Liver Injury In Nonalcoholic Steatohepatitis Research Articles

Targeting Extracellular RNA Mitigates Hepatic Lipotoxicity and Liver Injury in NASH.

Non-alcoholic steatohepatitis (NASH) is a clinically serious stage of non-alcoholic fatty liver disease (NAFLD). Histologically characterized by hepatocyte ballooning, immune cell infiltration, and fibrosis, NASH, at a molecular level, involves lipid-induced hepatocyte death and cytokine production. Currently, there are very few diagnostic biomarkers available to screen for NASH, and no pharmacological intervention is available for its treatment. In this study, we show that hepatocyte damage induced by lipotoxicity results in the release of extracellular RNAs (eRNAs), which serve as damage-associated molecular patterns (DAMPs) that stimulate the expression of pro-apoptotic and pro-inflammatory cytokines, aggravate inflammation, and lead to cell death in HepG2 cells. Furthermore, the inhibition of eRNA activity by RNase 1 significantly increases cellular viability and reduces NF-kB-mediated cytokine production. Similarly, RNase 1 administration significantly improves hepatic steatosis, inflammatory and injury markers in a murine NASH model. Therefore, this study, for the first time, underscores the therapeutic potential of inhibiting eRNA action as a novel strategy for NASH treatment.

Role of aerobic exercise in ameliorating NASH: Insights into the hepatic thyroid hormone signaling and circulating thyroid hormones.

Triiodothyronine (T3) administration significantly eliminates hepatic steatosis and also has a therapeutic effect on non-alcoholic steatohepatitis (NASH). However, the potential mechanism by which T3-mediated exercise improves NASH is unknow. This study aimed to explore the effect of aerobic exercise on liver injury in NASH. Aerobic exercise was conducted to explore the effects of exercise on liver injury in NASH model induced by Atherosclerotic (Ath) diet. Biochemical evaluations, histological staining and real-time PCR were first applied to confirm the amelioration effects of exercise on NASH. RNA-sequencing (RNA-seq) analysis for livers of each group were further used to identify the underlying mechanisms of aerobic exercise. Bioinformatics methods were used to explore the key functional pathways involved in the improvement of liver tissue in NASH mice by aerobic exercise. Aerobic exercise improved hepatic steatosis, lobular inflammation and fibrosis in NASH mice. multiple inflammation-related pathways were significantly enriched in the liver of NASH group and improved by aerobic exercise. The results of gene set variation analysis (GSVA) showed a higher enrichment score of T3 response signature in NASH mice with exercise. Increased Dio1 expression in the liver of NASH with exercise mice and increased circulating FT3 and FT4 levels upon aerobic exercise were confirmed. We found that aerobic exercise could significantly reduce hepatic lipid accumulation, inflammatory infiltration and fibrosis progression in the liver of NASH mice. Hepatic thyroid hormone signaling activation and circulating thyroid hormones is potentially involved in the amelioration effect of aerobatic exercise on NASH progression.

OC-02Activation of Liver X Receptor-α in the gut protects against liver injury in nonalcoholic steatohepatitis (NASH)

Introduction: Nonalcoholic steatohepatitis (NASH) is the most common form of progressive liver disease. The role of Liver X Receptor-α (LXRα), a nuclear hormone receptor involved in the regulation of hepatic lipid homeostasis and inflammation, has been studied in metabolic diseases, but its effects in the pathogenesis of NASH remain to be determined.

