bims-mepmim 2023-04-23 papers

bims-mepmim

Biomed News

on Metabolites in pathological microenvironments and immunometabolism

Issue of 2023–04–23
twenty-two papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism

Learning the metabolic language of cancer.
Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.
Pyruvate-supported flux through medium-chain ketothiolase promotes mitochondrial lipid tolerance in cardiac and skeletal muscles.
A rapid microglial metabolic response controls metabolism and improves memory.
Allosteric role of the citrate synthase homology domain of ATP citrate lyase.
Low production of mitochondrial reactive oxygen species after anoxia and reoxygenation in turtle hearts.
Isolation and Mass Spectrometry-Based Profiling of Major Lipids in Brown Adipose Tissue.
Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis.
Hepatocyte SREBP signaling mediates clock communication within the liver.
Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness.
Reduced methionine synthase expression results in uracil accumulation in mitochondrial DNA and impaired oxidative capacity.
Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.
Protocol for indirect and direct co-culture between human cancer cells and endothelial cells.
The proteomic landscape of genome-wide genetic perturbations.
Nitric oxide hinders club cell proliferation through Gdpd2 during allergic airway inflammation.
Cryo-EM structure of the polyphosphate polymerase VTC reveals coupling of polymer synthesis to membrane transit.
CX3CR1hi macrophages sustain metabolic adaptation by relieving adipose-derived stem cell senescence in visceral adipose tissue.
Very-long-chain fatty acids induce glial-derived sphingosine-1-phosphate synthesis, secretion, and neuroinflammation.
G-protein coupled receptor 19 (GPR19) knockout mice display sex-dependent metabolic dysfunction.
Distinct spatial immune microlandscapes are independently associated with outcomes in triple-negative breast cancer.
IL-18 Binding Protein-Producing Cells Attenuate Anemia in Murine Macrophage Activation Syndrome.
Amino acids downregulate SIRT4 to detoxify ammonia through the urea cycle.

Nature. 2023 Apr;616(7958): 670-671

Learning the metabolic language of cancer.

Minervo Perez, Jordan L Meier.



Keywords:  Cancer; Cell biology; Metabolism

DOI:  https://doi.org/10.1038/d41586-023-01024-x
Mol Cell. 2023 Apr 20. pii: S1097-2765(23)00213-7. [Epub ahead of print]83(8): 1340-1349.e7

Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.

Cong-Hui Yao, Joon Seok Park, Kiran Kurmi, Song-Hua Hu, Giulia Notarangelo, Joseph Crowley, Heidi Jacobson, Sheng Hui, Arlene H Sharpe, Marcia C Haigis.

  The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.

Keywords:  GPD; NAD; glycerol; glycerol-3-phosphate dehydrogenase; glycerol-3-phosphate shuttle; kidney cancer; lipids; metabolism; mitochondria

DOI:  https://doi.org/10.1016/j.molcel.2023.03.023
Cell Metab. 2023 Apr 11. pii: S1550-4131(23)00094-3. [Epub ahead of print]

Pyruvate-supported flux through medium-chain ketothiolase promotes mitochondrial lipid tolerance in cardiac and skeletal muscles.

Timothy R Koves, Guo-Fang Zhang, Michael T Davidson, Alec B Chaves, Scott B Crown, Jordan M Johnson, Dorothy H Slentz, Paul A Grimsrud, Deborah M Muoio.

  Even-chain acylcarnitine (AC) metabolites, most of which are generated as byproducts of incomplete fatty acid oxidation (FAO), are viewed as biomarkers of mitochondrial lipid stress attributable to one or more metabolic bottlenecks in the β-oxidation pathway. The origins and functional implications of FAO bottlenecks remain poorly understood. Here, we combined a sophisticated mitochondrial phenotyping platform with state-of-the-art molecular profiling tools and multiple two-state mouse models of respiratory function to uncover a mechanism that connects AC accumulation to lipid intolerance, metabolic inflexibility, and respiratory inefficiency in skeletal muscle mitochondria. These studies also identified a short-chain carbon circuit at the C4 node of FAO wherein reverse flux of glucose-derived acetyl CoA through medium-chain ketothiolase enhances lipid tolerance and redox stability in heart mitochondria by regenerating free CoA and NAD+. The findings help to explain why diminished FAO capacity, AC accumulation, and metabolic inflexibility are tightly linked to poor health outcomes.

