Internal Metabolites Research Articles

Impacts of paternal latitudinal origin on herbivore response strategies in Lythrum salicaria

Plants have evolved different strategies to withstand and respond to herbivore stress. The two main strategies plants employ under herbivore stress are known as tolerance and induced resistance. Tolerance is the ability to maintain high fitness despite experiencing high levels of herbivore damage. While induced resistance typically involves changing the concentrations of internal secondary metabolites as a response to initial herbivory and to prevent further damage. These strategies can lead to fitness differences between populations that vary by latitudinal origin. Previous studies have focused on the occurrence of herbivore response in general, whereas this study will make a direct comparison between response types and their fitness implications. Our study investigates whether eastern North American populations of Lythrum salicaria respond differently to herbivore stress. F1 crosses between L. salicaria individuals with different parental latitudinal ranges were grown in a common garden under two experimental herbivory treatments (pesticide sprayed vs. no pesticide) at the Queen’s University Biological Station. To identify differences in herbivore tolerance, we measured plant growth and development over 13 weeks and quantified herbivore damage, caused by the biocontrol beetle Galerucella calmariensis. A 48-hour feeding assay was then conducted to identify differences in herbivore resistance between populations. This assay provided beetles with a choice between young and old L. salicaria leaves from each experimental treatment. Our preliminary analysis of the feeding assay showed differences in leaf damage between parental lines. The initial analysis also showed that older leaves generally experienced more damage than younger leaves over the course of the 48-hour period. These results indicate population differences in herbivory stress response and differences in stress response effectiveness that varies with plant age. This experiment showed that L. salicaria individuals have varying degrees of herbivore stress response and effectiveness. However, further analysis that includes reproductive outputs and developmental differences will be completed to clearly identify the type of strategies L. salicaria populations with different parental latitudinal origins employ in response to herbivore damage.

The uptake, elimination, and toxicity of silver nanoparticles and silver ions in single-species and natural mixed-species bacterial biofilms

In nature, microorganisms predominantly exist as biofilms, which can reduce the bioavailability of toxic metals in water by adsorbing them. In this study, we investigated the adsorption, elimination, and toxicity of silver nanoparticles (AgNPs) and silver nitrate (AgNO3) in biofilms of the model bacterium Escherichia coli and those composed of natural mixed-species (NMS). Biofilms were exposed to different metal concentrations for 72 h, after which, they were transferred to a clean medium for 7 days to study the elimination of AgNPs and Ag+. We found that pseudo-second order kinetics predominated, and adsorption was jointly governed by external mass transfer and intraparticle diffusion. NMS biofilms were much more tolerant to AgNPs and Ag+ than E. coli biofilms, and Ag+ was much more toxic to both the biofilms than AgNPs. The concentrations of proteins, polysaccharides, and internal metabolites like respiratory chain dehydrogenase and cyclic diguanosine monophosphate were 0.3 to 3.7 fold greater in NMS biofilms than in E. coli biofilms, while those of reactive oxygen species and lactate dehydrogenase were 0.6 to 1.1 fold lower in NMS biofilms, highlighting a significant advantage that metal-exposed NMS biofilms enjoy in terms of both structure and metabolic function. These findings improve our understanding of the potential environmental risks of engineered nanoparticles and provide new insights into the bioremediation of nanoparticles-contaminated aquatic environments using biofilms.

Multi-Omics Analysis Revealed Increased De Novo Synthesis of Serine and Lower Activity of the Methionine Cycle in Breast Cancer Cell Lines.

A pipeline for metabolomics, based on UPLC-ESI-MS, was tested on two malignant breast cancer cell lines of the sub-types ER(+), PR(+), and HER2(3+) (MCF-7 and BCC), and one non-malignant epithelial cancer cell line (MCF-10A). This allowed us to quantify 33 internal metabolites, 10 of which showed a concentration profile associated with malignancy. Whole-transcriptome RNA-seq was also carried out for the three mentioned cell lines. An integrated analysis of metabolomics and transcriptomics was carried out using a genome-scale metabolic model. Metabolomics revealed the depletion of several metabolites that have homocysteine as a precursor, which was consistent with the lower activity of the methionine cycle caused by lower expression of the AHCY gene in cancer cell lines. Increased intracellular serine pools in cancer cell lines appeared to result from the over-expression of PHGDH and PSPH, which are involved in intracellular serine biosynthesis. An increased concentration of pyroglutamic acid in malignant cells was linked to the overexpression of the gene CHAC1.