Proteasome dysfunction under compromised redox metabolism dictates liver injury in NASH through ASK1/PPARγ binodal complementary modules

Incidence of hepatotoxicity following acute drug-induced proteasomal inhibition and development of chronic proteasome dysfunction in obesity and insulin resistance underscores the crucial importance of hepatic protein homeostasis albeit with an elusive molecular basis and therapeutic opportunities. Apart from lipotoxicity and endoplasmic reticulum (ER) stress, herein we report that hepatocytes are highly susceptible to proteasome-associated metabolic stress attune to altered redox homeostasis. Bortezomib-induced proteasomal inhibition caused severe hepatocellular injury independent of ER stress via proapoptotic Apoptosis Signal-regulating Kinase 1 (ASK1)- c-Jun N-terminal kinase (JNK1)- p38 signaling concomitant with inadequate peroxisome proliferator-activated receptor γ (PPARγ)- Nuclear factor erythroid 2-related factor 2 (Nrf2) -driven antioxidant response. Although inhibition of ASK1 rescued acute hepatotoxicity, hepatic depletion of PPARγ or its physiological activator pigment epithelium-derived factor (PEDF) further aggravated liver injury even under ASK1 inhibition, emphasizing that endogenous PPARγ driven antioxidant activity serves as a prerequisite for the favorable therapeutic outcome of ASK1 inhibition. Consequently, ASK1 inhibitor selonsertib and PPARγ agonist pioglitazone in pharmacological synergism ameliorated bortezomib-induced hepatotoxicity and significantly prolonged survival duration in mice. Moreover, we showed that proteasome dysfunction is associated with ASK1 activation and insufficient PPARγ/Nrf2-driven antioxidative response in a subset of human nonalcoholic steatohepatitis (NASH) patients and the preclinical NASH model. The latter remains highly responsive to the drug combination marked by revamped proteasomal activity and alleviated hallmarks of NASH such as steatosis, fibrosis, and hepatocellular death. We thus uncovered a pharmacologically amenable interdependent binodal molecular circuit underlying hepatic proteasomal dysfunction and associated oxidative injury.

Hydrogen-rich water protects against liver injury in nonalcoholic steatohepatitis through HO-1 enhancement via IL-10 and Sirt 1 signaling.

Nonalcoholic steatohepatitis (NASH) could progress to hepatic fibrosis in the absence of effective control. The purpose of our experiment was to investigate the protective effect of drinking water with a high concentration of hydrogen, namely, hydrogen-rich water (HRW), on mice with nonalcoholic fatty liver disease to elucidate the mechanism underlying the therapeutic action of molecular hydrogen. The choline-supplemented, l-amino acid-defined (CSAA) or the choline-deficient, l-amino acid-defined (CDAA) diet for 20 wk was used to induce NASH and fibrosis in the mice model and simultaneously treated with the high-concentration 7-ppm HRW for different periods (4 wk, 8 wk, and 20 wk). Primary hepatocytes were stimulated by palmitate to mimic liver lipid metabolism during fatty liver formation. Primary hepatocytes were cultured in a closed vessel filled with 21% O2 + 5% CO2 + 3.8% H2 and N2 as the base gas to verify the response of primary hepatocytes in a high concentration of hydrogen gas in vitro. Mice in the CSAA + HRW group had lower serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and milder histological damage. The inflammatory cytokines were expressed at lower levels in the HRW group than in the CSAA group. Importantly, HRW reversed hepatocyte fatty acid oxidation and lipogenesis as well as hepatic inflammation and fibrosis in preexisting hepatic fibrosis specimens. Molecular hydrogen inhibits the lipopolysaccharide-induced production of inflammation cytokines through increasing heme oxygenase-1 (HO-1) expression. Furthermore, HRW improved hepatic steatosis in the CSAA + HRW group. Sirtuin 1 (Sirt1) induction by molecular hydrogen via the HO-1/adenosine monophosphate activated protein kinase (AMPK)/peroxisome proliferator-activated receptor α (PPARα)/peroxisome proliferator-activated receptor γ (PPAR-γ) pathway suppresses palmitate-mediated abnormal fat metabolism. Orally administered HRW suppressed steatosis induced by CSAA and attenuated fibrosis induced by CDAA, possibly by reducing oxidative stress and the inflammation response.NEW & NOTEWORTHY The mRNA expression of inflammatory cytokines in the HRW group was lower than in the CSAA group. HRW reversed hepatocyte apoptosis as well as hepatic inflammation and fibrosis in NASH specimens. Molecular hydrogen inhibits LPS-induced inflammation via an HO-1/interleukin 10 (IL-10)-independent pathway. HRW improved hepatic steatosis in the CSAA + HRW group. Sirt1 induction by molecular hydrogen via the HO-1/AMPK/PPARα/PPARγ pathway suppresses palmitate-mediated abnormal fat metabolism.

