Sergey Tumanov

Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis

Succinate dehydrogenase (SDH) is a heterotetrameric nuclear-encoded complex responsible for the oxidation of succinate to fumarate in the tricarboxylic acid cycle. Loss-of-function mutations in any of the SDH genes are associated with cancer formation. However, the impact of SDH loss on cell metabolism and the mechanisms enabling growth of SDH-defective cells are largely unknown. Here, we generated Sdhb-ablated kidney mouse cells and used comparative metabolomics and stable-isotope-labelling approaches to identify nutritional requirements and metabolic adaptations to SDH loss. We found that lack of SDH activity commits cells to consume extracellular pyruvate, which sustains Warburg-like bioenergetic features. We further demonstrated that pyruvate carboxylation diverts glucose-derived carbons into aspartate biosynthesis, thus sustaining cell growth. By identifying pyruvate carboxylase as essential for the proliferation and tumorigenic capacity of SDH-deficient cells, this study revealed a metabolic vulnerability for potential future treatment of SDH-associated malignancies.

Fish oil supplements in New Zealand are highly oxidised and do not meet label content of n-3 PUFA

We evaluated the quality and content of fish oil supplements in New Zealand. All encapsulated fish oil supplements marketed in New Zealand were eligible for inclusion. Fatty acid content was measured by gas chromatography. Peroxide values (PV) and anisidine values (AV) were measured, and total oxidation values (Totox) calculated. Only 3 of 32 fish oil supplements contained quantities of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that were equal or higher than labelled content, with most products tested (69%) containing <67%. The vast majority of supplements exceeded recommended levels of oxidation markers. 83% products exceeded the recommended PV levels, 25% exceeded AV thresholds, and 50% exceeded recommended Totox levels. Only 8% met the international recommendations, not exceeding any of these indices. Almost all fish oil supplements available in the New Zealand market contain concentrations of EPA and DHA considerably lower than claimed by labels. Importantly, the majority of supplements tested exceeded the recommended indices of oxidative markers. Surprisingly, best-before date, cost, country of origin, and exclusivity were all poor markers of supplement quality.

Acetate Recapturing by Nuclear Acetyl-CoA Synthetase 2 Prevents Loss of Histone Acetylation during Oxygen and Serum Limitation

Summary Acetyl-CoA is a key metabolic intermediate with an important role in transcriptional regulation. The nuclear-cytosolic acetyl-CoA synthetase 2 (ACSS2) was found to sustain the growth of hypoxic tumor cells. It generates acetyl-CoA from acetate, but exactly which pathways it supports is not fully understood. Here, quantitative analysis of acetate metabolism reveals that ACSS2 fulfills distinct functions depending on its cellular location. Exogenous acetate uptake is controlled by expression of both ACSS2 and the mitochondrial ACSS1, and ACSS2 supports lipogenesis. The mitochondrial and lipogenic demand for two-carbon acetyl units considerably exceeds the uptake of exogenous acetate, leaving it to only sparingly contribute to histone acetylation. Surprisingly, oxygen and serum limitation increase nuclear localization of ACSS2. We find that nuclear ACSS2 recaptures acetate released from histone deacetylation for recycling by histone acetyltransferases. Our work provides evidence for limited equilibration between nuclear and cytosolic acetyl-CoA and demonstrates that ACSS2 retains acetate to maintain histone acetylation.

Serine one-carbon catabolism with formate overflow

Serine catabolism results in formate efflux that exceeds anabolic demands for purine synthesis. Serine catabolism to glycine and a one-carbon unit has been linked to the anabolic requirements of proliferating mammalian cells. However, genome-scale modeling predicts a catabolic role with one-carbon release as formate. We experimentally prove that in cultured cancer cells and nontransformed fibroblasts, most of the serine-derived one-carbon units are released from cells as formate, and that formate release is dependent on mitochondrial reverse 10-CHO-THF synthetase activity. We also show that in cancer cells, formate release is coupled to mitochondrial complex I activity, whereas in nontransformed fibroblasts, it is partially insensitive to inhibition of complex I activity. We demonstrate that in mice, about 50% of plasma formate is derived from serine and that serine starvation or complex I inhibition reduces formate synthesis in vivo. These observations transform our understanding of one-carbon metabolism and have implications for the treatment of diabetes and cancer with complex I inhibitors.

