bims-mepmim Biomed News
on Metabolites in pathological microenvironments and immunometabolism
Issue of 2023‒05‒07
twenty-one papers selected by
Erika Mariana Palmieri
NIH/NCI Laboratory of Cancer ImmunoMetabolism
  1. Acetylcarnitine shuttling links mitochondrial metabolism to histone acetylation and lipogenesis.
  2. Inter-organelle cross-talk supports acetyl-coenzyme A homeostasis and lipogenesis under metabolic stress.
  3. Dynamic protein deacetylation is a limited carbon source for acetyl-CoA-dependent metabolism.
  4. Neutrophil metabolomics in severe COVID-19 reveal GAPDH as a suppressor of neutrophil extracellular trap formation.
  5. Acetate regulates GAPDH acetylation and T helper 1 cell differentiation.
  6. Methionine restriction constrains lipoylation and activates mitochondria for nitrogenic synthesis of amino acids.
  7. Dendritic cell-intrinsic LKB1-AMPK/SIK signaling controls metabolic homeostasis by limiting the hepatic Th17 response during obesity.
  8. Targeting cellular respiration as a therapeutic strategy in glioblastoma.
  9. Osteoblast-intrinsic defect in glucose metabolism impairs bone formation in type II diabetic male mice.
  10. Metabolic homeostasis and growth in abiotic cells.
  11. Symport and antiport mechanisms of human glutamate transporters.
  12. The PRAK-NRF2 axis promotes the differentiation of Th17 cells by mediating the redox homeostasis and glycolysis.
  13. PPAR-γ regulates the effector function of human T helper 9 cells by promoting glycolysis.
  14. N-arachidonylglycine is a caloric state-dependent circulating metabolite which regulates human CD4+T cell responsiveness.
  15. Oxaloacetate regulates complex II respiration in brown fat: dependence on UCP1 expression.
  16. An evolving view of Complex II - non-canonical complexes, megacomplexes, respiration, signaling, and beyond.
  17. Knockdown of HMGB1 inhibits the crosstalk between oral squamous cell carcinoma cells and tumor-associated macrophages.
  18. Protocol for generation of multicellular spheroids through reduced gravity.
  19. The biochemistry and physiology of long-chain dicarboxylic acid metabolism.
  20. Massively parallel base editing to map variant effects in human hematopoiesis.
  21. Reduced hepatic bradykinin degradation accounts for cold-induced BAT thermogenesis and WAT browning in male mice.
  1. Sci Adv. 2023 May 03. 9(18): eadf0115
    Acetylcarnitine shuttling links mitochondrial metabolism to histone acetylation and lipogenesis.
    Luke T Izzo, Sophie Trefely, Christina Demetriadou, Jack M Drummond, Takuya Mizukami, Nina Kuprasertkul, Aimee T Farria, Phuong T T Nguyen, Nivitha Murali, Lauren Reich, Daniel S Kantner, Joshua Shaffer, Hayley Affronti, Alessandro Carrer, Andrew Andrews, Brian C Capell, Nathaniel W Snyder, Kathryn E Wellen.
      The metabolite acetyl-CoA is necessary for both lipid synthesis in the cytosol and histone acetylation in the nucleus. The two canonical precursors to acetyl-CoA in the nuclear-cytoplasmic compartment are citrate and acetate, which are processed to acetyl-CoA by ATP-citrate lyase (ACLY) and acyl-CoA synthetase short-chain 2 (ACSS2), respectively. It is unclear whether other substantial routes to nuclear-cytosolic acetyl-CoA exist. To investigate this, we generated cancer cell lines lacking both ACLY and ACSS2 [double knockout (DKO) cells]. Using stable isotope tracing, we show that both glucose and fatty acids contribute to acetyl-CoA pools and histone acetylation in DKO cells and that acetylcarnitine shuttling can transfer two-carbon units from mitochondria to cytosol. Further, in the absence of ACLY, glucose can feed fatty acid synthesis in a carnitine responsive and carnitine acetyltransferase (CrAT)-dependent manner. The data define acetylcarnitine as an ACLY- and ACSS2-independent precursor to nuclear-cytosolic acetyl-CoA that can support acetylation, fatty acid synthesis, and cell growth.
    DOI:  https://doi.org/10.1126/sciadv.adf0115
  2. Sci Adv. 2023 May 03. 9(18): eadf0138
    Inter-organelle cross-talk supports acetyl-coenzyme A homeostasis and lipogenesis under metabolic stress.