Altered Microbiota Diversity and Bile Acid Signaling in Cirrhotic and Noncirrhotic NASH-HCC.

OBJECTIVES:The precipitous increase in nonalcoholic steatohepatitis (NASH) is accompanied by a dramatic increase in the incidence of NASH-related hepatocellular carcinoma (HCC). HCC in NASH has a higher propensity to arise without pre-existing cirrhosis compared with other chronic liver diseases.METHODS:To identify the potential links between liver and gut in NASH-related hepatocarcinogenesis, we compared the gut microbiota and mediators of bile acid (BA) signaling in the absence or presence of cirrhosis through the analysis of stool and serum samples from patients with NASH non-HCC and NASH-HCC and healthy volunteers.RESULTS:Serum levels of total and individual BA were higher in NASH compared with healthy controls. Furthermore, serum levels of the primary conjugated BAs glycine-conjugated cholic acid, taurine-conjugated cholic acid, glycine-conjugated chenodeoxycholic acid, and taurine-conjugated chenodeoxycholic acid were significantly increased in cirrhotic vs noncirrhotic patients, independent of the occurrence of HCC. By contrast, serum FGF19 levels were higher in patients with NASH-HCC and associated with tumor markers as well as an attenuation of BA synthesis. Specific alterations in the gut microbiome were found for several bacteria involved in the BA metabolism including Bacteroides and Lactobacilli. Specifically, the abundance of Lactobacilli was associated with progressive disease, serum BA levels, and liver injury in NASH and NASH-HCC.DISCUSSION:Here, we demonstrate a clear association of the altered gut microbiota and primary conjugated BA composition in cirrhotic and noncirrhotic patients with NASH-HCC. Microbiota-associated alterations in BA homeostasis and farnesoid X receptor signaling, via FGF19, might thus contribute to fibrogenesis, liver injury, and tumorigenesis in NASH-HCC.

Green Tea Extract Treatment in Obese Mice with Nonalcoholic Steatohepatitis Restores the Hepatic Metabolome in Association with Limiting Endotoxemia-TLR4-NFκB-Mediated Inflammation.

Catechin-rich green tea extract (GTE) alleviates nonalcoholic steatohepatitis (NASH) by lowering endotoxin-TLR4 (Toll-like receptor-4)-NFκB (nuclear factor kappa-B) inflammation. This study aimed to define altered MS-metabolomic responses during high-fat (HF)-induced NASH that are restored by GTE utilizing livers from an earlier study in which GTE decreased endotoxin-TLR4-NFκB liver injury. Mice are fed a low-fat (LF) or HF diet for 12 weeks and then randomized to LF or HF diets containing 0% or 2% GTE for an additional 8 weeks. Global MS-based metabolomics and targeted metabolite profiling of catechins/catechin metabolites are evaluated. GTE in HF mice restores hepatic metabolites implicated in dyslipidemia insulin resistance, and inflammation. These include 122 metabolites: amino acids, lipids, nucleotides, vitamins, bile acids, flavonoids, xenobiotics, and carbohydrates. Hepatic amino acids, B-vitamins, and bile acids are inversely correlated with biomarkers of insulin resistance, liver injury, steatosis, and inflammation. Further, phosphatidylcholine metabolites are positively correlated with biomarkers of liver injury and NFκB inflammation. Thirteen catechin metabolites are identified in livers of GTE-treated mice, mostly as phase II conjugates of parental catechins or microbial-derived valerolactones. The defined anti-inflammatory/metabolic interactions advance an understanding of the mechanism by which GTE catechins protect against NFκB-mediated liver injury in NASH.

