Perspectives on cellular dysfunction in nonalcoholic steatohepatitis: a case of ‘multiorganelle failure’? Proceedings of a virtual workshop on nonalcoholic steatohepatitis

Keywords::

• cell biology
• history of medicine
• NAFLD
• NASH
• pathogenesis
• synonyms

Figure 1. A model of the pathogenesis of nonalcoholic steatohepatitis.

Insulin resistance occurs as an early inciting event in the pathogenesis of NASH. It is fully acceptable that, once triggered, the pathogenic cascade of events will proceed independent of the initial offending agent, insulin resistance, accounting for the finding that agents aimed at combating it will not necessarily result in the interruption of ongoing liver damage. This results in NASH from ‘multiorganelle failure’, as described in the proceedings of this special focus issue.

FFA: Free fatty acid; NAFLD: Nonalcoholic fatty liver disease; NASH: Nonalcoholic steatohepatitis; ROS: Reactive oxygen species.

Reprinted from [30].

Historical perspective

Nonalcoholic fatty liver disease (NAFLD) will be 60 years old next year: it was first reported in 1952 [1], and what we now allude to as nonalcoholic steatohepatitis (NASH) was described in 1962 by Thaler [2]. In 1979, Itoh described “five diabetic non-alcoholic women with micronodular cirrhosis” [3]. All aged over 50 years and obese, they displayed a phenotype that was later named NASH for the first time in 1980 by Ludwig [4]. In the following years, NASH was to be defined as ‘diabetic hepatitis’, ‘alcohol-like liver disease in the non-alcoholic’, ‘bright liver syndrome’ and ‘insulin-resistance-associated hepatic iron overload’, which can all be considered synonyms (Box 1)[5–8]. In 1999, Brunt proposed the most pragmatic classification for use in clinical practice [9], and in 2005 Kleiner introduced a semiquantitative scoring system designed to reduce interobserver variability among pathologists [10]. Pioneering studies had already identified mitochondrial uncoupling as a key finding in experimental fatty liver in 1954 [11]. Interestingly, the possibility of inhibiting its development with antioxidants – notably including α-tocopherol, which was proposed as early as 1965 [12] – remains a hotly debated issue [13]. A step forward in the ultrastructural pathology of NASH took place in 1999 when Caldwell first reported on the presence of inclusions of unknown composition in hepatic mitochondria in NASH [14]. This study promoted the pathogenic interpretation of NASH as a disease linked to altered energetic homeostasis, excess reactive oxygen species production and perturbed lipid composition. The demonstration of morphological correlates, whether at the light or ultrastructural level, is essential to refine our understanding of the disease process [15]. Since 2000, research has been shaped by more collective initiatives, featuring joint efforts from private foundations, the American and European Associations for the Study of the Liver, and the publication of the first book on NAFLD in Australia (Box 1)[16–22].

The diagnosis of NASH relies heavily on a clinical perspective, namely the exclusion of competing etiologies of steatogenic liver disease. Recognition of the components of the metabolic syndrome [22] has come to define the prehistological probability of NAFLD/NASH, thus reducing the number of potential liver biopsies. In particular, the detailed understanding of the metabolic derangements associated with NAFLD has led to proposals to rename NAFLD and NASH [23,24].

Nonalcoholic fatty liver disease is a common disorder consisting of simple steatosis, a relatively benign, nonprogressive condition, and NASH, a disease that infrequently improves spontaneously [25] and confers significant liver-related mortality [26]. However, both conditions are associated with excess cardiovascular mortality [25,26]. Therefore, there are two chief questions to be answered: what is the pathophysiologic underpinning of the inflammatory–fibrotic progression in NASH, and how is fat deposition in the liver connected to compromised vessel integrity and resultant premature cardiovascular mortality?

Preparation & rationale of this special focus issue: a virtual workshop

This special focus issue represents a ‘virtual workshop’, gathering authors from Europe, the USA and Australia. Guest editors (GEs) agreed on a certain number of topics to be developed, and invited distinguished authors to cover them extensively. All submissions underwent peer review. Notably, both the GEs and authors were left blind to reviewers’ identities. In some cases, GEs acted as authors or co-authors themselves.

The overriding emphasis of this compilation was to complete a systematic analysis of organelle dysfunction, both morphologically and molecularly, with the hope that this effort would effectively encapsulate the major factors in the pathophysiology of NASH. To this end, the lessons from common steatogenic conditions such as NAFLD and hepatitis C virus infection may well be interchangeable [27,28]. Chronic viral hepatitis C will tend to improve histologically once the hepatitis C virus is eradicated by successful antiviral regimens. By contrast, NASH will not invariably heal once insulin resistance is treated with insulin sensitizers. Moreover, therapeutic options acting via mechanisms other than insulin sensitization (e.g., vitamin E, presumably as an antioxidant) will prove beneficial in some cases. Taken together, these findings suggest that the pathogenesis of NASH is complex, with insulin resistance serving as an early, upstream factor in the subsequent inflammatory–fibrogenic cascade [29]. This is in full agreement with theories of pathogenesis based on experimental evidence (Figure 1)[30].

