Silvia Marin

Metabolic strategy of boar spermatozoa revealed by a metabolomic characterization

Metabolomic characteristics in boar spermatozoa were studied using [1,2‐13C2]glucose and mass isotopomer analysis. In boar spermatozoa, glycolysis was the main pathway of glucose utilization producing lactate/pyruvate, whereas no gluconeogenesis was seen. Slight glycogen synthesis through the direct pathway and some incorporation of pyruvate into the Krebs cycle also took place. Neither RNA ribose‐5‐phosphate nor fatty acid synthesis from glucose occurred despite the detection of pyruvate dehydrogenase activity. In contrast to the known metabolic activities in dog sperm, boar spermatozoa have low levels of energy production and biosynthetic activities suggesting two different metabolic profiles for the two different phenotypes.

Novel semisynthetic derivatives of betulin and betulinic acid with cytotoxic activity

A series of new imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid (14-29) have been synthesized. The new compounds were screened for in vitro cytotoxicity activity against human cancer cell lines HepG2, Jurkat and HeLa. A number of compounds have shown IC(50) values lower than 2 microM against the cancer cell lines tested and the vast majority has shown a better cytotoxicity profile than betulinic acid, including the betulin derivatives. N-Acylimidazole derivatives 26 and 27 (IC(50) 0.8 and 1.7 microM in HepG2 cells) and the C-3 carbamate derivative 16 (IC(50) 2.0 microM in HepG2 cells) were the most promising compounds. Based on the observed cytotoxicity, structure-activity relationships have been established.

Wheat germ extract decreases glucose uptake and RNA ribose formation but increases fatty acid synthesis in MIA pancreatic adenocarcinoma cells.

The fermented wheat germ extract with standardized benzoquinone composition has potent tumor propagation inhibitory properties. The authors show that this extract induces profound metabolic changes in cultured MIA pancreatic adenocarcinoma cells when the [1,2- 13 C 2 ]glucose isotope is used as the single tracer with biologic gas chromatography–mass spectrometry. MIA cells treated with 0.1, 1, and 10 mg/mL wheat germ extract showed a dose-dependent decrease in cell glucose consumption, uptake of isotope into ribosomal RNA (2.4%, 9.4%, and 28.0%), and release of 13 CO 2 . Conversely, direct glucose oxidation and ribose recycling in the pentose cycle showed a dose-dependent increase of 1.2%, 20.7%, and 93.4%. The newly synthesized fraction of cell palmitate and the 13 C enrichment of acetyl units were also significantly increased with all doses of wheat germ extract. The fermented wheat germ extract controls tumor propagation primarily by regulating glucose carbon redistribution between cell proliferation–related and cell differentiation–related macromolecules. Wheat germ extract treatment is likely associated with the phosphorylation and transcriptional regulation of metabolic enzymes that are involved in glucose carbon redistribution between cell proliferation–related structural and functional macromolecules (RNA, DNA) and the direct oxidative degradation of glucose, which have devastating consequences for the proliferation and survival of pancreatic adenocarcinoma cells in culture.

Dynamic profiling of the glucose metabolic network in fasted rat hepatocytes using [1,2-13C2]glucose

Recent studies in metabolic profiling have underscored the importance of the concept of a metabolic network of pathways with special functional characteristics that differ from those of simple reaction sequences. The characterization of metabolic functions requires the simultaneous measurement of substrate fluxes of interconnecting pathways. Here we present a novel stable isotope method by which the forward and reverse fluxes of the futile cycles of the hepatic glucose metabolic network are simultaneously determined. Unlike previous radio-isotope methods, a single tracer [1,2-13C2]D-glucose and mass isotopomer analysis is used. Changes in fluxes of substrate cycles, in response to several gluconeogenic substrates, in isolated fasted hepatocytes from male Wistar rats were measured simultaneously. Incubation with these substrates resulted in a change in glucose-6-phosphatase/glucokinase and glycolytic/gluconeogenic flux ratios. Different net redistributions of intermediates in the glucose network were observed, resulting in distinct metabolic phenotypes of the fasted hepatocytes in response to each substrate condition. Our experimental observations show that the constraints of concentrations of shared intermediates, and enzyme kinetics of intersecting pathways of the metabolic network determine substrate redistribution throughout the network when it is perturbed. These results support the systems-biology notion that network analysis provides an integrated view of the physiological state. Interaction between metabolic intermediates and glycolytic/gluconeogenic pathways is a basic element of cross-talk in hepatocytes, and may explain some of the difficulties in genotype and phenotype correlation.

