bims-mepmim Biomed News
on Metabolites in pathological microenvironments and immunometabolism
Issue of 2024–08–18
nineteen papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism
  1. ACLY and ACSS2 link nutrient-dependent chromatin accessibility to CD8 T cell effector responses.
  2. Broad-spectrum antibiotics disrupt homeostatic efferocytosis.
  3. LIFR regulates cholesterol-driven bidirectional hepatocyte-neutrophil cross-talk to promote liver regeneration.
  4. S-acylation of ATGL is required for lipid droplet homoeostasis in hepatocytes.
  5. Hypoxia-induced downregulation of PGK1 crotonylation promotes tumorigenesis by coordinating glycolysis and the TCA cycle.
  6. Defective mitochondrial COX1 translation due to loss of COX14 function triggers ROS-induced inflammation in mouse liver.
  7. Integration of epidemiological and blood biomarker analysis links haem iron intake to increased type 2 diabetes risk.
  8. OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response.
  9. Phosphorylation of Insig-2 mediates inhibition of fatty acid synthesis by polyunsaturated fatty acids.
  10. Macrophage-mediated myelin recycling fuels brain cancer malignancy.
  11. Intracellular pH differentially regulates transcription of metabolic and signaling pathways in normal epithelial cells.
  12. Differential utilization of vitamin B12-dependent and independent pathways for propionate metabolism across human cells.
  13. Protocol for monitoring fatty acid trafficking from lipid droplets to mitochondria in cultured cells.
  14. TET3-overexpressing macrophages promote endometriosis.
  15. The mitochondrial pyruvate carrier regulates adipose glucose partitioning in female mice.
  16. Phosphatidic acid is an endogenous negative regulator of PIEZO2 channels and mechanical sensitivity.
  17. Cullin 4B-RING E3 ligase negatively regulates the immunosuppressive capacity of mesenchymal stem cells by suppressing iNOS.
  18. Functional overlap of inborn errors of immunity and metabolism genes defines T cell metabolic vulnerabilities.
  19. Hydrogel crosslinking modulates macrophages, fibroblasts, and their communication, during wound healing.
  1. J Exp Med. 2024 Sep 02. pii: e20231820. [Epub ahead of print]221(9):
    ACLY and ACSS2 link nutrient-dependent chromatin accessibility to CD8 T cell effector responses.
    Irem Kaymak, McLane J Watson, Brandon M Oswald, Shixin Ma, Benjamin K Johnson, Lisa M DeCamp, Batsirai M Mabvakure, Katarzyna M Luda, Eric H Ma, Kin Lau, Zhen Fu, Brejnev Muhire, Susan M Kitchen-Goosen, Alexandra Vander Ark, Michael S Dahabieh, Bozena Samborska, Matthew Vos, Hui Shen, Zi Peng Fan, Thomas P Roddy, Gillian A Kingsbury, Cristovão M Sousa, Connie M Krawczyk, Kelsey S Williams, Ryan D Sheldon, Susan M Kaech, Dominic G Roy, Russell G Jones.
      Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.
    DOI:  https://doi.org/10.1084/jem.20231820
  2. Nat Metab. 2024 Aug 09.
    Broad-spectrum antibiotics disrupt homeostatic efferocytosis.
    Pedro H V Saavedra, Alissa J Trzeciak, Allie Lipshutz, Andrew W Daman, Anya J O'Neal, Zong-Lin Liu, Zhaoquan Wang, Jesús E Romero-Pichardo, Waleska Saitz Rojas, Giulia Zago, Marcel R M van den Brink, Steven Z Josefowicz, Christopher D Lucas, Christopher J Anderson, Alexander Y Rudensky, Justin S A Perry.
      The clearance of apoptotic cells, termed efferocytosis, is essential for tissue homeostasis and prevention of autoimmunity1. Although past studies have elucidated local molecular signals that regulate homeostatic efferocytosis in a tissue2,3, whether signals arising distally also regulate homeostatic efferocytosis remains elusive. Here, we show that large peritoneal macrophage (LPM) display impairs efferocytosis in broad-spectrum antibiotics (ABX)-treated, vancomycin-treated and germ-free mice in vivo, all of which have a depleted gut microbiota. Mechanistically, the microbiota-derived short-chain fatty acid butyrate directly boosts efferocytosis efficiency and capacity in mouse and human macrophages, and rescues ABX-induced LPM efferocytosis defects in vivo. Bulk messenger RNA sequencing of butyrate-treated macrophages in vitro and single-cell messenger RNA sequencing of LPMs isolated from ABX-treated and butyrate-rescued mice reveals regulation of efferocytosis-supportive transcriptional programmes. Specifically, we find that the efferocytosis receptor T cell immunoglobulin and mucin domain containing 4 (TIM-4, Timd4) is downregulated in LPMs of ABX-treated mice but rescued by oral butyrate. We show that TIM-4 is required for the butyrate-induced enhancement of LPM efferocytosis capacity and that LPM efferocytosis is impaired beyond withdrawal of ABX. ABX-treated mice exhibit significantly worse disease in a mouse model of lupus. Our results demonstrate that homeostatic efferocytosis relies on distal metabolic signals and suggest that defective homeostatic efferocytosis may explain the link between ABX use and inflammatory disease4-7.
    DOI:  https://doi.org/10.1038/s42255-024-01107-7
  3. Nat Metab. 2024 Aug 15.
    LIFR regulates cholesterol-driven bidirectional hepatocyte-neutrophil cross-talk to promote liver regeneration.
    Yalan Deng, Zilong Zhao, Marisela Sheldon, Yang Zhao, Hongqi Teng, Consuelo Martinez, Jie Zhang, Chunru Lin, Yutong Sun, Fan Yao, Michael A Curran, Hao Zhu, Li Ma.
      Liver regeneration is under metabolic and immune regulation. Despite increasing recognition of the involvement of neutrophils in regeneration, it is unclear how the liver signals to the bone marrow to release neutrophils after injury and how reparative neutrophils signal to hepatocytes to reenter the cell cycle. Here we report that loss of the liver tumour suppressor Lifr in mouse hepatocytes impairs, whereas overexpression of leukaemia inhibitory factor receptor (LIFR) promotes liver repair and regeneration after partial hepatectomy or toxic injury. In response to physical or chemical damage to the liver, LIFR from hepatocytes promotes the secretion of cholesterol and CXCL1 in a STAT3-dependent manner, leading to the efflux of bone marrow neutrophils to the circulation and damaged liver. Cholesterol, via its receptor ERRα, stimulates neutrophils to secrete hepatocyte growth factor to accelerate hepatocyte proliferation. Altogether, our findings reveal a LIFR-STAT3-CXCL1-CXCR2 axis and a LIFR-STAT3-cholesterol-ERRα-hepatocyte growth factor axis that form bidirectional hepatocyte-neutrophil cross-talk to repair and regenerate the liver.
    DOI:  https://doi.org/10.1038/s42255-024-01110-y
  4. Nat Metab. 2024 Aug 14.
    S-acylation of ATGL is required for lipid droplet homoeostasis in hepatocytes.
    Yuping Zheng, Jishun Chen, Vinitha Macwan, Charneal L Dixon, Xinran Li, Shengjie Liu, Yuyun Yu, Pinglong Xu, Qiming Sun, Qi Hu, Wei Liu, Brian Raught, Gregory D Fairn, Dante Neculai.
      Lipid droplets (LDs) are organelles specialized in the storage of neutral lipids, cholesterol esters and triglycerides, thereby protecting cells from the toxicity of excess lipids while allowing for the mobilization of lipids in times of nutrient deprivation. Defects in LD function are associated with many diseases. S-acylation mediated by zDHHC acyltransferases modifies thousands of proteins, yet the physiological impact of this post-translational modification on individual proteins is poorly understood. Here, we show that zDHHC11 regulates LD catabolism by modifying adipose triacylglyceride lipase (ATGL), the rate-limiting enzyme of lipolysis, both in hepatocyte cultures and in mice. zDHHC11 S-acylates ATGL at cysteine 15. Preventing the S-acylation of ATGL renders it catalytically inactive despite proper localization. Overexpression of zDHHC11 reduces LD size, whereas its elimination enlarges LDs. Mutating ATGL cysteine 15 phenocopies zDHHC11 loss, causing LD accumulation, defective lipolysis and lipophagy. Our results reveal S-acylation as a mode of regulation of ATGL function and LD homoeostasis. Modulating this pathway may offer therapeutic potential for treating diseases linked to defective lipolysis, such as fatty liver disease.
    DOI:  https://doi.org/10.1038/s42255-024-01085-w
  5. Nat Commun. 2024 Aug 12. 15(1): 6915
    Hypoxia-induced downregulation of PGK1 crotonylation promotes tumorigenesis by coordinating glycolysis and the TCA cycle.
    Zihao Guo, Yang Zhang, Haoyue Wang, Liming Liao, Lingdi Ma, Yiliang Zhao, Ronghui Yang, Xuexue Li, Jing Niu, Qiaoyun Chu, Yanxia Fu, Binghui Li, Chuanzhen Yang.
      Protein post-translational modifications (PTMs) are crucial for cancer cells to adapt to hypoxia; however, the functional significance of lysine crotonylation (Kcr) in hypoxia remains unclear. Herein we report a quantitative proteomics analysis of global crotonylome under normoxia and hypoxia, and demonstrate 128 Kcr site alterations across 101 proteins in MDA-MB231 cells. Specifically, we observe a significant decrease in K131cr, K156cr and K220cr of phosphoglycerate kinase 1 (PGK1) upon hypoxia. Enoyl-CoA hydratase 1 (ECHS1) is upregulated and interacts with PGK1, leading to the downregulation of PGK1 Kcr under hypoxia. Abolishment of PGK1 Kcr promotes glycolysis and suppresses mitochondrial pyruvate metabolism by activating pyruvate dehydrogenase kinase 1 (PDHK1). A low PGK1 K131cr level is correlated with malignancy and poor prognosis of breast cancer. Our findings show that PGK1 Kcr is a signal in coordinating glycolysis and the tricarboxylic acid (TCA) cycle and may serve as a diagnostic indicator for breast cancer.
    DOI:  https://doi.org/10.1038/s41467-024-51232-w
  6. Nat Commun. 2024 Aug 12. 15(1): 6914
    Defective mitochondrial COX1 translation due to loss of COX14 function triggers ROS-induced inflammation in mouse liver.
    Abhishek Aich, Angela Boshnakovska, Steffen Witte, Tanja Gall, Kerstin Unthan-Fechner, Roya Yousefi, Arpita Chowdhury, Drishan Dahal, Aditi Methi, Svenja Kaufmann, Ivan Silbern, Jan Prochazka, Zuzana Nichtova, Marcela Palkova, Miles Raishbrook, Gizela Koubkova, Radislav Sedlacek, Simon E Tröder, Branko Zevnik, Dietmar Riedel, Susann Michanski, Wiebke Möbius, Philipp Ströbel, Christian Lüchtenborg, Patrick Giavalisco, Henning Urlaub, Andre Fischer, Britta Brügger, Stefan Jakobs, Peter Rehling.
      Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.
    DOI:  https://doi.org/10.1038/s41467-024-51109-y
  7. Nat Metab. 2024 Aug 13.
    Integration of epidemiological and blood biomarker analysis links haem iron intake to increased type 2 diabetes risk.
    Fenglei Wang, Andrea J Glenn, Anne-Julie Tessier, Zhendong Mei, Danielle E Haslam, Marta Guasch-Ferré, Deirdre K Tobias, A Heather Eliassen, JoAnn E Manson, Clary Clish, Kyu Ha Lee, Eric B Rimm, Dong D Wang, Qi Sun, Liming Liang, Walter C Willett, Frank B Hu.
      Dietary haem iron intake is linked to an increased risk of type 2 diabetes (T2D), but the underlying plasma biomarkers are not well understood. We analysed data from 204,615 participants (79% females) in three large US cohorts over up to 36 years, examining the associations between iron intake and T2D risk. We also assessed plasma metabolic biomarkers and metabolomic profiles in subsets of 37,544 (82% females) and 9,024 (84% females) participants, respectively. Here we show that haem iron intake but not non-haem iron is associated with a higher T2D risk, with a multivariable-adjusted hazard ratio of 1.26 (95% confidence interval 1.20-1.33; P for trend <0.001) comparing the highest to the lowest quintiles. Haem iron accounts for significant proportions of the T2D risk linked to unprocessed red meat and specific dietary patterns. Increased haem iron intake correlates with unfavourable plasma profiles of insulinaemia, lipids, inflammation and T2D-linked metabolites. We also identify metabolites, including L-valine and uric acid, potentially mediating the haem iron-T2D relationship, highlighting their pivotal role in T2D pathogenesis.