    Ramya S Kuna, Avi Kumar, Karl A Wessendorf-Rodriguez, Hector Galvez, Courtney R Green, Grace H McGregor, Thekla Cordes, Reuben J Shaw, Robert U Svensson, Christian M Metallo.
      Proliferating cells rely on acetyl-CoA to support membrane biogenesis and acetylation. Several organelle-specific pathways are available for provision of acetyl-CoA as nutrient availability fluctuates, so understanding how cells maintain acetyl-CoA homeostasis under such stresses is critically important. To this end, we applied 13C isotope tracing cell lines deficient in these mitochondrial [ATP-citrate lyase (ACLY)]-, cytosolic [acetyl-CoA synthetase (ACSS2)]-, and peroxisomal [peroxisomal biogenesis factor 5 (PEX5)]-dependent pathways. ACLY knockout in multiple cell lines reduced fatty acid synthesis and increased reliance on extracellular lipids or acetate. Knockout of both ACLY and ACSS2 (DKO) severely stunted but did not entirely block proliferation, suggesting that alternate pathways can support acetyl-CoA homeostasis. Metabolic tracing and PEX5 knockout studies link peroxisomal oxidation of exogenous lipids as a major source of acetyl-CoA for lipogenesis and histone acetylation in cells lacking ACLY, highlighting a role for inter-organelle cross-talk in supporting cell survival in response to nutrient fluctuations.
    DOI:  https://doi.org/10.1126/sciadv.adf0138
  3. J Biol Chem. 2023 May 02. pii: S0021-9258(23)01800-8. [Epub ahead of print] 104772
    Dynamic protein deacetylation is a limited carbon source for acetyl-CoA-dependent metabolism.
    Ioana Soaita, Emily Megill, Daniel Kantner, Adam Chatoff, Yuen Jian Cheong, Philippa Clarke, Zoltan Arany, Nathaniel W Snyder, Kathryn E Wellen, Sophie Trefely.
      The ability of cells to store and rapidly mobilize energy reserves in response to nutrient availability is essential for survival. Breakdown of carbon stores produces acetyl-coenzyme-A (AcCoA), which fuels essential metabolic pathways and is also the acyl donor for protein lysine acetylation. Histones are abundant and highly acetylated proteins, accounting for 40% - 75% of cellular protein acetylation. Notably, histone acetylation is sensitive to AcCoA availability and nutrient replete conditions induce a substantial accumulation of acetylation on histones. Deacetylation releases acetate, which can be recycled to AcCoA, suggesting that deacetylation could be mobilized as an AcCoA source to feed downstream metabolic processes under nutrient depletion. While the notion of histones as a metabolic reservoir has been frequently proposed, experimental evidence has been lacking. Therefore, to test this concept directly, we used acetate-dependent ATP citrate lyase-deficient fibroblasts (Acly-/- MEFs) and designed a pulse-chase experimental system to trace deacetylation-derived acetate and its incorporation into AcCoA. We found that dynamic protein deacetylation in Acly-/- MEFs contributed carbons to AcCoA and proximal downstream metabolites. However, deacetylation had no significant effect on acyl-CoA pool sizes, and even at maximal acetylation, deacetylation transiently supplied less than 10% of cellular AcCoA. Together, our data reveal that although histone acetylation is dynamic and nutrient-sensitive, its potential for maintaining cellular AcCoA-dependent metabolic pathways is limited compared to cellular demand.
    Keywords:  Acetylation; acetate; acetyl-coenzyme A; histone; metabolism; stable isotope tracing
    DOI:  https://doi.org/10.1016/j.jbc.2023.104772
  4. Nat Commun. 2023 May 05. 14(1): 2610
    Neutrophil metabolomics in severe COVID-19 reveal GAPDH as a suppressor of neutrophil extracellular trap formation.
    Yafeng Li, Jessica S Hook, Qing Ding, Xue Xiao, Stephen S Chung, Marcel Mettlen, Lin Xu, Jessica G Moreland, Michalis Agathocleous.