Synthesis and structure–activity relationship study of novel cytotoxic carbamate and N-acylheterocyclic bearing derivatives of betulin and betulinic acid

Chemical transformation studies were conducted on betulin and betulinic acid, common plant-derived lupane-type triterpenes. The concise synthesis, via a stepwise approach, of betulin and betulinic acid carbamate and N-acylheterocyclic containing derivatives is described. All new compounds, as well as betulinic acid were tested in vitro for their cytotoxic activity. Most of the compounds have shown a better cytotoxic profile than betulinic acid, including the synthesized betulin derivatives. Compounds 25 and 32 were the most promising derivatives, being up to 12-fold more potent than betulinic acid against human PC-3 cell lines (IC(50) values of 1.1 and 1.8 microM, respectively).

COordination of Standards in MetabOlomicS (COSMOS): facilitating integrated metabolomics data access

Abstract Metabolomics has become a crucial phenotyping technique in a range of research fields including medicine, the life sciences, biotechnology and the environmental sciences. This necessitates the transfer of experimental information between research groups, as well as potentially to publishers and funders. After the initial efforts of the metabolomics standards initiative, minimum reporting standards were proposed which included the concepts for metabolomics databases. Built by the community, standards and infrastructure for metabolomics are still needed to allow storage, exchange, comparison and re-utilization of metabolomics data. The Framework Programme 7 EU Initiative ‘coordination of standards in metabolomics’ (COSMOS) is developing a robust data infrastructure and exchange standards for metabolomics data and metadata. This is to support workflows for a broad range of metabolomics applications within the European metabolomics community and the wider metabolomics and biomedical communities’ participation. Here we announce our concepts and efforts asking for re-engagement of the metabolomics community, academics and industry, journal publishers, software and hardware vendors, as well as those interested in standardisation worldwide (addressing missing metabolomics ontologies, complex-metadata capturing and XML based open source data exchange format), to join and work towards updating and implementing metabolomics standards.

New betulinic acid derivatives induce potent and selective antiproliferative activity through cell cycle arrest at the S phase and caspase dependent apoptosis in human cancer cells.

New semisynthetic derivatives of betulinic acid (BA) RS01, RS02 and RS03 with 18-45 times improved cytotoxic activity against HepG2 cells, were tested for their ability to induce apoptosis and cell cycle arrest in HepG2, HeLa and Jurkat cells. All the compounds induced significant increase in the population at the S phase more effectively than BA. RS01, RS02 and RS03 were also found to be potent inducers of apoptosis with RS01 being markedly more potent than BA, suggesting that the introduction of the imidazolyl moiety is crucial for enhancing the induction of apoptosis and the cell cycle arrest. The mechanism of apoptosis induction has been studied in HepG2 cells and found to be mediated by activation of the postmitochondrial caspases-9 and -3 cascade and possibly by mitochondrial amplification loop involving caspase-8. These facts were corroborated by detection of mitochondrial cytochrome c release and DNA fragmentation. Because RS01, RS02 and RS03 exhibited significant improved antitumor activity with respect to BA, they may be promising new agents for the treatment of cancer. In particular, RS01 is the most promising compound with an IC(50) value 45 times lower than BA on HepG2 cells and 61 times lower than the one found for the non-tumoral Chang liver cells.