    DOI:  https://doi.org/10.1038/s42255-024-01109-5
  8. Mol Cell. 2024 Aug 09. pii: S1097-2765(24)00618-X. [Epub ahead of print]
    OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response.
    Felix G Liang, Fereshteh Zandkarimi, Jaehoon Lee, Joshua L Axelrod, Ryan Pekson, Yisang Yoon, Brent R Stockwell, Richard N Kitsis.
      Ferroptosis, an iron-dependent form of nonapoptotic cell death mediated by lipid peroxidation, has been implicated in the pathogenesis of multiple diseases. Subcellular organelles play pivotal roles in the regulation of ferroptosis, but the mechanisms underlying the contributions of the mitochondria remain poorly defined. Optic atrophy 1 (OPA1) is a mitochondrial dynamin-like GTPase that controls mitochondrial morphogenesis, fusion, and energetics. Here, we report that human and mouse cells lacking OPA1 are markedly resistant to ferroptosis. Reconstitution with OPA1 mutants demonstrates that ferroptosis sensitization requires the GTPase activity but is independent of OPA1-mediated mitochondrial fusion. Mechanistically, OPA1 confers susceptibility to ferroptosis by maintaining mitochondrial homeostasis and function, which contributes both to the generation of mitochondrial lipid reactive oxygen species (ROS) and suppression of an ATF4-mediated integrated stress response. Together, these results identify an OPA1-controlled mitochondrial axis of ferroptosis regulation and provide mechanistic insights for therapeutically manipulating this form of cell death in diseases.
    Keywords:  ATF4; GPx4; OPA1; cell death; ferroptosis; integrated stress response; mitochondria; system X(c)(−); xCT
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.020
  9. Proc Natl Acad Sci U S A. 2024 Aug 20. 121(34): e2409262121
    Phosphorylation of Insig-2 mediates inhibition of fatty acid synthesis by polyunsaturated fatty acids.
    Jing Tian, Joseph L Goldstein, Shili Li, Marc M Schumacher, Michael S Brown.
      Insig-1 and Insig-2 are endoplasmic reticulum (ER) proteins that inhibit lipid synthesis by blocking transport of sterol regulatory element-binding proteins (SREBP-1 and SREBP-2) from ER to Golgi. In the Golgi, SREBPs are processed proteolytically to release their transcription-activating domains, which enhance the synthesis of fatty acids, triglycerides, and cholesterol. Heretofore, the two Insigs have redundant functions, and there is no rationale for two isoforms. The current data identify a specific function for Insig-2. We show that eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, inhibits fatty acid synthesis in human fibroblasts and rat hepatocytes by activating adenylate cyclase, which induces protein kinase A (PKA) to phosphorylate serine-106 in Insig-2. Phosphorylated Insig-2 inhibits the proteolytic processing of SREBP-1, thereby blocking fatty acid synthesis. Phosphorylated Insig-2 does not block the processing of SREBP-2, which activates cholesterol synthesis. Insig-1 lacks serine-106 and is not phosphorylated at this site. EPA inhibition of SREBP-1 processing was reduced by the replacement of serine-106 in Insig-2 with alanine or by treatment with KT5720, a PKA inhibitor. Inhibition did not occur in mutant human fibroblasts that possess Insig-1 but lack Insig-2. These data provide an Insig-2-specific mechanism for the long-known inhibition of fatty acid synthesis by polyunsaturated fatty acids.
    Keywords:  SREBP-1; cyclic AMP; fatty acid synthesis; mammalian cells; phosphorylated Insig-2
    DOI:  https://doi.org/10.1073/pnas.2409262121
  10. Cell. 2024 Aug 07. pii: S0092-8674(24)00824-9. [Epub ahead of print]
    Macrophage-mediated myelin recycling fuels brain cancer malignancy.
    Daan J Kloosterman, Johanna Erbani, Menno Boon, Martina Farber, Shanna M Handgraaf, Masami Ando-Kuri, Elena Sánchez-López, Bauke Fontein, Marjolijn Mertz, Marja Nieuwland, Ning Qing Liu, Gabriel Forn-Cuni, Nicole N van der Wel, Anita E Grootemaat, Luuk Reinalda, Sander I van Kasteren, Elzo de Wit, Brian Ruffell, Ewa Snaar-Jagalska, Kevin Petrecca, Dieta Brandsma, Alexander Kros, Martin Giera, Leila Akkari.