Keywords:  acylcarnitines; bioenergetics; exercise; fatty acid oxidation; heart; ketothiolase; metabolic flexibility; mitochondria; pyruvate; skeletal muscle

DOI:  https://doi.org/10.1016/j.cmet.2023.03.016
bioRxiv. 2023 Apr 04. pii: 2023.04.03.535373. [Epub ahead of print]

A rapid microglial metabolic response controls metabolism and improves memory.

Anne Drougard, Eric H Ma, Vanessa Wegert, Ryan Sheldon, Ilaria Panzeri, Naman Vatsa, Stefanos Apostle, Luca Fagnocchi, Judith Schaf, Klaus Gossens, Josephine Völker, Shengru Pang, Anna Bremser, Erez Dror, Francesca Giacona, Sagar, Michael X Henderson, Marco Prinz, Russell G Jones, J Andrew Pospisilik.

Chronic high-fat feeding triggers widespread metabolic dysfunction including obesity, insulin resistance, and diabetes. While these ultimate pathological states are relatively well understood, we have a limited understanding of how high-fat intake first triggers physiological changes. Here, we identify an acute microglial metabolic response that rapidly translates intake of high-fat diet (HFD) to a surprisingly beneficial effect on spatial and learning memory. Acute high-fat intake increases palmitate levels in cerebrospinal fluid and triggers a wave of microglial metabolic activation characterized by mitochondrial membrane activation, fission and metabolic skewing towards aerobic glycolysis. These effects are generalized, detectable in the hypothalamus, hippocampus, and cortex all within 1-3 days of HFD exposure. In vivo microglial ablation and conditional DRP1 deletion experiments show that the microglial metabolic response is necessary for the acute effects of HFD. 13 C-tracing experiments reveal that in addition to processing via β-oxidation, microglia shunt a substantial fraction of palmitate towards anaplerosis and re-release of bioenergetic carbons into the extracellular milieu in the form of lactate, glutamate, succinate, and intriguingly, the neuro-protective metabolite itaconate. Together, these data identify microglial cells as a critical nutrient regulatory node in the brain, metabolizing away harmful fatty acids and liberating the same carbons instead as alternate bioenergetic and protective substrates. The data identify a surprisingly beneficial effect of short-term HFD on learning and memory.

DOI: https://doi.org/10.1101/2023.04.03.535373
Nat Commun. 2023 04 19. 14(1): 2247

Allosteric role of the citrate synthase homology domain of ATP citrate lyase.

Xuepeng Wei, Kollin Schultz, Hannah L Pepper, Emily Megill, Austin Vogt, Nathaniel W Snyder, Ronen Marmorstein.

ATP citrate lyase (ACLY) is the predominant nucleocytosolic source of acetyl-CoA and is aberrantly regulated in many diseases making it an attractive therapeutic target. Structural studies of ACLY reveal a central homotetrameric core citrate synthase homology (CSH) module flanked by acyl-CoA synthetase homology (ASH) domains, with ATP and citrate binding the ASH domain and CoA binding the ASH-CSH interface to produce acetyl-CoA and oxaloacetate products. The specific catalytic role of the CSH module and an essential D1026A residue contained within it has been a matter of debate. Here, we report biochemical and structural analysis of an ACLY-D1026A mutant demonstrating that this mutant traps a (3S)-citryl-CoA intermediate in the ASH domain in a configuration that is incompatible with the formation of acetyl-CoA, is able to convert acetyl-CoA and OAA to (3S)-citryl-CoA in the ASH domain, and can load CoA and unload acetyl-CoA in the CSH module. Together, this data support an allosteric role for the CSH module in ACLY catalysis.

