Differences In Metabolic State Research Articles

A pan-cancer metabolic atlas of the tumor microenvironment.

Tumors are heterogeneous cellular environments with entwined metabolic dependencies. Here, we use a tumor transcriptome deconvolution approach to profile the metabolic states of cancer and non-cancer (stromal) cells in bulk tumors of 20 solid tumor types. We identify metabolic genes and processes recurrently altered in cancer cells across tumor types, highlighting pan-cancer upregulation of deoxythymidine triphosphate (dTTP) production. In contrast, the tryptophan catabolism rate-limiting enzymes IDO1 and TDO2 are highly overexpressed in stroma, raising the hypothesis that kynurenine-mediated suppression of antitumor immunity may be predominantly constrained by the stroma. Oxidative phosphorylation is the most upregulated metabolic process in cancer cells compared to both stromal cells and a large atlas of cancer cell lines, suggesting that the Warburg effect may be less pronounced in cancer cells invivo. Overall, our analysis highlights fundamental differences in metabolic states of cancer and stromal cells inside tumors and establishes a pan-cancer resource to interrogate tumor metabolism.

Metabolic imaging of human cumulus cells reveals associations among metabolic profiles of cumulus cells, patient clinical factors, and oocyte maturity

To determine whether fluorescence lifetime imaging microscopy (FLIM) detects differences in metabolic state among cumulus cell samples and whether their metabolic state is associated with patient age, body mass index (BMI), and antimüllerian hormone (AMH) level and maturity of the oocyte. Prospective observational study. Academic laboratory. Cumulus cell (CC) clusters from cumulus-oocyte complexes were collected from patients undergoing assisted reproductive technology treatment after oocyte retrieval and vitrified. Cumulus cell metabolism was assessed using FLIM to measure autofluorescence of nicotinamide adenine (phosphate) dinucleotide and flavine adenine dinucleotide, endogenous coenzymes essential for cellular respiration and glycolysis. Patient age, BMI, and AMH level and the maturity of the corresponding oocytes were recorded. Quantitative information from FLIM was obtained regarding metabolite concentrations from fluorescence intensity and metabolite enzyme engagement from fluorescence lifetimes. Associations were investigated between each FLIM parameter and oocyte maturity and patient age, BMI, and AMH. Variance between CC clusters within and between patients was determined. Of 619 CC clusters from 193 patients, 90 were associated with immature oocytes and 505 with metaphase II oocytes. FLIM enabled quantitative measurements of the metabolic state of CC clusters. These parameters were significantly correlated with patient age and AMH independently, but not with BMI. Cumulus cell nicotinamide adenine (phosphate) dinucleotide FLIM parameters and redox ratio were significantly associated with maturity of the enclosed oocyte. FLIM detects variations in the metabolic state of CCs, showing a greater variance among clusters from each patient than between patients. Fluorescence lifetime imaging microscopy can detect CC metabolic associations with patient age and AMH and variations between mature and immature oocytes, suggesting the potential utility of this technique to help identify superior oocytes.

Abstract PO-055: Pan-cancer metabolic profiling of the tumor microenvironment

Abstract Tumors are heterogeneous cellular environments with entwined metabolic dependencies. However, it is challenging to differentially profile metabolism in cancer and infiltrating non-malignant cells inside patient tumors. Here, we used a tumor transcriptome deconvolution approach to profile the metabolic states of cancer and non-cancer (stromal) cells in bulk tumors of 20 solid tumor types. This analysis highlighted metabolic genes and processes recurrently upregulated in cancer cells across tumor types, including the mitochondrial dicarboxylate carrier SLC25A10. Intriguingly, the tryptophan-degrading dioxygenase, IDO1, was highly recurrently overexpressed in stroma compared to cancer cells and normal tissue, suggesting that tryptophan-mediated suppression of antitumor immune response is predominantly constrained by the stroma. Oxidative phosphorylation was unexpectedly the most upregulated metabolic process in cancer cells compared to both stromal cells and a large atlas of cancer cell lines, suggesting that the warburg effect may be less pronounced in cancer cells in vivo. Overall, our analysis highlights fundamental differences in metabolic states of cancer and stromal cells inside tumors and establishes a pan-cancer resource to interrogate tumor metabolism. Citation Format: Neha Rohatgi, Umesh Ghoshdastider, Probhonjon Baruah, Anders Jacobsen Skanderup. Pan-cancer metabolic profiling of the tumor microenvironment [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-055.

