Search

The rising prevalence of nonalcoholic fatty liver disease (NAFLD) and NAFLD‐related cirrhosis in the United States and globally highlights the need to better understand the mechanisms causing progression of hepatic steatosis to fibrosing steatohepatitis and cirrhosis in a small proportion of patients with NAFLD. Accumulating evidence suggests that lipotoxicity mediated by hepatic free cholesterol (FC) overload is a mechanistic driver for necroinflammation and fibrosis, characteristic of nonalcoholic steatohepatitis (NASH), in many animal models and also in some patients with NASH. Diet, lifestyle, obesity, key genetic polymorphisms, and hyperinsulinemia secondary to insulin resistance are pivotal drivers leading to aberrant cholesterol signaling, which leads to accumulation of FC within hepatocytes. FC overload in hepatocytes can lead to ER stress, mitochondrial dysfunction, development of toxic oxysterols, and cholesterol crystallization in lipid droplets, which in turn lead to hepatocyte apoptosis, necrosis, or pyroptosis. Activation of Kupffer cells and hepatic stellate cells by hepatocyte signaling and cholesterol loading contributes to this inflammation and leads to hepatic fibrosis. Cholesterol accumulation in hepatocytes can be readily prevented or reversed by statins. Observational studies suggest that use of statins in NASH not only decreases the substantially increased cardiovascular risk, but may ameliorate liver pathology. Conclusion: Hepatic FC loading may result in cholesterol‐associated steatohepatitis and play an important role in the development and progression of NASH. Statins appear to provide significant benefit in preventing progression to NASH and NASH‐cirrhosis. Randomized controlled trials are needed to demonstrate whether statins or statin/ezetimibe combination can effectively reverse steatohepatitis and liver fibrosis in patients with NASH., Free cholesterol accumulates in the liver in some patients with NAFLF and promotes the necroinflammation and fibrosis typical of NASH. Cholesterol‐associated steatohepatitis (CASH) plays an important role in the development and progression of NASH. Randomized controlled trials are needed to demonstrate whether statins or statin/ezetimibe combination can effectively reverse steatohepatitis and liver fibrosis in patients with NASH.

Obesity and type 2 diabetes increase hepatocellular carcinoma (HCC) incidence in humans and accelerate diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. We investigated whether exercise reduces HCC development in obese/diabetic Alms1 mutant (foz/foz) mice and studied protective mechanisms.We measured HCC development in DEN-injected male foz/foz and wild-type (WT) littermates housed with or without an exercise wheel from week 4 until 12 or 24 weeks, and in foz/foz mice pair-fed to WT littermates. We also studied HCC development in DEN-injected Jnk1Exercising foz/foz mice maintained similar weight as WT mice up to 10 weeks, but then gained weight and were obese by 24 weeks; a similar body weight profile was obtained by pair-feeding foz/foz mice to WT. At 12 weeks, livers of exercising foz/foz mice exhibited fewer GST-pi positive hepatocytes than sedentary counterparts; by 24 weeks, fewer exercising foz/foz mice developed HCC (15% vs. 64%, p0.05). Conversely, pair-feeding foz/foz mice failed to reduce HCC incidence. In these insulin-resistant foz/foz mice, exercise failed to activate hepatic AMPK or Akt/mTORC1. Instead, it improved insulin sensitivity, ameliorated steatosis and liver injury, activated p53 to increase p27 expression, and prevented JNK activation. This was associated with suppression of hepatocellular proliferation. DEN-injected Jnk1Direct effects of exercise dampen proliferation of dysplastic hepatocytes to reduce 3-month dysplastic foci and 6-month incidence of DEN-induced HCC in obese, insulin-resistant mice. The effects of exercise that potentially slow hepatocarcinogenesis include p53-mediated induction of p27 and prevention of JNK activation.Fatty liver disease commonly occurs alongside obesity and diabetes, contributing to rapidly increasing rates of liver cancer throughout the world. Herein, we show that exercise reduces the incidence and progression of hepatocellular carcinoma in mouse models. The effect of exercise on cancer risk was shown to be independent of changes in weight. Exercise could be a protective mechanism against liver cancer in at-risk individuals.

Despite advances in antiviral therapy, molecular drivers of Hepatitis C Virus (HCV)-related liver disease remain poorly characterised. Chronic infection with HCV genotypes (1 and 3) differ in presentation of liver steatosis and virological response to therapies, both to interferon and direct acting antivirals. To understand what drives these clinically important differences, liver expression profiles of patients with HCV Genotype 1 or 3 infection (n = 26 and 33), alcoholic liver disease (n = 8), and no liver disease (n = 10) were analysed using transcriptome-wide microarrays. In progressive liver disease, HCV genotype was the major contributor to altered liver gene expression with 2151 genes differentially expressed >1.5-fold between HCV Genotype 1 and 3. In contrast, only 6 genes were altered between the HCV genotypes in advanced liver disease. Induction of lipogenic, lipolytic, and interferon stimulated gene pathways were enriched in Genotype 1 injury whilst a broad range of immune-associated pathways were associated with Genotype 3 injury. The results are consistent with greater lipid turnover in HCV Genotype 1 patients. Moreover, the lower activity in inflammatory pathways associated with HCV genotype 1 is consistent with relative resistance to interferon-based therapy. This data provides a molecular framework to explain the clinical manifestations of HCV-associated liver disease.

The underlining mechanisms of dietary cholesterol and nonalcoholic steatohepatitis (NASH) in contributing to hepatocellular carcinoma (HCC) remain undefined. Here we demonstrated that high-fat-non-cholesterol-fed mice developed simple steatosis, whilst high-fat-high-cholesterol-fed mice developed NASH. Moreover, dietary cholesterol induced larger and more numerous NASH-HCCs than non-cholesterol-induced steatosis-HCCs in diethylnitrosamine-treated mice. NASH-HCCs displayed significantly more aberrant gene expression-enriched signaling pathways and more non-synonymous somatic mutations than steatosis-HCCs (335 ± 84/sample vs 43 ± 13/sample). Integrated genetic and expressional alterations in NASH-HCCs affected distinct genes pertinent to five pathways: calcium, insulin, cell adhesion, axon guidance and metabolism. Some of the novel aberrant gene expression, mutations and core oncogenic pathways identified in cholesterol-associated NASH-HCCs in mice were confirmed in human NASH-HCCs, which included metabolism-related genes (ALDH18A1, CAD, CHKA, POLD4, PSPH and SQLE) and recurrently mutated genes (RYR1, MTOR, SDK1, CACNA1H and RYR2). These findings add insights into the link of cholesterol to NASH and NASH-HCC and provide potential therapeutic targets., Nonalcoholic steatohepatitis (NASH) and dietary cholesterol are risk factors for hepatocellular carcinoma (HCC). Here, the authors utilise mouse models to show that dietary cholesterol induces NASH by deregulating genes involved in metabolism, inflammation and calcium signaling to induce NASH-HCC.