DOI: https://doi.org/10.1038/s41467-023-37986-9
J Exp Biol. 2023 Apr 17. pii: jeb.245516. [Epub ahead of print]

Low production of mitochondrial reactive oxygen species after anoxia and reoxygenation in turtle hearts.

Amanda Bundgaard, Anja V Gruszczyk, Hiran A Prag, Catherine Williams, Angela McIntyre, Ilan M Ruhr, Andrew M James, Gina L J Galli, Michael P Murphy, Angela Fago.

  Extremely anoxia-tolerant animals, such as freshwater turtles, survive anoxia and reoxygenation without sustaining tissue damage to their hearts. In contrast, for mammals, the ischemia-reperfusion (IR) injury that leads to tissue damage during a heart attack is initiated by a burst of superoxide (O2.-) production from the mitochondrial respiratory chain upon reperfusion of ischemic tissue. Whether turtles avoid oxidative tissue damage because of an absence of mitochondrial superoxide production upon reoxygenation, or because the turtle heart is particularly protected against this damage, is unclear. Here, we investigate whether there was an increase in mitochondrial O2.- production upon the reoxygenation of anoxic red-eared slider turtle hearts in vivo and in vitro. This was done by measuring the production of H2O2, the dismutation product of O2.-, using the mitochondria-targeted mass-spectrometric probe in vivo MitoB, while in parallel assessing changes in the metabolites driving mitochondrial O2.- production succinate, ATP and ADP levels during anoxia and H2O2 consumption and production rates of isolated heart mitochondria. We found that there was no excess production of in vivo H2O2 during 1 h of reoxygenation in turtles after 3 h anoxia at room temperature, suggesting that turtle hearts most likely do not suffer oxidative injury after anoxia because their mitochondria produce no excess O2.- upon reoxygenation. Instead, our data support the conclusion that both the low levels of succinate accumulation and the maintenance of ADP levels in the anoxic turtle heart are key factors in preventing the surge of O2.- production upon reoxygenation.

Keywords:  Anoxia; Heart; Mitochondria; Oxidative damage; Reactive oxygen species; Turtle

DOI:  https://doi.org/10.1242/jeb.245516
Methods Mol Biol. 2023 ;2662 219-239

Isolation and Mass Spectrometry-Based Profiling of Major Lipids in Brown Adipose Tissue.

Dongliang Lu, Hideji Fujiwara, Irfan J Lodhi, Fong-Fu Hsu.

  Brown adipose tissue (BAT) is an important regulator of metabolic homeostasis through its role in adaptive thermogenesis and control of whole-body glucose metabolism. Lipids play multiple roles in BAT functions, including serving as a fuel source for thermogenesis, mediating inter-organelle cross talk, and acting as BAT-derived signaling molecules that influence systemic energy metabolism. Profiling of various lipids in BAT under distinct metabolic states could provide new insights into their roles in the biology of the thermogenic fat. In this chapter, we describe a step-by-step workflow starting from sample preparations to mass spectrometry-based analysis of fatty acids and phospholipids in BAT.

Keywords:  Brown adipose tissue; Folch method; Free fatty acid; Mass spectrometry; Phospholipid; Solid phase extraction; Sphingolipid

DOI:  https://doi.org/10.1007/978-1-0716-3167-6_20
Nat Commun. 2023 04 17. 14(1): 2194

Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis.

Ziheng Chen, I-Lin Ho, Melinda Soeung, Er-Yen Yen, Jintan Liu, Liang Yan, Johnathon L Rose, Sanjana Srinivasan, Shan Jiang, Q Edward Chang, Ningping Feng, Jason P Gay, Qi Wang, Jing Wang, Philip L Lorenzi, Lucas J Veillon, Bo Wei, John N Weinstein, Angela K Deem, Sisi Gao, Giannicola Genovese, Andrea Viale, Wantong Yao, Costas A Lyssiotis, Joseph R Marszalek, Giulio F Draetta, Haoqiang Ying.

Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.

