key: cord-353633-a4pu6rlu
authors: Perakakis, Nikolaos; Stefanakis, Konstantinos; Mantzoros, Christos S.
title: The role of omics in the pathophysiology, diagnosis and treatment of non-alcoholic fatty liver disease
date: 2020-07-23
journal: Metabolism
DOI: 10.1016/j.metabol.2020.154320
sha: 
doc_id: 353633
cord_uid: a4pu6rlu

Non-alcoholic fatty liver disease (NAFLD) is a multifaceted metabolic disorder, whose spectrum covers clinical, histological and pathophysiological developments ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and liver fibrosis, potentially evolving into cirrhosis, hepatocellular carcinoma and liver failure. Liver biopsy remains the gold standard for diagnosing NAFLD, while there are no specific treatments. An ever-increasing number of high-throughput Omics investigations on the molecular pathobiology of NAFLD at the cellular, tissue and system levels produce comprehensive biochemical patient snapshots. In the clinical setting, these applications are considerably enhancing our efforts toward obtaining a holistic insight on NAFLD pathophysiology. Omics are also generating non-invasive diagnostic modalities for the distinct stages of NAFLD, that remain though to be validated in multiple, large, heterogenous and independent cohorts, both cross-sectionally as well as prospectively. Finally, they aid in developing novel therapies. By tracing the flow of information from genomics to epigenomics, transcriptomics, proteomics, metabolomics, lipidomics and glycomics, the chief contributions of these techniques in understanding, diagnosing and treating NAFLD are summarized herein.

NAFLD is recognized as the most common liver disease in developed countries with its incidence continuously rising in parallel to the increasing incidences of obesity and type 2 diabetes (T2DM) [1] . Significant efforts are currently underway to delve deeper into the pathophysiology of the disease, to create non-invasive tools for its diagnosis and staging, as well as to develop highly effective and specific treatments.

The disease is characterized initially by hepatic lipid accumulation (nonalcoholic fatty liver; NAFL), that can often progress to non-alcoholic steatohepatitis (NASH), liver fibrosis or cirrhosis, as outlined in detail elsewhere in this special issue [1] . Several triggers and series of events stimulating necroinflammatory processes in the liver have been recognized and described as "the multiple hit" pathogenetic mechanism leading to advanced NASH [2] . Additionally, multiple factors (such as lack of physical activity, unhealthy nutrition, concomitant alcohol consumption and presence of other metabolic diseases) have been associated with increased risk of disease progression [3] . Still, we are not able to predict with great accuracy which patient and when will develop hepatic inflammation or fibrosis.

J o u r n a l P r e -p r o o f 13 assessed [43] , and histologically-proven GWAS [44] of more than 10000 total patients each, re-affirmed the robust clinical and histological genome-wide associations of PNPLA3, TM6SF2, GCKR, and HSD17B13. Novel fibroinflammatory loci in SLC30A10 and SLC39A8 were also studied [43] .

However, a bench-to-bedside conversion of current results through tangible diagnostic and therapeutic applications has yet to take place.

PNPLA3 rs738409 constitutes the chief NAFLD predictor, adjusted for known risk factors (OR=3.12) [21] . Yet its addition in an early predictive model of NAFLD, using routine variables, contributes trivially to score accuracy (area under the receiver operator characteristics (AUROC) = 0.866 and 0.872 respectively) [45] . PNPLA3 is included in the NASH Score (Aspartate transaminase (AST, insulin, PNPLA3 genotype) and the ClinLipMet Score (NASH Clin Score, metabolomic and lipidomic variables) with AUROCs of 0.778 and 0.866 respectively, yet its contribution to NASH prediction is not underlined [46] . In a more recent phenome-genome wide study of 27744 patients, clinical data (age, sex, BMI, blood counts) comprise a fitter NAFLD predictor than rs738409 genotyping (AUROC = 0.785 and 0.574 respectively) and have 34% higher sensitivity compared to genotyping alone. Even though rs738409 carriage was associated with 3.1 years earlier NAFLD detection, its addition does not markedly amplify the forecasting model (AUROC = 0.788) [17] .

Cumulative genetic risk scores (GRS) are frequently discussed (Table 2) . A GRS of previously mentioned SNPs in PNPLA3, TM6SF2, GCKR and MBOAT7 indicates a 3-fold increase in NAFLD risk starting from its second tertile [21] . When coupled with a multivariate insulin resistance score, a GRS J o u r n a l P r e -p r o o f 14 of PNPLA3 and TM6SF2 SNPs detects NAS ≥ 3 (AUROC=0.74) and fibrosis ≥ 3 when age included (AUROC = 0.82), yet GRS alone have AUROCs of ≤ 0.65 [41] . PNPLA3 and TM6SF2 polymorphisms are also included in a novel NASH score alongside diabetes, insulin resistance, AST and C-reactive protein levels, which predicts NASH more effectively compared to the older NASH Score (AUROCs of 0.787 and 0.729 respectively) and has enhanced accuracy in diabetic cohorts (AUROC = 0.835) [47] . Lastly, a recent investigation of 445452 individuals formulates a GRS comprising PNPLA3, TM6SF AND HSD17B13 SNPs. A maximum risk score of 6 designates a 26% increase in alanine aminotransferase (ALT) levels and a pronounced risk of cirrhosis and HCC (OR=12 and 29 respectively) [48] .

Genetic testing in NAFLD is currently not recommended by the American Association for the Study of Liver Diseases [49] . Thus far, genomics have not identified a consistent marker that reflects the accurate histologic features of NAFLD, partially owing to limited population sizes, inconsistent NAFLD validation methods and scarcity of biopsy-confirmed models. Most importantly, genomics are not dynamic processes, thus they cannot reflect the impact of environmental factors (high caloric intake, presence of obesity, T2DM etc.) in the pathogenesis and progression of NAFLD. Multi-gene models are promising tools of patient risk stratification but require additional validation.

PNPLA3 has also been investigated through early genetic interventions. Preand post-translational silencing of PNPLA3 in p.I148M knock-in mice J o u r n a l P r e -p r o o f 15 challenged with a sucrose-rich diet drastically improves intrahepatic fat profiles [50, 51] . Additionally, PNPLA3 silencing ameliorates steatosis and NAS independent of genotype, and improves inflammation, fibrosis and levels of acute phase, chemo-attractant and pro-fibrotic proteins, in p.I148M knockin mice fed a NASH-inducing diet [51] . However, PNPLA3 protects from hypercholesterolemia, gallstones, gout and acne in humans [52] , therefore future pilot studies must be carefully considered. TM6SF2 is another possible target, yet its substantial cardioprotective effects [53] indicate the complexity of such an approach, since the chief cause of death in NAFLD patients is cardiovascular disease [49] .

Patient genotyping prior to therapeutic scheme initiation implies compelling curative applications. For instance, PNPLA3 p.I148M carriers would not respond to statins and inhibitors of de novo lipogenesis, in contrast to TM6SF2 and GCKR carriers [54] . Moreover, HSD17B13 has already been implied as a therapeutic target [42] , while it was recently established that carriage of HSD17B13 rs6834314 diminishes the fibrotic effects of PNPLA3 rs738409, thus indicating a novel objective for pathway-targeting therapies [55] . Further research could uncover powerful hierarchical and therapeutic tools in NAFLD genomics, jumpstarting a new era of clinicogenomics.

J o u r n a l P r e -p r o o f 16 progression of NAFLD. Given the continuous evolvement of personalized medicine, we expect the information from genomics to be used in the near future for identifying the patients at higher risk for presenting a more aggressive course of disease and/or for potentially predicting the response to specific treatments. These may justify more intensive follow-ups or guide treatment decisions in high-risk populations. Sfince genomics are not dynamic processes and do not reflect the effects of environmental factors on NAFLD, we see less potentials for them as diagnostic markers and we expect for them to have only a small, if any, contribution in non-invasive diagnostic tests.

Finally, due to the technical and clinical challenges related to geneengineering as well as due to the complex pathophysiology of the disease, we recognize at the moment limited perspectives in the development of gene therapies for NAFLD.

Epigenomics investigates the epigenetic modifications on cellular genetical material. Several studies have assessed the impact of epigenetic modifications in the development and progress of NAFLD ( Figure 2 ) as well as in the association of NAFLD with other metabolic diseases by focusing on DNA methylation, histone modifications and miRNA expression profiles that can significantly affect transcriptional activity.

Significant alterations in DNA methylation are observed when moving from NAFL to NASH and liver fibrosis [56] [57] [58] . These changes include alterations J o u r n a l P r e -p r o o f in DNA methylation that affect the expression of genes involved in glucose, lipid or acetyl-coenzyme A (CoA) metabolism (PC, PLCG1, ACLY) [57] , insulin-like signaling (e.g. IGF-1, IGFBP2) and mitochondrial function (NADH dehydrogenase 6) [59] . Additionally, given that a specific DNA methylation signature develops with increasing age in humans, NASH seems to accelerate epigenetic age by promoting changes in methylation that are associated with hepatic collagen content [60] . An untargeted evaluation of DNA methylation in liver tissues of patients with NAFLD identified almost 70,000 CpG sites that were differentially methylated in patients with advanced liver fibrosis (F3-F4) compared to those with no or mild fibrosis (F0-F1). 76% of these sites were hypomethylated and 24% were hypermethylated in advanced liver fibrosis in NAFLD, whereas 7% of the reported methylations correlated with gene expression levels. The hypomethylation was associated with higher expression of genes related to tissue repair, liver inflammation, fibrosis and carcinogenesis (e.g. FGFR2, COL1A1, CASP1, CCR7, CCL5)

whereas the hypermethylation with lower expression of genes involved in lipid and aminoacid metabolism [56, 61] . These findings were also replicated in other studies [62] . DNA methylation seems to be particularly involved in the activation of hepatic stellate cells and their differentiation to myofibroblast that are crucial procedures for hepatic fibrogenesis [63, 64] . Changes in methylation of specific genes have been linked with these processes. Genes promoting fibrogenesis such as TGFβ1 and PDGFα are hypomethylated and thus highly expressed whereas genes inhibiting hepatic stellate cells activation such as the PPARα, PPARδ and PPARγ are hypermethylated and J o u r n a l P r e -p r o o f 18 thus lower expressed in the liver of patients with advanced fibrosis compared to the ones with mild disease [65, 66] .

Apart from their involvement in NAFLD development and progress, hepatic alterations in DNA methylation may be associated with systemic metabolic outcomes. Specifically, a case-control study in NAFLD patients has shown that increased methylation in specific sites of the promoter of the PPARGC1A, which encodes the major regulator of mitochondrial biogenesis, is associated with lower mRNA expression of PPARGC1A in the liver, lower ratio of mitochondrial to nuclear DNA and increased insulin resistance, thus linking hepatic mitochondrial dysfunction of NAFLD with peripheral insulin resistance [67] . In agreement with the above finding, a study following untargeted procedures has identified 30 methylations that are affecting mRNA expression of hepatic genes involved in insulin signaling that are also highly correlated with fasting insulin independently of the presence of T2DM, thus providing further evidence for the relation of hepatic DNA methylation with insulin sensitivity [58] . Finally, a recent study focused on the identification of differentially methylated regions that form networks which are associated with the progression of NAFLD. They identified two important networks, one that included genes that affect cytoskeleton organization, transcriptional activity and cell proliferation and another that was associated with metabolic pathways. The CpG methylation levels in both networks were affected by age and fasting plasma glucose levels and for the second network the changes in methylation levels were partially corrected by controlling weight and blood glucose levels [68] .