There has been a concerted effort to change the nomenclature of non-alcoholic fatty liver disease (NAFLD) to metabolic dysfunction-associated fatty liver disease (MAFLD), and one wonders if it is appropriate and timely to bid adieu to the good old term. Numerous reasons have been put forth to justify this, as outlined in a recently published statement proposing the change. However, there are considerable flaws in the proposal, and changing NAFLD to MAFLD is unlikely to move the field forward.We have serious misgivings on this matter.o begin with, the statement by the MAFLD consensus group that the term NAFLD was coined by Ludwig and colleagues is factually incorrect. Although the histological features of NAFLD were first described decades ago, Klatskin and his colleagues, in 1979, were the first to use the term ‘non-alcoholic liver disease’.Later, while reporting similar findings, Ludwig and colleagues coined the term “non-alcoholic steatohepatitis (NASH)”.Although an association between metabolic syndrome (MS) and NAFLD has been established and NAFLD termed the hepatic manifestation of MS, this generalisation has since been questioned, since the complex heterogeneity of this entity precludes any single postulation to explain its pathogenesis. Besides, individuals with normal BMI also develop NAFLD, and studies in non-Caucasian populations have shown that a significant proportion of patients with NAFLD do not have insulin resistance (IR).Further, even with elevated hepatic triacylglycerol and diacylglycerol content, hepatic IR has not been observed in murine models,and dissociation of hepatic steatosis from IR has also been noted in a subset of individuals. Importantly, in subjects with PNPLA3 polymorphisms,steatosis occurs independently of IR and serum lipid concentration. Further, increased serum bile acid levels also seem to be independently associated with NASH in non-diabetics.NAFLD is also associated with gut dysbiosis independent of BMI and IR. Finally, “soft” drink consumption has also been linked to the development of fatty liver independent of obesity, diabetes and hyperlipidemia.and last but not least, cigarette smoking too has been found to be an independent risk factor in NAFLD progression. Thus it is clear that pathogenesis of NAFLD is multifactorial and complex, involving multiple and divergent pathways.In Medicine, a change of name of any disease has significant implications for both medical professionals and patients.The term ‘NAFLD’ aptly describes individuals who have fatty liver but neither consume significant amounts of alcohol nor had any other reason for fatty liver. Research in NAFLD to date has failed to pinpoint any factor as the sole cause for hepatic steatosis and NAFLD still encompasses a spectrum of disorders, metabolic syndrome being a part – maybe a major part – of that spectrum with a relatively barren treatment armamentarium. Will changing nomenclature address these concerns? We fear it might paradoxically misdirect therapeutics in the direction of MS alone which may ultimately turn out to be a red herring. The consensus group found multiple faults with the term NAFLD; these include: i) NAFLD is a disease of exclusion instead of being defined by inclusion, ii) NAFLD is a vastly heterogeneous entity and cannot be managed as one single condition and iii) patients with NAFLD do consume alcohol and the impact of alcohol, albeit in non-significant amounts, is under scrutiny. In Medicine, naming a disease through exclusion has been acceptable since time immemorial. Non-Hodgkin lymphoma encompasses vastly diverse malignancies and yet the terminology very effectively delineates those disorders from Hodgkin lymphoma. Regarding heterogeneity, it is not clear how a mere change of name would make the entity more homogeneous. If the word “metabolic” in MAFLD is meant as a reference to MS, it would be a rejection of much of the scientific evidence gathered on NAFLD pathogenesis. In contrast to the assertion of the proponents of MAFLD, who after splitting “nonalcoholic” into - ‘non’ and ‘alcoholic’, suggest that the word “non” trivializes the problem while the word “alcoholic” demeans the patient, we believe that the word ‘nonalcoholic’ does go a long way in destigmatizing the patient. The European Liver Patients Association (ELPA) is believed to have expressed displeasure with the term NAFLD to the European Commission in 2018, and suggested that a change in nomenclature was required. The degree of assertion and the rationale for such a suggestion are unclear; it is also unclear whether the diverse pathogenesis of NAFLD – especially in non-Caucasians – was considered in the decision. Further, an opinion on the impact of non-significant alcohol intake on hepatic steatosis is also unclear as acknowledged in the consensus statement. Notably, metabolic abnormalities and BMI are well described risk factors for alcoholic liver disease too,but it is unclear why both conditions more or less related to alcohol should be brought under the umbrella of MAFLD. The change of nomenclature has also been argued against since NAFLD is treated by cardiologists, diabetologists and primary care providers in addition to hepatologists; a name change could create unnecessary clinical confusion and coding issues. The heterogeneity of NAFLD and presence of multiple pathophysiological pathways inherent to its progression implies that the time is ripe to classify NAFLD in a novel way that takes into account the various pathophysiological processes. We wish to propose the ‘MEGA-D’ classification emphasising the ‘mega diversity’ or heterogeneity in NAFLD where NAFLD remains the umbrella entity with different subgroups under it (NAFLD-M: Metabolic syndrome associated NAFLD, NAFLD-E: Environmental Stressor Related NAFLD, NAFLD-G: Genetic Factor Associated NAFLD, NAFLD-A: Bile Acid Dysregulation Related NAFLD, NAFLD-D: Gut Dysbiosis Related NAFLD) representing separate pathways culminating in hepatic steatosis. We feel, instead of semantic juggling, collaborative efforts should be launched worldwide to better understand the vast heterogeneity in NAFLD across populations and ethnicities and explore its different pathophysiologic mechanisms, with the sole purpose of modifying disease progression, bolstering the treatment arsenal and curbing this epidemic.

Background In clinical trials, hepatitis C virus (HCV) salvage treatment with sofosbuvir/velpatasvir/voxilaprevir (SOF/VEL/VOX) achieved an SVR12 rate of >95% in NS5A-experienced participants. Lower SVR12 rates have been reported in real-world studies, particularly for genotype (GT)3 infection and cirrhosis. We determined the efficacy and safety of SOF/VEL/VOX in a large real-world cohort. Methods We assessed the efficacy of salvage SOF/VEL/VOX for HCV infection in NS5A-inhibitor experienced participants with cirrhosis and portal hypertension, prior liver transplantation (LT) or severe extra-hepatic manifestations. SOF/VEL/VOX was available via an early access program. The primary outcome was SVR12. Secondary outcome was frequency of adverse events (AE). Findings Ninety-seven participants were included. Median age was 58, 82% were male, 78% had cirrhosis, most with portal hypertension (61%, n = 46/76), and 18% had prior-LT. Of the cirrhotic participants, 96% were Child-Turcotte-Pugh class A, and 4% were class B. Of the 72% with GT3, 76% were also cirrhotic. By intention-to-treat analysis, SVR12 rate was 85% (n = 82/97). Per protocol, the SVR12 rate was 90%, including 91% in GT1 (GT1a n = 18/18, GT1b n = 2/4), 89% in GT3 (n = 59/66) and 100% in GT6 (n = 3/3). SVR12 in participants with GT3 and cirrhosis was 90%. No predictors of non-SVR12 were identified. There were 4 serious AEs including 1 death and 3 hepatic decompensation events. NS5A resistance-associated substitutions detected at baseline did not affect SVR12. Conclusions This real-world study confirms high efficacy of SOF/VEL/VOX for the treatment of difficult-to-cure NS5A-inhibitor experienced patients, including those with GT3 and cirrhosis. Treatment was well tolerated in most; however, serious AEs can occur in those with advanced liver disease.

Background and aims Clinical syndromes associated with biallelic mutations of bile acid (BA) transporters usually present in childhood. Subtle mutations may underlie intrahepatic cholestasis of pregnancy (ICP) and oral contraceptive steroid (OCS) induced cholestasis. In five women with identified genetic mutations of such transporters, with eight observed pregnancies complicated by ICP, we examined relationships between transporter mutations, clinical phenotypes, and treatment outcomes. Methods Gene mutation analysis for BA transporter deficiencies was performed using Next Generation/Sanger sequencing, with analysis for gene deletions/duplications. Results Intrahepatic cholestasis of pregnancy was early-onset (9-32 weeks gestation) and severe (peak BA 74-370 μmol/L), with premature delivery (28+1 -370 weeks gestation) in 7/8 pregnancies, in utero passage of meconium in 4/8, but overall good perinatal outcomes, with no stillbirths. There was generally no response to ursodeoxycholic acid and variable responses to rifampicin and chelation therapies; naso-biliary drainage appeared effective in 2/2 episodes persisting post-partum in each of the two sisters. Episodic jaundice occurring spontaneously or provoked by non-specific infections, and OCS-induced cholestasis, had previously occurred in 3/5 women. Two cases showed biallelic heterozygosity for several ABCB11 mutations, one was homozygous for an ABCB4 mutation and a fourth case was heterozygous for another ABCB4 mutation. Conclusions Early-onset or recurrent ICP, especially with previous spontaneous or OCS-induced episodes of cholestasis and/or familial cholestasis, may be attributable to transporter mutations, including biallelic mutations of one or more transporters. Response to standard therapies for ICP is often incomplete; BA sequestering therapy or naso-biliary drainage may be effective. Optimized management can produce good outcomes despite premature birth and evidence of fetal compromise.

