The traditional view is that cancer cells predominately produce ATP by glycolysis, rather than by oxidation of energy-providing substrates. Mitochondrial uncoupling -the continuing reduction of oxygen without ATP synthesis -has recently been shown in leukemia cells to circumvent the ability of oxygen to inhibit glycolysis, and may promote the metabolic preference for glycolysis by shifting from pyruvate oxidation to fatty acid oxidation (FAO). Here we have demonstrated that pharmacologic inhibition of FAO with etomoxir or ranolazine inhibited proliferation and sensitized human leukemia cells -cultured alone or on bone marrow stromal cells -to apoptosis induction by ABT-737, a molecule that releases proapoptotic Bcl-2 proteins such as Bak from antiapoptotic family members. Likewise, treatment with the fatty acid synthase/lipolysis inhibitor orlistat also sensitized leukemia cells to ABT-737, which supports the notion that fatty acids promote cell survival. Mechanistically, we generated evidence suggesting that FAO regulates the activity of Bak-dependent mitochondrial permeability transition. Importantly, etomoxir decreased the number of quiescent leukemia progenitor cells in approximately 50% of primary human acute myeloid leukemia samples and, when combined with either ABT-737 or cytosine arabinoside, provided substantial therapeutic benefit in a murine model of leukemia. The results support the concept of FAO inhibitors as a therapeutic strategy in hematological malignancies. IntroductionMore than half a century ago, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that circumvents the Pasteur effect, i.e., the inhibition of anaerobic fermentation by oxygen (1). However, we have recently demonstrated that in leukemia cells, mitochondrial uncoupling - the continuing reduction of oxygen without the synthesis of ATP - could mimic the Warburg effect in the absence of permanent, transmissible alterations to the oxidative capacity of cells (2). This metabolic pattern was observed when leukemia cells were cultured on feeder layers of bone marrow-derived mesenchymal stromal cells (MSCs). MSCs have previously been reported to support both normal and malignant hematopoiesis (reviewed in refs. 3-5) and have become an important component in the in vitro modeling of the bone marrow microenvironment. Leukemia cells cultured on MSC feeder layers demonstrated increased lactate generation, and, most curiously, decreased mitochondrial membrane potential in the presence of a transient (6-8 hour) increase in oxygen consumption. Additionally, this uncoupled phenotype appeared to be associated with the antiapoptotic effect of MSC feeder layers, and we hypothesized a shift away from the complete oxidation of glucose. This concept has already been alluded to by Lynen (6), and by Ronzoni and Ehrenfest in experiments using the prototypical protonophore 2,4-dinitrophenol, and suggests a metabolic shift to fatty acid oxidation (FAO) rather than pyruvate oxidation (2, 7). Although i...

In response to myocardial infarction (MI), cardiac macrophages regulate inflammation and scar formation. We hypothesized that macrophages undergo polarization state changes over the MI time course and assessed macrophage polarization transcriptomic signatures over the first week of MI. C57BL/6 J male mice (3–6 months old) were subjected to permanent coronary artery ligation to induce MI, and macrophages were isolated from the infarct region at days 1, 3, and 7 post-MI. Day 0, no MI resident cardiac macrophages served as the negative MI control. Whole transcriptome analysis was performed using RNA-sequencing on n = 4 pooled sets for each time. Day 1 macrophages displayed a unique pro-inflammatory, extracellular matrix (ECM)-degrading signature. By flow cytometry, day 0 macrophages were largely F4/80highLy6Clow resident macrophages, whereas day 1 macrophages were largely F4/80lowLy6Chigh infiltrating monocytes. Day 3 macrophages exhibited increased proliferation and phagocytosis, and expression of genes related to mitochondrial function and oxidative phosphorylation, indicative of metabolic reprogramming. Day 7 macrophages displayed a pro-reparative signature enriched for genes involved in ECM remodeling and scar formation. By triple in situ hybridization, day 7 infarct macrophages in vivo expressed collagen I and periostin mRNA. Our results indicate macrophages show distinct gene expression profiles over the first week of MI, with metabolic reprogramming important for polarization. In addition to serving as indirect mediators of ECM remodeling, macrophages are a direct source of ECM components. Our study is the first to report the detailed changes in the macrophage transcriptome over the first week of MI. Electronic supplementary materialThe online version of this article (10.1007/s00395-018-0686-x) contains supplementary material, which is available to authorized users.

Hematologic malignancies are frequently associated with cardiac pathologies. Mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a subset of acute myeloid leukemia patients, causing metabolic and epigenetic derangements. We have now discovered that altered metabolism in leukemic cells has a profound effect on cardiac metabolism. Combining mathematical modeling and in vivo as well as ex vivo studies, we found that increased amounts of the oncometabolite D-2-hydroxyglutarate (D2-HG), produced by IDH2 mutant leukemic cells, cause contractile dysfunction in the heart. This contractile dysfunction is associated with impaired oxidative decarboxylation of α-ketoglutarate, a redirection of Krebs cycle intermediates, and increased ATP citrate lyase (ACL) activity. Increased availability of D2-HG also leads to altered histone methylation and acetylation in the heart. We propose that D2-HG promotes cardiac dysfunction by impairing α-ketoglutarate dehydrogenase and induces histone modifications in an ACL-dependent manner. Collectively, our results highlight the impact of cancer cell metabolism on function and metabolism of the heart. D-2-hydroxyglutarate | IDH2 | metabolism | cardiomyopathy | flux rate analysis M etabolic dysregulation in cancer cells changes the way nutrients are consumed and macromolecules are produced to meet the increased demands for cell growth. Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are common and are described in several cancer types (i.e., gliomas and acute myeloid leukemia). IDH mutations lead to increased production and accumulation of the oncometabolite D-2-hydroxyglutarate (D2-HG) through a neomorphic enzymatic function (1). WT IDH1/2 catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), while reducing NADP + to NADPH either in the cytosol and peroxisome (IDH1), or in mitochondria (IDH2). In this reaction, D2-HG is produced in small amounts but converted back to its structural homolog α-KG by D2-HG dehydrogenase. Common features of tumors with IDH1/2 mutations are abnormal histone and DNA methylation, connecting metabolic changes with epigenetic control of gene expression (2). In hematologic malignancies, IDH1/2 are often co-mutated with epigenetic regulatory genes encoding enzymes that are important in DNA hydroxymethylation (i.e., tet methylcytosine dioxygenase 2, TET2) and methylation (i.e., DNA methyltransferase 3 A, DNMT3A) (3). Accumulation of D2-HG contributes to leukemogenesis, likely due to inhibition of α-KG-dependent dioxygenases, including histone lysine demethylases (KDMs) and TET2 (4). This hypothesis has been supported by recent reports linking the hypermethylation phenotype in cancer cells to IDH, fumarate hydratase, and succinate dehydrogenase mutations (5, 6).The starting point for the present work were reports that myeloid malignancies are associated with cardiac pathologies, which are commonly considered a side effect of chemotherapy (7). Other recent reports suggest that systemically produced D2-HG by neomorphic ...

Carbohydrate Response Element Binding Protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.