      Tumors growing in metabolically challenged environments, such as glioblastoma in the brain, are particularly reliant on crosstalk with their tumor microenvironment (TME) to satisfy their high energetic needs. To study the intricacies of this metabolic interplay, we interrogated the heterogeneity of the glioblastoma TME using single-cell and multi-omics analyses and identified metabolically rewired tumor-associated macrophage (TAM) subpopulations with pro-tumorigenic properties. These TAM subsets, termed lipid-laden macrophages (LLMs) to reflect their cholesterol accumulation, are epigenetically rewired, display immunosuppressive features, and are enriched in the aggressive mesenchymal glioblastoma subtype. Engulfment of cholesterol-rich myelin debris endows subsets of TAMs to acquire an LLM phenotype. Subsequently, LLMs directly transfer myelin-derived lipids to cancer cells in an LXR/Abca1-dependent manner, thereby fueling the heightened metabolic demands of mesenchymal glioblastoma. Our work provides an in-depth understanding of the immune-metabolic interplay during glioblastoma progression, thereby laying a framework to unveil targetable metabolic vulnerabilities in glioblastoma.
    Keywords:  cancer immunity; cholesterol; glioblastoma; lipid metabolism; macrophages; myelin recycling; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.cell.2024.07.030
  11. J Biol Chem. 2024 Aug 09. pii: S0021-9258(24)02159-8. [Epub ahead of print] 107658
    Intracellular pH differentially regulates transcription of metabolic and signaling pathways in normal epithelial cells.
    Ricardo Romero-Moreno, Brandon J Czowski, Lindsey Harris, Jessamine F Kuehn, Katharine A White.
      Intracellular pH (pHi) dynamics regulate normal cell function, and dysregulated pHi dynamics is an emerging hallmark of cancer (constitutively increased pHi) and neurodegeneration (constitutively decreased pHi). However, the molecular mechanisms by which pHi dynamics regulate cell biology are poorly understood. Here, we discovered that altering pHi in normal human breast epithelial cells triggers global transcriptional changes. We identified 176 genes differentially regulated by pHi, with pHi-dependent genes clustering in signaling and glycolytic pathways. Using various normal epithelial cell models, we showed pH-dependent Notch1 expression, with increased protein abundance at high pHi. This resulted in pH-dependent downstream signaling, with increased Notch1 signaling at high pHi. We also found that high pHi increased the expression of glycolytic enzymes and regulators of pyruvate fate, including lactate dehydrogenase and pyruvate dehydrogenase kinase. These transcriptional changes were sufficient to alter lactate production, with high pHi shifting these normal epithelial cells toward a glycolytic metabolism and increasing lactate production. Thus, pHi dynamics transcriptionally regulate signaling and metabolic pathways in normal epithelial cells. Our data reveal new molecular regulators of pHi-dependent biology and a role for increased pHi in driving the acquisition of cancer-associated signaling and metabolic changes in normal human epithelial cells.
    Keywords:  Intracellular pH; Notch1 signaling; glycolysis; lactate; metabolism; pyruvate
    DOI:  https://doi.org/10.1016/j.jbc.2024.107658
  12. J Biol Chem. 2024 Aug 09. pii: S0021-9258(24)02163-X. [Epub ahead of print] 107662
    Differential utilization of vitamin B12-dependent and independent pathways for propionate metabolism across human cells.
    Harsha Gouda, Yuanyuan Ji, Sneha Rath, David Watkins, David Rosenblatt, Vamsi Mootha, Jace W Jones, Ruma Banerjee.
