Physiologically relevant culture medium Plasmax improves human placental trophoblast stem cell function.

Human trophoblast cultures provide powerful tools to model key processes of placental development. In vitro trophoblast studies to date have relied on commercial media that contains nonphysiological levels of nutrients, and the impact of these conditions on trophoblast metabolism and function is unknown. Here, we show that the physiological medium (Plasmax) with nutrient and metabolite concentrations recapitulating human plasma improves human trophoblast stem cell (hTSC) proliferation and differentiation compared with standard medium (DMEM-F12). hTSCs cultured in Plasmax-based medium also show altered glycolytic and mitochondrial metabolism, as well as reduced S-adenosylmethionine/S-adenosyl-homocysteine ratio compared with DMEM-F12-based medium. These findings demonstrate the importance of the nutritional environment for phenotyping cultured human trophoblasts.

Hepatic glutamine synthetase controls N5-methylglutamine in homeostasis and cancer.

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a ß-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite.

Mesenchymal stromal cells cultured in physiological conditions sustain citrate secretion with glutamate anaplerosis.

Bone marrow mesenchymal stromal cells (MSCs) have immunomodulatory and regenerative potential. However, culture conditions govern their metabolic processes and therapeutic efficacy. Here we show that culturing donor-derived MSCs in Plasmax™, a physiological medium with the concentrations of nutrients found in human plasma, supports their proliferation and stemness, and prevents the nutritional stress induced by the conventional medium DMEM. The quantification of the exchange rates of metabolites between cells and medium, untargeted metabolomics, stable isotope tracing and transcriptomic analysis, performed at physiologically relevant oxygen concentrations (1%O2), reveal that MSCs rely on a high rate of glucose to lactate conversion, coupled with parallel anaplerotic fluxes from glutamine and glutamate to support citrate synthesis and secretion. These distinctive traits of MSCs shape the metabolic microenvironment of the bone marrow niche and can influence nutrient cross-talks under physiological and pathological conditions.

A map of the altered glioma metabolism.

The frequent occurrence of neomorphic isocitrate dehydrogenase 1 (IDH1) mutations in low-grade glioma led to an IDH-centric classification of these tumors. However, exploiting metabolic alterations of glioma for diagnostic imaging and treatment has marginally improved patients' prognosis. Here we discuss the nutritional microenvironment of glioma, shaped by the distinctive dependence of the brain on glucose and ketone bodies for energy, and on amino acids for neurotransmission. We highlight the progress in metabolic applications for glioma diagnosis and therapy, and present a map that streamlines the rewired glioma metabolism. The map illustrates the altered reactions in central carbon and nitrogen metabolism that drive glioma biology, and represent metabolic vulnerabilities with translational potential.

An IDH1-vitamin C crosstalk drives human erythroid development by inhibiting pro-oxidant mitochondrial metabolism.

The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation.

Caspase-10 inhibits ATP-citrate lyase-mediated metabolic and epigenetic reprogramming to suppress tumorigenesis.

Caspase-10 belongs to the class of initiator caspases and is a close homolog of caspase-8. However, the lack of caspase-10 in mice and limited substrate repertoire restricts the understanding of its physiological functions. Here, we report that ATP-citrate lyase (ACLY) is a caspase-10 substrate. Caspase-10 cleaves ACLY at the conserved Asp1026 site under conditions of altered metabolic homeostasis. Cleavage of ACLY abrogates its enzymatic activity and suppresses the generation of acetyl-CoA, which is critical for lipogenesis and histone acetylation. Thus, caspase-10-mediated ACLY cleavage results in reduced intracellular lipid levels and represses GCN5-mediated histone H3 and H4 acetylation. Furthermore, decline in GCN5 activity alters the epigenetic profile, resulting in downregulation of proliferative and metastatic genes. Thus caspase-10 suppresses ACLY-promoted malignant phenotype. These findings expand the substrate repertoire of caspase-10 and highlight its pivotal role in inhibiting tumorigenesis through metabolic and epigenetic mechanisms.

Cyclin F-Dependent Degradation of RBPJ Inhibits IDH1R132H-Mediated Tumorigenesis.

Cyclin F is a substrate recognition subunit of Skp1-Cul1-F-box protein (SCF) E3 ubiquitin ligase complex. Although there have been reports describing the role of cyclin F in the genotoxic stress response, its function under conditions of altered metabolic homeostasis remain unexplored. Here we report that cyclin F is induced upon metabolic stress in a FOXO1-dependent manner. Under metabolic stress conditions, cyclin F mediated polyubiquitylation of RBPJ at Lys315, leading to its proteasomal degradation. RBPJ regulated the expression of IDH1, which is often mutated to an oncogenic form IDH1R132H in cancers. Thus, metabolic stress-induced cyclin F attenuated the oncogenic functions of IDH1R132H in an RBPJ-dependent manner. Studies in mouse tumor models indicated that abrogation of cyclin F expression facilitates IDH1R132H-mediated tumorigenesis and metastasis. In addition, increased IDH1R132H levels correlated with reduced cyclin F levels in increasing grades of glioma. These findings highlight a novel aspect of cyclin F functions in inhibiting tumorigenesis and provide mechanistic insights into regulation of IDH1R132H Significance: These findings reveal mechanistic insights into the key role of the cyclin F-RBPJ axis in response to metabolic stress in cancer cells. Cancer Res; 78(22); 6386-98. ©2018 AACR.

Effect of pesticides on the aggregation of mutant huntingtin protein.

The classical reports on neurodegeneration concentrate on studying disruption of signalling cascades. Although it is now well recognized that misfolding and aggregation of specific proteins are associated with a majority of these diseases, their role in aggravating the symptoms is not so well understood. Huntington's disease (HD) is a neurodegenerative disorder that results from damage to complex II of mitochondria. In this work, we have studied the effect of mitochondrial complex I inhibitors, viz. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone, and complex II inhibitor, viz. 3-nitropropionic acid, on the aggregation of mutant huntingtin (mthtt) protein, whose misfolding and aggregation results in cellular abnormalities characteristic of HD. All three inhibitors were found to accelerate the aggregation of mthtt in vitro, although the amounts of aggregates formed were different in all cases. Thus, apart from their effect on mitochondrial viability, these neurotoxins are capable of interfering with the protein aggregation process and thus, hastening the onset of the disease.