J o u r n a l P r e -p r o o f 19 Histone modifications are also important epigenetic changes that affect transcriptional activity and refer to several posttranslational procedures such as acetylation, phosphorylation, methylation and ubiquitination. Among them, acetylation status has been most vigorously studied and is considered the net result of histone acetylation by histone acetyltransferases (HATs) and histone deacetylation by histone deacetylases (HDACs) [69] . Acetylation neutralizes lysin's positive charge, thus leading to a looser chromatin structure that facilitates transcription. The transcriptional coactivator p300 belongs to HAT proteins, whereas sirtuins belong to NAD-dependent class III HDACs.

Glucose-induced activation of p300 increases the transcription of carbohydrate-responsive element-binding protein (ChREBP) resulting in the stimulation of lipogenic genes through histone acetylation and thus promoting the development of NAFLD [69] . Tannic acid attenuated the effects of p300 leading to a decrease in the lipogenesis-related genes and to an improvement of NAFLD in mice [70] . Similarly, inhibition of cdk4 protein reduces the formation of C/EBPα -p300 complexes reducing liver steatosis and correcting age-associated liver changes [71] . P300 may also be involved in the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts [72] . Among the Sirtuins, especially Sirtuin 1 (SIRT1) has been shown to participate in the regulation of hepatic metabolism and insulin sensitivity, with hepatic deletion of SIRT1 resulting in steatosis and inflammation, whereas overexpression of SIRT1 protecting from NAFLD [73] [74] [75] . Similarly, SIRT3 deficiency leads to insulin resistance, hyperlipidemia and steatohepatitis in mice [76] whereas a polymorphism of SIRT3 is associated with metabolic syndrome and NAFLD in humans [77] . HDAC3, a member of J o u r n a l P r e -p r o o f 20 human class I HDACs has been also implicated with circadian metabolic rhythm and its deletion leads to hepatic steatosis in mouse liver [78] .

Altogether, there is increasing evidence that both DNA methylation as well as histone modifications play a crucial role in the development and progress of NAFLD.

An important question is whether the numerous epigenetic changes observed in NAFLD in combination with the highly dynamic nature of epigenome can serve as diagnostic or prognostic markers of NAFLD or even as markers of treatment response. Very few studies have addressed this point so far. One of them that included a small number of patients with NAFLD has shown that alterations in hepatic DNA methylation may be sufficiently reflected in plasma.

In this study, % of plasma DNA methylation of PPARγ was used to identify subjects with severe (Kleiner 3-4) vs mild fibrosis (Kleiner 1-2). The threshold of methylation was 81% and demonstrated 83% sensitivity and 93% specificity to differentiate between the two above categories [79] . Although validation of the findings in largest and more heterogenous cohorts is needed, it is one of the first studies to suggest that changes in DNA methylation can have beyond their pathophysiological significance also value as potential noninvasive biomarkers for the diagnosis and staging of NAFLD. A second study has also investigated differences in methylation of DNA from peripheral blood leukocytes that may be able to discriminate between NASH vs NAFL. The study identified six genes that their methylation correlates with lobular inflammation and can be potentially used as diagnostic biomarkers (with the J o u r n a l P r e -p r o o f 21 best one showing 99% specificity and 71% sensitivity) for differentiating NASH from NAFLD [80] . Finally, in a large epigenome-wide association study consisting of 3,400 participants of European ancenstry, 22 CpGs were found in peripheral blood to be associated with hepatic fat as assessed by computed tomography or ultrasound imaging and among them one was also associated with risk for new-onset T2DM [81] . Nevertheless, significantly more and larger studies are needed in order to decide whether epigenetic changes in the blood can have any diagnostic or prognostic value.

Targeting epigenomic changes for the treatment of diseases seems to be theoretically a very attractive option for a number of reasons. First, epigenomic alterations are involved in fundamental pathophysiological mechanisms of a disease through their impact on gene expression. Second, epigenetic changes are also highly dynamic and reversible, thus correction of them holds promise for reversal to "normal status". For example, after bariatric surgery, robust changes in methylation have been reported. Hypermethylation and consequently reduction of transcriptional activity of PTPRE (encoding protein-tyrosine phosphatase epsilon) may improve hepatic insulin sensitivity [57] . Exercise may also reduce methylation and improve mRNA levels of mitochondrial genes, thus improving mitochondrial function [59] . Third, it may be possible to combine treatments targeting epigenome with other drugs, having in that case a complementary role. Such treatments are currently being developed mainly against cancer diseases and consist of pharmacological DNA methylation inhibitors (DNMTis), HDAC inhibitors (HDACis) or activators. In NAFLD very few of them have been tested so far.

J o u r n a l P r e -p r o o f 22 Among them, the SIRT1 activator, resveratrol, improved dyslipidemia and steatohepatitis in a mouse model of atherogenic NAFLD [82] . Several ongoing human clinical trials (two of them in Phase III) are currently investigating the efficacy of resveratrol in human NAFLD but results from the first studies have shown no significant effect on hepatic fat content [83] [84] [85] . Other medications are currently also being developed and have shown promising results in animal studies, such as the 3-deazaneplanocin A (DZNep) [86] or the HDAC inhibitor SAHA but none of them is in a more advanced stage of clinical development [87] .

Epigenomics are one of the least investigated "omics" in NAFLD. The few studies performed so far have shown that the extensive hepatic epigenetic modifications observed in NAFLD are causally related to the disease. It is still unclear though how accurately the hepatic epigenome is reflected in peripheral blood and thus it is questionable whether epigenetic changes studied at the level of peripheral blood cells may have any significant diagnostic value. Given also the limited amount of information from pathophysiological studies investigating the dynamic of epigenetic changes with NAFLD development and progression, it is probably premature to expect any drugs to be developed in order to target exclusively epigenome of NAFLD in the near future. 

Genetic predisposition determines NAFLD transcriptomic signatures, as shown by recent investigations on the carriage of PNPLA3 I148M on transcriptome variability [88] . As described above, transcriptomics are also linked to epigenetic modifications in NAFLD [56, 57] .

Through global microarray transcriptional snapshots, NAFLD and especially NASH are characterized by overexpression of genes associated with lipid metabolism [89] [90] [91] [92] , acute phase regulators of insulin sensitivity [93] , cellular division [89] , DNA [89] and tissue repair [56] , extracellular matrix organization [91, [94] [95] [96] , immune function [95] cellular adhesion and migration [91, 92, 96] , signal transduction [91] , P53 signaling [92] and cancer progression [96] among others ( Figure 2 ). Conversely, genes modulating mitochondrial function [93] oxidative, glucose, fatty acid and aminoacid or protein metabolism [56, 91-93, 96, 97] as well as insulin signaling and transcription factors [91, 94] are downregulated. and underexpression of PZP mRNAs [92] . Hepatic transcriptomes of NAFLD are distinct from those of normal and healthy obese subjects, yet a 132-gene signature, with extracellular matrix remodeling and immune system genes as its main components, distinguishes NASH from simple steatosis [95] . More specifically, the transcriptional snapshot of NASH is defined by upregulation of PDGF, STAT, HNF-3 and SMAD-4 pathway-related genes [97] , as well as downregulation of BNIP1, IGFBP1 [97] , SLC25A48, C4ORF48 [95] SDC4, ATF3, various inflammatory suppressors [94] and amino acid-metabolizing and reactive oxygen species (ROS) scavenger genes [97] . Interleukin-32 (IL-32) was recently ascertained as the most significantly upregulated transcript in advanced NAFLD and NASH (fold-change = 2.3 vs non-NASH), correlating with lipid accumulation and disease severity [88] . Additionally, several cancerrelated genes, such as AKR1B10, KRT23 and GPC3, are enhanced in NASH compared with NAFL or controls, by up to 155-fold and 460-fold respectively [96] .

Concerning fibrosis, cross-sectional transcriptome analysis of hepatic cells defines 3820 and 2980 differentially expressed transcripts for the inflammatory and fibrotic states, respectively, and further underlines 16 fibrosis-defining genetic routes, pertaining to cytokine and extracellular matrix receptor interaction, focal adhesion and the PI3K-Akt signaling pathway [98] .

Differentially expressed microarray signals, such as UBE2V1, BNIP3L, RP11-128N14.5, linked to oxidative stress, inflammation, apoptosis and fibrogenesis, are upregulated in NAS ≥ 5 and fibrosis stages 3-4 [99] .

J o u r n a l P r e -p r o o f Likewise, transcriptomics pinpoint dermatopontin, a propagator of extracellular matrix remodeling, which is robustly expressed (by 800-fold) in murine stellate cells, enhanced in human fibrotic livers and implicated in NAFLD fibrogenesis [100] .

Minor molecules such as micro-RNAs (miRNA) along with long noncoding RNAs (lncRNA) perform a variety of epigenetic and post-transcriptional functions, thus significantly affecting transcriptional activity in numerous diseases and among them also NAFLD [101] . For this reason, they are also considered important epigenetic modifiers. These processes are thoroughly delineated through large panels of differential miRNA expression [102] [103] [104] while the distinct NAFLD-related roles of miRNA subspecies were recently reviewed [101, 105] . Of note, microRNA-122 (miR-122), deriving from chromosome 18, represents 70% of hepatic miRNA and has been implicated in cholesterol metabolism and liver cancer [106] . Cheung et al. first mapped a 63% underexpression of liver miR-122 in NASH patients compared with controls, through an evaluation of a 474-miRNA panel that identified 46 differentially expressed miRNA species and their corresponding targets [104] .

MiR-122 was subsequently associated with steatosis severity [107] . The (a,b), miR-125b (associated with metabolic syndrome, atherogenesis and circulation) [103] as well as miR-34a, miR-16 (associated with cancer) [108] , miR-21 and miR-451 [107] are upregulated in both NAFLD and NASH.

Moreover, miR-301a, miR-34a and miR-375 alter lipid and carbohydrate J o u r n a l P r e -p r o o f 26 metabolism, reflect NAFLD severity and are implicated in hepatocellular carcinomas [102] .

Expression levels of lesser non-coding molecules have also been investigated [99, 109, 110] . Global hepatic RNA profiling has identified 535 lncRNA and 760 mRNA species overexpressed in NAFLD, whereas 1200 lncRNA and 725 mRNA species are underexpressed. These enriched RNAs chiefly belong to small molecule and organic acid metabolic pathways [109] . Functional transcriptome research of 4383 lncRNA species by Atanasovka et al.

pinpoints elevated levels of hepatic lncRNA RP11-484N16.1 in NASH, and robustly associates it with NASH grade, lobular inflammation and NAS, while its knockdown in vitro aggravates hepatocellular growth and viability [110] .

