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Abstract Lactate accumulation and acidification in tumours are a cancer hallmark associated with the Warburg effect. Lactic acidosis correlates with cancer malignancy, and the benefit it offers to tumours has been the subject of numerous hypotheses. Strikingly, lactic acidosis enhances cancer cell survival to environmental glucose depletion by repressing high-rate glycolysis and lactic fermentation, and promoting an oxidative metabolism involving reactivated respiration. We used real-time NMR to evaluate how cytosolic lactate accumulation up to 40 mM and acidification up to pH 6.5 individually impact glucose consumption, lactate production and pyruvate evolution in isolated cytosols. We used a reductive cell-free system (CFS) to specifically study cytosolic metabolism independently of other Warburg-regulatory mechanisms found in the cell. We assessed the impact of lactate and acidification on the Warburg metabolism of cancer cytosols, and whether this effect extended to different cytosolic phenotypes of lactic fermentation and cancer. We observed that moderate acidification, independently of lactate concentration, drastically reduces the glucose consumption rate and halts lactate production in different lactic fermentation phenotypes. In parallel, for Warburg-type CFS lactate supplementation induces pyruvate accumulation at control pH, and can maintain a higher cytosolic pyruvate pool at low pH. Altogether, we demonstrate that intracellular acidification accounts for the direct repression of lactic fermentation by the Warburg-associated lactic acidosis.

Background & Aims: Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers. Methods: Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting. Results: Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca2+ signaling. Conclusions: HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca2+ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed. Impact and implications: Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication.