K. A. Lockman

Oxidative stress rather than triglyceride accumulation is a determinant of mitochondrial dysfunction in in vitro models of hepatic cellular steatosis

There is still debate about the relationship between fat accumulation and mitochondrial function in nonalcoholic fatty liver disease. It is a critical question as only a small proportion of individuals with steatosis progress to steatohepatitis. In this study, we focused on defining (i) the effects of triglyceride accumulation and reactive oxygen species (ROS) on mitochondrial function (ii) the contributions of triglyceride, ROS and subsequent mitochondrial impairment on the metabolism of energy substrates.

Carvedilol use is associated with improved survival in patients with liver cirrhosis and ascites

BACKGROUND & AIMS Carvedilol, a non-selective beta-blocker (NSBB) with additional anti-alpha 1 receptor activity, is a potent portal hypotensive agent and has been used as prophylaxis against variceal bleeding. However, its safety in patients with decompensated liver cirrhosis and ascites is still disputed. In this study, we examined whether long-term use of carvedilol in patients with ascites is a risk factor for mortality. METHODS A single-centre retrospective analysis of 325 consecutive patients with liver cirrhosis and ascites presenting to our Liver Unit between 1st of January 2009 to 31st August 2012 was carried out. The primary outcome was all-cause and liver-specific mortality in patients receiving or not receiving carvedilol as prophylaxis against variceal bleeding. RESULTS The final cohort after propensity score matching comprised 264 patients. Baseline ascites severity and UK end-stage liver disease (UKELD) score between carvedilol (n=132) and non-carvedilol (n=132) treated patient groups were comparable. Median follow-up time was 2.3years. Survival at the end of the follow-up was 24% and 2% for the carvedilol and the non-carvedilol groups respectively (log-rank p<0.0001). The long-term survival was significantly better in carvedilol than non-carvedilol group (log-rank p<0.001). The survival difference remained significant after adjusting for age, gender, ascites severity, aetiology of cirrhosis, previous variceal bleed, spontaneous bacterial peritonitis prophylaxis, serum albumin and UKELD with hazard ratio of 0.59 (95% confidence interval [CI]: 0.44, 0.80; p=0.001), suggesting a 41% reduction in mortality risk. When stratified by the severity of ascites, carvedilol therapy resulted in hazard ratio of 0.47 (95% CI: 0.29, 0.77; p=0.003) in those with mild ascites. Even with moderate or severe ascites, carvedilol use was not associated with increased mortality risk. CONCLUSION Long-term carvedilol therapy is not harmful in patients with decompensated cirrhosis and ascites. LAY SUMMARY The safety of carvedilol and other non-selective beta-blocker drugs in patients with liver cirrhosis and ascites is still debated. In this study, we have shown that carvedilol therapy in these patients was associated with reduced risk of mortality, particularly in those with mild ascites. We concluded that low dose, chronic treatment with carvedilol in patients with liver cirrhosis and ascites is not detrimental.

Proteomic profiling of cellular steatosis with concomitant oxidative stress in vitro.

BackgroundNutrient excess underpins the development of nonalcoholic fatty liver disease (NAFLD). The ensuing metabolic derangement is characterised by increased cellular respiration, oxidative stress and mitochondrial impairment. We have previously recapitulated these events in an in vitro cellular steatosis model. Here, we examined the distinct patterns of protein expression involved using a proteomics approach.MethodsHuman hepatoblastoma C3A cells were treated with a combination of energy substrates; lactate (L), pyruvate (P), octanoate (O) and ammonia (N). Proteins extracts were trypsinized and analyzed on a capillary HPLC OrbitrapXL mass spectrometer. Proteins were quantified using a label-free intensity based approach. Functional enrichment analysis was performed using ToppCluster via Gene Ontology (GO) database.ResultsOf the 1327 proteins identified, 104 were differentially expressed between LPON and untreated cells (defined as: ≥2 peptides; fold change ≥1.5; p-value <0.05). Seventy of these were upregulated with LPON. Functional enrichment analysis revealed enhanced protein biosynthesis accompanied by downregulation of histones H2A type 1-A, H1.2, H1.5 and H1.0I in LPON cells. Lipid binding annotations were also enriched as well as proteins involved in cholesterol synthesis, uptake and efflux. Increased expression of aldo-keto reductase family 1, member C1 and C3 suggests enhanced sterol metabolism and increased ROS-mediated lipid peroxidation.ConclusionsThe surge of energy substrates diverts free fatty acid metabolism towards pathways that can mitigate lipotoxicity. The histones depletion may represent an adaptation to increased protein synthesis. However, this can also expose DNA to oxidative stress thus should be explored further in the context of NAFLD progression.