Digoxin improves steatohepatitis with differential involvement of liver cell subsets in mice through inhibition of PKM2 transactivation.

The cardiac glycoside digoxin was identified as a potent suppressor of pyruvate kinase isoform 2-hypoxia-inducible factor-α (PKM2-HIF-1α) pathway activation in liver injury mouse models via intraperitoneal injection. We have assessed the therapeutic effects of digoxin to reduce nonalcoholic steatohepatitis (NASH) by the clinically relevant oral route in mice and analyzed the cellular basis for this effect with differential involvement of liver cell subsets. C57BL/6J male mice were placed on a high-fat diet (HFD) for 10 wk and started concurrently with the gavage of digoxin (2.5, 0.5, 0.125 mg/kg twice a week) for 5 wk. Digoxin significantly reduced HFD-induced hepatic damage, steatosis, and liver inflammation across a wide dosage range. The lowest dose of digoxin (0.125 mg/kg) showed significant protective effects against liver injury and sterile inflammation. Consistently, digoxin attenuated HIF-1α sustained NLRP3 inflammasome activation in macrophages. We have reported for the first time that PKM2 is upregulated in hepatocytes with hepatic steatosis, and digoxin directly improved hepatocyte mitochondrial dysfunction and steatosis. Mechanistically, digoxin directly bound to PKM2 and inhibited PKM2 targeting HIF-1α transactivation without affecting PKM2 enzyme activation. Thus, oral digoxin showed potential to therapeutically inhibit liver injury in NASH through the regulation of PKM2-HIF-1α pathway activation with involvement of multiple cell types. Because of the large clinical experience with oral digoxin, this may have significant clinical applicability in human NASH.NEW & NOTEWORTHY This study is the first to assess the therapeutic efficacy of oral digoxin on nonalcoholic steatohepatitis (NASH) in a high-fat diet (HFD) mouse model and to determine the divergent of cell type-specific effects. Oral digoxin reduced liver damage, steatosis, and inflammation in HFD mice. Digoxin attenuated hypoxia-inducible factor (HIF)-1α axis-sustained inflammasome activity in macrophages and hepatic oxidative stress response in hepatocytes via the regulation of PKM2-HIF-1α axis pathway activation. Oral digoxin may have significant clinical applicability in human NASH.

Adropin protects against liver injury in nonalcoholic steatohepatitis via the Nrf2 mediated antioxidant capacity

Adropin, a secretory signal peptide, has shown beneficial effects on improving glucose homeostasis and dyslipidemia. However, whether this peptide affects nonalcoholic steatohepatitis (NASH) has remained unclear. In this study, the serum adropin levels, liver injury and oxidative stress were measured in diet-induced NASH mice. Adropin knock-out mice and palmitate treated primary hepatic cells were used to investigate the influence of adropin on liver injury. Our results show that serum adropin levels were decreased and negatively correlated with liver injury in NASH mice. Knockout of adropin significantly exacerbated hepatic steatosis, inflammatory responses and fibrosis in mice after either methionine-choline deficient diet (MCD) or western diet (WD) feeding. And the treatment with adropin bioactive peptides ameliorated NASH progression in mice. Adropin alleviated hepatocyte injury by upregulating the expression of Gclc, Gclm, and Gpx1 in a manner dependent on Nrf2 transcriptional activity and by increasing the glutathione (GSH) levels. And adropin significantly increased CBP expression and promoted its binding with Nrf2, which enhanced Nrf2 transcriptional activity. Furthermore, AAV8-mediated overexpression of hepatic Nrf2 expression functionally restored the liver injury induced by adropin-deficiency MCD-fed mice. These findings provide evidence that adropin activates Nrf2 signaling and plays a protective role in liver injury of NASH and therefore might represent a novel target for the prevention and treatment of NASH.

Effect of sitagliptin on hepatic histological activity and fibrosis of nonalcoholic steatohepatitis patients: a 1-year randomized control trial.