Relevance of the MEK/ERK Signaling Pathway in the Metabolism of Activated Macrophages: A Metabolomic Approach

The activation of immune cells in response to a pathogen involves a succession of signaling events leading to gene and protein expression, which requires metabolic changes to match the energy demands. The metabolic profile associated with the MAPK cascade (ERK1/2, p38, and JNK) in macrophages was studied, and the effect of its inhibition on the specific metabolic pattern of LPS stimulation was characterized. A [1,2-[13C]2]glucose tracer-based metabolomic approach was used to examine the metabolic flux distribution in these cells after MEK/ERK inhibition. Bioinformatic tools were used to analyze changes in mass isotopomer distribution and changes in glucose and glutamine consumption and lactate production in basal and LPS-stimulated conditions in the presence and absence of the selective inhibitor of the MEK/ERK cascade, PD325901. Results showed that PD325901-mediated ERK1/2 inhibition significantly decreased glucose consumption and lactate production but did not affect glutamine consumption. These changes were accompanied by a decrease in the glycolytic flux, consistent with the observed decrease in fructose-2,6-bisphosphate concentration. The oxidative and nonoxidative pentose phosphate pathways and the ratio between them also decreased. However, tricarboxylic acid cycle flux did not change significantly. LPS activation led to the opposite responses, although all of these were suppressed by PD325901. However, LPS also induced a small decrease in pentose phosphate pathway fluxes and an increase in glutamine consumption that were not affected by PD325901. We concluded that inhibition of the MEK/ERK cascade interferes with central metabolism, and this cross-talk between signal transduction and metabolism also occurs in the presence of LPS.

Metabolic Reprogramming and Dependencies Associated with Epithelial Cancer Stem Cells Independent of the Epithelial‐Mesenchymal Transition Program

In solid tumors, cancer stem cells (CSCs) can arise independently of epithelial‐mesenchymal transition (EMT). In spite of recent efforts, the metabolic reprogramming associated with CSC phenotypes uncoupled from EMT is poorly understood. Here, by using metabolomic and fluxomic approaches, we identify major metabolic profiles that differentiate metastatic prostate epithelial CSCs (e‐CSCs) from non‐CSCs expressing a stable EMT. We have found that the e‐CSC program in our cellular model is characterized by a high plasticity in energy substrate metabolism, including an enhanced Warburg effect, a greater carbon and energy source flexibility driven by fatty acids and amino acid metabolism and an essential reliance on the proton buffering capacity conferred by glutamine metabolism. An analysis of transcriptomic data yielded a metabolic gene signature for our e‐CSCs consistent with the metabolomics and fluxomics analyses that correlated with tumor progression and metastasis in prostate cancer and in 11 additional cancer types. Interestingly, an integrated metabolomics, fluxomics, and transcriptomics analysis allowed us to identify key metabolic players regulated at the post‐transcriptional level, suggesting potential biomarkers and therapeutic targets to effectively forestall metastasis. Stem Cells 2016;34:1163–1176

Metabolic network adaptations in cancer as targets for novel therapies

Metabolite concentrations and fluxes are the system variables that characterize metabolism. The systematic study of metabolite profiles is known as metabolomics; however, knowledge of the complete set of metabolites may not be enough to predict distinct phenotypes. A complete understanding of metabolic processes requires detailed knowledge of enzyme-controlled intracellular fluxes. These can be estimated through quantitative measurements of metabolites at different times or by analysing the stable isotope patterns obtained after incubation with labelled substrates. We have identified distinct intracellular fluxes associated with metabolic adaptations accompanying cancer. The maintenance of an imbalance between fluxes for the oxidative and non-oxidative PPP (pentose phosphate pathway) has been shown to be critical for angiogenesis and cancer cell survival. Mouse NIH 3T3 cells transformed by different mutated K-ras oncogenes have differential routing of glucose to anaerobic glycolysis, the PPP and the Krebs cycle. These results indicate that knowledge of metabolic fingerprints associated with an altered genetic profile could be exploited in the rational design of new therapies. We conclude that the understanding of the multifactorial nature of metabolic adaptations in cancer may open new ways to develop novel multi-hit antitumoral therapies.