      Severe COVID-19 is characterized by an increase in the number and changes in the function of innate immune cells including neutrophils. However, it is not known how the metabolome of immune cells changes in patients with COVID-19. To address these questions, we analyzed the metabolome of neutrophils from patients with severe or mild COVID-19 and healthy controls. We identified widespread dysregulation of neutrophil metabolism with disease progression including in amino acid, redox, and central carbon metabolism. Metabolic changes in neutrophils from patients with severe COVID-19 were consistent with reduced activity of the glycolytic enzyme GAPDH. Inhibition of GAPDH blocked glycolysis and promoted pentose phosphate pathway activity but blunted the neutrophil respiratory burst. Inhibition of GAPDH was sufficient to cause neutrophil extracellular trap (NET) formation which required neutrophil elastase activity. GAPDH inhibition increased neutrophil pH, and blocking this increase prevented cell death and NET formation. These findings indicate that neutrophils in severe COVID-19 have an aberrant metabolism which can contribute to their dysfunction. Our work also shows that NET formation, a pathogenic feature of many inflammatory diseases, is actively suppressed in neutrophils by a cell-intrinsic mechanism controlled by GAPDH.
    DOI:  https://doi.org/10.1038/s41467-023-37567-w
  5. Mol Biol Cell. 2023 May 03. mbcE23020070
    Acetate regulates GAPDH acetylation and T helper 1 cell differentiation.
    Xizhong Jing, Junfang Lyu, Jianhua Xiong.
      The short-chain fatty acid metabolite acetyl-coenzyme A (acetyl-CoA) has emerged as a major signal transducer that can broadly affect cell fate and function at least partly by influencing acetylation of key proteins. The mechanism by which acetyl-CoA regulates CD4+ T cell fate determination remains poorly understood. Herein, we report that acetate modulates glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acetylation and CD4+ T helper 1 (Th1) cell differentiation by altering acetyl-CoA levels. Our transcriptome profiling shows that acetate is a robust positive regulator of CD4+ T cell gene expression typical of glycolysis. We further show that acetate potentiates GAPDH activity, aerobic glycolysis and Th1 polarization through regulation of GAPDH acetylation levels. This acetate-dependent GAPDH acetylation occurs in a dose- and time-dependent manner, while decreasing acetyl-CoA levels by fatty acid oxidation (FAO) inhibition results in a decline in acetyl-GAPDH levels. Thus, acetate functions as a potent metabolic regulator in CD4+ T cells by promoting GAPDH acetylation and Th1 cell fate decision.
    DOI:  https://doi.org/10.1091/mbc.E23-02-0070
  6. Nat Commun. 2023 May 02. 14(1): 2504
    Methionine restriction constrains lipoylation and activates mitochondria for nitrogenic synthesis of amino acids.
    Wen Fang, Liu Jiang, Yibing Zhu, Sen Yang, Hong Qiu, Jiou Cheng, Qingxi Liang, Zong-Cai Tu, Cunqi Ye.
      Methionine restriction (MR) provides metabolic benefits in many organisms. However, mechanisms underlying the MR-induced effect remain incompletely understood. Here, we show in the budding yeast S. cerevisiae that MR relays a signal of S-adenosylmethionine (SAM) deprivation to adapt bioenergetic mitochondria to nitrogenic anabolism. In particular, decreases in cellular SAM constrain lipoate metabolism and protein lipoylation required for the operation of the tricarboxylic acid (TCA) cycle in the mitochondria, leading to incomplete glucose oxidation with an exit of acetyl-CoA and α-ketoglutarate from the TCA cycle to the syntheses of amino acids, such as arginine and leucine. This mitochondrial response achieves a trade-off between energy metabolism and nitrogenic anabolism, which serves as an effector mechanism promoting cell survival under MR.
    DOI:  https://doi.org/10.1038/s41467-023-38289-9
  7. JCI Insight. 2023 May 04. pii: e157948. [Epub ahead of print]
    Dendritic cell-intrinsic LKB1-AMPK/SIK signaling controls metabolic homeostasis by limiting the hepatic Th17 response during obesity.
    Hendrik Jp van der Zande, Eline C Brombacher, Joost M Lambooij, Leonard R Pelgrom, Anna Zawistowska-Deniziak, Thiago A Patente, Graham A Heieis, Frank Otto, Arifa Ozir-Fazalalikhan, Maria Yazdanbakhsh, Bart Everts, Bruno Guigas.
