Total Pyruvate Research Articles

TMET-06. METABOLIC FATE OF OXALOACETATE IN GLIOBLASTOMA

Abstract Oxaloacetate (OAA) inhibits the Warburg effect by reducing the glucose-derived lactate levels in human cancer cell lines. In dose ranging IC50 experiments with human hepatocellular carcinoma (HCC) cell lines, OAA inhibited cell growth in the concentration range 1.7 - 2.4 mM. Previously, we showed that in the presence of OAA, [U-13C]glucose derived lactate was reduced by 48.8% in GBM cells. OAA also increased survival in mouse models of GBM. Here, we are showing an evidence for the direct utilization of OAA by Glioblastoma (GBM) cells, using uniformly 13C-labeled oxaloacetate ([U-13C]OAA) as a metabolic tracer. Patient-derived GBM cells were grown in DMEM medium containing 11.0 mM glucose and 2.0 mM glutamine. When cells reached confluency, were treated with 4.0 mM [U-13C]OAA in the above DMEM medium. GC-MS based 13C isotopomer analysis showed that [U-13C]OAA entered the cells and get metabolized to generate distinct 13C-labeled isotopomers of aspartate, citrate, glutamate, malate and fumarate. Upon entering TCA cycle, [U-13C]OAA generated M+4 aspartate (3.0%) and citrate (3.2%), and M+3 glutamate (4.1%). During the first turn of the TCA cycle, it produced M+2 aspartate (13.9%), malate (10.5%), and fumarate (8.8%), and after multiple turns, [U-13C]OAA led to the production of the following 13C-labeled isotopomers: M+2 citrate (21.5%), M+2 glutamate (17.2%), M+1 glutamate (5.6%) and M+4 glutamate (2.2%). In addition, [U-13C]OAA also produced [U-13C]pyruvate (17.0%) through PEPCK/PK/ or malic enzyme which further generated similar amount of [U-13C]lactate (17.0%) and [U-13C]alanine (12%). These data demonstrate that GBM cells utilize [U-13C]OAA to perturb TCA cycle and glycolytic pathways, and support our previous observation that OAA-derived pyruvate lead to an increase in the total pyruvate pool and associated decrease in glycolytic flux including the glucose-derived lactate. OAA may metabolically reduce tumor growth in difficult to treat cancers.

Identifying the Metabolic Signatures of PPARD-Overexpressing Gastric Tumors.

Peroxisome proliferator-activated receptor delta (PPARD) is a nuclear receptor known to play an essential role in regulation of cell metabolism, cell proliferation, inflammation, and tumorigenesis in normal and cancer cells. Recently, we found that a newly generated villin-PPARD mouse model, in which PPARD is overexpressed in villin-positive gastric progenitor cells, demonstrated spontaneous development of large, invasive gastric tumors as the mice aged. However, the role of PPARD in regulation of downstream metabolism in normal gastric and tumor cells is elusive. The aim of the present study was to find PPARD-regulated downstream metabolic changes and to determine the potential significance of those changes to gastric tumorigenesis in mice. Hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy, nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry were employed for metabolic profiling to determine the PPARD-regulated metabolite changes in PPARD mice at different ages during the development of gastric cancer, and the changes were compared to corresponding wild-type mice. Nuclear magnetic resonance spectroscopy-based metabolomic screening results showed higher levels of inosine monophosphate (p = 0.0054), uracil (p = 0.0205), phenylalanine (p = 0.017), glycine (p = 0.014), and isocitrate (p = 0.029) and lower levels of inosine (p = 0.0188) in 55-week-old PPARD mice than in 55-week-old wild-type mice. As the PPARD mice aged from 10 weeks to 35 weeks and 55 weeks, we observed significant changes in levels of the metabolites inosine monophosphate (p = 0.0054), adenosine monophosphate (p = 0.009), UDP-glucose (p = 0.0006), and oxypurinol (p = 0.039). Hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy performed to measure lactate flux in live 10-week-old PPARD mice with no gastric tumors and 35-week-old PPARD mice with gastric tumors did not reveal a significant difference in the ratio of lactate to total pyruvate plus lactate, indicating that this PPARD-induced spontaneous gastric tumor development does not require glycolysis as the main source of fuel for tumorigenesis. Liquid chromatography-mass spectrometry-based measurement of fatty acid levels showed lower linoleic acid, palmitic acid, oleic acid, and steric acid levels in 55-week-old PPARD mice than in 10-week-old PPARD mice, supporting fatty acid oxidation as a bioenergy source for PPARD-expressing gastric tumors.

