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Background & Aims Nonalcoholic steatohepatitis (NASH) is commonly observed in patients with type 2 diabetes, and thiazolidinediones (TZD) are considered a potential therapy for NASH. Although TZD increase insulin sensitivity and partially reduce steatosis and alanine aminotransferase, the efficacy of TZD on resolving liver pathology is limited. In fact, TZD may activate peroxisome proliferator-activated receptor gamma (PPARγ) in hepatocytes and promote steatosis. Therefore, we assessed the role that hepatocyte-specific PPARγ plays in the development of NASH, and how it alters the therapeutic effects of TZD on the liver of mice with diet-induced NASH. Methods Hepatocyte-specific PPARγ expression was knocked out in adult mice before and after the development of NASH induced with a high fat, cholesterol, and fructose (HFCF) diet. Results HFCF diet increased PPARγ expression in hepatocytes, and rosiglitazone further activated PPARγ in hepatocytes of HFCF-fed mice in vivo and in vitro. Hepatocyte-specific loss of PPARγ reduced the progression of HFCF-induced NASH in male mice and increased the benefits derived from the effects of TZD on extrahepatic tissues and non-parenchymal cells. RNAseq and metabolomics indicated that HFCF diet promoted inflammation and fibrogenesis in a hepatocyte PPARγ–dependent manner and was associated with dysregulation of hepatic metabolism. Specifically, hepatocyte-specific loss of PPARγ plays a positive role in the regulation of methionine metabolism, and that could reduce the progression of NASH. Conclusions Because of the negative effect of hepatocyte PPARγ in NASH, inhibition of mechanisms promoted by endogenous PPARγ in hepatocytes may represent a novel strategy that increases the efficiency of therapies for NAFLD., Graphical abstract

Background & Aims Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. Methods Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. Results TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. Conclusions Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress., Graphical abstract

Background and Aims GAS6 signaling, through the TAM receptor tyrosine kinases AXL and MERTK, participates in chronic liver pathologies. Here, we addressed GAS6/TAM involvement in Non-Alcoholic SteatoHepatitis (NASH) development. Methods GAS6/TAM signaling was analyzed in cultured primary hepatocytes, hepatic stellate cells (HSC) and Kupffer cells (KCs). Axl-/-, Mertk-/- and wild-type C57BL/6 mice were fed with Chow, High Fat Choline-Deficient Methionine-Restricted (HFD) or methionine-choline-deficient (MCD) diet. HSC activation, liver inflammation and cytokine/chemokine production were measured by qPCR, mRNA Array analysis, western blotting and ELISA. GAS6, soluble AXL (sAXL) and MERTK (sMERTK) levels were analyzed in control individuals, steatotic and NASH patients. Results In primary mouse cultures, GAS6 or MERTK activation protected primary hepatocytes against lipid toxicity via AKT/STAT-3 signaling, while bemcentinib (small molecule AXL inhibitor BGB324) blocked AXL-induced fibrogenesis in primary HSCs and cytokine production in LPS-treated KCs. Accordingly; bemcentinib diminished liver inflammation and fibrosis in MCD- and HFD-fed mice. Upregulation of AXL and ADAM10/ADAM17 metalloproteinases increased sAXL in HFD-fed mice. Transcriptome profiling revealed major reduction in fibrotic- and inflammatory-related genes in HFD-fed mice after bemcentinib administration. HFD-fed Mertk-/- mice exhibited enhanced NASH, while Axl-/- mice were partially protected. In human serum, sAXL levels augmented even at initial stages, whereas GAS6 and sMERTK increased only in cirrhotic NASH patients. In agreement, sAXL increased in HFD-fed mice before fibrosis establishment, while bemcentinib prevented liver fibrosis/inflammation in early NASH. Conclusion AXL signaling, increased in NASH patients, promotes fibrosis in HSCs and inflammation in KCs, while GAS6 protects cultured hepatocytes against lipotoxicity via MERTK. Bemcentinib, by blocking AXL signaling and increasing GAS6 levels, reduces experimental NASH, revealing AXL as an effective therapeutic target for clinical practice., Graphical abstract

