A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we ...developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium HPLM). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis—an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.
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•Systematic development of human plasma-like medium (HPLM)•Culture in HPLM has widespread effects on cellular metabolism•Culture in HPLM reveals uric acid as an endogenous inhibitor of UMPS•Uric acid can influence cellular sensitivity to 5-fluorouracil
Mimicking the metabolite composition of human plasma in culture extensively alters the metabolic landscape of cells and highlights the potential to uncover new metabolite-drug interactions.
An in vivo model of antiangiogenic therapy allowed us to identify genes upregulated by bevacizumab treatment, including Fatty Acid Binding Protein 3 (FABP3) and FABP7, both of which are involved in ...fatty acid uptake. In vitro, both were induced by hypoxia in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. There was a significant lipid droplet (LD) accumulation in hypoxia that was time and O2 concentration dependent. Knockdown of endogenous expression of FABP3, FABP7, or Adipophilin (an essential LD structural component) significantly impaired LD formation under hypoxia. We showed that LD accumulation is due to FABP3/7-dependent fatty acid uptake while de novo fatty acid synthesis is repressed in hypoxia. We also showed that ATP production occurs via β-oxidation or glycogen degradation in a cell-type-dependent manner in hypoxia-reoxygenation. Finally, inhibition of lipid storage reduced protection against reactive oxygen species toxicity, decreased the survival of cells subjected to hypoxia-reoxygenation in vitro, and strongly impaired tumorigenesis in vivo.
Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the ...electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells.
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•Use of 2H stable isotopes to trace NAD(P)H metabolism in cells•Quantification of pentose phosphate pathway contribution to cytosolic NADPH•Reporter system to distinguish cytosolic and mitochondrial NADPH•Resolve direction of otherwise identical compartmentalized redox reactions
NAD(P)H cofactors are not transported across cell membranes, with metabolic shuttles required for transport of reducing equivalents between the mitochondria and cytosol. Lewis et al. develop an approach to resolve NADPH-dependent pathways in these distinct metabolic compartments and show that directionality of serine/glycine interconversions can be determined within intact cells.
Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better ...understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD+/NADH ratio. This change in NAD+/NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD+/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD+ to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD+ is in excess of the demand for ATP.
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•PDH activation suppresses cell proliferation by reducing the NAD+/NADH ratio•Insufficient ATP demand slows mitochondria NAD+ regeneration in proliferating cells•Uncoupling mitochondrial respiration from ATP synthesis can increase proliferation•Aerobic glycolysis reflects increased cell demand for NAD+ relative to ATP turnover
Aerobic glycolysis is associated with proliferation in many biological contexts, yet what drives this phenotype has not been fully explained. Luengo et al. show that cells engage in aerobic glycolysis when the demand for NAD+ exceeds the demand for ATP, which leads to impaired NAD+ regeneration by mitochondrial respiration.
Cancer has myriad effects on metabolism that include both rewiring of intracellular metabolism to enable cancer cells to proliferate inappropriately and adapt to the tumor microenvironment, and ...changes in normal tissue metabolism. With the recognition that fluorodeoxyglucose‐positron emission tomography imaging is an important tool for the management of many cancers, other metabolites in biological samples have been in the spotlight for cancer diagnosis, monitoring, and therapy. Metabolomics is the global analysis of small molecule metabolites that like other ‐omics technologies can provide critical information about the cancer state that are otherwise not apparent. Here, the authors review how cancer and cancer therapies interact with metabolism at the cellular and systemic levels. An overview of metabolomics is provided with a focus on currently available technologies and how they have been applied in the clinical and translational research setting. The authors also discuss how metabolomics could be further leveraged in the future to improve the management of patients with cancer.
The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase
, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis
. Depletion of tetrahydrofolate ...causes cell death by suppressing DNA and RNA production
. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials
, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy
. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR-Cas9-based screen
. This screen yielded FTCD, which encodes an enzyme-formimidoyltransferase cyclodeaminase-that is required for the catabolism of the amino acid histidine
, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.
Variants in the triggering receptor expressed on myeloid cells 2 (TREM2) gene are associated with increased risk for late-onset AD. Genetic loss of or decreased TREM2 function impairs the microglial ...response to amyloid-β (Aβ) plaques, resulting in more diffuse Aβ plaques and increased peri-plaque neuritic dystrophy and AD-tau seeding. Thus, microglia and TREM2 are at a critical intersection of Aβ and tau pathologies in AD. Since genetically decreasing TREM2 function increases Aβ-induced tau seeding, we hypothesized that chronically increasing TREM2 signaling would decrease amyloid-induced tau-seeding and spreading. Using a mouse model of amyloidosis in which AD-tau is injected into the brain to induce Aβ-dependent tau seeding/spreading, we found that chronic administration of an activating TREM2 antibody increases peri-plaque microglial activation but surprisingly increases peri-plaque NP-tau pathology and neuritic dystrophy, without altering Aβ plaque burden. Our data suggest that sustained microglial activation through TREM2 that does not result in strong amyloid removal may exacerbate Aβ-induced tau pathology, which may have important clinical implications.
The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1 mechanistic target of rapamycin (mTOR) complex 1, and is associated with human disease. We performed ...quantitative proteomic analyses of rapidly isolated lysosomes and found that nutrient levels and mTOR dynamically modulate the lysosomal proteome. Upon mTORC1 inhibition, NUFIP1 (nuclear fragile X mental retardation-interacting protein 1) redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to microtubule-associated proteins 1A/1B light chain 3B (LC3B). The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes.
Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand ...tumor metabolism. To measure the extracellular nutrient levels available to tumors, we utilized quantitative metabolomics methods to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors.
Rett syndrome (RTT) is caused by mutations of MECP2, a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show, using an isogenic human embryonic stem cell model ...of RTT, that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells.
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•Genetically engineered hESC model of RTT•RTT neurons show deficits in morphology, AKT signaling, and physiology•RTT neurons display reduced global transcription and translation•Boosting translation via AKT/mTOR ameliorates disease-related deficits
Modeling of Rett syndrome using hESC-derived neurons shows that MECP2 has a broad transcriptional activation role, contrasting with previous indications that it functions as a transcriptional repressor.
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