Mitochondria in steatohepatitis

For the first time in history, populations in affluent countries may concomitantly indulge in rich food and physical idleness. Various combinations of obesity, diabetes, and hypertriglyceridemia, with insulin resistance as the common feature, cause hepatic steatosis, which can trigger necroinflammation and fibrosis. Patients with "primary" steatohepatitis exhibit ultrastructural mitochondrial lesions, decreased activity of respiratory chain complexes, and have impaired ability to resynthesize ATP after a fructose challenge. Mitochondria play a major role in fat oxidation and energy production but also leak reactive oxygen species (ROS) and are the main cellular source of ROS. In patients with steatosis, mitochondrial ROS may oxidize hepatic fat deposits, as suggested in animal models. Lipid peroxidation products impair the flow of electrons along the respiratory chain, which may cause overreduction of respiratory chain components, further increasing mitochondrial ROS formation and lipid peroxidation. Another vicious circle could involve ROS-induced depletion of antioxidants, impairing ROS inactivation. Blood vitamin E is decreased in some obese children with steatohepatitis, and serum transaminases improve after vitamin E supplementation. Steatohepatitis is also caused by alcohol abuse, drugs, and other causes. In "secondary" steatohepatitis, mitochondrial ROS formation is further increased as the causative disease itself directly increases ROS or first impairs respiration, which secondarily increases mitochondrial ROS formation. This "second hit" could cause more lipid peroxidation, cytokine induction, Fas ligand induction, and fibrogenesis than in primary steatohepatitis.     

Mitochondrial injury in steatohepatitis

Rich diet and lack of exercise are causing a surge in obesity, insulin resistance and steatosis, which can evolve into steatohepatitis. Patients with non-alcoholic steatohepatitis have increased lipid peroxidation, increased tumour necrosis factor-alpha (TNF-alpha) and increased mitochondrial beta-oxidation rates. Their in-vivo ability to re-synthesize ATP after a fructose challenge is decreased, and their hepatic mitochondria exhibit ultrastructural lesions, depletion of mitochondrial DNA and decreased activity of respiratory chain complexes. Although the mechanisms for these effects is unknown, the basal cellular formation of reactive oxygen species (ROS) may oxidize fat deposits to cause lipid peroxidation, which damages mitochondrial DNA, proteins and cardiolipin to partially hamper the flow of electrons within the respiratory chain. This flow may be further decreased by TNF-alpha, which can release cytochrome c from mitochondria. Concomitantly, the increased mitochondrial fatty acid beta-oxidation rate augments the delivery of electrons to the respiratory chain. Due to the imbalance between a high electron input and a restricted outflow, electrons may accumulate within complexes I and III, and react with oxygen to form the superoxide anion radical. Increased mitochondrial ROS formation could in turn directly oxidize mitochondrial DNA, proteins and lipids, enhance lipid peroxidation-related mitochondrial damage, trigger hepatic TNF-alpha formation and deplete antioxidants, thus further blocking electron flow and further increasing mitochondrial ROS formation. Mitochondrial dysfunction plays an important role in liver lesions, through the ROS-induced release of both biologically active lipid peroxidation products and cytokines. In particular, the up-regulation of both TNF-alpha and Fas triggers mitochondrial membrane permeability and apoptosis. The ingestion of apoptotic bodies by stellate cells stimulates fibrogenesis, which is further activated by lipid peroxidation products and high leptin levels. Chronic apoptosis is compensated by increased cell proliferation, which, together with oxidative DNA damage, may cause gene mutations and cancer.     

Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: mechanisms and pathophysiological role

Due to the worldwide surge in obesity and type 2 diabetes, the increased incidence of nonalcoholic fatty liver disease (NAFLD) is a major concern for the public health. Indeed, NAFLD encompasses a large spectrum of conditions ranging from fatty liver to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis in some patients. A better understanding of the mechanisms involved in fatty liver and its progression into NASH is important in order to develop efficient drugs able to alleviate these liver diseases. Although numerous investigations pointed to reactive oxygen species (ROS) as key players in the progression of fatty liver to NASH, their exact source is still uncertain. Besides the mitochondrial respiratory chain, cytochrome P450 2E1 (CYP2E1) has recently emerged as another potentially important cause of ROS overproduction. Indeed, higher hepatic CYP2E1 expression and activity have been frequently observed in the context of obesity and NAFLD. It is currently unknown why CYP2E1 is enhanced in these dysmetabolic diseases, although increased hepatic levels of fatty acids and insulin resistance might play a role. Nonetheless, higher hepatic CYP2E1 could play a significant role in the pathophysiology of NASH by inducing lipid peroxidation and oxidative damage of key cellular components. Moreover, CYP2E1-mediated overproduction of ROS could promote hepatic insulin resistance, which can further aggravate fatty liver. Since a significant amount of CYP2E1 can be located within liver mitochondria, higher levels of CYP2E1 in NAFLD could also have detrimental effects on mitochondrial function. Finally, increased CYP2E1 activity during NAFLD could enhance the susceptibility of some patients to the hepatotoxicity of different xenobiotics through the CYP2E1-mediated generation of harmful reactive metabolites.     