OC-007 Oxidative Stress Rather than Triglyceride Accumulation Perturbs Glutathione Metabolism in an in Vitro Model of Cellular Steatosis

Introduction Oxidative stress is the central to molecular events leading to the progression of simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD). We have previously shown that an in vitro cellular steatosis model using C3A cells treated with energy substrates; lactate (L), pyruvate (P), octanoate (O) and ammonia (N), recapitulates the sequence of events in dietary-induced NAFLD; namely enhanced acute respiration and reactive oxygen species (ROS) formation leading to mitochondrial impairment. In contrast, treatment with oleate results in similar triglyceride accumulation but with relatively low ROS. Using a combined microarray, proteomic and metabolomic approach, we aimed to explore how triglyceride accumulation and enhanced ROS affect glutathione metabolism in our in vitro cell model. Methods C3A cells were treated with either LPON or oleate for 72 hours. Microarray RNA expression was measured using Illumina® Whole Human Genome BeadChip H12 Microarray. For proteomics, peptides were analysed by liquid chromatography (LC) coupled mass spectrometry (MS) (Agilent HPLC/OrbitrapXL). Data were quantified label-free using Progenesis LC-MS and MASCOT. For metabolomics, LC separation was performed using hydrophilic interaction chromatography with a ZIC–HILIC. MS was performed using Orbitrap Exactive with HESI 2 probe. Raw LC/MS data were processed with XCMS Centwave and mzMatch. Results LPON led to 2-fold downregulation of GCLC (encodes glutamate-cysteine ligase catalytic subunit, the rate limiting enzyme for glutathione synthesis) and upregulation of GPX1 and TXNDC12. Expression of GCLC and TXNDC12 was unchanged with oleate. Metabolomics confirmed that oxidised glutathione, glutathione disulfide, was higher in LPON- than oleate-treated cells. Among glutathione S-transferase genes, GSTA1 was unchanged with oleate but was upregulated by LPON (2.4-fold). Similarly, GSTT1, GSTK1 and GSTO1 were significantly increased by LPON. In contrast, MGST2 expression was higher in oleate than LPON-treated cells. Finally, proteomics showed that microsomal glutathione S-transferase 2 was downregulated by 2.5-fold by LPON. Conclusion Our data show that increased ROS formation rather than triglyceride accumulation alters glutathione metabolism. Such alterations may influence susceptibility to further insults, particularly those accelerating glutathione depletion, for example, paracetamol overdose. Disclosure of Interest None Declared

Comparison of the effects of long-term pioglitazone and rosiglitazone therapy on liver enzymes in type 2 diabetes

AimsInsulin resistance is a major factor influencing the progression of non-alcoholic fatty liver disease (NAFLD). Hence, insulin sensitisers such as thiazolidinediones (TZDs) are a potential treat...

Electronic-nose breath print distinguishes non-alcoholic fatty liver disease from healthy lean control: A pilot study

Sensor 1, Sensor 2, Sensor 3 and Sensor 4 identified NAFLD cirrhosis patients with AUC 0.96 (standard error=0.043; p<0.001), 0.89 (standard error=0.046; p<0.001), 0.98 (standard error =0.016; p<0.001) and 0.96 (standard error=0.022; p<0.001) respectively eNose was able to differentiate between healthy from; non-cirrhotic NAFLD (p<0.001, CVV 96.8%) and NAFLD cirrhotic (p<0.001, CVV 95.1%). This method, designed to reflect the generalization property of the k-nearest neighbour’s (k-NN) classifier, scored a classification rate of 96%. METHODS

PMO-130 Altered acetyl-coa metabolism in hepatic mitochondrial impairment in in vitro models of hepatic cellular steatosis

