bims-mepmim Biomed News
on Metabolites in pathological microenvironments and immunometabolism
Issue of 2022–04–24
53 papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism
  1. Therapeutic Targeting Hypoxia-Inducible Factor (HIF-1) in Cancer: Cutting Gordian Knot of Cancer Cell Metabolism.
  2. Effects of Glucose Metabolism, Lipid Metabolism, and Glutamine Metabolism on Tumor Microenvironment and Clinical Implications.
  3. The mitochondrial pyruvate carrier regulates memory T cell differentiation and antitumor function.
  4. Mitochondrial and metabolic alterations in cancer cells.
  5. Mitochondrial respiratory quiescence: A new model for examining the role of mitochondrial metabolism in development.
  6. Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis.
  7. High-dimensionality reduction clustering of complex carbohydrates to study lung cancer metabolic heterogeneity.
  8. The Role of Metabolic Dysfunction in T-Cell Exhaustion During Chronic Viral Infection.
  9. Impacts and mechanisms of metabolic reprogramming of tumor microenvironment for immunotherapy in gastric cancer.
  10. Complexity of subcellular metabolism: strategies for compartment-specific profiling.
  11. Adenylate Kinase Isozyme 3 Regulates Mitochondrial Energy Metabolism and Knockout Alters HeLa Cell Metabolism.
  12. Immunometabolism-fit: How exercise and training can modify T cell and macrophage metabolism in health and disease.
  13. Improving Mitochondrial Function in Viral Infection: Targeting Cellular Metabolism.
  14. Targeting pancreatic cancer with combinatorial treatment of CPI-613 and inhibitors of lactate metabolism.
  15. The gut microbial metabolite formate exacerbates colorectal cancer progression.
  16. Glycometabolic reprogramming-mediated proangiogenic phenotype enhancement of cancer-associated fibroblasts in oral squamous cell carcinoma: role of PGC-1α/PFKFB3 axis.
  17. KLF7 Alleviates Atherosclerotic Lesions and Inhibits Glucose Metabolic Reprogramming in Macrophages by Regulating HDAC4/miR-148b-3p/NCOR1.
  18. Inhibition of the succinyl dehydrogenase complex in acute myeloid leukemia leads to a lactate-fuelled respiratory metabolic vulnerability.
  19. 2D Transition Metal Dichalcogenides Trigger Trained Immunity in Human Macrophages through Epigenetic and Metabolic Pathways.
  20. Cellular metabolic adaptations in rheumatoid arthritis and their therapeutic implications.
  21. Cancer's camouflage: Microvesicle shedding from cholesterol-rich tumor plasma membranes might blindfold first-responder immunosurveillance strategies.
  22. Mitochondria Transfer to CD4+ T Cells May Alleviate Rheumatoid Arthritis by Suppressing Pro-Inflammatory Cytokine Production.
  23. Does a Hypertrophying Muscle Fibre Reprogramme its Metabolism Similar to a Cancer Cell?
  24. ATP-citrate lyase (ACLY) inhibitors as therapeutic agents: a patenting perspective.
  25. Type I interferon antagonism of the JMJD3-IRF4 pathway modulates macrophage activation and polarization.
  26. Targeting oncometabolism to maximize immunotherapy in malignant brain tumors.
  27. The blood-brain barrier-a metabolic ecosystem.
  28. iTRAQ-Based Quantitative Proteomics Reveals the Energy Metabolism Alterations Induced by Chlorogenic Acid in HepG2 Cells.
  29. The goodies of chelated fat: iron-regulated lipid droplet biogenesis precedes and preserves mitophagy.
  30. The BCAT1 CXXC Motif Provides Protection against ROS in Acute Myeloid Leukaemia Cells.
  31. Methylglyoxal Induces Inflammation, Metabolic Modulation and Oxidative Stress in Myoblast Cells.
  32. Impact of Drp1-Mediated Mitochondrial Dynamics on T Cell Immune Modulation.
  33. CoA in Health and Disease.
  34. Slc38a9 Deficiency Induces Apoptosis and Metabolic Dysregulation and Leads to Premature Death in Zebrafish.
  35. Untargeted Metabolomics of Slc13a5 Deficiency Reveal Critical Liver-Brain Axis for Lipid Homeostasis.
  36. Prognosis and Dissection of Immunosuppressive Microenvironment in Breast Cancer Based on Fatty Acid Metabolism-Related Signature.
  37. Dimethyloxalylglycine (DMOG), a Hypoxia Mimetic Agent, Does Not Replicate a Rat Pheochromocytoma (PC12) Cell Biological Response to Reduced Oxygen Culture.
  38. Iron plays a role in sulfasalazine-induced ferroptosis with autophagic flux blockage in K7M2 osteosarcoma cells.
  39. Mitochondrial oxidative phosphorylation is dispensable for survival of CD34+ chronic myeloid leukemia stem and progenitor cells.
  40. Chronically Elevated O-GlcNAcylation Limits Nitric Oxide Production and Deregulates Specific Pro-Inflammatory Cytokines.
  41. Carnitines as Mitochondrial Modulators of Oocyte and Embryo Bioenergetics.
  42. Preclinical studies for improving radiosensitivity of non-small cell lung cancer cell lines by combining glutaminase inhibition and senolysis.
  43. SLC38A10 Regulate Glutamate Homeostasis and Modulate the AKT/TSC2/mTOR Pathway in Mouse Primary Cortex Cells.
  44. Titanium dioxide nanoparticles induced reactive oxygen species (ROS) related changes of metabolomics signatures in human normal bronchial epithelial (BEAS-2B) cells.
  45. Pancreatic Stellate Cells Prolong Ex Vivo Islet Viability and Function and Improve Engraftment.
  46. Branched-Chain Amino Acid Deprivation Decreases Lipid Oxidation and Lipogenesis in C2C12 Myotubes.
  47. Pharmacological intervention of cholesterol sulfate-mediated T cell exclusion promotes antitumor immunity.
  48. Interaction between macrophages and ferroptosis.
  49. Lysophosphatidylcholine acyltransferase 1 controls mitochondrial reactive oxygen species generation and survival of retinal photoreceptor cells.
  50. Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK-STAT signaling in macrophages by perturbing lipid raft structures.
  51. Effects of Arachidonic Acid and Its Metabolites on Functional Beta-Cell Mass.
  52. COVID-19 and One-Carbon Metabolism.
  53. Modeling the measurement bias in interstitial glucose concentrations derived from microdialysis in skeletal muscle.
  1. Front Genet. 2022 ;13 849040
    Therapeutic Targeting Hypoxia-Inducible Factor (HIF-1) in Cancer: Cutting Gordian Knot of Cancer Cell Metabolism.
    Abhilasha Sharma, Sonam Sinha, Neeta Shrivastava.
      Metabolic alterations are one of the hallmarks of cancer, which has recently gained great attention. Increased glucose absorption and lactate secretion in cancer cells are characterized by the Warburg effect, which is caused by the metabolic changes in the tumor tissue. Cancer cells switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis due to changes in glucose degradation mechanisms, a process known as "metabolic reprogramming". As a result, proteins involved in mediating the altered metabolic pathways identified in cancer cells pose novel therapeutic targets. Hypoxic tumor microenvironment (HTM) is anticipated to trigger and promote metabolic alterations, oncogene activation, epithelial-mesenchymal transition, and drug resistance, all of which are hallmarks of aggressive cancer behaviour. Angiogenesis, erythropoiesis, glycolysis regulation, glucose transport, acidosis regulators have all been orchestrated through the activation and stability of a transcription factor termed hypoxia-inducible factor-1 (HIF-1), hence altering crucial Warburg effect activities. Therefore, targeting HIF-1 as a cancer therapy seems like an extremely rational approach as it is directly involved in the shift of cancer tissue. In this mini-review, we present a brief overview of the function of HIF-1 in hypoxic glycolysis with a particular focus on novel therapeutic strategies currently available.
    Keywords:  cancer; cancer therapies; clinical outcomes; genomic alterations; hypoxia-induced tumor microenvironment; metabolic reprogramming; metabolism; warburg effect
    DOI:  https://doi.org/10.3389/fgene.2022.849040
  2. Biomolecules. 2022 Apr 14. pii: 580. [Epub ahead of print]12(4):
    Effects of Glucose Metabolism, Lipid Metabolism, and Glutamine Metabolism on Tumor Microenvironment and Clinical Implications.
    Longfei Zhu, Xuanyu Zhu, Yan Wu.
      In recent years, an increasingly more in depth understanding of tumor metabolism in tumorigenesis, tumor growth, metastasis, and prognosis has been achieved. The broad heterogeneity in tumor tissue is the critical factor affecting the outcome of tumor treatment. Metabolic heterogeneity is not only found in tumor cells but also in their surrounding immune and stromal cells; for example, many suppressor cells, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and tumor-associated T-lymphocytes. Abnormalities in metabolism often lead to short survival or resistance to antitumor therapy, e.g., chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Using the metabolic characteristics of the tumor microenvironment to identify and treat cancer has become a great research hotspot. This review systematically addresses the impacts of metabolism on tumor cells and effector cells and represents recent research advances of metabolic effects on other cells in the tumor microenvironment. Finally, we introduce some applications of metabolic features in clinical oncology.
    Keywords:  fatty acid metabolism; glutaminolysis; glycolysis; immunotherapy; metabolism; tumor microenvironment
    DOI:  https://doi.org/10.3390/biom12040580
  3. Cell Metab. 2022 Apr 18. pii: S1550-4131(22)00127-9. [Epub ahead of print]
    The mitochondrial pyruvate carrier regulates memory T cell differentiation and antitumor function.
    Mathias Wenes, Alison Jaccard, Tania Wyss, Noelia Maldonado-Pérez, Shao Thing Teoh, Anouk Lepez, Fabrice Renaud, Fabien Franco, Patrice Waridel, Céline Yacoub Maroun, Benjamin Tschumi, Nina Dumauthioz, Lianjun Zhang, Alena Donda, Francisco Martín, Denis Migliorini, Sophia Y Lunt, Ping-Chih Ho, Pedro Romero.
      Glycolysis, including both lactate fermentation and pyruvate oxidation, orchestrates CD8+ T cell differentiation. However, how mitochondrial pyruvate metabolism and uptake controlled by the mitochondrial pyruvate carrier (MPC) impact T cell function and fate remains elusive. We found that genetic deletion of MPC drives CD8+ T cell differentiation toward a memory phenotype. Metabolic flexibility induced by MPC inhibition facilitated acetyl-coenzyme-A production by glutamine and fatty acid oxidation that results in enhanced histone acetylation and chromatin accessibility on pro-memory genes. However, in the tumor microenvironment, MPC is essential for sustaining lactate oxidation to support CD8+ T cell antitumor function. We further revealed that chimeric antigen receptor (CAR) T cell manufacturing with an MPC inhibitor imprinted a memory phenotype and demonstrated that infusing MPC inhibitor-conditioned CAR T cells resulted in superior and long-lasting antitumor activity. Altogether, we uncover that mitochondrial pyruvate uptake instructs metabolic flexibility for guiding T cell differentiation and antitumor responses.
    Keywords:  T cell memory; chimeric antigen receptor T cell therapy; immunometabolism; mitochondrial pyruvate carrier; tumor-infiltrating lymphocyte metabolism
    DOI:  https://doi.org/10.1016/j.cmet.2022.03.013
  4. Eur J Cell Biol. 2022 Apr 13. pii: S0171-9335(22)00028-0. [Epub ahead of print]101(3): 151225
    Mitochondrial and metabolic alterations in cancer cells.
    Jacopo Di Gregorio, Sabrina Petricca, Roberto Iorio, Elena Toniato, Vincenzo Flati.
      Metabolic alterations have been observed in many cancer types. The deregulated metabolism has thus become an emerging hallmark of the disease, where the metabolism is frequently rewired to aerobic glycolysis. This has led to the concept of "metabolic reprogramming", which has therefore been extensively studied. Over the years, it has been characterized the enhancement of aerobic glycolysis, where key mutations in some of the enzymes of the TCA cycle, and the increased glucose uptake, are used by cancer cells to achieve a "metabolic phenotype" useful to gain a proliferation advantage. Many studies have highlighted in detail the signaling pathways and the molecular mechanisms responsible for the glycolytic switch. However, glycolysis is not the only metabolic process that cancer cells rely on. Oxidative Phosphorylation (OXPHOS), gluconeogenesis or the beta-oxidation of fatty acids (FAO) may be involved in the development and progression of several tumors. In some cases, these metabolisms are even more crucial than aerobic glycolysis for the tumor survival. This review will focus on the contribution of these alterations of metabolism to the development and survival of cancers. We will also analyze the molecular mechanisms by which the balance between these metabolic processes may be regulated, as well as some of the therapeutical approaches that can derive from their study.
    Keywords:  Amino acids; Cancer; Fatty acids; Metabolism; Mitochondria; OXPHOS
    DOI:  https://doi.org/10.1016/j.ejcb.2022.151225
  5. Semin Cell Dev Biol. 2022 Apr 18. pii: S1084-9521(22)00122-7. [Epub ahead of print]
    Mitochondrial respiratory quiescence: A new model for examining the role of mitochondrial metabolism in development.
    Helin Hocaoglu, Matthew Sieber.
      Mitochondria are vital organelles with a central role in all aspects of cellular metabolism. As a means to support the ever-changing demands of the cell, mitochondria produce energy, drive biosynthetic processes, maintain redox homeostasis, and function as a hub for cell signaling. While mitochondria have been widely studied for their role in disease and metabolic dysfunction, this organelle has a continually evolving role in the regulation of development, wound repair, and regeneration. Mitochondrial metabolism dynamically changes as tissues transition through distinct phases of development. These organelles support the energetic and biosynthetic demands of developing cells and function as key structures that coordinate the nutrient status of the organism with developmental progression. This review will examine the mechanisms that link mitochondria to developmental processes. We will also examine the process of mitochondrial respiratory quiescence (MRQ), a novel mechanism for regulating cellular metabolism through the biochemical and physiological remodeling of mitochondria. Lastly, we will examine MRQ as a system to discover the mechanisms that drive mitochondrial remodeling during development.
    Keywords:  Cancer; Drosophila; Metabolism; Mitochondria; Oocytes; Quiescence; Reprogramming; Stem cells
    DOI:  https://doi.org/10.1016/j.semcdb.2022.03.040
  6. Cell Death Dis. 2022 Apr 19. 13(4): 370
    Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis.
    Xuetian Yue, Jianming Wang, Chun-Yuan Chang, Juan Liu, Xue Yang, Fan Zhou, Xia Qiu, Vrushank Bhatt, Jessie Yanxiang Guo, Xiaoyang Su, Lanjing Zhang, Zhaohui Feng, Wenwei Hu.
      LIF, a multifunctional cytokine, is frequently overexpressed in many types of solid tumors, including breast cancer, and plays an important role in promoting tumorigenesis. Currently, how LIF promotes tumorigenesis is not well-understood. Metabolic reprogramming is a hallmark of cancer cells and a key contributor to cancer progression. However, the role of LIF in cancer metabolic reprogramming is unclear. In this study, we found that LIF increases glucose uptake and drives glycolysis, contributing to breast tumorigenesis. Blocking glucose uptake largely abolishes the promoting effect of LIF on breast tumorigenesis. Mechanistically, LIF overexpression enhances glucose uptake via activating the AKT/GLUT1 axis to promote glycolysis. Blocking the AKT signaling by shRNA or its inhibitors greatly inhibits glycolysis driven by LIF and largely abolishes the promoting effect of LIF on breast tumorigenesis. These results demonstrate an important role of LIF overexpression in glucose metabolism reprogramming in breast cancers, which contributes to breast tumorigenesis. This study also reveals an important mechanism underlying metabolic reprogramming of breast cancers, and identifies LIF and its downstream signaling as potential therapeutic targets for breast cancers, especially those with LIF overexpression.
    DOI:  https://doi.org/10.1038/s41419-022-04820-x
  7. Adv Cancer Res. 2022 ;pii: S0065-230X(22)00031-8. [Epub ahead of print]154 227-251
    High-dimensionality reduction clustering of complex carbohydrates to study lung cancer metabolic heterogeneity.
    Lindsey R Conroy, Josephine E Chang, Qi Sun, Harrison A Clarke, Michael D Buoncristiani, Lyndsay E A Young, Robert J McDonald, Jinze Liu, Matthew S Gentry, Derek B Allison, Ramon C Sun.
      The tumor microenvironment contains a heterogeneous population of stromal and cancer cells that engage in metabolic crosstalk to ultimately promote tumor growth and contribute to progression. Due to heterogeneity within solid tumors, pooled mass spectrometry workflows are less sensitive at delineating unique metabolic perturbations between stromal and immune cell populations. Two critical, but understudied, facets of glucose metabolism are anabolic pathways for glycogen and N-linked glycan biosynthesis. Together, these complex carbohydrates modulate bioenergetics and protein-structure function, and create functional microanatomy in distinct cell populations within the tumor heterogeneity. Herein, we combine high-dimensionality reduction and clustering (HDRC) analysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and demonstrate its ability for the comprehensive assessment of tissue histopathology and metabolic heterogeneity in human FFPE sections. In human lung adenocarcinoma (LUAD) tumor tissues, HDRC accurately clusters distinct regions and cell populations within the tumor microenvironment, including tumor cells, tumor-infiltrating lymphocytes, cancer-associated fibroblasts, and necrotic regions. In-depth pathway enrichment analyses revealed unique metabolic pathways are associated with each distinct pathological region. Further, we highlight the potential of HDRC analysis to study complex carbohydrate metabolism in a case study of lung cancer disparity. Collectively, our results demonstrate the promising potentials of HDRC of pixel-based carbohydrate analysis to study cell-type and regional-specific stromal signaling within the tumor microenvironment.
    Keywords:  Clustering analysis; Glycogen; Lung cancer; MALDI imaging; N-linked glycans
    DOI:  https://doi.org/10.1016/bs.acr.2022.02.005
  8. Front Immunol. 2022 ;13 843242
    The Role of Metabolic Dysfunction in T-Cell Exhaustion During Chronic Viral Infection.
    Kehong Zheng, Xiaojun Zheng, Wei Yang.
      T cells are important components of adaptive immunity that protect the host against invading pathogens during infection. Upon recognizing the activation signals, naïve and/or memory T cells will initiate clonal expansion, trigger differentiation into effector populations and traffic to the inflamed sites to eliminate pathogens. However, in chronic viral infections, such as those caused by human immunodeficiency virus (HIV), hepatitis B and C (HBV and HCV), T cells exhibit impaired function and become difficult to clear pathogens in a state known as T-cell exhaustion. The activation and function persistence of T cells demand for dynamic changes in cellular metabolism to meet their bioenergetic and biosynthetic demands, especially the augmentation of aerobic glycolysis, which not only provide efficient energy generation, but also fuel multiple biochemical intermediates that are essential for nucleotide, amino acid, fatty acid synthesis and mitochondria function. Changes in cellular metabolism also affect the function of effectors T cells through modifying epigenetic signatures. It is widely accepted that the dysfunction of T cell metabolism contributes greatly to T-cell exhaustion. Here, we reviewed recent findings on T cells metabolism under chronic viral infection, seeking to reveal the role of metabolic dysfunction played in T-cell exhaustion.
    Keywords:  PD-1; T-cell exhaustion; chronic viral infection; glycolysis; metabolism
    DOI:  https://doi.org/10.3389/fimmu.2022.843242
  9. Cell Death Dis. 2022 Apr 20. 13(4): 378
    Impacts and mechanisms of metabolic reprogramming of tumor microenvironment for immunotherapy in gastric cancer.
    Lin Zhao, Yuanyuan Liu, Simiao Zhang, Lingyu Wei, Hongbing Cheng, Jinsheng Wang, Jia Wang.
      Metabolic disorders and abnormal immune function changes occur in tumor tissues and cells to varying degrees. There is increasing evidence that reprogrammed energy metabolism contributes to the development of tumor suppressive immune microenvironment and influences the course of gastric cancer (GC). Current studies have found that tumor microenvironment (TME) also has important clinicopathological significance in predicting prognosis and therapeutic efficacy. Novel approaches targeting TME therapy, such as immune checkpoint blockade (ICB), metabolic inhibitors and key enzymes of immune metabolism, have been involved in the treatment of GC. However, the interaction between GC cells metabolism and immune metabolism and how to make better use of these immunotherapy methods in the complex TME in GC are still being explored. Here, we discuss how metabolic reprogramming of GC cells and immune cells involved in GC immune responses modulate anti-tumor immune responses, as well as the effects of gastrointestinal flora in TME and GC. It is also proposed how to enhance anti-tumor immune response by understanding the targeted metabolism of these metabolic reprogramming to provide direction for the treatment and prognosis of GC.
    DOI:  https://doi.org/10.1038/s41419-022-04821-w
  10. Curr Opin Biotechnol. 2022 Apr 13. pii: S0958-1669(22)00044-1. [Epub ahead of print]75 102711
    Complexity of subcellular metabolism: strategies for compartment-specific profiling.
    Tushar H More, Karsten Hiller.
      Subcellular compartmentalization provides cells with tremendous advantages for the operation of cellular metabolism. Spatial separation of metabolism generates microenvironments with distinct concentrations of metabolites and cofactors allowing the cell to execute otherwise thermodynamically exclusive reactions simultaneously. Moreover, compartmentalization is also involved in the fine-tuned regulation of gene expression. Thus, the elucidation of compartment-specific metabolic processes has become essential for a thorough understanding of cellular metabolism. The limited availability of reliable methods to inspect metabolic activities in subcellular compartments has been a major impediment in this field. During the last decade, progress has been greatly extended through methodological advances in subcellular metabolic profiling. This review summarizes recent strategies applied for the direct and indirect assessment of compartment-specific metabolism. We review contemporary techniques, recent advances, limitations, and future trends in the field of subcellular metabolomics.
    DOI:  https://doi.org/10.1016/j.copbio.2022.102711
  11. Int J Mol Sci. 2022 Apr 13. pii: 4316. [Epub ahead of print]23(8):
    Adenylate Kinase Isozyme 3 Regulates Mitochondrial Energy Metabolism and Knockout Alters HeLa Cell Metabolism.
    Koichi Fujisawa, Maina Wakazaki, Aya Matsuzaki, Toshihiko Matsumoto, Naoki Yamamoto, Takafumi Noma, Taro Takami.
      The balance between oxidative phosphorylation and glycolysis is important for cancer cell growth and survival, and changes in energy metabolism are an emerging therapeutic target. Adenylate kinase (AK) regulates adenine nucleotide metabolism, maintaining intracellular nucleotide metabolic homeostasis. In this study, we focused on AK3, the isozyme localized in the mitochondrial matrix that reversibly mediates the following reaction: Mg2+ GTP + AMP ⇌ Mg2+ GDP + ADP. Additionally, we analyzed AK3-knockout (KO) HeLa cells, which showed reduced proliferation and were detected at an increased number in the G1 phase. A metabolomic analysis showed decreased ATP; increased glycolytic metabolites such as glucose 6 phosphate (G6P), fructose 6 phosphate (F6P), and phosphoenolpyruvate (PEP); and decreased levels of tricarboxylic acid (TCA) cycle metabolites in AK3KO cells. An intracellular ATP evaluation of AK3KO HeLa cells transfected with ATeam plasmid, an ATP sensor, showed decreased whole cell levels. Levels of mitochondrial DNA (mtDNA), a complementary response to mitochondrial failure, were increased in AK3KO HeLa cells. Oxidative stress levels increased with changes in gene expression, evidenced as an increase in related enzymes such as superoxide dismutase 2 (SOD2) and SOD3. Phosphoenolpyruvate carboxykinase 2 (PCK2) expression and PEP levels increased, whereas PCK2 inhibition affected AK3KO HeLa cells more than wild-type (WT) cells. Therefore, we concluded that increased PCK2 expression may be complementary to increased GDP, which was found to be deficient through AK3KO. This study demonstrated the importance of AK3 in mitochondrial matrix energy metabolism.