DOI: https://doi.org/10.1038/s41467-023-37924-9
J Clin Invest. 2023 Apr 17. pii: e163018. [Epub ahead of print]133(8):

Hepatocyte SREBP signaling mediates clock communication within the liver.

Dongyin Guan, Hosung Bae, Dishu Zhou, Ying Chen, Chunjie Jiang, Cam Mong La, Yang Xiao, Kun Zhu, Wenxiang Hu, Trang Minh Trinh, Panpan Liu, Ying Xiong, Bishuang Cai, Cholsoon Jang, Mitchell A Lazar.

  Rhythmic intraorgan communication coordinates environmental signals and the cell-intrinsic clock to maintain organ homeostasis. Hepatocyte-specific KO of core components of the molecular clock Rev-erbα and -β (Reverb-hDKO) alters cholesterol and lipid metabolism in hepatocytes as well as rhythmic gene expression in nonparenchymal cells (NPCs) of the liver. Here, we report that in fatty liver caused by diet-induced obesity (DIO), hepatocyte SREBP cleavage-activating protein (SCAP) was required for Reverb-hDKO-induced diurnal rhythmic remodeling and epigenomic reprogramming in liver macrophages (LMs). Integrative analyses of isolated hepatocytes and LMs revealed that SCAP-dependent lipidomic changes in REV-ERB-depleted hepatocytes led to the enhancement of LM metabolic rhythms. Hepatocytic loss of REV-ERBα and β (REV-ERBs) also attenuated LM rhythms via SCAP-independent polypeptide secretion. These results shed light on the signaling mechanisms by which hepatocytes regulate diurnal rhythms in NPCs in fatty liver disease caused by DIO.

Keywords:  Epigenetics; Hepatology; Homeostasis; Metabolism

DOI:  https://doi.org/10.1172/JCI163018
Cell Metab. 2023 Apr 12. pii: S1550-4131(23)00126-2. [Epub ahead of print]

Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness.

Li Gu, Yahui Zhu, Kosuke Watari, Maiya Lee, Junlai Liu, Sofia Perez, Melinda Thai, Joshua E Mayfield, Bichen Zhang, Karina Cunha E Rocha, Fuming Li, Laura C Kim, Alexander C Jones, Igor H Wierzbicki, Xiao Liu, Alexandra C Newton, Tatiana Kisseleva, Jun Hee Lee, Wei Ying, David J Gonzalez, Alan R Saltiel, M Celeste Simon, Michael Karin.

  Insulin inhibits gluconeogenesis and stimulates glucose conversion to glycogen and lipids. How these activities are coordinated to prevent hypoglycemia and hepatosteatosis is unclear. Fructose-1,6-bisphosphatase (FBP1) is rate controlling for gluconeogenesis. However, inborn human FBP1 deficiency does not cause hypoglycemia unless accompanied by fasting or starvation, which also trigger paradoxical hepatomegaly, hepatosteatosis, and hyperlipidemia. Hepatocyte FBP1-ablated mice exhibit identical fasting-conditional pathologies along with AKT hyperactivation, whose inhibition reversed hepatomegaly, hepatosteatosis, and hyperlipidemia but not hypoglycemia. Surprisingly, fasting-mediated AKT hyperactivation is insulin dependent. Independently of its catalytic activity, FBP1 prevents insulin hyperresponsiveness by forming a stable complex with AKT, PP2A-C, and aldolase B (ALDOB), which specifically accelerates AKT dephosphorylation. Enhanced by fasting and weakened by elevated insulin, FBP1:PP2A-C:ALDOB:AKT complex formation, which is disrupted by human FBP1 deficiency mutations or a C-terminal FBP1 truncation, prevents insulin-triggered liver pathologies and maintains lipid and glucose homeostasis. Conversely, an FBP1-derived complex disrupting peptide reverses diet-induced insulin resistance.

Keywords:  AKT; FBP1; hepatomegaly; hepatosteatosis

DOI:  https://doi.org/10.1016/j.cmet.2023.03.021
PNAS Nexus. 2023 Apr;2(4): pgad105

Reduced methionine synthase expression results in uracil accumulation in mitochondrial DNA and impaired oxidative capacity.