From fatty liver to fibrosis:A tale of “second hit”

Although much is known about how fat accumulates in the liver,much remains unknown about how this causes sustained hepatocellular injury.The consequences of injury are recognized as nonalcoholic steatohepatitis(NASH) and progressive fibrosis.The accumulation of fat within the hepatocytes sensitizes the liver to injury from a variety of causes and the regenerative capacity of a fatty liver is impaired.An additional stressor is sometimes referred to as a "second hit" in a paradigm that identifies the accumulation of fat as the "first hit".Possible candidates for the second hit include increased oxidative stress,lipid peroxidation and release of toxic products such as malondialdehyde and 4-hydroxynonenal,decreased antioxidants,adipocytokines,transforming growth factor(TGF)-β,Fas ligand,mitochondrial dysfunction,fatty acid oxidation by CYPs(CYP 2E1,4A10 and 4A14),and peroxisomes,excess iron,small intestinal bacterial overgrowth,and the generation of gut-derived toxins such as lipopolysaccharide and ethanol.Oxidative stress is one of the most popular proposed mechanisms of hepatocellular injury.Previous studies have specifically observed increased plasma and tissue levels of oxidative stress markers and lipid peroxidation products,with reduced hepatic and plasma levels of antioxidants.There is also some indirect evidence of the benefit of antioxidants such as vitamin E,S-adenosylmethionine,betaine,phlebotomy to remove iron,and N-acetylcysteine in NASH.However,a causal relationship or a pathogenic link between NASH and oxidative stress has not been established so far.A number of sources of increased reactive oxygen species production have been established in NASH that include proinflammatory cytokines such as tumor necrosis factor(TNF)-α,iron overload,overburdened and dysfunctional mitochondria,CYPs,and peroxisomes.Briefly,the pathogenesis of NASH is multifactorial and excess intracellular fatty acids,oxidant stress,ATP depletion,and mitochondrial dysfunction are important causes of hepatocellular injury in the stea     

Drug-induced steatohepatitis

Drugs rarely cause steatohepatitis, but amiodarone, perhexiline, and DH, have unequivocally been found to independently induce the histologic picture of alcoholic liver disease or NASH. All three agents have similar pathogenetic mechanisms of hepatotoxicity, targeting mitochondrial ATP production and fatty acid catabolism. Other drugs that occasionally cause steatohepatitis, most importantly steroid hormones, likely exacerbate the pathogenetic mechanisms leading to NASH. Similar to NASH, lipid peroxidation resulting from mitochondrial injury may account for all of the histologic findings in drug-induced steatohepatitis. Further research should determine the mechanisms by which drug-induced steatosis, a benign lesion, evolves to steatohepatitis and progressive fibrosis.     

Drugs and steatohepatitis

In addition to the usual associations with insulin resistance, type 2 diabetes, central obesity, and hypertriglyceridemia, nonalcoholic steatohepatitis (NASH) has been associated with several drugs and toxins. However, drug-induced liver disease is a relatively uncommon cause of steatohepatitis. The term drug-induced steatohepatitis is preferred when the association appears to result from a direct toxic effect of the drug on the liver. For some agents implicated as causing cirrhosis or fatty liver disorders, the association may be coincidental because NASH is a common component of the insulin resistance (or metabolic) syndrome. In other instances, corticosteroids, tamoxifen, and estrogens may precipitate NASH in predisposed persons by exacerbating insulin resistance, central obesity, diabetes, and hypertriglyceridemia, and methotrexate may worsen hepatic fibrosis in NASH. Drug-induced steatohepatitis is associated with prolonged therapy (more than 6 months) and possibly drug accumulation, which in the case of perhexiline maleate is favored by a genetic polymorphism of CYP2D6 that leads to slow perhexiline oxidation. The toxic mechanism appears to involve mitochondrial injury, which causes steatosis because of impaired beta-oxidation of fatty acids, and leads to generation of reactive oxygen species and ATP depletion. Thus, drug-induced steatohepatitis may provide clues to injurious events in the more common metabolic forms of NASH. A clinical feature of some types of drug-induced steatohepatitis is progression after discontinuation of the causative agent. It follows that early recognition of hepatotoxicity is crucial to prevent the development of severer forms of liver disease and improve the clinical outcome.     