    Keywords:  GTP metabolism; adenylate kinase 3; nucleotide metabolism; phosphoenolpyruvate
    DOI:  https://doi.org/10.3390/ijms23084316
  12. Exerc Immunol Rev. 2022 ;28 29-46
    Immunometabolism-fit: How exercise and training can modify T cell and macrophage metabolism in health and disease.
    José Cesar Rosa-Neto, Fábio Santos Lira, Jonathan Peter Little, Graham Landells, Hashim Islam, Bénédicte Chazaud, David B Pyne, Ana Maria Teixeira, Helena Batatinha, Barbara Moura Antunes, Luciele Guerra Minuzzi, Jana Palmowski, Richard J Simpson, Karsten Krüger.
       BACKGROUND: The term immunometabolism describes cellular and molecular metabolic processes that control the immune system and the associated immune responses. Acute exercise and regular physical activity have a substantial influence on the metabolism and the immune system, so that both processes are closely associated and influence each other bidirectionally.
    SCOPE OF REVIEW: We limit the review here to focus on metabolic phenotypes and metabolic plasticity of T cells and macrophages to describe the complex role of acute exercise stress and regular physical activity on these cell types. The metabolic and immunological consequences of the social problem of inactivity and how, conversely, an active lifestyle can break this vicious circle, are then described. Finally, these aspects are evaluated against the background of an aging society.
    MAJOR CONCLUSIONS: T cells and macrophages show high sensitivity to changes in their metabolic environment, which indirectly or directly affects their central functions. Physical activity and sedentary behaviour have an important influence on metabolic status, thereby modifying immune cell phenotypes and influencing immunological plasticity. A detailed understanding of the interactions between acute and chronic physical activity, sedentary behaviour, and the metabolic status of immune cells, can help to target the dysregulated immune system of people who live in a much too inactive society.
    Keywords:  Exercise; Immune System; Inflammation; Metabolism; Sedentary behavior
  13. Am J Respir Cell Mol Biol. 2022 Apr 20.
    Improving Mitochondrial Function in Viral Infection: Targeting Cellular Metabolism.
    Brydie R Huckestein, John F Alcorn.
      
    DOI:  https://doi.org/10.1165/rcmb.2022-0096ED
  14. PLoS One. 2022 ;17(4): e0266601
    Targeting pancreatic cancer with combinatorial treatment of CPI-613 and inhibitors of lactate metabolism.
    Simone Kumstel, Tim Schreiber, Lea Goldstein, Jan Stenzel, Tobias Lindner, Markus Joksch, Xianbin Zhang, Edgar Heinz Uwe Wendt, Maria Schönrogge, Bernd Krause, Brigitte Vollmar, Dietmar Zechner.
      Pancreatic cancer is the fourth leading cause of cancer death, with a 5-year survival rate of 10%. A stagnant high mortality rate over the last decades highlights the need for innovative therapeutic approaches. Pancreatic tumors pursue an altered metabolism in order to maintain energy generation under low nutrient influx and hypoxic conditions. Targeting these metabolic strategies might therefore be a reasonable therapeutic approach for pancreatic cancer. One promising agent is CPI- 613, a potent inhibitor of two enzymes of the tricarboxylic acid cycle. The present study evaluated the anti-cancerous efficacy of CPI-613 in combination with galloflavin, a lactate dehydrogenase inhibitor or with alpha-cyano-4-hydroxycinnamic acid, an inhibitor of monocarboxylate transporters. The efficacy of both combination therapies was tested in vitro on one human and two murine pancreatic cancer cell lines and in vivo in an orthotopic pancreatic cancer model. Tumor progression was evaluated by MRI and 18F-FDG PET-CT. Both combinatorial treatments demonstrated in vitro a significant inhibition of pancreatic cancer cell proliferation and induction of cell death. In contrast to the in vitro results, both combination therapies did not significantly reduce tumor growth in vivo. The in vitro results suggest that a combined inhibition of different metabolic pathways might be a promising approach for cancer therapy. However, the in vivo experiments indicate that applying a higher dosage or using other drugs targeting these metabolic pathways might be more promising.
    DOI:  https://doi.org/10.1371/journal.pone.0266601
  15. Nat Metab. 2022 Apr 18.
    The gut microbial metabolite formate exacerbates colorectal cancer progression.
    Dominik Ternes, Mina Tsenkova, Vitaly Igorevich Pozdeev, Marianne Meyers, Eric Koncina, Sura Atatri, Martine Schmitz, Jessica Karta, Maryse Schmoetten, Almut Heinken, Fabien Rodriguez, Catherine Delbrouck, Anthoula Gaigneaux, Aurelien Ginolhac, Tam Thuy Dan Nguyen, Lea Grandmougin, Audrey Frachet-Bour, Camille Martin-Gallausiaux, Maria Pacheco, Lorie Neuberger-Castillo, Paulo Miranda, Nikolaus Zuegel, Jean-Yves Ferrand, Manon Gantenbein, Thomas Sauter, Daniel Joseph Slade, Ines Thiele, Johannes Meiser, Serge Haan, Paul Wilmes, Elisabeth Letellier.
      The gut microbiome is a key player in the immunomodulatory and protumorigenic microenvironment during colorectal cancer (CRC), as different gut-derived bacteria can induce tumour growth. However, the crosstalk between the gut microbiome and the host in relation to tumour cell metabolism remains largely unexplored. Here we show that formate, a metabolite produced by the CRC-associated bacterium Fusobacterium nucleatum, promotes CRC development. We describe molecular signatures linking CRC phenotypes with Fusobacterium abundance. Cocultures of F. nucleatum with patient-derived CRC cells display protumorigenic effects, along with a metabolic shift towards increased formate secretion and cancer glutamine metabolism. We further show that microbiome-derived formate drives CRC tumour invasion by triggering AhR signalling, while increasing cancer stemness. Finally, F. nucleatum or formate treatment in mice leads to increased tumour incidence or size, and Th17 cell expansion, which can favour proinflammatory profiles. Moving beyond observational studies, we identify formate as a gut-derived oncometabolite that is relevant for CRC progression.
    DOI:  https://doi.org/10.1038/s42255-022-00558-0
  16. Br J Cancer. 2022 Apr 20.
    Glycometabolic reprogramming-mediated proangiogenic phenotype enhancement of cancer-associated fibroblasts in oral squamous cell carcinoma: role of PGC-1α/PFKFB3 axis.
    Xiang Li, Erhui Jiang, Hui Zhao, Yang Chen, Yuming Xu, Chunyu Feng, Ji Li, Zhengjun Shang.
       BACKGROUND: Angiogenesis is a key rate-limiting step in the process of tumour progression. Cancer-associated fibroblasts (CAFs), the most abundant component OSCC stroma, play important roles in pro-angiogenesis. Recently, the stroma "reverse Warburg effect" was proposed, and PFKFB3 has been brought to the forefront as a metabolic enzyme regulating glycometabolism. However, it remains unclear whether glycometabolism reprogramming is involved in promoting the angiogenesis of CAFs.
    METHODS: CAFs and paracancerous fibroblasts (PFs) were isolated from OSCC and adjacent tissues. We detected the pro-angiogenesis and glycometabolism phenotype of three pairs of fibroblasts. Targeted blockage of PFKFB3 or activation of PGC-1α signal was used to investigate the effect of glycolysis on regulating angiogenesis of CAFs in vitro and vivo.
    RESULTS: CAFs exhibited metabolic reprogramming and enhanced proangiogenic phenotype compared with PFs. Inhibition of PFKFB3-dependent glycolysis impaired proangiogenic factors (VEGF-A, PDGF-C and MMP9) expression in CAFs. Furthermore, CAFs proangiogenic phenotype was regulated by glycometabolism through the PGC-1α/PFKFB3 axis. Consistently, PGC-1α overexpression or PFKFB3 knockdown in CAFs slowed down tumour development by reducing tumour angiogenesis in the xenograft model.
    CONCLUSION: CAFs of OSCC are characterised with glycometabolic reprogramming and enhanced proangiogenic phenotypes. Our findings suggest that activating PGC-1α signalling impairs proangiogenic phenotype of CAFs by blocking PFKFB3-driven glycolysis.
    DOI:  https://doi.org/10.1038/s41416-022-01818-2
  17. Gerontology. 2022 Apr 19. 1-20
    KLF7 Alleviates Atherosclerotic Lesions and Inhibits Glucose Metabolic Reprogramming in Macrophages by Regulating HDAC4/miR-148b-3p/NCOR1.
    Fangyuan Chen, Juanli Li, Tao Zheng, Tao Chen, Zuyi Yuan.
       OBJECTIVES: Atherosclerosis (AS) remains a major contributor to death worldwide. This study sought to explore the role of Krüppel-like factor 7 (KLF7) in AS lesions via regulating glucose metabolic reprogramming (GMR) in macrophages.
    METHODS: AS mouse and cell models were established via high-fat-diet feeding and oxidized low-density lipoprotein (ox-LDL) induction. KLF7, histone deacetylase 4 (HDAC4), miR-148b-3p, and nuclear receptor corepressor 1 (NCOR1) expressions in aortic tissue and cells were detected via reverse transcription quantitative polymerase chain reaction or Western blotting. Parameters of AS lesions and mouse metabolism were detected via hematoxylin-eosin, oil red O, and Masson staining, assay kits, glucose tolerance test, and enzymatic analysis. Peritoneal macrophages of mice were isolated and cellular metabolism was detected via Seahorse metabolic flux analysis, assay kits, ELISA, and Western blotting. Bindings among KLF7, HDAC4, microRNA (miR)-148b-3p, and NCOR1 were testified via the dual-luciferase assay and chromatin immunoprecipitation assay.
    RESULTS: KLF7 was poorly expressed in AS mice and ox-LDL-induced RAW264.7 cells. KLF7 overexpression attenuated AS lesions and rescued metabolic abnormities in AS mice, and reduced glucose intake and GMR in ox-LDL-induced RAW264.7 cells. Mechanically, KLF7 bound to the HDAC4 promoter to activate HDAC4. HDAC4 reduced H3 and H4 acetylation levels in the miR-148b promoter to inhibit miR-148b-3p and promote NCOR1 transcription. HDAC4 downregulation abolished the protective role of KLF7 overexpression in AS mice and ox-LDL-induced RAW264.7 cells via the miR-148b-3p/NCOR1 axis.
    CONCLUSION: KLF7 bound to the HDAC4 promoter to activate HDAC4, inhibit miR-148b-3p via reducing acetylation level, and promote NCOR1 transcription, thereby limiting GMR in macrophages and alleviating AS lesions.
    Keywords:  Atherosclerosis; HDAC4; Histone deacetylase; Krüppel-like factor 7; Macrophage metabolic reprogramming
    DOI:  https://doi.org/10.1159/000524029
  18. Nat Commun. 2022 Apr 19. 13(1): 2013
    Inhibition of the succinyl dehydrogenase complex in acute myeloid leukemia leads to a lactate-fuelled respiratory metabolic vulnerability.
    Ayşegül Erdem, Silvia Marin, Diego A Pereira-Martins, Marjan Geugien, Alan Cunningham, Maurien G Pruis, Isabel Weinhäuser, Albert Gerding, Barbara M Bakker, Albertus T J Wierenga, Eduardo M Rego, Gerwin Huls, Marta Cascante, Jan Jacob Schuringa.
