Revisiting the metabolic syndrome and paving the way for micrornas in non-alcoholic fatty liver disease

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1 REVIEW ARTICLE Revisiting the metabolic syndrome and paving the way for micrornas in non-alcoholic fatty liver disease Duarte M. S. Ferreira 1, *, Andre L. Sim~ao 1, Cecılia M. P. Rodrigues 1,2 and Rui E. Castro 1,2 1 Instituto de Investigacß~ao do Medicamento (imed.ulisboa), Faculdade de Farmacia, Universidade de Lisboa, Lisbon, Portugal 2 Department of Biochemistry and Human Biology, Faculdade de Farmacia, Universidade de Lisboa, Lisbon, Portugal Keywords apoptosis biomarkers; ER stress; gut microbiota; insulin resistance; metabolism; mirnas; NAFLD; oxidative stress Correspondence R. E. Castro, Instituto de Investigacß~ao do Medicamento (imed.ulisboa), Faculdade de Farmacia, Universidade de Lisboa, Av. Prof. Gama Pinto, Lisbon, Portugal Fax: Tel: ruieduardocastro@ff.ul.pt *Present address Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (Received 15 September 2013, revised 16 March 2014, accepted 3 April 2014) doi: /febs Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of stages from simple steatosis to non-alcoholic steatohepatitis, which can progress to fibrosis, cirrhosis and, ultimately, hepatocellular carcinoma. Despite being one of the most common chronic liver diseases, NAFLD pathogenesis remains largely unknown. In this review, we discuss the key molecular mechanisms involved in NAFLD development and progression, focusing on the emerging role of micrornas. NAFLD is intrinsically related to obesity and the metabolic syndrome. Changes in lipid metabolism increase free fatty acids in blood, which in turn induces peripheral insulin resistance and increases oxidative and endoplasmic reticulum stress. Although not yet considered in the diagnosis of NAFLD, recent reports also reinforce the crucial role of apoptosis in disease progression via activation of either death receptor or mitochondrial pathways and p53. In addition, the role of gut microbiota and the gut liver axis has been recently associated with NAFLD. Finally, there is an accumulating and growing body of evidence supporting the role of micrornas in NAFLD pathogenesis and progression, as well as hinting at their use as biomarkers or therapeutic tools. The ultimate goal is to review different molecular pathways that may underlie NAFLD pathogenesis in the hope of finding targets for new and efficient therapeutic interventions. Introduction Non-alcoholic fatty liver disease (NAFLD) is defined as the accumulation of fat within hepatocytes exceeding 5%, in the absence of significant alcohol intake (20 gday 1 for men and 10 gday 1 for women), viral infection or any other specific aetiology of liver disease [1]. In addition, NAFLD includes a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Although simple Abbreviations AGO, argonauts; ALT, alanine transaminase; ATF6, activating transcription factor 6; ER, endoplasmic reticulum; FFA, free fatty acids; HFD, high-fat diet; INSR, insulin receptor; IRE1a, inositol requiring enzyme 1a; IR, insulin resistance; IRS-1, insulin receptor substrate 1; JNK, c- Jun NH 2 -terminal kinase; mirisc, mirna-induced silencing complex; mirna or mir, microrna; NAFLD, non-alcoholic fatty liver disease; NAMPT, nicotinamide phosphoribosyltransferase; NASH, non-alcoholic steatohepatitis; NF-jB, nuclear factor kappa B; PERK, doublestranded RNA-activated protein kinase-like ER kinase; PI3K, phosphoinositide-3-kinase; PPAR, peroxisome proliferator activator receptor; PTEN, phosphatase and tensin homologue detected on chromosome 10; PTPs, protein tyrosine phosphatases; PUMA, p53-upregulated modulator of apoptosis; ROS, reactive oxygen species; SIRT1, sirtuin 1; SREBP2, sterol regulatory element-binding protein 2; TLR, toll-like receptor; TNF, tumour necrosis factor; UPR, unfolded protein response; XBP1, X-box-binding protein 1. FEBS Journal 281 (2014) ª 2014 FEBS 2503

2 D. M. S. Ferreira et al. steatosis is characterized by a relatively favourable clinical course, NASH more frequently progresses to cirrhosis and hepatocellular carcinoma, leading to liver-related morbidity and mortality [2,3]. In NAFLD patients, histological characterization of liver biopsies is key in identifying steatosis and liver injury, including hepatocyte ballooning, inflammation, and fibrosis. In steatosis, portal inflammation is usually mild or absent and there is almost no ballooning injury. If fibrosis is present, it should be limited to mild periportal or perisinusoidal fibrosis. On the other hand, NASH diagnosis implies the presence of ballooning injury, as indicated by enlarged cells whose cytoplasm becomes irregularly clumped with nonvesiculated areas. It is usually still possible to identify steatotic vacuoles in ballooned cells, although they should not fill the cytoplasm [4]. In addition, most balloon cells contain Mallory Denk bodies, found near the nucleus, which comprise hyperphosphorylated and misfolded cytokeratin 8 and 18 filaments [5]. Early in the disease, fibrosis is mild and inflammation is lobular and associated with steatosis and fibrosis. Later, steatosis is spread all over the liver and inflammation may become prominent. Periportal fibrosis may then occur and extend into the surrounding parenchyma. Eventually, hepatocytes trapped by collagen from fibrosis will undergo apoptosis, and regeneration will create solid nodules of hepatocytes, in such a way that the end stage may resemble cirrhosis. Taking such information into consideration, Kleiner et al. [6] proposed a widely used scoring system, named the NAFLD activity score. It is defined as the sum of the steatosis (0 3), lobular inflammation (0 3) and ballooning (0 2) scores. Fibrosis was not included in this classification because it is less reversible and a result of the disease activity. Briefly, cases with NAFLD activity score of 0 2 are considered not diagnostic of NASH and cases with scores 5 or higher are diagnosed as NASH. Cases with activity scores of 3 4 are considered at an early stage of NASH [6]. NAFLD is strongly associated with obesity, insulin resistance (IR), diabetes, dyslipidaemia, increased blood pressure, hypercholesterolaemia, and a proinflammatory state [7,8]. IR is a common characteristic feature of NAFLD. Several studies have reported that insulin resistant