Summary: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder worldwide. This historical narrative traces the evolution from basic descriptions of fatty liver in the nineteenth century to our contemporary understanding of NAFLD in the twentieth and twenty-first centuries. A detailed historiographic review of fatty liver from 1800s onwards was performed alongside a brief review of contemporary associations. Archived published literature dating back to the 1800s describe clinicopathological features of fatty liver. In the nineteenth century, doyens of medicine associated fatty liver with alcohol, malnutrition or wasting conditions, and subsequently adiposity, unhealthy diets and sedentary lifestyle. Microscopically, fatty liver was described when 5% or more hepatocytes were distended with fat. Recommendations to reverse fatty liver included reducing consumption of fat, sugar, starchy carbohydrates and alcohol, plus increasing physical exercise. Fatty liver was associated with liver fibrosis and cirrhosis in the late 1800s, and with diabetes in the early 1900s. The diagnostic labels NAFLD and non-alcoholic steatohepatitis (NASH) were introduced in the late 1900s. Metabolic dysfunction-associated fatty liver disease (MAFLD) was recently proposed to update the nosology of fatty liver, recognising the similar metabolic pathogenesis evident in individuals with typical NAFLD and those with heterogenous “secondary” co-factors including alcohol and other aetiologies. Fatty liver has emerged from being considered a disorder of nutritional extremes or alcohol excess to contemporary recognition as a complex metabolic disorder that risks progression to cirrhosis and hepatocellular carcinoma. The increasing prevalence of NAFLD and our growing understanding of its lifestyle and metabolic determinants justify the current exercise of re-examining the evolution of this common metabolic disorder.

Non-alcoholic fatty liver disease (NAFLD) is by now the most prevalent liver disease worldwide. The non-proteogenic amino acid l-citrulline (L-Cit) has been shown to protect mice from the development of NAFLD. Here, we aimed to further assess if L-Cit also attenuates the progression of a pre-existing diet-induced NAFLD and to determine molecular mechanisms involved. Female C57BL/6J mice were either fed a liquid fat-, fructose- and cholesterol-rich diet (FFC) or control diet (C) for 8 weeks to induce early stages of NASH followed by 5 more weeks with either FFC-feeding +/- 2.5 g L-Cit/kg bw or C-feeding. In addition, female C57BL/6J mice were either pair-fed a FFC +/- 2.5 g L-Cit/kg bw +/- 0.01 g/kg bw i.p. N(ω)-hydroxy-nor-l-arginine (NOHA) or C diet for 8 weeks. The protective effects of supplementing L-Cit on the progression of a pre-existing NAFLD were associated with an attenuation of 1) the increased translocation of bacterial endotoxin and 2) the loss of tight junction proteins as well as 3) arginase activity in small intestinal tissue, while no marked changes in intestinal microbiota composition were prevalent in small intestine. Treatment of mice with the arginase inhibitor NOHA abolished the protective effects of L-Cit on diet-induced NAFLD. Our results suggest that the protective effects of L-Cit on the development and progression of NAFLD are related to alterations of intestinal arginase activity and intestinal permeability., Graphical abstract Image 1, Highlights • l-citrulline diminished progression of non-alcoholic fatty liver disease (NAFLD). • l-citrulline protects from fructose-induced small intestinal barrier dysfunction. • NASH development is associated with a loss of arginase activity in small intestine. • l-citrulline improves intestinal arginase activity in diet-induced NAFLD. • Arginase inhibitor attenuates effects of l-citrulline on NAFLD development.

Summary Among the large population of patients with non-alcoholic fatty liver disease (NAFLD), identifying those with advanced disease remains challenging. Many patients are diagnosed late, following the development of liver-related complications, leading to poor clinical outcomes. Accumulating evidence suggests that using non-invasive tests for liver fibrosis in patients with metabolic risk factors improves the detection of patients in need of specialised management and is cost-effective. Because of the vast number of patients requiring evaluation, the active participation of general practitioners and physicians who manage patients with metabolic disorders, such as diabetologists, is crucial; this calls for the increased awareness of NAFLD beyond liver clinics. Non-invasive case-finding strategies will need to be further validated and generalised for upcoming drug therapies to have the required impact on the worldwide burden of NAFLD.

Background & Aims Thrombospondin 1 (TSP1) is a multifunctional matricellular protein. We previously showed that TSP1 has an important role in obesity-associated metabolic complications, including inflammation, insulin resistance, cardiovascular, and renal disease. However, its contribution to obesity-associated non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD or NASH) remains largely unknown; thus, we aimed to determine its role. Methods High-fat diet or AMLN (amylin liver NASH) diet-induced obese and insulin-resistant NAFLD/NASH mouse models were utilised, in addition to tissue-specific Tsp1-knockout mice, to determine the contribution of different cellular sources of obesity-induced TSP1 to NAFLD/NASH development. Results Liver TSP1 levels were increased in experimental obese and insulin-resistant NAFLD/NASH mouse models as well as in obese patients with NASH. Moreover, TSP1 deletion in adipocytes did not protect mice from diet-induced NAFLD/NASH. However, myeloid/macrophage-specific TSP1 deletion protected mice against obesity-associated liver injury, accompanied by reduced liver inflammation and fibrosis. Importantly, this protection was independent of the levels of obesity and hepatic steatosis. Mechanistically, through an autocrine effect, macrophage-derived TSP1 suppressed Smpdl3b expression in liver, which amplified liver proinflammatory signalling (Toll-like receptor 4 signal pathway) and promoted NAFLD progression. Conclusions Macrophage-derived TSP1 is a significant contributor to obesity-associated NAFLD/NASH development and progression and could serve as a therapeutic target for this disease. Lay summary Obesity-associated non-alcoholic fatty liver disease is a most common chronic liver disease in the Western world and can progress to liver cirrhosis and cancer. No treatment is currently available for this disease. The present study reveals an important factor (macrophage-derived TSP1) that drives macrophage activation and non-alcoholic fatty liver disease development and progression and that could serve as a therapeutic target for non-alcoholic fatty liver disease/steatohepatitis., Graphical abstract, Highlights • Obesity induces TSP1 expression in several tissues, such as adipose tissue and liver. • Macrophages are an important cellular source of increased TSP1 in mouse NASH livers. • Deletion of TSP1 in macrophages protects mice from diet-induced NASH. • SMPDL3B negatively regulates TSP1-mediated macrophage activation.

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., Highlights • Numerous non-invasive NAFLD biomarkers are developed but none is broadly applicable • OMICS enrich the understanding, diagnosis and treatment of NAFLD, NASH and fibrosis • Genomics and epigenomics pinpoint NAFLD polymorphisms and DNA methylation loci • Transcriptomics and proteomics prove the diagnostic value of miRNAs and peptides • Metabolomics, lipidomics and glycomics offer robust panels and therapeutic insight

Summary Identifying the most effective therapeutic intervention in patients with NAFLD is challenging. Precise stratification in clinical trials is key to ensuring the inclusion of patients who will benefit (and not those who will be harmed) and/or in whom the natural history can be improved. Clinical trials in NAFLD can provide useful information about the individual components that underlie this complex metabolic disorder and the concomitant medications that could interfere with responses to an experimental intervention. However, to date, clinical trial reporting for NAFLD has been suboptimal, limiting our understanding. Frequently dysmetabolic comorbidities and/or daily habits are not reported or adequately accounted for. Herein, we suggest new strategies to integrate the spectra of comorbidities usually present in patients with NAFLD, accounting for the impact of lifestyle, to develop personalised therapeutic approaches. First, the mechanism of action of the drug being explored should be considered. Second, the same proportion of patients with relevant metabolic comorbidities should be maintained from phase II to III clinical trials, if such comorbidities are expected to impact on the treatment response. Third, innovative trial designs, such as the adaptative, umbrella or basket strategies, could be used to increase the efficiency of clinical trials, potentially benefiting patients while reducing costs and enhancing the likelihood of finding a real benefit of the therapy being studied. Finally, alcohol intake and daily exercise should be assessed objectively not only in the screening period but also during follow-up.