      Metabolic programs can differ substantially across genetically distinct subtypes of acute myeloid leukemia (AML). These programs are not static entities but can change swiftly as a consequence of extracellular changes or in response to pathway-inhibiting drugs. Here, we uncover that AML patients with FLT3 internal tandem duplications (FLT3-ITD+) are characterized by a high expression of succinate-CoA ligases and high activity of mitochondrial electron transport chain (ETC) complex II, thereby driving high mitochondrial respiration activity linked to the Krebs cycle. While inhibition of ETC complex II enhances apoptosis in FLT3-ITD+ AML, cells also quickly adapt by importing lactate from the extracellular microenvironment. 13C3-labelled lactate metabolic flux analyses reveal that AML cells use lactate as a fuel for mitochondrial respiration. Inhibition of lactate transport by blocking Monocarboxylic Acid Transporter 1 (MCT1) strongly enhances sensitivity to ETC complex II inhibition in vitro as well as in vivo. Our study highlights a metabolic adaptability of cancer cells that can be exploited therapeutically.
    DOI:  https://doi.org/10.1038/s41467-022-29639-0
  19. Small. 2022 Apr 18. e2107816
    2D Transition Metal Dichalcogenides Trigger Trained Immunity in Human Macrophages through Epigenetic and Metabolic Pathways.
    Guotao Peng, Sandeep Keshavan, Lucia Delogu, Yuyoung Shin, Cinzia Casiraghi, Bengt Fadeel.
      Trained immunity is a recently described phenomenon whereby cells of the innate immune system undergo long-term epigenetic and/or metabolic reprogramming following a short-term interaction with microbes or microbial products. Here, it is shown that 2D transition metal dichalcogenides (TMDs) trigger trained immunity in primary human monocyte-derived macrophages. First, aqueous dispersions of 2D crystal formulations of MoS2 and WS2 are tested, and no cytotoxicity is found despite avid uptake of these materials by macrophages. However, when macrophages are pre-exposed to TMDs, followed by a resting period, this causes a marked modulation of immune-specific gene expression upon subsequent challenge with a microbial agent (i.e., bacterial lipopolysaccharides). Specifically, MoS2 triggers trained immunity through an epigenetic pathway insofar as the histone methyltransferase inhibitor methylthioadenosine reverses these effects. Furthermore, MoS2 triggers an elevation of cyclic adenosine monophosphate (cAMP) levels in macrophages and increased glycolysis is also evidenced in cells subjected to MoS2 training, pointing toward a metabolic rewiring of the cells. Importantly, it is observed that MoS2 triggers the upregulation of Mo-dependent enzymes in macrophages, thus confirming that Mo is bioavailable in these cells. In conclusion, MoS2 is identified as a novel inducer of trained immunity. Thus, TMDs could potentially be harnessed as immunomodulatory agents.
    Keywords:  2D materials; epigenetic regulation; macrophages; trained immunity
    DOI:  https://doi.org/10.1002/smll.202107816
  20. Nat Rev Rheumatol. 2022 Apr 19.
    Cellular metabolic adaptations in rheumatoid arthritis and their therapeutic implications.
    Ursula Fearon, Megan M Hanlon, Achilleas Floudas, Douglas J Veale.
      Activation of endothelium and immune cells is fundamental to the initiation of autoimmune diseases such as rheumatoid arthritis (RA), and it results in trans-endothelial cell migration and synovial fibroblast proliferation, leading to joint destruction. In RA, the synovial microvasculature is highly dysregulated, resulting in inefficient oxygen perfusion to the synovium, which, along with the high metabolic demands of activated immune and stromal cells, leads to a profoundly hypoxic microenvironment. In inflamed joints, infiltrating immune cells and synovial resident cells have great requirements for energy and nutrients, and they adapt their metabolic profiles to generate sufficient energy to support their highly activated inflammatory states. This shift in metabolic capacity of synovial cells enables them to produce the essential building blocks to support their proliferation, activation and invasiveness. Furthermore, it results in the accumulation of metabolic intermediates and alteration of redox-sensitive pathways, affecting signalling pathways that further potentiate the inflammatory response. Importantly, the inflamed synovium is a multicellular tissue, with cells differing in their metabolic requirements depending on complex cell-cell interactions, nutrient supply, metabolic intermediates and transcriptional regulation. Therefore, understanding the complex interplay between metabolic and inflammatory pathways in synovial cells in RA will provide insight into the underlying mechanisms of disease pathogenesis.
    DOI:  https://doi.org/10.1038/s41584-022-00771-x
  21. Eur J Cell Biol. 2022 Mar 23. pii: S0171-9335(22)00022-X. [Epub ahead of print]101(2): 151219
    Cancer's camouflage: Microvesicle shedding from cholesterol-rich tumor plasma membranes might blindfold first-responder immunosurveillance strategies.
    Peter S Coleman, Risa A Parlo.
      Intermediary metabolism of tumors is characterized, in part, by a dysregulation of the cholesterol biosynthesis pathway at its rate-controlling enzyme providing the molecular basis for tumor membranes (mitochondria, plasma membrane) to become enriched with cholesterol (Bloch, 1965; Feo et al., 1975; Brown and Goldstein, 1980; Goldstein and Brown, 1990). Cholesterol enriched tumor mitochondria manifest preferential citrate export, thereby providing a continuous supply of substrate precursor for the tumor's dysregulated cholesterogenesis via a "truncated" Krebs/TCA cycle (Kaplan et al., 1986; Coleman et al., 1997). Proliferating tumors shed elevated amounts of plasma membrane-derived extracellular vesicles (pmEV) compared with normal tissues (van Blitterswijk et al., 1979; Black, 1980). Coordination of these metabolic phenomena in tumors supports the enhanced intercalation of cholesterol within the plasma membrane lipid bilayer's cytoplasmic face, the promotion of outward protrusions from the plasma membrane, and the evolution of cholesterol enriched pmEV. The pmEV shed by tumors possess elevated cholesterol and concentrated cell surface antigen clusters found on the tumor cells themselves (Kim et al., 2002). Upon exfoliation, saturation of the extracellular milieu with tumor-derived pmEV could allow early onset mammalian immune surveillance mechanisms to become "blind" to an evolving cancer and lose their ability to detect and initiate strategies to destroy the cancer. However, a molecular mechanism is lacking that would help explain how cholesterol enrichment of the pmEV inner lipid bilayer might allow the tumor cell to evade the host immune system. We offer a hypothesis, endorsed by published mathematical modeling of biomembrane structure as well as by decades of in vivo data with diverse cancers, that a cholesterol enriched inner bilayer leaflet, coupled with a logarithmic expansion in surface area of shed tumor pmEV load relative to its derivative cancer cell, conspire to force exposure of otherwise unfamiliar membrane integral protein domains as antigenic epitopes to the host's circulating immune surveillance system, allowing the tumor cells to evade destruction. We provide elementary numerical estimations comparing the amount of pmEV shed from tumor versus normal cells.
    Keywords:  Decoying immunosurveillance; Tumor cholesterogenesis; Vesicle exfoliation
    DOI:  https://doi.org/10.1016/j.ejcb.2022.151219
  22. Immunometabolism. 2022 ;pii: e220009. [Epub ahead of print]4(2):
    Mitochondria Transfer to CD4+ T Cells May Alleviate Rheumatoid Arthritis by Suppressing Pro-Inflammatory Cytokine Production.
    Rocky Giwa, Jonathan R Brestoff.
      CD4+ T cells contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). These cells infiltrate the joints of RA patients and produce cytokines, including Tumor necrosis factor (TNF)-α, that drive joint inflammation and bone destruction. Although biologic therapeutics targeting T cells and TNF-α have benefited patients suffering from RA, some patients are refractory to these therapies, develop antibodies that neutralize these biologics, or develop undesirable side effects. Recent studies indicate that CD4+ T cell cytokine production is regulated in part by specific metabolic modules, suggesting that immunometabolic pathways could represent a novel therapeutic strategy for T cell-mediated diseases such as RA. Wu et al. (2021) demonstrate that mitochondrial function is impaired in CD4+ T cells from RA patients, leading to reduced levels of various citric acid cycle metabolites (e.g., aspartate) that regulate TNF-α production. Treatment of RA-associated T cells with purified mitochondria was sufficient to restore these metabolic defects, limit production of numerous pro-inflammatory cytokines such as TNF-α and IL-17A, and reduce the development of RA-like disease in a humanized mouse model. These data suggest that T cells can be metabolically "re-engineered" ex vivo with exogenous mitochondria and that this mitochondria transfer approach confers anti-inflammatory properties that may reduce disease severity in RA and possibly other rheumatologic diseases. Increasing our understanding of how intercellular mitochondria transfer occurs may identify novel biological pathways that can be targeted therapeutically or harnessed to support cell engineering.
    Keywords:  CD4 T cells; mitochondria; rheumatoid arthritis
    DOI:  https://doi.org/10.20900/immunometab20220009
  23. Sports Med. 2022 Apr 23.
    Does a Hypertrophying Muscle Fibre Reprogramme its Metabolism Similar to a Cancer Cell?
    Henning Wackerhage, Ivan J Vechetti, Philipp Baumert, Sebastian Gehlert, Lore Becker, Richard T Jaspers, Martin Hrabě de Angelis.
      In 1924, Otto Warburg asked "How does the metabolism of a growing tissue differ from that of a non-growing tissue?" Currently, we know that proliferating healthy and cancer cells reprogramme their metabolism. This typically includes increased glucose uptake, glycolytic flux and lactate synthesis. A key function of this reprogramming is to channel glycolytic intermediates and other metabolites into anabolic reactions such as nucleotide-RNA/DNA synthesis, amino acid-protein synthesis and the synthesis of, for example, acetyl and methyl groups for epigenetic modification. In this review, we discuss evidence that a hypertrophying muscle similarly takes up more glucose and reprogrammes its metabolism to channel energy metabolites into anabolic pathways. We specifically discuss the functions of the cancer-associated enzymes phosphoglycerate dehydrogenase and pyruvate kinase muscle 2 in skeletal muscle. In addition, we ask whether increased glucose uptake by a hypertrophying muscle explains why muscularity is often negatively associated with type 2 diabetes mellitus and obesity.
    DOI:  https://doi.org/10.1007/s40279-022-01676-1
  24. Expert Opin Ther Pat. 2022 Apr 18.
    ATP-citrate lyase (ACLY) inhibitors as therapeutic agents: a patenting perspective.
    Carlotta Granchi.
       INTRODUCTION: ATP citrate lyase (ACLY) is a key enzyme in cellular metabolism, being the main source of acetyl-Coenzyme A, an important precursor for fatty acid, cholesterol and isoprenoid biosynthesis, and it is also involved in protein acetylation. Its expression changes are related to hyperlipidemia and cardiovascular diseases. Other studies have shown that ACLY is closely related to the occurrence of cancer: the increase in lipid synthesis provides the necessary building blocks for cell growth and division. Therefore, finding effective ACLY inhibitors has very important application prospects for lipid-related pathologies and cancer.
    AREAS COVERED: : This review covers patents concerning ACLY inhibitors and alternative strategies to modulate ACLY activity, with their potential therapeutic applications.
    EXPERT OPINION: In recent years ACLY as a drug target has become a hot spot in the research of innovative drugs for disorders of glucose and lipid metabolism. Many types of small-molecule ACLY inhibitors have been discovered, but few ACLY inhibitors proved to be highly effective in vitro and in vivo, since their main limitations were low cell penetration and low affinity to ACLY. The search for new effective ACLY inhibitors is of great significance and has broad application prospects for the treatment of hyperlipidemia and cancer.
    Keywords:  ATP citrate lyase; cancer; inhibitors; lipid metabolism
    DOI:  https://doi.org/10.1080/13543776.2022.2067478
  25. Cell Rep. 2022 Apr 19. pii: S2211-1247(22)00480-6. [Epub ahead of print]39(3): 110719
    Type I interferon antagonism of the JMJD3-IRF4 pathway modulates macrophage activation and polarization.
    Kevin Ming-Chin Lee, Adrian A Achuthan, David P De Souza, Tanya J Lupancu, Katrina J Binger, Man K S Lee, Yangsong Xu, Malcolm J McConville, Nicole A de Weerd, Dragana Dragoljevic, Paul J Hertzog, Andrew J Murphy, John A Hamilton, Andrew J Fleetwood.
