Glycolysis | EPFL Graph Search

Glycolysis is the metabolic pathway that converts glucose () into pyruvate, and in most organisms, occurs in the liquid part of cells, the cytosol. The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH). Glycolysis is a sequence of ten reactions catalyzed by enzymes. The wide occurrence of glycolysis in other species indicates that it is an ancient metabolic pathway. Indeed, the reactions that make up glycolysis and its parallel pathway, the pentose phosphate pathway, can occur in the oxygen-free conditions of the Archean oceans, also in the absence of enzymes, catalyzed by metal ions, meaning this is a plausible prebiotic pathway for abiogenesis. The most common type of glycolysis is the Embden–Meyerhof–Parnas (EMP) pathway, which was discovered by Gustav Embden, Otto Meyerhof, and Jakub Karol Parnas. Glycolysis also refers to other pathways, such as the Entner–Doudoroff p

Class I histone deacetylases (HDAC1-3) are histone lysine delactylases

Alexander Lund Nielsen, Di Zhang

Lysine L-lactylation [K(L-la)] is a newly discovered histone mark stimulated under conditions of high glycolysis, such as the Warburg effect. K(L-la) is associated with functions that are different from the widely studied histone acetylation. While K(L-la) can be introduced by the acetyltransferase p300, histone delactylases enzymes remained unknown. Here, we report the systematic evaluation of zinc- and nicotinamide adenine dinucleotide-dependent histone deacetylases (HDACs) for their ability to cleave epsilon-N-L-lactyllysine marks. Our screens identified HDAC1-3 and SIRT1-3 as delactylases in vitro. HDAC1-3 show robust activity toward not only K(L-la) but also K(D-la) and diverse short-chain acyl modifications. We further confirmed the de-L-lactylase activity of HDACs 1 and 3 in cells. Together, these data suggest that histone lactylation is installed and removed by regulatory enzymes as opposed to spontaneous chemical reactivity. Our results therefore represent an important step toward full characterization of this pathway's regulatory elements.

2022

GLUT1 and GLUT3 in non-small cell lung cancer

Mark Jonathan Masin

While altered metabolism is a well-established tumor cell trait, the specifics of metabolic rewiring during cancer development and progression are yet to be fully understood. The Warburg effect, or aerobic glycolysis, has been described in a wide array of malignancies and has long been considered as an inefficient mean to fill energetic requirements. Emerging evidence however supports a role that extends beyond that, with glycolytic intermediates diverting to anabolic pathways to allow growth and proliferation. Importantly, the first and most critical rate-limiting step in glycolysis is glucose uptake. Fittingly, high affinity, high capacity glucose transporters such as GLUT1 and to a lesser extent GLUT3, are upregulated in cancer. Given the centrality of glycolysis in tumor biology and reported clinical observations relating to glucose transporter expression patterns, the present work focuses on characterizing the role of GLUT1 and GLUT3 in the context of non-small cell lung cancer (NSCLC), the most prevalent histological subtype of lung cancer. Linking glucose metabolism and a program involved in early steps of metastasis, part of the findings uncovers an aberrant expression of GLUT3 as integral to the epithelial-mesenchymal transition (EMT) in NSCLC. Indeed, a unique association between GLUT3 and a mesenchymal status is observed in a panel of human NSCLC cell lines. Furthermore, GLUT3 expression is increased during EMT by a mechanism that involves direct binding of EMT mediating transcription factor ZEB1 to the GLUT3 (SLC2A3) gene. Data also supports a functional role of GLUT3 in proliferation, as inhibiting GLUT3 expression reduces glucose import and proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. In an analysis of a large microarray data collection of human NSCLC samples, GLUT3 expression is found to correlate with EMT markers, and is prognostic of poor overall survival. Taken collectively, the data reveal an important role for GLUT3 in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Owing to the restricted expression of this transporter in healthy individuals, its presence in lung tumor cells may represent a noteworthy prospect for targeted therapy. The contrastingly ubiquitous nature of GLUT1 in normal and transformed tissue warrants in vivo investigation to determine expression patterns and contribution tumor development. To that end, generation of autochthonous lung cancer mouse models allows assessment of the effect of tumor exclusive GLUT1 loss. Ensuing preliminary results allude to a possible role of GLUT1 in specific histological lesions. The significance of this specificity is currently under investigation.