Background Liver regeneration following partial hepatectomy (PHx) is a complicated process involving multiple organs and several types of signaling networks. The bile acid-activated metabolic pathways occupy an auxiliary yet important chapter in the entire biochemical story. PHx is characterized by rapid but transient bile acid overload in the liver, which constitutes the first wave of proliferative signaling in the remnant hepatocytes. Bile acids trigger hepatocyte proliferation through activation of several nuclear receptors. Following biliary passage into the intestines, enterocytes reabsorb the bile acids, which results in the activation of farnesoid X receptor (FXR), the consequent excretion of fibroblast growth factor (FGF)19/FGF15, and its release into the enterohepatic circulation. FGF19/FGF15 subsequently binds to its cognate receptor, fibroblast growth factor receptor 4 (FGFR4) complexed with β-klotho, on the hepatocyte membrane, which initiates the second wave of proliferative signaling. Because some bile acids are toxic, the remnant hepatocytes must resolve the potentially detrimental state of bile acid excess. Therefore, the hepatocytes orchestrate a bile acid detoxification and elimination response as a protective mechanism in concurrence with the proliferative signaling. The response in part results in the excretion of (biotransformed) bile acids into the canalicular system, causing the bile acids to end up in the intestines. Relevance for patients Recently, FXR agonists have been shown to promote regeneration via the gut-liver axis. This type of pharmacological intervention may prove beneficial for patients with hepatobiliary tumors undergoing PHx. In light of these developments, the review provides an in-depth account of the pathways that underlie post-PHx liver regeneration in the context of bile acid homeostasis in the liver and the gut-liver signaling axis.

Non-alcoholic steatohepatitis (NASH) is strongly associated with overnutrition, insulin resistance, and predisposition to type 2 diabetes. To critically analyze the translational significance of currently used animal models of NASH, we reviewed articles published during the last 3 years that studied NASH pathogenesis using mouse models. Among 146 articles, 34 (23%) used models in which overnutrition was reported, and 36 (25%) demonstrated insulin resistance, with or without glucose intolerance. Half the articles contained no information on whether mice exhibited overnutrition or insulin resistance. While 75 papers (52%) reported > 2-fold increase of serum/plasma alanine aminotransferase (ALT) compared with controls, ALT levels were near normal or not reported in 48%. Liver pathology was assessed by a pathologist with an interest in liver pathology in 53% of articles published in gastroenterology/hepatology journals, versus 43-44% in other journals. While there appears to be a trend to use models that are potentially relevant to the pathogenesis of human NASH, journals currently publish data on mouse models in which overnutrition and insulin resistance do not occur, without ALT increase or appropriate analysis of NASH pathology. We recommend that investigators, reviewers, and journal editors carefully consider the validity of NASH models in current use and that moves are made to reach a consensus on what the minimal criteria should be.

Liver progenitor cells have the potential to repair and regenerate a diseased liver. The success of any translational efforts, however, hinges on thorough understanding of the fate of these cells after transplant, especially in terms of long-term safety and efficacy. Here, we report transplantation of a liver progenitor population isolated from human fetal livers into immune-permissive mice with follow-up up to 36 weeks after transplant. We found that human progenitor cells engraft and differentiate into functional human hepatocytes in the mouse, producing albumin, alpha-1-antitrypsin, and glycogen. They create tight junctions with mouse hepatocytes, with no evidence of cell fusion. Interestingly, they also differentiate into functional endothelial cell and bile duct cells. Transplantation of progenitor cells abrogated carbon tetrachloride-induced fibrosis in recipient mice, with downregulation of procollagen and anti-smooth muscle actin. Paradoxically, the degree of engraftment of human hepatocytes correlated negatively with the anti-fibrotic effect. Progenitor cell expansion was most prominent in cirrhotic animals, and correlated with transcript levels of pro-fibrotic genes. Animals that had resolution of fibrosis had quiescent native progenitor cells in their livers. No evidence of neoplasia was observed, even up to 9 months after transplantation. Human fetal liver progenitor cells successfully attenuate liver fibrosis in mice. They are activated in the setting of liver injury, but become quiescent when injury resolves, mimicking the behavior of de novo progenitor cells. Our data suggest that liver progenitor cells transplanted into injured livers maintain a functional role in the repair and regeneration of the liver.

We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ≥0.5%, whereas mice on lower-cholesterol diets developed only simple steatosis. Hepatic cholesterol crystals and crown-like structures also developed at a dietary cholesterol concentration ≥0.5%. Crown-like structures consisted of activated Kupffer cells (KCs) staining positive for NLRP3 and activated caspase 1, which surrounded and processed cholesterol crystal-containing remnant LDs of dead hepatocytes. The KCs processed LDs at the center of crown-like structures in the extracellular space by lysosomal enzymes, ultimately transforming into lipid-laden foam cells. When HepG2 cells were exposed to LDL cholesterol, they developed cholesterol crystals in LD membranes, which caused activation of THP1 cells (macrophages) grown in coculture; upregulation of TNF-alpha, NLRP3, and interleukin 1beta (IL1β) mRNA; and secretion of IL-1beta. In conclusion, cholesterol crystals form on the LD membrane of hepatocytes and cause activation and cholesterol loading of KCs that surround and process these LDs by lysosomal enzymes.

Background Entecavir and tenofovir potently suppress hepatitis B virus (HBV) replication so that serum HBV DNA levels 20% of cases for unclear reasons. Aim We tested whether 4-week viral load (VL) assessment could improve 96-week treatment outcome. Methods Data on all chronic hepatitis B patients treated with entecavir or tenofovir between 2005 and 2014 were entered prospectively. Full data capture included pre-treatment, weeks 4, 24, 48 and 96 HBV DNA titre, HBeAg, age, gender, antiviral agent and dose escalation. Compliance data were compiled from pharmacy records, doctors’ letters and clinic bookings/attendance. Time to achieve complete viral suppression (HBV DNA < 20 IU/mL) was graphed using Kaplan–Meier curves. Factors affecting this were examined using a multivariate Cox Proportional Hazard model. Results Among 156 patients treated, 72 received entecavir and 84 tenofovir. Pre-treatment HBV DNA titre, 4-week assessment and compliance impacted significantly on time to complete viral suppression. At 96 weeks, 90% of those assessed as compliant by 4-week HBV DNA had complete viral suppression versus 50% followed by 6-month VL estimation. Continuing care by the same physician was related to 4-week VL testing and optimal compliance. Conclusions Medium-term outcomes of HBV antiviral therapy are improved by early on-treatment VL testing, facilitating patient engagement and improved compliance. The observation that 90% complete viral suppression after 2 years monotherapy is achievable in a routine clinic setting questions the need for combination therapy in HBV cases with suboptimal response.

Objective Nonalcoholic steatohepatitis (NASH) is the outcome of interactions between overnutrition, energy metabolism, and adipose function. Obeticholic acid (OCA) improves steatosis in patients but for unknown reasons does not resolve NASH pathology. This study therefore investigated OCA effects in Wt mice, which develop obesity with atherogenic dietary feeding, and appetite-dysregulated, Alms1 mutant foz/foz mice fed the same diet, which develop metabolic obesity and diabetes. Methods OCA (1 mg/kg) was administered orally to female foz/foz mice and Wt littermates from weaning until 28 weeks. Adipose indices, glucose tolerance, and fatty liver pathology were studied. Experiments were repeated with OCA 10 mg/kg. Results OCA reduced body weight and hepatic lipids and improved glucose disposal only in Wt mice. OCA limited Wt adipose expansion, altered morphometry in favor of small adipocytes, enhanced expression of genes indicating adipose browning, and reduced crown-like structure number in visceral adipose tissue. foz/foz mice showed more crown-like structures in all compartments; OCA failed to alter adipose morphometry, browning, inflammation, or improve NASH severity, even at 10 mg/kg. Conclusions OCA improved adipose indices, glucose tolerance, and steatosis in a milder metabolic phenotype but failed to improve these factors in morbidly obese diabetic mice. These results help explain OCA's limited efficacy to reverse human NASH.