Background & Aims The precise determination of non-alcoholic fatty liver disease (NAFLD) onset is challenging. Thus, the initial hepatic responses to fat accumulation, which may be fundamental to our understanding of NAFLD evolution and clinical outcomes, are largely unknown. Herein, we chronologically mapped the immunologic and metabolic changes in the liver during the early stages of fatty liver disease in mice and compared this with human NAFLD samples. Methods Liver biopsies from patients with NAFLD (NAFLD activity score [NAS] 2–3) were collected for gene expression profiling. Mice received a high-fat diet for short periods to mimic initial steatosis and the hepatic immune response was investigated using a combination of confocal intravital imaging, gene expression, cell isolation, flow cytometry and bone marrow transplantation assays. Results We observed major immunologic changes in patients with NAS 2–3 and in mice in the initial stages of NAFLD. In mice, these changes significantly increased mortality rates upon drug-induced liver injury, as well as predisposing mice to bacterial infections. Moreover, deletion of Toll-like receptor 4 in liver cells dampened tolerogenesis, particularly in Kupffer cells, in the initial stages of dietary insult. Conclusion The hepatic immune system acts as a sentinel for early and minor changes in hepatic lipid content, mounting a biphasic response upon dietary insult. Priming of liver immune cells by gut-derived Toll-like receptor 4 ligands plays an important role in liver tolerance in initial phases, but continuous exposure to insults may lead to damage and reduced ability to control infections. Lay summary Fatty liver is a very common form of hepatic disease, leading to millions of cases of cirrhosis every year. Patients are often asymptomatic until becoming very sick. Therefore, it is important that we expand our knowledge of the early stages of disease pathogenesis, to enable early diagnosis. Herein, we show that even in the early stages of fatty liver disease, there are significant alterations in genes involved in the inflammatory response, suggesting that the hepatic immune system is disturbed even following minor and undetectable changes in liver fat content. This could have implications for the diagnosis and clinical management of fatty liver disease., Graphical abstract, Highlights • Hepatic immune response is already altered in liver biopsies from patients with mild NAFLD. • We designed a novel mouse model to mimic mild NAFLD, enabling the chronological mapping of liver changes. • This revealed an increased mortality rate upon secondary liver damage and a window of increased susceptibility to infection. • NAFLD diagnosis may be significantly improved by a more profound investigation of changes in hepatic immunology. • These data could guide customized nutritional and therapeutic interventions at different stages of NAFLD.

Background While non-alcoholic fatty liver disease (NAFLD) has been well characterised in patients with diabetes mellitus (DM), less is known about NAFLD in non-DM patients. We investigated the clinical characteristics of NAFLD patients with and without DM and accuracy of the NAFLD fibrosis score (NFS) in these two NAFLD groups. Methods Clinical, biochemical and histological variables were evaluated in this prospective cross-sectional study of 503 patients with biopsy proven NAFLD. Comparisons between patients with and without DM were analysed. NFS was correlated with liver histology to assess its robustness in patients with and without DM. Results There were 503 biopsy proven NAFLD patients with 48% of the cohort being diabetic. Relative to patients without DM, patients with DM were older (52 vs. 46 years, p, Highlights • Patients with diabetes have more severe NAFLD based on histology. • Severe NAFLD can occur in a considerable proportion of non-diabetic NAFLD patients. • The NAFLD fibrosis score may be less accurate in non-diabetics.