EPFL2015

Multidisciplinary Analysis of the Metabolic Shift to Lactate Consumption in CHO Cell Culture

Francesca Zagari

Mammalian cells represent the most widely used host system for the industrial production of recombinant therapeutic proteins. In order to increase productivity, chemically defined media are often used and optimized to provide the cells with the necessary nutrients. However, a deregulated cell metabolism is often observed in these culture conditions. In particular, carbon sources are fast and inefficiently consumed with a consequent accumulation of byproducts, mainly lactate and ammonia. Lactate, in particular, causes a decrease of the medium pH which can be detrimental for cells growth and productivity. Its metabolic profile is normally characterized by a first production phase, which is associated to the cells exponential growth. Afterwards, when the cells enter into the stationary phase, a shift to net lactate consumption can occur under optimal culture conditions. However, such a metabolic shift is not easily controlled, since the mechanisms modulating lactate production in cell culture are still under investigation. This work aims to understand which factors could influence the lactate metabolic shift in CHO cell culture, focusing, in particular, on the mitochondrial role. To this purpose, cell lines with opposite lactate profiles were compared. The initial lactate production phase was common to all the fast growing cultures and was concomitant to a rapid glutamine consumption. After glutamine depletion, two different scenario occurred. In one case, the cells started to consume lactate until complete depletion. Alternatively, lactate continued to be accumulated, causing a decrease of media pH. The mitochondrial oxidative capacity was hypothesized as a possible cause of the different lactate profile. Indeed, mitochondrial membrane potential and oxygen consumption measurements highlighted a correlation between a reduced oxidative metabolism and a state of high lactate production. This correlation was confirmed by evaluating other cell lines or media compositions which resulted in the same divergence of lactate metabolism. Afterwards, the expression of selected genes was analysed in correlation with the observed lactate profile. Among 22 genes, two were identified as significantly downregulated (absolute fold change ≥2) in conditions of high lactate accumulation; namely, the mitochondrial aspartate-glutamate carrier (aralar1) and the translocase of the inner mitochondrial membrane 8 (timm8a). Aralar1, in particular, is an important component of the malate-aspartate shuttle (MAS). This system promotes the recycling of the cytosolic NADH pool, which is necessary for maintaining the glycolytic flux. Alternatively, NADH can be oxidised through pyruvate conversion into lactate, catalysed by the lactate dehydrogenase enzyme. Therefore, an inefficient MAS activity can engender an increased lactate accumulation. Finally, stable cell lines, which overexpressed aralar1 or timm8a, were generated. Clones derived from a lactate-producing cell line showed an improvement of lactate metabolism. In particular, they switched more easily to lactate consumption compared to the untransfected control, while maintaining a similar glucose consumption rate. On the other hand, no impact of the transgene expression was observed in the clones derived from the lactate-consuming cell line, since the switch to lactate consumption was maintained and no other effect on cell growth or glucose metabolism was detected. The data obtained indicate that lactate consumption was most likely promoted by a better NADH oxidation through the malate-aspartate shuttle, which resulted in a more efficient link between glycolysis and the mitochondrial oxidative metabolism. In conclusion, the results presented in this thesis indicate that the mitochondrial oxidative capacity plays a central role in the control of lactate production. Media composition and intrinsic cell characteristics have both an impact on mitochondrial activity. Moreover, the expression of specific genes can also be influenced by the culture media. In particular, aralar1 and timm8a have been identified as promising targets for the generation of a host cell line with an improved lactate metabolism.

EPFL2012