Metabolic Markers of Insulin Resistance in Feline Hypertrophied Myocardium. Insulin Resistance in Heart Failure

Investigation of myocardial tissue concentration of ATP, glucose transporters 1 and 4, pyruvate dehydrogenase, hexokinase 2, insulin receptor and adropin proteins, determining metabolic changes and possible insulin resistance in feline myocardium with hypertrophic phenotype. This is the pilot study for metabolic markers determination. Eighteen cats were studied, divided into 3 groups: Without cardiac disease (n = 5), cats with hypertrophic cardiomyopathy (n = 8), cats with chronic kidney disease and secondary myocardial hypertrophy (n = 5). Animals in the study were diagnosed for the primary disease by standard methods and algorithms. Cats were euthanized due to the end-stage chronic kidney disease, refractory heart failure or by owners’ will. The myocardium samples were obtained immediately after death. Samples for the metabolic study were taken from the apical part of the left ventricular free wall and fixed in liquid nitrogen at once and were stored in -80°C refrigerator. Studied proteins concentrations were analyzed in a specialized research laboratory, using ELISA kits, provided by Cloud-Clone Corp. (USA), included: Total ATP, pyruvate dehydrogenase, hexokinase II, Adropin, insulin receptor, GLUT1 and GLUT4. In the group with HCM, we discovered that levels of ATP, pyruvate dehydrogenase and adropin were severely suppressed in comparison to healthy cats, while GLUT1 and GLUT4 did not change. The concentration of hexokinase 2 and insulin receptor proteins significantly increased. In the group of secondary myocardial hypertrophy, suppression of most studied proteins was admitted, except insulin receptor. In conclusion, we discovered metabolic remodeling and development of insulin resistance in observed diseases with hypertrophy phenotype. We observed depression of pivotal enzymes proteins, limiting energy restoration potency for cardio myocytes.

가공방법에 따른 마늘껍질의 품질 특성

Garlic peel has been found to have various beneficial effects including antioxidant activities, antithrombosis activities and cancer inhibition. This study was to analyze the quality characteristics and antioxidant activities of garlic peel processed using different methods (control, roasting, steaming, roasting after steaming and blanching) to determine the most appropriate processing method for garlic peel to be used as a functional food ingredient. The lightness color values was highest in the control group, redness was highest in the roasting after steaming group, and yellowness was highest in the roasting group. The total pyruvate content of the roasting after steaming group garlic peel was 37.05 mg/100 g, which was higher than those of the other groups. The total polyphenol and total flavonoid contents of garlic peel ranged 47.60 to 172.53 mg/100 g and 3.43 to 17.63 mg/100 g, respectively. The highest total polyphenol content and total flavonoid content were observed for the roasting after steaming group. 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity was also the highest in the roasting after steaming group (81.28%). These results indicate that the method used to process garlic peel affects its quality, and that roasting after steaming is the most appropriate processing method, as it achieves the highest antioxidant activity.