Analysis of Fatty Acid Metabolism Using Stable Isotope Tracers and Mass Spectrometry

Cells can synthesize fatty acids by ligating multiple acetyl units from acetyl-CoA. This is followed by desaturation and elongation reactions to produce a variety of fatty acids required for proper cellular functioning. Alternatively, exogenous lipid sources can contribute to cellular fatty acid pools. Here, we present a method based on incorporation of (13)C-carbon from labeled substrates into fatty acids and subsequent mass spectrometry analysis. The resulting labeling patterns can be used to determine (1) (13)C-enrichment of lipogenic acetyl-CoA, (2) the relative contributions of synthesis and uptake, and (3) absolute fatty acid fluxes. We begin by providing a background and general principles regarding the use of stable isotopes to study fatty acid metabolism. We then proceed with detailing procedures for sample preparation and both GC-MS and LC-MS analysis of isotope incorporation. Finally, we discuss the interpretation of the resulting fatty acid-labeling patterns.

Metabolite profiling of symbiont and host during thermal stress and bleaching in a model cnidarian-dinoflagellate symbiosis

ABSTRACT Bleaching (dinoflagellate symbiont loss) is one of the greatest threats facing coral reefs. The functional cnidarian–dinoflagellate symbiosis, which forms coral reefs, is based on the bi-directional exchange of nutrients. During thermal stress this exchange breaks down; however, major gaps remain in our understanding of the roles of free metabolite pools in symbiosis and homeostasis. In this study we applied gas chromatography–mass spectrometry (GC-MS) to explore thermally induced changes in intracellular pools of amino and non-amino organic acids in each partner of the model sea anemone Aiptasia sp. and its dinoflagellate symbiont. Elevated temperatures (32°C for 6 days) resulted in symbiont photoinhibition and bleaching. Thermal stress induced distinct changes in the metabolite profiles of both partners, associated with alterations to central metabolism, oxidative state, cell structure, biosynthesis and signalling. Principally, we detected elevated pools of polyunsaturated fatty acids (PUFAs) in the symbiont, indicative of modifications to lipogenesis/lysis, membrane structure and nitrogen assimilation. In contrast, reductions of multiple PUFAs were detected in host pools, indicative of increased metabolism, peroxidation and/or reduced translocation of these groups. Accumulations of glycolysis intermediates were also observed in both partners, associated with photoinhibition and downstream reductions in carbohydrate metabolism. Correspondingly, we detected accumulations of amino acids and intermediate groups in both partners, with roles in gluconeogenesis and acclimation responses to oxidative stress. These data further our understanding of cellular responses to thermal stress in the symbiosis and generate hypotheses relating to the secondary roles of a number of compounds in homeostasis and heat-stress resistance. Summary: Thermally induced modifications to free metabolite pools of amino and non-amino organic acids are characterised in a model system for reef-building corals, in both symbiont and host.

Recent advances in expanding the coverage of the lipidome

Graphical abstract

LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells

ABSTRACT Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here, we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 – but not of its homologues LPP1 or LPP2 – diminishes the cells ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes the ability of melanoma cells to invade. Our results demonstrate that LPP3 is the key enzyme in the breakdown of LPA by melanoma cells, and confirm the importance of attractant breakdown in LPA-mediated cell steering. This article has an associated First Person interview with the first author of the paper. Highlighted Article: Melanoma cells can create and follow their own gradients of attractant, via a new mechanism by which tumour cells may undergo metastasis.

Triglycerides Promote Lipid Homeostasis during Hypoxic Stress by Balancing Fatty Acid Saturation

Summary Lipid droplets, which store triglycerides and cholesterol esters, are a prominent feature of clear cell renal cell carcinoma (ccRCC). Although their presence in ccRCC is critical for sustained tumorigenesis, their contribution to lipid homeostasis and tumor cell viability is incompletely understood. Here we show that disrupting triglyceride synthesis compromises the growth of both ccRCC tumors and ccRCC cells exposed to tumor-like conditions. Functionally, hypoxia leads to increased fatty acid saturation through inhibition of the oxygen-dependent stearoyl-CoA desaturase (SCD) enzyme. Triglycerides counter a toxic buildup of saturated lipids, primarily by releasing the unsaturated fatty acid oleate (the principal product of SCD activity) from lipid droplets into phospholipid pools. Disrupting this process derails lipid homeostasis, causing overproduction of toxic saturated ceramides and acyl-carnitines as well as activation of the NF-κB transcription factor. Our work demonstrates that triglycerides promote homeostasis by “buffering” specific fatty acids.

Corrigendum: Fish oil supplements in New Zealand are highly oxidised and do not meet label content of n-3 PUFA

Corrigendum: Fish oil supplements in New Zealand are highly oxidised and do not meet label content of n-3 PUFA