Internal standard metabolites for estimating origin blood volume of bloodstains

The volume of blood leaked from blood vessels may change due to evaporation of water under the natural influence of the external environment. Bloodstains and dried blood spots (DBS), which describes blood dried in the external environment, are similar in their production and their metabolite quantification profiles. In both bloodstain metabolite analysis in the forensic science field and DBS metabolite analysis in the clinical field, it is important to determine the volume of the origin blood as this affects metabolite quantification results. Therefore, the purpose of this study is to discover the internal standard metabolites that have quantitatively proportional relationships with origin blood volume and maintain constant concentrations even as the age of the bloodstain increases. As a result, the concentrations of L-isoleucine and L-phenylalanine increased in proportion to the origin blood volume of the bloodstain. The differences in concentration of L-isoleucine were significant in all volume comparisons except in the comparison between 65 μL and 85 μL. The differences in concentration of L-phenylalanine were significant in all volume comparisons except between 65 μL and 45 μL and between 65 μL and 85 μL. In addition, it was confirmed that both metabolites tended to maintain constant concentrations without being affected by bloodstain age as the volume became smaller. These internal standard metabolites can be used for estimating the origin blood volume of bloodstains during metabolite analysis of bloodstains and DBS and could provide a volume criterion for standardization when comparing metabolite quantification between samples.

The applications and challenges of liquid chromatography tandem mass spectrometry in maternal and child health

Due to its ultra-high sensitivity, specificity and throughput, liquid chromatography tandem mass spectrometry (LC-MS/MS) has become an important analytical tool in clinical laboratories in quantifying various small molecules, such as vitamins, bile acids, steroids and other internal metabolites relevant to maternal diseases. As an effective means of screening and diagnosing diseases in preventive medicine, LC-MS/MS has been widely used in maternal and child health, contributing to the reduction of the incidence of maternal and child diseases and premature morbidity and mortality. At present, LC-MS/MS is an emerging and powerful platform in laboratory testing in China, facing both challenges and opportunities. In this article, the representative applications in the field of maternal and child health are summarized and discussed, along with the major hurdles of LC-MS/MS in clinical recognition and implementation.

Dynamics of internal isoprenoid metabolites in young Picea abies (Norway spruce) shoots during drought stress conditions in springtime

Currently, large areas of Picea abies (Norway spruce) stands in Europe are increasingly affected by drought and heat waves. Moreover, early spring drought has occurred with much higher frequency. Our work focuses on physiological changes induced by drought in four-year-old spruce seedlings during shoot elongation. We investigated drought effect on photosynthetic rate, concentration of abscisic acid and its metabolites, amount and composition of monoterpenes in needles of seedlings from five different provenances (altitude range 550–1280 m above sea level) in Western Carpathians. Spruce seedlings subjected to one-month drought stress of moderate intensity (about 50% of soil water content at the end of experiment) showed significant reduction of CO2 uptake and increased concentration of ABA related to untreated controls. Induced drought affected needle monoterpene content and composition. Observed changes in drought-induced physiological parameters were influenced by seedling provenance. The provenance from 920 m above sea level showed the greatest sensitivity to drought with significantly highest ABA content and, at the same time, a clear decline of CO2 uptake and amounts of total monoterpenes. Our results indicating intra-specific provenance-related variability in physiological response of spruce seedlings to drought may provide a basis for improved reforestation strategies in drought risk areas.

Strategies for studying in vivo biochemical formation pathways and multilevel distributions of sulfanilamide metabolites in food (2012–2022)

Sulfonamide metabolites are a major source of food pollution worldwide. However, the formation of internal sulfanilamide metabolites has only been investigated for selected compounds. In this paper, the fragmentation mechanism and characteristic ions of sulfonamide metabolites are reviewed using density functional theory and Q-Orbitrap high-resolution mass spectrometry. The result of the protonation site, rearrangement and bond breaking induced fragmentations at C6H6NO2S+m/z 156.01138, C6H6NO+m/z 108.04439, and C6H6N+m/z 92.04948. Mass shifts are calculated for derivative metabolites, including hydrogenation, acetylation, oxidation, glucosylation, glucosidation, sulfation, deamination, formylation, desulfonation and O-aminomethylation. Given their homologous series, it is demonstrated that similar metabolic reactions occur for all sulfonamides. The suspicious sulfonamide metabolites are confirmed by d-labelling experiments and reference standards. This is the first review of the latest advances in the field of sulfonamide metabolite prediction (2012–2022), and scheme design for metabolite multirresidue screening, as well as the challenges in the mass spectrometry evolution.

ATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis.

The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biology. Synthetic cellular systems are envisioned as out-of-equilibrium enzymatic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metabolism. Importantly, gaining tight control over the external medium is essential to avoid thermodynamic equilibrium due to nutrient depletion or waste buildup in a closed compartment (e.g., a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g., of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymatic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable l-arginine breakdown. In addition, we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium composition and to achieve sustainable glycerol 3-phosphate synthesis.

Development of an electrochemical flow-through cell for the fast and easy generation of isotopically labeled metabolite standards.

In drug development, metabolite standards of new chemical entities are required for a comprehensive safety evaluation. Stable isotope-labeled internal metabolite standards at the milligram scale, which are difficult and expensive to synthesize in common bottom-up approaches, are necessary for metabolite quantification using liquid chromatography/mass spectrometry. A preparative electrochemical flow-through cell is presented here as a powerful tool for the cheap and straightforward synthesis of milligram amounts of isotopically labeled metabolite standards. The developed cell scales up established, so-called "coulometric" electrochemical cells. Problems like electrode fouling and cross contamination between syntheses are addressed by the use of exchangeable working electrodes. The applicability of the developed cell for the synthesis of metabolite standards is demonstrated using isotopically labeled acetaminophen as a model system for the generation of a biologically relevant phase II metabolite.

Environmental pesticide concentrations in air and pregnant women’s urinary pesticide metabolites in the Infants’ Environmental Health Study (ISA)

BACKGROUND AND AIM: We evaluated associations between environmental pesticide concentrations in air and specific urinary pesticide metabolites among pregnant women from the Infants' Environmental Health Study (ISA) in Matina County, Costa Rica. METHODS: We used a Bayesian spatiotemporal model to extrapolate concentrations of the fungicide pyrimethanil and the insecticide chlorpyrifos measured with polyurethane foam passive air samplers (n=48, from 12 schools) across space and time. Using mixed models, we compared them with urinary specific-gravity corrected pesticide metabolite concentrations: 4-hydroxypyrimethanil (OHP, metabolite of pyrimethanil) and 3,5,6-trichloro-2-pyridinol (TCPy, metabolite of chlorpyrifos), repeatedly obtained among 451 pregnant women from the ISA Study (n=951). Pyrimethanil in air, OHP, and TCPy were log-transformed prior to statistical analysis to normalize residuals of models. We considered several covariables. RESULTS:Median (p10-p90) concentrations were: pyrimethanil = 1.33 (0.27-17.0) ng/m3, chlorpyrifos = 15.62 (4.38 - 24.19) ng/m3, OHP = 0.39 (0.06- 2.75) ug/L, and TCPy = 1.63 (0.75-4.27) µg/L. A 10% increase in pyrimethanil in air was associated with a 5.7% (95% CI 4.6, 6.8) increase in urinary OHP (μg/L), women who lived near banana plantations had higher OHP, whilst frequent consumption of rice and beans was associated with lower OHP. In addition, each 1 ng/m3 increase in chlorpyrifos in air was associated with a 1.5% increase in TCPy (μg/L), and women who worked in agriculture tended to have higher TCPy. CONCLUSIONS:Despite the limitations of having limited data on pesticide air concentrations in space and time, and biomonitoring of pesticides with short half-lives, the results of this study indicate environmental air concentrations of pyrimethanil and chlorpyrifos explained to some extent internal pesticide metabolite concentrations in pregnant women. Our results suggest that inhalation of these pesticides is one of the pathways of environmental exposure. Passive air sampling of current-use pesticides seems a promising method to monitor environmental pesticide exposure. KEYWORDS: passive air sampling, pesticides, environmental exposure, biomonitoring

Inter-study and time-dependent variability of metabolite abundance in cultured red blood cells