subjects with NAFLD have reduced insulin sensitivity in the liver, skeletal muscle and adipose tissues [9]. In the adipose tissue, insulin inhibits lipolysis; upon IR development, the adipose tissue does not respond to insulin-inhibition of lipolysis, increasing the release of free fatty acids (FFAs) to the blood. In the liver, the presence of increased lipolysis and/or fat intake promotes hepatic triacylglycerol synthesis [10]. Finally, increased circulating plasma levels of triacylglycerol and FFAs contribute to IR in the skeletal muscle and peripheral tissues. The muscle and the liver take up these FFAs, saturating their oxidative capacity and accumulating them as ectopic fat, mainly as intramyocellular and hepatic lipids, respectively [11]. Several studies have also demonstrated the existence of high levels of endoplasmic reticulum (ER) stress markers and a reduced unfolded protein response (UPR) in NAFLD, hinting at a likely role for ER stress during disease pathogenesis. When the cell is faced with unfolded proteins, the UPR becomes crucial to restore ER homeostasis. UPR decreases the amount of protein entering the ER and reinforces its capacity to fold and degrade proteins [12]. However, in several diseases, including NAFLD, there is an inability to resolve ER stress, leading to sustained activation of the UPR; knockdown of proteins of the UPR pathway results in ER stress and hepatic steatosis as a result of the inability to oxidize FFAs. Thus, the UPR appears to have an important role in promoting lipid homeostasis by maintaining ER homeostasis subsequent to ER stress. Moreover, the exacerbation of hepatic steatosis, in the context of NAFLD, might lead to UPR impairment, reducing its ability to restore ER homeostasis [13]. Hepatocyte apoptosis is considered to be a pivotal event in several types of liver injury, including NA- FLD. In the liver, apoptotic bodies are phagocytized by stellate cells and Kupffer cells, the resident macrophages. Engulfment of apoptotic bodies by Kupffer cells promotes the generation of death ligands, including Fas ligand, and tumour necrosis factor (TNF)-a. These death ligands then promote hepatocyte apoptosis in a feed-forward loop. In addition, apoptotic cells also produce profibrogenic factors, such as transforming growth factor b1 and type I collagen, and release nucleotides that bind to purinergic receptors on macrophages and hepatic stellate cells to activate them further [14]. The continued and sustained induction of hepatocyte apoptosis culminates in hepatic inflammation and fibrosis. Apoptosis has not yet been included as criteria in the classification of NAFLD, despite the convincing correlation with NAFLD severity. More recently, the role of gut microbiota and micrornas (mirnas or mirs) in NAFLD pathogenesis has been described. Several studies have highlighted the functional role of gut microbiota in weight management, development of obesity and the metabolic syndrome, including NAFLD [15,16]. On the other hand, mirnas are a large family of RNAs (approximately 21 nucleotides in length) that act as gene expression regulators at the post-transcriptional level, controlling several crucial cellular processes in 2504 FEBS Journal 281 (2014) ª 2014 FEBS

3 D. M. S. Ferreira et al. eukaryotes. Several studies have shown that abnormal changes in mirna expression are often associated with cell growth and apoptosis, inflammation, and even human pathologies, including NAFLD. For example, mir-122 is primarily expressed in hepatocytes and represents approximately 50% of the total liver mirnas, with estimates ranging from to copies per 10 pg of total liver RNA [17]. mir- 122 directly and indirectly controls the expression of multiple targets regulating hepatic function. Of note, mice lacking mir-122 develop normally, although they quickly progress into steatohepatitis [18]. The role of mirnas in the pathogenesis of liver diseases, particularly NAFLD, is continuously expanding, and their use as potential therapeutic targets is gaining interest. In addition, this class of small RNAs may be able to serve as markers to discriminate disease from the normal tissue or for prognostic purposes. This is particularly relevant for NAFLD because present screening methods use ultrasound, and computerized tomography scans that are time-consuming, although inaccurate and unreliable. Furthermore, the current standard of care for treating NAFLD patients relies almost solely on lifestyle interventions and there is a lack of consensus regarding the most effective and appropriate pharmacologic therapy. As such, the development of mirna-based therapeutics could be of great advantage for NAFLD. This review summarizes some of the most recent findings on the molecular mechanisms governing NA- FLD pathogenesis and progression. These include new concepts on the role of IR, oxidative and ER stress, apoptosis, gut microbiota and particularly mirnas on NAFLD development. NAFLD epidemiology In Europe, the prevalence of NAFLD ranges from 26% to 40% in patients with chronic liver disease [19]. In particular, in the Barcelona and DIONYSOS studies, the prevalence of NAFLD was approximately 26%. In the SHIP study, this value was slightly higher, at 30.4%, whereas, in the RISC study, 33% of patients had very high probabilities of the disease [20 22]. Unexpectedly, NAFLD has a high prevalence in obese children; the Raine cohort analyzed 1170 Australian adolescents and 12.8% had NAFLD [23]. Nevertheless, male individuals aged between 40 and 65 years have a higher prevalence of NAFLD, which indicates that NAFLD increases with age [24]. The Rotterdam study found that the prevalence of NAFLD in the elderly was high, at 35.1% (2811 participants from the Netherlands with a mean age of years). This value decreased to 24.3% for participants aged > 85 years. This could reflect changes in the dietary composition or intake, which varies significantly with advanced age [25]. The prevalence of NAFLD is increasing worldwide in parallel with the increase in obesity and type 2 diabetes; indeed, NAFLD is particularly prevalent in type 2 diabetic patients [26]. This assumption was confirmed by two major European epidemiological studies, which reported prevalence rates of NAFLD of % in type 2 diabetic patients [27,28]. These studies indicated that 50% of adults in the European Union with type 2 diabetes