PB1974 PYRUVATE KINASE DEFICIENCY IN FOUR CHILDREN WITH TWO UNPUBLISHED MUTATIONS

Background:Pyruvate kinase is a key enzyme of anaerobic glycolysis which helps keeping the energy level of red blood cells and their viability in circulation. The genetic heterogeneity of pyruvate kinase deficiency (PKD) is high and, to this day, over 250 different mutations have been identified.Aims:We aimed to emphasize the importance of genetic testing in patients with unexplained non‐immune hemolytic anemia.Methods:Four patients who had been followed because of non‐spherocytic hemolytic anemia and in whom no causative enzyme deficiencies were identified were referred for targeted next‐generation sequencing using the Oxford Red Cell Panel, a 50‐gene panel for inherited anemias.Results:We studied four patients (2 females, and 2 males) with evidence of chronic hemolytic anemia aged between 4 years and 24 years. Three of the four patients had jaundice, pallor and hepatosplenomegaly, while one patient had growth retardation and hypotonicity. One patient had gallstones and also undergone splenectomy at the age of two. While homozygous mutation was detected in three of four patients, only one patient had a compound heterozygous mutations (c.602G > A, c.1675C > G). The five identified mutations are single nucleotide gene changes. Two of the five mutations (c.602G > A, and c.172C > T) are previously unpublished.Summary/Conclusion:The clinical presentation of PKD ranges from frequent transfusion in neonates to mild jaundice in adulthood. Total pyruvate kinase in liver and red cell (PKLR) gene sequencing is necessary for the characterization of all patients with PKD and for genetic counselling. We also report two previously unpublished mutations in PKLR.

Melatonin Therapy Modulates Cerebral Metabolism and Enhances Remyelination by Increasing PDK4 in a Mouse Model of Multiple Sclerosis.

Metabolic disturbances have been implicated in demyelinating diseases including multiple sclerosis (MS). Melatonin, a naturally occurring hormone, has emerged as a potent neuroprotective candidate to reduce myelin loss and improve MS outcomes. In this study, we evaluated the effect of melatonin, at both physiological and pharmacological doses, on oligodendrocytes metabolism in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Results showed that melatonin decreased neurological disability scores and enhanced remyelination, significantly increasing myelin protein levels including MBP, MOG, and MOBP. In addition, melatonin attenuated inflammation by reducing pro-inflammatory cytokines (IL-1β and TNF-α) and increasing anti-inflammatory cytokines (IL-4 and IL-10). Moreover, melatonin significantly increased brain concentrations of lactate, N-acetylaspartate (NAA), and 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR). Pyruvate dehydrogenase kinase-4 (PDK-4) mRNA and protein expression levels were also increased in melatonin-treated, compared to untreated EAE mice. However, melatonin significantly inhibited active and total pyruvate dehydrogenase complex (PDC), an enzyme under the control of PDK4. In summary, although PDC activity was reduced by melatonin, it caused a reduction in inflammatory mediators while stimulating oligodendrogenesis, suggesting that oligodendrocytes are forced to use an alternative pathway to synthesize fatty acids for remyelination. We propose that combining melatonin and PDK inhibitors may provide greater benefits for MS patients than the use of melatonin therapy alone.

NEPHROPROTECTIVE ROLE OF ZINC AGAINST THE AMMONIUM SULFATE TOXICITY IN MALE ALBINO RATS

Objective: Intention of the present study is to investigate the protective role of zinc against ammonium sulfate (AS) toxicity in renal tissue by evaluating certain biochemical activities of albino rats.
 Methods: Rats were divided into four groups, namely control, ammonia, zinc, and ammonia + zinc. Rats were exposed to AS (18.3 mg/kg body weight) or zinc chloride (4 mg/kg body weight) or both through intraperitoneally for 7-day experimentation with 24-h time interval.
 Results: AS-administered rats showed significant increased levels of ammonia, urea, glutamine, glutamine synthetase, free amino acids, and lactate dehydrogenase and decreased levels of total proteins, pyruvate, succinate dehydrogenases, malate dehydrogenase, and biochemical activities when compared with control. Supplementation of zinc mitigated AS-induced oxidative stress and restored all the biochemical parameter activities. Zinc administered to normal rats did not exhibit any significant changes in any of the parameters studied.
 Conclusion: From the study, it concluded that zinc cotreatment with AS has effectively recovered the mitochondrial enzyme activities and ammonia metabolic biochemical parameters in renal tissue of rat treated with AS.