BackgroundCultured human red blood cells (RBCs) provide a powerful ex vivo assay platform to study blood-stage malaria infection and propagation. In recent years, high-resolution metabolomic methods have quantified hundreds of metabolites from parasite-infected RBC cultures under a variety of perturbations. In this context, the corresponding control samples of the uninfected culture systems can also be used to examine the effects of these perturbations on RBC metabolism itself and their dependence on blood donors (inter-study variations).MethodsTime-course datasets from five independent studies were generated and analysed, maintaining uninfected RBCs (uRBC) at 2% haematocrit for 48 h under conditions originally designed for parasite cultures. Using identical experimental protocols, quadruplicate samples were collected at six time points, and global metabolomics were employed on the pellet fraction of the uRBC cultures. In total, ~ 500 metabolites were examined across each dataset to quantify inter-study variability in RBC metabolism, and metabolic network modelling augmented the analyses to characterize the metabolic state and fluxes of the RBCs.ResultsTo minimize inter-study variations unrelated to RBC metabolism, an internal standard metabolite (phosphatidylethanolamine C18:0/20:4) was identified with minimal variation in abundance over time and across all the samples of each dataset to normalize the data. Although the bulk of the normalized data showed a high degree of inter-study consistency, changes and variations in metabolite levels from individual donors were noted. Thus, a total of 24 metabolites were associated with significant variation in the 48-h culture time window, with the largest variations involving metabolites in glycolysis and synthesis of glutathione. Metabolic network analysis was used to identify the production of superoxide radicals in cultured RBCs as countered by the activity of glutathione oxidoreductase and synthesis of reducing equivalents via the pentose phosphate pathway. Peptide degradation occurred at a rate that is comparable with central carbon fluxes, consistent with active degradation of methaemoglobin, processes also commonly associated with storage lesions in RBCs.ConclusionsThe bulk of the data showed high inter-study consistency. The collected data, quantification of an expected abundance variation of RBC metabolites, and characterization of a subset of highly variable metabolites in the RBCs will help in identifying non-specific changes in metabolic abundances that may obscure accurate metabolomic profiling of Plasmodium falciparum and other blood-borne pathogens.

Metabolomic analysis of coelomic fluids reveals the physiological mechanisms underlying evisceration behavior in the sea cucumber Apostichopus japonicus

Evisceration is a peculiar behavior that happens frequently in the processes of aquaculture and transportation of sea cucumbers. This behavior involves a complex physiological process and results in the expulsion of the digestive tract and other viscera. However, studies on evisceration are insufficient, and the identification of the internal regulatory metabolites and potential pathways of evisceration in the sea cucumber are therefore likely to provide a scientific basis for this specific behavior. In this study, ultraperformance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was performed on the coelomic fluids that ejected by evisceration Apostichopus japonicus and in the coeloms of normal A. japonicus to detect changes in metabolites and metabolic pathways in the process of evisceration behavior. In total, nine metabolites were found to have increased and eleven to have decreased in the ejected coelomic fluids of evisceration sea cucumbers. These metabolites included phosphatidylethanolamine, glucosylceramide, L-tryptophan, carbamic acid, and cyclohexylamine. In addition, enrichment of metabolic pathway analyses revealed five significantly changed signaling pathways: Glycosyl-phosphatidyl-inositol (GPI) -anchor biosynthesis, regulation of autophagy, sphingolipid metabolism, nitrogen metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. These results contribute valuable data on the potential physiological mechanisms underlying evisceration behavior of A. japonicus, which could have important implications for the aquaculture and transportation of sea cucumbers.

Recommendationsfor the content and management of Certificates of Analysis for reference standards from the GCCfor bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held inNew Orleans, LA, USA on 5April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysisfor reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysisfor reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolitesand critical reagents are discussed.

Effects of Sevoflurane Inhalation Anesthesia on the Intestinal Microbiome in Mice.

In recent years, more and more attention has been paid to intestinal microbiome. Almost all operations will go through the anesthesia process, but it is not clear whether the intervention of anesthesia alone will affect the change in the intestinal microbiome. The purpose of this study was to verify the effect of sevoflurane inhalation anesthesia on the intestinal microbiome. The animal in the experimental group was used to provide sevoflurane inhalation anesthesia for 4 hours. The control group was not intervened. The feces of the experimental group and the control group were collected on the 1st, 3rd, 7th and 14th days after anesthesia. Sevoflurane inhalation anesthesia will cause changes in the intestinal microbiome of mice. It appears on the 1st day after anesthesia and is most obvious on the 7th day. The specific manifestation is that the abundance of microbiome and the diversity of the microbiome is reduced. At the same time, Untargeted metabonomics showed that compared with the control group, the experimental group had more increased metabolites related to the different microbiome, among which 5-methylthioadenosine was related to the central nervous system. Subsequently, the intestinal microbiome diversity of mice showed a trend of recovery on the 14th day. At the genus level, the fecal samples obtained on the 14th day after anesthesia exhibited significantly increased abundances of Bacteroides, Alloprevotella, and Akkermansia and significantly decreased abundances of Lactobacillus compared with the samples obtained on the 1st day after anesthesia. However, the abundance of differential bacteria did not recover with the changing trend of diversity. Therefore, we believe that sevoflurane inhalation anesthesia is associated with changes in the internal microbiome and metabolites, and this change may be completed through the brain-gut axis, while sevoflurane inhalation anesthesia may change the intestinal microbiome for as long as 14 days or longer.