Mechanisms mediating nutritional effects on embryonic survival in pigs

The inconsistency of data from experiments designed to show nutritional effects on embryonic survival is perplexing. However, a number of experimental models have provided some insight into the mechanisms that potentially mediate interactions between nutrition, metabolic state and embryonic survival. The developing ovarian follicle provides the maturational environment for the oocyte, and differences in follicular maturation are associated with differences in the ability of these follicles to support oocyte maturation. In turn, the rate of oocyte maturation and the maturational state of the oocyte immediately before ovulation may contribute to differences in embryonic survival. Therefore, evidence that nutritionally induced differences in metabolic state affect follicular development and the maturational state of the oocyte in the late follicular phase may constitute a mechanism by which nutrition affects the very early stages of conceptus development. Once ovulation occurs, the process of fertilization and early cleavage occurs in the environment of the oviduct. Nutritional state might affect the secretory and motile activity of the oviduct both directly, by influencing the physiology of the oviductal cells, or indirectly by affecting the secretion of key regulatory hormones. Thus evidence for nutritionally dependent effects on plasma progesterone concentrations in early pregnancy and associations with differences in embryonic survival may be partly mediated at the oviductal stage of development. Nutritional effects on circulating progesterone concentrations may also affect the uterine environment. However, the metabolic state of the gilt or sow, or specific nutrients in the diet, may directly affect the integrity of the endometrium and thus affect embryonic survival at this stage of development.

Effects of nest type and sex on blood saccharide profiles in Humboldt penguins (Spheniscus humboldti): Implications for habitat conservation.

Reproductive success of endangered Humboldt penguin (Spheniscus humboldti) colonies in Peru has been associated with nesting habitat type, presumably due to differences in environmental exposure and activity patterns that may affect energy demands and metabolism. Gas chromatography and mass spectrometry were used to determine serum concentrations of 19 saccharides from 30 Humboldt penguins nesting at Punta San Juan, Peru in order to evaluate differences in metabolic state between penguins nesting in a sheltered burrow or crevice (n = 17) and those in exposed surface nests (n = 13). Univariate and multivariate statistical analyses identified serum saccharides (arabinose, maltose, glucose-6-phosphate, and levoglucosenone in particular) that were nest-dimorphic with substantial differences between surface- and sheltered-nesting penguins. Four sugars (arabinose, xylose, fructose-6-phosphate, and sucrose) had ≥ 2-fold difference in concentration between nest types. Seven saccharides were in the top five subsets generated by discriminant analysis; four of these are simple sugars (D-glucopyranose, α ⇄ β; D-glucose; D-maltose; and D-mannose) and three are derivatives (glucose 6-phosphate, levoglucosenone, and N-acetylglucosamine). D-ribose had the highest information values (generated from weight-of-evidence values) followed by glucose 6-phosphate, levoglucosenone, and D-galactose. Sex was not a significant predictor of saccharide concentration. Levoglucosenone, which is a metabolite of the environmental contaminant levoglucosan, was significantly higher in surface-nesting penguins, reflecting a higher rate of exposure in non-sheltered penguins. Differences in the saccharide profiles of surface- and sheltered-nesting Humboldt penguins likely reflect increased metabolic requirements of surface-nesters at Punta San Juan. Conservation of appropriate sheltered-nesting habitat for penguins is essential for sustained reproductive success and colony health.