      Obesity-associated metabolic inflammation drives the development of insulin resistance and type 2 diabetes, notably through modulating innate and adaptive immune cells in metabolic organs. The nutrient sensor liver kinase B1 (LKB1) has recently been shown to control cellular metabolism and T cell priming functions of dendritic cells (DCs). Here, we report that hepatic DCs from high-fat diet (HFD)-fed obese mice display increased LKB1 phosphorylation and that LKB1 deficiency in DCs (CD11cΔLKB1) worsened HFD-driven hepatic steatosis and impaired glucose homeostasis. Loss of LKB1 in DCs was associated with increased expression of T helper 17-polarizing cytokines and accumulation of hepatic IL-17A+ T helper cells in HFD-fed mice. Importantly, IL-17A neutralization rescued metabolic perturbations in HFD-fed CD11cΔLKB1 mice. Mechanistically, deficiency of the canonical LKB1 target AMPK in HFD-fed CD11cΔAMPKα1 mice recapitulated neither the hepatic Th17 phenotype nor the disrupted metabolic homeostasis, suggesting the involvement of other and/or additional LKB1 downstream effectors. We indeed provide evidence that the control of Th17 responses by DCs via LKB1 is actually dependent on both AMPKalpha1 and AMPK-related salt-inducible kinase(s) signaling. Altogether, our data reveal a key role for LKB1 signaling in DCs in protection against obesity-induced metabolic dysfunctions by limiting hepatic Th17 responses.
    Keywords:  Dendritic cells; Immunology; Metabolism; Obesity; T cells
    DOI:  https://doi.org/10.1172/jci.insight.157948
  8. Oncotarget. 2023 May 04. 14 419-425
    Targeting cellular respiration as a therapeutic strategy in glioblastoma.
    Enyuan Shang, Trang Thi Thu Nguyen, Mike-Andrew Westhoff, Georg Karpel-Massler, Markus D Siegelin.
      While glycolysis is abundant in malignancies, mitochondrial metabolism is significant as well. Mitochondria harbor the enzymes relevant for cellular respiration, which is a critical pathway for both regeneration of reduction equivalents and energy production in the form of ATP. The oxidation of NADH2 and FADH2 are fundamental since NAD and FAD are the key components of the TCA-cycle that is critical to entertain biosynthesis in cancer cells. The TCA-cycle itself is predominantly fueled through carbons from glucose, glutamine, fatty acids and lactate. Targeting mitochondrial energy metabolism appears feasible through several drug compounds that activate the CLPP protein or interfere with NADH-dehydrogenase, pyruvate-dehydrogenase, enzymes of the TCA-cycle and mitochondrial matrix chaperones. While these compounds have demonstrated anti-cancer effects in vivo, recent research suggests which patients most likely benefit from such treatments. Here, we provide a brief overview of the status quo of targeting mitochondrial energy metabolism in glioblastoma and highlight a novel combination therapy.
    Keywords:  carbon tracing; central carbon metabolism; glioblastoma; lactate; metabolism
    DOI:  https://doi.org/10.18632/oncotarget.28424
  9. Elife. 2023 May 05. pii: e85714. [Epub ahead of print]12
    Osteoblast-intrinsic defect in glucose metabolism impairs bone formation in type II diabetic male mice.
    Fangfang Song, Won Dong Lee, Tyler Marmo, Xing Ji, Chao Song, Xueyang Liao, Rebecca Seeley, Lutian Yao, Haoran Liu, Fanxin Long.
      Skeletal fragility is associated with type 2 diabetes mellitus (T2D), but the underlying mechanism is not well understood. Here, in a mouse model for youth-onset T2D, we show that both trabecular and cortical bone mass are reduced due to diminished osteoblast activity. Stable isotope tracing in vivo with 13C-glucose demonstrates that both glycolysis and glucose fueling of the TCA cycle are impaired in diabetic bones. Similarly, Seahorse assays show suppression of both glycolysis and oxidative phosphorylation by diabetes in bone marrow mesenchymal cells as a whole, whereas single-cell RNA sequencing reveals distinct modes of metabolic dysregulation among the subpopulations. Metformin not only promotes glycolysis and osteoblast differentiation in vitro, but also improves bone mass in diabetic mice. Finally, osteoblast-specific overexpression of either Hif1a, a general inducer of glycolysis, or Pfkfb3 which stimulates a specific step in glycolysis, averts bone loss in T2D mice. The study identifies osteoblast-intrinsic defects in glucose metabolism as an underlying cause of diabetic osteopenia, which may be targeted therapeutically.