Summary Non-alcoholic fatty liver disease affects 20–40% of the population. Its active form, non-alcoholic steatohepatitis (NASH), is characterised by hepatocyte injury, liver inflammation, and progression of fibrosis, and has emerged as one of the most important causes of liver failure and hepatocellular carcinoma. Weight reduction of 10% by dietary restriction and regular exercise is sufficient to reverse NASH in most patients, but in practice this reduction is often not achieved. Available drugs such as vitamin E, pioglitazone, and pentoxifylline have borderline efficacy, but are limited by potential side-effects and toxicities, and do not improve liver fibrosis. However, basic and translational research has improved our understanding of the pathophysiology of NASH, thereby identifying several promising new treatment targets. Several drugs are in phase 2 and 3 development and could enter clinical practice in the near future. In this Review, we discuss the pathogenesis, treatment evaluation, existing therapies, and potential new treatments for NASH.

BACKGROUND AND AIM While gender differences in hepatocellular carcinoma (HCC) are profound, the mechanism is unclear. Using castration and hormone replacement strategies, we tested whether these gender differences are attributable to testosterone or estradiol/progesterone effects on cell cycle regulators and p53. METHODS We studied dysplastic liver and HCCs in intact and castrated diethylnitrosamine-injected C57BL/6J male and female mice, with or without hormonal replacement. Effects of sex steroids on proliferation and survival of primary hepatocytes and primary HCC cells were also characterized. RESULTS Diethylnitrosamine-injected female mice displayed fewer dysplastic foci and slower onset of HCC than male mice, with smaller/more differentiated tumors and fewer metastases. Castration of diethylnitrosamine-injected male mice reduced cyclin E kinase and augmented hepatocyte apoptosis compared with intact male mice; estradiol/progesterone enhanced these effects. In intact female mice, cyclin E kinase activity was less than in males; testosterone administered to ovariectomized female mice upregulated cyclin E, increased cyclin E kinase, and accelerated hepatocarcinogenesis. In vitro, testosterone increased expression of cell cycle regulators (cyclin D1, cyclin E, and cyclin-dependent kinase 2) and reduced p53 and p21, which enhanced hepatocyte viability. In contrast, estradiol both suppressed hepatocyte cell cycle markers, upregulated p53 and reduced viability of hepatocytes and HCC cells. CONCLUSIONS Testosterone is the positive regulator of hepatocyte cell cycle via cyclin E, while estradiol plays a negative role by effects of p53 and p21. Together, both sex hormones determine the male predominance of gender differences in hepatocarcinogenesis.

Overnutrition, usually with obesity and genetic predisposition, lead to insulin resistance, which is an invariable accompaniment of nonalcoholic fatty liver disease (NAFLD). The associated metabolic abnormalities, pre- or established diabetes, hypertension and atherogenic dyslipidemia (clustered as metabolic syndrome) tend to be worse for nonalcoholic steatohepatitis (NASH), revealing it as part of a continuum of metabolic pathogenesis. The origins of hepatocellular injury and lobular inflammation which distinguish NASH from simple steatosis have intrigued investigators, but it is now widely accepted that NASH results from liver lipotoxicity. The key issue is not the quantity of liver fat but the type(s) of lipid molecules that accumulate, and how they are "packaged" to avoid subcellular injury. Possible lipotoxic mediators include free (unesterified) cholesterol, saturated free fatty acids, diacylglycerols, lysophosphatidyl-choline, sphingolipids and ceramide. Lipid droplets are intracellular storage organelles for non-structural lipid whose regulation is influenced by genetic polymorphisms, such as PNPLA3. Cells unable to sequester chemically reactive lipid molecules undergo mitochondrial injury, endoplasmic reticulum (ER) stress and autophagy, all processes of interest for NASH pathogenesis. Lipotoxicity kills hepatocytes by apoptosis, a highly regulated, non-inflammatory form of cell death, but also by necrosis, necroptosis and pyroptosis; the latter involve mitochondrial injury, oxidative stress, activation of c-Jun N-terminal kinase (JNK) and release of danger-associated molecular patterns (DAMPs). DAMPs stimulate innate immunity by binding pattern recognition receptors, such as Toll-like receptor 4 (TLR4) and the NOD-like receptor protein 3 (NLRP3) inflammasome, which release a cascade of pro-inflammatory chemokines and cytokines. Thus, lipotoxic hepatocellular injury attracts inflammatory cells, particularly activated macrophages which surround ballooned hepatocytes as crown-like structures. In both experimental and human NASH, livers contain cholesterol crystals which are a second signal for NLRP3 activation; this causes interleukin (IL)-1β and IL18 secretion to attract and activate macrophages and neutrophils. Injured hepatocytes also liberate plasma membrane-derived extracellular vesicles; these have been shown to circulate in NASH and to be pro-inflammatory. The way metabolic dysfunction leads to lipotoxicity, innate immune responses and the resultant pattern of cellular inflammation in the liver are likely also relevant to hepatic fibrogenesis and hepatocarcinogenesis. Pinpointing the key molecules involved pharmacologically should eventually lead to effective pharmacotherapy against NASH.

The aim of this study was to evaluate cenicriviroc (CVC), a dual antagonist of C-C chemokine receptor types 2 and 5, for treatment of nonalcoholic steatohepatitis (NASH) with liver fibrosis. A randomized, double-blind, multinational phase 2b study enrolled subjects with NASH, a nonalcoholic fatty liver disease activity score [NAS] ≥4, and liver fibrosis (stages 1-3, NASH Clinical Research Network) at 81 clinical sites. Subjects (N = 289) were randomly assigned CVC 150 mg or placebo. Primary outcome was ≥2-point improvement in NAS and no worsening of fibrosis at year 1. Key secondary outcomes were: resolution of steatohepatitis and no worsening of fibrosis; improvement in fibrosis by ≥1 stage and no worsening of steatohepatitis. Biomarkers of inflammation and adverse events were assessed. Full study recruitment was achieved. The primary end point of NAS improvement in the intent-to-treat population and resolution of steatohepatitis was achieved in a similar proportion of subjects on CVC (N = 145) and placebo (N = 144) (16% vs 19%, P = 0.52 and 8% vs 6%, P = 0.49, respectively). However, the fibrosis end point was met in significantly more subjects on CVC than placebo (20% vs 10%; P = 0.02). Treatment benefits were greater in those with higher disease activity and fibrosis stage at baseline. Biomarkers of systemic inflammation were reduced with CVC. Safety and tolerability of CVC were comparable to placebo. Conclusions: After 1 year of CVC treatment, twice as many subjects achieved improvement in fibrosis and no worsening of steatohepatitis compared with placebo. Given the urgent need to develop antifibrotic therapies in NASH, these findings warrant phase 3 evaluation. ClinicalTrials.gov no: NCT02217475 (CENTAUR). This article is protected by copyright. All rights reserved.

Non-alcoholic steatohepatitis (NASH) is strongly associated with overnutrition, insulin resistance, and predisposition to type 2 diabetes. To critically analyze the translational significance of currently used animal models of NASH, we reviewed articles published during the last 3 years that studied NASH pathogenesis using mouse models. Among 146 articles, 34 (23%) used models in which overnutrition was reported, and 36 (25%) demonstrated insulin resistance, with or without glucose intolerance. Half the articles contained no information on whether mice exhibited overnutrition or insulin resistance. While 75 papers (52%) reported 2-fold increase of serum/plasma alanine aminotransferase (ALT) compared with controls, ALT levels were near normal or not reported in 48%. Liver pathology was assessed by a pathologist with an interest in liver pathology in 53% of articles published in gastroenterology/hepatology journals, versus 43-44% in other journals. While there appears to be a trend to use models that are potentially relevant to the pathogenesis of human NASH, journals currently publish data on mouse models in which overnutrition and insulin resistance do not occur, without ALT increase or appropriate analysis of NASH pathology. We recommend that investigators, reviewers, and journal editors carefully consider the validity of NASH models in current use and that moves are made to reach a consensus on what the minimal criteria should be.