Introduction Increased ketogenesis, in the presence of unaltered β-oxidation, is a feature of human steatohepatitis. This is thought to be attributable to decreased acetyl-coA entry to tricarboxylic acid cycle with mitochondrial impairment. In this study, we examined the diversion of acetyl-coA towards free fatty acid (FFA) biosynthesis and mevalonate pathways (including vitamin D3, steroids hormones and bile acids) in the presence of mitochondrial dysfunction and triglyceride accumulation. Methods Human hepatoblastoma C3A cells were treated with; oleate or various combinations of octanoate (O), lactate (L), pyruvate (P) and ammonia (N) for 72 h. Metabolites that correspond to the intermediates of FFA biosynthesis, mevalonate pathways were measured using metabolomics study. Results We have previously shown that LPON treatment, but not oleate, affected mitochondrial function as evidenced by decreased respiration and ROS formation with concomitant enhanced ketogenesis despite the similarities in triglyceride accumulation. Using metabolomics analysis, we identified three metabolites that correspond to FFA biosynthesis, three were bile acids and three were the derivatives of steroid hormones and vitamin D3 synthesis. We also identified mevalonate and 7-dehydrodemosterol, the intermediates of cholesterol biosynthesis. The concentrations of FFA biosynthesis intermediates were higher with LPON compared with oleate (3-oxo-tetradecanoate (p=0.005) and 3-oxo-hexadecanoate (p=0.02)). Although mevalonate (p=0.37) and 7-dehydrodesmosterol (p=0.46) levels were higher with oleate than that seen with LPON, these differences did not reach statistical significance. In contrast, bile acids were significantly elevated with oleate than LPON ((taurocholate (p=0.002), glycocholate (p=0.001), (6RS)-22-oxo-23,24,25,26,27-pentanorvitamin D3 6,19-sulphur dioxide adduct (p=0.04) and 1,25-dihydroxy-2,4-dinor-1,3-secovitamin D3 (p=0.0006). Conclusion These data suggest that, aside from enhanced ketogenesis, impaired mitochondrial function is also associated with acetyl-coA diversion towards FFA synthesis, but not mevalonate pathways. These differences are likely to reflect cellular demand in the presence of decreased ATP formation with mitochondrial dysfunction. Competing interests None declared.

Oleate upregulates lectin galactoside-binding soluble 2 (LGALS2) in in vitro model of cellular steatosis

Introduction Galectin-2 (LGALS2) has been shown to co-localise with and bind to lymphotoxin-α (LTA); a cytokine that have been associated with insulin resistance. Thus, LGALS2 has been implicated in metabolic syndrome traits. The association between a common polymorphism of LGALS2 with myocardial infarction has further supported this notion. However, a recent study also demonstrates a contrasting finding of lower fasting insulin and glucose levels with LGALS2. The association between LGALS and the hepatic manifestation of insulin resistance, non-alcoholic fatty liver disease (NAFLD), has not been examined. Here, the authors investigated in vitro whether hepatic steatosis influenced the expression of LGALS2. Methods Human hepatoblastoma HepG2/C3A cells were pretreated for 3 days with oleate (0.25 mM) or octanoate (2 mM) to induce triglyceride accumulation. The authors have previously demonstrated that the addition of gluconeogenic substrates; lactate (L), pyruvate (P) and ammonia (N) to octanoate resulted in increased cellular steatosis that manifests many of the key features associated with steatohepatitis such as impaired mitochondrial structure/function, enhanced oxidative stress, decreased PTEN expression and altered cell cycle. LGALS2 mRNA expression was measured using quantitative real time PCR. Insulin resistance was determined by measuring concentration of glucose after a 4 h incubation of rinsed pretreated cells in the presence of insulin (0–10 nM). Results As previously demonstrated, all pretreatment induced significant intracellular triglyceride accumulation. The authors found that oleate upregulated LGALS2 expression. In contrast, the expression of LGALS2 was unchanged with LPON. Despite a higher triglyceride accumulation with octanoate, LGALS2 mRNA expression was also unaltered (oleate 1.24±0.06, octanoate 1.19±0.04, LPON 1.09±0.06, untreated 0.95±0.02 fold change from β-actin, p=0.0006). Glucose concentration in oleate showed a stepwise reduction with increasing insulin concentration (insulin 0 nM: 1.23±0.21 μg/gTP/h; 10 nM: 0.92±0.15 μg/gTP/h, where TP, total protein) contrasting to the unchanged glucose with LPON (Insulin 0 nM: 1.94±0.28 μg/gTP/h; 10 nM: 2.05±0.25 μg/gTP/h). Conclusion This data demonstrate that different FFA induces different LGALS2 expression. The presence of cellular steatosis per se or triglyceride concentration does not influence LGALS2 expression. Similar to the recent study, the upregulation of LGALS2 with oleate is associated with lower glucose concentration with preserved insulin sensitivity.