Identified and validated through whole serum transcriptome analysis, lncRNA RP11-128N14.5 has been linked to NASH diagnosis and is upregulated in NAS ≥ 5 [99] . Furthermore, transport RNA (tRNA) profiling pinpoints the differential expression levels of several anticodons for lysine, aspartate and glutamate in cirrhosis, in addition to various mitochondrial and amino acid tRNAs in cirrhosis versus NASH and NASH cirrhosis versus normal respectively [102] . The same study has also compared the abundance of 392 several small RNA molecules (ribosomal, small nuclear and nucleolar) between healthy, NASH and cirrhotic patients [102] .

Various predictive modalities involving gene expression parameters, targeted measurements and miRNA panels with increased functionality are based on transcriptomics. In general, the reported accuracies for many of these tests J o u r n a l P r e -p r o o f 27 have been high for diagnosing NAFLD or NAFL from healthy status but rather low for differentiating NAFL from NASH or for diagnosing advanced fibrosis.

Additionally, even the most promising studies, have not been further tested or have performed poorly in other cohorts.

Among them, predictive models based on miRNAs have in theory higher chances to be robust and reproducible, since miRNAs can withstand numerous freeze-thaw cycles and long-term storage before degrading [111] .

Several studies have identified miR-122 as a potential diagnostic biomarker.

Most of them have shown that miR-122 alone or in combination with other miRNAs (e.g. miR-1290, miR-27, miR-192, miR-34, miR-99a) can accurately predict the presence of NAFLD or NAFL, but they all perform inadequately when trying to differentiate NAFL from NASH [103, 108, [112] [113] [114] (Table 2) .

with other proteins in their algorithm. Among them, NIS4 TM , which includes miR-34a, alpha-2-macroglobulin, chitinase-3-like protein 1, has been recently tested in a population with type 2 diabetes (n=275), showing an AUROC performance of 0.801 for diagnosing advanced NASH (NAS≥4) and significant fibrosis (F≥2), which was also higher than in other tests, such as FIB4 (0.704), ELF (0.704) or Fibrometer (0.678) but probably still suboptimal [115] .

Additionally, the same test demonstrated 76% sensitivity and specificity in detecting NASH patients at risk of cirrhosis [116] . Another model integrating miR-122, miR-192, miR-21 and CK-18 fragments, can differentiate NASH from NAFL (AUROC = 0.83) [117] (Table 2) .

New frontiers, such as circular RNA (circRNA) transcriptomics, are being investigated in animals. Researchers have already mapped and correlated J o u r n a l P r e -p r o o f 28 circRNA species and their corresponding transcriptional pathways in mouse models of NASH, showcasing their associations with metabolism [118] and stellate cell activation [119] . Yet whether these results could yield meaningful conclusions that can be extrapolated in human cohorts remains unclear.

Other prospective research questions in diagnostic NAFLD transcriptomics concern alternative RNA splicing [120] and the scrutiny of unexplored transcriptional components, such as CD24 [92] .

Curative and bedside implications of transcriptomics in NAFLD are considerably limited. From an interspecies aspect, the human and murine NAFLD transcriptomes are largely dissimilar, yet their likelihood increases in murine models following high-fat diets [90] . Hence the transcriptional signatures of NAFLD or NASH as well as the organic transcriptomic responses to relevant treatments for liver disease can be evaluated by numerous available pre-clinical in vitro prototypes and animal models [121] .

For instance, mapping of the murine NASH transcriptome demonstrated an increase in the levels of Tsukushi, a novel adipose-related thermogenetic and metabolic hepatokine. Subsequent silencing of Tsukushi improved NASH and attenuated the hepatic transcriptional alterations associated with it [122] .

Transcriptomics were also implemented in recent murine models assessing the NAFLD-related role of Poly rC binding protein 1, a cytosolic iron chaperone, whose deficiency exacerbates steatosis and inflammation, increases lipid biosynthesis and oxidative stress pathway expression levels, and is treated via reduced iron intake and vitamin E supplementation [123] .

Lastly, in view of the COVID-19 pandemic, the expression levels of 4 SARS-J o u r n a l P r e -p r o o f 29 COV-2 entry proteins were assessed by liver transcriptomics in human and murine NAFLD and NASH cohorts, with no significant changes being detected [124] . Future transcriptomic modalities should approach all different phases and components of transcription, kindle research for new remedies, and investigate the associations of liver disease with various pathologies precipitated by its metabolic milieu.

Transcriptomics have been widely used and have contributed significantly to our understanding of the pathophysiology of NAFLD. Transcriptomics, with the exception of miRNAs, do not add significant value in non-invasive diagnostic scores, since their hepatic profile is not adequately reflected in peripheral blood. With the rapid improvement of available technologies and data analysis, transcriptomics are now routinely used in order to identify potential mechanistic pathways affected by drug candidates for the treatment of NAFLD.

Proteomics refers to the investigation of "proteome", thus to all proteins expressed by a cell. The proteome, similar to transcriptome and epigenome, is highly dynamic. Cell activation, trafficking, differentiation or transformation as well as tissue type have a major impact on the proteome. Additionally, posttranslational modifications (phosphorylation, acetylation, methylation, glycosylation etc.) as well as alternative splicing render proteome much more J o u r n a l P r e -p r o o f 30 complex than transcriptome, with more than 120,000 proteins being expressed by a human cell. Finally, the proteome is downstream of genome and transcriptome, and thus it is stronger associated with the final phenotype [125] .

Proteomic analyses have been performed for cell line -characterization or for protein investigation and identification in tissues under specific conditions or treatments, but their use has been in general rather limited, especially for biomarker or drug discovery [126] . There are several reasons for this. First, significant technical limitations exist that restrict the number of proteins that can be detected and can be identified with certainty. The first efforts involved two-dimensional gel electrophoresis and were able to detect some few dozens of spots that were subsequently matched to specific proteins with the use of MS. Later approaches managed to detect more protein peaks without though being able to identify the corresponding proteins. Advances in methods have enabled in the last years the identification of some thousands of proteins but still the largest part of proteome cannot be investigated with certainty with the available technologies. Second, reliable quantification of the levels of proteins, especially in blood has been very challenging. In plasma, three classes of proteins mainly exist: a) the highly abundant proteins, that include human serum albumin (approximately 50% of protein mass), apolipoproteins, coagulation markers and acute phase proteins of the immune system, that are measured in mg to ug/ml level, b) the organ-tissue secreted proteins without functional role in the circulation such as ALT, AST or troponins, measured in ug to ng/ml, c) the signaling molecules, such as cytokines or insulin, measured at ng to pg/ml level. This extreme range While keeping in mind the above limitations, we next present the efforts to identify pathophysiological pathways and develop diagnostic models for NAFLD with the use of proteomics.

Several studies have investigated hepatic proteome alone or in combination with blood proteome, either in animal models or in humans with NAFLD, aiming to answer fundamental pathophysiological questions [127] [128] [129] . In one recent study, the levels and cellular distributions of 6,000 liver proteins and 16,000 phosphopeptides have been assessed in the liver of mice developing hepatic steatosis due to high fat diet (HFD). This provided important J o u r n a l P r e -p r o o f 32 fundamental information about the reorganization of organelles, lipid accumulation and cellular dysfunction that occurs with nutrient overload [129] .

In another study, proteomics in liver biopsies identified almost 220 proteins that their levels significantly differ in patients with NAFLD compared to obese metabolically healthy individuals. The proteins that their levels were increased in NAFLD were involved in PPAR-signaling and extracellular matrix-receptor interactions whereas the ones that their levels were reduced, were mainly localized in mitochondria and participated in oxidative phosphorylation [128] .

In a combined hepatic phosphoproteome and serum proteome analysis of 67 biopsy-proven NAFLD subjects, the ASK1-MAPK pathway that is activated by IL-10 was recognized as important for liver fibrosis, indicated by its strong association with higher % of hepatic collagen. In serum, alpha-2 macroglobulin and coagulation factor V were strongly associated with hepatic collagen [127] . It has thus been suggested that these pathways can potentially serve as therapeutic targets. In another approach, proteomic differences in liver tissues of obese patients with vs without T2DM were investigated. The analysis identified 850 proteins, from which 27 were significantly different between the two groups. Levels of proteins involved in methionine metabolism and especially of glutathione were reduced whereas levels of other proteins involved in oxidative stress were increased in the T2DM group [130] . Expanding on complications of the disease, the proteome of liver tissues with NAFLD from mice developing HCC has been assessed.

Differences in tumor suppressor genes and oncogenes were observed, which were further investigated in human samples with NAFLD and HCC. Among them S100A11, which is secreted by cancer cells and stimulates cell J o u r n a l P r e -p r o o f 33 proliferation and migration, was identified and associated with high-grade HCC and poor prognosis [128] .

A small number of studies has aimed to develop diagnostic models with the use of proteomics (Table 3) . When evaluating the results of these studies, we have to keep in mind the limitations and challenges related to such procedures, that are described below (8. Conclusions -Challenges -Perspectives). In one of the early efforts, combined genomic/proteomic analysis involving liver tissues for assessing gene expression and serum samples for assessing proteins was performed in patients with different stages of NAFLD. A SELDI-TOF mass spectrometry (MS) identified approximately 300 protein peaks with 16 of them being significantly different between groups. At that timepoint, it was not possible to identify in which proteins these peaks corresponded to but their masses were compared with the masses of 1,440 serum proteins, which led to the identification of fibrinogen γ as one possible biomarker [131] . In another study including eighty morbidly obese patients, SELDI-TOF MS has identified in serum three protein peaks that their intensity significantly increased with the severity of NAFLD, i.e. when moving from steatosis to NASH. None of these peaks correlated with liver function tests or metabolic parameters and all of them returned to normal after bariatric surgery. With immunoSELDI assay these peaks were recognized as alpha-and beta-hemoglobin subunits [132] . One of the most comprehensive studies so far was able to identify 1,700 serum proteins in a population with NAFLD. Among them 55 were significantly different between NALF and NASH with fibrosis stage 3 or 4 (F3/F4) and 15 between NASH vs J o u r n a l P r e -p r o o f 34 NASH F3/F4 group. These proteins were involved in immune system regulation and inflammation, in coagulation cascades (including fibrinogen γ demonstrated by previous studies), in cell growth and proliferation (including IGFBP2, IGFBP3, also shown in targeted approaches to be relevant [133] ) and many of them were apolipoproteins (demonstrated also in [134] ) and blood carrier proteins. Six (fibrinogen β chain, retinol binding protein 4, serum amyloid P component, lumican, transgelin 2, and CD5 antigen-like) were selected to create a panel for the non-differential diagnosis of NAFLD and its stages, ranging from healthy liver status to NAFL, NASH or NASH with F3/F4

showing good to very good diagnostic accuracy (AUROCs 1.0, 0.83, 0.86, 0.91, respectively). This was though a study with small sample size and its findings have not been replicated so far [135] . In another approach, diagnostic models for differentiating between NAFLD vs healthy controls among the obese population undergoing bariatric surgery were developed. These models combined genomic information and especially the PNPLA3 genotype, phenomic data (i.e. selected biochemical measurements such as glucose, insulin, lipid profile, ALT) and proteomic data. Specifically, a proteomic analysis identified 1129 proteins with 30 of them being significantly different between the two groups. Among them was aminoacylase-1, sex hormonebinding globulin, Galectin-3, antithrombin III and the hepatocyte growth factor.

