Metabolic remodeling: a pyruvate transport affair

Metabolic remodeling is a major determinant for many cell fate decisions, and a switch from respiration to aerobic glycolysis is generally considered as a hallmark of cancer cell transformation. Pyruvate is a key metabolite at the major junction of carbohydrate metabolism between cytosolic glycolysis and the mitochondrial Krebs cycle. In this issue of The EMBO Journal, Bender et al show that yeast cells regulate pyruvate uptake into mitochondria, and thus its metabolic fate, by expressing alternative pyruvate carrier complexes with different activities.     

Effect of (R)‐salbutamol on the switch of phenotype and metabolic pattern in LPS‐induced macrophage cells

Evidence demonstrates that M1 macrophage polarization promotes inflammatory disease. Here, we discovered that (R)‐salbutamol, a β₂ receptor agonist, inhibits and reprograms the cellular metabolism of RAW264.7 macrophages. (R)‐salbutamol significantly inhibited LPS‐induced M1 macrophage polarization and downregulated expressions of typical M1 macrophage cytokines, including monocyte chemotactic protein‐1 (MCP‐1), interleukin‐1β (IL‐1β) and tumour necrosis factor α (TNF‐α). Also, (R)‐salbutamol significantly decreased the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and reactive oxygen species (ROS), while increasing the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. In contrast, (S)‐salbutamol increased the production of NO and ROS. Bioenergetic profiles showed that (R)‐salbutamol significantly reduced aerobic glycolysis and enhanced mitochondrial respiration. Untargeted metabolomics analysis demonstrated that (R)‐salbutamol modulated metabolic pathways, of which three metabolic pathways, namely, (a) phenylalanine metabolism, (b) the pentose phosphate pathway and (c) glycerophospholipid metabolism were the most noticeably impacted pathways. The effects of (R)‐salbutamol on M1 polarization were inhibited by a specific β₂ receptor antagonist, ICI‐118551. These findings demonstrated that (R)‐salbutamol inhibits the M1 phenotype by downregulating aerobic glycolysis and glycerophospholipid metabolism, which may propose (R)‐salbutamol as the major pharmacologically active component of racemic salbutamol for the treatment of inflammatory diseases and highlight the medicinal value of (R)‐salbutamol.     

Inhibition of Anaplerotic Glutaminolysis Underlies Selenite Toxicity in Human Lung Cancer

Large clinical trials and model systems studies suggest that the chemical form of selenium dictates chemopreventive and chemotherapeutic efficacy. Selenite induces excess ROS production, which mediates autophagy and eventual cell death in non‐small cell lung cancer adenocarcinoma A549 cells. As the mechanisms underlying these phenotypic effects are unclear, the clinical relevance of selenite for cancer therapy remains to be determined. The authors' previous stable isotope‐resolved metabolomics and gene expression analysis showed that selenite disrupts glycolysis, the Krebs cycle, and polyamine metabolism in A549 cells, potentially through perturbed glutaminolysis, a vital anaplerotic process for proliferation of many cancer cells. Herein, the role of the glutaminolytic enzyme glutaminase 1 (GLS1) in selenite's toxicity in A549 cells and in patient‐derived lung cancer tissues is investigated. Using [¹³C₆]‐glucose and [¹³C₅,¹⁵N₂]‐glutamine tracers, selenite's action on metabolic networks is determined. Selenite inhibits glutaminolysis and glutathione synthesis by suppressing GLS1 expression, and blocks the Krebs cycle, but transiently activates pyruvate carboxylase activity. Glutamate supplementation partially rescues these anti‐proliferative and oxidative stress activities. Similar metabolic perturbations and necrosis are observed in selenite‐treated human patients' cancerous lung tissues ex vivo. The results support the hypothesis that GLS1 suppression mediates part of the anti‐cancer activity of selenite both in vitro and ex vivo.     