Exercise-Induced Interleukin-6 and Metabolic Responses in Hot, Temperate, and Cold Conditions

The purpose of this study was to determine the effects of exercise in hot, cold, and temperate environments on plasma interleukin-6 (IL-6). Eleven recreationally trained males (age = 25 ± 4 years, height = 178 ± 5 cm, weight = 79.4 ± 13.5 kg, body fat = 14.7 ± 3.6%, VO2 peak = 54.6 ± 11.5 ml kg-1 min-1) performed a 1 hr cycling bout in hot (33 °C), cold (7 °C), and temperate (20 °C) environments at 60% of Wmax followed by 3 hr of supine recovery in temperate conditions. Expired gases were measured every 15 min during exercise and once every hour during recovery. Heart rate was continuously measured throughout the trials. Blood samples were obtained from the antecubital vein pre-exercise, immediately post-exercise, and 3 hr post-exercise. Blood samples were analyzed for plasma concentrations of IL-6 using a commercial ELISA kit. Plasma IL-6 concentrations were significantly higher immediately post-exercise (14.8 ± 1.6 pg ml-1, p = 0.008) and 3 hr post-exercise (14.8 ± 0.9 pg ml-1, p = 0.018) compared to pre-exercise (11.4 ± 2.4 pg ml-1), across all trials. There were no differences in plasma IL-6 concentrations (p = 0.207) between temperature conditions.Oxygen consumption and heart rate were higher and respiratory exchange ratio was lower in the hot compared to other conditions (p < 0.05). These data indicate that the temperature in which exercise occurs does not affect acute plasma IL-6 response despite differences in metabolic state.

Abstract B144: Elucidating germinal center B cell metabolic states and their influence on selection

Abstract A majority of B cell lymphomas originate from germinal center (GC) B cells. While somatic hypermutation in these cells can increase antibody affinity in response to infection, it can also cause collateral damage to DNA giving rise to cancer in this highly proliferative cell type. Successfully mutated antibody with increased affinity leads to increased antigen capture and presentation and subsequently increased follicular T cell help. T cell help in the light zone of the germinal center initiates a program of GC B cell proliferation with increased T cell help shortening the S phase and controlling dwell time in the dark zone where GC B cells proliferate. However, the mechanism underlying the number of cell cycles a selected B cell performs before returning to the light zone is elusive. We hypothesize that the number of cell cycles a selected GC B cell goes through is metabolically controlled by a mechanism in which a GC B cell is metabolically charged to perform a given number of divisions based on the strength of the selecting T cell signal. Once the metabolic charge is exhausted cells would return to the light zone for another round of selection. This metabolic charge could entail a) storage of metabolites, b) selection-controlled access to metabolites via temporal, e.g. transcriptional control of metabolite transporters or c) regulation of mitochondria function and metabolic enzymes of aerobic respiration. We are currently creating various mouse models to define the underlying mechanisms involved. To determine the contribution of metabolite transporters we will delete the main glucose transporter Glut1 in GC B cells using both constitutive deletion with AID-Cre and inducible deletion using AID-CreERT2. We are also constructing an IgHG1-IRES-CreERT2 mouse as an alternative to AID-CreERT2 and to potentially evaluate differences between IgM+ and IgG1+ cells. Using metabolic assays we will evaluate the status of glucose uptake, oxidative phosphorylation and mitochondrial function both by flow cytometric and microscopic methods. Using an established in vivo system in which we can selectively target a population of GC B cells for selection by loading them with antigen we will determine differences in metabolic states of selected and unselected GC B cells. Using an in vivo cell tracing assay in which mCherry fluorescent protein is diluted with cell division upon doxycycline treatment we will be able to determine differences in metabolic states of cells having highly proliferated or just having been selected. Our studies will show whether GC B cells are subject to the Warburg effect similar to malignant tumors and might in part explain the origin of the metabolic reprogramming seen in GC B cell-derived lymphomas. Understanding the underlying metabolic processes of GC B cells will not only help explain the emergence of B cell lymphoma but also their dependence on certain metabolic pathways. Specific factors important for metabolic regulation such as, but not limited to Glut1, will also be identified. These insights could eventually open up new avenues for B cell lymphoma treatment and particularly augment vaccination approaches both in tumor immunology and elsewhere. Citation Format: Harald Hartweger, Shlomo Finkin, Michel C. Nussenzweig. Elucidating germinal center B cell metabolic states and their influence on selection [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B144.