Background/purposeDipeptidyl peptidase 4 (DPP-4) expression is directly associated with hepatic lipogenesis and liver injury in nonalcoholic steatohepatitis (NASH). This study has been designed to elucidate the histological improvement of NASH with the DPP-4 inhibitor sitagliptin.Materials and methodsIn this open-label randomized control trial, paired liver biopsy was taken from 40 NASH patients. Sitagliptin 100 mg was given once daily to the SL group and no sitagliptin was given to the L group for 1 year. Patients from both groups were encouraged to exercise moderately and advised to avoid saturated fat, excessive sugar, soft drinks, fast food, and refined carbohydrates to reduce weight.ResultsSteatosis improved in the SL group (from 2.3±0.6 to 1.2±0.8; P=0.000) and the L group (from 2.1±0.6 to 1.6±0.9; P=0.008), ballooning decreased from 1.8±0.6 to 1.3±06 (P=0.002) in the SL group, but not in the L group. Nonalcoholic fatty liver disease activity score (NAS) attenuated in both groups: the SL group (from 5.8±0.9 to 3.9±1.4; P=0.000) and the L group (from 5.3±0.6 to 4.6±1.2; P=0.009). NAS improvement was much higher in the SL group (1.9±1.4) than in the L group (0.7±1.1) (P=0.006), with NAS improving by ≥2 in 13 patients from the SL group and five patients from the L group (P=0.01). Improvement was irrespective of diabetes. Regression analysis explored that sitagliptin had odds of 6.38 and weight reduction had odds of 4.51 for NAS reduction.ConclusionSitagliptin 100 mg once daily for 1 year ameliorates NAS by improving steatosis and ballooning, irrespective of diabetes. Sitagliptin has stronger efficacy than that of weight reduction.

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

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. 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. 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. 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. 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.

Mechanisms of Liver Injury in Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a disorder marked by alterations in hepatic lipid homeostasis as well as liver injury in the form of cell death, inflammation and fibrosis. Research into the pathophysiology of NASH is dynamic. New concepts from the fields of cell biology, microbiology, immunology and genetics are being tested for their applicability to NASH; discoveries in each of these areas are enriching our understanding of this complex disease. This review summarizes how recent developments from the bench and bedside are merging with more traditional concepts to reshape our view of NASH pathogenesis. Highlights include human studies that emphasize the role of de novo lipogenesis in NASH and experimental work uncovering a role for the inflammasome in NASH. Genetic predispositions to NASH are being clarified, and intestinal microbiome is emerging as a determinant of fatty liver. These unique ideas are now taking their place within an integrated picture of NASH pathogenesis.

Oxidized metabolites of linoleic acid as biomarkers of liver injury in nonalcoholic steatohepatitis

The rising prevalence of obesity in the last few decades has been accompanied by an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD encompasses a spectrum of liver pathology from isolated hepatic steatosis to steatohepatitis and advanced fibrosis. Dietary habits characterized by consumption of high-caloric, lipid-rich diets play a major role in the development of NAFLD. Recent studies have uncovered the importance of certain components of the diet. In this review, we will focus on the growing evidence for a central role of n-6 polyunsaturated fatty acids. We will discuss novel findings linking oxidative stress and increased production of reactive oxygen species in the liver to oxidation of n-6 polyunsaturated fatty acids and production of specific lipid oxidation metabolites. In particular, we will highlight the potential role of these metabolites as noninvasive markers to diagnose and monitor the extent of liver damage in patients with NAFLD.

Antioxidant Therapy and Drugs Interfering with Lipid Metabolism: Could They Be Effective in NAFLD Patients?