Logistic regression models for differentiating between NAFLD and healthy status were trained by using each type of data individually (i.e. genomic, However, the utility of such algorithms is questionable given that ultrasound or other simpler algorithms (e.g. fatty liver index, etc.) show similar or higher sensitivity and specificity. Additionally, diagnosing simply NAFLD does not initiate treatment decisions [136] .

Other studies performed first a proteomic analysis in a relevant condition or in another species and then tested their findings in patients with NAFLD. For example, in one study, an HCC pig model was used and a proteomic analysis in serum was performed. The subsequent analysis identified the serum interalpha-trypsin inhibitor heavy chain 4 as the most important protein corresponding with NAFLD progression and HCC development. This was further tested in human serum samples and showed that the levels of the proteins were higher in NAFLD patients with HCC compared to those without [137] . Similarly, in another study a proteomic analysis in plasma of patients with liver cirrhosis vs healthy ones was performed. This identified 57 significantly different proteins which were involved in immune system regulation, inflammation, coagulation and fibrinolysis. Several of these proteins were subsequently tested in NAFLD patients and were significantly different in people with NAFLD and normal glucose tolerance compared to NAFLD with T2DM [138] .

Proteomic studies have rather limited contribution to drug development against NAFLD so far. Most of the treatment targets have been identified by animal studies supported by human physiology or epidemiological studies. 

Proteomics have not been widely used in NAFLD research. The proteins and signaling pathways involved in the pathophysiology of the disease have been almost exclusively identified by targeted "hypothesis-driven" approaches based on data from mechanistic animal, observational human and in some cases large epidemiological studies. This is probably due to methodological challenges and technical limitations related to proteomics. We expect in the near future proteomics to be much more frequently applied in NAFLD studies.

We expect that the main contribution of proteomics will not be in direct development of non-invasive diagnostic scores but rather in the identification of novel molecules and/or pathways with important role in the pathophysiology of the disease and with diagnostic and therapeutic potentials.

Metabolomics refers to the investigation of small molecules and metabolic products, such as aminoacids, fatty acids and carbohydrates. Lipidomics are J o u r n a l P r e -p r o o f 38 considered part of metabolomics and refer to the investigation of cellular lipids. Numerous studies have investigated the metabolome and lipidome in mouse models or patients with NASH. The evidence linking bile acid homeostasis as well as gut microbiome with the development and progress of NAFLD have been recently reviewed elsewhere [156, 157] . In this section, we will focus particularly on the robust and extensive changes that are observed both in the hepatic (Figure 3 ) as well as in the circulating lipidome, which have led to the development of numerous diagnostic models for NAFLD as well as to the identification of novel therapeutic targets.

The development of NAFLD is strongly associated with obesity and is characterized by increased lipid accumulation due to higher uptake of elevated FFA by unrestricted adipose tissue lipolysis and due to increased [164] [165] [166] . In non-parenchymal liver cells, SFA stimulate the production and secretion of proinflammatory and profibrotic cytokines from Kupffer cells and induce proinflammatory M1 polarization of macrophages [166, 167] . Additionally, they stimulate the secretion of chemokines from hepatic stellate cells that recruit more macrophages in the liver [166, 167] .

Similar to SFA, lysophosphatidylcholines (LPC) have unfavorable hepatic effects. Specifically, they stimulate ER stress, cause mitochondrial dysfunction and increase apoptosis [168] . Additionally, they stimulate the release of hepatic extracellular vesicles from hepatocytes that trigger inflammatory procedures [169] . Moreover, the increased activity of the enzyme phospholipase A2 (PLA2) that catalyzes the formation of LPC from PC, leads to the rapid depletion of PC which affects hepatocyte membrane integrity and results in hepatocyte apoptosis, high release of lipotoxic lipids and increased inflammation [170] . Additionally, PC deficiency reduces VLDL secretion resulting in higher intrahepatic lipid degradation and formation of toxic intermediates. Furthermore, the low PC/PE ratio affects lipid droplet stability, leading to the formation of larger lipid droplets. Finally, LPC is metabolized by the enzyme autotaxin to phospholipid lysophosphatidic acid which stimulates liver fibrosis and development of HCC [171] . Ceramides are a type of sphingolipids whose hepatic levels are increased in NAFLD and correlate positively with disease severity [46, [159] [160] [161] [162] [163] . In hepatocytes, ceramide promotes insulin resistance by inhibiting Akt-mediated insulin signaling, impairs beta-oxidation, induces ROS production, ER stress and proinflammatory cytokine secretion, enhances de novo lipogenesis, cholesterol synthesis and triglyceride accumulation and induces apoptosis [164, [172] [173] [174] [175] . Finally, ceramide stimulates fibrogenesis and angiogenesis by increasing extracellular matrix deposition and secretion of pro-angiogenic factors by hepatic stellate cells [176] . [159, 162, 163] . The increased ω-6 concentrations lead to the synthesis of eicosanoids by their enzymatic oxidation (especially of AA but also of EPA and dihomo-γ-linolenic acid) with proinflammatory properties such as prostanglandines, thromboxanes and leukotrienes which induce hepatic inflammation. This occurs in expense of the synthesis of SPMs that mainly act to restore normal cell function and thus reduce chronic inflammation and fibrosis [178, 179] .

Finally, the high ω-6 to ω-3 ratio is associated with an impaired FADS1 activity that can affect cell membrane phospholipid composition resulting in membrane deficiency, cell necrosis and extracellular deposition of lipotoxic lipids that can aggravate hepatic injury [162, 163] .

Many studies have reported several diagnostic models based on metabolomics, lipidomics alone or combined with other biochemical and clinical parameters for the diagnosis and staging of NAFLD (Table 4 ). The best studies use populations of biopsy-proven NAFLD to create non-invasive algorithms based on blood measurements. The models aim to diagnose advanced fibrosis (≥3) (in most studies), differentiate NAFLD from healthy status, distinguish between NASH and NAFL or NASH vs non-NASH (in many studies), or detect the presence of fibrosis independently of its stage (in few studies). Challenges related to the development of these models are [180] . Based on the above findings and methodology, the OWLiver® test was created, which includes 25 triglycerides and aims to discriminate NASH from NAFL patients. The test, which was initially trained in white patients without T2DM, performed poorly in a multiethnic cohort of patients with T2DM (AUROC=0.69). This shows that patient heterogeneity is an important factor and that tests developed in non-diabetic populations may not be as accurate in patients with T2DM [181] . In another study, information from metabolomics and lipidomics were combined with biochemical and genotypic measurements to create an algorithm for differentiating NASH from non-NASH (i.e. combined healthy and NAFL). 223 subjects were used to train the algorithm and 95 to validate it. The final model consisted of AST, insulin, PNPLA3 genotype, glutamate, two aminoacids (glycine and isoleucine) and two lipids (LysoPC 16:0 and PE 40:6) and was able to differentiate NASH from non-NASH with 85% sensitivity and 72% specificity [46] .However, the non-NASH population consisted both of healthy subjects (53%) and of NAFL (47%). It is though important to be able to distinguish accurately NAFL from healthy status. Additionally, sensitivity and specificity in most tests is high when healthy subjects are included, but the same tests may not perform as well when NAFL is directly compared with NASH.

In a smaller proof-of-concept study a different approach was followed. A lipidomics, glycomics and targeted hormonal analysis was performed. Results were analyzed with different machine learning methods and 19 different diagnostic models were reported for classifying the subjects simultaneously to healthy or NAFL or NASH group. The models consisted of varying number of J o u r n a l P r e -p r o o f 44 variables (from 10 up to 29) and demonstrated high sensitivities and specificities [144] . The variables included in the models were mainly lipids and often adiponectin, which its protective role against steatosis, inflammation and liver fibrosis is well established [152, 153, 182] . A validation cohort did not exist, but 2/3 of the study population was used to develop the models and 1/3 to validate it, with this procedure being repeated 100 times with several slices of data. This approach helps to control against random results but it still does not address population heterogeneity. Nevertheless, the ability to diagnose between three and not two conditions (healthy, NAFL, NASH) can be attractive for large screenings of high-risk population in primary care setting and without the need for any additional imaging [144] and should be further evaluated in future studies.

Several studies have attempted to develop non-invasive algorithms for differentiating between advanced (stage 3-4) vs not advanced liver fibrosis (stage 0-2). In one of the most comprehensive studies so far, a cohort of 156 subjects was initially used to develop an algorithm after a metabolomic and lipidomic analysis, which finally consisted of 10 metabolites/lipids. Among the 10 parameters, six were cholesterol-derived precursors of steroid hormones which were significantly reduced in advanced fibrosis, one was the primary conjugated bile acid, glycopholate, that was increased in advanced fibrosis and one the aminoacid taurine that is associated with bile acid conjugation and its levels were decreased in fibrosis. Finally, the other two parameters were palmitoleate acid and fucose, which were both higher in advanced fibrosis. Increased fucosylation, as described below (s. glycomics) is one of the main findings of studies that investigated alterations in circulating glycome sensitivity and 79% specificity for detecting advanced fibrosis, which was higher compared to FIB-4 index (39% specificity) and NAFLD fibrosis score (59% specificity) [183] .

Finally, very few studies have tried to develop such models in pediatric population. The most comprehensive one so far included 222 children and adolescent with biopsy-or MRI-proven NAFLD and 337 without the disease.

Metabolomics identified 11 metabolites of interest which together with waist circumference, whole-body insulin sensitivity index and triglycerides could detect the presence of NAFLD with 97% specificity and 73% sensitivity [184] .

NAFLD is considered an unmet clinical need. Several treatments that are currently in the pipeline restore many of the abnormalities observed in the lipidome. Specifically, agonists of farnesoid X receptor (FXR) reduce lipotoxicity by promoting mitochondrial beta-oxidation, decreasing de novo lipogenesis and stimulating cholesterol excretion [142] . In Phase 2 clinical trials and in an interim analysis of an ongoing phase 3 clinical trial, obeticholic acid reduced hepatic steatosis, inflammation and fibrosis compared to placebo [142, [185] [186] [187] .

Dual PPARα and PPARδ agonists (e.g. elafibranor) also stimulate mitochondrial and peroxisome β-oxidation as well as omega-oxidation [188- J o u r n a l P r e -p r o o f 46 191 ]. PPARγ agonists reduce circulating FFA and consequently the flow of SFA to the liver, they improve insulin sensitivity and guide macrophage and Kupffer cell polarization from the proinflammatory M1 phenotype towards to the anti-inflammatory M2 one [142, 167] . Various selective PPARγ modulators, dual PPARα/γ agonists (e.g. Saroglitazar) or Pan-PPAR agonists (e.g. lanifibranor) have shown promising results in human studies and are currently under evaluation in phase 2 or phase 3 clinical trials [142, [192] [193] [194] .

In this context, it was recently announced, that lanifibranor met the primary endpoint of a reduction in steatosis activity fibrosis score, including NASH resolution without worsening of fibrosis in a Phase II clinical trial, thus being the first study that met both FDA and EMA regulatory endpoints for accelerated approval [195] ACC inhibitors (e.g. firsocostat) attenuate the function of acetyl-coA carboxylase (ACC) that catalyzes the conversion of acetyl-CoA to malonyl-CoA. Consequently, malonyl-CoA is reduced and hepatic de novo lipogenesis as well as mitochondrial FA beta oxidation are downregulated [142, 196] . In humans, ACC inhibitors reduce hepatic steatosis and on-going clinical trials will evaluate their effect also on inflammation and liver fibrosis [142, 196] .