Thermoregulatory and metabolic responses of Japanese quail to hypoxia

Common responses to hypoxia include decreased body temperature (T_b) and decreased energy metabolism. In this study, the effects of hypoxia and hypercapnia on T_b and metabolic oxygen consumption (V.O₂) were investigated in Japanese quail (Coturnix japonica). When exposed to hypoxia (15, 13, 11 and 9% O₂), T_b decreased only at 11% and 9% O₂ compared to normoxia; quail were better able to maintain T_b during acute hypoxia after a one-week acclimation to 10% O₂. V.O₂ also decreased during hypoxia, but at 9% O₂ this was partially offset by increased anaerobic metabolism. T_b and V.O₂ responses to 9% O₂ were exaggerated at lower ambient temperature (T_a), reflecting a decreased lower critical temperature during hypoxia. Conversely, hypoxia had little effect on T_b or V.O₂ at higher T_a (36 °C). We conclude that Japanese quail respond to hypoxia in much the same way as mammals, by reducing both T_b and V.O₂. No relationship was found between the magnitudes of decreases in T_b and V.O₂ during 9% O₂, however. Since metabolism is the source of heat generation, this suggests that Japanese quail increase thermolysis to reduce T_b. During hypercapnia (3, 6 and 9% CO₂), T_b was reduced only at 9% CO₂ while V.O₂ was unchanged.     

In vivo detection of protein cysteine sulfenylation in plastids

Protein cysteine thiols are post‐translationally modified under oxidative stress conditions. Illuminated chloroplasts are one of the important sources of hydrogen peroxide (H₂O₂) and are highly sensitive to environmental stimuli, yet a comprehensive view of the oxidation‐sensitive chloroplast proteome is still missing. By targeting the sulfenic acid YAP1C‐trapping technology to the plastids of light‐grown Arabidopsis cells, we identified 132 putatively sulfenylated plastid proteins upon H₂O₂ pulse treatment. Almost half of the sulfenylated proteins are enzymes of the amino acid metabolism. Using metabolomics, we observed a reversible decrease in the levels of the amino acids Ala, Asn, Cys, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr and Val after H₂O₂ treatment, which is in line with an anticipated decrease in the levels of the glycolysis and tricarboxylic acid metabolites. Through the identification of an organelle‐tailored proteome, we demonstrated that the subcellular targeting of the YAP1C probe enables us to study in vivo cysteine sulfenylation at the organellar level. All in all, the identification of these oxidation events in plastids revealed that several enzymes of the amino acid metabolism rapidly undergo cysteine oxidation upon oxidative stress.     

Metabolomic and flux‐balance analysis of age‐related decline of hypoxia tolerance in Drosophila muscle tissue

The fruitfly Drosophila melanogaster is increasingly used as a model organism for studying acute hypoxia tolerance and for studying aging, but the interactions between these two factors are not well known. Here we show that hypoxia tolerance degrades with age in post‐hypoxic recovery of whole‐body movement, heart rate and ATP content. We previously used ¹H NMR metabolomics and a constraint‐based model of ATP‐generating metabolism to discover the end products of hypoxic metabolism in flies and generate hypotheses for the biological mechanisms. We expand the reactions in the model using tissue‐ and age‐specific microarray data from the literature, and then examine metabolomic profiles of thoraxes after 4 h at 0.5% O₂ and after 5 min of recovery in 40‐ versus 3‐day‐old flies. Model simulations were constrained to fluxes calculated from these data. Simulations suggest that the decreased ATP production during reoxygenation seen in aging flies can be attributed to reduced recovery of mitochondrial respiration pathways and concomitant overdependence on the acetate production pathway as an energy source.     

Energy metabolism of Chironomus anthracinus (Diptera: Chironomidae) from the profundal zone of Lake Esrom,Denmark, as a function of body size,temperature and oxygen concentration

The profundal zone of Lake Esrom, Denmark has a dense population of Chironomus anthracinus, which survives 2–4 months of oxygen depletion each summer during stratification. The metabolism of 3rd and 4th instar larvae was examined in regard to variation in biomass and temperature. Respiration at air saturation was described by a curvilinear multiple regression relating oxygen consumption to individual AFDW and temperature. At 10 °C and varying oxygen regimes the O₂ consumption and CO₂ production of 4th instar larvae were almost unaltered from saturation to about 3 mg O₂ l^–1, but decreased steeply below this level. The respiratory quotient increased from 0.82 at saturation to about 3.4 at oxygen concentrations near 0.5 mg O₂ l^–1. This implied a shift from aerobic to partially anaerobic metabolism. At 0.5 mg O₂ l^–1 the total energy production equalled 20% of the rate at saturation of which more than one third was accounted for by anaerobic degradation of glycogen. This corresponded to a daily loss of 12 µg mg AFDW^–1 or approximately 5% of the body reserves. At unchanged metabolic rate the glycogen store would last three weeks, but long term oxygen deficiency causes a further suppression of the energy metabolism in C. anthracinus.     