Effects of parity and periconceptional metabolic state of Holstein–Friesian dams on the glucose metabolism and conformation in their newborn calves

The metabolic state of pregnant mammals influences the offspring’s development and risk of metabolic disease in postnatal life. The metabolic state in a lactating dairy cow differs immensely from that in a non-lactating heifer around the time of conception, but consequences for their calves are poorly understood. The hypothesis of this study was that differences in metabolic state between non-lactating heifers and lactating cows during early pregnancy would affect insulin-dependent glucose metabolism and development in their neonatal calves. Using a mixed linear model, concentrations of glucose, IGF-I and non-esterified fatty acids (NEFAs) were compared between 13 non-lactating heifers and 16 high-yielding dairy cows in repeated blood samples obtained during the 1st month after successful insemination. Calves born from these dams were weighed and measured at birth, and subjected to intravenous glucose and insulin challenges between 7 and 14 days of age. Eight estimators of insulin-dependent glucose metabolism were determined: glucose and insulin peak concentration, area under the curve and elimination rate after glucose challenge, glucose reduction rate after insulin challenge, and quantitative insulin sensitivity check index. Effects of dam parity and calf sex on the metabolic and developmental traits were analysed in a two-way ANOVA. Compared with heifers, cows displayed lower glucose and IGF-I and higher NEFA concentrations during the 1st month after conception. However, these differences did not affect developmental traits and glucose homeostasis in their calves: birth weight, withers height, heart girth, and responses to glucose and insulin challenges in the calves were unaffected by their dam’s parity. In conclusion, differences in the metabolic state of heifers and cows during early gestation under field conditions could not be related to their offspring’s development and glucose homeostasis.

Glucose regulates enzymatic sources of mitochondrial NADPH in skeletal muscle cells; a novel role for glucose‐6‐phosphate dehydrogenase

Reduced nicotinamide adenine dinucleotide (NADPH) is a functionally important metabolite required to support numerous cellular processes. However, despite the identification of numerous NADPH-producing enzymes, the mechanisms underlying how the organellar pools of NADPH are maintained remain elusive. Here, we have identified glucose-6-phosphate dehydrogenase (G6PDH) as an important source of NADPH in mitochondria. Activity analysis, submitochondrial fractionation, fluorescence microscopy, and protease sensitivity assays revealed that G6PDH is localized to the mitochondrial matrix. 6-ANAM, a specific G6PDH inhibitor, depleted mitochondrial NADPH pools and increased oxidative stress revealing the importance of G6PDH in NADPH maintenance. We also show that glucose availability and differences in metabolic state modulate the enzymatic sources of NADPH in mitochondria. Indeed, cells cultured in high glucose (HG) not only adopted a glycolytic phenotype but also relied heavily on matrix-associated G6PDH as a source of NADPH. In contrast, cells exposed to low-glucose (LG) concentrations, which displayed increased oxygen consumption, mitochondrial metabolic efficiency, and decreased glycolysis, relied predominantly on isocitrate dehydrogenase (ICDH) as the principal NADPH-producing enzyme in the mitochondria. Culturing glycolytic cells in LG for 48 h decreased G6PDH and increased ICDH protein levels in the mitochondria, further pointing to the regulatory role of glucose. 2-Deoxyglucose treatment also prevented the increase of mitochondrial G6PDH in response to HG. The role of glucose in regulating enzymatic sources of mitochondrial NADPH pool maintenance was confirmed using human myotubes from obese adults with a history of type 2 diabetes mellitus (post-T2DM). Myotubes from post-T2DM participants failed to increase mitochondrial G6PDH in response to HG in contrast to mitochondria in myotubes from control participants (non-T2DM). Hence, we not only identified a matrix-associated G6PDH but also provide evidence that metabolic state/glucose availability modulate enzymatic sources of NADPH.

Long-term Population Dynamics and in situ Physiology in Activated Sludge Systems with Enhanced Biological Phosphorus Removal Operated with and without Nitrogen Removal