might eventually develop NAFLD. Regarding obesity, the Finnish type 2 diabetes survey, which collected information from 2849 patients (aged years), identified that body mass index has a strong correlation with alanine aminotransferase (ALT) and aspartate aminotransferase levels, as well as NAFLD activity score [29]. Finally, in parallel with dysfunctional lipid metabolism and IR, obesity contributes to the development of the metabolic syndrome, which is a key risk factor for triggering NAFLD [7,8]. Apart from its significant health issues, NAFLD already represents an important economic burden for European countries, with patients having up to 26% higher overall healthcare costs at 5-year follow-up [30]. As such, NAFLD constitutes a major potential threat to public health both in terms of health and of economical factors, and more suitable forms of treatment are urgently needed. NAFLD pathogenesis For a long time, NAFLD pathogenesis was explained on the basis of the two hits hypothesis. The first hit was the reversible deposit of triacylglycerols in hepatocytes that lead to the development of steatosis and sensitize hepatocytes for the second hit. In the second hit, FFAs mobilized from the adipose tissue undergo oxidation in the mitochondria of hepatocytes. If the FFAs supply exceeds the liver metabolic capacity, triacylglycerol accumulation occurs, leading to oxidative stress- and cytokine-induced liver injury [31]. Despite the two hits hypothesis no longer being truly considered as a result of its rather simplistic view, when considering more recent data, it is still useful with respect to revising the main mechanisms involved in NAFLD pathogenesis. Several factors have been recently described as playing a key role in NAFLD pathogenesis, including adipokines (adiponectin, leptin and resistin) and cytokines (such as TNF-a, interleukin-6 and FEBS Journal 281 (2014) ª 2014 FEBS 2505

4 D. M. S. Ferreira et al. interleukin-1b), which are secreted by adipocytes or inflammatory cells infiltrating into the adipose tissue in insulin-resistant states. The adipocytokines exert a cross-talk between adipose, skeletal muscle and hepatic tissues. In NASH patients, adiponectin serum levels are decreased, and inversely correlated with hepatic IR and fat content, as well as with the extent of fibrosis [32]. In addition, high levels of TNF-a and low levels of adiponectin have been proposed as independent predictors of NASH in human patients because low serum adiponectin is associated with more extensive necroinflammation [33]. By contrast, resistin serum levels are increased in patients with NASH and a decrease in resistin levels could be positively correlated with improvement of hepatic insulin sensitivity and decreased hepatic fat content [34]. Finally, leptin has been positively correlated with hepatic fat content but not with inflammation or fibrosis in NASH patients [35]. Kupffer cells and hepatic stellate cells are recruited by cytokines, which contribute to the progression of NAFLD from steatosis to NASH by increasing inflammation and fibrosis [36]. They can also affect the insulin signalling pathway, thus also playing a role in the development of IR. Overall, overload of FFAs in the liver and changes in adipokines and inflammatory cytokines profiles lead to IR, oxidative stress, ER impairment and apoptosis [37]. These four metabolic syndrome-associated processes are now acknowledged as well established pathogenic factors for NAFLD development and progression, and its characterization continues to unravel. More recently, intestinal microbiota and, particularly, mirnas have been described as new and crucial players in the development of the metabolic syndrome and NAFLD [38,39]. Recent updates on the role of IR during NAFLD Under physiological conditions, in adipose and skeletal muscle tissues, the insulin signalling pathway initiates by insulin binding to the insulin receptor (INSR), thus allowing its tyrosine transphosphorylation and activation (Fig. 1, top). This is followed by the sequential activation of insulin receptor substrate 1 (IRS-1), phosphoinositol-3-kinase (PI3K) and AKT. This last kinase activates glucose transporter type 4, found in cytoplasmic vesicles, which moves to the plasma membrane to allow glucose uptake [40]. In addition, PI3K activates phosphodiesterases that degrade camp and decrease its amount in the cell. Low levels of camp induce protein kinase A activation, which activates lipoprotein lipase [41]. In the liver, the engagement of INSR activates a different substrate, insulin receptor substrate 2, through which PI3K and AKT phosphorylate and inactivate glycogen synthase kinase-3. Its inactivation then releases glycogen synthase, which increases the synthesis of glycogen [42]. On the other hand, in the presence of FFAs and TNF-a, IRS-1 is phosphorylated at Ser312 (in humans; Ser307 in mice), rather than tyrosine, disturbing the physiological insulin signalling pathway and contributing to IR in adipose and skeletal tissues (Fig. 1, bottom) [43,44]. In this context, cell glucose uptake is interrupted and glucose is retained in the extracellular space, inducing hyperglycaemia and stimulating further release of insulin from pancreatic b cells. Furthermore, in the adipose tissue, IR also leads to increased camp levels, which activate protein kinase A and, ultimately, lipoprotein lipase, resulting in triacylglycerol degradation and FFAs release into the bloodstream. Instead, in the liver, IR decreases glycogen synthesis and increases glycolysis, gluconeogenesis and the release of glucose into the blood stream. In addition, insulin also stimulates the expression of lipogenic genes, thus determining the synthesis of fatty acids. Overall, IR leads to a condition in which normal insulin levels fail to maintain euglycaemia, upon which higher levels of insulin are needed [45]. Several studies have suggested an important role of c-jun NH 2 -terminal kinase (JNK) in inducing IR. Mice subjected to a high-fat diet (HFD) display obesity in wild-type animals but not in jnk1 knockout mice. Additionally, jnk1 knockout mice show decreased IRS-1 phosphorylation at the inhibitory Ser307 site and increased phosphorylation at the activator Tyr1172 and Tyr1222 sites, thus resulting in lower IR. Moreover, HFD-induced hepatocyte injury and steatosis are suppressed in jnk1 knockout mice [46] and, on the other hand, jnk2 knockout mice under HFD present increased levels of hepatocyte injury, obesity and IR, despite hepatic steatosis being similar to that of wild-type mice. Furthermore, these mice have higher JNK1 activity, suggesting that JNK1 overcompensates the loss of JNK2 and, by doing so, promotes liver damage and IR [47]. A recent study in morbid obese patients with different stages of NAFLD showed increased IR with decreased phosphorylation of INSR, IRS and AKT, alongside disease progression, in agreement with an increase in JNK phosphorylation [3]. Furthermore, acute oxidative stress has been described to activate JNK in the nucleus, whereas chronic oxidative stress activates JNK in the cytosol. As a result of this differential activation, chronic oxidative stress induces IR and glucose intolerance in muscle and adipose tissues, whereas acute oxidative stress increases AKT 2506 FEBS Journal 281 (2014) ª 2014 FEBS

