Enrique Sentandreu

The early metabolomic response of adipose tissue during acute cold exposure in mice

To maintain core body temperature in cold conditions, mammals activate a complex multi-organ metabolic response for heat production. White adipose tissue (WAT) primarily functions as an energy reservoir, while brown adipose tissue (BAT) is activated during cold exposure to generate heat from nutrients. Both BAT and WAT undergo specific metabolic changes during acute cold exposure. Here, we use an untargeted metabolomics approach to characterize the initial metabolic response to cold exposure in multiple adipose tissue depots in mice. Results demonstrate dramatically distinct metabolic responses during cold exposure in BAT and WAT. Amino acids, nucleotide pathways, and metabolites involved in redox regulation were greatly affected 4 hours post-exposure in BAT, while no polar metabolites were observed to significantly change in WAT depots up to 6 hours post exposure. Lipid metabolism was activated early (2 hours) in both BAT and the subcutaneous WAT depots, with the most striking change being observed in the modulation of diglyceride and monoglyceride levels in BAT. Overall, these data provide a timeline of global thermogenic metabolism in adipose depots during acute cold exposure. We have highlighted differences in visceral and subcutaneous WAT thermogenic metabolism and demonstrate the distinct metabolism of BAT during cold exposure.

Combinatorial treatment with natural compounds in prostate cancer inhibits prostate tumor growth and leads to key modulations of cancer cell metabolism

High-throughput screening of a natural compound library was performed to identify the most efficacious combinatorial treatment on prostate cancer. Ursolic acid, curcumin and resveratrol were selected for further analyses and administered in vivo via the diet, either alone or in combination, in a mouse allograft model of prostate cancer. All possible combinations of these natural compounds produced synergistic effects on tumor size and weight, as predicted in the screens. A subsequent untargeted metabolomics and metabolic flux analysis using isotopically labeled glutamine indicated that the compound combinations modulated glutamine metabolism. In addition, ASCT2 levels and STAT3, mTORC1 and AMPK activity were modulated to a greater extent by the combinations compared to the individual compounds. Overall, this approach can be useful for identifying synergistic combinations of natural compounds for chemopreventive and therapeutic interventions.Prostate cancer: Combination of natural compounds limits tumor growthCombinations of two molecules found naturally in edible plants synergistically help reduce tumor growth in a mouse model of prostate cancer. Stefano Tiziani and John DiGiovanni from the University of Texas at Austin, and colleagues screened a library of 142 natural compounds for the effects of each molecule, alone or in combination, on the viability of cells from mouse and human prostate cancer cell lines. Amongst other promising combinations, the researchers identified ursolic acid and curcumin as the most promising combination for inhibiting tumor growth. (These compounds are found naturally in apple peels and turmeric, respectively.) In mice with implanted prostate tumors, the two compounds synergistically reduced tumor volume and weight, while in cell culture the researchers showed that the compound-combination strategy modulated metabolism of a critical amino acid and other cell signaling pathways.

RpeakChrom: Novel R package for the automated characterization and optimization of column efficiency in high-performance liquid chromatography analysis

Characterization of chromatographic columns using the traditional van Deemter method is limited by the necessity of calculating extra‐column variance, issue particularly relevant when modeling asymmetrical peaks eluted from monolithic columns. A novel R package that implements Parabolic Variance Modified Gaussian approach for accurate peak modeling, van Deemter equation and two alternatives approaches, based on van Deemter, has been developed to calculate the height equivalent to a theoretical plate (HETP). To assess package capabilities conventional packed reverse‐phase and monolithic HPLC columns were characterized. Peaks eluted from the monolithic column showed a high value of factor asymmetry due, in part, to the contribution of extra‐column factors. Such deviation can be circumvented by the two alternatives approaches implemented in the R‐package. Furthermore, increased values of eddy diffusion and mass transfer kinetics terms in HETP were observed for the packed column, while accuracy was below 9% in all cases. These results showed the usefulness of the R‐package for both modeling chromatographic peaks and assessing column efficiency. The RpeakChrom package could become a helpful tool for testing new stationary phases during column development and to evaluate column during its lifetime. This R tool is freely available from CRAN (https://CRAN.R-project.org/package=RpeakChrom).