Quantitative fluorescence in situ hybridization (FISH) and the combination of FISH with microautoradiography (MAR) were used in order to study the long-term population dynamics (2.5 years) and the in situ physiology in two parallel activated sludge pilot systems with enhanced biological phosphorus removal (EBPR). The two systems received the same influent wastewater, but were differently operated (with and without nitrogen removal, respectively). Both systems showed a significant P removal that increased when different substrates (phosphorus (P), acetate and glucose, respectively) were added to the influent wastewater. Rhodocyclus-related bacteria were present in both systems in significant numbers (ranging from 4 to 28%) throughout the whole period. This supports the hypothesis that these bacteria occur in significant numbers in different types of well-operating EBPR activated sludge processes. However, we observed a lower correlation (< 0.5) for the amount of Rhodocyclus-related bacteria to the P content in activated sludge than previous studies (> 0.9). The Actinobacteria were the only additional group of bacteria which showed a similar degree of correlation to the P content in activated sludge as the Rhodocyclus-related bacteria--but only for the system without nitrogen removal. Significant amounts (< or = 12%) of glycogen-accumulating bacteria (GAOs) were detected in the system with nitrogen removal (but not in the other system), but had no, in contrast to previous observations, apparent negative effect on the overall EBPR performance. FISH-MAR indicated that a significant part of the Betaproteobacteria (part of them identified as Rhodocyclus-related bacteria) as well as the Actinobacteria were able to take up 33Pi, [3H]-acetate and [3H]-glucose under anaerobic-aerobic conditions. The contribution of anoxic 33Pi uptake under alternating anaerobic-anoxic conditions was significantly lower. Interestingly, not all Rhodocyclus-related bacteria showed uptake of these three radioactive substrates. This may be due to differences in metabolic state, physiological potential or genotype, not detectable by the present probe set for Rhodocyclus-related bacteria. Comparison of the 33Pi, [3H]-acetate and [3H]-glucose uptake by activated sludge after different fixation and incubation procedures showed that a part of the observed 33Pi uptake may have been caused by a combination of a biological and chemical or biologically induced chemical P adsorption.

Phenolic Compounds as a Tool of Bioindication for Novel Forest Decline at Numerous Spruce Tree Sites in Germany

Abstract Within a project that applied biochemical criteria to the diagnosis of damage to Norway spruce, 43 sites in western and eastern Germany showing only moderate tree damage were screened for the amounts of methanol soluble phenolic compounds in spruce needles. The concentrations of most of the main compounds-especially catechin-positively correlated with needle loss and the altitude of the site. It was also found that it is necessary to differenti­ate between trees younger and older than 60 years of age. The correlations between the increase of the phenolic compounds studied and the needle loss or the damage class are stronger in the younger trees, possibly implicating differences in metabolic state or distur­bances in protectective mechanisms in the older trees.

The annual cycle of Erinaceus europaeus L. As a model for a further study of cytochemical heterogeneity in purkinje neuron nuclei

The cytochemical heterogeneity of nuclei in Purkinje cells, which reflects differences in metabolic states within the population, has been studied by comparing cerebellar hemispheres of the hedgehog during periods of activity and hibernation. Cerebellar granule cells and hepatocytes served as controls for diploid cells. Three different cytochemical stains (Feulgen reaction, Propidium iodide and Hoechst 33342 fluorochromes) were used in order to evaluate by microdensitometry and microfluorometry how much the heterogeneity is dependent on the variations in DNA content and degree of chromatin compaction. In the active period, Feulgen-DNA values of Purkinje cells were more widely dispersed than in control cells. Some values (18–40% of the population) exceeded (hyperdiploid) the diploid value. With Propidium iodide and Hoechst 33342 (under conditions for quantitative evaluation of DNA), a few values also exceeded the maximum value for control cells. We conclude that there is nuclear heterogeneity (diploid-hyperdiploid) in the Purkinje neuron population during normal activity. The percentage of hyperdiploid nuclei changes according to the technique used; in particular, the comparison of the different stains indicates that a different chromatin compaction is mainly responsible for hyperdiploid Feulgen-DNA values, but in a few hyperdiploid cells there is an actual surplus of DNA. In view of the modalities of detection of DNA by the different cytochemical approaches utilized, this may be mainly single-stranded DNA. During hibernation nuclear heterogeneity is still present in Purkinje neurons, but with a shift of the distribution towards lower values; hyperdiploid nuclei were not detected with all the procedures used. Moreover, when Propidium iodide is used at the concentration suitable for qualitative studies, the dye intercalation is lower than during activity. Data suggest that both decreased DNA and increased chromatin compaction occur in Purkinje cells, thus pointing to a lower metabolic activity of these neurons in hibernating animals. The changes in diploid granule cells were always less than those in Purkinje neurons and could be ascribed to a further increase in chromatin compaction only, not requiring changes in DNA content.