Objective Adipose inflammation and dysfunction underlie metabolic obesity. Exercise improves glycemic control and metabolic indices, but effects on adipose function and inflammation are less clear. Accordingly, it was hypothesized that exercise improves adipose morphometry to reduce adipose inflammation in hyperphagic obese mice. Methods Alms1 mutant foz/foz mice housed in pairs were fed an atherogenic or chow diet; half the cages were fitted with a computer-monitored wheel for voluntary exercise. Insulin-induced AKT-phosphorylation, adipocyte size distribution, and inflammatory recruitment were studied in visceral versus subcutaneous depots, and severity of fatty liver disease was determined. Results Exercise prevented obesity and diabetes development in chow-fed foz/foz mice and delayed their onset in atherogenic-fed counterparts. Insulin-stimulated phospho-AKT levels in muscle were improved with exercise, but not in adipose or liver. Exercise suppressed adipose inflammatory recruitment, particularly in visceral adipose, associated with an increased number of small adipocyte subpopulations, and enhanced expression of beige adipocyte factor PRDM16 in subcutaneous fat. In atherogenic-fed foz/foz mice liver, exercise suppressed development of nonalcoholic steatohepatitis and related liver fibrosis. Conclusions Exercise confers metabo-protective effects in atherogenic-fed hyperphagic mice by preventing early onset of obesity and diabetes in association with enhanced muscle insulin sensitivity, improved adipose morphometry, and suppressed adipose and liver inflammation.

Free cholesterol (FC) accumulates in non-alcoholic steatohepatitis (NASH) but not in simple steatosis. We sought to establish how FC causes hepatocyte injury.In NASH-affected livers from diabetic mice, subcellular FC distribution (filipin fluorescence) was established by subcellular marker co-localization. We loaded murine hepatocytes with FC by incubation with low-density lipoprotein (LDL) and studied the effects of FC on JNK1 activation, mitochondrial injury and cell death and on the amplifying roles of the high-mobility-group-box 1 (HMGB1) protein and the Toll-like receptor 4 (TLR4).In NASH, FC localized to hepatocyte plasma membrane, mitochondria and ER. This was reproduced in FC-loaded hepatocytes. At 40 μM LDL, hepatocyte FC increased to cause LDH leakage, apoptosis and necrosis associated with JNK1 activation (c-Jun phosphorylation), mitochondrial membrane pore transition, cytochrome c release, oxidative stress (GSSG:GSH ratio) and ATP depletion. Mitochondrial swelling and crystae disarray were evident by electron microscopy. Jnk1(-/-) and Tlr4(-/-) hepatocytes were refractory to FC lipotoxicity; JNK inhibitors (1-2 μM CC-401, CC-930) blocked apoptosis and necrosis. Cyclosporine A and caspase-3 inhibitors protected FC-loaded hepatocytes, confirming mitochondrial cell death pathways; in contrast, 4-phenylbutyric acid, which improves ER folding capacity did not protect FC-loaded hepatocytes. HMGB1 was released into the culture medium of FC-loaded wild type (WT) but not Jnk1(-/-) or Tlr4(-/-) hepatocytes, while anti-HMGB1 anti-serum prevented JNK activation and FC lipotoxicity in WT hepatocytes.These novel findings show that mitochondrial FC deposition causes hepatocyte apoptosis and necrosis by activating JNK1; inhibition of which could be a novel therapeutic approach in NASH. Further, there is a tight link between JNK1-dependent HMGB1 secretion from lipotoxic hepatocytes and a paracrine cytolytic effect on neighbouring cholesterol-loaded hepatocytes operating via TLR4.

Background & Aims Perpetuate liver inflammation is crucial in the pathogenesis of non-alcoholic steatohepatitis (NASH). Expression of CXCL10, a pro-inflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 plays a role in NASH was unknown. We aimed to investigate the functional and clinical impact of CXCL10 in NASH. Methods Cxcl10 gene-deleted ( Cxcl10 −/− ) and C57BL/6 wild type (WT) mice were fed a methionine- and choline-deficient (MCD) diet for 4 or 8weeks. In other experiments, we injected neutralizing anti-CXCL10 mAb into MCD-fed WT mice. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 control subjects. Results WT mice, fed the MCD diet, developed steatohepatitis with higher hepatic CXCL10 expression. Cxcl10 −/− mice were refractory to MCD-induced steatohepatitis. We further revealed that CXCL10 was associated with the induction of important pro-inflammatory cytokines (TNF-α, IL-1β, and MCP-1) and activation of the NF-κB pathway. CXCL10 was linked to steatosis through upregulation of the lipogenic factors SREBP-1c and LXR, and also to oxidative stress (upregulation of CYP2E1 and C/EBPβ). Blockade of CXCL10 protected against hepatocyte injury in vitro and against steatohepatitis development in mice. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, the circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. Conclusions We demonstrate for the first time that CXCL10 plays a pivotal role in the pathogenesis of experimental steatohepatitis. CXCL10 maybe a potential non-invasive biomarker for NASH patients.

Lipotoxicity associated with insulin resistance is central to nonalcoholic steatohepatitis (NASH) pathogenesis. To date, only weight loss fully reverses NASH pathology, but mixed peroxisome proliferator-activated receptor-alpha/delta (PPAR-α/δ) agonists show some efficacy. Seladelpar (MBX-8025), a selective PPAR-δ agonist, improves atherogenic dyslipidemia. We therefore used this agent to test whether selective PPAR-δ activation can reverse hepatic lipotoxicity and NASH in an obese, dyslipidemic, and diabetic mouse model. From weaning, female Alms1 mutant (foz/foz) mice and wild-type littermates were fed an atherogenic diet for 16 weeks; groups (n = 8-12) were then randomized to receive MBX-8025 (10 mg/kg) or vehicle (1% methylcellulose) by gavage for 8 weeks. Despite minimally altering body weight, MBX-8025 normalized hyperglycemia, hyperinsulinemia, and glucose disposal in foz/foz mice. Serum alanine aminotransferase ranged 300-600 U/L in vehicle-treated foz/foz mice; MBX-8025 reduced alanine aminotransferase by 50%. In addition, MBX-8025 normalized serum lipids and hepatic levels of free cholesterol and other lipotoxic lipids that were increased in vehicle-treated foz/foz versus wild-type mice. This abolished hepatocyte ballooning and apoptosis, substantially reduced steatosis and liver inflammation, and improved liver fibrosis. In vehicle-treated foz/foz mice, the mean nonalcoholic fatty liver disease activity score was 6.9, indicating NASH; MBX-8025 reversed NASH in all foz/foz mice (nonalcoholic fatty liver disease activity score 3.13). Conclusion: Seladelpar improves insulin sensitivity and reverses dyslipidemia and hepatic storage of lipotoxic lipids to improve NASH pathology in atherogenic diet-fed obese diabetic mice. Selective PPAR-δ agonists act independently of weight reduction, but counter lipotoxicity related to insulin resistance, thereby providing a novel therapy for NASH. (Hepatology Communications 2017;1:663-674).

Dysplastic hepatocytes (DH) represent altered hepatocytes with potential for malignant transformation. To date, most research on pathways to hepatocarcinogenesis has focused on use of “hepatoma” cell lines derived from hepatocellular carcinoma (HCC). We describe a novel technique for deriving/culturing DH and demonstrate their utility for functional studies in vitro, compared to primary hepatocytes (PH) and HCC. PH and DH were prepared by portal vein collagenase perfusion from C57BL/6J mice. DH were subsequently subjected to FACS. HCC from diethylnitrosamine (DEN)-injected mice were mechanically isolated. Cell cycle analyses were performed by flow cytometry and PCNA immunohistochemistry. To establish utility of DH, we studied pathways of p53 turnover, apoptosis and cell proliferation using pfithrin-α (PFT) and nutlin-3. Like PH, DH were minimally proliferative compared to HCC. Only 30±0.03% of DH were in G2/M phase versus 51±0.01% of HCC; this difference corroborated with PCNA-immunostaining of dysplastic nodules from DEN-injected mice. In DH and HCC, nutlin-3 suppressed p53 mRNA, induced p53 and mdm2 activation but paradoxically resulted in increased anti-apoptotic and proliferative activity. Primary murine DH display distinctive biological characteristics compared with PH and HCC. As an intermediate cell type to HCC, they offer a new pathobiologically relevant primary cell culture system with which to interrogate the molecular changes in hepatocarcinogenesis.