Reversal of indoleamine 2,3-dioxygenase–mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

Abstract 3: Amino acid profiles indicate dependence on different metabolic pathways between leukemia subtypes

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Despite the growing body of evidence that the tumor microenvironment protects leukemia cells from chemotherapeutic stresses (1-3), the effect of many extracellular metabolites remains largely unknown. To explore the influence of extracellular metabolites on different leukemia subtypes, cells were treated for 24 hours in vitro with either a supplemental amino acid or amino acid derivative. From this initial screening, a subset of metabolites were chosen for metabolomics analysis. Mass spectrometry (UPLC-MS/MS) was performed on intracellular fractions to identify metabolic differences that resulted from supplementation. Metabolite profiles were also compared between leukemia cell types, namely AML, pre-B cell ALL, and T cell ALL. Of the metabolites tested, lysine and 4-hydroxyphenylpyruvate, an intermediate of tyrosine and phenylalanine metabolism, had the greatest impact on global amino acid profiles. In AML and T cell ALL cell lines, intracellular glutamate, glutamine, proline, and aspartate were increased relative to their respective controls. These amino acids can enter the tricarboxylic acid (TCA) cycle as either α-ketoglutarate or oxaloacetate, suggesting a central role of the TCA cycle in both AML and T cell ALL metabolism. Interestingly, these metabolites were not significantly increased in pre-B cell ALL, signifying the inverse it true for pre-B cells. This observation provides metabolomics evidence that is consistent with a previous study that reported downregulated expression of TCA cycle related genes in pre-B cell ALL (4). Our findings indicate that uptake and metabolism of amino acids and their derivatives is distinct for different leukemia types. Moreover, supplementation with a single metabolite can result in global changes in intracellular metabolite profiles, suggesting an influence not only as an energy substrate, but on overall metabolic pathway activity. Specifically, we conclude that the TCA cycle is more active in AML and T cell ALL and can be modulated by changing the extracellular environment, while pre-B cells are less sensitive to amino acid modulation. (1) Meads MB, et al. Clin Cancer Res 2008;14(9):2519-2526. (2) Ayala F, et al. Leukemia 2009;23:2233-2241. (3) Konopleva M, et al. Drug Resist Updat 2009;12:103-113. (4) Boag JM, et al. Leukemia 2006;20:1731-1737. Citation Format: Shannon R. Sweeney, Enrique Sentandreu, Stefano Tiziani. Amino acid profiles indicate dependence on different metabolic pathways between leukemia subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3.

Deletion of the neural tube defect-associated gene Mthfd1l disrupts one-carbon and central energy metabolism in mouse embryos

One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by Mthfd1l deletion are not known. Here, we performed untargeted metabolomics on whole Mthfd1l-null and wildtype mouse embryos in combination with isotope tracer analysis in mouse embryonic fibroblast (MEF) cell lines to identify Mthfd1l deletion–induced disruptions in 1C metabolism, glycolysis, and the TCA cycle. We found that maternal formate supplementation largely corrects these disruptions in Mthfd1l-null embryos. Serine tracer experiments revealed that Mthfd1l-null MEFs have altered methionine synthesis, indicating that Mthfd1l deletion impairs the methyl cycle. Supplementation of Mthfd1l-null MEFs with formate, hypoxanthine, or combined hypoxanthine and thymidine restored their growth to wildtype levels. Thymidine addition alone was ineffective, suggesting a purine synthesis defect in Mthfd1l-null MEFs. Tracer experiments also revealed lower proportions of labeled hypoxanthine and inosine monophosphate in Mthfd1l-null than in wildtype MEFs, suggesting that Mthfd1l deletion results in increased reliance on the purine salvage pathway. These results indicate that disruptions of mitochondrial 1C metabolism have wide-ranging consequences for many metabolic processes, including those that may not directly interact with 1C metabolism.

