Browsing by Subject "Pyruvate Dehydrogenase Complex"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
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 The Role of Pyruvate Dehydrogenase in Cell Growth(2014-07-28) Rajagopalan, Kartik N.; Amatruda, James F.; DeBerardinis, Ralph J.; Brown, Michael S.; Lum, LawrenceOtto Warburg's observation that tumor cells have increased rates of glucose uptake and lactate secretion in comparison to normal cells spawned his notion that tumors have dysfunctional mitochondria. However, in addition to metabolizing glucose to lactate, tumors in vivo exhibit mitochondrial glucose oxidation, indicating activity of pyruvate dehydrognase (PDH), which gates entry of glucose derived carbon into the tricarboxylic acid (TCA) cycle. To test whether cells require glucose oxidation for proliferation, the work in this thesis establishes a model wherein PDH activity is suppressed using RNA interference. Small hairpin RNAs against the transcript encoding the PDHE1α protein were cloned into a retroviral vector which allowed doxycycline-inducible control of expression. Metabolism of cancer cells was studied in vitro using a combination of metabolomics and metabolic flux analysis. Growth in monolayer culture was performed in medium containing lipid-replete serum as well as serum in which lipids had been depleted. As expected, suppression of PDH activity reduced flow of carbon from glucose to the TCA cycle and to de novo fatty acid synthesis. Surprisingly, H460 lung cancer cells could tolerate a 60% reduction of PDH flux without any significant effect on proliferation rate, as long as lipids were present in the medium. Further examination of the effects of PDH silencing on the overall network of central carbon metabolism revealed enhanced channeling of carbon from glutamine to fatty acids and an increase in scavenging free fatty acids. Lipid depletion caused a reduction in growth rate of PDH deficient cells, and this defect was completely rescued by supplying free fatty acids to the medium. Together the data indicate that proliferating cells exhibit PDH activity that allows transfer of glucose carbon to citrate and the TCA cycle as well as ultimately into fatty acids. Importantly, suppression of PDH activity limits growth in conditions in which cancer cells do not have access to extracellular lipids. This work illustrates that compensatory pathways that sustain cell proliferation are activated during suppression of mitochondrial oxidation of glucose.