Katarina E Heyden, Joanna L Fiddler, Yuwen Xiu, Olga V Malysheva, Michal K Handzlik, Whitney N Phinney, Linsey Stiles, Sally P Stabler, Christian M Metallo, Marie A Caudill, Martha S Field.

  Adequate thymidylate [deoxythymidine monophosphate (dTMP) or the "T" base in DNA] levels are essential for stability of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Folate and vitamin B12 (B12) are essential cofactors in folate-mediated one-carbon metabolism (FOCM), a metabolic network which supports synthesis of nucleotides (including dTMP) and methionine. Perturbations in FOCM impair dTMP synthesis, causing misincorporation of uracil (or a "U" base) into DNA. During B12 deficiency, cellular folate accumulates as 5-methyltetrahdryfolate (5-methyl-THF), limiting nucleotide synthesis. The purpose of this study was to determine how reduced levels of the B12-dpendent enzyme methionine synthase (MTR) and dietary folate interact to affect mtDNA integrity and mitochondrial function in mouse liver. Folate accumulation, uracil levels, mtDNA content, and oxidative phosphorylation capacity were measured in male Mtr+/+ and Mtr+/- mice weaned onto either a folate-sufficient control (C) diet (2 mg/kg folic acid) or a folate-deficient (FD) diet (lacking folic acid) for 7 weeks. Mtr heterozygosity led to increased liver 5-methyl-THF levels. Mtr+/- mice consuming the C diet also exhibited a 40-fold increase in uracil in liver mtDNA. Mtr+/- mice consuming the FD diet exhibited less uracil accumulation in liver mtDNA as compared to Mtr+/+ mice consuming the FD diet. Furthermore, Mtr+/- mice exhibited 25% lower liver mtDNA content and a 20% lower maximal oxygen consumption rates. Impairments in mitochondrial FOCM are known to lead to increased uracil in mtDNA. This study demonstrates that impaired cytosolic dTMP synthesis, induced by decreased Mtr expression, also leads to increased uracil in mtDNA.

Keywords:  DNA; folate; methionine synthase; uracil; vitamin B12

DOI:  https://doi.org/10.1093/pnasnexus/pgad105
Sci Adv. 2023 04 21. 9(16): eadf9284

Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.

Subhadip Mukhopadhyay, Joel Encarnación-Rosado, Elaine Y Lin, Albert S W Sohn, Huan Zhang, Joseph D Mancias, Alec C Kimmelman.

Pancreatic ductal adenocarcinoma (PDAC) cells maintain a high level of autophagy, allowing them to thrive in an austere microenvironment. However, the processes through which autophagy promotes PDAC growth and survival are still not fully understood. Here, we show that autophagy inhibition in PDAC alters mitochondrial function by losing succinate dehydrogenase complex iron sulfur subunit B expression by limiting the availability of the labile iron pool. PDAC uses autophagy to maintain iron homeostasis, while other tumor types assessed require macropinocytosis, with autophagy being dispensable. We observed that cancer-associated fibroblasts can provide bioavailable iron to PDAC cells, promoting resistance to autophagy ablation. To overcome this cross-talk, we used a low-iron diet and demonstrated that this augmented the response to autophagy inhibition therapy in PDAC-bearing mice. Our work highlights a critical link between autophagy, iron metabolism, and mitochondrial function that may have implications for PDAC progression.

DOI: https://doi.org/10.1126/sciadv.adf9284
STAR Protoc. 2023 Apr 21. pii: S2666-1667(23)00135-1. [Epub ahead of print]4(2): 102177

Protocol for indirect and direct co-culture between human cancer cells and endothelial cells.

Yichen Guo, Bronte Miller, Michael Heim, Ana Gutierrez-Garcia, Renata Jaskula-Sztul, Bin Ren, Mary Kathryn Sewell-Loftin.