Regarding LPC accumulation, animal studies have shown that interruption of the generation of LPC from PC with inhibition of PLA2 results in normalization of PC levels, prevention of NAFLD and reversal of NASH and fibrosis [158, 197] .

Several treatments are targeting also ceramide levels by aiming to reduce ceramide synthesis (de novo or through hydrolysis from sphingomyelin) or J o u r n a l P r e -p r o o f 47 increase ceramide degradation. The reduction of ceramide synthesis can be achieved by inhibiting relevant enzymes (i.e. palmitoyltransferase) but it should be limited only to hepatic ceramides, since global reduction of ceramides can negatively affect nervous system [198] . Alternatively, lower ceramide synthesis can be achieved by blocking intestinal FXR activation [199] . However, in that case, FXR blocking should be limited to the intestine and not affect FXR activation in the liver which acts beneficially. In other approaches, reduction of the accumulation of specific ceramides (i.e. C16:0 ceramide) is targeted with the use of several inhibitors (e.g. DES-1 70) or degradation of ceramides is stimulated by enzymes (e.g. acid ceraminidase).

These have till now been tested only in animal studies and not in humans.

Many treatments have targeted to restore the increased ω-6/ω-3 ratio by ω-3 supplementation. Although the findings from animal studies had been very promising, and the first results from human studies had shown an improvement in liver steatosis, later studies could not demonstrate any benefit in steatosis, necro-inflammation or fibrosis in patients with NASH [142, [200] [201] [202] . Newer experimental strategies are focusing not on the supplementation of ω3 but on restoration of the ω-6/ω-3 ratio with the use of ω-3 desaturase that leads to the production of ω-3 from ω-6 PUFAs, showing beneficial effects in steatosis and necro-inflammation in animal studies [203] .

Finally, different experimental approaches are aiming to decrease proinflammatory eicosanoids and increase the concentrations of SPMs.

Specifically, some of them are targeting the inhibition of PLA2α enzyme that catalyzes the cascade leading to AA and lysophospholipids generation, showing promising results in animal studies [197, 204] . Others are targeting J o u r n a l P r e -p r o o f 48 the production of proinflammatory lipids by inhibiting the enzyme 5-LOX [205, 206] which catalyzes the lipooxygenation of AA to leukotriene lipids or they aim to block the binding of leukotrienes to their receptors [207] . The 5-LOX inhibitor tipelukast has been approved by FDA for a phase IIa clinical trial.

Finally, other approaches are aiming to increase the SPMs RvD1, RvE1, protectin DX and maresin-1 leading to robust improvement of fibrosis in animal models of NAFLD [164, 208, 209] . Given that SPMs are rapidly inactivated by oxidoreductases and that SPM receptors are downregulated in obesity, recent efforts are also focusing on improving the pharmacologic properties of them by creating either resistant to deactivation analogues [210] or by enhancing their delivery on their site of function.

Altogether, most drugs in on-going clinical trials are targeting, among other mechanisms, hepatic lipidome in order to improve NAFLD. Additionally, there is a significant number of medications that have shown beneficial effects on animal models of NAFLD by improving hepatic metabolome/lipidome and remain to be further clinically tested.

Metabolomics/Lipidomics are one of the most investigated omics in NAFLD. 

Glycomics refers to the comprehensive investigation of glycome, i.e. of glycan structures that circulate either as free glycans or they are bound to proteins (glycoproteins), lipids (glycolipids) or phospholipids (glycophospholipids).

Glycome is highly dynamic as it is affected by transcriptome, proteome, environmental factors (nutrition) and cellular secretory machinery [125, 211] .

Glycans can affect protein morphology and interaction with other proteins as well as regulate nutrient storage and sequestration [212] . Additionally, they can protect cell stability and facilitate cell to cell interactions [212] .

Glycosylation is the process of the formation of the glycoconjugates (glycoproteins or glycolipids) [213] which can happen both intra-and extracellularly. In the extracellular glycosylation, enzymes secreted mainly J o u r n a l P r e -p r o o f 50 from liver hepatocytes and platelets (i.e. glycosyltransferases) are involved [213] . Changes in glycan profile have been observed in numerous inflammatory diseases and in different types of cancer and have been often linked causally with the pathogenesis and progression of these diseases [213] [214] [215] .

In NAFLD, few glycomic studies have been performed so far (Table 5 ). These have either aimed to assess the circulating glycome in untargeted approaches or they have focused on the identification of glycans related to specific proteins (i.e. haptoglobin, transferrin, IgG2, IgA1, alpha-1 antitrypsin, ceruloplasmin). The findings of these studies suggest that in NAFLD and during its progression from NAFL to NASH and liver fibrosis higher concentrations in fucosylated, sialylated and agalactosylated glycans are observed. Sialic acids in glycolipids or glycoproteins have diverse functions, including formation of a protective cell surface barrier, involvement in interactions of white blood cells with endothelial lining of blood vessels, recognition by pathogens and toxins and facilitation of cell migration by some cancers [216] . Circulating sialic acid levels have been positively associated with metabolic syndrome and with NAFLD [217, 218] . Fucosylated glycans are also involved in a variety of physiological and pathological procedures, including cell adhesion and migration, angiogenesis, malignancy, tumor metastasis as well as immune cell development and regulation [219] .

Consequently changes in fucosylation have been reported in numerous inflammatory conditions, such as in rheumatoid arthritis, chronic pancreatitis, sickle cell disease and Crohn disease [219] . In the liver, fucosylation serves as a signal for the secretion of fucosylated glycoproteins from normal J o u r n a l P r e -p r o o f hepatocytes into bile [219] . In ballooning hepatocytes, which are observed in NASH, the fucosylation-related sorting machinery is dysfunctional which may result in the secretion of the fucosylated glycoproteins in the sera instead of bile [219] . Similarly, hypogalactosylation (lack of galactose in the formed glycoprotein or glycolipid) especially of IgG has been associated with inflammatory response and with a number of autoimmune diseases [213] .

Most of the glycomic studies in NAFLD have tried to identify glycans or glycoproteins that can serve as blood biomarkers for differentiating between NAFL and NASH or for detection of the presence of liver fibrosis and its stage ( Table 5 ). The diagnostic accuracy of most of these tests has been limited, with some reporting higher accuracy for diagnosing NASH and others for diagnosing advanced fibrosis. This suggests that although changes in circulating glycome/glycoproteins are observed in NAFLD, these are not sufficient in order to be used alone for the development of diagnostic models of the disease and thus combination with other clinical or biochemical parameters are needed. In this context, fucosylated-haptoglobin showed below 70% accuracy at differentiating NAFL from NASH, but 76-81% when combined with Mac2bp [220] . Similarly, in our study, which detected with the use of mass-spectrometry the highest number of glycans so far, the concentrations of glycans in serum were able to differentiate between the presence of liver fibrosis or not with 76% sensitivity and 74% specificity [144] .

Glycans alone could poorly differentiate between NAFL and NASH but when combined with lipid species in models of 20 variables (18 lipid species and 2 J o u r n a l P r e -p r o o f 52 glycans) the sensitivity and specificity for discriminating between NASH, NAFL or healthy liver status increases significantly.

Regarding potential treatments, no study thus far has investigated whether targeting glycome can be a therapeutic option in NAFLD. Treatments aiming to decrease fucosylation are currently under evaluation in autoimmune diseases and cancer. In this context, inhibition of fucosylation with a 2-deoxyd-galactose, a fucosylation inhibitor, reduces inflammation in rheumatoid arthritis by decreasing inflammatory macrophages and Th17 cells in the lymph nodes as well as by reducing the levels of TNFα, interleukin-6 (IL-6), and antibodies to type II collagen in the serum [221] . Similarly, inhibition of fucosylation by 2-fluorofucose suppresses the proliferation, migration and tumor formation of HepG2 liver cells [222] . Whether similar interventions can be beneficial in NAFLD by reducing inflammation, fibrosis or HCC formation should be addressed in future studies.

Glycomics are one of the least investigated "omics" in NAFLD. Given the strong relation between glycoprotein-glycolipid formation and liver function, more studies assessing the potentials of glycans in biomarker discovery and drug development are needed. performed. Thus, these models often do not account for population heterogeneity, which is crucial for the accuracy all non-invasive diagnostic panels. This has been repeatedly shown in studies that focused on patients with overweight, obesity and especially with T2DM. Several and diverse biomarker panels (including SteatoTest, ActiTest, NashTest-2, cytokeratin-18, FibroTest, OWLiver tests) were evaluated but none of them demonstrated optimal performance for diagnosing NASH or liver fibrosis especially in patients with T2DM [5, 13, 181, 223, 224] . Third, very few tests have been evaluated prospectively. This is a very important step, which even other simpler algorithms or imaging modalities have failed to pass [225] .

Additionally, depending on their mechanism of function, medications may have different levels of impact on the metabolic and lipid profile in the liver which may affect circulating metabolome/lipidome and its ability to reflect the stage of the disease through a diagnostic algorithm originally designed in untreated patients.

Altogether, diagnostic models based on omics have yet to reach exceptional levels of performance, while satisfactory study outcomes need to be cautiously scrutinized and repeatedly validated into large and diverse patient cohorts both cross-sectionally as well as prospectively. To this direction, FDA has recently approved the parallel evaluation of diagnostic biomarker panels [239] Metab J o u r n a l P r e -p r o o f (rs738409) impairs PUFA transfer from DAGs to PCs, thus increasing PUFA in TG and DAG. TM6SF2 E167K (rs58542926) impairs PUFA synthesis, increases polyunsaturated FFAs and prevents PUFA incorporation into TGs and PCs. Both mechanisms lead to impaired VLDL synthesis and lipid droplet hydrolysis. GCKR P446L (rs780094) incites glycolysis, glycogen deposition and de novo lipogenesis by disinhibiting glucokinase. Epigenetic modifications characteristic of NAFLD progression include CpG site hypermethylation, thus reduced expression, of genes pertaining to lipid and aminoacid metabolism and stellate cell inhibition. Hypomethylation, thus increased expression, of genes pertaining to tissue repair, inflammation, carcinogenesis and fibrogenesis, increases insulin resistance and further propagates the disease. Methylation levels of cytoskeletal, transcriptional, proliferation-related and metabolic genes are affected by age, fasting glucose levels and body weight. At the histone level, depletion of sirtuins 1 and 3 and HDAC3 may propagate NASH and increase susceptibility to MetS, insulin resistance and hyperlipidemia. On the other hand, the glucose-activated HAT p300 activates ChREBP and thus precipitates stellate cell activation, elevates lipogenic gene expression and expedites steatosis, though these effects can be attenuated by tannic acid. Finally, the NAFLD transcriptome is characterized by overexpression of lipid metabolism, cellular stress, division and adhesion, extracellular matrix production and repair, cancer progression and immunomodulatory genes, whereas several pro-metabolic and insulin signaling genes are downregulated. miRNAs, especially miR-122, miR-192 and miR-34a, are linked to steatosis, cholesterol metabolism, liver cancer, atherogenesis and MetS, whereas other noncoding molecules, such as lncRNAs, are indicators of NASH grade and hepatocellular viability. The uncontrolled lipolysis from adipose tissue, the increased dietary intake of TG and the upregulated de novo lipogenesis observed in NAFLD leads to elevated SFA, LPC, Ceramides and ω6/ω3 PUFA ratio. SFA stimulate the secretion of inflammatory cytokines via TLR4 and apoptosis via TRAIL2, whereas they increase oxidative stress, ER stress and impair β-oxidation in the mitochondria of hepatocytes. In stellate cells, they stimulate macrophage recruitment, whereas in Kupffer cells and macrophages SFA induce their polarization to the M1 proinflammatory state. Increased activation of PLA2 enzyme leads to formation of LPC and depletion of PC. PC are important for lipid droplet stability and their deficiency leads to large droplet formation and inadequate VLDL secretion. High LPC are also triggering mechanisms of impaired β-oxidation, apoptosis, fibrosis and HCC. High hepatic ceramide concentrations increase cholesterol synthesis and TG accumulation, promote insulin resistance by blocking Akt-mediated insulin signaling, induce the secretion of proinflammatory cytokines and stimulate apoptosis by increasing ROS generation, ER stress and β-oxidation impairment. In hepatic stellate cells, they increase extracellular matrix deposition and pro-angiogenic factors secretion promoting fibrogenesis. Finally, the high ω6/ω3 ratio leads to increased synthesis of proinflammatory molecules, such as prostaglandins, leukotrienes, thromboxanes in expense of the synthesis of anti-inflammatory SPMs, thus resulting in a pro-inflammatory