This review is part of a special issue dealing with various aspects of non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). We will focus on promising treatments of NASH with antioxidants and drugs that interfere with lipid metabolism.The other therapies of interest, such as diet, behavioral changes, and insulin sensitizers are presented elsewhere. Oxidative stress is believed to play a key role in the pathogenesis of NASH and other liver diseases. Antioxidants aimed at improving chronic alcoholic or viral liver diseases have been an object of study for some time. However, only a few high quality, randomized, versus placebo-controlled, double-blinded trials have been carried out to assess these drugs. Vitamin E is currently the most widely assessed antioxidant. Several questions need to be answered, including long-term tolerance and efficacy of vitamin E in particular subsets, such as diabetes and NASH-related cirrhosis. Other antioxidants are promising, and should be assessed using the standards of evidence-based medicine. NAFLD frequently coexists with hyperlipidemia and carries an increased risk of cardiovascular disease (CVD). Furthermore, altered lipid metabolism is thought to be central to the pathogenesis of liver injury in NASH. Therefore, lipid-lowering drugs are attractive therapeutic tools in the treatment of NAFLD. Statins have ameliorated surrogate markers of steatosis in several randomized controlled trials, but their impact on liver histology is unknown. They have, however, been found to be the only class of lipid-lowering drugs that reduces cardiovascular risk in NAFLD. Preliminary evidence suggests that ezetimibe, an inhibitor of intestinal and hepatic cholesterol absorption, may improve liver histology, but its impact on the risk of CVD and on clinical outcome remains to be determined. Despite strong experimental evidence supporting the use of omega-3 polyunsaturated fatty acids in NAFLD, the studies published on humans have consisted of small sample sizes and had a number of methodological flaws, including the absence of post-treatment histology. Association of antioxidants and/or lipid-lowering drugs plus other drugs of interest in NASH, such as insulin sensitizers, warrant investigation. However, as promising as these drug treatments may continue to be, they should be associated with diet and modifications in lifestyle.

Antioxidant Therapy and Drugs Interfering with Lipid Metabolism: Could They Be Effective in NAFLD Patients?

This review is part of a special issue dealing with various aspects of non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). We will focus on promising treatments of NASH with antioxidants and drugs that interfere with lipid metabolism.The other therapies of interest, such as diet, behavioral changes, and insulin sensitizers are presented elsewhere. Oxidative stress is believed to play a key role in the pathogenesis of NASH and other liver diseases. Antioxidants aimed at improving chronic alcoholic or viral liver diseases have been an object of study for some time. However, only a few high quality, randomized, versus placebo-controlled, double-blinded trials have been carried out to assess these drugs. Vitamin E is currently the most widely assessed antioxidant. Several questions need to be answered, including long-term tolerance and efficacy of vitamin E in particular subsets, such as diabetes and NASH-related cirrhosis. Other antioxidants are promising, and should be assessed using the standards of evidence-based medicine. NAFLD frequently coexists with hyperlipidemia and carries an increased risk of cardiovascular disease (CVD). Furthermore, altered lipid metabolism is thought to be central to the pathogenesis of liver injury in NASH. Therefore, lipid-lowering drugs are attractive therapeutic tools in the treatment of NAFLD. Statins have ameliorated surrogate markers of steatosis in several randomized controlled trials, but their impact on liver histology is unknown. They have, however, been found to be the only class of lipid-lowering drugs that reduces cardiovascular risk in NAFLD. Preliminary evidence suggests that ezetimibe, an inhibitor of intestinal and hepatic cholesterol absorption, may improve liver histology, but its impact on the risk of CVD and on clinical outcome remains to be determined. Despite strong experimental evidence supporting the use of omega-3 polyunsaturated fatty acids in NAFLD, the studies published on humans have consisted of small sample sizes and had a number of methodological flaws, including the absence of post-treatment histology. Association of antioxidants and/or lipid-lowering drugs plus other drugs of interest in NASH, such as insulin sensitizers, warrant investigation. However, as promising as these drug treatments may continue to be, they should be associated with diet and modifications in lifestyle.