// Ning Zhang 1,2 , Eagle S. H. Chu 1,3 , Jingwan Zhang 1,3 , Xiaoxing Li 1,3 , Qiaoyi Liang 1,3 , Jie Chen 2 , Minhu Chen 2 , Narci Teoh 4 , Geoffrey Farrell 4 , Joseph J.Y. Sung 1,3 and Jun Yu 1 1 Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China 2 Department of Gastroenterology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China 3 Gastrointestinal Cancer Biology & Therapeutics Laboratory, CUHK-Shenzhen Research Institute, Shenzhen, China 4 Australian National University Medical School at The Canberra Hospital, Canberra, Australia Correspondence: Jun Yu, email: // Keywords : PPARα, tumor suppressor, nuclear factor-kappa B, signaling pathway, hepatocellular carcinoma Received : June 16, 2014 Accepted : July 11, 2014 Published : July 13, 2014 Abstract Peroxisome proliferator-activated receptor alpha (PPARα) ligands have been reported to suppress cancer growth. However, the role of PPARα in hepatocarcinogenesis remains unclear. We investigated the functional significance of PPARα in HCC. PPARα-knockout (PPARα -/- ) mice were more susceptible to diethylnitrosamine (DEN)-induced HCC at 6 months compared with wild-type (WT) littermates (80% versus 43%, P < 0.05). In resected HCCs, TUNEL-positive apoptotic cells were significantly less in PPARα -/- mice than in WT mice ( P < 0.01), commensurate with a reduction in cleaved caspase-3 and caspase-7 protein expression. Ki-67 staining showed increased cell proliferation in PPARα -/- mice ( P < 0.01), with concomitant up-regulation of cyclin-D1 and down-regulation of p15. Moreover, ectopic expression of PPARα in HCC cells significantly suppressed cell proliferation and induced apoptosis. The anti-tumorigenic function of PPARα was mediated via NF-κB as evidenced by inhibition of NF-κB promoter activity, diminution of phosphor-p65, phosphor-p50 and BCL2 levels, and enhancing IkBα protein. Chromatin immunoprecipitation analysis confirmed PPARαdirectly binds to the IkBα promoter. In conclusion, PPARα deficiency enhances susceptibility to DEN-initiated HCC. PPARα suppresses tumor cell growth by inhibiting cell proliferation and inducing cell apoptosis via direct targeting IκBα and NF-κB signaling pathway.

Coffee is one of the most popular beverages in the world. Several studies consistently show that coffee drinkers with chronic liver disease have a reduced risk of cirrhosis and a lower incidence of hepatocellular carcinoma regardless of primary etiology. With the increasing prevalence of non-alcoholic fatty liver disease (NAFLD) worldwide, there is renewed interest in the effect of coffee intake on NAFLD severity and positive clinical outcomes. This review gives an overview of growing epidemiological and clinical evidence which indicate that coffee consumption reduces severity of NAFLD. These studies vary in methodology, and potential confounding factors have not always been completely excluded. However, it does appear that coffee, and particular components other than caffeine, reduce NAFLD prevalence and inflammation of non-alcoholic steatohepatitis. Several possible mechanisms underlying coffee's hepatoprotective effects in NAFLD include antioxidative, anti-inflammatory, and antifibrotic effects, while a chemopreventive effect against hepatocarcinogenesis seems likely. The so-far limited data supporting such effects will be discussed, and the need for further study is highlighted.

steatosis. 1 Thus, whereas NAFLD is a risk factor for developing diabetes during 3- to 5-year follow-up (hazard ratio, 3–4), 70% of cases with NASH are known to have type 2 diabetes (T2D), or this is revealed by oral glucose tolerance testing. 3 Glucoseintolerance,T2D,anda family history of T2D are also risk factors for fibrotic progression of NASH. 3,4 Along the same lines, the prevalence of NAFLD is proportional to the number of components of metabolic syndrome, but with NASH pathology, 85% of patients have established metabolic syndrome, 1–3 defined by the International Diabetes Federation as central obesity plus 2 or more of glucose intolerance, high blood pressure, and atherogenic dyslipidemia.

Background: There are strong links between obesity, diabetes and hepatocellular carcinoma (HCC), but molecular mechanisms remain unclear. Aim: We tested the proposed involvement of NF-κB, IL-6/STAT3 and Akt/mTORC1 before onset (at 3 months) and at onset (6 months) of accelerated hepatocarcinogenesis in DEN-injected obese and diabetic foz/foz compared to lean wildtype (Wt) mice, and also studied the hepatocyte proliferative response to DNA damage between the obese and lean lines. Methods: Male foz/foz and Wt littermates fed normal chow were DEN-injected (10mg/kg i.p.) at age 12-15 days. To test the effect of mTOR inhibitor on growth of dysplastic hepatocytes, a separate cohort of DEN-injected foz/foz mice was administered rapamycin (4 mg/kg body weight/day). Results: foz/foz mice developed obesity, hyperinsulinemia, diabetes, adipokine dysregulation and fatty liver, without increased serum or liver TNF-α or serum IL-6. All DEN-injected foz/foz mice developed HCC by 6 mths vs. 0/10 lean Wt. At 3 mths, there were more dysplastic hepatocytes in DEN-injected foz/foz than Wt, with increased liver injury (serum ALT), hepatocyte apoptosis (M30-positive cells) and proliferation (cyclin D1, cyclin E, PCNA), but neither NF-κB nor STAT3 activation. foz/foz livers exhibited upregulation of DNA damage sensors ATM and ATR, with inadequate cell cycle checkpoint controls (CHK1, CHK2, p53, p21). Akt and mTORC1 were highly activated in livers from foz/foz vs. Wt mice. Despite such activation, rapamycin failed to reduce growth of dysplastic hepatocytes. Conclusions: Accelerated DEN-induced HCC in obese/diabetic mice is linked to enhanced growth of dysplastic hepatocytes that cannot be attributed to NF-κB or IL-6/STAT3 activation, nor to sustained mTORC1 activation. The critical mechanism for obesity-enhanced hepatocarcinogenesis lies in the disconnection between hepatocellular injury with DNA damage, and an unrestrained proliferative response. Relevance for patients: This study supports the epidemiological data linking obesity, diabetes and fatty liver disease with increased risk for developing HCC. The findings also suggest that mTORC1 inhibition may not be beneficial in the prevention of obesity-related hepatocarcinogenesis.

Background and aims : TLR9 deletion protects against steatohepatitis due to choline–amino acid depletion and high-fat diet. We measured TLR9 in human non-alcoholic steatohepatitis (NASH) livers, and tested whether TLR9 mediates inflammatory recruitment in three murine models of non-alcoholic fatty liver disease (NAFLD). Methods : We assayed TLR mRNA in liver biopsies from bariatric surgery patients. Wild-type ( Wt ), appetite-dysregulated Alms1 mutant (foz/foz), Tlr9 −/− , and Tlr9 −/− . foz/foz C57BL6/J mice and bone marrow (BM) chimeras were fed 0.2% cholesterol, high-fat, high sucrose (atherogenic[Ath]) diet or chow, and NAFLD activity score (NAS)/NASH pathology, macrophage/neutrophil infiltration, cytokines/chemokines, and cell death markers measured in livers. Results : Hepatic TLR9 and TLR4 mRNA were increased in human NASH but not simple steatosis, and in Ath-fed foz/foz mice with metabolic syndrome-related NASH. Ath-fed Tlr9 −/− mice showed simple steatosis and less Th1 cytokines than Wt. Tlr9 −/− . foz/foz mice were obese and diabetic, but necroinflammatory changes were less severe than Tlr9 +/+ .foz/foz mice. TLR9-expressing myeloid cells were critical for Th1 cytokine production in BM chimeras. BM macrophages from Tlr9 −/− mice showed M2 polarization, were resistant to M1 activation by necrotic hepatocytes/other pro-inflammatory triggers, and provoked less neutrophil chemotaxis than Wt . Livers from Ath-fed Tlr9 −/− mice appeared to exhibit more markers of necroptosis [receptor interacting protein kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] than Wt , and ∼25% showed portal foci of mononuclear cells unrelated to NASH pathology. Conclusion: Our novel clinical data and studies in overnutrition models, including those with diabetes and metabolic syndrome, clarify TLR9 as a pro-inflammatory trigger in NASH. This response is mediated via M1-macrophages and neutrophil chemotaxis.