      Metabolic adaptations can directly influence the scope and scale of macrophage activation and polarization. Here we explore the impact of type I interferon (IFNβ) on macrophage metabolism and its broader impact on cytokine signaling pathways. We find that IFNβ simultaneously increased the expression of immune-responsive gene 1 and itaconate production while inhibiting isocitrate dehydrogenase activity and restricting α-ketoglutarate accumulation. IFNβ also increased the flux of glutamine-derived carbon into the tricarboxylic acid cycle to boost succinate levels. Combined, we identify that IFNβ controls the cellular α-ketoglutarate/succinate ratio. We show that by lowering the α-ketoglutarate/succinate ratio, IFNβ potently blocks the JMJD3-IRF4-dependent pathway in GM-CSF and IL-4 activated macrophages. The suppressive effects of IFNβ on JMJD3-IRF4-dependent responses, including M2 polarization and GM-CSF-induced inflammatory pain, were reversed by supplementation with α-ketoglutarate. These results reveal that IFNβ modulates macrophage activation and polarization through control of the cellular α-ketoglutarate/succinate ratio.
    Keywords:  CP: Immunology; CP: Metabolism; granulocyte macrophage colony-stimulating factor; interleukin-4; isocitrate dehydrogenase; macrophage polarization; metabolism; succinate; type I interferon; α-ketoglutarate
    DOI:  https://doi.org/10.1016/j.celrep.2022.110719
  26. Oncogene. 2022 Apr 16.
    Targeting oncometabolism to maximize immunotherapy in malignant brain tumors.
    Joshua D Bernstock, Kyung-Don Kang, Neil V Klinger, Hannah E Olsen, Sam Gary, Stacie K Totsch, Gelare Ghajar-Rahimi, David Segar, Eric M Thompson, Victor Darley-Usmar, Bryan T Mott, Luca Peruzzotti-Jametti, Gregory K Friedman.
      Brain tumors result in significant morbidity and mortality in both children and adults. Recent data indicate that immunotherapies may offer a survival benefit after standard of care has failed for malignant brain tumors. Modest results from several late phase clinical trials, however, underscore the need for more refined, comprehensive strategies that incorporate new mechanistic and pharmacologic knowledge. Recently, oncometabolism has emerged as an adjunct modality for combinatorial treatment approaches necessitated by the aggressive, refractory nature of high-grade glioma and other progressive malignant brain tumors. Manipulation of metabolic processes in cancer and immune cells that comprise the tumor microenvironment through controlled targeting of oncogenic pathways may be utilized to maximize the efficacy of immunotherapy and improve patient outcomes. Herein, we summarize preclinical and early phase clinical trial research of oncometabolism-based therapeutics that may augment immunotherapy by exploiting the biochemical and genetic underpinnings of brain tumors. We also examine metabolic pathways related to immune cells that target tumor cells, termed "tumor immunometabolism". Specifically, we focus on glycolysis and altered glucose metabolism, including glucose transporters, hexokinase, pyruvate dehydrogenase, and lactate dehydrogenase, glutamine, and we discuss targeting arginase, adenosine, and indoleamine 2,3-dioxygenase, and toll-like receptors. Lastly, we summarize future directions targeting metabolism in combination with emerging therapies such as oncolytic virotherapy, vaccines, and chimeric antigen receptor T cells.
    DOI:  https://doi.org/10.1038/s41388-022-02312-y
  27. EMBO J. 2022 Apr 19. e111189
    The blood-brain barrier-a metabolic ecosystem.
    Marco Castro, Michael Potente.
      A functional blood-brain barrier relies on a tightly controlled interplay between endothelial cells, pericytes, and astrocytes, which together form the neurovascular unit. Recent work by Lee et al (2022) discovers endothelial cell-derived lactate as a crucial metabolic fuel for brain pericytes, revealing a new way of CNS vascular communication that links nutrient metabolism to blood-brain barrier function.
    DOI:  https://doi.org/10.15252/embj.2022111189
  28. Nutrients. 2022 Apr 18. pii: 1676. [Epub ahead of print]14(8):
    iTRAQ-Based Quantitative Proteomics Reveals the Energy Metabolism Alterations Induced by Chlorogenic Acid in HepG2 Cells.
    Shoko Takahashi, Kenji Saito, Xuguang Li, Huijuan Jia, Hisanori Kato.
      Epidemiological studies have suggested that coffee consumption is associated with a decrease in the risk of developing obesity and diabetes; however, the detailed mechanisms underlying these effects of coffee consumption remain poorly understood. In this study, we examined the effects of chlorogenic acid on energy metabolism in vitro. Hepatocellular carcinoma G2 (HepG2) cells were cultured in a medium containing chlorogenic acid. Chlorogenic acid increased the activity of mitochondrial enzymes, including citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase (MDH), which are involved in the tricarboxylic acid (TCA) cycle. Proteome analysis using the isobaric tags for the relative and absolute quantitation (iTRAQ) method revealed the upregulation of proteins involved in the glycolytic system, electron transport system, and ATP synthesis in mitochondria. Therefore, we propose a notable mechanism whereby chlorogenic acid enhances energy metabolism, including the TCA cycle, glycolytic system, electron transport, and ATP synthesis. This mechanism provides important insights into understanding the beneficial effects of coffee consumption.
    Keywords:  HepG2 cells; chlorogenic acid; coffee; energy metabolism; lipid metabolism; proteomics
    DOI:  https://doi.org/10.3390/nu14081676
  29. EMBO J. 2022 Apr 22. e111238
    The goodies of chelated fat: iron-regulated lipid droplet biogenesis precedes and preserves mitophagy.
    Marc Liesa.
      The role of iron-regulated mitophagy in lipid metabolism is unclear. Recent work by Long, Sanchez-Martinez et al (2022) shows that iron chelation induces a primary change in lipid metabolism that promotes fat accumulation and precedes mitophagy.
    DOI:  https://doi.org/10.15252/embj.2022111238
  30. Antioxidants (Basel). 2022 Mar 31. pii: 683. [Epub ahead of print]11(4):
    The BCAT1 CXXC Motif Provides Protection against ROS in Acute Myeloid Leukaemia Cells.
    James Hillier, Gemma J Allcott, Laura A Guest, Wayne Heaselgrave, Alex Tonks, Myra E Conway, Amy L Cherry, Steven J Coles.
      The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a 'redox-switch' in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise H2O2 in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, TrxRD1. Our data show that the expression of TrxRD1 was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia.
    Keywords:  AML; BCAT1; CXXC-motif; ROS; antioxidant; cysteine; leukaemia; myeloid
    DOI:  https://doi.org/10.3390/antiox11040683
  31. Toxins (Basel). 2022 Apr 07. pii: 263. [Epub ahead of print]14(4):
    Methylglyoxal Induces Inflammation, Metabolic Modulation and Oxidative Stress in Myoblast Cells.
    Sota Todoriki, Yui Hosoda, Tae Yamamoto, Mayu Watanabe, Akiyo Sekimoto, Hiroshi Sato, Takefumi Mori, Mariko Miyazaki, Nobuyuki Takahashi, Emiko Sato.
      Uremic sarcopenia is a serious clinical problem associated with physical disability and increased morbidity and mortality. Methylglyoxal (MG) is a highly reactive, dicarbonyl uremic toxin that accumulates in the circulatory system in patients with chronic kidney disease (CKD) and is related to the pathology of uremic sarcopenia. The pathophysiology of uremic sarcopenia is multifactorial; however, the details remain unknown. We investigated the mechanisms of MG-induced muscle atrophy using mouse myoblast C2C12 cells, focusing on intracellular metabolism and mitochondrial injury. We found that one of the causative pathological mechanisms of uremic sarcopenia is metabolic flow change to fatty acid synthesis with MG-induced ATP shortage in myoblasts. Evaluation of cell viability revealed that MG showed toxic effects only in myoblast cells, but not in myotube cells. Expression of mRNA or protein analysis revealed that MG induces muscle atrophy, inflammation, fibrosis, and oxidative stress in myoblast cells. Target metabolomics revealed that MG induces metabolic alterations, such as a reduction in tricarboxylic acid cycle metabolites. In addition, MG induces mitochondrial morphological abnormalities in myoblasts. These changes resulted in the reduction of ATP derived from the mitochondria of myoblast cells. Our results indicate that MG is a pathogenic factor in sarcopenia in CKD.
    Keywords:  chronic kidney disease; metabolic alteration; methylglyoxal; myoblast cell; sarcopenia
    DOI:  https://doi.org/10.3390/toxins14040263
  32. Front Immunol. 2022 ;13 873834
    Impact of Drp1-Mediated Mitochondrial Dynamics on T Cell Immune Modulation.
    Jun Song, Xiaofang Yi, Ruolin Gao, Li Sun, Zhixuan Wu, Shuling Zhang, Letian Huang, Chengbo Han, Jietao Ma.
      In recent years, various breakthroughs have been made in tumor immunotherapy that have contributed to prolonging the survival of tumor patients. However, only a subset of patients respond to immunotherapy, which limits its use. One reason for this is that the tumor microenvironment (TME) hinders the migration and infiltration of T cells and affects their continuous functioning, resulting in an exhausted phenotype. Therefore, clarifying the mechanism by which T cells become exhausted is of significance for improving the efficacy of immunotherapy. Several recent studies have shown that mitochondrial dynamics play an important role in the immune surveillance function of T cells. Dynamin-related protein 1 (Drp1) is a key protein that mediates mitochondrial fission and maintains the mitochondrial dynamic network. Drp1 regulates various activities of T cells in vivo by mediating the activation of a series of pathways. In addition, abnormal mitochondrial dynamics were observed in exhausted T cells in the TME. As a potential target for immunotherapy, in this review, we describe in detail how Drp1 regulates various physiological functions of T cells and induces changes in mitochondrial dynamics in the TME, providing a theoretical basis for further research.
    Keywords:  T cell exhaustion; dynamin-related protein 1; immunotherapy; mitochondrial dynamics; tumor microenvironment
    DOI:  https://doi.org/10.3389/fimmu.2022.873834
  33. Int J Mol Sci. 2022 Apr 15. pii: 4371. [Epub ahead of print]23(8):
    CoA in Health and Disease.
    Pawel Dobrzyn.
      Coenzyme A (CoA) and its thioester derivatives are crucial components of numerous biosynthetic and degradative pathways of the cellular metabolism (including fatty acid synthesis and oxidation, the Krebs cycle, ketogenesis, cholesterol and acetylcholine biosynthesis, amino acid degradation, and neurotransmitter biosynthesis), post-translational modifications of proteins, and the regulation of gene expression [...].
    DOI:  https://doi.org/10.3390/ijms23084371
  34. Int J Mol Sci. 2022 Apr 11. pii: 4200. [Epub ahead of print]23(8):
    Slc38a9 Deficiency Induces Apoptosis and Metabolic Dysregulation and Leads to Premature Death in Zebrafish.
    Xiya Wu, Jianyang Chen, Chengdong Liu, Xuan Wang, Huihui Zhou, Kangsen Mai, Gen He.
      Eukaryotic cells control nutritional homeostasis and determine cell metabolic fate through a series of nutrient transporters and metabolic regulation pathways. Lysosomal localized amino acid transporter member 9 of the solute carrier family 38 (SLC38A9) regulates essential amino acids' efflux from lysosomes in an arginine-regulated fashion. To better understand the physiological role of SLC38A9, we first described the spatiotemporal expression pattern of the slc38a9 gene in zebrafish. A quarter of slc38a9-/- mutant embryos developed pericardial edema and died prematurely, while the remaining mutants were viable and grew normally. By profiling the transcriptome of the abnormally developed embryos using RNA-seq, we identified increased apoptosis, dysregulated amino acid metabolism, and glycolysis/gluconeogenesis disorders that occurred in slc38a9-/- mutant fish. slc38a9 deficiency increased whole-body free amino acid and lactate levels but reduced glucose and pyruvate levels. The change of glycolysis-related metabolites in viable slc38a9-/- mutant fish was ameliorated. Moreover, loss of slc38a9 resulted in a significant reduction in hypoxia-inducible gene expression and hypoxia-inducible factor 1-alpha (Hif1α) protein levels. These results improved our understanding of the physiological functions of SLC38A9 and revealed its indispensable role in embryonic development, metabolic regulation, and stress adaption.