Complications, morbidity and mortality of nonalcoholic fatty liver disease

The multiple-hit pathogenesis of nonalcoholic fatty liver disease (NAFLD)

Established and emerging factors affecting the progression of nonalcoholic fatty liver disease

Diagnostic accuracy and reliability of ultrasonography for the detection of fatty liver: a meta-analysis

Clinical value of liver ultrasound for the diagnosis of nonalcoholic fatty liver disease in overweight and obese patients

Non-invasive diagnosis of hepatic steatosis

Individual patient data meta-analysis of controlled attenuation parameter (CAP) technology for assessing steatosis

Controlled Attenuation Parameter (CAP) with the XL Probe of the Fibroscan((R)): A Comparative Study with the M Probe and Liver Biopsy

Diagnostic value of MRI-PDFF for hepatic steatosis in patients with non-alcoholic fatty liver disease: a metaanalysis

Noninvasive Assessment of Liver Disease in Patients With Nonalcoholic Fatty Liver Disease

Advances in non-invasive biomarkers for the diagnosis and monitoring of non-alcoholic fatty liver disease

Diagnostic Value of CK-18, FGF-21, and Related Biomarker Panel in Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis

Limited value of plasma cytokeratin-18 as a biomarker for NASH and fibrosis in patients with nonalcoholic fatty liver disease

PNPLA3, the triacylglycerol synthesis/hydrolysis/storage dilemma, and nonalcoholic fatty liver disease

Human PNPLA3-I148M variant increases hepatic retention of polyunsaturated fatty acids

Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease

A common variant in PNPLA3 is associated with age at diagnosis of NAFLD in patients from a multi-ethnic biobank

Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis

Genome-wide scan revealed that polymorphisms in the PNPLA3, SAMM50, and PARVB genes are associated with development and progression of nonalcoholic fatty liver disease in Japan

Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits

Evaluation of Polygenic Determinants of Non-Alcoholic Fatty Liver Disease (NAFLD) By a Candidate Genes Resequencing Strategy

GWAS and enrichment analyses of non-alcoholic fatty liver disease identify new traitassociated genes and pathways across eMERGE Network

A common variant in PNPLA3, which encodes adiponutrin, is associated with liver fat content in humans

A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity

PNPLA3 variants specifically confer increased risk for histologic nonalcoholic fatty liver disease but not metabolic disease

The association of genetic variability in patatin-like phospholipase domain-containing protein 3 (PNPLA3) with histological severity of nonalcoholic fatty liver disease

Genetic polymorphisms of the human PNPLA3 gene are strongly associated with severity of non-alcoholic fatty liver disease in Japanese

Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: a multicenter biopsy-based study

Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci

Genetic determinants of steatosis and fibrosis progression in paediatric nonalcoholic fatty liver disease

Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of nonalcoholic fatty liver disease associated hepatocellular carcinoma

Association Between PNPLA3 rs738409 C>G Variant and Liver-Related Outcomes in Patients With Nonalcoholic Fatty Liver Disease

Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease

Impaired hepatic lipid synthesis from polyunsaturated fatty acids in TM6SF2 E167K variant carriers with NAFLD

TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with nonalcoholic fatty liver disease

Genetic variation in transmembrane 6 superfamily member 2 and the risk of nonalcoholic fatty liver disease and histological disease severity

MBOAT7 rs641738 variant and hepatocellular carcinoma in non-cirrhotic individuals

An intronic variant in the GCKR gene is associated with multiple lipids

Common variant in the glucokinase regulatory gene rs780094 and risk of nonalcoholic fatty liver disease: a metaanalysis

Update on NAFLD genetics: From new variants to the clinic

A Pathophysiologic Approach Combining Genetics and Insulin Resistance to Predict the Severity of Nonalcoholic Fatty Liver Disease

A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease

Genome-wide and Mendelian randomisation studies of liver MRI yield insights into the pathogenesis of steatohepatitis

Genome-wide association study of non-alcoholic fatty liver and steatohepatitis in a histologically-characterised cohort

Prediction of non-alcoholic fatty liver disease and liver fat using metabolic and genetic factors

Noninvasive Detection of Nonalcoholic Steatohepatitis Using Clinical Markers and Circulating Levels of Lipids and Metabolites

Development and Validation of a Scoring System, Based on Genetic and Clinical Factors, to Determine Risk of Steatohepatitis in Asian Patients with Nonalcoholic Fatty Liver Disease

Combined Effect of PNPLA3, TM6SF2, and HSD17B13 Variants on Risk of Cirrhosis and Hepatocellular Carcinoma in the General Population

The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases

Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis

Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice

Phenome-wide association studies across large population cohorts support drug target validation

The dual and opposite role of the TM6SF2-rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: A meta-analysis

Leveraging Human Genetics to Identify Potential New Treatments for Fatty Liver Disease

Attenuated effect of PNPLA3 on hepatic fibrosis by HSD17B13 in Japanese patients with non-alcoholic fatty liver disease

Relationship between methylome and transcriptome in patients with nonalcoholic fatty liver disease

DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery

Human liver epigenetic alterations in non-alcoholic steatohepatitis are related to insulin action

Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease

DNA methylation signatures reflect aging in patients with nonalcoholic steatohepatitis

Targetedbisulfite sequence analysis of the methylation of CpG islands in genes encoding PNPLA3, SAMM50, and PARVB of patients with non-alcoholic fatty liver disease

Identification of the genomic region under epigenetic regulation during non-alcoholic fatty liver disease progression

Hepatic stellate cell transdifferentiation involves genome-wide remodeling of the DNA methylation landscape

MeCP2 controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis

Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease

Multigenerational epigenetic adaptation of the hepatic wound-healing response

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: impact of liver methylation of the peroxisome proliferator-activated receptor gamma coactivator 1alpha promoter

Identification of differentially methylated region (DMR) networks associated with progression of nonalcoholic fatty liver disease

Epigenetics in non-alcoholic fatty liver disease

Tannic acid, a novel histone acetyltransferase inhibitor, prevents non-alcoholic fatty liver disease both in vivo and in vitro model

Elimination of Age-Associated Hepatic Steatosis and Correction of Aging Phenotype by Inhibition of cdk4-C/EBPalpha-p300 Axis

P300 Acetyltransferase Mediates Stiffness-Induced Activation of Hepatic Stellate Cells Into Tumor-Promoting Myofibroblasts

SirT1 gain of function increases energy efficiency and prevents diabetes in mice

Sirt1 protects against high-fat diet-induced metabolic damage

Hepatocytespecific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation

SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome

Epigenetic mechanisms underlying the link between non-alcoholic fatty liver diseases and nutrition

A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism

Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in nonalcoholic fatty liver disease

Altered DNA Methylation Sites in Peripheral Blood Leukocytes from Patients with Simple Steatosis and Nonalcoholic Steatohepatitis (NASH)

A Peripheral Blood DNA Methylation Signature of Hepatic Fat Reveals a Potential Causal Pathway for Nonalcoholic Fatty Liver Disease

Dietary resveratrol alters lipid metabolismrelated gene expression of mice on an atherogenic diet

Effects of resveratrol supplementation on liver fat content in overweight and insulinresistant subjects: A randomized, double-blind, placebo-controlled clinical trial

No effect of resveratrol on VLDL-TG kinetics and insulin sensitivity in obese men with nonalcoholic fatty liver disease

Comparison of Calorie-Restricted Diet and Resveratrol Supplementation on Anthropometric Indices, Metabolic Parameters, and Serum Sirtuin-1 Levels in Patients With Nonalcoholic Fatty Liver Disease: A Randomized Controlled Clinical Trial

A Proof-of-Concept for Epigenetic Therapy of Tissue Fibrosis: Inhibition of Liver Fibrosis Progression by 3-Deazaneplanocin A

Gene expression profile-based drug screen identifies SAHA as a novel treatment for NAFLD

Liver transcriptomics highlights interleukin-32 as novel NAFLD-related cytokine and candidate biomarker

Human hepatic gene expression signature of non-alcoholic fatty liver disease progression, a meta-analysis

Comparison of Gene Expression Patterns Between Mouse Models of Nonalcoholic Fatty Liver Disease and Liver Tissues From Patients

Hepatic gene expression profiles differentiate presymptomatic patients with mild versus severe nonalcoholic fatty liver disease

A comprehensive bioinformatics analysis on multiple Gene Expression Omnibus datasets of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis

Hepatic gene expression in histologically progressive nonalcoholic steatohepatitis

Hepatic gene expression in patients with obesity-related non-alcoholic steatohepatitis

Hepatic transcriptome signatures in patients with varying degrees of nonalcoholic fatty liver disease compared with healthy normal-weight individuals

Gene expression profiling unravels cancer-related hepatic molecular signatures in steatohepatitis but not in steatosis

Gene expression profiling of non-alcoholic steatohepatitis using gene set enrichment analysis

Transcriptomic Profiling of Obesity-Related Nonalcoholic Steatohepatitis Reveals a Core Set of Fibrosis-Specific Genes

Serum coding and non-coding RNAs as biomarkers of NAFLD and fibrosis severity

Interspecies NASH disease activity whole-genome profiling identifies a fibrogenic role of PPARalpha-regulated dermatopontin

Role of Noncoding RNA in Development of Nonalcoholic Fatty Liver Disease

A micro-RNA expression signature for human NAFLD progression

Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis

Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression

miRNA Signature in NAFLD: A Turning Point for a Non-Invasive Diagnosis

Liver-specific microRNA-122: Biogenesis and function

Associations between circulating microRNAs (miR-21, miR-34a, miR-122 and miR-451) and non-alcoholic fatty liver

Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease

Genome-wide analysis of long noncoding RNA expression profiles in patients with non-alcoholic fatty liver disease

A liver-specific long noncoding RNA with a role in cell viability is elevated in human nonalcoholic steatohepatitis

Impact of long-term storage and freeze-thawing on eight circulating microRNAs in plasma samples

A pilot study of serum microRNAs panel as potential biomarkers for diagnosis of nonalcoholic fatty liver disease

The Circulating Micro-RNAs (-122, -34a and -99a) as Predictive Biomarkers for Non-Alcoholic Fatty Liver Diseases

miRNAs in patients with non-alcoholic fatty liver disease: A systematic review and meta-analysis

GENFIT-Announces-Results-of-NIS4-an-Investigational-Non-Invasive-NASH-Diagnostic-at-AASLD-2019.html

Validation of NIS4 algorithm for detection of NASH at risk of cirrhosis in 467 NAFLD patients prospectively screened for inclusion in the RESOLVE-IT trial

Performance of Serum microRNAs -122, -192 and -21 as Biomarkers in Patients with Non-Alcoholic Steatohepatitis

CircRNA expression pattern and circRNA-miRNA-mRNA network in the pathogenesis of nonalcoholic steatohepatitis

Comprehensive circular RNA expression profiles in a mouse model of nonalcoholic steatohepatitis

Alternative RNA Splicing in the Pathogenesis of Liver Disease

Mechanisms of NAFLD development and therapeutic strategies

Mapping the molecular signatures of diet-induced NASH and its regulation by the hepatokine Tsukushi

Iron chaperone PCBP1 protects murine liver from lipid peroxidation and steatosis

No evidence for an increased liver uptake of SARS-CoV-2 in metabolic associated fatty liver disease

Omics, big data and machine learning as tools to propel understanding of biological mechanisms and to discover novel diagnostics and therapeutics

Revisiting biomarker discovery by plasma proteomics

An exploratory study examining how nano-liquid chromatography-mass spectrometry and phosphoproteomics can differentiate patients with advanced fibrosis and higher percentage collagen in non-alcoholic fatty liver disease

Proteomic analysis to identify differentially expressed proteins between subjects with metabolic healthy obesity and non-alcoholic fatty liver disease

Organellar Proteomics and Phospho-Proteomics Reveal Subcellular Reorganization in Diet-Induced Hepatic Steatosis

Identification of liver proteins altered by type 2 diabetes mellitus in obese subjects

A genomic and proteomic study of the spectrum of nonalcoholic fatty liver disease

Serum proteomic profiling of obese patients: correlation with liver pathology and evolution after bariatric surgery

Targeted Analysis of Three Hormonal Systems Identifies Molecules Associated with the Presence and Severity of NAFLD

A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

Serum proteomics and biomarker discovery across the spectrum of nonalcoholic fatty liver disease

A multi-component classifier for nonalcoholic fatty liver disease (NAFLD) based on genomic, proteomic, and phenomic data domains

Elevated levels of circulating ITIH4 are associated with hepatocellular carcinoma with nonalcoholic fatty liver disease: from pig model to human study

Plasma proteome profiling discovers novel proteins associated with non-alcoholic fatty liver disease

Pharmacotherapy of obesity: Available medications and drugs under investigation

Obesity as a Disease

Pharmacotherapy of type 2 diabetes: An update

Current and emerging pharmacological options for the treatment of nonalcoholic steatohepatitis

Metabolic regulation of activins in healthy individuals and in obese patients undergoing bariatric surgery

Non-invasive diagnosis of non-alcoholic steatohepatitis and fibrosis with the use of omics and supervised learning: A proof of concept study

Circulating levels of gastrointestinal hormones in response to the most common types of bariatric surgery and predictive value for weight loss over one year: Evidence from two independent trials

Shortterm treatment with high dose liraglutide improves lipid and lipoprotein profile and changes hormonal mediators of lipid metabolism in obese patients with no overt type 2 diabetes mellitus: a randomized, placebo-controlled, cross-over, double-blind clinical trial

Circulating levels of the components of the GH/IGF-1/IGFBPs axis total and intact IGF-binding proteins (IGFBP) 3 and IGFBP 4 and total IGFBP 5, as well as PAPPA, PAPPA2 and Stanniocalcin-2 levels are not altered in response to energy deprivation and/or metreleptin administration in humans

Free IGF-1, Intact IGFBP-4, and PicoPAPP-A are Altered in Acute Myocardial Infarction Compared to Stable Coronary Artery Disease and Healthy Controls

Follistatins in glucose regulation in healthy and obese individuals

Physiology of Activins/Follistatins: Associations With Metabolic and Anthropometric Variables and Response to Exercise

Regulation of the activins-follistatins-inhibins axis by energy status: Impact on reproductive function

Association of Adipokines with Development and Progression of Nonalcoholic Fatty Liver Disease

Adiponectin administration prevents weight gain and glycemic profile changes in diet-induced obese immune deficient Rag1-/-mice lacking mature lymphocytes

The Role of Glicentin and Oxyntomodulin in Human Metabolism: New Evidence and New Directions

Quantitative Proteomics and Mechanistic Modeling of Transporter-Mediated Disposition in Nonalcoholic Fatty Liver Disease

Gut microbial metabolites in obesity, NAFLD and T2DM

Bile acids and nonalcoholic fatty liver disease: Molecular insights and therapeutic perspectives

Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease

A lipidomic analysis of nonalcoholic fatty liver disease

The plasma lipidomic signature of nonalcoholic steatohepatitis

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic

Hepatic and serum lipid signatures specific to nonalcoholic steatohepatitis in murine models

Metabolism dysregulation induces a specific lipid signature of nonalcoholic steatohepatitis in patients

Bioactive Lipid Species and Metabolic Pathways in Progression and Resolution of Nonalcoholic Steatohepatitis

Non-alcoholic steatohepatitis: emerging molecular targets and therapeutic strategies

New insights into the role and mechanism of c-Jun-N-terminal kinase signaling in the pathobiology of liver diseases

Effect of modulation of PPAR-gamma activity on Kupffer cells M1/M2 polarization in the development of non-alcoholic fatty liver disease

Micromolar changes in lysophosphatidylcholine concentration cause minor effects on mitochondrial permeability but major alterations in function

Lipid-Induced Signaling Causes Release of Inflammatory Extracellular Vesicles From Hepatocytes

The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis

Hepatocyte autotaxin expression promotes liver fibrosis and cancer

Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice

CerS2 haploinsufficiency inhibits beta-oxidation and confers susceptibility to diet-induced steatohepatitis and insulin resistance

Obesity-induced CerS6-dependent C16:0 ceramide production promotes weight gain and glucose intolerance

Hepatic cannabinoid-1 receptors mediate diet-induced insulin resistance by increasing de novo synthesis of long-chain ceramides

Acidic sphingomyelinase controls hepatic stellate cell activation and in vivo liver fibrogenesis

Biosynthesis and bioavailability of long-chain polyunsaturated fatty acids in non-alcoholic fatty liver disease

5-lipoxygenase activating protein signals adipose tissue inflammation and lipid dysfunction in experimental obesity

The specialized proresolving lipid mediator maresin 1 protects hepatocytes from lipotoxic and hypoxia-induced endoplasmic reticulum stress

Metabolomic-based noninvasive serum test to diagnose nonalcoholic steatohepatitis: Results from discovery and validation cohorts

Use of a metabolomic approach to non-invasively diagnose non-alcoholic fatty liver disease in patients with type 2 diabetes mellitus

Fatty liver in lipodystrophy: A review with a focus on therapeutic perspectives of adiponectin and/or leptin replacement

Serum metabolites detect the presence of advanced fibrosis in derivation and validation cohorts of patients with non-alcoholic fatty liver disease

Development of a Plasma Screening Panel for Pediatric Nonalcoholic Fatty Liver Disease Using Metabolomics

Farnesoid X nuclear receptor ligand obeticholic acid for noncirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebocontrolled trial

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Obeticholic acid for the treatment of nonalcoholic steatohepatitis: Expectations and concerns

Hepatoprotective effects of the dual peroxisome proliferator-activated receptor alpha/delta agonist, GFT505, in rodent models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis

The selective peroxisome proliferator-activated receptor-delta agonist seladelpar reverses nonalcoholic steatohepatitis pathology by abrogating lipotoxicity in diabetic obese mice

GENFIT-Announces-Results-from-Interim-Analysis-of-RESOLVE-IT-Phase-3-Trial-of-Elafibranor-in-Adults-with-NASH-and-Fibrosis.html

Agonist of the Peroxisome Proliferator-Activated Receptor-alpha anddelta, Induces Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsening

Selective Tissue Distribution Mediates Tissue-Dependent PPARgamma Activation and Insulin Sensitization by INT131, a Selective PPARgamma Modulator

Dual PPARalpha/gamma agonist saroglitazar improves liver histopathology and biochemistry in experimental NASH models

Effect of saroglitazar 2 mg and 4 mg on glycemic control, lipid profile and cardiovascular disease risk in patients with type 2 diabetes mellitus: a 56-week, randomized, double blind, phase 3 study (PRESS XII study)

lanifibranor-meets-the-primary-andkey-secondary-endpoints-in-the-Phase-IIb-NATIVE-clinical-trial-in-non-alcoholicsteatohepatitis-NASH.html

Acetyl-CoA Carboxylase Inhibitor GS-0976 for 12 Weeks Reduces Hepatic De Novo Lipogenesis and Steatosis in Patients With Nonalcoholic Steatohepatitis

ASB14780, an Orally Active Inhibitor of Group IVA Phospholipase A2, Is a Pharmacotherapeutic Candidate for Nonalcoholic Fatty Liver Disease

Elevation of 20-carbon long chain bases due to a mutation in serine palmitoyltransferase small subunit b results in neurodegeneration

Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction

Omega-3 supplementation and non-alcoholic fatty liver disease: a systematic review and meta-analysis

No significant effects of ethyl-eicosapentanoic acid on histologic features of nonalcoholic steatohepatitis in a phase 2 trial

Effects of purified eicosapentaenoic and docosahexaenoic acids in nonalcoholic fatty liver disease: results from the Welcome* study

Molecular interplay between Delta5/Delta6 desaturases and longchain fatty acids in the pathogenesis of non-alcoholic steatohepatitis

Group IVA phospholipase A(2) deficiency prevents CCl4-induced hepatic cell death through the enhancement of autophagy

5-lipoxygenase deficiency reduces hepatic inflammation and tumor necrosis factor alpha-induced hepatocyte damage in hyperlipidemia-prone ApoE-null mice

Protection from hepatic lipid accumulation and inflammation by genetic ablation of 5-lipoxygenase

LTB4 promotes insulin resistance in obese mice by acting on macrophages, hepatocytes and myocytes

Posttreatment with Protectin DX ameliorates bleomycin-induced pulmonary fibrosis and lung dysfunction in mice

17(R)-resolvin D1 ameliorates bleomycin-induced pulmonary fibrosis in mice

Proresolving actions of a new resolvin D1 analog mimetic qualifies as an immunoresolvent