Effects of Bacillus thuringiensis Cry1C toxin on the metabolic rate of Cry1C resistant and susceptible Spodoptera exigua (Lepidoptera: Noctuidae)

Abstract. The effects of Bacillus thuringiensis (Bt) Cry1C toxin on the metabolic rate of Cry1C resistant and susceptible Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) are investigated using closed‐system respirometry. Mechanisms of resistance to the Bt toxin may be associated with an energetic cost that can be measured as an increase in metabolic rate compared with Bt‐susceptible insects. This hypothesis is tested using third‐ and fifth‐instar larvae and 1–7‐day‐old pupae. Metabolic rate is measured as the amount of O₂ consumed and CO₂ produced. V?_O2 and V?_CO2 (mL g^?1 h^?1) of third‐instar Cry1C resistant larvae reared continuously on a diet containing 320 µg Cry1C toxin per g diet (CryonT) are significantly greater than third‐instar Cry1C resistant larvae reared on toxin for 5 days and reared thereafter on untreated diet (Cry5dT), Cry1C resistant larvae reared on untreated diet (CryReg) and the susceptible parental strain (SeA) reared on untreated diet. There are no differences in V?_O2 and V?_CO2 (mL g^?1 h^?1) among treatment groups for fifth‐instar larvae. CryonT larvae and pupae weigh significantly less than larvae and pupae receiving other treatments. Smaller body mass may be an important biological cost to individuals exposed continuously to Bt toxin. One‐day‐old pupae of all treatment groups exhibit a high V?_O2 (mean approximately 0.174 mL g^?1 h^?1) with CryonT having a significantly greater value than all other treatments; there are no differences among the other treatments. Pupal metabolic rates of all treatment groups decline to a minimum between days 2 and 4 then increase linearly between days 4 and 7 until adult emergence. These results demonstrate no difference in metabolic rates, and possibly fitness costs, between resistant (CryReg and Cry5dT) and susceptible (SeA) S. exigua except when larvae were reared continuously on toxin (CryonT).     

Responses to K deficiency and waterlogging interact via respiratory and nitrogen metabolism

K deficiency and waterlogging are common stresses that can occur simultaneously and impact on crop development and yield. They are both known to affect catabolism, with rather opposite effects: inhibition of glycolysis and higher glycolytic fermentative flux, respectively. But surprisingly, the effect of their combination on plant metabolism has never been examined precisely. Here, we applied a combined treatment (K availability and waterlogging) to sunflower (Helianthus annuus L.) plants under controlled greenhouse conditions and performed elemental quantitation, metabolomics, and isotope analyses at different sampling times. Whereas separate K deficiency and waterlogging caused well‐known effects such as polyamines production and sugar accumulation, respectively, waterlogging altered K‐induced respiration enhancement (via the C₅‐branched acid pathway) and polyamine production, and K deficiency tended to suppress waterlogging‐induced accumulation of Krebs cycle intermediates in leaves. Furthermore, the natural ¹⁵N/¹⁴N isotope composition (δ¹⁵N) in leaf compounds shows that there was a change in nitrate circulation, with less nitrate influx to leaves under low K availablity combined with waterlogging and more isotopic dilution of lamina nitrates under high K. Our results show that K deficiency and waterlogging effects are not simply additive, reshape respiration as well as nitrogen metabolism and partitioning, and are associated with metabolomic and isotopic biomarkers of potential interest for crop monitoring.     