Background and Aim By array-comparative genomic hybridization, we demonstrated cyclin E as one of seven genes associated with hepatocellular carcinoma (HCC) development in Ku70 DNA repair-deficient mice. We therefore explored the hypothesis that during hepatocarcinogenesis, cyclin E kinase can overcome the inhibitory effects of p53 and establish whether abnormal miRNA(mi-R)-34, a co-regulator of cyclin E and p53, can account for their interactions as “drivers” of HCC. Methods Dysplastic hepatocytes (DNs) and HCCs were generated from diethylnitrosamine (DEN)-injected C57BL/6 male mice at 3–12 months. Results Cyclin E/cdk2 was barely expressed in normal liver, but was readily detected in dysplastic hepatocytes, localizing to glutathione-S transferase pi-form positive cells dissected by laser-dissection. Cyclin E kinase activity preceded cyclin D1, proliferating cell nuclear antigen expression in DNs and HCCs despite maximal p53 and p21 expression. We confirmed that cyclin E, rather than cyclin D1, is the proliferative driver in hepatocarcinogenesis by immunoprecipitation experiments demonstrating preferential binding of p21 to cyclin D1, allowing cyclin E-mediated “escape” from G1/S checkpoint. We then showed cyclin E was responsible for regulating wild-type p53 by knockdown experiments in primary HCC cells; cyclin E-knockdown increased p53 and p21, diminished anti-apoptotic Bcl-XL and reduced cell viability. Conversely, blocking p53 augmented cyclin E, Bcl-XL expression and increased proliferation. Physiological interactions between cyclin E/p53/p21 were confirmed in primary hepatocytes. miR-34a,c were upregulated in dysplastic murine, human liver and HCCs compared with normal liver, and appeared to be linked to cyclin E/p53. Conclusion Upregulation of functionally active cyclin E via miR34 with loss of p53 function is associated with cell-cycle checkpoint failure increasing proliferative drive that favors hepatocarcinogenesis.

Background & Aims We have recently showed that hyperinsulinemia promotes hepatic free cholesterol (FC) accumulation in obese, insulin-resistant Alms1 mutant ( foz/foz ) mice with NASH. Here we tested whether cholesterol-lowering drugs reduce stress-activated c-Jun N-terminal kinase (JNK) activation, hepatocyte injury/apoptosis, inflammation, and fibrosis in this metabolic syndrome NASH model. Methods Female foz/foz and WT mice were fed HF (0.2% cholesterol) 16weeks, before adding ezetimibe (5mg/kg), atorvastatin (20mg/kg), or both to diet, another 8weeks. Hepatic lipidomic analysis, ALT, liver histology, Sirius Red morphometry, hepatic mRNA and protein expression and immunohistochemistry (IHC) for apoptosis (M30), macrophages (F4/80), and polymorphs (myeloperoxidase) were determined. Results In mice with NASH, ezetimibe/atorvastatin combination normalized hepatic FC but did not alter saturated free fatty acids (FFA) and had minimal effects on other lipids; ezetimibe and atorvastatin had similar but less profound effects. Pharmacological lowering of FC abolished JNK activation, improved serum ALT, apoptosis, liver inflammation/NAFLD activity score, designation as "NASH", macrophage chemotactic protein-1 expression, reduced macrophage and polymorph populations, and liver fibrosis. Conclusions Cholesterol lowering with ezetimibe/atorvastatin combination reverses hepatic FC but not saturated FFA accumulation. This dampens JNK activation, ALT release, hepatocyte apoptosis, and inflammatory recruitment, with reversal of steatohepatitis pathology and liver fibrosis. Ezetimibe/statin combination is a potent, mechanism-based treatment that could reverse NASH and liver fibrosis.

Background: Alms1 mutant (foz/foz) mice develop hyperphagic obesity, diabetes, metabolic syndrome, and fatty liver (steatosis). High-fat (HF) feeding converts pathology from bland steatosis to nonalcoholic steatohepatitis (NASH) with fibrosis, which leads to cirrhosis in humans. Objective: We sought to establish how dietary composition contributes to NASH pathogenesis. Design and Methods: foz/foz mice were fed HF diet or chow 24 weeks, or switched HF to chow after 12 weeks. Serum ALT, NAFLD activity score (NAS), fibrosis severity, neutrophil, macrophage and apoptosis immunohistochemistry, uncoupling protein (UCP)2, ATP, NF-κB activation/expression of chemokines/adhesion molecules/fibrogenic pathways were determined. Result: HF intake upregulated liver fatty acid and cholesterol transporter, CD36. Dietary switch expanded adipose tissue and decreased hepatomegaly by lowering triglyceride, cholesterol ester, free cholesterol and diacylglyceride content of liver. There was no change in lipogenesis or fatty acid oxidation pathways; instead, CD36 was suppressed. These diet-induced changes in hepatic lipids improved NAS, reduced neutrophil infiltration, normalized UCP2 and increased ATP; this facilitated apoptosis with a change in macrophage phenotype favoring M2 cells. Dietary switch also abrogated NF-κB activation and chemokine/adhesion molecule expression, and arrested fibrosis by dampening stellate cell activation. Conclusion: Reversion to a physiological dietary composition after HF feeding in foz/foz mice alters body weight distribution but not obesity. This attenuates NASH severity and fibrotic progression by suppressing NF-κB activation and reducing neutrophil and macrophage activation. However, adipose inflammation persists and is associated with continuing apoptosis in the residual fatty liver disease. Taken together, these findings indicate that other measures, such as weight reduction, may be required to fully reverse obesity-related NASH.

NAFLD--regarded as a consequence of the modern sedentary, food-abundant lifestyle prevalent in the West--was recorded in Japan nearly 50 years ago and its changing epidemiology during the past three decades is well-documented. NAFLD, and its pathologically more severe form NASH, occur in genetically susceptible people who are over-nourished. Asian people are particularly susceptible, partly owing to body composition differences in fat and muscle. Community prevalence ranges between 20% (China), 27% (Hong Kong), and 15-45% (South Asia, South-East Asia, Korea, Japan and Taiwan). This Review presents emerging data on genetic polymorphisms that predispose Asian people to NAFLD, NASH and cirrhosis, and discusses the clinical and pathological outcomes of these disorders. NAFLD is unlikely to be less severe in Asians than in other populations, but the associated obesity and diabetes pandemics have occurred more recently in Asia than in Europe and the USA, and occur with reduced degrees of adiposity. Cases of cryptogenic cirrhosis and hepatocellular carcinoma have also been attributed to NAFLD. Public health efforts to curb over-nutrition and insulin resistance are needed to prevent and/or reverse NAFLD, as well as its adverse health outcomes of type 2 diabetes, cardiovascular events, cirrhosis and liver cancer.

Fatty liver diseases are complications of the metabolic syndrome associated with obesity, insulin resistance and low grade inflammation. Our aim was to uncover mechanisms contributing to hepatic complications in this setting. We used foz/foz mice prone to obesity, insulin resistance and progressive fibrosing non-alcoholic steatohepatitis (NASH). Foz/foz mice are hyperphagic but wild-type (WT)-matched calorie intake failed to protect against obesity, adipose inflammation and glucose intolerance. Obese foz/foz mice had similar physical activity level but reduced energy expenditure. Thermogenic adaptation to high-fat diet (HFD) or to cold exposure was severely impaired in foz/foz mice compared with HFD-fed WT littermates due to lower sympathetic tone in their brown adipose tissue (BAT). Intermittent cold exposure (ICE) restored BAT function and thereby improved glucose tolerance, decreased fat mass and liver steatosis. We conclude that failure of BAT adaptation drives the metabolic complications of obesity in foz/foz mice, including development of liver steatosis. Induction of endogenous BAT function had a significant therapeutic impact on obesity, glucose tolerance and liver complications and is a potential new avenue for therapy of non-alcoholic fatty liver disease (NAFLD).