5 D. M. S. Ferreira et al. Fig. 1. The insulin signalling pathway under physiological and insulin resistance conditions. Under physiological conditions (top), insulin binds to INSR, which allows its tyrosine autophosphorylation and activation with latter phosphorylation in tyrosine and the activation of IRS. This is followed by the activation of PI3K and AKT. This last kinase activates glucose transporter type 4 (GLUT-4), which is found in vesicles in the cytoplasm and moves to the plasma membrane to allow glucose uptake. In addition, AKT phosphorylates and inactivates glycogen synthase kinase-3 (GSK-3). GSK-3 inactivation then releases glycogen synthase (GS) and allows its activation to increase the glycogen synthesis in the liver. FFAs and TNF-a interfere with the insulin signalling pathway and contribute to IR (bottom). The increase in FFAs and TNF-a leads to ROS production that activates JNK1. JNK1 then phosphorylates IRS serine residues rather than tyrosine residues. IRS serine phosphorylation decreases AKT phosphorylation, which impairs GLUT-4 activation and translocation to the plasma membrane. In addition, it also decreases glycogen synthesis and increases glycolysis, with the release of glucose into the blood stream inducing hyperglycaemia that stimulates the release of insulin from pancreatic b cells. Of note, PTPs may also dephosphorylate INSR and IRS-1, thus further impairing and negatively regulating insulin signalling. GP, glycogen phosphorylase. phosphorylation and reverses hyperglycaemia-induced IR, restoring insulin stimulation of glucose uptake [48]. Apart from JNK, other serine/threonine kinases, such as inhibitor of nuclear factor kappa B (NF-jB), kinase b and protein kinase C-h are also capable of phosphorylating IRS-1 Ser307. Indeed, these proinflammatory kinases are activated in the skeletal muscle of insulin-resistant subjects, leading to decreased AKT phosphorylation and impairing glucose transporter type 4 activation and translocation to the plasma membrane [49]. Notably, a recent study using knock-in mice with impaired phosphorylation of IRS-1 Ser307 showed that this phosphorylation is important for maintaining normal insulin signalling; Ser307 appears to contribute to insulin sensitivity via PI3K, allowing its physiological binding to IRS1. It was also demonstrated that upregulation of Ser307 phosphorylation during IR in vivo appears to have an adaptive role instead of a pathological one [44]. With continuous insult, this adaptive response might become a pathological trigger. Several recent studies have highlighted the role of protein tyrosine phosphatases (PTPs) in NAFLD. Concerning IR, PTPs such as PTP1B dephosphorylate FEBS Journal 281 (2014) ª 2014 FEBS 2507

6 D. M. S. Ferreira et al. the INSR and IRS-1, thus impairing and negatively regulating insulin signalling. Obese, liver-specific PTP1B knockout mice show an improvement in glucose tolerance, as well as in lipid homeostasis, compared to wild-type animals [50]. In addition, liverspecific PTP1B knockout mice also show decreased serum triacylglycerol levels, concomitantly with decreased lipogenic gene expression [51]. From a therapeutic perspective, a curcumin-enriched diet has been shown to inhibit PTP1B, which prevents hepatic steatosis in fructose-fed rats, suggesting that PTP1B knockdown may be a suitable approach for treating NAFLD [52]. Furthermore, it is now established that antagonizing PTPs significantly improves the insulin response and increases INSR phosphorylation and activation [53]. Curiously, knockdown of carcinoembryonic antigen-related cell adhesion molecule, a PTP substrate, leads to the development of NASH in mice [54] because it mediates an important link between inflammation and IR in obesity and NAFLD [55]. Oxidative stress leading to NAFLD Oxidative stress constitutes another important pathogenic factor for NAFLD (Fig. 2). Upon an excessive supply of FFAs to the liver, mitochondrial and peroxisomal b-oxidation of FFAs increase. This leads to the formation of reactive oxygen species (ROS), which induce hepatocyte toxicity, inflammation and fibrosis. Moreover, patients with NASH display decreased mitochondrial respiratory chain complexes, leading to inefficient ATP production [56], further emphasizing the role of oxidative stress and mitochondrial dysfunction in NASH and NAFLD progression. Animal models have also confirmed this; in ob/ob mice, increased FFA b-oxidation in the mitochondria and peroxisomes leads to oxidative stress and, ultimately, to IR [57]. Furthermore, excessive b-oxidation increases ROS levels, leading to depletion of mtdna. This severely affects mitochondrial function, not only impairing the synthesis of enzymes involved in the mitochondrial Fig. 2. Cell death, oxidative stress and endoplasmic reticulum stress interplay in NAFLD pathogenesis. Excessive supply of FFAs to the liver increases mitochondrial and peroxisomal b-oxidation of FFAs. This increases intracellular ROS levels, which then decrease mitochondrial respiratory chain complexes, leading to inefficient ATP production and depletion of mitochondrial DNA. In addition, patients with NASH display high levels of TNF-a in the blood that also leads to increased ROS production and JNK1 phosphorylation. NAFLD patients have also exhibit high levels of ER stress in the liver as a result of reduced UPR. In obesity and NAFLD, there is an inability to resolve ER