Respiratory metabolism of Hydromedion sparsutum and Perimylops antarcticus (Col., Perimylopidae) from South Georgia

Summary Metabolic rates were studied in adults and larvae of the two phytophagous beetles Hydromedion sparsutum and Perimylops antarcticus (Col., Perimylopidae) indigenous to the Sub-Antarctic island of South Georgia. From the regression lines for log₁₀ metabolic rate on temperature no difference in oxygen consumption was found between adults and larvae of Hydromedion or adults of Perimylops from lower sites near sea level and upper sites at about 200 m. Elevated rates of metabolism, however, was found in larvae of Perimylops from upper sites. The slopes of the regression lines of corresponding upper and lower sites adults or larvae were similar in all cases. The metabolic rates were of the same level in adults of both species and in the larvae, but generally higher in adults than in larvae. The activation energies, calculated from Arrhenius plots, varied from 6.7 to 13.6 kcal.mol^–1 in larvae and from 10.7 to 11.3 kcal.mol^–1 in adults. The low values may be interpreted as an expression of cold adapted metabolism. Compared with published data on phytophagous beetles, the metabolic rates of the two species from South Georgia are comparable to two alpine species, but lower than the rates of an Arctic species.     

Allometric relationship between body mass and aerobic metabolism in zebrafish Danio rerio

The relationship between body mass (M) and metabolic rate was investigated through the assessment of active (R_A) and standard (R_S) metabolic rate at different life stages in zebrafish Danio rerio (5 day‐old larvae, 2 month‐old juveniles and 6 month‐old adults). Scaling exponents and constants were assessed for standard (R_S = 0·273M^0·965 in mgO₂ g^?1 h^?1) and active metabolic rate (R_A = 0·799M^0·926 in mgO₂ g^?1 h^?1). These data provide the basis for further experiments regarding the effects of environmental factors on aerobic metabolism throughout the life cycle of this species.     

CO2 enhances effects of hypoxia on mortality,development, and gene expression in cowpea bruchid, Callosobruchus maculatus

Modified atmosphere based on lack of O₂ offers a safe, residue-free alternative to chemical fumigants for pest control in stored grains. In this study, we intended to determine whether elevated CO₂ (at a biologically achievable level) has an enhanced suppressive effect over low O₂ atmosphere alone on the cowpea bruchid (Callosobruchus maculatus), a storage pest of cowpea and other legumes. Experiments were performed under two modified atmospheric conditions, (1) 2% O₂ + 18% CO₂ + 80% N₂ and (2) 2% O₂ + 98% N₂. Both hypoxic environments significantly affected the development and survival of all insect developmental stages. Eggs were most vulnerable to hypoxia, particularly at the early stage (4–6 h old), surviving only up to a maximum of 2 days in both treatments. These were followed by adults, pupae and larvae, in order of decreasing susceptibility. The 3rd and 4th instar larvae were most resilient to hypoxia and could survive up to 20 days of low O₂. The presence of 18% CO₂ significantly increased the mortality of adults, the later stage of eggs, as well as 1st and 4th instar larvae caused by hypoxia. However, the surviving insects exhibited faster development, evidenced by their earlier emergence from cowpea seeds compared to those without CO₂. One interesting observation was the frequent, premature opening of the emergence windows in the 4th instar larvae when CO₂ was involved. This phenomenon was not observed at all in insects stressed by low O₂ alone. Differential expression profiling of metabolic genes and proteolytic activity of midgut digestive enzymes suggested that the rate of metabolic activity could contribute in part to the difference in insect development and survival under hypoxia in the presence and absence of CO₂.     

Do elevated atmospheric CO2 and O3 affect food quality and performance of folivorous insects on silver birch?

