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.