Browsing by Subject "Pyruvic Acid"
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Item Cell-Type Specific Requirement for Pyruvate Dehydrogenase in Hematopoiesis and Leukemia Development(August 2021) Jun, Sojeong; Xu, Jian; Chuang, David; Pan, Duojia; Agathocleous, MichalisWhen I joined the lab, it was already established that pyruvate dehydrogenase complex (PDC) was required for double positive (DP) T cells, and for the initiation of T- cell acute lymphoblastic leukemia (T-ALL). However, it was not known if lymphoid-specific deletion of PDHA1 using lymphoid-specific Cre line (CD2Cre, CD4Cre) would provide us with a similar phenotype as pan-hematopoietic deletion of PDHA1 using Mx1Cre, and we did not know the metabolic consequences of PDHA1 deletion in the thymus versus bone marrow. Our metabolomics analysis led us to investigate the contribution of glucose in the thymus versus bone marrow, leading us to develop low cell number isotope-tracing method in hematopoietic stem and progenitor cells (HSPCs) after U-13C glucose tracing. The conclusion that PDC is required in DP and T-ALL cells was previously established via flow and transplantation analysis. However, it was not known 1) the metabolic consequences of PDHA1 deletion after hematopoietic deletion, and 2) whether the thymus or bone marrow primarily uses glucose as an energy source using in vivo tracing method. U-13C glucose tracing followed by metabolomics analysis in the bone marrow also provided evidence that HSCs do not preferentially utilize glucose, at least evidenced from the metabolites we were able to examine, but this question needs to be further investigated with more extensive low-cell number metabolomics experiments that can detect more metabolites. My work in collaboration with S. Mahesula also arrived at a conceptually novel discovery that the role of PDC in the thymus is to prevent upstream metabolites from accumulating, rather than to generate acetyl-CoA to fuel the TCA cycle. Moreover, I showed that PDC is needed to maintain NAD+/NADH homeostasis in the thymus. Here, I present that PDC was not required to generate acetyl-CoA or maintain levels of TCA cycle metabolites but was required to prevent pyruvate accumulation and to maintain glutathione levels and redox homeostasis. Moreover, I present results highlighting the efforts to rescue metabolic defects seen in the PDHA1-deficient mice. My work shows the importance of employing in vivo approaches to characterize the metabolic requirement of DP thymocytes and T-ALL cells, and through this work we could draw a conclusive result that some metabolic characteristics of cancer cells may be inherited from the metabolism of the normal cell type of origin.Item Propionate Increase Hyperpolarized H13CO3- Signal in Perfused Mouse Hearts(2014-02-04) Purmal, Colin; Malloy, Craig R.; Sherry, A. Dean; Merritt, Matthew E.BACKGROUND: As early as 2008, MR imaging of [1-13C]pyruvate and its metabolites, including bicarbonate, in post-ischemic pig hearts was reported (1). Since the method does not use ionizing radiation, there is widespread interest in applications in other fields including oncology (2). In the heart, pyruvate is oxidized to acetyl-CoA and CO2. Oxidation of hyperpolarized (HP) [1-13C]pyruvate to HP [13C]bicarbonate is reduced in injured myocardium, and the presence of preserved flux through pyruvate dehydrogenase (PDH) may identify viable myocardium (1). However, oxidation of alternative substrates normally present in the blood also reduces the appearance of HP [13C]bicarbonate even in healthy myocardium (3). Propionate, a short-chain three-carbon fatty acid normally present in the blood, is known to activate PDH, and it is under study as a nutritional therapy for heart failure (4). The efficacy of propionate for restoring PDH flux in hearts supplied with high concentrations of glucose and fatty acids was studied using 13C NMR isotopomer analysis paired with experiments using HP [1-13C]pyruvate. 