An improved enzyme nanoparticles based amperometric pyruvate biosensor for detection of pyruvate in serum

The nanoparticles of commercially available pyruvate oxidase (POx) from Aerococcus species were prepared by desolvation method, which were then characterized and covalently immobilized onto gold electrode (AuE) to construct an improved model of amperometric pyruvate biosensor. The POxNPs/Au electrode was analyzed morphologically by scanning electron microscopy (SEM). On the other hand, cyclic voltammetry studies (CV) and electrochemical impedance spectroscopy (EIS) helped in deciphering the electrochemical properties of the electrode at different stages of construction. The biosensor showed optimum response within 7.5 s, at a potential of 0.28 V, pH 5.5 and 35 °C. A linear relationship was observed between biosensor response i.e. current (μA) and pyruvate concentration in the range, 0.01 μM - 5000 μM, with a lower detection limit of 0.67 μM. The analytical recovery of added pyruvate in sera was 99.0% and 99.5% within and between batch coefficient of variation (CV) were 0.045% and 0.040% respectively. The working electrode displayed an excellent correlation coefficient (R2 = 0.99%) between levels of pyruvate in sera, as detected by the standard spectrophotometric method and the present biosensor. The biosensor was utilized for detection of total pyruvate level in sera of apparently healthy individuals and patients suffering from cardiogenic stress, more specifically cardiac failure. The activity of the biosensor deteriorated by 25%, after its regular use over a period of 240 days, while being stored dry at 4°C.

High-level expression of Aerococcus viridans pyruvate oxidase in Escherichia coli by optimization of vectors and induction conditions.

This study demonstrates that a highly soluble pyruvate oxidase can be obtained in recombinant Escherichia coli by optimizing vectors and induction conditions. The pyruvate oxidase yield achieved is the highest reported so far, which provides a convenient and cost-saving way to produce pyruvate oxidase. This research promotes pyruvate oxidase application in the pharmaceutical and biochemical industries.

HIF1 mediates a switch in pyruvate kinase isoforms after myocardial infarction.

Alternative splicing of RNA is an underexplored area of transcriptional response. We expect that early changes in alternatively spliced genes may be important for responses to cardiac injury. Hypoxia inducible factor 1 (HIF1) is a key transcription factor that rapidly responds to loss of oxygen through alteration of metabolism and angiogenesis. The goal of this study was to investigate the transcriptional response after myocardial infarction (MI) and to identify novel, hypoxia-driven changes, including alternative splicing. After ligation of the left anterior descending artery in mice, we observed an abrupt loss of cardiac contractility and upregulation of hypoxic signaling. We then performed RNA sequencing on ischemic heart tissue 1 and 3 days after infarct to assess early transcriptional changes and identified 89 transcripts with altered splicing. Of particular interest was the switch in Pkm isoform expression (pyruvate kinase, muscle). The usually predominant Pkm1 isoform was less abundant in ischemic hearts, while Pkm2 and associated splicing factors (hnRNPA1, hnRNPA2B1, Ptbp1) rapidly increased. Despite increased Pkm2 expression, total pyruvate kinase activity remained reduced in ischemic myocardial tissue. We also demonstrated HIF1 binding to PKM by chromatin immunoprecipitation, indicating a direct role for HIF1 in mediating this isoform switch. Our study provides a new, detailed characterization of the early transcriptome after MI. From this analysis, we identified an HIF1-mediated alternative splicing event in the PKM gene. Pkm1 and Pkm2 play distinct roles in glycolytic metabolism and the upregulation of Pkm2 is likely to have important consequences for ATP synthesis in infarcted cardiac muscle.

Abstract 58: Metformin impacts IGF-1R/ Akt signaling and energy metabolism in lung adenocarcinoma