Management of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is one of the most common hepatic disorders in the United States, but uncertainty remains as to the optimal way to manage it. Using the case of Mr T, a 60-year-old man with obesity, diabetes mellitus, and increased serum transaminase levels, an evidence-based approach to diagnosis and treatment is discussed. Diagnosis of NAFLD is based on patient clinical profile and risk factors for metabolic syndrome, the exclusion of other liver diseases, radiologic imaging and sometimes biopsy. At this point in Mr T's disease, the most important step is differentiation between simple steatosis and nonalcoholic steatohepatitis (NASH). Simple steatosis has a benign natural history, but NASH is progressive and may lead to cirrhosis, liver failure, and liver cancer. An evidence-based approach to treatment is limited by lack of large randomized trials, particularly of combinations of therapies, but weight loss, exercise, and medical therapies targeted at the mechanism of liver injury in NASH are recommended. Improved noninvasive diagnostic tests, a clearer understanding of the natural history of NAFLD, and large, well-designed clinical trials are needed.

Hepatocyte growth factor overexpression ameliorates liver inflammation and fibrosis in a mouse model of nonalcoholic steatohepatitis

Hepatocyte growth factor (HGF) is a potent growth factor involved in liver regeneration that has various effects on epithelial and nonepithelial cells. Although it has been demonstrated that HGF can reduce liver inflammation or fibrosis caused by pharmaceutical or chemical insult, no examination of its effect on liver injury in nonalcoholic steatohepatitis (NASH) has been reported. To examine the effect of HGF on liver injury in NASH, we generated a murine steatohepatitis model on an HGF overexpression transgenic (Tg) background, and fed the mice a methionine- and choline-deficient diet (MCD). In mice fed the MCD diet, serum ALT levels and the inflammation score for the Tg mice were significantly lower than those for the wild-type (Wt) control mice (P<0.01). The index of lipid peroxidation increased in the liver of the Wt mice as demonstrated by thiobarbituric acid-reactive substances. Furthermore, the liver fibrosis in Tg mice was dramatically suppressed in comparison to that in Wt mice. The gene expression of matrix metalloprotease-13 in the Tg mice was significantly increased in comparison to that of the Wt mice. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay showed the apoptotic cells to significantly decrease in number in the Tg mice in comparison to the Wt mice fed the MCD diet (P<0.01). Hepatocyte growth factor ameliorated liver inflammation and fibrosis in a murine model of NASH as a result of the anti-oxidative and anti-apoptotic effect, and the induction of fibrinolysis.

Lipotoxicity in NASH

Exposure to a sedentary lifestyle, fat-rich and fiber-poor diets, positive caloric imbalance and an extended life disrupt the metabolic homeostasis causing non-alcoholic fatty liver disease. A subset of patients with this emerging public health problem may develop non-alcoholic steatohepatitis (NASH) and progress to cirrhosis and liver cancer [1]. Understanding the molecular mechanisms promoting liver injury in NASH is not only of biomedical and public health interest, but also key to develop new avenues for specific treatment interventions. In this Snapshot article, we will outline current evolving concepts of signaling cascades that may be linked to liver injury in NASH. It is important to acknowledge that a significant amount of information has been obtained from in vitro and animal studies that may not entirely reflect the situation in the liver of patients with NASH. In addition, multiple pathways leading to apoptosis may be operational in NASH patients at the same time, but the relative contribution of each one is unknown. Nevertheless, targeting apoptotic pathways in NASH may represent a viable therapeutic strategy particularly in the context of acute liver injury augmented by fatty liver or in the setting of transplantation using highly steatotic donor livers. Major sources of hepatic saturated fatty acids (SFAs) are adipose tissue, diet, and de novo lipogenesis from glucose. Under physiological conditions, SFA are transported to mitochondria for b-oxidation or esterified for either excretion in very low density lipoproteins or storage as lipid droplets. SFA can be released from lipid droplets via macrolipophagy (Fig. 1A). Multiple mechanisms are concurrently operative to produce liver injury in hepatocytes overwhelmed by SFA, primarily from adipocyte lipolysis, and free cholesterol from de novo synthesis [1,2]. These lipids, and in particular SFA, can activate a variety of intracellular responses resulting in lipotoxic stress in the endoplasmic reticulum (ER) and mitochondria, respectively. As a consequence, apoptosis occurs which represents a key pathogenic feature of NASH. Metabolic stress from lipids has also been linked to macrolipophagy dysfunction, presumably at the level of autophagosome-lysosome fusion [3]. Whether this promotes additional lipid accumulation or increased lipolysis in NASH remains to be addressed.