    Keywords:  SLC38A9; amino acid homeostasis; apoptosis; glycolysis; hypoxia
    DOI:  https://doi.org/10.3390/ijms23084200
  35. Metabolites. 2022 Apr 14. pii: 351. [Epub ahead of print]12(4):
    Untargeted Metabolomics of Slc13a5 Deficiency Reveal Critical Liver-Brain Axis for Lipid Homeostasis.
    Sofia Milosavljevic, Kevin E Glinton, Xiqi Li, Cláudia Medeiros, Patrick Gillespie, John R Seavitt, Brett H Graham, Sarah H Elsea.
      Though biallelic variants in SLC13A5 are known to cause severe encephalopathy, the mechanism of this disease is poorly understood. SLC13A5 protein deficiency reduces citrate transport into the cell. Downstream abnormalities in fatty acid synthesis and energy generation have been described, though biochemical signs of these perturbations are inconsistent across SLC13A5 deficiency patients. To investigate SLC13A5-related disorders, we performed untargeted metabolic analyses on the liver, brain, and serum from a Slc13a5-deficient mouse model. Metabolomic data were analyzed using the connect-the-dots (CTD) methodology and were compared to plasma and CSF metabolomics from SLC13A5-deficient patients. Mice homozygous for the Slc13a5tm1b/tm1b null allele had perturbations in fatty acids, bile acids, and energy metabolites in all tissues examined. Further analyses demonstrated that for several of these molecules, the ratio of their relative tissue concentrations differed widely in the knockout mouse, suggesting that deficiency of Slc13a5 impacts the biosynthesis and flux of metabolites between tissues. Similar findings were observed in patient biofluids, indicating altered transport and/or flux of molecules involved in energy, fatty acid, nucleotide, and bile acid metabolism. Deficiency of SLC13A5 likely causes a broader state of metabolic dysregulation than previously recognized, particularly regarding lipid synthesis, storage, and metabolism, supporting SLC13A5 deficiency as a lipid disorder.
    Keywords:  SLC13A5; SLC13A5 deficiency; bile acid metabolism; citrate transport; lipid synthesis; lipid utilization; liver-brain axis; untargeted metabolomics
    DOI:  https://doi.org/10.3390/metabo12040351
  36. Front Immunol. 2022 ;13 843515
    Prognosis and Dissection of Immunosuppressive Microenvironment in Breast Cancer Based on Fatty Acid Metabolism-Related Signature.
    Yuhui Tang, Wenwen Tian, Jindong Xie, Yutian Zou, Zehao Wang, Ning Li, Yan Zeng, Linyu Wu, Yue Zhang, Song Wu, Xiaoming Xie, Lu Yang.
      Fatty acid metabolism has been deciphered to augment tumorigenesis and disease progression in addition to therapy resistance via strengthened lipid synthesis, storage, and catabolism. Breast cancer is strongly associated with the biological function of fatty acid metabolism owing to the abundant presence of adipocytes in breast tissue. It has been unraveled that tumor cells exhibit considerable plasticity based on fatty acid metabolism, responding to extra-tumoral and a range of metabolic signals, in which tumor microenvironment plays a pivotal role. However, the prognostic significance of fatty acid metabolism in breast cancer remains to be further investigated. Alongside these insights, we retrieved 269 reliable fatty acid metabolism-related genes (FMGs) and identified the landscape of copy number variations and expression level among those genes. Additionally, 11 overall survival-related FMGs were clarified by univariate Cox hazards regression analysis in The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) databases. Subsequently, a prognostic signature based on 6 overall survival (OS)-related FMGs was generated using Lasso Cox hazards regression analysis in TCGA dataset and was validated in two external cohorts. The correlation between the signature and several essential clinical parameters, including T, N, and PAM50 subtypes, was unveiled by comparing the accumulating signature value in various degrees. Furthermore, an optimal nomogram incorporating the signature, age, and American Joint Committee on Cancer (AJCC) stage was constructed, and the discrimination was verified by C-index, the calibration curve, and the decision curve analysis. The underlying implications for immune checkpoints inhibitors, the landscape of tumor immune microenvironment, and the predictive significance in therapy resistance to diverse strategies were depicted ultimately. In conclusion, our findings indicate the potential prognostic connotation of fatty acid metabolism in breast cancer, supporting novel insights into breast cancer patients' prognosis and administrating effective immunotherapy.
    Keywords:  breast cancer; fatty acid metabolism; immunotherapy; prognostic signature; therapy resistance; tumor immune microenvironment
    DOI:  https://doi.org/10.3389/fimmu.2022.843515
  37. Biomolecules. 2022 Apr 03. pii: 541. [Epub ahead of print]12(4):
    Dimethyloxalylglycine (DMOG), a Hypoxia Mimetic Agent, Does Not Replicate a Rat Pheochromocytoma (PC12) Cell Biological Response to Reduced Oxygen Culture.
    RuoLi Chen, Mohammad Alkataan Ahmed, Nicholas Robert Forsyth.
      Cells respond to reduced oxygen availability predominately by activation of the hypoxia-inducible factor (HIF) pathway. HIF activation upregulates hundreds of genes that help cells survive in the reduced oxygen environment. The aim of this study is to determine whether chemical-induced HIF accumulation mimics all aspects of the hypoxic response of cells. We compared the effects of dimethyloxalylglycine (DMOG) (a HIF stabiliser) on PC12 cells cultured in air oxygen (20.9% O2, AO) with those cultured in either intermittent 20.9% O2 to 2% O2 (IH) or constant 2% O2 (CN). Cell viability, cell cycle, HIF accumulation, reactive oxygen species (ROS) formation, mitochondrial function and differentiation were used to characterise the PC12 cells and evaluate the impact of DMOG. IH and CN culture reduced the increase in cell numbers after 72 and 96 h and MTT activity after 48 h compared to AO culture. Further, DMOG supplementation in AO induced a dose-dependent reduction in the increase in PC12 cell numbers and MTT activity. IH-cultured PC12 cells displayed increased and sustained HIF-1 expression over 96 h. This was accompanied by increased ROS and mitochondrial burden. PC12 cells in CN displayed little changes in HIF-1 expression or ROS levels. DMOG (0.1 mM) supplementation resulted in an IH-like HIF-1 profile. The mitochondrial burden and action potential of DMOG-supplemented PC12 cells did not mirror those seen in other conditions. DMOG significantly increased S phase cell populations after 72 and 96 h. No significant effect on PC12 cell differentiation was noted with IH and CN culture without induction by nerve growth factor (NGF), while DMOG significantly increased PC12 cell differentiation with and without NGF. In conclusion, DMOG and reduced oxygen levels stabilise HIF and affect mitochondrial activity and cell behaviour. However, DMOG does not provide an accurate replication of the reduced oxygen environments.
    Keywords:  HIF; PC12 cells; cell cycle; differentiation; hypoxia; mitochondria; proliferation
    DOI:  https://doi.org/10.3390/biom12040541
  38. Metallomics. 2022 Apr 20. pii: mfac027. [Epub ahead of print]
    Iron plays a role in sulfasalazine-induced ferroptosis with autophagic flux blockage in K7M2 osteosarcoma cells.
    Junyu Liu, Chenge Lou, Chenxiao Zhen, Yijia Wang, Peng Shang, Huanhuan Lv.
      Osteosarcoma is the most common primary bone malignancy in children and young adults with very poor prognosis. It is of great importance to develop targeted therapeutic strategies for osteosarcoma. Sulfasalazine (SAS) is an FDA-approved drug for the treatment of Crohn disease, rheumatoid arthritis and inflammatory bowel disease. It acts as an inhibitor of cystine/glutamate system which is important for cellular GSH synthesis and maintenance of GPx4 activity. Nowadays, SAS has been repurposed as an antitumor drug for inducing ferroptosis in cancers. This study aimed to uncover the role of iron in SAS-induced ferroptotic cell death in K7M2 osteosarcoma cells. Herein, SAS led to an iron-dependent cell death mode in K7M2 cells accompanied with decreased antioxidant defense and increased production of cytosolic and lipid ROS. Results also showed that iron supplement with FAC or FAS exacerbated the declined cell viability of SAS-treated K7M2 cells while in the case of iron depletion, it weakened such suppression. Furthermore, iron promoted SAS-induced alterations on cell cycle, cytoskeleton, mitochondria morphology and function, and redox system. Iron also induced the dysfunction of autophagic activity in SAS-treated K7M2 cells. In conclusion, our study uncovered the essential role of iron in SAS's effects on K7M2 cells and provided the potential combined therapy of inhibition on antioxidant defense and increase on oxidative potential which further disturbed redox status in tumor cells.
    Keywords:  Osteosarcoma; autophagy; ferroptosis; iron metabolism; sulfasalazine
    DOI:  https://doi.org/10.1093/mtomcs/mfac027
  39. Cell Death Dis. 2022 Apr 20. 13(4): 384
    Mitochondrial oxidative phosphorylation is dispensable for survival of CD34+ chronic myeloid leukemia stem and progenitor cells.
    Jin-Song Yan, Meng-Ying Yang, Xue-Hong Zhang, Chen-Hui Luo, Cheng-Kan Du, Yue Jiang, Xuan-Jia Dong, Zhang-Man Wang, Li-Xue Yang, Yi-Dong Li, Li Xia, Ying Lu.
      Chronic myeloid leukemia (CML) are initiated and sustained by self-renewing malignant CD34+ stem cells. Extensive efforts have been made to reveal the metabolic signature of the leukemia stem/progenitor cells in genomic, transcriptomic, and metabolomic studies. However, very little proteomic investigation has been conducted and the mechanism regarding at what level the metabolic program was rewired remains poorly understood. Here, using label-free quantitative proteomic profiling, we compared the signature of CD34+ stem/progenitor cells collected from CML individuals with that of healthy donors and observed significant changes in the abundance of enzymes associated with aerobic central carbonate metabolic pathways. Specifically, CML stem/progenitor cells expressed increased tricarboxylic acid cycle (TCA) with decreased glycolytic proteins, accompanying by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis activity. Administration of the well-known OXPHOS inhibitor metformin eradicated CML stem/progenitor cells and re-sensitized CD34+ CML cells to imatinib in vitro and in patient-derived tumor xenograft murine model. However, different from normal CD34+ cells, the abundance and activity of OXPHOS protein were both unexpectedly elevated with endoplasmic reticulum stress induced by metformin in CML CD34+ cells. The four major aberrantly expressed protein sets, in contrast, were downregulated by metformin in CML CD34+ cells. These data challenged the dependency of OXPHOS for CML CD34+ cell survival and underlined the novel mechanism of metformin. More importantly, it suggested a strong rationale for the use of tyrosine kinase inhibitors in combination with metformin in treating CML.
    DOI:  https://doi.org/10.1038/s41419-022-04842-5
  40. Front Immunol. 2022 ;13 802336
    Chronically Elevated O-GlcNAcylation Limits Nitric Oxide Production and Deregulates Specific Pro-Inflammatory Cytokines.
    Lara K Abramowitz, John A Hanover.