Objective Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is associated with an enhanced risk for liver and cardiovascular diseases and mortality. NAFLD can progress from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH). However, the mechanisms predisposing to this progression remain undefined. Notably, hepatic mitochondrial dysfunction is a common finding in patients with NASH. Due to a lack of appropriate experimental animal models, it has not been evaluated whether this mitochondrial dysfunction plays a causative role for the development of NASH. Methods To determine the effect of a well-defined mitochondrial dysfunction on liver physiology at baseline and during dietary challenge, C57BL/6J-mtFVB/N mice were employed. This conplastic inbred strain has been previously reported to exhibit decreased mitochondrial respiration likely linked to a non-synonymous gene variation (nt7778 G/T) of the mitochondrial ATP synthase protein 8 (mt-ATP8). Results At baseline conditions, C57BL/6J-mtFVB/N mice displayed hepatic mitochondrial dysfunction characterized by decreased ATP production and increased formation of reactive oxygen species (ROS). Moreover, genes affecting lipid metabolism were differentially expressed, hepatic triglyceride and cholesterol levels were changed in these animals, and various acyl-carnitines were altered, pointing towards an impaired mitochondrial carnitine shuttle. However, over a period of twelve months, no spontaneous hepatic steatosis or inflammation was observed. On the other hand, upon dietary challenge with either a methionine and choline deficient diet or a western-style diet, C57BL/6J-mtFVB/N mice developed aggravated steatohepatitis as characterized by lipid accumulation, ballooning of hepatocytes and infiltration of immune cells. Conclusions We observed distinct metabolic alterations in mice with a mitochondrial polymorphism associated hepatic mitochondrial dysfunction. However, a second hit, such as dietary stress, was required to cause hepatic steatosis and inflammation. This study suggests a causative role of hepatic mitochondrial dysfunction in the development of experimental NASH., Highlights • C57BL/6J-mtFVB/N mice (mt-ATP8, nt7778 G/T) display hepatic mitochondrial dysfunction. • C57BL/6J-mtFVB/N mice display alterations in hepatic energy metabolism. • C57BL/6J-mtFVB/N mice show no spontaneous hepatic steatosis or inflammation. • C57BL/6J-mtFVB/N mice are susceptible to diet induced NASH. • Study demonstrates causative role of mitochondrial dysfunction for NASH development.

Background and aim It is recognized that nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), may develop after pancreaticoduodenectomy (PD). However, the mechanism of NASH development remains unclear. This study aimed to examine the changes in gene expression associated with NASH occurrence following PD. Methods The expression of genes related to fatty acid/triglyceride (FA/TG) metabolism and inflammatory signaling was examined using liver samples obtained from 7 post-PD NASH patients and compared with 6 healthy individuals and 32 conventional NASH patients. Results The livers of post-PD NASH patients demonstrated significant up-regulation of the genes encoding CD36, FA-binding proteins 1 and 4, acetyl-coenzyme A carboxylase α, diacylglycerol acyltransferase 2, and peroxisome proliferator-activated receptor (PPAR) γ compared with normal and conventional NASH livers. Although serum apolipoprotein B (ApoB) and TG were decreased in post-PD NASH patients, the mRNAs of ApoB and microsomal TG transfer protein were robustly increased, indicating impaired TG export from the liver as very-low-density lipoprotein (VLDL). Additionally, elevated mRNA levels of myeloid differentiation primary response 88 and superoxide dismutases in post-PD NASH livers suggested significant activation of innate immune response and augmentation of oxidative stress generation. Conclusions Enhanced FA uptake into hepatocytes and lipogenesis, up-regulation of PPARγ, and disruption of VLDL excretion into the circulation are possible mechanisms of steatogenesis after PD. General significance These results provide a basis for understanding the pathogenesis of NAFLD/NASH following PD., Highlights • The mechanism of NASH development after pancreaticoduodenectomy (PD) was unclear. • The gene expression involved in fatty acid uptake and lipogenesis was increased. • PPARγ and its target genes were up-regulated in post-PD NASH livers. • Impaired triglyceride excretion from the liver was suggested in post-PD NASH. • This study proposes possible mechanisms of steatogenesis after PD.