Abstract Introduction: Clinical outcomes for type II diabetes patients diagnosed with NSCLC are superior for those receiving metformin relative to other diabetic medications, though the mechanism for this effect remains incompletely elucidated. We hypothesized that metformin induces changes in Akt-based signaling via AMPK that increases glycolytic flux, and adversely affects survival in cell-line models of lung adenocarcinoma. The objective for these studies is to investigate therapeutic strategies involving metformin that have the potential to synergize with strategies targeting IGF-1R/Akt signaling. Methods: The lung adenocarcinoma cell lines, A549 and H358, were treated with 0, 1, 5, 10 mM metformin for 24, 48, and 72 hours and evaluated for changes in expression and activity of IGF-1R/Akt signaling, as well as the expression of 17 enzymes involved in glycolytic and oxidative energy metabolism, all using Luminex® immunobead assays. In parallel, MTT assays were performed for up to 5 days to document changes in cellular proliferation/ death rates. Extracellular acidification was also monitored as a measure of glycolytic flux. All findings were collated and statistically evaluated using SPSS v19 (Chicago, IL) using independent t-tests or one-way ANOVA. Results: We observed that metformin decreased IGF-1R/Akt signaling while increasing the expression of the protein levels of the signaling intermediates. Treatment with metformin increased the expression of Akt, IRS-1, PTEN, GSK3alpha and TSC2 (p<0.05 for all). Further, metformin treatment decreased the mean ratio of phospho-/total pyruvate dehydrogenase and increased the expression of ATP Synthase (both p<0.05), and were associated (p<0.05) with a dose-dependent increase in extracellular acidification. Lower doses (0.1 mM) of metformin did not have a significant impact on cell proliferation at 5 days, but a dose-dependent decrease in cell numbers was observed, with 37% and 29% reduction in cell numbers documented (p<0.05) at 5 mM for the H358 and A549 cells, respectively. Conclusion: This study links changes with Akt signaling with changes in glycolytic flux and oxidative metabolism. This study suggests metformin may make an attractive adjunct for advanced NSCLC patients receiving therapy that mechanistically involves targeting Akt signaling and metabolic control, such as IGF1R inhibition. Citation Format: Gabriela C. Lobato, Steve Li, Imad Tarhoni, Wen-Rong Lie, Jeffrey A. Borgia. Metformin impacts IGF-1R/ Akt signaling and energy metabolism in lung adenocarcinoma. [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 58.

데치기 처리에 따른 마늘종의 이화학적 품질 특성 변화

This study was performed to investigate changes in the quality characteristics and chemical compounds in garlic shoots by blanching for different time (0, 0.5, 1, 2, 3, and 4 min). The color (L, a, and b values), texture profile (hardness, springiness, chewiness, gumminess, and cohesiveness), total vitamin C, total sugar, total polyphenol and flavonoid, chlorophyll a and b contents, total pyruvate and thiosulfinate contents of the blanched garlic shoots were examined. As the blanching time increased, the L and b values decreased, while the a value increased. Hardness, chewiness, springiness and gumminess of the blanched shoots were significantly lowered with the increasing blanching time. Vitamin C content of raw garlic shoot was 1.62 mg/100 g while that of the blanched garlic shoots was 0.16~0.24 mg/100 g, implying vitamin C loss into blanching water and destruction by heat. The total sugar and polyphenol contents were fluctuated during blanching, but over 75% of their initial levels were retained under all blanching conditions. The total flavonoid contents increased as the blanching time increased. The chlorophyll a content did not show significant change with blanching time, but the chlorophyll b content significantly decreased. These results suggest that blanching for 2 min could be the best for retaining beneficial compounds and desirable quality of garlic shoot.

냉동다진마늘에 대한 전자선 살균 효과와 생리활성 성분의 안정성

Recently, the market for ready-to-use vegetables has increased largely due to consumer demands, which led to the production of minimally-processed frozen crushed garlic products. This study was designed to determine the effect of electron beam irradiation (0-7 kGy) on microbial decontamination, bioactive compounds, and antimicrobial activities of frozen crushed garlic obtained from Korea and China. The microbial counts (total bacteria, yeasts & molds) were reduced from log 3-4 CFU/g to non-detectable levels as a result of irradiation at 4 kGy. Irradiation treatment at 4 kGy did not affect the amount of alliin, allicin, total pyruvate, and total thiosulfinate of crushed garlic, however, this dose induced insignificant changes in antibacterial activities against pathogenic bacteria including Bacillus subtilis, Staphylococcus aureus, Salmonella enteritidis, Vibrio parahaemolyticus, and Yersinia enterocolitica. Therefore, electron beam irradiation less than 4 kGy can be considered suitable to improve the microbial decontamination without altering the biological activity of frozen garlics.