Metabolomic analysis of Schoenoplectus juncoides reveals common markers of acetolactate synthase inhibition among paddy weeds

Despite the increase in pressure for reducing the use of chemical pesticides in agriculture, herbicides remain one of the efficient tools for augmenting food production. Various herbicide-resistant weeds against most herbicidal modes of action (MoA) are emerging worldwide, and therefore, the necessity of developing herbicides with novel MoA is increasing. Toward this, rigid methods of determining MOA that can be applied for various weeds species are required. Despite the existence of weed species with resistance to acetolactate synthase (ALS) inhibitors, inhibition of ALS remains one of the most widely used herbicidal MoAs containing more than 50 commercial active ingredients. Here, we aimed to identify marker metabolites that are indicative of ALS inhibition. We performed non-targeted and targeted metabolomics using ALS inhibitor-treated Schoenoplectus juncoides. We identified internal metabolite markers for ALS inhibition, with excellent selectivity for ALS inhibitors and herbicides with different MOAs in various weed species. These markers will enable us to evaluate ALS inhibitory activity of chemicals in vivo in a wide variety of weed species.

In Silico Prediction of Metabolic Fluxes in Cancer Cells with Altered S-adenosylmethionine Decarboxylase Activity.

This paper investigates the redistribution of metabolic fluxes in the cell with altered activity of S-adenosylmethionine decarboxylase (SAMdc, EC: 4.1.1.50), the key enzyme of the polyamine cycle and the common target for antitumor therapy. To address these goals, a stoichiometric metabolic model was developed that includes five metabolic pathways: polyamine, methionine, methionine salvage cycles, folic acid cycle, and the pathway of glutathione and taurine synthesis. The model is based on 51 reactions involving 57 metabolites, 31 of which are internal metabolites. All calculations were performed using the method of Flux Balance Analysis. The outcome indicates that the inactivation of SAMdc results in a significant increase in fluxes through the methionine, the taurine and glutathione synthesis, and the folate cycles. Therefore, when using therapeutic agents inactivating SAMdc, it is necessary to consider the possibility of cellular tumor metabolism reprogramming. S-adenosylmethionine affects serine methylation and activates serine-dependent de novo ATP synthesis. Methionine-depleted cell becomes methionine-dependent, searching for new sources of methionine. Inactivation of SAMdc enhances the transformation of S-adenosylmethionine to homocysteine and then to methionine. It also intensifies the transsulfuration process activating the synthesis of glutathione and taurine.

Application of a combined aggregate exposure pathway and adverse outcome pathway (AEP-AOP) approach to inform a cumulative risk assessment: A case study with phthalates

Advancements in measurement and modeling capabilities are providing unprecedented access to estimates of chemical exposure and bioactivity. With this influx of new data, there is a need for frameworks that help organize and disseminate information on chemical hazard and exposure in a manner that is accessible and transparent. A case study approach was used to demonstrate integration of the Adverse Outcome Pathway (AOP) and Aggregate Exposure Pathway (AEP) frameworks to support cumulative risk assessment of co-exposure to two phthalate esters that are ubiquitous in the environment and that are associated with disruption of male sexual development in the rat: di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DnBP). A putative AOP was developed to guide selection of an in vitro assay for derivation of bioactivity values for DEHP and DnBP and their metabolites. AEPs for DEHP and DnBP were used to extract key exposure data as inputs for a physiologically based pharmacokinetic (PBPK) model to predict internal metabolite concentrations. These metabolite concentrations were then combined using in vitro-based relative potency factors for comparison with an internal dose metric, resulting in an estimated margin of safety of ~13,000. This case study provides an adaptable workflow for integrating exposure and toxicity data by coupling AEP and AOP frameworks and using in vitro and in silico methodologies for cumulative risk assessment.