Essentials of Glycobiology. Cold Spring Harbor (NY)2015. p

Essentials of Glycobiology. Cold Spring Harbor (NY)2015. p

Glycosylation in health and disease

Glycans in Acquired Human Diseases

Glycosylation Changes in Cancer

Sialic Acids and Other Nonulosonic Acids

Serum Sialic Acid Level Is Significantly Associated with Nonalcoholic Fatty Liver Disease in a Nonobese Chinese Population: A Cross-Sectional Study

Association between Serum Sialic Acid Levels and Nonalcoholic Fatty Liver Disease: A Cross-Sectional Study

Unmasking Fucosylation: from Cell Adhesion to Immune System Regulation and Diseases

A novel noninvasive diagnostic method for nonalcoholic steatohepatitis using two glycobiomarkers

Inhibition of fucosylation reshapes inflammatory macrophages and suppresses type II collagen-induced arthritis

Inhibition of fucosylation by 2-fluorofucose suppresses human liver cancer HepG2 cell proliferation and migration as well as tumor formation

Performance of Plasma Biomarkers and Diagnostic Panels for Nonalcoholic Steatohepatitis and Advanced Fibrosis in Patients With Type 2 Diabetes

Performance of the SteatoTest, ActiTest, NashTest and FibroTest in a multiethnic cohort of patients with type 2 diabetes mellitus

Change in hepatic fat content measured by MRI does not predict treatment-induced histological improvement of steatohepatitis

Performance of biomarkers FibroTest, ActiTest, SteatoTest, and NashTest in patients with severe obesity: meta analysis of individual patient data

ADAPT: An Algorithm Incorporating PRO-C3 Accurately Identifies Patients With NAFLD and Advanced Fibrosis

Comparison of laboratory tests, ultrasound, or magnetic resonance elastography to detect fibrosis in patients with nonalcoholic fatty liver disease: A meta-analysis

Enhanced liver fibrosis test for the non-invasive diagnosis of fibrosis in patients with NAFLD: A systematic review and meta-analysis

Accuracy of FibroScan Controlled Attenuation Parameter and Liver Stiffness Measurement in Assessing Steatosis and Fibrosis in Patients With Nonalcoholic Fatty Liver Disease

Diagnostic Accuracy of Noninvasive Fibrosis Models to Detect Change in Fibrosis Stage

Noninvasive Tests Accurately Identify Advanced Fibrosis due to NASH: Baseline Data From the STELLAR Trials

Genetic signatures in choline and 1-carbon metabolism are associated with the severity of hepatic steatosis

Efficacy of serum miRNA test as a noninvasive method to diagnose nonalcoholic steatohepatitis: a systematic review and meta-analysis

Serum proteomic analysis revealed diagnostic value of hemoglobin for nonalcoholic fatty liver disease

Proteomic analysis of serum in patients with non-alcoholic steatohepatitis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Serum proteome of nonalcoholic fatty liver disease: a multimodal approach to discovery of biomarkers of nonalcoholic steatohepatitis

Phosphoproteomic biomarkers predicting histologic nonalcoholic steatohepatitis and fibrosis

Mapping of the circulating metabolome reveals alpha-ketoglutarate as a predictor of morbid obesity-associated non-alcoholic fatty liver disease

Prediction of non-alcoholic fatty-liver disease and liver fat content by serum molecular lipids

Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease progression

Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis

Urinary metabolomics analysis identifies key biomarkers of different stages of nonalcoholic fatty liver disease

Serum C16:1n7/C16:0 ratio as a diagnostic marker for non-alcoholic steatohepatitis

Fatty acids in non-alcoholic steatohepatitis: Focus on pentadecanoic acid

Accurate non-invasive diagnosis and staging of non-alcoholic fatty liver disease using the urinary steroid metabolome

Serum metabolomic profile and potential biomarkers for severity of fibrosis in nonalcoholic fatty liver disease

Lipidomic-based Analysis of Plasma to Diagnose Hepatocellular Ballooning in Patients with Nonalcoholic Fatty Liver Disease: A Multicenter Study

Use of non-invasive serum glycan markers to distinguish non-alcoholic steatohepatitis from simple steatosis

N-glycan based biomarker distinguishing non-alcoholic steatohepatitis from steatosis independently of fibrosis

Serum protein N-glycans profiling for the discovery of potential biomarkers for nonalcoholic steatohepatitis

Use of Mac-2 binding protein as a biomarker for nonalcoholic fatty liver disease diagnosis

Serum progranulin as an independent marker of liver fibrosis in patients with biopsy-proven nonalcoholic fatty liver disease

Identification of a support vector machine-based biomarker panel with high sensitivity and specificity for nonalcoholic steatohepatitis

Derivation and validation of a scoring system for predicting nonalcoholic steatohepatitis in Taiwanese patients with severe obesity

Nonalcoholic fatty liver disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the severely obese

Noninvasive assessment of hepatic steatosis and fibrosis in patients with severe obesity

Non-alcoholic steatohepatitis: a non-invasive diagnosis by analysis of exhaled breath

How adiponectin, leptin, and ghrelin orchestrate together and correlate with the severity of nonalcoholic fatty liver disease

A newly noninvasive model for prediction of non-alcoholic fatty liver disease: utility of serum prolactin levels

A new composite model including metabolic syndrome, alanine aminotransferase and cytokeratin-18 for the diagnosis of non-alcoholic steatohepatitis in morbidly obese patients

A noninvasive clinical scoring model predicts risk of nonalcoholic steatohepatitis in morbidly obese patients

Serum ferritin is a clinical biomarker in Japanese patients with nonalcoholic steatohepatitis (NASH) independent of HFE gene mutation

Serum thioredoxin levels as a predictor of steatohepatitis in patients with nonalcoholic fatty liver disease

A simple clinical scoring system using ferritin, fasting insulin, and type IV collagen 7S for predicting steatohepatitis in nonalcoholic fatty liver disease

Combined Serum Biomarkers in Non-Invasive Diagnosis of Non-Alcoholic Steatohepatitis

A 4-polymorphism risk score predicts steatohepatitis in children with nonalcoholic fatty liver disease

A population-based study on the prevalence of NASH using scores validated against liver histology

Serum adipokine levels predictive of liver injury in non-alcoholic fatty liver disease

Could inflammatory markers help diagnose nonalcoholic steatohepatitis?

Retinol-binding protein 4: a promising circulating marker of liver damage in pediatric nonalcoholic fatty liver disease

Serum levels of hyaluronic acid and tissue metalloproteinase inhibitor-1 combined with age predict the presence of nonalcoholic steatohepatitis in a pilot cohort of subjects with nonalcoholic fatty liver disease

A diagnostic model to differentiate simple steatosis from nonalcoholic steatohepatitis based on the likelihood ratio form of Bayes theorem

A novel diagnostic biomarker panel for obesity-related nonalcoholic steatohepatitis (NASH)

Usefulness of a combined evaluation of the serum adiponectin level, HOMA-IR, and serum type IV collagen 7S level to predict the early stage of nonalcoholic steatohepatitis

Accuracy of proton magnetic resonance for diagnosing non-alcoholic steatohepatitis: a meta-analysis

Plasma Pentraxin3 is a novel marker for nonalcoholic steatohepatitis (NASH)

Validation of terminal peptide of procollagen III for the detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease

Diagnostic value of serum prolidase enzyme activity to predict the liver histological lesions in non-alcoholic fatty liver disease: a surrogate marker to distinguish steatohepatitis from simple steatosis

Prediction of Nonalcoholic Fatty Liver Disease Via a Novel Panel of Serum Adipokines

Predictive factors for nonalcoholic steatohepatitis (NASH) in patients with nonalcoholic fatty liver disease (NAFLD)

MACK-3 (combination of hoMa, Ast and CK18): A promising novel biomarker for fibrotic non-alcoholic steatohepatitis

Noninvasive assessment of NAFLD as systemic disease-A machine learning perspective

Non-invasive diagnosis of non-alcoholic steatohepatitis by combined serum biomarkers

Meta-analysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity

Soluble forms of extracellular cytokeratin 18 may differentiate simple steatosis from nonalcoholic steatohepatitis

Clinical model for distinguishing nonalcoholic steatohepatitis from simple steatosis in patients with nonalcoholic fatty liver disease

Development and validation of a novel non-invasive test for diagnosing fibrotic non-alcoholic steatohepatitis in patients with biopsy-proven non-alcoholic fatty liver disease

Clinical usefulness of biochemical markers of liver fibrosis in patients with nonalcoholic fatty liver disease

Diagnostic performance of FibroTest, SteatoTest and ActiTest in patients with NAFLD using the SAF score as histological reference

Hyaluronic acid levels can predict severe fibrosis and platelet counts can predict cirrhosis in patients with nonalcoholic fatty liver disease

Serum markers detect the presence of liver fibrosis: a cohort study

Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease

Direct comparison of the specialised blood fibrosis tests FibroMeter(V2G) and Enhanced Liver Fibrosis score in patients with non-alcoholic fatty liver disease from tertiary care centres

Step layered combination of noninvasive fibrosis models improves diagnostic accuracy of advanced fibrosis in nonalcoholic fatty liver disease

Development and Validation of Hepamet Fibrosis Scoring System-A Simple, Noninvasive Test to Identify Patients With Nonalcoholic Fatty Liver Disease With Advanced Fibrosis

Low circulating levels of dehydroepiandrosterone in histologically advanced nonalcoholic fatty liver disease

Validation of Serum Test for Advanced Liver Fibrosis in Patients With Nonalcoholic Steatohepatitis

Comparison of FIB-4 index, NAFLD fibrosis score and BARD score for prediction of advanced fibrosis in adult patients with non-alcoholic fatty liver disease: A meta-analysis study

NIKEI: a new inexpensive and non-invasive scoring system to exclude advanced fibrosis in patients with NAFLD

The development of the pediatric NAFLD fibrosis score (PNFS) to predict the presence of advanced fibrosis in children with nonalcoholic fatty liver disease

Simple noninvasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease

Noninvasive markers of fibrosis in nonalcoholic fatty liver disease: Validating the European Liver Fibrosis Panel and exploring simple markers

Hyaluronic acid, an accurate serum marker for severe hepatic fibrosis in patients with non-alcoholic fatty liver disease

Development of Serum Marker Models to Increase Diagnostic Accuracy of Advanced Fibrosis in Nonalcoholic Fatty Liver Disease: The New LINKI Algorithm Compared with Established Algorithms

Classification of patients with non-alcoholic fatty liver disease using rapid immunoassay of serum type IV collagen compared with liver histology and other fibrosis markers

A PDGFRbeta-based score predicts significant liver fibrosis in patients with chronic alcohol abuse, NAFLD and viral liver disease

An Evaluation of the Collagen Fragments Related to Fibrogenesis and Fibrolysis in Nonalcoholic Steatohepatitis

A gene-based model for staging liver fibrosis

A biomarker panel for non-alcoholic steatohepatitis (NASH) and NASH-related fibrosis

New discriminant score to predict the fibrotic stage of non-alcoholic steatohepatitis in Japan

The diagnostic accuracy of liver fibrosis in non-viral liver diseases using acoustic radiation force impulse elastography: A systematic review and meta-analysis

Magnetic resonance elastography for staging liver fibrosis in non-alcoholic fatty liver disease: a diagnostic accuracy systematic review and individual participant data pooled analysis