Hepatitis C Virus Antiviral Drug Resistance and Salvage Therapy Outcomes Across Australia.

Background: Hepatitis C virus (HCV) infection can now be cured with well-tolerated direct-acting antiviral (DAA) therapy. However, a potential barrier to HCV elimination is the emergence of resistance-associated substitutions (RASs) that reduce the efficacy of antiviral drugs, but real-world studies assessing the clinical impact of RASs are limited. Here, an analysis of the impact of RASs on retreatment outcomes for different salvage regimens in patients nationally who failed first-line DAA therapy is reported. Methods: We collected data from 363 Australian patients who failed first-line DAA therapy, including: age, sex, fibrosis stage, HCV genotype, NS3/NS5A/NS5B RASs, details of failed first-line regimen, subsequent salvage regimens, and treatment outcome. Results: Of 240 patients who were initially retreated as per protocol, 210 (87.5%) achieved sustained virologic response (SVR) and 30 (12.5%) relapsed or did not respond. The SVR rate for salvage regimens that included sofosbuvir/velpatasvir/voxilaprevir was 94.3% (n = 140), sofosbuvir/velpatasvir 75.0% (n = 52), elbasvir/grazoprevir 81.6% (n = 38), and glecaprevir/pibrentasvir 84.6% (n = 13). NS5A RASs were present in 71.0% (n = 210) of patients who achieved SVR and in 66.7% (n = 30) of patients who subsequently relapsed. NS3 RASs were detected in 20 patients (20%) in the SVR group and 1 patient in the relapse group. NS5B RASs were observed in only 3 patients. Cirrhosis was a predictor of relapse after retreatment, as was previous treatment with sofosbuvir/velpatasvir. Conclusions: In our cohort, the SVR rate for sofosbuvir/velpatasvir/voxilaprevir was higher than with other salvage regimens. The presence of NS5A, NS5B, or NS3 RASs did not appear to negatively influence retreatment outcomes.

Role of Cholesterol-Associated Steatohepatitis in the Development of NASH.

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.

Exercise retards hepatocarcinogenesis in obese mice independently of weight control.

BACKGROUND & AIMS: 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. METHODS: 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 Jnk1-/-.foz/foz mice generated by cross breeding, as well as their genetic controls. Dysplastic hepatocytes were identified by glutathione-S-transferase pi form (GST-pi) immunohistochemistry, liver nodules were counted, and HCC was analysed by histopathology. RESULTS: Exercising 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%, p <0.05). 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 Jnk1-/-.foz/foz mice failed to develop liver tumours or HCC at 24 weeks. CONCLUSIONS: Direct 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. LAY SUMMARY: 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.

Cholesterol Crystals in Hepatocyte Lipid Droplets Are Strongly Associated With Human Nonalcoholic Steatohepatitis.

It is unclear what drives the development of fibrosing nonalcoholic steatohepatitis (NASH). We aimed to determine whether cholesterol crystallization within hepatocyte lipid droplets (LDs) distinguishes patients with fibrosing NASH from patients with isolated hepatic steatosis and to study pathways leading to cholesterol accumulation in hepatocyte LDs. Patients with fibrosing NASH (n = 16) were compared to patients with isolated steatosis (n = 14). Almost all patients with fibrosing NASH had free cholesterol staining by filipin (16/16) and cholesterol crystals (15/16) in hepatocyte LDs, mostly in association with the LD membrane, compared to only 3/14 with cholesterol crystals and 3/14 with faint filipin staining in patients with isolated steatosis (P < 0.05). We were unable to identify significant differences in the expression of genes in liver tissue related to cholesterol homeostasis or LD proteins between patients with fibrosing NASH and isolated steatosis. Human hepatoma cell line (HepG2) cells were supplemented with low-density lipoprotein (LDL)-cholesterol and oleic acid to develop large LDs, similar to those observed in patients with NASH. Fluorescent markers were used to track the uptake and intracellular trafficking of LDL-cholesterol. LDL-cholesterol was taken up by HepG2 cells and transported through the endosomal-lysosomal compartment directly to LDs, suggesting direct contact sites between late endosomes and LDs. Exposure of HepG2 cells to LDL-cholesterol resulted in a high concentration of cholesterol and cholesterol crystallization in LDs. Conclusion: Excess cholesterol is stored in the liver primarily within hepatocyte LDs where it can crystallize. Our findings are best explained by direct transport of cholesterol from late endosomes/lysosomes to LDs in hepatocytes. We found a strong association between the presence of LD cholesterol crystals and the development of fibrosing NASH in humans, suggesting a causal relationship.

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion.

OBJECTIVE AND BACKGROUND: Activation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice. METHODS: Plasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury. RESULTS: In patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects. CONCLUSION: HMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.

Activation of brown adipose tissue enhances the efficacy of caloric restriction for treatment of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is the form of nonalcoholic fatty liver disease that can evolve into cirrhosis. Lifestyle modifications achieving 10% weight loss reverse NASH, but there are no effective approved drug treatments. We previously identified defective adaptive thermogenesis as a factor contributing to metabolic syndrome and hepatic steatosis. We have now tested whether increasing nonshivering thermogenesis can improve preexisting NASH in mice. In high-fat diet-fed foz/foz mice with established NASH, treatment with ß3AR agonist restored brown adipose tissue (BAT) function, decreased body weight, improved glucose tolerance, and reduced hepatic lipid content compared to untreated counterparts, but had no impact on liver inflammation or on nonalcoholic fatty liver disease activity score (NAS). Similarly, ß3AR agonist did not alter liver pathology in other steatohepatitis models, including MCD diet-fed diabetic obese db/db mice. Caloric restriction alone alleviated the hepatic inflammatory signature in foz/foz mice. Addition of a ß3AR agonist to mice subjected to caloric restriction enhanced weight loss and glucose tolerance, and improved liver steatosis, hepatocellular injury, and further reduced liver inflammation. These changes contributed to a significantly lower NAS score such as no (0/9) animals in this group fulfilled the criteria for NASH pathology compared to eight out of ten mice under caloric restriction alone. In conclusion, ß3AR agonist counteracts features of the metabolic syndrome and alleviates steatosis, but does not reverse NASH. However, when coupled with weight loss therapy, BAT stimulation provides additional therapeutic advantages and reverses NASH.

Pathogenesis of NASH: How Metabolic Complications of Overnutrition Favour Lipotoxicity and Pro-Inflammatory Fatty Liver Disease.

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)-1ß 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.

Mouse models of non-alcoholic steatohepatitis: A reflection on recent literature.

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.

Post-hepatectomy liver regeneration in the context of bile acid homeostasis and the gut-liver signaling axis.

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 ß-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.

TLR9 is up-regulated in human and murine NASH: pivotal role in inflammatory recruitment and cell survival.

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.

Deriving and testing of dysplastic murine hepatocytes: A new platform in liver cancer research.

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.

Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.

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 (IL1ß) 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.

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

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-1ß, active caspase-1 and IL-1ß increased at 24weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1ß, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1ß expression, plasma IL-1ß, 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-1ß; 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-1ß, 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.

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

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).

Defective adaptive thermogenesis contributes to metabolic syndrome and liver steatosis in obese mice.

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).

Obeticholic acid improves adipose morphometry and inflammation and reduces steatosis in dietary but not metabolic obesity in mice.

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.

Obesity and diabetes accelerate hepatocarcinogenesis via hepatocyte proliferation independent of NF-κB or Akt/mTORC1.

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.

Testosterone regulation of cyclin E kinase: A key factor in determining gender differences in hepatocarcinogenesis.

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.