    Keywords:  medicine; mouse
    DOI:  https://doi.org/10.7554/eLife.85714
  10. Proc Natl Acad Sci U S A. 2023 May 09. 120(19): e2300687120
    Metabolic homeostasis and growth in abiotic cells.
    Amir Akbari, Bernhard O Palsson.
      Metabolism constitutes the core chemistry of life. How it began on the early Earth and whether it had a cellular origin are still uncertain. A leading hypothesis for life's origins postulates that metabolism arose from geochemical CO2-fixing pathways, driven by inorganic catalysts and energy sources, long before enzymes or genes existed. The acetyl-CoA pathway and the reductive tricarboxylic acid cycle are considered ancient reaction networks that hold relics of early carbon-fixing pathways. Although transition metals can promote many steps of these pathways, whether they form a functional metabolic network in abiotic cells has not been demonstrated. Here, we formulate a nonenzymatic carbon-fixing network from these pathways and determine its functional feasibility in abiotic cells by imposing fundamental physicochemical constraints. Using first principles, we show that abiotic cells can sustain a steady carbon-fixing cycle that performs a systemic function over a relatively narrow range of conditions. Furthermore, we find that in all feasible steady states, the operation of the cycle elevates the osmotic pressure, leading to volume expansion. These results suggest that achieving homeostatic metabolic states under prebiotic conditions was possible, but challenging, and volume growth was a fundamental property of early metabolism.
    Keywords:  alkaline hydrothermal vents; carbon-fixing cycles; emergent properties; metabolic homeostasis; origin of metabolism
    DOI:  https://doi.org/10.1073/pnas.2300687120
  11. Nat Commun. 2023 May 04. 14(1): 2579
    Symport and antiport mechanisms of human glutamate transporters.
    Biao Qiu, Olga Boudker.
      Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.
    DOI:  https://doi.org/10.1038/s41467-023-38120-5
  12. Proc Natl Acad Sci U S A. 2023 May 09. 120(19): e2212613120
    The PRAK-NRF2 axis promotes the differentiation of Th17 cells by mediating the redox homeostasis and glycolysis.
    Ziheng Zhao, Yan Wang, Yuhan Gao, Yurong Ju, Ye Zhao, Zhaofei Wu, Shuaixin Gao, Boyang Zhang, Xuewen Pang, Yu Zhang, Wei Wang.
      Oxidative stress is a key feature in both chronic inflammation and cancer. P38 regulated/activated protein kinase (PRAK) deficiency can cause functional disorders in neutrophils and macrophages under high oxidative stress, but the precise mechanisms by which PRAK regulates reactive oxygen species (ROS) elimination and its potential impact on CD4+ T helper subset function are unclear. The present study reveals that the PRAK-NF-E2-related factor 2(NRF2) axis is essential for maintaining the intracellular redox homeostasis of T helper 17(Th17) cells, thereby promoting Th17 cell differentiation and antitumor effects. Through mechanistic analysis, we identify NRF2 as a novel protein substrate of PRAK and find that PRAK enhances the stability of the NRF2 protein through phosphorylation NRF2 Serine(S) 558 independent of protein ubiquitination. High accumulation of cellular ROS caused by loss of PRAK disrupts both glycolysis and PKM2-dependent phosphorylation of STAT3, which subsequently impairs the differentiation of Th17 cells. As a result, Prak knockout (KO) mice display significant resistance to experimental autoimmune encephalomyelitis (EAE) but impaired antitumor immunity in a MC38 tumor model. This work reveals that the PRAK-NRF2-mediated antioxidant pathway is a metabolic checkpoint that controls Th17-cell glycolysis and differentiation. Targeting PRAK is a promising strategy for maintaining an active ROS scavenging system and may lead to potent Th17 cell antitumor immunity.
    Keywords:  NRF2; PRAK; ROS; Th17; glycolysis
    DOI:  https://doi.org/10.1073/pnas.2212613120
  13. Nat Commun. 2023 04 29. 14(1): 2471
    PPAR-γ regulates the effector function of human T helper 9 cells by promoting glycolysis.
    Nicole L Bertschi, Oliver Steck, Fabian Luther, Cecilia Bazzini, Leonhard von Meyenn, Stefanie Schärli, Angela Vallone, Andrea Felser, Irene Keller, Olivier Friedli, Stefan Freigang, Nadja Begré, Susanne Radonjic-Hoesli, Cristina Lamos, Max Philip Gabutti, Michael Benzaquen, Markus Laimer, Dagmar Simon, Jean-Marc Nuoffer, Christoph Schlapbach.