P53 The contrasting effect of octanoate and oleate on phosphatase and tensin homologue expression in in vitro model of steatosis using HepG2/C3A cells

Introduction The tumour suppressor phosphatase and tensin homologue (PTEN) is mutated or deleted in several human cancers including hepatocellular carcinoma. PTEN-deficient mice demonstrated triglyceride accumulation, steatohepatitis, progressing to liver fibrosis and hepatocellular carcinoma. Similarly, reduced PTEN expression with free fatty acid (FFA) oleate has been shown to promote hepatic steatosis. In other cancer, mitochondrial respiration defect with enhanced glycolysis and NADH formation has been suggested to be a key event in PTEN downregulation. Aim Our aims were to examine whether i) medium chain FFA octanoate altered PTEN expression ii) PTEN downregulation with FFA was associated with hepatic mitochondrial dysfunction. Method Human hepatoblastoma cell line HepG2/C3A was pretreated for 3 days with oleate (0.25 mM) or octanoate (2 mM). PTEN expression was determined using quantitative real time PCR. Mitochondrial function was measured using BDTM oxygen biosensor in the presence of 2,4 dinitrophenol. Lactate and pyruvate concentrations were measured in the supernatant to determine glycolytic activity and NADH/NAD+ ratio. Intracellular lipid accumulation was confirmed with triglyceride concentrations. Experiments were done in triplicate to n=3. Results are expressed in mean±SEM. Differences between groups were analysed by one-way ANOVA. Results We have previously demonstrated that oleate and octanoate pretreatment resulted in a similar intracellular triglyceride accumulation. In this study, we have found that despite similarities in triglyceride concentration, PTEN expression was lower in octanoate pretreated cells (octanoate 0.84±0.06, oleate 1.18±0.12, untreated 1.19±0.12 fold change from b-actin, p=0.04). However, octanoate pretreatment was not associated with impaired respiration (octanoate 0.24±0.01, oleate 0.20±0.02, untreated 0.28±0.01 AFU/gTP (gram of total protein)/min). Nevertheless, reduced PTEN expression with octanoate was associated with increased glycolysis (octanoate 315.2±42.91, oleate 100.9±14.09, untreated 145.3±8.83 μmol/gTP/hr, p=0.0001) with raised NADH/NAD ratio (octanoate 17.3±1.4, oleate 13.8±2.9 untreated 17.3±1.4; p=0.007). Conclusion To our knowledge, the effect of octanoate on PTEN expression has not been previously shown. In contrast to the previous finding, our data demonstrate that octanoate, not oleate, downregulates PTEN expression. Differences in glycolysis hence redox potential may have influenced the disparity in PTEN expression between these FFA. Octanoate has recently been proposed to be beneficial in weight loss and diabetes. However, our findings suggest that it may not have a favourable effect on the progression of nonalcoholic fatty liver disease.

PWE-002 Cell growth is impaired in a cellular model of non-alcoholic fatty liver disease, but is unaffected by simple steatosis

Introduction Non-alcoholic fatty liver disease (NAFLD) is an important cause of cirrhosis. Cirrhosis can be considered as a state where disordered growth leads to inadequate repair and a risk of malignancy. We sought to study the growth characteristics of C3A cells in an in vitro model of NAFLD. Methods C3A cells (well differentiated hepatoblastoma cells) were preconditioned for 72 h in control media, media containing oleate (which leads to steatosis, but not insulin resistance) or LPON media containing lactate (L), pyruvate (P), octanoate (O) and ammonia (N) (leading to steatosis, insulin resistance and increased reactive oxygen species). The cells were passaged and re-incubated in the appropriate media. Cells were harvested daily for cell counting and total protein estimation for 5 days. Cell cycle analysis and apoptosis estimation were performed on day 3. Results Growth curves were established, showing by both cell counting and protein estimation that growth was significantly reduced in the NAFLD model (p< 0.0001) compared to control media and oleate (Abstract 002 Figure 1). Abstract PWE-002 Figure 1 Growth curve. Cell cycle analysis showed that cells grown in LPON did not accumulate in G0/G1, but appeared to accumulate in S phase (Abstract 002 Figure 2). Cells grown in oleate cycled normally. There was an increase in apoptosis in both oleate and LPON groups (26.1 and 27.7% cells undergoing apoptosis, compared to 13.5% of control cells, p<0.0001). Abstract PWE-002 Figure 2 Cell cycle analysis. Conclusion NAFLD-induced cells showed significantly impaired growth compared with both control and simple steatotic cells in this model. This was not wholly attributable to an increase in apoptosis, as there was a similar increase in apoptosis with steatosis. While simple fat accumulation increased apoptosis, there was in addition, impairment of cell growth in our NAFLD model, which may be due to formation of reactive oxygen species, causing DNA damage and cell cycle arrest.