxidize lipids, this aggravates ROS overproduction by the respiratory chain (arrow 2).

Involvement of ROS and RNS

Several MRC complexes including COX are sensitive to ROS and RNS.227,228 In addition, COX can be inhibited by low levels of NO229 and inactivated by 4-hydroxynonenal (4-HNE), a reactive aldehyde generated during lipid peroxidation.230 ROS overproduction within mitochondria could be favored by higher CYP2E1 expression and lower levels of mitochondrial GSH (mtGSH) (Fig. 4).

Overproduction of TNF-α and Interferons

TNF-α is able to impair MRC activity, possibly by inducing hypoxia inducible factor-1α (HIF-1α) and mtDNA damage.5,57,231,232 In addition, Kupffer cell-derived interferons could also impair MRC activity.20


Some lipid derivatives such as FAs can directly inhibit several enzymes involved in MRC and OXPHOS.12,233,234 Moreover, saturated FAs are able to activate c-Jun N-terminal kinase and trigger the mitochondrial membrane permeability transition, thus inducing mitochondrial release of cytochrome c and apoptosis.40,235

Other Mechanisms

Reduced adiponectin action in liver could be involved, since this adipokine seems to control MRC activity.236 Plasma adiponectin levels are indeed decreased in NAFLD, and especially in NASH.184,202,237 Moreover, lower hepatic expression of AdipoR1 and AdipoR2 is found in NASH,52,238-240 although other studies reported increased expression.237,241 Another mechanism could be higher activity of forkhead box O1 transcription factor (FoxO1), linked to IR.27 Indeed, FoxO1 activation is able to reduce the content of different MRC polypeptides, possibly by decreasing heme synthesis and impairing PGC1α activity.172 Reduced PGC1α activity could also be secondary to oxidative stress and inflammation, as previously mentioned (Fig. 4).197,198

Significance for Pharmacology and Toxicology

Pharmacology of NAFLD

Numerous drugs are currently tested in order to alleviate fatty liver and NASH. These treatments can have different pharmacological effects including improvement of insulin sensitivity, stimulation of lipid oxidation, as well as reduction of DNL, oxidative stress, and inflammation.63,242,243 Regarding mtFAO, some investigations showed that fatty liver could be alleviated by further stimulating the already enhanced capacity for lipid oxidation.75,77

Drug-induced stimulation of mtFAO could have, however, deleterious consequence over the long term if this is not associated with concomitant improvement of MRC. Indeed, an imbalance between mtFAO and MRC activity induces ROS overproduction,5,7,17,63,171 as previously mentioned. Hence, it will be important in the future to find therapeutic strategies able to restore both mtFAO and MRC activity in a coordinated manner. Increasing the activity of PGC1α and sirtuins in NAFLD could be an interesting option since this should activate the whole program of mitochondrial biogenesis.244-246 Finally, enhancing mtFAO in liver can also alleviate IR, which could be dependent or not on the reduction in hepatic lipids.247,248

NAFLD and Drug-Induced Liver Injury

Obese patients are consuming on average more drugs than nonobese individuals.249 However, numerous drugs can impair mitochondrial function, or more broadly, lipid homeostasis.12,17,250 Excessive alcohol consumption is also able to impair mitochondrial function and lipid homeostasis.12,17,251 Thus, NAFLD could worsen during the prolonged exposure of such xenobiotics. Using rodent models with preexisting hepatic steatosis and/or NASH, an aggravation of liver lesions was observed with phenobarbital, rosiglitazone, and pentoxifylline.17,252 In addition, clinical studies suggested that NASH could be induced, or aggravated, in obese individuals treated with drugs such as tamoxifen, methotrexate, irinotecan, and nucleoside reverse transcriptase inhibitors (NRTIs) such as stavudine and didanosine.17,253,254 NAFLD aggravation was also shown in rodents with ethanol,54,255 and in ducks force-fed with corn contaminated with mycotoxins.256

Obese individuals with NAFLD could also be more prone to develop drug-induced acute hepatitis. This has been suggested for halothane and acetaminophen.17,254 For these drugs, which undergo CYP2E1-mediated biotransformation into highly toxic metabolites, increased liver injury could be secondary to CYP2E1 induction.39,254,257 Underlying mitochondrial dysfunction could also lead to higher susceptibility to drug-induced acute hepatitis, although further investigations are needed to confirm this hypothesis.

Summary, Outlook, and Remaining Issues

Although studies dealing with mitochondrial dysfunctions in NAFLD present some discrepancies, a frequent finding is the significant alteration of MRC activity in NASH. Importantly, moderate impairment of MRC activity can already be observed in simple fatty liver. Hence, MRC activity could progressively decline during the progression of NAFLD. In contrast, mtFAO is stimulated (or at least preserved) in fatty liver and NASH, most probably as a compensatory mechanism in order to restrain fat accumulation. This imbalance between mtFAO and MRC is leading to ROS overproduction by way of enhanced leakage of electrons from the MRC.5,7,17,63,171 It is likely that this event triggers a vicious cycle since mitochondrial ROS are able to oxidatively damage nearby MRC enzymes and mtDNA. If this scheme is correct, restoring MRC activity in NAFLD could be a major goal to achieve in order to alleviate oxidative stress. Since mitochondrial ROS could play a significant role in cell death, inflammation, and fibrosis,258-260 developing drugs improving both mtFAO and MRC activity could provide major benefits beyond the improvement of fatty liver. In contrast, physicians should be aware that numerous drugs are able to induce mitochondrial dysfunction,12,17,250 which could aggravate NAFLD in some patients.

Several important issues remain regarding mitochondrial adaptations and dysfunctions in NAFLD. For instance, investigations are needed to determine which lipid(s) can alter mitochondrial function, either directly or indirectly. Interestingly, cholesterol could be an attractive candidate. Indeed, increased mitochondrial cholesterol during NAFLD could reduce mitochondrial transport of GSH, thus inducing lower mtGSH levels and oxidative stress (Fig. 4).18,50,261 Because members of the Bcl-2 family can also regulate mtGSH,262 further studies are required to identify all the factors able to reduce mtGSH levels in NAFLD. Interestingly, altered mitochondrial levels of cholesterol may also disturb the production of some oxysterols,263 which could play a role in the pathophysiology of NAFLD.264 Concerning lipid-induced oxidative stress, investigations should also be carried out to compare the ability of the main endogenous FAs to increase CYP2E1 expression (Fig. 4).39

The natural history of NAFLD is another major issue. Indeed, although progression from isolated fatty liver to NASH has been reported,265,266 it is not certain whether this evolution occurs in all patients and thus NASH could not always be preceded by simple steatosis.267 Investigations will be necessary to determine whether mitochondrial alterations in NASH are different when this disease is preceded or not by simple fatty liver. It is also noteworthy that animal NAFLD seldom reproduce all the features of human NAFLD, although some studies reported animal models of liver lesions with close resemblance to human NASH.268-270 These models should be useful to study mitochondrial dysfunctions and other key events involved in the pathophysiology of NAFLD. Finally, investigations are required to improve histological classification of human and experimental NAFLD. This may avoid some discrepancies between studies dealing with NAFLD pathogenesis (Table 1).


We would like to apologize to the researchers whose articles have not been cited in the present review because of space limitation. Note: Only the first five references in the introduction section of this article are available below. The remaining references are available as Supporting Material 1.