      T helper 9 (TH9) cells promote allergic tissue inflammation and express the type 2 cytokines, IL-9 and IL-13, as well as the transcription factor, PPAR-γ. However, the functional role of PPAR-γ in human TH9 cells remains unknown. Here, we demonstrate that PPAR-γ drives activation-induced glycolysis, which, in turn, promotes the expression of IL-9, but not IL-13, in an mTORC1-dependent manner. In vitro and ex vivo experiments show that the PPAR-γ-mTORC1-IL-9 pathway is active in TH9 cells in human skin inflammation. Additionally, we find dynamic regulation of tissue glucose levels in acute allergic skin inflammation, suggesting that in situ glucose availability is linked to distinct immunological functions in vivo. Furthermore, paracrine IL-9 induces expression of the lactate transporter, MCT1, in TH cells and promotes their aerobic glycolysis and proliferative capacity. Altogether, our findings uncover a hitherto unknown relationship between PPAR-γ-dependent glucose metabolism and pathogenic effector functions in human TH9 cells.
    DOI:  https://doi.org/10.1038/s41467-023-38233-x
  14. iScience. 2023 May 19. 26(5): 106578
    N-arachidonylglycine is a caloric state-dependent circulating metabolite which regulates human CD4+T cell responsiveness.
    Allison M Meadows, Kim Han, Komudi Singh, Antonio Murgia, Ben D McNally, James A West, Rebecca D Huffstutler, Tiffany M Powell-Wiley, Yvonne Baumer, Julian L Griffin, Michael N Sack.
      Caloric deprivation interventions such as intermittent fasting and caloric restriction ameliorate metabolic and inflammatory disease. As a human model of caloric deprivation, a 24-h fast blunts innate and adaptive immune cell responsiveness relative to the refed state. Isolated serum at these time points confers these same immunomodulatory effects on transformed cell lines. To identify serum mediators orchestrating this, metabolomic and lipidomic analysis was performed on serum extracted after a 24-h fast and re-feeding. Bioinformatic integration with concurrent peripheral blood mononuclear cells RNA-seq analysis implicated key metabolite-sensing GPCRs in fasting-mediated immunomodulation. The putative GPR18 ligand N-arachidonylglycine (NAGly) was elevated during fasting and attenuated CD4+T cell responsiveness via GPR18 MTORC1 signaling. In parallel, NAGly reduced inflammatory Th1 and Th17 cytokines levels in CD4+T cells isolated from obese subjects, identifying a fasting-responsive metabolic intermediate that may contribute to the regulation of nutrient-level dependent inflammation associated with metabolic disease.
    Keywords:  Human metabolism; Immunology; Lipidomics; Metabolomics; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2023.106578
  15. Am J Physiol Cell Physiol. 2023 May 01.
    Oxaloacetate regulates complex II respiration in brown fat: dependence on UCP1 expression.
    Ritu Som, Brian D Fink, Liping Yu, William I Sivitz.
      We previously found that skeletal muscle mitochondria incubated at low membrane potential (ΔΨ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low, accumulate oxaloacetate (OAA) in amounts sufficient to inhibit complex II respiration. We proposed a mechanism wherein low ΔΨ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance we carried out studies of mice with inherently different levels of IBAT mitochondrial inner membrane potential. Isolated complex II (succinate)-energized IBAT mitochondria from obesity resistant 129SVE mice compared to obesity prone C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower ΔΨ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower ΔΨ, exhibited much lower respiration, 2-fold greater OAA concentrations, much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared to the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater ΔΨ, ROS, and NADH, but equal or greater O2 flux compared to WT mitochondria. GDP addition, compared to no GDP, increased ΔΨ and complex II respiration in wildtype mice associated with much less OAA. Respiration on complex I substrates followed the more classical dynamics of greater respiration at lower ΔΨ. These findings support the above-mentioned mechanism for OAA- and ΔΨ-dependent complex II respiration and support its physiological relevance.
    Keywords:  brown adipose tissue; mitochondria; mitochondrial complex II; oxaloacetate; succinate dehydrogenase
    DOI:  https://doi.org/10.1152/ajpcell.00565.2022
  16. J Biol Chem. 2023 Apr 27. pii: S0021-9258(23)01789-1. [Epub ahead of print] 104761
    An evolving view of Complex II - non-canonical complexes, megacomplexes, respiration, signaling, and beyond.