Genetics & lipidomics

The recent epidemic explosion of NAFLD could directly result from ‘junk food’ consumption [31], but a role for genetics in the development of NAFLD is supported by family clusters [32] and ethnic susceptibility of the disease [33,34]. Until 2004, the study of candidate genes was the only technique available, and very few studies feature multivariate analysis or have been reproduced [35]. In recent years, the genome-wide association study (GWAS) technique has become available. However, the GWAS approach provides a high false-positivity rate, and probably misses rare gene variants [35]. Reduced export capacity of lipids from the hepatocyte into the bloodstream was reported in experimental fatty liver as early as 1968 [36]. Among the various candidate genes, those associated with the function to export lipids from hepatocytes into the bloodstream, such as the ApoB and microsomal triglyceride transfer protein (MTP) genes, are of acknowledged significance for the development of human NAFLD [37,38]. Therefore, it seems that genetic assessments based on the known pathophysiology of NASH are more likely to be fruitful than random genome-wide assays in establishing meaningful associations and providing direction for therapeutic investigation.

One of the most exciting findings is that a nonsynonymous sequence variation that substitutes methionine for isoleucine at codon 148 in the gene encoding patatin-like phospholipase domain-containing 3 (PNPLA3) is associated with steatosis and aminotransferase values [39]. The gene varies proportionately in populations at risk for NASH, and increases the risk of alcoholic cirrhosis [40]. Although the detailed mechanistic link between the I148M variant and liver disease is unclear, it can be predicted on the grounds of the biological function of PNPLA3. This enzyme is associated with the endoplasmic reticulum and lipid droplets, and catalyzes either triglyceride deposition or hydrolysis [40].

Further to mechanisms regulating steatogenesis, the chemical composition of intrahepatocytic fat is a major determinant of liver injury. Palmitic acid, a saturated fatty acid, is more noxious to hepatocytic cells in vitro than oleic acid, although the latter is more steatogenic than the former [41]. Hepatic and plasma lipidomic analysis will probably provide essential and synthetic information on both genetics and lifestyle. On these grounds, it is a very exciting line of research that is likely to promote our understanding of the steps further to lipid accumulation, namely inflammation and fibrogenesis [42], although to date the field remains nascent.

Inflammation & fibrogenesis

Inflammation and fibrogenesis are closely inter-related phenomena that represent major potential targets in NASH research. Due to adaptations induced by evolutionary pressures to cope with both infections and food shortage, the study of insects and their ‘fat body’ may provide clues as to how deranged metabolic function may turn into inflammation [43,44]. NASH patients will typically display far lower degrees of inflammatory changes than patients with chronic hepatitis C [45], suggesting less profound activation of the immune system. Nevertheless, studies have demonstrated that inflammation is the force driving both the development of NASH and its subsequent evolution to fibrosis–cirrhosis [46] and, thus, is potentially a major therapeutic target in NASH [47].

The production of collagen in the liver results from multiple factors: activation of endogenous fibroblasts, stimulation of bone marrow fibrocytes and ductular reaction by the proinflammatory milieu of secreted adipokines and cytokines, oxidative stress, and lipotoxicity [48]. In addition, Toll-like receptors and nuclear receptors presumably play a role [48]. Inhibition/apoptosis of collagen-producing cells and/or manipulation of the proinflammatory milieu represents another key aim in NASH and all other fibrosing liver diseases.

Impaired energy production

Mitochondrial uncoupling, first identified in the 1950s [11], was later confirmed by functional [49] and ultrastructural evaluation [14]. Moreover, steatosis reduces hepatic blood flow and parenchymal microcirculation [50,51]. Taken together, these findings point to the fatty liver as a ‘weakened’ organ. The significance, adaptive versus causative, of mitochondrial abnormalities still remains elusive [52]. However, irrespective of its origin, mitochondrial dysfunction in NASH contributes to lipid accumulation, oxidative stress and enhanced susceptibility to either necrotic or apoptotic cell death [53,54]. Therefore, any consistent theory on NASH should keep disturbed energetic homeostasis central [53]. For instance, mediated by metabolism of keratins, close relationships link ballooning, Mallory–Denk body (MDB) formation and apoptosis [54]. Hepatocyte apoptosis is a key mechanism for the progression of NAFLD, in which both the extrinsic (induced by saturated fatty acids) and the intrinsic pathways are activated [55]. Serum levels of cytokeratin 18 and soluble Fas (circulating markers of liver cell apoptosis) may assist the clinician in noninvasive diagnosis of NASH [25,55]. Moreover, apoptosis may contribute to ballooning, thus creating a self-perpetuating process that would increase hepatocytes’ susceptibility to further apoptosis [54].