      Propionic acid links the oxidation of branched-chain amino acids and odd-chain fatty acids to the TCA cycle. Gut microbes ferment complex fiber remnants, generating high concentrations of short chain fatty acids, acetate, propionate and butyrate, which are shared with the host as fuel sources. Analysis of vitamin B12-dependent propionate utilization in skin biopsy samples has been used to characterize and diagnose underlying inborn errors of cobalamin (or B12) metabolism. In these cells, the B12-dependent enzyme, methylmalonyl-CoA mutase (MMUT), plays a central role in funneling propionate to the TCA cycle intermediate, succinate. Our understanding of the fate of propionate in other cell types, specifically, the involvement of the β-oxidation-like and methylcitrate pathways, is limited. In this study, we have used [14C]-propionate tracing in combination with genetic ablation or inhibition of MMUT, to reveal the differential utilization of the B12-dependent and independent pathways for propionate metabolism in fibroblast versus colon cell lines. We demonstrate that itaconate can be used as a tool to investigate MMUT-dependent propionate metabolism in cultured cell lines. While MMUT gates the entry of propionate carbons into the TCA cycle in fibroblasts, colon-derived cell lines exhibit a quantitatively significant or exclusive reliance on the β-oxidation-like pathway. Lipidomics and metabolomics analyses reveal that propionate elicits pleiotropic changes, including an increase in odd-chain glycerophospholipids, and perturbations in the purine nucleotide cycle and arginine/nitric oxide metabolism. The metabolic rationale and the regulatory mechanisms underlying the differential reliance on propionate utilization pathways at a cellular, and possibly tissue level, warrant further elucidation.
    DOI:  https://doi.org/10.1016/j.jbc.2024.107662
  13. STAR Protoc. 2024 Aug 13. pii: S2666-1667(24)00401-5. [Epub ahead of print]5(3): 103236
    Protocol for monitoring fatty acid trafficking from lipid droplets to mitochondria in cultured cells.
    Gregory E Miner, Sarah Cohen.
      Intracellular trafficking of fatty acids (FAs) between organelles is critical for cells to adjust their metabolism in response to stimuli such as exercise, fasting, and cold exposure. Here, we describe a protocol to monitor trafficking of FAs from lipid droplets to mitochondria. We describe the labeling of organelles in cultured C2C12 myoblasts with transfection and dyes. We detail a pulse-chase labeling paradigm using a fluorescent FA analog, live-cell imaging to visualize trafficking of FAs, and steps to quantify FA trafficking. For complete details on the use and execution of this protocol, please refer to Miner et al.1.
    Keywords:  Cell Biology; Metabolism; Microscopy
    DOI:  https://doi.org/10.1016/j.xpro.2024.103236
  14. J Clin Invest. 2024 Aug 14. pii: e181839. [Epub ahead of print]
    TET3-overexpressing macrophages promote endometriosis.
    Haining Lv, Beibei Liu, Yangyang Dai, Feng Li, Stefania Bellone, Yuping Zhou, Ramanaiah Mamillapalli, Dejian Zhao, Muthukumaran Venkatachalapathy, Yali Hu, Gordon G Carmichael, Da Li, Hugh S Taylor, Yingqun Huang.
      Endometriosis is a debilitating, chronic inflammatory disease affecting ~10% of reproductive age women worldwide with no cure. While macrophages have been intrinsically linked to the pathophysiology of endometriosis, targeting them therapeutically has been extremely challenging due to their high heterogeneity and because these disease-associated macrophages (DAMs) can be either pathogenic or protective. Here, we reported identification of pathogenic macrophages characterized by TET3 overexpression in human endometriosis lesions. We showed that factors from the disease microenvironment upregulated TET3 expression transforming macrophages into pathogenic DAMs. TET3 overexpression stimulated pro-inflammatory cytokine production via a feedback mechanism involving inhibition of let-7 miRNA expression. Remarkably, these cells relied on TET3 overexpression for survival, hence vulnerable to TET3 knockdown. We demonstrated that Bobcat339, a synthetic cytosine derivative, triggered TET3 degradation both in human and mouse macrophages. This degradation was dependent on a VHL E3 ubiquitin ligase whose expression was also upregulated in TET3-overexpressing macrophages. Furthermore, depleting TET3-overexpressing macrophages either through myeloid-specific Tet3 ablation or using Bobcat339 strongly inhibited endometriosis progression in mice. Our results defined TET3-overexpressing macrophages as key pathogenic contributors to and attractive therapeutic targets for endometriosis. Our findings may also be applicable to other chronic inflammatory diseases where DAMs have important roles.
    Keywords:  Inflammation; Macrophages; Obstetrics/gynecology; Pain; Reproductive biology
    DOI:  https://doi.org/10.1172/JCI181839
  15. Mol Metab. 2024 Aug 11. pii: S2212-8778(24)00136-4. [Epub ahead of print] 102005
    The mitochondrial pyruvate carrier regulates adipose glucose partitioning in female mice.
    Christopher E Shannon, Terry Bakewell, Marcel J Fourcaudot, Iriscilla Ayala, Annie A Smelter, Edgar A Hinostroza, Giovanna Romero, Mara Asmis, Leandro C Freitas Lima, Martina Wallace, Luke Norton.