  The cross talk between cancer cells and endothelial cells (ECs) within the tumor microenvironment plays a critical role in tumor progression, recurrence, and cancer stemness. Here, we present a protocol containing two in vitro approaches to study such interactions. We first describe an indirect co-culture system to study the regulation of stemness markers in cancer cells by secreted factors from ECs. We then detail a direct co-culture system to study juxtracrine communications between the cell types. For complete details on the use and execution of this protocol, please refer to Sewell-Loftin et al.1 and Guo et al.2.

Keywords:  Biotechnology and Bioengineering; Cancer; Cell Biology; Cell Culture; Cell-based Assays; Microscopy; Molecular Biology; Tissue Engineering

DOI:  https://doi.org/10.1016/j.xpro.2023.102177
Cell. 2023 Apr 17. pii: S0092-8674(23)00300-8. [Epub ahead of print]

The proteomic landscape of genome-wide genetic perturbations.

Christoph B Messner, Vadim Demichev, Julia Muenzner, Simran K Aulakh, Natalie Barthel, Annika Röhl, Lucía Herrera-Domínguez, Anna-Sophia Egger, Stephan Kamrad, Jing Hou, Guihong Tan, Oliver Lemke, Enrica Calvani, Lukasz Szyrwiel, Michael Mülleder, Kathryn S Lilley, Charles Boone, Georg Kustatscher, Markus Ralser.

  Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

Keywords:  Saccharomyces cerevisiae; data-independent acquisition; deletion; functional genomics; functional proteomics; gene annotation; high throughput; knockout; quantitative proteomics; systems biology

DOI:  https://doi.org/10.1016/j.cell.2023.03.026
FEBS Open Bio. 2023 Apr 20.

Nitric oxide hinders club cell proliferation through Gdpd2 during allergic airway inflammation.

Qing Yue, Kuan Li, Zhaoyu Song, Qi Wang, Jianhai Wang, Xue Li, Yu Li, Qiuyang Zhang, Yu Zhu, Huaiyong Chen.

  Excessive nitric oxide (NO) is often observed in the airways of patients with severe asthma. Here, we show that the NO donor diethylamine NONOate impairs the proliferative capacity of mouse club cells and induces club cell apoptosis, cell cycle arrest, and alterations in lipid metabolism. Our data suggest that NO inhibits club cell proliferation via upregulation of Gdpd2 (glycerophosphodiester phosphodiesterase domain containing 2). During OVA challenge, apoptotic club cells are observed, but surviving club cells continue to proliferate. OVA exposure induces Gdpd2 expression; Gdpd2 knockout promotes the proliferation of club cells but inhibits goblet cell differentiation. Elimination of airway NO was found to inhibit goblet cell differentiation from club cells during OVA challenge. Our data reveal that excessive NO might be related to airway epithelial damage in severe asthma, and suggest that blockade of the NO-Gdpd2 pathway may be beneficial for airway epithelial restoration.

Keywords:  Ins1p1; epithelial progenitor cells; glycerin; lipid metabolism; nitric oxide

DOI:  https://doi.org/10.1002/2211-5463.13617
EMBO J. 2023 Apr 17. e113320

Cryo-EM structure of the polyphosphate polymerase VTC reveals coupling of polymer synthesis to membrane transit.

Wei Liu, Jiening Wang, Véronique Comte-Miserez, Mengyu Zhang, Xuejing Yu, Qingfeng Chen, Henning Jacob Jessen, Andreas Mayer, Shan Wu, Sheng Ye.

  The eukaryotic vacuolar transporter chaperone (VTC) complex acts as a polyphosphate (polyP) polymerase that synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the vacuolar membrane to maintain an intracellular phosphate (Pi ) homeostasis. To discover how the VTC complex performs its function, we determined a cryo-electron microscopy structure of an endogenous VTC complex (Vtc4/Vtc3/Vtc1) purified from Saccharomyces cerevisiae at 3.1 Å resolution. The structure reveals a heteropentameric architecture of one Vtc4, one Vtc3, and three Vtc1 subunits. The transmembrane region forms a polyP-selective channel, likely adopting a resting state conformation, in which a latch-like, horizontal helix of Vtc4 limits the entrance. The catalytic Vtc4 central domain is located on top of the pseudo-symmetric polyP channel, creating a strongly electropositive pathway for nascent polyP that can couple synthesis to translocation. The SPX domain of the catalytic Vtc4 subunit positively regulates polyP synthesis by the VTC complex. The noncatalytic Vtc3 regulates VTC through a phosphorylatable loop. Our findings, along with the functional data, allow us to propose a mechanism of polyP channel gating and VTC complex activation.