Opposite to necrosis and apoptosis, autophagy is predominantly a cell survival pathway: it involves the channeling of cell organelles to the lysosome for degradation. Therefore, autophagy serves both a catabolic function during limited nutrient availability and allows the removal of malfunctioning subcellular structures. Decreased autophagy in NAFLD may promote the development of steatosis, insulin resistance, the progression to NASH and facilitate the primary liver cancer complication [56]. A decrease in autophagy occurs in aging, which is of interest given the age dependency of NAFLD [57]. Moreover, autophagy is a regulatory pathway of the innate immune system and fibrogenesis [56].

Ballooning: cytoskeleton

Hepatocellular ballooning, a feature required for the diagnosis of NASH, is associated with a substantially increased risk of developing cirrhosis and liver-related mortality. Ballooning, accumulation of microsteatosis and dilation of endoplasmic reticulum result from the rearrangement of the intermediate filament cytoskeleton [58]. Injury to multiple organelles, including fat droplets and endoplasmic reticulum, may contribute to cellular ballooning [59]. Therefore, the presence of ballooned cells probably indicates a more profound dysfunction of the cytoskeleton, spanning from the presence of MDBs, altered locomotion, defective intracellular trafficking or increased mitotic errors [60]. The presence of MDBs is of great diagnostic utility in that it decreases the likelihood of acute viral hepatitis and acute cholestasis [60]. Moreover, they could serve as a model suitable to explore protein aggregation in common neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease [60].

Conclusion

Although much work remains, a clear picture emerges from this special focus issue. NASH can no longer be considered a disease associated with mitochondrial changes alone; rather, virtually all cell organelles appear to be involved, probably owing to a central metabolic defect of impaired energetic homeostasis. This special focus issue demonstrates the fine interplay among altered energetic homeostasis, metabolically induced steatogenesis, lipidomics and lipotoxicity, adipokines, cytokines and oxidative stress. A structured and, as yet, incompletely characterized process linking these mechanisms will eventually result in damage to multiple subcellular constituents, a process that could be termed ‘multiorganelle failure’. Other avenues of investigation include further elucidation of the roles of Toll-like receptors, nuclear receptors, organelle dysfunction and cell death cascades in leading to inflammatory changes and secondary fibrosis. These two processes modulate the differences between an asymptomatic, nonprogressive condition (steatosis) and a fibrosing liver disease conducive to liver- and cardiovascular-related mortality.

Future therapeutic studies should aim at reversing energetic dysfunction and restoring the normal structure and function of cell organelles in NAFLD.

Box 1. Historical overview of research on the disease, including synonyms of nonalcoholic fatty liver disease.

1952: Zelman reports on the liver in the obese [1].

1954: Dianzani identifies mitochondrial uncoupling in experimental fatty liver [11].

1962: Thaler describes what we now allude to as NASH [2].

1965: Antioxidant use is proposed [12].

1979: Itoh described “five diabetic non-alcoholic women with micronodular cirrhosis” [3].

1980: Ludwig names NASH for the first time [4].

1985: Batman uses the definition “diabetic hepatitis” [5].

1988: Diehl defines NAFLD as “alcohol-like liver disease in the non-alcoholic” [6].

1995: Lonardo reports on “bright liver syndrome”. The associations with gallstones and atherosclerosis are highlighted [7].

1999: Mendler reports on a seemingly novel disease, the “insulin-resistance-associated hepatic iron overload”, probably NAFLD, observed in a population at high risk for hereditary hemochromatosis [8].

1999: Brunt’s histological classification is proposed [9].

1999: Caldwell reports on the intramitochondrial inclusions [14].

2000: Falk Symposium “NASH & ASH” (Den Haag, The Netherlands) [16].

2002: AGA technical review on NAFLD [17].

2003: AASLD single-topic conference [18].

2005: The first book entirely devoted to NAFLD is published in Australia [19].

2005: Kleiner’s semiquantitative histological scoring system [10].

2006: EASL Monothematic Conference (Lisboa, Portugal) [20].

2009: EASL Monothematic Conference (Bologna, Italy) [21].

2010: NAFLD Evidence-Based Guidelines of AISF [22].

Financial & competing interests disclosure

Stephen Caldwell has consulting disclosures for Abbott in March 2010 and research support from Nordic Natural and Wellstat Diagnostics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Notes

AASLD: American Association for the Study of Liver Disease; AGA: American Gastroenterological Association; AISF: Associazione Italiana per lo Studio del Fegato (Italian Association for the Study of the Liver); ASH: Alcoholic steatohepatitis; EASL: European Association for the Study of the Liver; NAFLD: Nonalcoholic fatty liver disease; NASH: Nonalcoholic steatohepatitis.