       OBJECTIVE: The mitochondrial pyruvate carrier (MPC) occupies a critical node in intermediary metabolism, prompting interest in its utility as a therapeutic target for the treatment of obesity and cardiometabolic disease. Dysregulated nutrient metabolism in adipose tissue is a prominent feature of obesity pathophysiology, yet the functional role of adipose MPC has not been explored. We investigated whether the MPC shapes the adaptation of adipose tissue to dietary stress in female and male mice.
    METHODS: The impact of pharmacological and genetic disruption of the MPC on mitochondrial pathways of triglyceride assembly (lipogenesis and glyceroneogenesis) was assessed in 3T3L1 adipocytes and murine adipose explants, combined with analyses of adipose MPC expression in metabolically compromised humans. Whole-body and adipose-specific glucose metabolism were subsequently investigated in male and female mice lacking adipocyte MPC1 (Mpc1AD-/-) and fed either standard chow, high-fat western style, or high-sucrose lipid restricted diets for 24 weeks, using a combination of radiolabeled tracers and GC/MS metabolomics.
    RESULTS: Treatment with UK5099 or siMPC1 impaired the synthesis of lipids and glycerol-3-phosphate from pyruvate and blunted triglyceride accumulation in 3T3L1 adipocytes, whilst MPC expression in human adipose tissue was negatively correlated with indices of whole-body and adipose tissue metabolic dysfunction. Mature adipose explants from Mpc1AD-/- mice were intrinsically incapable of incorporating pyruvate into triglycerides. In vivo, MPC deletion restricted the incorporation of circulating glucose into adipose triglycerides, but only in female mice fed a zero fat diet, and this associated with sex-specific reductions in tricarboxylic acid cycle pool sizes and compensatory transcriptional changes in lipogenic and glycerol metabolism pathways. However, whole-body adiposity and metabolic health were preserved in Mpc1AD-/- mice regardless of sex, even under conditions of zero dietary fat.
    CONCLUSION: These findings highlight the greater capacity for mitochondrially driven triglyceride assembly in adipose from female versus male mice and expose a reliance upon MPC-gated metabolism for glucose partitioning in female adipose under conditions of dietary lipid restriction.
    Keywords:  Adipose; glyceroneogenesis; lipogenesis; mitochondria; sexual dimorphism
    DOI:  https://doi.org/10.1016/j.molmet.2024.102005
  16. Nat Commun. 2024 Aug 15. 15(1): 7020
    Phosphatidic acid is an endogenous negative regulator of PIEZO2 channels and mechanical sensitivity.
    Matthew Gabrielle, Yevgen Yudin, Yujue Wang, Xiaoyang Su, Tibor Rohacs.
      Mechanosensitive PIEZO2 ion channels play roles in touch, proprioception, and inflammatory pain. Currently, there are no small molecule inhibitors that selectively inhibit PIEZO2 over PIEZO1. The TMEM120A protein was shown to inhibit PIEZO2 while leaving PIEZO1 unaffected. Here we find that TMEM120A expression elevates cellular levels of phosphatidic acid and lysophosphatidic acid (LPA), aligning with its structural resemblance to lipid-modifying enzymes. Intracellular application of phosphatidic acid or LPA inhibits PIEZO2 but not PIEZO1 activity. Extended extracellular exposure to the non-hydrolyzable phosphatidic acid and LPA analog carbocyclic phosphatidic acid (ccPA) also inhibits PIEZO2. Optogenetic activation of phospholipase D (PLD), a signaling enzyme that generates phosphatidic acid, inhibits PIEZO2 but not PIEZO1. Conversely, inhibiting PLD leads to increased PIEZO2 activity and increased mechanical sensitivity in mice in behavioral experiments. These findings unveil lipid regulators that selectively target PIEZO2 over PIEZO1, and identify the PLD pathway as a regulator of PIEZO2 activity.
    DOI:  https://doi.org/10.1038/s41467-024-51181-4
  17. Cell Death Differ. 2024 Aug 14.
    Cullin 4B-RING E3 ligase negatively regulates the immunosuppressive capacity of mesenchymal stem cells by suppressing iNOS.