      Inflammation is the immune response to harmful stimuli, including pathogens, damaged cells and toxic compounds. However, uncontrolled inflammation can be detrimental and contribute to numerous chronic inflammatory diseases, such as insulin resistance. At the forefront of this response are macrophages, which sense the local microenvironment to respond with a pro-inflammatory, M1-polarized phenotype, or anti-inflammatory, M2-polarized phenotype. M1 macrophages upregulate factors like pro-inflammatory cytokines, to promote inflammatory signaling, and inducible Nitric Oxide Synthase (iNOS), to produce nitric oxide (NO). The generated NO can kill microorganisms to protect the body, but also signal back to the macrophage to limit pro-inflammatory cytokine production to maintain macrophage homeostasis. Thus, the tight regulation of iNOS in macrophages is critical for the immune system. Here, we investigated how elevation of the nutrient-sensitive posttranslational modification, O-GlcNAc, impacts M1 polarized macrophages. We identified increased gene expression of specific pro-inflammatory cytokines (Il-6, Il-1β, Il-12) when O-GlcNAc cycling was blocked. We further uncovered an interaction between O-GlcNAc and iNOS, with iNOS being an OGT target in vitro. Analysis of M1 polarized bone marrow derived macrophages deficient in the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), revealed decreased iNOS activity as measured by a reduction in NO release. Further, elevated O-GlcNAc acted on Il-6 expression through the iNOS pathway, as iNOS inhibitior L-NIL raised wildtype Il-6 expression similar to OGA deficient cells but had no further effect on the hyper-O-GlcNAcylated cells. Thus O-GlcNAc contributes to macrophage homeostasis through modulation of iNOS activity.
    Keywords:  O-GlcNAcase; O-GlcNAcylation; cytokines ; iNOS; inflammation; macrophages
    DOI:  https://doi.org/10.3389/fimmu.2022.802336
  41. Antioxidants (Basel). 2022 Apr 08. pii: 745. [Epub ahead of print]11(4):
    Carnitines as Mitochondrial Modulators of Oocyte and Embryo Bioenergetics.
    Martina Placidi, Giovanna Di Emidio, Ashraf Virmani, Angela D'Alfonso, Paolo Giovanni Artini, Anna Maria D'Alessandro, Carla Tatone.
      Recently, the importance of bioenergetics in the reproductive process has emerged. For its energetic demand, the oocyte relies on numerous mitochondria, whose activity increases during embryo development under a fine regulation to limit ROS production. Healthy oocyte mitochondria require a balance of pyruvate and fatty acid oxidation. Transport of activated fatty acids into mitochondria requires carnitine. In this regard, the interest in the role of carnitines as mitochondrial modulators in oocyte and embryos is increasing. Carnitine pool includes the un-esterified l-carnitine (LC) and carnitine esters, such as acetyl-l-carnitine (ALC) and propionyl-l-carnitine (PLC). In this review, carnitine medium supplementation for counteracting energetic and redox unbalance during in vitro culture and cryopreservation is reported. Although most studies have focused on LC, there is new evidence that the addition of ALC and/or PLC may boost LC effects. Pathways activated by carnitines include antiapoptotic, antiglycative, antioxidant, and antiinflammatory signaling. Nevertheless, the potential of carnitine to improve energetic metabolism and oocyte and embryo competence remains poorly investigated. The importance of carnitine as a mitochondrial modulator may suggest that this molecule may exert a beneficial role in ovarian disfunctions associated with metabolic and mitochondrial alterations, including PCOS and reproductive aging.
    Keywords:  acetyl-l-carnitine (ALC); beta-oxidation; bioenergetics; embryo; l-carnitine (LC); mitochondria; oocyte; propionyl-l-carnitine (PLC)
    DOI:  https://doi.org/10.3390/antiox11040745
  42. Transl Oncol. 2022 Apr 19. pii: S1936-5233(22)00090-0. [Epub ahead of print]21 101431
    Preclinical studies for improving radiosensitivity of non-small cell lung cancer cell lines by combining glutaminase inhibition and senolysis.
    Masaki Fujimoto, Ritsuko Higashiyama, Hironobu Yasui, Koya Yamashita, Osamu Inanami.
      Glutamine metabolism, known as glutaminolysis, is abnormally activated in many cancer cells with KRAS or BRAF mutations or active c-MYC. Glutaminolysis plays an important role in the proliferation of cancer cells with oncogenic mutations. In this study, we characterized radiation-induced cell death, which was enhanced by glutaminolysis inhibition in non-small cell lung cancer A549 and H460 cell lines with KRAS mutation. A clonogenic survival assay revealed that treatment with a glutaminase inhibitor, CB839, enhanced radiosensitivity. X-irradiation increased glutamate production, mitochondrial oxygen consumption, and ATP production, whereas CB839 treatment suppressed these effects. The data suggest that the enhancement of glutaminolysis-dependent energy metabolism for ATP production is important for survival after X-irradiation. Evaluation of the cell death phenotype revealed that glutaminolysis inhibitory treatment with CB839 or a low-glutamine medium significantly promoted the proliferation of β-galactosidase-positive and IL-6/IL-8 secretory cells among X-irradiated tumor cells, corresponding to an increase in the senescent cell population. Furthermore, treatment with ABT263, a Bcl-2 family inhibitor, transformed senescent cells into apoptotic cells. The findings suggest that combination treatment with a glutaminolysis inhibitor and a senolytic drug is useful for efficient radiotherapy.
    Keywords:  Apoptosis; Glutaminolysis; Radiation; Senescence; Senolytic drug
    DOI:  https://doi.org/10.1016/j.tranon.2022.101431
  43. Front Cell Dev Biol. 2022 ;10 854397
    SLC38A10 Regulate Glutamate Homeostasis and Modulate the AKT/TSC2/mTOR Pathway in Mouse Primary Cortex Cells.
    Rekha Tripathi, Tanya Aggarwal, Frida A Lindberg, Anna H Klemm, Robert Fredriksson.
      Glutamate acts as a critical regulator of neurotransmitter balance, recycling, synaptic function and homeostasis in the brain and glutamate transporters control glutamate levels in the brain. SLC38A10 is a member of the SLC38 family and regulates protein synthesis and cellular stress responses. Here, we uncover the role of SLC38A10 as a transceptor involved in glutamate-sensing signaling pathways that control both the glutamate homeostasis and mTOR-signaling. The culture of primary cortex cells from SLC38A10 knockout mice had increased intracellular glutamate. In addition, under nutrient starvation, KO cells had an impaired response in amino acid-dependent mTORC1 signaling. Combined studies from transcriptomics, protein arrays and metabolomics established that SLC38A10 is involved in mTOR signaling and that SLC38A10 deficient primary cortex cells have increased protein synthesis. Metabolomic data showed decreased cholesterol levels, changed fatty acid synthesis, and altered levels of fumaric acid, citrate, 2-oxoglutarate and succinate in the TCA cycle. These data suggests that SLC38A10 may act as a modulator of glutamate homeostasis, and mTOR-sensing and loss of this transceptor result in lower cholesterol, which could have implications in neurodegenerative diseases.
    Keywords:  SLC38A10; mTOR; primary cortex cultures; solute carriers transporter; starvation
    DOI:  https://doi.org/10.3389/fcell.2022.854397
  44. Toxicol Appl Pharmacol. 2022 Apr 14. pii: S0041-008X(22)00165-X. [Epub ahead of print]444 116020
    Titanium dioxide nanoparticles induced reactive oxygen species (ROS) related changes of metabolomics signatures in human normal bronchial epithelial (BEAS-2B) cells.
    Jiahe Zhang, Jiaqi Shi, Shuo Han, Pai Zheng, Zhangjian Chen, Guang Jia.
      Titanium dioxide often enters the respiratory tract in the form of nano-dust in occupational sites. Metabolomics may be a promising method for studying the toxicology of nano titanium dioxide. The intention of this study was to explore the possible impact of titanium dioxide nanoparticles (TiO2 NPs) on the metabolomics signatures of human normal bronchial epithelial (BEAS-2B) cells and to search for investigate the role of reactive oxygen species (ROS). In this study, BEAS-2B cells were treated by TiO2 NPs (0, 25, 50 and 100 μg/mL) for 48 h. The metabolites extracted from BEAS-2B cells were determined by untargeted metabolomics technique, and the differential metabolites and metabolic pathways were discovered by using multivariate analysis. Intracellular ROS was detected by DCFH-DA probe and flow cytometry method. Machine learning was used to explore the relationship between ROS and metabolomics changes. Totally, seventy-six differential metabolites and the steroid biosynthesis pathway of BEAS-2B cells were observed after treatment of TiO2 NPs. Lipid and lipid-like metabolites were the most significant classes among the metabolite products induced by TiO2 NPs. TiO2 NPs resulted in a dose-dependent increase in intracellular ROS levels, and correlated with most of the differential metabolites. In conclusion, TiO2 NPs increased the level of the oxidative stress, which could contribute to the altered signature represented by lipid metabolism in BEAS-2B cells.
    Keywords:  Lipid metabolism; Liquid chromatography; Metabolomics; Reactive oxygen pecies; Titanium dioxide nanoparticles
    DOI:  https://doi.org/10.1016/j.taap.2022.116020
  45. Stem Cells Transl Med. 2022 Apr 19. pii: szac018. [Epub ahead of print]
    Pancreatic Stellate Cells Prolong Ex Vivo Islet Viability and Function and Improve Engraftment.
    Pradyut K Paul, Rahul Das, Travis J Drow, Arnaldo H de Souza, Appakalai N Balamurugan, Dawn Belt Davis, Jacques Galipeau.
      Preserving islet health and function is critical during pretransplant culture to improve islet transplantation outcome and for ex vivo modeling of diabetes for pharmaceutical drug discovery. The limited islet engraftment potential is primarily attributable to loss of extracellular matrix (ECM) support and interaction. Multipotent cells with ECM depositing competency improve islet survival during short coculture period. However, role of pancreatic stellate cells (PSCs) and their ECM support in preserving ex vivo islet physiology remains largely unknown. Here, we report novel cytoprotective effects of culture-adapted porcine PSCs and role of their ECM-mediated intercellular communication on pig, mouse and human islets ex vivo. Using direct-contact coculture system, we demonstrate that porcine PSCs preserve and significantly prolong islet viability and function from 7 ± 3 days to more than 28 ± 5 (P < .001) days in vitro. These beneficial effects of PSCs on islet health are not species-specific. Using NSC47924 to specifically inhibit 37/67 kDa laminin receptor (LR), we identified that LR-mediated intercellular communication is essential for PSCs to protect functional viability of islets in vitro. Finally, our results demonstrate that PSC co-transplantation improved function and enhanced capacity of syngeneic islets to reverse hyperglycemia in mice with preexisiting diabetes. Cumulatively, our findings unveil novel effects of culture-adapted PSCs on islet health likely mirroring in vivo niche interaction. Furthermore, islet and PSC coculture may aid in development of ex vivo diabetes modeling and also suggests that a combined islet-PSC tissue engineered implant may significantly improve islet transplantation outcome.
    Keywords:  direct coculture; engraftment; extracellular matrix; islet transplantation; laminin receptor; pancreatic islets; pancreatic stellate cells; trophic effects
    DOI:  https://doi.org/10.1093/stcltm/szac018
  46. Metabolites. 2022 Apr 05. pii: 328. [Epub ahead of print]12(4):
    Branched-Chain Amino Acid Deprivation Decreases Lipid Oxidation and Lipogenesis in C2C12 Myotubes.
    Sira Karvinen, Vasco Fachada, Ulla-Maria Sahinaho, Satu Pekkala, Juulia H Lautaoja, Sakari Mäntyselkä, Perttu Permi, Juha J Hulmi, Mika Silvennoinen, Heikki Kainulainen.
      Impaired lipid metabolism is a common risk factor underlying several metabolic diseases such as metabolic syndrome and type 2 diabetes. Branched-chain amino acids (BCAAs) that include valine, leucine and isoleucine have been proven to share a role in lipid metabolism and hence in maintaining metabolic health. We have previously introduced a hypothesis suggesting that BCAA degradation mechanistically connects to lipid oxidation and storage in skeletal muscle. To test our hypothesis, the present study examined the effects of BCAA deprivation and supplementation on lipid oxidation, lipogenesis and lipid droplet characteristics in murine C2C12 myotubes. In addition, the role of myotube contractions on cell metabolism was studied by utilizing in vitro skeletal-muscle-specific exercise-like electrical pulse stimulation (EPS). Our results showed that the deprivation of BCAAs decreased both lipid oxidation and lipogenesis in C2C12 myotubes. BCAA deprivation further diminished the number of lipid droplets in the EPS-treated myotubes. EPS decreased lipid oxidation especially when combined with high BCAA supplementation. Similar to BCAA deprivation, high BCAA supplementation also decreased lipid oxidation. The present results highlight the role of an adequate level of BCAAs in healthy lipid metabolism.