stress, leading to the accumulation of unfolded proteins in the ER lumen, as well as to the suppression of insulin signalling pathway through IRE- 1a/TNF receptor associated protein 2 (TRAF2)-dependent activation of JNK1 and the subsequent Ser307 phosphorylation of IRS-1. In addition, JNK1 cooperates with ER stress-induced expression of CAAT/enhancer binding homologous protein (CHOP) to upregulate PUMA. PUMA then activates BAX, which translocates to mitochondria, causing mitochondrial dysfunction and caspase-dependent apoptosis. Furthermore, toxic saturated FFAs, such as palmitic acid, stimulate protein phosphatase 2A activity and promote FOXO dephosphorylation and activation. One of the transcriptional targets of FOXO3a is BIM, which further increases FFA-dependent apoptosis. Finally, in response to ER stress, IRE1a can directly interact with TRAF2 and bind to the IKK complex, thus activating NF-jB. Nuclear NF-jB then induces TNF-a expression, contributing to the inflammatory response observed in NASH patients. 2A, phosphatase 2A FEBS Journal 281 (2014) ª 2014 FEBS

7 D. M. S. Ferreira et al. respiratory chain, but also inducing steatosis and liver lesion. mtdna depletion has also been described in NASH patients [58]. In addition, FFAs activate the transcription factor peroxisome proliferator activator receptor (PPAR)a, which induces the expression of genes involved in FFA b-oxidation, further increasing ROS production and, ultimately, inducing IR, lipolysis and FFAs uptake by the liver. In vitro studies have shown that TNF-a is also capable of increasing ROS levels, thus inhibiting enzymes involved in the mitochondrial respiratory chain [59]. Indeed, a positive correlation between high levels of TNF-a in the blood and a reduced activity of the mitochondrial respiratory chain has been found in NASH patients [56]. These high levels of TNF-a are released from adipose tissue or even hepatocytes and Kupfer cells as a result of NF-jB activation during lipolysis [60]. In parallel with its cytotoxic effects, ROS can also mediate physiological intracellular signalling and act as a second messenger. In that regard, PTPs have been shown to be oxidized by ROS and, thus, act as ROS effectors [61,62]. Very few studies have addressed the potential role of PTP oxidation in NAFLD pathogenesis. A key study demonstrated that mice lacking glutathione peroxidase 1, one of the enzymes involved in the elimination of physiological ROS, were protected from HFD-induced IR, and that the increased insulin signalling correlated with enhanced oxidation of the PTP family member phosphatase and tensin homologue detected on chromosome 10 (PTEN) [63]. More recently, glutathione peroxidase 1 knockout mice were also shown to exhibit decreased hepatic steatosis and liver damage accompanied by decreased glucoseinduced insulin secretion which, in turn, was associated with elevated pancreatic PTP oxidation, including PTPN2 oxidation [64]. Furthermore, PTP-a was shown to mediate the effects of ROS on IR in patients with Fanconi anaemia [65]. In summary, the main consequences of oxidative stress in hepatocytes are lipid peroxidation, ER stress, cell degeneration, cell death, increased expression of pro-inflammatory cytokines, liver stellate cell activation and fibrogenesis. As such, oxidative stress is one of the main mechanisms involved in the progression of NAFLD from simple steatosis to NASH and, furthermore, to more advanced lesions. Unravelling the role of ER stress in NAFLD pathogenesis ER homeostasis is kept under control by three main proteins, namely inositol requiring enzyme 1a (IRE1a), double-stranded RNA-activated protein kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6) [66,67]. These are activated upon the UPR to increase the protein folding capacity of the ER (Fig. 2). If the ER stress is too severe, the UPR is not capable of re-establishing protein folding, and apoptosis is engaged [68,69]. It is now well established that ER stress plays a key role during NAFLD pathogenesis. For example, activated IRE1a induces the splicing of X-box-binding protein 1 (XBP1), which prompts the transcription of several genes involved in protein folding and maturation [70], and mice deficient in XBP1 exhibit ER stress, hyperactivation of JNK and reduced activation of the insulin signalling pathway in the liver [71]. In addition, IRE1a liver-specific knockout mice develop steatosis as a consequence of repressed expression of metabolic transcriptional regulators and triacylglycerol biosynthesis enzymes [13]. In parallel with IRE1a/XBP1, pharmacological induction of ER stress in ATF6 knockout mice leads to the formation and accumulation of lipid droplets in the liver, as well as steatosis [72]. Furthermore, ATF6 knockout mice also show decreased glucose tolerance and insulin secretion, suggesting that ATF6 might also participate in the pathogenesis of diabetes type 2 [73]. Regarding PERK, knockout mice were shown to acquire hyperglycaemia and hyperinsulinaemia, likely as a result of the loss of the effects of PERK in pancreatic b-cells proliferation [74,75]. Moreover, deletion of PERK also inhibits the expression of fatty acid synthase, ATP-citrate lyase and stearoyl-coa desaturase- 1, which are critical players in lipid metabolism [76]. Curiously, PTP1B has been identified as a crucial linker between ER stress and IR in obese patients as a result of its signal transduction control of obesity and diabetes type 2 pathophysiology [77]. For example, liver-specific PTP1B knockout mice on a HFD display an improvement on the insulin signalling pathway and reduced triacylglycerol and cholesterol levels. These mice have also decreased phosphorylation levels of PERK and JNK2, indicating that PTP1B inhibition may improve ER stress [51]. Indeed, PERK is a direct target of PTP1B, which has also been shown to increase accumulation of unfolded proteins and, as a consequence, activate cell death, by inducing PERK dephosphorylation [78]. FFAs may also contribute to cell death and apoptosis, during NASH, through ER stress; FFAs are known to assemble into saturated phospholipids that integrate the ER membrane. A high accumulation of saturated phospholipids in the ER membrane, as a result of high FFA levels, decreases membrane stiffness, contributing to its loss of functionality and ER FEBS Journal 281 (2014) ª 2014 FEBS 2509