Oxidative stress,cardiolipin and mitochondrial dysfunction in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease(NAFLD) is today considered the most common form of chronic liver disease, affecting a high proportion of the population worldwide. NAFLD encompasses a large spectrum of liver damage, ranging from simple steatosis to steatohepatitis, advanced fibrosis and cirrhosis. Obesity, hyperglycemia, type 2 diabetes and hypertriglyceridemia are the most important risk factors. The pathogenesis of NAFLD and its progression to fibrosis and chronic liver disease is still unknown. Accumulating evidence indicates that mitochondrial dysfunction plays a key role in the physiopathology of NAFLD, although the mechanisms underlying this dysfunction are still unclear. Oxidative stress is considered an important factor in producing lethal hepatocyte injury associated with NAFLD. Mitochondrial respiratory chain is the main subcellular source of reactive oxygen species(ROS), which may damage mitochondrial proteins, lipids and mitochondrial DNA. Cardiolipin, a phospholipid located at the level of the inner mitochondrial membrane, plays an important role in several reactions and processes involved in mitochondrial bioenergetics as well as in mitochondrial dependent steps of apoptosis. This phospholipid is particularly susceptible to ROS attack. Cardiolipin peroxidation has been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions, including NAFLD. In this review, we focus on the potential roles played by oxidative stress and cardiolipin alterations in mitochondrial dysfunction associated with NAFLD.     

Melatonin protects against lipid‐induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice

Lipid generates reactive oxygen species (ROS) in consequence to mitochondrial fission followed by inflammation in propagating hepatic fibrosis. The interaction of SIRT1/Mitofusin2 is critical for maintaining mitochondrial integrity and functioning, which is disrupted upon excess lipid infiltration during the progression of steatohepatitis. The complex interplay between hepatic stellate cells and steatotic hepatocytes is critically regulated by extracellular factors including increased circulating free fatty acids during fibrogenesis. Melatonin, a potent antioxidant, protects against lipid‐mediated mitochondrial ROS generation. Lipotoxicity induces disruption of SIRT1 and Mitofusin2 interaction leading to mitochondrial morphological disintegration in hepatocytes. Further, fragmented mitochondria leads to mitochondrial permeability transition pore opening, cell cycle arrest and apoptosis and melatonin protects against all these lipotoxicity‐mediated dysfunctions. These impaired mitochondrial dynamics also enhances the cellular glycolytic flux and reduces mitochondrial oxygen consumption rate that potentiates ROS production. High glycolytic flux generates metabolically unfavorable milieu in hepatocytes leading to inflammation, which is abrogated by melatonin. The melatonin‐mediated protection against mitochondrial dysfunction was also observed in high‐fat diet (HFD)‐fed mice through restoration of enzymatic activities associated with respiratory chain and TCA cycle. Subsequently, melatonin reduces hepatic fat deposition and inflammation in HFD‐fed mice. Thus, melatonin disrupts the interaction between steatotic hepatocyte and stellate cells, leading to the activation of the latter to abrogate collagen deposition. Altogether, the results of the current study document that the pharmacological intervention with low dose of melatonin could abrogate lipotoxicity‐mediated hepatic stellate cell activation and prevent the fibrosis progression.     

Pivotal role of superoxide anion and beneficial effect of antioxidant molecules in murine steatohepatitis

Nonalcoholic fatty liver disease, frequently associated with obesity, can lead to nonalcoholic steatohepatitis (NASH) and cirrhosis. The pathophysiology of NASH is poorly understood, and no effective treatment is available. In view of a potential deleterious role for reactive oxygen species (ROS), we investigated the origin of ROS overproduction in NASH. Mitochondrial production of ROS and its alterations in the presence of antioxidant molecules were studied in livers from ob/ob mice that bear a mutation of the leptin gene and develop experimental NASH. N-acetyl-cysteine and the superoxide dismutase (SOD) mimics ambroxol, manganese [III] tetrakis (5,10,15,20 benzoic acid) (MnTBAP), and copper [II] diisopropyl salicylate (CuDIPS) were used to target different checkpoints of the oxidative cascade to determine the pathways involved in ROS production. Liver mitochondria from ob/ob mice generated more O(2)*- than those of lean littermates (P <.01). Ex vivo, all three SOD mimics decreased O(2)*- generation (P <.001) and totally inhibited lipid peroxidation (P <.001) versus untreated ob/ob mice. Those modifications were associated with in vivo improvements: MnTBAP and CuDIPS reduced weight (P <.02) and limited the extension of histological liver steatosis by 30% and 52%, respectively, versus untreated ob/ob mice. In conclusion, these data demonstrate deleterious effects of superoxide anions in NASH and point at the potential interest of nonpeptidyl mimics of SOD in the treatment of NASH in humans.     