13C NMR is a standard method for measuring fluxes in metabolic pathways. METHODS AND RESULTS: Hearts excised from fed C57/bl6 mice were perfused in Langendorff mode using a mixture of acetate (2 mM), glucose (8.25 mM), and with and without propionate (2 mM). O2 consumption was not changed for the two different perfusion conditions. Isotopomer analysis of extracts of the freeze-clamped hearts indicated that carboxylation of propionate was very active, as expected, and glucose oxidation was minimal. For HP experiments, the perfused heart was located inside a 10 mm cryogenically-cooled probe paired with a 14.1 Tesla nuclear magnetic resonance spectrometer. After addition of hyperpolarized pyruvate, NMR signals from lactate, alanine, bicarbonate, CO2, aspartate, malate, acetyl-carnitine, and glutamate were detected in real time and in a highly reproducible manner. The presence of propionate increased appearance of HP [13C]bicarbonate 37-fold. This is the first application of hyperpolarization with detection using a cryogenically-cooled probe. CONCLUSION: In the presence of a high concentration of a competing substrate, propionate stimulates PDH flux in perfused mouse hearts as measured by the appearance of hyperpolarized [13C]bicarbonate from metabolism of hyperpolarized [1-13C]pyruvate. REFERENCES 1. Golman K, Petersson JS, Magnusson P, Johansson E, Akeson P, Chai CM, Hansson G, Månsson S. Cardiac metabolism measured noninvasively by hyperpolarized 13C MRI. Magn Reson Med. 2008; 59: 1005-13. PMID: 18429038 2. Harrison CE, DeBerardinis RJ, Jindal AK, Yang C, Sherry AD, Malloy CR. Analysis of mitochondrial metabolism in cancer cells by combining hyperpolarization and isotopomer analysis. Proc Int Soc Magn Reson Med 2010;18:569. 3. Moreno KX, Sabelhaus SM, Merritt ME, Sherry AD, Malloy CR. Competition of Pyruvate with Physiological Substrates for Oxidation by the Heart: Implications for Studies with Hyperpolarized [1-13C]Pyruvate. Am J Physiol Heart Circ Physiol. 2010; 298: H1556 - 64. PMID: 20207817 4. Lango R, Smoleski RT, Rogowski J, Siebert J, Wujtewicz M, S?omi?ska EM, Lysiak-Szyd?owska W, Yacoub MH. Propionyl-L-carnitine improves hemodynamics and metabolic markers of cardiac perfusion during coronary surgery in diabetic patients. Cardiovasc Drugs Ther. 2005; 19: 267-75. PMID: 16187006Item Technical Development of Hyperpolarized [1-13C]Pyruvate Imaging for Clinical Translation(December 2021) Ma, Junjie; Madhuranthakam, Ananth; Park, Jae Mo; Malloy, Craig R.; Pinho, Marco Da Cunha; Chopra, RajivMagnetic resonance imaging with hyperpolarized [1-13C]pyruvate is an emerging tool for assessing in vivo metabolism noninvasively. Over the past few years since the first clinical study with prostate cancer patients, translational studies using this imaging technique have been focused on demonstrating the feasibility of applying to healthy subjects and patients under several pathophysiological conditions. However, there are multiple remaining technical limitations to overcome for translating the technique to clinics. In this study, I made three technical advances in imaging hyperpolarized [1-13C]pyruvate and products in humans. First, I measured in vivo T2* of hyperpolarized signals, one of the key parameters that directly affect 13C acquisition methods and image quality. Measuring the in vivo T2*'s can be useful for optimizing the acquisition parameters and improving the signal-to-noise ratio. I proposed a dynamic 13C metabolite-selective multi-echo spiral imaging sequence for the T2* measurement of hyperpolarized 13C-labeled metabolites. The feasibility and reproducibility of the method were confirmed by phantom and rat studies. Moreover, from healthy volunteers, in vivo T2*s of hyperpolarized [1-13C]pyruvate, [1-13C]lactate and [13C]bicarbonate were measured from cardiac tissue compartments using the sequence. Second, I developed a cardiac-gated multi-phase 13C imaging sequence for the human heart. To demonstrate cyclic changes in cardiac metabolic profiles seen by hyperpolarized signals, dual-phase cardiac imaging of hyperpolarized [1-13C]pyruvate, [1-13C]lactate, [1-13C]alanine and [13C]bicarbonate was conducted at end-systole (ES) and end-diastole (ED) on the short-axis and vertical long-axis planes. Significantly smaller myocardial signal of [13C]bicarbonate relative to [1-13C]lactate was observed at ED compared to that at ES (p < 0.05). Third, a patch-based algorithm (PA) was developed to improve spatial resolution of hyperpolarized 13C images by exploiting the co-registered high-resolution 1H images. The spatial resolution of hyperpolarized 13C imaging is genuinely poor, compromising the overall image conspicuity and limiting accurate assessment of the hyperpolarized 13C metabolites. The PA was validated in simulation and phantom studies, and was further applied to low-resolution human brain metabolite maps of hyperpolarized [1-13C]pyruvate and [1-13C]lactate with three compartment segmentation (grey matter, white matter and cerebrospinal fluid). The results demonstrated that the PA can enhance low-resolution hyperpolarized 13C images in terms of spatial resolution and contrast while preserving quantification accuracy and intra-compartment signal inhomogeneity.