Bile secretion in hemoglobin-free perfused rat liver.

Hemoglobin-free perfused rat liver was demonstrated to be a suitable experimental model in studying bile secretion. Bile flow slowly decreased to more than 3 h of perfusion. Despite differences in metabolic states, the bile flow was the same in the recirculating as in the nonrecirculating mode of perfusion. Sulfobromophthalein stimulated bile flow at high rates of infusion. In bile, the ratio conjugated to unconjugated sulfobromophthalein also increased with sulfobromophthalein infusion rate. The access of [14C]insulin, [14C] sucrose, and inorganic [32P] phosphate from perfusate into bile was restricted. Bile flow, secretion of taurocholate and sulfobromophthalein, and bile pressure are compared with values from anesthetized animals and from isolated livers perfused with medium containing erythrocytes.

Reversible Abolition of Normal Morphology in Hydra

THE experimental evidence concerning head and foot regeneration in hydra has been interpreted most consistently in terms of one or more gradients of unknown biochemical nature running along the length of the animal1–5. There is also a considerable literature implicating underlying variations in “metabolic activity” in the setting up of gradients in hydra and other developing systems6–9, but the relationship of this activity to problems of pattern formation in animal development is ill defined, because it is difficult to explain how quantitative differences in metabolic state (characterized biochemically) could be reliably distributed and translated into the qualitative differences of various cell types (see refs 10–12 for models for this process). Hydra might be particularly suited to studies in this area, because its axial pattern is simple and relatively well characterized5,13, and regeneration and pattern regulation do not appear to require cell division13. The changing fates of cells within a single, organized population could ideally be followed under defined environmental influences on the metabolism.

Studies on controlled and released respiration in animal and vegetal halves of the embryo of the sea urchin, Paracentrotus lividus

Summary Eggs of the sea urchin Paracentrotus lividus were separated in the 16–32 cell stage in animal and vegetal halves. These were allowed to develop, and the respiration of the halves was measured in the microrespirometer of Linderstrom-Lang and Holter. The measurements referred to advanced cleavage stages (≥128 cells) and to the mesenchyme blastula stage or beginning gastrula stage. The medium was either pure sea water or sea water within the addition of 5×10 −5 M 2,4-dinitrophenol. In the first medium the “controlled” in the latter the “released” respiration was measured. A survey of the results is given in Figs. 1 and 2. In both stages which were subject to examination, the respiratory control ratios (released respiration over controlled respiration) in animal halves were high, about 2.9, a value corresponding to that found immediately after fertilization in whole eggs suspended in normal sea water [14]. In advanced cleavage stage, the respiratory rate seemed to be higher in vegetal than in animal halves. Addition of dinitrophenol caused a certain decrease in the respiratory rate (Fig. 1). Vegetal halves in the mesenchyme blastula state (Fig. 2), on the other hand, showed a respiratory rate not significantly different from that in animal halves. Upon addition of dinitrophenol only a rather low increase in the respiratory rate occurred (Fig. 2). Controls were made in order to ascertain that the changes in respiratory rate were not due to damage caused by the process of separating the halves. As the released respiration expresses the maximal electron transport capacity [24], it may also be considered an approximate measure of the amount of respiratory enzyme chains present in the embryos. Thus, a considerable synthesis of these enzymes must take place in the isolated animal halves. This is in agreement with the increasing incorporation of amino acids in isolated animal halves, demonstrated by Markman [21]. The results with isolated vegetal halves at mesenchyme blastula stage indicate, on the other hand, that the amount of respiratory enzyme chains is lower than in animal halves, which again corresponds to studies on incorporation of labeled amino acids [21]. The different values of the respiratory control ratio in animal and vegetal halves indicate differences in metabolic state. A high value of the ratio is in keeping with an accumulation of energy-rich compounds, whereas lower values indicate that a certain steady state has been approached. The interactions between animal and vegetal regions which display themselves in the normal development are discussed in the light of the results obtained. Financial support was given from the Swedish Natural Sciences Research Council, the Swedish Cancer Society and the Consiglio Nazionale delle Recerche (Rome). These grants are most gratefully acknowledged.