Nonalcoholic steatohepatitis (NASH) is the outcome of interactions between overnutrition, energy metabolism, and adipose function. Obeticholic acid (OCA) improves steatosis in patients but for unknown reasons does not resolve NASH pathology. This study therefore investigated OCA effects in Wt mice, which develop obesity with atherogenic dietary feeding, and appetite-dysregulated, Alms1 mutant foz/foz mice fed the same diet, which develop metabolic obesity and diabetes.OCA (1 mg/kg) was administered orally to female foz/foz mice and Wt littermates from weaning until 28 weeks. Adipose indices, glucose tolerance, and fatty liver pathology were studied. Experiments were repeated with OCA 10 mg/kg.OCA reduced body weight and hepatic lipids and improved glucose disposal only in Wt mice. OCA limited Wt adipose expansion, altered morphometry in favor of small adipocytes, enhanced expression of genes indicating adipose browning, and reduced crown-like structure number in visceral adipose tissue. foz/foz mice showed more crown-like structures in all compartments; OCA failed to alter adipose morphometry, browning, inflammation, or improve NASH severity, even at 10 mg/kg.OCA improved adipose indices, glucose tolerance, and steatosis in a milder metabolic phenotype but failed to improve these factors in morbidly obese diabetic mice. These results help explain OCA's limited efficacy to reverse human NASH.

Background & Aims NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. Methods We fed foz/foz and wild-type mice an atherogenic diet for 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks and determined the effects on liver fibrosis. Results In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro , cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. Conclusion MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. Lay summary Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.

Background There are strong links between obesity, diabetes and hepatocellular carcinoma (HCC), but molecular mechanisms remain unclear. Aim We tested the proposed involvement of NF-κB, IL-6/STAT3 and Akt/mTORC1 before onset (at 3 months) and at onset (6 months) of accelerated hepatocarcinogenesis in DEN-injected obese and diabetic foz/foz compared to lean wildtype (Wt) mice, and also studied the hepatocyte proliferative response to DNA damage between the obese and lean lines. Methods Male foz/foz and Wt littermates fed normal chow were DEN-injected (10mg/kg i.p.) at age 12-15 days. To test the effect of mTOR inhibitor on growth of dysplastic hepatocytes, a separate cohort of DEN-injected foz/foz mice was administered rapamycin (4 mg/kg body weight/day). Results foz/foz mice developed obesity, hyperinsulinemia, diabetes, adipokine dysregulation and fatty liver, without increased serum or liver TNF-α or serum IL-6. All DEN-injected foz/foz mice developed HCC by 6 mths vs. 0/10 lean Wt. At 3 mths, there were more dysplastic hepatocytes in DEN-injected foz/foz than Wt, with increased liver injury (serum ALT), hepatocyte apoptosis (M30-positive cells) and proliferation (cyclin D1, cyclin E, PCNA), but neither NF-κB nor STAT3 activation. foz/foz livers exhibited upregulation of DNA damage sensors ATM and ATR, with inadequate cell cycle checkpoint controls (CHK1, CHK2, p53, p21). Akt and mTORC1 were highly activated in livers from foz/foz vs. Wt mice. Despite such activation, rapamycin failed to reduce growth of dysplastic hepatocytes. Conclusions Accelerated DEN-induced HCC in obese/diabetic mice is linked to enhanced growth of dysplastic hepatocytes that cannot be attributed to NF-κB or IL-6/STAT3 activation, nor to sustained mTORC1 activation. The critical mechanism for obesity-enhanced hepatocarcinogenesis lies in the disconnection between hepatocellular injury with DNA damage, and an unrestrained proliferative response. Relevance for patients This study supports the epidemiological data linking obesity, diabetes and fatty liver disease with increased risk for developing HCC. The findings also suggest that mTORC1 inhibition may not be beneficial in the prevention of obesity-related hepatocarcinogenesis.

Background and Aim: Steatosis accentuates the severity of hepatic ischemia–reperfusion injury (IRI). 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (“statins”) protect the heart and brain against post-ischemic injury, without necessarily lowering serum cholesterol. We tested whether 10-day or 1-day atorvastatin administration protects livers with fatty change or non-alcoholic steatohepatitis (NASH) against IRI. Methods: Mice with dietary or genetic simple steatosis (SS) or NASH were subjected to 60 min of partial hepatic ischemia/24-h reperfusion, with/without atorvastatin administered with food (5 mg/kg body weight) for 10 days, or injected intravenously (5 mg/kg) 24 h before ischemia. Liver injury, Toll-like receptor-4 (TLR4), cytokines/chemokines, endothelial nitric oxide synthase (eNOS), activation and thromboxane B2 production were determined. Results: Atorvastatin conferred 70–90% hepatic protection against IRI in obese animals with SS or NASH, in which IRI was accentuated twofold to fivefold. IRI markedly upregulated TLR4 and activated nuclear factor-κB (NF-κB); atorvastatin abrogated these effects, as well as activating eNOS. Atorvastatin dampened the post-ischemic induction of thromboxane B2, macrophage inflammatory protein-1a, monocyte chemotactic protein-1, tumor necrosis factor-α, interleukin (IL)-12 p40, γ-interferon, IL-6, and adhesion molecules (vascular cell adhesion molecule-1, E-selectin, vascular endothelial–cadherin), and reduced macrophage and neutrophil recruitment. There was no reduction in serum cholesterol that could explain these effects, and hepatic cholesterol was normal in these mice. A single 24-h injection of atorvastatin conferred equivalent hepatoprotection. Conclusion: Statins exert major hepatoprotection against IRI in lean, fatty, and NASH livers that is not due to cholesterol removal. Rather, statins downregulate TLR4 to prevent NF-κB activation, with resultant suppression of adhesion molecules, chemokines/cytokines, and thromboxane B2 production. Short-term statin treatment is an effective, readily-available preventive agent against hepatic IRI, irrespective of obesity and fatty liver disease.

While non-alcoholic fatty liver disease (NAFLD) is highly prevalent (15% to 45%) in modern societies, only 10% to 25% of cases develop hepatic fibrosis leading to cirrhosis, end-stage liver disease or hepatocellular carcinoma. Apart from pre-existing fibrosis, the strongest predictor of fibrotic progression in NAFLD is steatohepatitis or non-alcoholic steatohepatitis (NASH). The critical features other than steatosis are hepatocellular degeneration (ballooning, Mallory hyaline) and mixed infl ammatory cell infi ltration. While much is understood about the relationship of steatosis to metabolic factors (over-nutrition, insulin resistance, hyperglycemia, metabolic syndrome, hypoadiponectinemia), less is known about infl ammatory recruitment, despite its importance for the perpetuation of liver injury and fi brogenesis. In this review, we present evidence that liver infl ammation has prognostic signifi cance in NAFLD. We then consider the origins and components of liver infl ammation in NASH. Hepatocytes injured by toxic lipid molecules (lipotoxicity) play a central role in the recruitment of innate immunity involving Toll-like receptors (TLRs), Kupffer cells (KCs), lymphocytes and neutrophils and possibly infl ammasome. The key pro-infl ammatory signaling pathways in NASH are nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK). The downstream effectors include adhesion molecules, chemokines, cytokines and the activation of cell death pathways leading to apoptosis. The upstream activators of NF-κB and JNK are more contentious and may depend on the experimental model used. TLRs are strong contenders. It remains possible that infl ammation in NASH originates outside the liver and in the gut microbiota that prime KC/TLR responses, infl amed adipose tissue and circulating infl ammatory cells. We briefl y review these mechanistic considerations and project their implications for the effective treatment of NASH. (Gut Liver 2012;6:149-171)