A Survey of Orbitrap All Ion Fragmentation Analysis Assessed by an R MetaboList Package to Study Small-Molecule Metabolites

Leukemia cell and melanoma tumor tissue extracts were studied for small (mostly m/z < 250) polar metabolites by LC-ESI-HRMSn analysis powered by a hybrid Quadrupole-Orbitrap. MS data were simultaneously acquired in fast polarity switching mode operating in MS1 and MS/MS (All Ion Fragmentation, AIF) full-scan analyses at high mass resolution. Positive metabolite assignments were achieved by AIF analysis considering at least two characteristic transitions. Targeted metabolite profiling was achieved by the relative quantification of 18 metabolites through spiking of their respective deuterated counterparts. Manual data processing of MS1 and AIF scans were compared for the accurate determination of natural metabolites and their deuterated analogs by chromatographic alignment and peak area integration. Evaluation of manual and automated (MetaboList R package) AIF data processing yielded comparable results. The versatility of AIF analysis also enabled the untargeted metabolite profiling of leukemia and melanoma samples in which 22 and 53 compounds were, respectively, identified outside those studied by labeling. The main limitation of this method was that low abundance metabolites with scan rates below 8 scans/peak could not be accurately quantified by AIF analysis. The combination of AIF analysis with MetaboList R package represents an opportunity to move towards automated, faster, and more global metabolomics approaches supported by an entirely flexible open source data processing platform freely available from Comprehensive R Archive Network (CRAN, https://CRAN.R-project.org/package=MetaboList).

Abstract 12: Combinatorial intervention with natural compounds induces key metabolic modulations for prostate cancer prevention and treatment

Prostate cancer is one of the three most relevant cancer types in men, and it ranks second in death rate and first in newly diagnosed cancer cases according to the US cancer statistics 2015. In recent years, the impact of nutrition on cancer prevention has been increasingly recognized. Accordingly the study of natural compounds for cancer prevention and treatment has drawn attention, mainly for the low toxicity to normal tissues. In this study, a natural compound library of 140 compounds was screened on cultured mouse prostate tumor cells from HiMyc mice (HMVP-2 cells), and ATP and reactive oxygen species (ROS) bioluminescence measurements were performed. Based on ATP and ROS results, three hits, ursolic acid (UA), curcumin (CURC) and resveratrol (RES) were selected for more in depth metabolomic and lipidomic analyses. High-resolution liquid chromatography mass spectrometry (HPLC-MS) and magnetic resonance spectroscopy (MRS) were applied for large scale untargeted metabolic and lipidomic profiling of intra and extracellular prostate cancer extracts after treatment with the three drugs and their combinations at different time points. We next performed an in vivo study on an allograft mouse model of prostate cancer by injecting HMVP-2 spheroids into FVB/N mice. After 12 hours of treatment, 115 metabolites in KEGG database and 12 classes of lipids (664 features) were included in the study. Principal component analysis (PCA) showed that the combination of UA and CURC exerts the most profound metabolic perturbation in HMVP2 cells compared to the individual treatment or the combination of other selected hits. Further data mining showed that the CURC+UA had a synergistic effect on cell metabolism by altering metabolic pathways associated with alanine, aspartate, proline and glutamate metabolism. Moreover, key intermediates in glycerophospholipid and ceramide metabolism were highly perturbed in CURC +UA indicating a relevant response of lipid mechanism to treatment with the combination of these agents. The in vivo mouse model produces palpable tumors within 10-14 days post injection. Dietary administration of CURC+UA and UA+RES showed significant inhibition of tumor growth compared to the administration of the individual compounds, with CURC+UA yielding the most effective combination. In summary, amongst the 140 screened compounds, CURC, UA and RES exerted the most prominent metabolic effects on prostate cancers cells, and the combined CURC+UA treatment showed a synergistic effect on cell metabolism and significantly affected key metabolic pathways active in mitochondria, most likely via lipid metabolisms. Citation Format: Xiyuan Lu, Bo Wang, Achinto Saha, Enrique Sentandreu, Alessia Lodi, John DiGiovanni, Stefano Tiziani. Combinatorial intervention with natural compounds induces key metabolic modulations for prostate cancer prevention and treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 12.