Mitochondrial abnormalities in non-alcoholic steatohepatitis.

BACKGROUND/AIMS: We assessed mitochondrial morphology by electron microscopy and the prevalence of a mitochondrial gene deletion in patients with non-alcoholic steatohepatitis (NASH), alcohol-related liver disease and non-fatty liver diseases. Respiratory chain function using a cytoplasmic hybrid (cybrid) assay was further studied in NASH patients and healthy controls. METHODS: Electron microscopy was performed in 26 specimens. Fifteen patients were studied by polymerase chain reaction to detect a 520-bp deletion product of the mitochondrial genome (dmtDNA). Cybrids were created by fusion of platelets with anaerobic neuroblastoma cells in six NASH patients and 12 controls. RESULTS: Eight of ten NASH, one of seven alcoholics and two of nine other patients had linear crystalline inclusions in megamitochondria (p<0.05). Three of five patients with alcohol-related liver disease had dmtDNA compared to one of five NASH patients and one of five non-steatohepatitis controls. Cybrid respiratory chain function in platelets was not different from that of controls. CONCLUSIONS: Respiratory chain dysfunction, if present in NASH, is not expressed in platelet-derived mitochondria. In contrast to alcohol-related liver disease with active drinking, NASH patients do not commonly express the 5-kb mitochondrial DNA gene deletion in liver tissue. As previously described in early alcohol-related liver disease, crystalline inclusions of unknown composition are seen in hepatic mitochondria in NASH. Their presence suggests either an adaptive process or mitochondrial injury.     

A myriad of pathways to NASH

Nonalcoholic steatohepatitis (NASH) is defined histopathologically by the presence of macrovesicular steatosis, cellular ballooning, and inflammation. NASH represents a complex multifactorial disease that typically occurs within the context of the metabolic syndrome. NASH lacks homogeneity, and other forms of NASH can present atypically. Less than 50% of patients with NASH respond to pharmacologic treatment, which speaks to this heterogeneity. The authors discuss drugs, disease entities, and nutritional states that can cause or exacerbate underlying NASH indirectly through worsening insulin resistance or directly by interfering with lipid metabolism, promoting oxidative injury, or activating inflammatory pathways.     

Drug-induced fatty liver disease: An overview of pathogenesis and management

Over the past decades, many drugs have been identified, that can potentially induce steatohepatitis in the predisposed individual. Classically this has been incriminated to amiodarone, perhexiline, and 4,4’-diethylaminoethoxyhexestrol (DH), all of which have been found to independently induce the histologic picture of non-alcoholic steatohepatitis (NASH). Pathogenetic mechanisms of hepatotoxicity although still evolving, demonstrate that mitochondrial dysfunction, deranged ATP production and fatty acid catabolism likely play an important role. Drugs like steroid hormones can exacerbate the pathogenetic mechanisms that lead to NASH, and other drugs like tamoxifen, cisplatin and irenotecan have been shown to precipitate latent fatty liver as well. Further research aiming to elucidate the pathogenesis of drug-induced steatosis and steatohepatitis is needed in order to better design therapeutic targets.     

Nonalcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD), the most common liver disorder in the Western world, is a clinico-histopathological entity in which excessive triglyceride accumulation in the liver occurs. Non-alcoholic steatohepatitis (NASH) represents the necroinflammatory form, which can lead to advanced liver fibrosis, cirrhosis, and hepatocellular carcinoma. The pathogenesis of NAFLD/NASH is complex but increased visceral adiposity plus insulin resistance with increased free fatty acids release play an initial key role for the onset and perpetuation of liver steatosis. Further events in the liver include oxidative stress and lipid peroxidation, decreased antioxidant defences, early mitochondrial dysfunction, iron accumulation, unbalance of adipose-derived adipokines with a chronic proinflammatory status, and gut-derived microbial adducts. New gene polymorphisms increasing the risk of fatty liver, namely APOC3 and PNPLA3, have been lately identified allowing further insights into the pathogenesis of this condition. In our review pathophysiological, genetic, and essential diagnostic and therapeutic aspects of NAFLD are examined with future trends in this field highlighted.     