    T M Iverson, Prashant K Singh, Gary Cecchini.
      Mitochondrial Complex II is traditionally studied for its participation in two key respiratory processes: the electron transport chain and the Krebs cycle. There is now a rich body of literature explaining how Complex II contributes to respiration. However, more recent research shows that not all of the pathologies associated with altered Complex II activity clearly correlate with this respiratory role. Complex II activity has now been shown to be necessary for a range of biological processes peripherally-related to respiration, including metabolic control, inflammation, and cell fate. Integration of findings from multiple types of studies suggests that Complex II both participates in respiration and controls multiple succinate-dependent signal transduction pathways. Thus, the emerging view is that the true biological function of Complex II is well beyond respiration. This review uses a semi-chronological approach to highlight major paradigm shifts that occurred over time. Special emphasis is given to the more recently identified functions of Complex II and its subunits because these findings have infused new directions into an established field.
    Keywords:  Complex II; Krebs cycle; bacterial chemotaxis; cancer metabolism; hypoxia; inflammation; ischemia-reperfusion; paraganglioma; pheochromocytoma; respirasome; reverse electron transfer; succinate dehydrogenase; succinate signaling
    DOI:  https://doi.org/10.1016/j.jbc.2023.104761
  17. Int Immunopharmacol. 2023 May 02. pii: S1567-5769(23)00580-5. [Epub ahead of print]119 110259
    Knockdown of HMGB1 inhibits the crosstalk between oral squamous cell carcinoma cells and tumor-associated macrophages.
    Jinlin Wen, Panpan Yin, Ying Su, Feng Gao, Yanlin Wu, Wenbin Zhang, Peng Chi, Jiahui Chen, Xinyan Zhang.
      Tumor-associated macrophages (TAMs), the major component of the tumor microenvironment (TME), play distinctly different roles in different tumors. High mobility group box 1 (HMGB1), a nonhistone protein in the nucleus, can perform functions during inflammation and cancers. However, the role of HMGB1 in the crosstalk between oral squamous cell carcinoma (OSCC) cells and TAMs remains unclear. Here, we established a coculture system of TAMs and OSCC cells to explore the bidirectional effect and potential mechanism of HMGB1 in OSCC cell-TAM interactions. Our results showed that HMGB1 was significantly upregulated in OSCC tissues and positively associated with tumor progression, immune cell infiltration and macrophage polarization. Then, knocking down HMGB1 in OSCC cells inhibited the recruitment and polarization of cocultured TAMs. Moreover, the knockdown of HMGB1 in macrophages not only suppressed polarization, but also inhibited cocultured OSCC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, macrophages secreted higher levels of HMGB1 than OSCC cells, and dampening endogenous HMGB1 reduced HMGB1 secretion. Both OSCC cell-generated and macrophage-endogenous HMGB1 may regulate TAM polarization by promoting receptor TLR4 expression and NF-κB/p65 activation and enhancing IL-10/TGF-β expression. HMGB1 in OSCC cells may regulate macrophage recruitment via IL-6/STAT3. In addition, TAM-derived HMGB1 may affect aggressive phenotypes of cocultured OSCC cells by regulating the immunosuppressive microenvironment through the IL-6/STAT3/PD-L1 and IL-6/NF-κB/MMP-9 pathways. In conclusion, HMGB1 may regulate the crosstalk between OSCC cells and TAMs, including modulating macrophage polarization and attraction, enhancing cytokine secretion, and remodeling and creating an immunosuppressive TME to further affect OSCC progression.
    Keywords:  Crosstalk; HMGB1; Metastasis; OSCC; TAMs; TME
    DOI:  https://doi.org/10.1016/j.intimp.2023.110259
  18. STAR Protoc. 2023 May 02. pii: S2666-1667(23)00222-8. [Epub ahead of print]4(2): 102264
    Protocol for generation of multicellular spheroids through reduced gravity.
    Dylan A Zinn, Christine Mehner, Tushar Patel.