    Ruiqi Yu, Hong Han, Shuxian Chu, Liping Qin, Mengying Du, Yanyan Ma, Yufeng Wang, Wei Jiang, Yu Song, Yongxin Zou, Molin Wang, Qiao Liu, Baichun Jiang, Yaoqin Gong, Gongping Sun.
      Mesenchymal stem cells (MSCs) are multipotent stem cells that can exert immunomodulatory capacity upon stimulation with pro-inflammatory cytokines. Our previous work has identified Cullin 4B (CUL4B), a scaffold protein in the CUL4B-RING E3 ligase (CRL4B) complex, as a key regulator in the differentiation of MSCs. Here, we demonstrate the critical role of CUL4B in regulating the immunosuppressive function of MSCs. When stimulated with pro-inflammatory cytokines, MSCs lacking CUL4B display enhanced immunosuppressive capacity, which is mediated by the elevated inducible nitric oxide synthase (iNOS). TGF-β signaling can suppress iNOS by inhibiting its transcription as well as promoting its protein degradation. We show that the CRL4B complex cooperates with PRC2 complex and HDACs to repress transcription of Dlx1 and Pmepa1, two inhibitors of TGF-β signaling, leading to decreased expression and accelerated degradation of iNOS. Our study unveils the CRL4B complex as a potential therapeutic target in promoting the immunosuppressive capacity of MSCs.
    DOI:  https://doi.org/10.1038/s41418-024-01359-6
  18. Sci Immunol. 2024 Aug 16. 9(98): eadh0368
    Functional overlap of inborn errors of immunity and metabolism genes defines T cell metabolic vulnerabilities.
    Andrew R Patterson, Gabriel A Needle, Ayaka Sugiura, Erin Q Jennings, Channing Chi, KayLee K Steiner, Emilie L Fisher, Gabriella L Robertson, Caroline Bodnya, Janet G Markle, Ryan D Sheldon, Russell G Jones, Vivian Gama, Jeffrey C Rathmell.
      Inborn errors of metabolism (IEMs) and immunity (IEIs) are Mendelian diseases in which complex phenotypes and patient rarity have limited clinical understanding. Whereas few genes have been annotated as contributing to both IEMs and IEIs, immunometabolic demands suggested greater functional overlap. Here, CRISPR screens tested IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable previously unappreciated crossover. Analysis of IEMs showed that N-linked glycosylation and the hexosamine pathway enzyme Gfpt1 are critical for T cell expansion and function. Further, T helper (TH1) cells synthesized uridine diphosphate N-acetylglucosamine more rapidly and were more impaired by Gfpt1 deficiency than TH17 cells. Screening IEI genes found that Bcl11b promotes the CD4 T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. Thus, a high degree of functional overlap exists between IEM and IEI genes, and immunometabolic mechanisms may underlie a previously underappreciated intersection of these disorders.
    DOI:  https://doi.org/10.1126/sciimmunol.adh0368
  19. Nat Commun. 2024 Aug 09. 15(1): 6820
    Hydrogel crosslinking modulates macrophages, fibroblasts, and their communication, during wound healing.
    Sergei Butenko, Raji R Nagalla, Christian F Guerrero-Juarez, Francesco Palomba, Li-Mor David, Ronald Q Nguyen, Denise Gay, Axel A Almet, Michelle A Digman, Qing Nie, Philip O Scumpia, Maksim V Plikus, Wendy F Liu.
      Biomaterial wound dressings, such as hydrogels, interact with host cells to regulate tissue repair. This study investigates how crosslinking of gelatin-based hydrogels influences immune and stromal cell behavior and wound healing in female mice. We observe that softer, lightly crosslinked hydrogels promote greater cellular infiltration and result in smaller scars compared to stiffer, heavily crosslinked hydrogels. Using single-cell RNA sequencing, we further show that heavily crosslinked hydrogels increase inflammation and lead to the formation of a distinct macrophage subpopulation exhibiting signs of oxidative activity and cell fusion. Conversely, lightly crosslinked hydrogels are more readily taken up by macrophages and integrated within the tissue. The physical properties differentially affect macrophage and fibroblast interactions, with heavily crosslinked hydrogels promoting pro-fibrotic fibroblast activity that drives macrophage fusion through RANKL signaling. These findings suggest that tuning the physical properties of hydrogels can guide cellular responses and improve healing, offering insights for designing better biomaterials for wound treatment.
    DOI:  https://doi.org/10.1038/s41467-024-50072-y
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