Lipid-induced oxidative stress causes steatohepatitis in mice fed an atherogenic diet

Recently, nonalcoholic steatohepatitis (NASH) was found to be correlated with cardiovascular disease events independently of the metabolic syndrome. The aim of this study was to investigate whether an atherogenic (Ath) diet induces the pathology of steatohepatitis necessary for the diagnosis of human NASH and how cholesterol and triglyceride alter the hepatic gene expression profiles responsible for oxidative stress. We investigated the liver pathology and plasma and hepatic lipids of mice fed the Ath diet. The hepatic gene expression profile was examined with microarrays and real-time polymerase chain reactions. The Ath diet induced dyslipidemia, lipid peroxidation, and stellate cell activation in the liver and finally caused precirrhotic steatohepatitis after 24 weeks. Cellular ballooning, a necessary histological feature defining human NASH, was observed in contrast to existing animal models. The addition of a high-fat component to the Ath diet caused hepatic insulin resistance and further accelerated the pathology of steatohepatitis. A global gene expression analysis revealed that the Ath diet up-regulated the hepatic expression levels of genes for fatty acid synthesis, oxidative stress, inflammation, and fibrogenesis, which were further accelerated by the addition of a high-fat component. Conversely, the high-fat component down-regulated the hepatic gene expression of antioxidant enzymes and might have increased oxidative stress. CONCLUSION: The Ath diet induces oxidative stress and steatohepatitis with cellular ballooning. The high-fat component induces insulin resistance, down-regulates genes for antioxidant enzymes, and further aggravates the steatohepatitis. This model suggests the critical role of lipids in causing oxidative stress and insulin resistance leading to steatohepatitis.     

Non-alcoholic steatohepatitis: review of a growing medical problem

Non-alcoholic steatohepatitis (NASH) is a metabolic liver disorder that is seen in 2-6% of the general population. It manifests itself by elevated liver enzymes, frequently without symptoms. The histological findings include steatosis, inflammation, fibrosis, and cirrhosis. Three case reports are presented to illustrate features of NASH. A two-hit model has been proposed in the pathogenesis of NASH. The first hit is hepatic steatosis. A hypercaloric diet with high levels of carbohydrates and saturated fatty acids results in elevated plasma free fatty acids (FFA) and expands the adipose tissue. Insulin resistance develops and augments steatosis. Oxidation of FFA yields toxic free radicals, resulting in lipid peroxidation. They cause the second hits: increased oxidative stress on hepatocytes and induction of pro-inflammatory cytokines. When the antioxidant capacities of the liver are insufficient, mitochondrial dysfunction and tumor necrosis factor alpha (TNF-alpha) cause inflammation and fibrosis. Treatment consists of life style modifications, particularly weight loss and exercise. Many drugs have been tried in the treatment of NASH. The insulin-sensitizing drugs metformin, rosiglitazone, and pioglitazone, and the antioxidant vitamin E show promising results. Further investigation of therapeutic options is needed to direct the choice of therapy in the future.     

Hepatic AdipoR2 signaling plays a protective role against progression of nonalcoholic steatohepatitis in mice

It is unclear how hepatic adiponectin resistance and sensitivity mediated by the adiponectin receptor, AdipoR2, contributes to the progression of nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the roles of hepatic AdipoR2 in NASH, using an animal model. We fed C57BL/6 mice a methionine-deficient and choline-deficient (MCD) diet for up to 8 weeks and analyzed changes in liver pathology caused by either an AdipoR2 short hairpin RNA-expressing adenovirus or an AdipoR2-overexpressing adenovirus. Inhibition of hepatic AdipoR2 expression aggravated the pathological state of NASH at all stages: fatty changes, inflammation, and fibrosis. In contrast, enhancement of AdipoR2 expression in the liver improved NASH at every stage, from the early stage to the progression of fibrosis. Inhibition of AdipoR2 signaling in the liver diminished hepatic peroxisome proliferator activated receptor (PPAR)-alpha signaling, with decreased expression of acyl-CoA oxidase (ACO) and catalase, leading to an increase in lipid peroxidation. Hepatic AdipoR2 overexpression had the opposite effect. Reactive oxygen species (ROS) accumulation in liver increases hepatic production of transforming growth factor (TGF)-beta1 at all stages of NASH; adiponectin/AdipoR2 signaling ameliorated TGF-beta-induced ROS accumulation in primary cultured hepatocytes, by enhancing PPAR-alpha activity and catalase expression. CONCLUSION: The adiponectin resistance and sensitivity mediated by AdipoR2 in hepatocytes regulated steatohepatitis progression by changing PPAR-alpha activity and ROS accumulation, a process in which TGF-beta signaling is implicated. Thus, the liver AdipoR2 signaling pathway could be a promising target in treating NASH.