Extracellular signal-regulated kinases 1/2 suppression aggravates transforming growth factor-beta1 hepatotoxicity: a potential mechanism for liver injury in methionine-choline deficient-diet-fed mice

Hepatocyte cell death is a characteristic indication in the development of non-alcoholic steatohepatitis (NASH); however, the underlying mechanism is still unclear. In this study, we examined the potential mechanism(s) involved in the development of liver injury using a methionine-choline deficient (MCD) diet feeding NASH model. Male C57BL6/J mice were fed MCD and methionine-choline sufficient (MCS) diet for two weeks before being killed. Our results showed that MCD diet feeding resulted in fatty liver and liver injury, evidenced by increased hepatic triglyceride (TG), plasma alanine aminotransferases and hepatic thiobarbituric acid reactive substances levels in MCD-fed mice. Furthermore, we found that MCD diet feeding caused remarkable suppression of hepatic extracellular signal-regulated kinases (ERK) 1/2 activation and increased transforming growth factor (TGF)-beta1 levels in plasma and the liver tissue. In vitro investigations showed that intracellular MEK/ERK1/2 activation status played a critical role in the determination of sensitivity of hepatocytes to TGF-beta1-induced cell death. HepG2 cells, otherwise resistant to TGF-beta1 killing due to high level of ERK1/2 activation, was sensitized by U0126, a specific MEK/ERK1/2 inhibitor, to TGF-beta1 cytotoxicity. H4IIEC3 cells, which have lower level of constitutive ERK1/2 activity, are sensitive to TGF-beta1-induced cell death. Lastly, we demonstrated that administration of epidermal growth factor, a strong ERK1/2 activator, to MCD-fed mice attenuated liver injury without affecting hepatic TG accumulation. Our findings demonstrated that hepatic ERK1/2 inactivation aggravates TGF-beta1-induced hepatotoxicity, which may contribute, at least in part, to the initiation of liver injury in NASH.

A randomized controlled trial of high-dose ursodesoxycholic acid for nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a prevalent liver disease associated with increased morbidity and mortality. Ursodeoxycholic acid (UDCA) may have antioxidant, anti-inflammatory, and antifibrotic properties and may reduce liver injury in NASH. To date, no studies have assessed the efficacy and safety of high-dose UDCA (HD-UDCA) in patients with NASH. We conducted a 12-month, randomized, double-blind, placebo-controlled multicenter trial to evaluate the efficacy and safety of HD-UDCA (28-35 mg/kg per day) in 126 patients with biopsy-proven NASH and elevated alanine aminotransferase (ALT) levels. The primary study end point was reduction in ALT levels from baseline in patients treated with HD-UDCA compared with placebo. Secondary study end points were the proportion of patients with ALT normalization, relative reduction in the scores of serum markers of fibrosis and hepatic inflammation, and safety and tolerability. HD-UDCA significantly reduced mean ALT levels -28.3% from baseline after 12 months compared with -1.6% with placebo (p<0.001). At the end of the trial, ALT levels normalized (≤35 IU/L) in 24.5% of patients treated with HD-UDCA and in 4.8% of patients who received placebo (p=0.003). Both results were not accounted for by changes in weight during the trial. HD-UDCA significantly reduced the FibroTest® serum fibrosis marker (p<0.001) compared with placebo. HD-UDCA also significantly improved markers of glycemic control and insulin resistance. There were no safety issues in this population. Treatment with HD-UDCA was safe, improved aminotransferase levels, serum fibrosis markers, and selected metabolic parameters. Studies with histologic end points are warranted.