    Keywords:  electrical pulse stimulation; in vitro exercise; metabolic health; nuclear magnetic resonance spectroscopy; protein supplementation; skeletal muscle
    DOI:  https://doi.org/10.3390/metabo12040328
  47. Biochem Biophys Res Commun. 2022 Apr 10. pii: S0006-291X(22)00558-7. [Epub ahead of print]609 183-188
    Pharmacological intervention of cholesterol sulfate-mediated T cell exclusion promotes antitumor immunity.
    Takaaki Tatsuguchi, Takehito Uruno, Yuki Sugiura, Kounosuke Oisaki, Daisuke Takaya, Daiji Sakata, Yoshihiro Izumi, Takaya Togo, Yuko Hattori, Kazufumi Kunimura, Tetsuya Sakurai, Teruki Honma, Takeshi Bamba, Masafumi Nakamura, Motomu Kanai, Makoto Suematsu, Yoshinori Fukui.
      Effective cancer immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute special microenvironments that exclude T cells and resist immunotherapy. Cholesterol sulfate (CS) is a product of sulfotransferase SULT2B1b and acts as an endogenous inhibitor of DOCK2, a Rac activator essential for migration and activation of lymphocytes. We have recently shown that cancer-derived CS prevents tumor infiltration by effector T cells. Therefore, SULT2B1b may be a therapeutic target to dampen CS-mediated immune evasion. Here, we identified 3β-hydroxy-5-cholenoic acid (3β-OH-5-Chln) as a cell-active inhibitor of SULT2B1b. 3β-OH-5-Chln inhibited the cholesterol sulfotransferase activity of SULT2B1b in vitro and suppressed CS production from cancer cells expressing SULT2B1b. In vivo administration of 3β-OH-5-Chln locally reduced CS level in murine CS-producing tumors and increased infiltration of CD8+ T cells. When combined with immune checkpoint blockade or antigen-specific T cell transfer, 3β-OH-5-Chln suppressed the growth of CS-producing tumors. These results demonstrate that pharmacological inhibition of SULT2B1b can promote antitumor immunity through suppressing CS-mediated T cell exclusion.
    Keywords:  Cancer immunotherapy; Cholesterol sulfate; DOCK2; Immune evasion; SULT2B1b; T cell exclusion
    DOI:  https://doi.org/10.1016/j.bbrc.2022.04.035
  48. Cell Death Dis. 2022 Apr 16. 13(4): 355
    Interaction between macrophages and ferroptosis.
    Yan Yang, Yu Wang, Lin Guo, Wen Gao, Ting-Li Tang, Miao Yan.
      Ferroptosis, a newly discovered iron-dependent cell death pathway, is characterized by lipid peroxidation and GSH depletion mediated by iron metabolism and is morphologically, biologically and genetically different from other programmed cell deaths. Besides, ferroptosis is usually found accompanied by inflammatory reactions. So far, it has been found participating in the development of many kinds of diseases. Macrophages are a group of immune cells that widely exist in our body for host defense and play an important role in tissue homeostasis by mediating inflammation and regulating iron, lipid and amino acid metabolisms through their unique functions like phagocytosis and efferocytosis, cytokines secretion and ROS production under different polarization. According to these common points in ferroptosis characteristics and macrophages functions, it's obvious that there must be relationship between macrophages and ferroptosis. Therefore, our review aims at revealing the interaction between macrophages and ferroptosis concerning three metabolisms and integrating the application of certain relationship in curing diseases, mostly cancer. Finally, we also provide inspirations for further studies in therapy for some diseases by targeting certain resident macrophages in distinct tissues to regulate ferroptosis. FACTS: Ferroptosis is considered as a newly discovered form characterized by its nonapoptotic and iron-dependent lipid hydroperoxide, concerning iron, lipid and amino acid metabolisms. Ferroptosis has been widely found playing a crucial part in various diseases, including hepatic diseases, neurological diseases, cancer, etc. Macrophages are phagocytic immune cells, widely existing and owning various functions such as phagocytosis and efferocytosis, cytokines secretion and ROS production. Macrophages are proved to participate in mediating metabolisms and initiating immune reactions to maintain balance in our body. Recent studies try to treat cancer by altering macrophages' polarization which damages tumor microenvironment and induces ferroptosis of cancer cells. OPEN QUESTIONS: How do macrophages regulate ferroptosis of other tissue cells specifically? Can we use the interaction between macrophages and ferroptosis in treating diseases other than cancer? What can we do to treat diseases related to ferroptosis by targeting macrophages? Is the use of the relationship between macrophages and ferroptosis more effective than other therapies when treating diseases?
    DOI:  https://doi.org/10.1038/s41419-022-04775-z
  49. J Biol Chem. 2022 Apr 19. pii: S0021-9258(22)00398-2. [Epub ahead of print] 101958
    Lysophosphatidylcholine acyltransferase 1 controls mitochondrial reactive oxygen species generation and survival of retinal photoreceptor cells.
    Katsuyuki Nagata, Daisuke Hishikawa, Hiroshi Sagara, Masamichi Saito, Sumiko Watanabe, Takao Shimizu, Hideo Shindou.
      Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production, but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species (ROS) levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases.
    Keywords:  apoptosis; membrane phospholipids; photoreceptor cells; retinal degeneration; saturated fatty acid
    DOI:  https://doi.org/10.1016/j.jbc.2022.101958
  50. Mol Oncol. 2022 Apr 22.
    Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK-STAT signaling in macrophages by perturbing lipid raft structures.
    Mohamad K Hammoud, Raimund Dietze, Jelena Pesek, Florian Finkernagel, Annika Unger, Tim Bieringer, Andrea Nist, Thorsten Stiewe, Aditya M Bhagwat, W Andreas Nockher, Silke Reinartz, Sabine Müller-Brüsselbach, Johannes Graumann, Rolf Müller.
      Survival of ovarian carcinoma is associated with the abundance of immunosuppressed CD163high CD206high tumor-associated macrophages (TAMs) and high levels of arachidonic acid (AA) in the tumor microenvironment. Here, we show that both associations are functionally linked. Transcriptional profiling revealed that high CD163 and CD206/MRC1 expression in TAMs is strongly associated with an inhibition of cytokine-triggered signaling, mirrored by an impaired transcriptional response to interferons and IL-6 in monocyte-derived macrophages by AA. This inhibition of pro-inflammatory signaling is caused by dysfunctions of the cognate receptors, indicated by the inhibition of JAK1, JAK2, STAT1 and STAT3 phosphorylation, and by the displacement of the interferon receptor IFNAR1, STAT1 and other immune-regulatory proteins from lipid rafts. AA exposure led to a dramatic accumulation of free AA in lipid rafts, which appears to be mechanistically crucial, as the inhibition of its incorporation into phospholipids did not affect the AA-mediated interference with STAT1 phosphorylation. Inhibition of interferon-triggered STAT1 phosphorylation by AA was reversed by water-soluble cholesterol, known to prevent the perturbation of lipid raft structure by AA. These findings suggest that the pharmacologic restoration of lipid raft functions in TAMs may contribute to the development new therapeutic approaches.
    Keywords:  STAT; arachidonic acid; interferon; lipid rafts; macrophage; ovarian cancer microenvironment
    DOI:  https://doi.org/10.1002/1878-0261.13221
  51. Metabolites. 2022 Apr 12. pii: 342. [Epub ahead of print]12(4):
    Effects of Arachidonic Acid and Its Metabolites on Functional Beta-Cell Mass.
    Karin J Bosma, Cecilia E Kaiser, Michelle E Kimple, Maureen Gannon.
      Arachidonic acid (AA) is a polyunsaturated 20-carbon fatty acid present in phospholipids in the plasma membrane. The three primary pathways by which AA is metabolized are mediated by cyclooxygenase (COX) enzymes, lipoxygenase (LOX) enzymes, and cytochrome P450 (CYP) enzymes. These three pathways produce eicosanoids, lipid signaling molecules that play roles in biological processes such as inflammation, pain, and immune function. Eicosanoids have been demonstrated to play a role in inflammatory, renal, and cardiovascular diseases as well type 1 and type 2 diabetes. Alterations in AA release or AA concentrations have been shown to affect insulin secretion from the pancreatic beta cell, leading to interest in the role of AA and its metabolites in the regulation of beta-cell function and maintenance of beta-cell mass. In this review, we discuss the metabolism of AA by COX, LOX, and CYP, the roles of these enzymes and their metabolites in beta-cell mass and function, and the possibility of targeting these pathways as novel therapies for treating diabetes.
    Keywords:  arachidonic acid; beta-cell mass; eicosanoids; prostaglandins
    DOI:  https://doi.org/10.3390/metabo12040342
  52. Int J Mol Sci. 2022 Apr 10. pii: 4181. [Epub ahead of print]23(8):
    COVID-19 and One-Carbon Metabolism.
    Joanna Perła-Kaján, Hieronim Jakubowski.
      Dysregulation of one-carbon metabolism affects a wide range of biological processes and is associated with a number of diseases, including cardiovascular disease, dementia, neural tube defects, and cancer. Accumulating evidence suggests that one-carbon metabolism plays an important role in COVID-19. The symptoms of long COVID-19 are similar to those presented by subjects suffering from vitamin B12 deficiency (pernicious anemia). The metabolism of a cell infected by the SARS-CoV-2 virus is reshaped to fulfill the need for massive viral RNA synthesis, which requires de novo purine biosynthesis involving folate and one-carbon metabolism. Many aspects of host sulfur amino acid metabolism, particularly glutathione metabolism underlying antioxidant defenses, are also taken over by the SARS-CoV-2 virus. The purpose of this review is to summarize recent findings related to one-carbon metabolism and sulfur metabolites in COVID-19 and discuss how they inform strategies to combat the disease.
    Keywords:  S-adenosylhomocysteine; S-adenosylmethionine; choline; cysteine; folate; glutathione; homocysteine; methionine; methionine sulfoxide; purine biosynthesis
    DOI:  https://doi.org/10.3390/ijms23084181
  53. Physiol Rep. 2022 Apr;10(8): e15252
    Modeling the measurement bias in interstitial glucose concentrations derived from microdialysis in skeletal muscle.
    Hugo Angleys, Leif Østergaard.
      Muscle tissue utilizes glucose as a fuel during exercise and stores glucose in form of glycogen during rest. The associated glucose transport includes delivery of glucose from blood plasma into the interstitial space and subsequent, GLUT-4 facilitated diffusion into muscle cells. The extent to which the vascular endothelium acts as a barrier to glucose transport, however, remains debated. While accurate measurements of interstitial glucose concentration (IGC) are key to resolve this debate, these are also challenging as removal of interstitial fluid may perturb glucose transport and therefore bias IGC measurements. We developed a three-compartment model to infer IGC in skeletal muscle from its local metabolism and blood flow. The model predicts that IGC remains within 5% of that of blood plasma during resting conditions but decreases more as metabolism increases. Next, we determined how microdialysis protocols affect IGC. Our model analysis suggests that microdialysis-based IGC measurements underestimate true values. Notably, reported increases in muscle capillary permeability surface area product (PS) to glucose under the condition of elevated metabolism may owe in part to such measurements bias. Our study demonstrates that microdialysis may be associated with significant measurement bias in the context of muscle IGC assessment. Reappraising literature data with this bias in mind, we find that muscle capillary endothelium may represent less of a barrier to glucose transport in muscle than previously believed. We discuss the impact of glucose removal on the microdialysis relative recovery and means of correcting microdialysis IGC values.
    Keywords:  biophysical modeling; endothelial glucose transport; interstitial glucose monitoring; microdialysis; muscle metabolism
    DOI:  https://doi.org/10.14814/phy2.15252
This page is being maintained by Thomas Krichel. It was last updated on 2024‒11‒22 at 4:20.