Keywords:  PP-InsP; VTC; coupled polymerase and translocase; cryo-EM; polyP

DOI:  https://doi.org/10.15252/embj.2022113320
Cell Rep. 2023 Apr 20. pii: S2211-1247(23)00435-7. [Epub ahead of print]42(5): 112424

CX3CR1hi macrophages sustain metabolic adaptation by relieving adipose-derived stem cell senescence in visceral adipose tissue.

Zixin Zhou, Huiying Zhang, Yan Tao, Haipeng Jie, Jingyuan Zhao, Jinhao Zang, Huijie Li, Yalin Wang, Tianci Wang, Hui Zhao, Yuan Li, Chun Guo, Faliang Zhu, Haiting Mao, Lining Zhang, Fengming Liu, Qun Wang.

  Adipose-derived stem cells (ASCs) drive healthy visceral adipose tissue (VAT) expansion via adipocyte hyperplasia. Obesity induces ASC senescence that causes VAT dysfunction and metabolic disorders. It is challenging to restrain this process by biological intervention, as mechanisms of controlling VAT ASC senescence remain unclear. We demonstrate that a population of CX3CR1hi macrophages is maintained in mouse VAT during short-term energy surplus, which sustains ASCs by restraining their senescence, driving adaptive VAT expansion and metabolic health. Long-term overnutrition induces diminishment of CX3CR1hi macrophages in mouse VAT accompanied by ASC senescence and exhaustion, while transferring CX3CR1hi macrophages restores ASC reservoir and triggers VAT beiging to alleviate the metabolic maladaptation. Mechanistically, visceral ASCs attract macrophages via MCP-1 and shape their CX3CR1hi phenotype via exosomes; these macrophages relieve ASC senescence by promoting the arginase1-eIF5A hypusination axis. These findings identify VAT CX3CR1hi macrophages as ASC supporters and unravel their therapeutic potential for metabolic maladaptation to obesity.

Keywords:  CP: Immunology; CP: Metabolism; CX3CR1; adipose-derived stem cell; cellular senescence; macrophage; obesity; visceral adipose tissue

DOI:  https://doi.org/10.1016/j.celrep.2023.112424
Cell Metab. 2023 Apr 14. pii: S1550-4131(23)00127-4. [Epub ahead of print]

Very-long-chain fatty acids induce glial-derived sphingosine-1-phosphate synthesis, secretion, and neuroinflammation.

Hyung-Lok Chung, Qi Ye, Ye-Jin Park, Zhongyuan Zuo, Jung-Wan Mok, Oguz Kanca, Sudhir Gopal Tattikota, Shenzhao Lu, Nobert Perrimon, Hyun Kyoung Lee, Hugo J Bellen.

  VLCFAs (very-long-chain fatty acids) are the most abundant fatty acids in myelin. Hence, during demyelination or aging, glia are exposed to higher levels of VLCFA than normal. We report that glia convert these VLCFA into sphingosine-1-phosphate (S1P) via a glial-specific S1P pathway. Excess S1P causes neuroinflammation, NF-κB activation, and macrophage infiltration into the CNS. Suppressing the function of S1P in fly glia or neurons, or administration of Fingolimod, an S1P receptor antagonist, strongly attenuates the phenotypes caused by excess VLCFAs. In contrast, elevating the VLCFA levels in glia and immune cells exacerbates these phenotypes. Elevated VLCFA and S1P are also toxic in vertebrates based on a mouse model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Indeed, reducing VLCFA with bezafibrate ameliorates the phenotypes. Moreover, simultaneous use of bezafibrate and fingolimod synergizes to improve EAE, suggesting that lowering VLCFA and S1P is a treatment avenue for MS.