      Multicellular spheroids are useful models for drug testing or studying tumor biology, but their production requires specialized approaches. Here, we present a protocol to produce viable spheroids by slow rotation around a horizontal axis using standard culture tubes. We describe steps for both seed and starter culture, and maintenance and expansion of spheroids. We detail assessment of spheroid size, count, viability, and immunohistochemistry. This protocol reduces gravitational forces that lead to cell clumping and is amenable to high-throughput use.
    Keywords:  Cancer; Cell Biology; Cell culture; Cell-based Assays; Organoids
    DOI:  https://doi.org/10.1016/j.xpro.2023.102264
  19. Biochem J. 2023 May 15. 480(9): 607-627
    The biochemistry and physiology of long-chain dicarboxylic acid metabolism.
    Pablo Ranea-Robles, Sander M Houten.
      Mitochondrial β-oxidation is the most prominent pathway for fatty acid oxidation but alternative oxidative metabolism exists. Fatty acid ω-oxidation is one of these pathways and forms dicarboxylic acids as products. These dicarboxylic acids are metabolized through peroxisomal β-oxidation representing an alternative pathway, which could potentially limit the toxic effects of fatty acid accumulation. Although dicarboxylic acid metabolism is highly active in liver and kidney, its role in physiology has not been explored in depth. In this review, we summarize the biochemical mechanism of the formation and degradation of dicarboxylic acids through ω- and β-oxidation, respectively. We will discuss the role of dicarboxylic acids in different (patho)physiological states with a particular focus on the role of the intermediates and products generated through peroxisomal β-oxidation. This review is expected to increase the understanding of dicarboxylic acid metabolism and spark future research.
    DOI:  https://doi.org/10.1042/BCJ20230041
  20. Cell. 2023 Apr 27. pii: S0092-8674(23)00332-X. [Epub ahead of print]
    Massively parallel base editing to map variant effects in human hematopoiesis.
    Jorge D Martin-Rufino, Nicole Castano, Michael Pang, Emanuelle I Grody, Samantha Joubran, Alexis Caulier, Lara Wahlster, Tongqing Li, Xiaojie Qiu, Anna Maria Riera-Escandell, Gregory A Newby, Aziz Al'Khafaji, Santosh Chaudhary, Susan Black, Chen Weng, Glen Munson, David R Liu, Marcin W Wlodarski, Kacie Sims, Jamie H Oakley, Ross M Fasano, Ramnik J Xavier, Eric S Lander, Daryl E Klein, Vijay G Sankaran.
      Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.
    Keywords:  base editing; differentiation; functional screens; genome engineering; hematopoiesis; hematopoietic stem cell; primary cells; single-cell genomics
    DOI:  https://doi.org/10.1016/j.cell.2023.03.035
  21. Nat Commun. 2023 May 02. 14(1): 2523
    Reduced hepatic bradykinin degradation accounts for cold-induced BAT thermogenesis and WAT browning in male mice.
    Fei Xiao, Haizhou Jiang, Zi Li, Xiaoxue Jiang, Shanghai Chen, Yuguo Niu, Hanrui Yin, Yousheng Shu, Bo Peng, Wei Lu, Xiaoying Li, Zhigang Li, Shujue Lan, Xiaoyan Xu, Feifan Guo.
      An important role for liver in the regulation of adipose tissue thermogenesis upon cold exposure has been suggested; however, the underlying mechanisms remain incompletely defined. Here, we identify elevated serum bradykinin levels in response to acute cold exposure in male mice. A bolus of anti-bradykinin antibodies reduces body temperature during acute cold exposure, whereas bradykinin has the opposite effect. We demonstrate that bradykinin induces brown adipose tissue thermogenesis and white adipose tissue browning, and bradykinin increases uncoupling protein 1 (UCP1) expression in adipose tissue. The bradykinin B2 receptor (B2R), adrenergic signaling and nitric oxide signaling are involved in regulating bradykinin-increased UCP1 expression. Moreover, acute cold exposure inhibits hepatic prolyl endopeptidase (PREP) activity, causing reduced liver bradykinin degradation and increased serum bradykinin levels. Finally, by blocking the breakdown of bradykinin, angiotensin-converting enzyme inhibitors (ACEIs) increase serum bradykinin levels and induce brown adipose tissue thermogenesis and white adipose tissue browning via B2R. Collectively, our data provide new insights into the mechanisms underlying organ crosstalk in whole-body physiology control during cold exposure and also suggest bradykinin as a possible anti-obesity target.
    DOI:  https://doi.org/10.1038/s41467-023-38141-0
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