The individual and combined effects of elevated CO₂ and O₃ on the foliar chemistry of silver birch (Betula pendula Roth) and on the performance of five potential birch‐defoliating insect herbivore species (two geometrid moths, one lymantrid moth and two weevils) were examined. Elevated CO₂ decreased the water concentration in both short‐ and long‐shoot leaves, but the effect of CO₂ on the concentration of nitrogen and individual phenolic compounds was mediated by O₃ treatment, tree genotype and leaf type. Elevated O₃ increased the total carbon concentration only in short‐shoot leaves. Bioassays showed that elevated CO₂ increased the food consumption rate of juvenile Epirrita autumnata and Rheumaptera hastata larvae fed with short‐ and long‐shoot leaves in spring and mid‐summer, respectively, but had no effect on the growth of larvae. The contribution of leaf quality variables to the observed CO₂ effects indicate that insect compensatory consumption may be related to leaf age. Elevated CO₂ increased the food preference of only two tested species: Phyllobius argentatus (CO₂ alone) and R. hastata (CO₂ combined with O₃). The observed stimulus was dependent on tree genotype and the measured leaf quality variables explained only a portion of the stimulus. Elevated O₃ decreased the growth of flush‐feeding young E. autumnata larvae, irrespective of CO₂ concentration, apparently via reductions in general food quality. Therefore, the increasing tropospheric O₃ concentration could pose a health risk for juvenile early‐season birch folivores in future. In conclusion, the effects of elevated O₃ were found to be detrimental to the performance of early‐season insect herbivores in birch whereas elevated CO₂ had only minor effects on insect performance despite changes in food quality related foliar chemistry.     

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.     

Comparative iTRAQ‐Based Quantitative Proteomic Analysis of Pelteobagrus vachelli Liver under Acute Hypoxia: Implications in Metabolic Responses

More and more frequently these days, aquatic ecosystems are being stressed by nutrient enrichment, pollutants, and global warming, leading to a serious depletion in oxygen concentrations. Although a sudden, significant lack of oxygen will result in mortality, fishes can have an acute behavior (e.g., an increase in breathing rate, reduction in swimming frequency) and physiology responses (e.g., increase in oxygen delivery, and reduction in oxygen consumption) to hypoxia, which allows them to maintain normal physical activity. Therefore, in order to shed further light on the molecular mechanisms of hypoxia adaptation in fishes, the authors conduct comparative quantitative proteomics on Pelteobagrus vachelli livers using iTRAQ. The research identifies 511 acute hypoxia‐responsive proteins in P. vachelli. Furthermore, comparison of several of the diverse key pathways studied (e.g., peroxisome pathway, PPAR signaling pathway, lipid metabolism, glycolysis/gluco‐neogenesis, and amino acid metabolism) help to articulate the different mechanisms involved in the hypoxia response of P. vachelli. Data from proteome analysis shows that P. vachelli can have an acute reaction to hypoxia, including detoxification of metabolic by‐products and oxidative stress in light of continued metabolic activity (e.g., peroxisomes), an activation in the capacity of catabolism to get more energy (e.g., lipolysis and amino acid catabolism), a depression in the capacity of biosynthesis to reduce energy consumption (e.g., biosynthesis of amino acids and lipids), and a shift in the aerobic and anaerobic contributions to total metabolism. The observed hypoxia‐related changes in the liver proteome of the fish can help to understand or can be related to the hypoxia‐related response that takes place in similar conditions in the liver or other proteomes of mammals.     

Reactive oxygen species‐induced changes in glucose and lipid metabolism contribute to the accumulation of cholesterol in the liver during aging

Aging is a major risk factor for many chronic diseases due to increased vulnerability to external stress and susceptibility to disease. Aging is associated with metabolic liver disease such as nonalcoholic fatty liver. In this study, we investigated changes in lipid metabolism during aging in mice and the mechanisms involved. Lipid accumulation was increased in liver tissues of aged mice, particularly cholesterol. Increased uptake of both cholesterol and glucose was observed in hepatocytes of aged mice as compared with younger mice. The mRNA expression of GLUT2, GK, SREBP2, HMGCR, and HMGCS, genes for cholesterol synthesis, was gradually increased in liver tissues during aging. Reactive oxygen species (ROS) increase with aging and are closely related to various aging‐related diseases. When we treated HepG2 cells and primary hepatocytes with the ROS inducer, H₂O₂, lipid accumulation increased significantly compared to the case for untreated HepG2 cells. H₂O₂ treatment significantly increased glucose uptake and acetyl‐CoA production, which results in glycolysis and lipid synthesis. Treatment with H₂O₂ significantly increased the expression of mRNA for genes related to cholesterol synthesis and uptake. These results suggest that ROS play an important role in altering cholesterol metabolism and consequently contribute to the accumulation of cholesterol in the liver during the aging process.