In human alloreactive CD4⁺ T-cells, dichloroacetate inhibits aerobic glycolysis, induces apoptosis and favors differentiation towards the regulatory T-cell subset instead of effector T-cell subsets.

Although kidney transplantation is the best therapy for end-stage renal disease, rejection remains a concern, and currently available immunosuppressive agents contribute to morbidity and mortality. Thus, novel immunosuppressive drugs are required. Dichloroacetate (DCA) is already used in the treatment of congenital lactic acidosis and characterized by limited toxicity. As DCA inhibits aerobic glycolysis, which is a prerequisite for CD4+ T-cell proliferation and differentiation into effector T-cells, its possible immunosuppressive role in mixed lymphocyte reaction (MLR), a model of alloreactivity, was investigated. Glucose and lactate concentrations were measured in the supernatants, and cell proliferation was assessed immunoenzymatically. CD4+ T‑cells were then isolated from the MLRs and the expression of cleaved caspase‑3, various enzymes involved in glycolysis, and the signature transcription factors of CD4+ T‑cell subsets were evaluated by western blotting. In MLRs, DCA decreased glucose consumption and aerobic glycolysis, while it exerted a negligible effect on cell proliferation. In CD4+ T‑cells, DCA induced apoptosis, and decreased the expression of glucose trasporter‑1, hexokinase II, lactate dehydrogenase‑A and phosphorylated pyruvate dehydrogenase, while it increased total pyruvate dehydrogenase. In addition, DCA increased the expression of transcription factor forkhead box P3, whereas it decreased the expression of T‑box transcription factor TBX21, trans‑acting T-cell-specific transcription factor GATA‑3 and retinoic acid receptor related orphan receptor‑γt. In conclusion, in alloreactive CD4+ T‑cells, DCA inhibits aerobic glycolysis, induces apoptosis and favors differentiation towards the regulatory T‑cell subset. These characteristics render it a promising immunosuppressive agent in the field of transplantation.

자이언트흑마늘의 유효성분 분석

Garlic has been consumed for a long time as a food in Korea. Among commercial garlic products, aged black garlic is known to contain bioactive ingredients. Giant garlic is large garlic with a mild and sweet flavor. However, there have been few studies on the bioactive effects of giant garlic. This study analyzed valid substances of giant black garlic (GBG), giant fresh garlic (GFG), normal black garlic (NBG), and normal fresh garlic (NFG). Moisture content of GBG decreased in comparison with that of GFG. Ash, crude protein, crude lipid, total pyruvate, S-allylcysteine, and S-allylmercaptocysteine contents were the highest in GBG. The pH values of GBG, GFG, NBG, and NFG were 3.77, 5.97, 3.94, and 6.10, respectively. Acidity of GBG was higher than that of GFG. Total sugar content of GBG increased while reducing sugar content was the same as that of GFG. Composition of amino acids except for histidine, lysine, and arginine in GBG was higher than that of GFG, and free amino acids excluding arginine, cysteine, lysine, phosphoethanolamine, and urea were higher than those of GFG. In minerals, Cu, Fe, and Mg contents were higher than those of GFG.

Comparison of Biochemical Composition and Antimicrobial Activity of Southern-Type Garlic Grown in the Eastern and Western Regions of Jeju

Components and physiological activity of crops are strongly affected by the natural environments of the growth regions. In this study, we carried out soil analysis and determined the allicin, alliin, total pyruvate, total phenol and flavonoid, and reducing sugar contents of garlic from garlic-cultivating regions in the eastern and western portions of Jeju Province. Significantly higher contents of macronutrients were found in the soil and garlic from the eastern region of Jeju compared to western region, but micronutrients were higher in the western region than eastern region. In addition, the alliin and total phenolic contents were higher in the western region than in the eastern region. Also, allicin, total pyruvate, total flavonoid content, and ɑ-glucosidase inhibitory activity of the eastern region samples were higher than those of the western region in Jeju. This study promotes our understanding of the different components of garlic according to the cultivation regions of Jeju.