8 D. M. S. Ferreira et al. stress [79]. ER stress ultimately activates TNF-a, in a NF-jB-dependent manner, leading to the inflammatory response observed in NASH patients [80]. Curiously, in an animal model of dietary ingestion of FFAs, liver injury, ER stress and increased caspase-3 activity developed long before increased TNF-a circulating levels and body fat developed [37]. Additionally, there are well known physical and functional links between the ER and mitochondria, where ER-mitochondrial coupling may promote mitochondrial respiration and be influenced by ER stress [81]. In addition, chronic or severe ER stress may modify cellular metabolism and mitochondrial respiration [82]. Therefore, it is natural that mitochondrial dysfunction and apoptosis in NAFLD also involves ER stress. Consolidating view on the role of apoptosis during NAFLD pathogenesis Hepatocyte apoptosis is a crucial event in several liver diseases, including NAFLD (Fig. 2). In more severe stages of NAFLD, namely NASH, patients display a significant increase in hepatocyte levels of caspase-3 and -7, as well as overall apoptosis [2,3]. In addition, apoptosis and NF-jB activity are increased in NASH patients, correlating with inflammation and fibrosis but not with steatosis [83]. Moreover, the increased expression of death receptors in NAFLD, namely Fas, TNF receptor 1 and TNF-related apoptosis-inducing ligand, all correlate with increased hepatocyte apoptosis. Furthermore, TNF receptor 1 knockout mice fed a high-carbohydrate diet display less steatosis and liver injury compared to wild-type controls [84]. Finally, TNF receptor 1, Fas and TNF-related apoptosisinducing ligand have all been described to activate JNK1 in response to bile acids or FFAs [85]. Apart from activation of the death receptor pathway of apoptosis, several NAFLD human and animal studies have demonstrated that hepatocytes also display both structural and functional abnormalities in mitochondria and, as a consequence, mitochondrial-dependent apoptosis. In particular, in the setting of NAFLD, mitochondria become enlarged and develop crystalline inclusions that change its structure, in parallel with the enhanced production of ROS, accumulation of lipid peroxides and release of cytochrome c into the cytoplasm [86]. Regarding proteins of the BCL-2 family, it was found that both BAX and BCL- 2 expression are increased in NASH patients. Of note, BCL-2 is not expressed under physiological conditions in hepatocytes, suggesting that its activation during NAFLD progression may represent an adaptive phenomenon to resist to apoptosis in response to obesity-related stress. Nevertheless, in these same patients, apoptosis was still evident, suggesting that increased expression of BCL-2 is either insufficient to antagonize apoptosis or prevents a worse scenario, where the levels of apoptosis in liver tissue could further compromise its functions [87]. More recently, it was shown that p53-induced apoptosis may be critical in NAFLD pathogenesis. In an animal model of NASH, insulin-like growth factor-1 was decreased with disease progression, resulting in increased p53 levels. p53 was then suggested to mediate mitochondrial cell death pathways, possibly also being responsible for increasing TNF-related apoptosis-inducing ligand receptor expression, thereby linking intrinsic and exogenous apoptosis pathways during NASH [88]. FFAs and free cholesterol, derived from lipotoxicity, appear to be the main inducers of the mitochondrial pathway of apoptosis in NAFLD [89]. In addition, high levels of free cholesterol also increase hepatocyte susceptibility to TNF-a and Fas, in nutritional and genetic models of hepatic steatosis. In this context, increased triacylglycerol levels also increase hepatic inflammation and TNF-a expression by activating NFjB [90]. One mechanism by which FFAs induce apoptosis involves the stimulation of protein phosphatase 2A that promotes FOXO3a dephosphorylation and activation. One of the transcriptional targets of FOXO3a is the BCL-2-interacting mediator of cell death, which amplifies FFA-dependent apoptosis [91]. FFAs also appear to activate JNK1 and phosphorylate c-jun, leading to increased p53-upregulated modulator of apoptosis (PUMA) transcription and apoptosis during lipogenic hepatocyte injury [92]. Moreover, JNK is activated in a HFD animal model, resulting in BAX activation without changes in BCL-2 or BCL-x L. This imbalance of pro- and anti-apoptotic proteins of the BCL-2 family further contributes to an increase in hepatocyte apoptosis during NAFLD [93]. Feeding NAFLD with gut microbiota Recent years have seen a focus on the role of gut microbiota in the development of obesity, IR and NAFLD. It is now fully accepted that the metabolic activities of gut microbiota have a huge impact on host nutrient absorption and energy homeostasis; host and microbiota face a win win situation where the bacteria obtain nutrients and the host obtains vitamin K, intestinal wall preservation and protection against invasion by alien microbes [94]. However, changes in gut microbiota composition, known as dysbiosis, are now associated with different clinical conditions, including obesity, NAFLD or even autoimmune 2510 FEBS Journal 281 (2014) ª 2014 FEBS

9 D. M. S. Ferreira et al. diseases [16]. For example, germ-free mice under HFD are resistant to obesity [95]. However, if the gut microbiota is transferred from conventional to germ-free mice, adipogenesis is promoted [96]. In addition, HFD-induced obesity in rats was shown to occur in parallel with changes in gut microbiota and concomitant toll-like receptor (TLR)4 activation. This leads to gastrointestinal inflammation, hyperphagia and obesity [97]. Indeed, obesity may result, at least in part, from increased systemic levels of lipopolysaccharide, which was reduced after antibiotic treatment in HFD-fed mice and ob/ob mice [98]. Apart from lipopolysaccharide, several in vivo studies have further suggested that gut microbiota can trigger hepatic steatosis either by increasing monosaccharide absorption, bacterial hepatotoxic bioproducts or by modulating bile acid metabolism [16]. In this regard, it was recently shown that dietary- or genetic obesity-induced alterations in gut microbiota, increases intestinal levels of deoxycholic acid, a toxic, pro-apoptotic bile acid, shown to target mirnas in the liver [99]. Deoxycholic acid then enters the enterohepatic circulation and activates hepatic stellate cells, which in turn secrete various inflammatory and tumour-promoting factors in the liver [100]. As such, changes in gut microbiota appear to be able to induce NAFLD and the metabolic syndrome via modulation of the inflammasome activity. The inflammasome is a large multimeric structure that regulates caspase-1 activation. Once the inflammasome is assembled in the cytosol, caspase-1 is activated and released to promote the cleavage and maturation of pro-inflammatory cytokines, which induces a sustained inflammation [101]. NASH patients were shown to have bacterial overgrowth concomitantly with TNF-a, further indicating that NASH is also associated with dysbiosis and systemic inflammation [102]. In the gut, dysbiosis induces the abnormal accumulation of bacterial products in the portal vein that travel to the liver as consequence of its first pass properties. If the liver is already conditioned by lipid accumulations, the presence of toxic bacterial products, TLR agonists and inflammasome activity are sufficient to induce NAFLD progression [103]; it was recently demonstrated that TLR2 and palmitic acid cooperatively activate the inflammasome in Kupffer cells to increase the production of cytokines that induce animal NASH development. Moreover, TLR2 knockout mice show less liver inflammation and fibrosis [104]. Exposure of hepatocytes to FFAs and lipopolysaccharide also induces the release of interleukin-1b from hepatocytes to activate the inflammasome in immune cells, such as macrophages, further contributing to NASH-associated inflammation [105]. Given the continued increase in the burden of liver diseases, huge efforts are being made to unravel the molecular alterations that trigger liver pathogenesis and progression. As such, mirnas have been found to be deregulated in several human diseases and their use as potential therapeutic targets is gaining interest. In the liver, the deregulation of particular sets of mir- NAs has already been associated with most liver diseases, particularly NAFLD. Biogenesis and mode of action mirnas are initially transcribed as long precursor molecules termed primary mirnas (Fig. 3). These are synthesized either by RNA polymerase II transcription or from the cleavage of introns in protein-coding genes [106]. Primary mirnas are then processed in a twostep sequence via the actions of Drosha and Dicer, Rnase III enzymes. First, nuclear Drosha processes the primary mirna into an approximately 70-nucleotide precursor hairpin (precursor mirna), which undergoes cytoplasm export via Exportin 5. In the cytoplasm, the precursor mirna is further processed by Dicer into an approximately 21-nucleotide mirna/ mirna* duplex, which will form a complex with the argonaut (AGO) proteins to form the mirna-induced silencing complex (mirisc). One mirna strand acts as the active strand, whereas the other one, also known as passenger strand or mirna*, is released and, in most cases, degraded. Efficient mrna targeting by mirnas requires base-pairing with the seed region of the mirna, a region formed by two to eight nucleotides at the 5 0 end of an mirna [107]. Most mirnas bind with an imperfect base-pair match with the sequences in the UTR of target mrnas, thereby inhibiting protein synthesis by repressing translation or inducing mrna deadenylation. Although the mechanisms by which mirnas induce translational repression are diverse and still not fully understood, mirna-mediated mrna deadenylation is more or less established, being mediated by GW182 proteins, upon interaction with AGOs. Although the N-terminal region of GW182 interacts with AGO via its GW repeats, the C- terminal region interacts with the poly(a) binding protein and recruits deadenylases [108]. mirna-induced mrna deadenylation may ultimately lead to the decay of target mrnas via the recruitment of decapping machinery. Finally, when AGO2 is present in the mirisc and it encounters mrnas that have sites almost matching to mirna, these mrnas are FEBS Journal 281 (2014) ª 2014 FEBS 2511

10 D. M. S. Ferreira et al. Fig. 3. mirna synthesis and mechanism of action. mirnas are processed from precursor molecules (pri-mirna). The pri-mirnas can be produced by two different mechanisms, including the action of RNA polymerase II transcription or the cleavage of introns from proteincoding genes. Next, pri-mirnas are processed by Drosha in the nucleus into an approximately 70-nucleotide precursor hairpin (pre-mirna), which is exported to the cytoplasm via Exportin 5. In the cytoplasm, pre-mirna is processed by Dicer into an approximately 21-nucleotide mirna/mirna* duplex from where the mature mirna will then form a complex with AGO and GW182 (mirisc). The other strand (passenger strand or mirna*) is released and often degraded. The majority of the mirnas base-pair imperfectly with sequences in the UTR of target mrnas, which inhibits protein synthesis by either repressing translation or promoting mrna deadenylation and decay. Deadenylation of mrnas is mediated by GW182 proteins, which interact with AGOs and act downstream. When mirisc-containing AGO2 encounters mrnas bearing sites almost perfectly complementary to mirna, these mrnas are cleaved endonucleolytically and degraded. degraded by endonuclear cleavage, a typical mechanism in plants [109]. mirnas as crucial regulators of NAFLD pathogenesis and progression Because of their crucial role in lipid metabolism, cell growth, differentiation, apoptosis and inflammation, mirnas are now being extensively studied as important regulators of NAFLD pathogenesis (Table 1). Several microarray or profiling experiments have already shown that mirnas are differentially expressed in human NASH, as well as after geneticand/or diet-induced mouse models of NASH [110]. Furthermore, NASH severity and susceptibility to NASH could be determined by variations in mirna expression response [111]. For example, in ob/ob mice, 11 mirnas were found to be deregulated in NAFLD compared to control mice. Among these, eight mir- NAs (mir-34a, -31, -103, -107, -194, p, -221 and -200a) were upregulated and three mirnas (mir-29c, -451 and -21) were downregulated [112]. NAFLD pathogenesis in the low-density lipoprotein receptor knockout mice is also associated with modulation of the hepatocyte mirna profile, including