Celine Filippi

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.

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 72u2005h. 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 3u2005days with oleate (0.25u2005mM) or octanoate (2u2005mM). 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.