Abstract 1158: Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay

Abstract Objective: The purpose of this study was to evaluate the feasibility of monitoring lactate uptake and utilization in intact human cancer cells using a dynamic 1H-NMR assay and [3-13C]-lactate. This would provide a simple, real-time method for monitoring enzyme kinetics in cells as an alternative to spectrophotometry - which cannot be applied to intact cells - or dynamic nuclear polarisation-NMR, which requires complex mathematical modelling. The ability to better understand and measure lactate metabolism in cancer cells could be applied to monitor MCT1 inhibition. Methods: 3×10^7 MDA-MB-231 human breast cancer cells were suspended in 400μl serum-free DMEM plus 100μl D2O in a 5mm NMR tube. A mixture of 10mM [3-13C]-lactate combined with varying concentrations of unlabelled 12C-pyruvate (0mM, 5mM or 10mM) was added and proton spectra were acquired every minute for 30 minutes on a 500MHz Bruker spectrometer. For comparison, cells were also incubated with 10mM [3-13C]-pyruvate and either 0mM or 10mM 12C-lactate. Results: The presence of 13C-pyruvate was detected in real-time following addition of [3-13C]-lactate indicating that 13C-label had been exchanged from lactate to pyruvate via the enzyme lactate dehydrogenase. The optimisation of reagent concentrations, however, revealed that this observation was dependent on the addition of unlabelled 12C-pyruvate and the apparent rate of 13C-label exchange was proportional to the concentration of 12C-pyruvate added. The data show that no 13C-pyruvate signal is detected when 10mM 13C-lactate is added alone. However, 6% of the 10mM 13C-lactate is exchanged with 13C-pyruvate when 5mM 12C-pyruvate is added (rate = 1.1×10-3mM s-1). This increases to 12% when the 12C-pyruvate concentration was increased to 10mM (rate = 2.2×10-3 mM s-1). The total pyruvate concentration (12C + 13C-pyruvate) remained constant, which suggests that there is no net production of pyruvate. In comparison, the 13C-pyruvate experiment was less dependent on the addition of unlabelled 12C-lactate for 13C-label exchange to be observed. Conclusion: The fact that unlabelled pyruvate was required for a reaction to be observed following addition of 13C-lactate suggests that the position of the isotopic equilibrium favours the label to remain in the lactate pool. Therefore a large amount of unlabelled pyruvate is required for the exchange process to be observed. This would also explain why the addition of unlabelled lactate was not required in the 13C-pyruvate experiment, since to reach isotopic equilibrium, a large proportion of the 13C-pyruvate needs to immediately exchange with the naturally larger lactate pool. Nevertheless, these findings demonstrate the feasibility of using this 1H-NMR assay to measure [3-13C]-lactate uptake in real-time. This could inform on metabolic pathway activity in response to MCT1 inhibition in intact cells. Citation Format: Emily G. Wholey, Harold G. Parkes, Paul Workman, Martin O. Leach, Mounia Beloueche-Babari. Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1158. doi:10.1158/1538-7445.AM2015-1158

Moderate DNA damage promotes metabolic flux into PPP via PKM2 Y-105 phosphorylation: a feature that favours cancer cells

Pyruvate kinase M2, an important metabolic enzyme, promotes aerobic glycolysis (Warburg effect) to facilitate cancer cell proliferation. Unravelling the status of this important glycolytic pathway enzyme under sub-lethal doses of etoposide, a commonly used anti-proliferative genotoxic drug to induce mild/moderate DNA damage in HeLa cells as a model system and discern its effect on: PKM2 expression, phosphorylation, dimer: tetramer ratio, activity and associated effects, was pertinent. Protein expression and phosphorylation of PKM2 from HeLa cells was estimated using Western blotting. Same protein lysate was also used to estimate total pyruvate kinase activity and the total dimer: tetramer content evaluated using glycerol gradient ultra-centrifugation. Intracellular PEP was estimated manually using standard curve; while NADPH was assessed by NADPH estimation kit. Unpaired t test and two-way-ANOVA was used for statistical analysis. A relative decrease in PKM2 expression and a subsequent dose and time dependent increase in Y105-phosphorylation were observed. A concomitant increase in PKM2 dimer content and Y105-phosphorylation responsible for reduced PKM2 activity promoted PEP accumulation and NADPH production, representing increased metabolic flux into PPP, a feature that favours cancer cells. It was apparent that the sub-lethal doses of etoposide induced inadequate damage to DNA in cancer cells in culture promoted pro-survival conditions due to Y105-phosphorylation of PKM2, its stable dimerization and inactivation, a unique association not known earlier, indicating what might happen in tumour revivals or recurrences.