a five-fold decrease in mir-216 and mir-302a expression [113]. Another study, using different diet-induced NASH models in mice, has also identified between 1% and 3% changes in mirna expression profiles during steatohepatitis, including variations in mir-705, -1224, -182, -183 and mir-199a-3p [114]. In addition, rats fed either a standard diet with high fructose, a HFD or a diet with high levels of fats and fructose, all showed significant downregulation of mir-122, -451 and mir- 27, as well as upregulation of mir-200a, -200b and mir-429 [115]. One of the first reports in human NASH livers identified 46 mirnas to be under- or overexpressed compared to control samples [39]. Taken together, these findings support the participation of mirnas in the pathophysiological processes of NAFLD. mir-122 is a liver specific mirna playing a key regulatory role in lipid and fatty acid metabolism, as well as cholesterol accumulation. mir-122 is a posttranscriptional regulator of CYP7A1, the rate-limiting enzyme controlling bile acid synthesis in human hepatocytes [116]. Recent studies showed that mir-122 knockout mice accumulate triacylglycerol in the liver, as a result of the upregulation of enzymes responsible for triacylglycerol synthesis and storage. In addition, 2512 FEBS Journal 281 (2014) ª 2014 FEBS

11 D. M. S. Ferreira et al. Table 1. Summary of dysregulated mirnas in NAFLD. NAFLD mirnas Downregulation Upregulation Humans mir-26b [39] ; mir-28 [39] ; mir-30d [39] ; mir-92b [39] ; mir-122 [39] ;mir-126 [39] ; mir-139 [39] ; mir-145 [39] ; mir-188 [39] ; mir-191* [39] ; mir-198 [39] ; mir-203 [39] ; mir-223 [39] ; mir-296-5p [125] ; mir-361 [39] ; mir-375 [39] ; mir-563 [39] ; mir-574 [39] ; mir-601 [39] ; mir-617 [39] ; mir-641 [39] ; mir-671 [39] ; mir-765 [39] ; mir-768-5p [39] Animal models mir-15b [155] ; mir-21 [112] ; mir-27 [115] ; mir-29c [111,112] ; mir-33a [123a] ; mir-122 [18a,111,115,117a,130,151] ; mir-155 [131a] ; mir-192 [111] mir-203 [111] ; mir-216 [113] ; mir-302a [113] ; mir-467b [132] ; mir-451 [112,115] mir-15b [155b] ; mir-16 [39,152b] ; mir-21 [39,154b] ; mir-23a [39] ; mir-23b [39] ; mir-24 [39] ; mir-27b [39] ; mir-34a [2,39,152b,154b] ; mir-99b [39] ; mir-100 [39] ; mir-122 [152b,154b] ; mir-125b [39] ; mir-127 [39] ; mir-128a [39] ; mir-128b [39] ; mir-146b [39] ; mir-181b [39] ; mir-199a [39] ; mir-199 a * [39] ; mir-200a [39] ; mir-214 [39] ; mir-221 [39,129] ; mir-222 [39,129] ; mir-224 [39] ; mir-451 [154b] ; mir-455 [39] mir-21 [130] ; mir-31 [112] ; mir-33a [120a,122] ; mir-34a [111,112,134,137,137a,140,140a,151,151b] ; mir-103 [112] ; mir-107 [112] ; mir-122 [147b,150b,151b] ; mir-155 [111,130] ; mir-181a [151,151b] ; mir-182 [114] ; mir-183 [114] ; mir-192 [151b] ; mir-194 [112] ; mir-199a-3p [114] ; mir-200a [112,115] ; mir-200b [111,115,151] ; mir-221 [111,112,130,151,151b] ; mir-222 [130] ; mir-335-5p [112] ; mir-429 [115] ; mir-705 [114] ; mir-1224 [114] a From mirna-specific in vivo silencing or knockout studies. b From plasma. mir-122 knockout mice also display hepatic inflammation, progressive fibrosis and, ultimately, hepatocellular carcinoma [117]. mir-122 also regulates fibrogenic factors, including Kruppel-like factor 6 that targets transforming growth factor b1. As such, when inhibited, mir-122 leads to activation of hepatic stellate cells and fibrogenic processes. mir-122 knockout mice show evidences of steatosis and abnormal levels of very-low-density lipoproteins and high-density lipoproteins [18]. mir-33a plays a key role in bile acid synthesis, fatty acid oxidation and cholesterol homeostasis [118,119]. In particular, when cellular cholesterol levels decrease, mir-33a expression is co-induced with sterol regulatory element-binding protein 2 (SREBP2) mrna. mir-33a silencing promoted regression of atherosclerosis in mice, which suggests that mir-33a acts in synergy with SREBP2 to regulate cholesterol homeostasis [120]. A recent study showed that cholesterol might repress mir-33a levels to increase CYP7A1 expression, as well as cholesterol efflux transporters. By contrast, SREBP2 and mir-33a activation downregulate cholesterol efflux transporters and bile acid synthesis, which results in increased intrahepatic cholesterol [121]; as such, any imbalance in this regulatory circuit will increase hepatocyte lipid content and, ultimately, induce NAFLD. This appears to be the case in hepatic stellate cells exposed to free cholesterol; suppression of PPAR c signalling accompanying hepatic stellate cells activation was shown to enhance both SREBP2 and mir-33a signalling. As a consequence, there is greater accumulation of free cholesterol in hepatic stellate cells, further sensitizing them to TGFb-induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH [122]. SREBP1 is also a target of mir-33, and is significantly enhanced in mir-33 knockout mice. Unexpectedly, these mice develop obesity and liver steatosis by a SREBP1-dependent mechanism. It was suggested that the sustained, continuous absence of mir-33 in the liver may have detrimental effects as opposed to its temporal inhibition in pathological settings [123]. mir-33a and -33b have been also described to inhibit genes involved in fatty acid metabolism and insulin signalling in hepatocytes [124], further implying their pathogenic role during NAFLD. mir-296-5p was recently identified as a negative regulator of PUMA expression during hepatocyte lipoapoptosis. In NASH patients, hepatic mir-296-5p levels are reduced and associated with increased PUMA expression, confirming an inverse association between both [125]. These results suggest that lipoapoptosis, a key mediator of liver injury in NAFLD, results at least in part from mir-296-5p downregulation. In turn, lipogenesis is targeted by mir-1 and mir-206. These mirnas directly repress LXRa, thereby inhibiting the expression of LXRa target genes, such as SREBP1c, fatty acid synthase, carbohydrate responsive elementbinding protein and acetyl-coa carboxylase 1. Accodingly, mir-1/mir-206 attenuates LXRa-induced lipogenesis, hepatic steatosis and NASH [126]. mir-21 is intrinsically connected with NAFLD because it represents a crucial factor affecting PTEN expression during the metabolic syndrome, both in the human liver and in primary human hepatocytes. Indeed, excessive circulating unsaturated FFAs, such FEBS Journal 281 (2014) ª 2014 FEBS 2513