Application of nuclear magnetic resonance for analyzing metabolic characteristics of winter diatom blooms

Abstract We compared two metabolome profiles of a small centric diatom species, Stephanodiscus hantzschii Grun., grown under conditions with enriched nutrients but different temperatures. This species proliferates in eutrophic rivers during winter. We investigated the population dynamics and internal metabolite changes of Stephanodiscus by performing a simple culture experiment at different temperatures (5 and 15°C). We applied the 1H nuclear magnetic resonance (NMR) technique to fully grown cells to obtain the metabolite profiles of S. hantzschii. Growth rates were significantly different at different temperature conditions (0.99 ± 0.11 day−1 at 15°C and 0.21 ± 0.12 day−1 at 5°C, n = 10). Characterized metabolites included saturated and unsaturated fatty acids, AXP (including AMP, ADP and ATP), and UDP-glucose and UDP-galactose, all of which are important for energy metabolism. These metabolites were abundant within S. hantzschii cells grown at 15°C but were not prolific in those grown at 5°C. Furthermore, other 1H NMR spectrum uncovered very little amounts of metabolites. Based on these observations of cell growth rate, although required nutrients were supplied, colder temperatures suppressed population growth through the deactivation of various internal metabolisms. Thus, winter proliferation of this species is opportunistic, implying that survival success led to dominance in freshwater ecosystems with neither resource competition nor grazing pressure.

HIF1/2-exerted control over glycolytic gene expression is not functionally relevant for glycolysis in human leukemic stem/progenitor cells

BackgroundHypoxia-inducible factors (HIF)1 and 2 are transcription factors that regulate the homeostatic response to low oxygen conditions. Since data related to the importance of HIF1 and 2 in hematopoietic stem and progenitors is conflicting, we investigated the chromatin binding profiles of HIF1 and HIF2 and linked that to transcriptional networks and the cellular metabolic state.MethodsGenome-wide ChIPseq and ChIP-PCR experiments were performed to identify HIF1 and HIF2 binding sites in human acute myeloid leukemia (AML) cells and healthy CD34+ hematopoietic stem/progenitor cells. Transcriptome studies were performed to identify gene expression changes induced by hypoxia or by overexpression of oxygen-insensitive HIF1 and HIF2 mutants. Metabolism studies were performed by 1D-NMR, and glucose consumption and lactate production levels were determined by spectrophotometric enzyme assays. CRISPR-CAS9-mediated HIF1, HIF2, and ARNT−/− lines were generated to study the functional consequences upon loss of HIF signaling, in vitro and in vivo upon transplantation of knockout lines in xenograft mice.ResultsGenome-wide ChIP-seq and transcriptome studies revealed that overlapping HIF1- and HIF2-controlled loci were highly enriched for various processes including metabolism, particularly glucose metabolism, but also for chromatin organization, cellular response to stress and G protein-coupled receptor signaling. ChIP-qPCR validation studies confirmed that glycolysis-related genes but not genes related to the TCA cycle or glutaminolysis were controlled by both HIF1 and HIF2 in leukemic cell lines and primary AMLs, while in healthy human CD34+ cells these loci were predominantly controlled by HIF1 and not HIF2. However, and in contrast to our initial hypotheses, CRISPR/Cas9-mediated knockout of HIF signaling did not affect growth, internal metabolite concentrations, glucose consumption or lactate production under hypoxia, not even in vivo upon transplantation of knockout cells into xenograft mice.ConclusionThese data indicate that, while HIFs exert control over glycolysis but not OxPHOS gene expression in human leukemic cells, this is not critically important for their metabolic state. In contrast, inhibition of BCR-ABL did impact on glucose consumption and lactate production regardless of the presence of HIFs. These data indicate that oncogene-mediated control over glycolysis can occur independently of hypoxic signaling modules.

Integrated TORC1 and PKA signaling control the temporal activation of glucose-induced gene expression in yeast

The growth rate of a yeast cell is controlled by the target of rapamycin kinase complex I (TORC1) and cAMP-dependent protein kinase (PKA) pathways. To determine how TORC1 and PKA cooperate to regulate cell growth, we performed temporal analysis of gene expression in yeast switched from a non-fermentable substrate, to glucose, in the presence and absence of TORC1 and PKA inhibitors. Quantitative analysis of these data reveals that PKA drives the expression of key cell growth genes during transitions into, and out of, the rapid growth state in glucose, while TORC1 is important for the steady-state expression of the same genes. This circuit design may enable yeast to set an exact growth rate based on the abundance of internal metabolites such as amino acids, via TORC1, but also adapt rapidly to changes in external nutrients, such as glucose, via PKA.