국내산 및 중국산 냉동 다진 마늘의 품질 특성 평가

시판되는 냉동 다진 마늘의 품질 특성을 파악하기 위하여 냉동 다진 마늘 국내산 4종과 중국산 2종을 수집하여 품질 특성을 조사하였다. 냉동 다진 마늘의 수분 함량은 국내산이 64.95±0.09~72.06±0.11%, 중국산이 83.80±0.10~90.93±0.23%의 범위로 측정되었고 중국산 제품의 수분 함량이 유의적으로 높은 것으로 나타났다. 해동 감량은 국내산 냉동 다진 마늘이 0.33±0.07~0.70±0.22%, 중국산 냉동 다진 마늘은 8.89±0.66~10.26±0.77%로 측정되어 국내산에 비해 약 10배 이상 높은 값을 보였으며, 해동 감량은 각 시료의 수분 함량과 양의 상관관계(r=0.918, P<0.01)를 보이는 것으로 나타났다. 냉동 다진 마늘의 total pyruvate 함량은 국내산의 경우 118.69±5.97~224.81±9.92 μmol/g으로 중국산이 75.27±5.91~79.35±1.79 μmol/g인데 비하여 유의적으로 높은 것으로 나타났다. Allicin 함량은 국내산이 8.91±0.34~13.09±0.35 mg/g으로 중국산 5.91±0.08~6.05±0.27 mg/g에 비해 유의적으로 높은 것으로 나타났고, total thiosulfinate 함량은 0.60±0.07~1.33±0.12 O.D./g으로 측정되었다. Allicin 함량과 total thiosulfinate 함량은 유의적인 양의 상관관계(r=0.892, P<0.01)를 보였다. 전자코에 의한 냉동 다진 마늘의 향기 패턴 분석은 국내산과 중국산과의 차이가 없는 것으로 나타났다.

Kinetic and perfusion modeling of hyperpolarized (13)C pyruvate and urea in cancer with arbitrary RF flip angles.

The accurate detection and characterization of cancerous tissue is still a major problem for the clinical management of individual cancer patients and for monitoring their response to therapy. MRI with hyperpolarized agents is a promising technique for cancer characterization because it can non-invasively provide a local assessment of the tissue metabolic profile. In this work, we measured the kinetics of hyperpolarized [1-(13)C] pyruvate and (13)C-urea in prostate and liver tumor models using a compressed sensing dynamic MRSI method. A kinetic model fitting method was developed that incorporated arbitrary RF flip angle excitation and measured a pyruvate to lactate conversion rate, Kpl, of 0.050 and 0.052 (1/s) in prostate and liver tumors, respectively, which was significantly higher than Kpl in healthy tissues [Kpl =0.028 (1/s), P<0.001]. Kpl was highly correlated to the total lactate to total pyruvate signal ratio (correlation coefficient =0.95). We additionally characterized the total pyruvate and urea perfusion, as in cancerous tissue there is both existing vasculature and neovascularization as different kinds of lesions surpass the normal blood supply, including small circulation disturbance in some of the abnormal vessels. A significantly higher perfusion of pyruvate (accounting for conversion to lactate and alanine) relative to urea perfusion was seen in cancerous tissues (liver cancer and prostate cancer) compared to healthy tissues (P<0.001), presumably due to high pyruvate uptake in tumors.