Keywords:  NF-κB activation; VLCFA β-oxidation; fingolimod; lipid metabolism; multiple sclerosis; myelin lipid; neurodegeneration; neuroinflammation; sphingolipid; sphingosine 1-phosphate

DOI:  https://doi.org/10.1016/j.cmet.2023.03.022
Sci Rep. 2023 Apr 15. 13(1): 6134

G-protein coupled receptor 19 (GPR19) knockout mice display sex-dependent metabolic dysfunction.

Bellina A S Mushala, Bingxian Xie, Ian J Sipula, Michael W Stoner, Dharendra Thapa, Janet R Manning, Paramesha Bugga, Amber M Vandevender, Michael J Jurczak, Iain Scott.

G-protein coupled receptors (GPCRs) mediate signal transduction from the cellular surface to intracellular metabolic pathways. While the function of many GPCRs has been delineated previously, a significant number require further characterization to elucidate their cellular function. G-protein coupled receptor 19 (GPR19) is a poorly characterized class A GPCR which has been implicated in the regulation of circadian rhythm, tumor metastasis, and mitochondrial homeostasis. In this report, we use a novel knockout (KO) mouse model to examine the role of GPR19 in whole-body metabolic regulation. We show that loss of GPR19 promotes increased energy expenditure and decreased activity in both male and female mice. However, only male GPR19 KO mice display glucose intolerance in response to a high fat diet. Loss of GPR19 expression in male mice, but not female mice, resulted in diet-induced hepatomegaly, which was associated with decreased expression of key fatty acid oxidation genes in male GPR19 KO livers. Overall, our data suggest that loss of GPR19 impacts whole-body energy metabolism in diet-induced obese mice in a sex-dependent manner.

DOI: https://doi.org/10.1038/s41598-023-33308-7
Nat Commun. 2023 04 18. 14(1): 2215

Distinct spatial immune microlandscapes are independently associated with outcomes in triple-negative breast cancer.

Jodi M Carter, Saranya Chumsri, Douglas A Hinerfeld, Yaohua Ma, Xue Wang, David Zahrieh, David W Hillman, Kathleen S Tenner, Jennifer M Kachergus, Heather Ann Brauer, Sarah E Warren, David Henderson, Ji Shi, Yi Liu, Heikki Joensuu, Henrik Lindman, Roberto A Leon-Ferre, Judy C Boughey, Minetta C Liu, James N Ingle, Krishna R Kalari, Fergus J Couch, Keith L Knutson, Matthew P Goetz, Edith A Perez, E Aubrey Thompson.

The utility of spatial immunobiomarker quantitation in prognostication and therapeutic prediction is actively being investigated in triple-negative breast cancer (TNBC). Here, with high-plex quantitative digital spatial profiling, we map and quantitate intraepithelial and adjacent stromal tumor immune protein microenvironments in systemic treatment-naïve (female only) TNBC to assess the spatial context in immunobiomarker-based prediction of outcome. Immune protein profiles of CD45-rich and CD68-rich stromal microenvironments differ significantly. While they typically mirror adjacent, intraepithelial microenvironments, this is not uniformly true. In two TNBC cohorts, intraepithelial CD40 or HLA-DR enrichment associates with better outcomes, independently of stromal immune protein profiles or stromal TILs and other established prognostic variables. In contrast, intraepithelial or stromal microenvironment enrichment with IDO1 associates with improved survival irrespective of its spatial location. Antigen-presenting and T-cell activation states are inferred from eigenprotein scores. Such scores within the intraepithelial compartment interact with PD-L1 and IDO1 in ways that suggest prognostic and/or therapeutic potential. This characterization of the intrinsic spatial immunobiology of treatment-naïve TNBC highlights the importance of spatial microenvironments for biomarker quantitation to resolve intrinsic prognostic and predictive immune features and ultimately inform therapeutic strategies for clinically actionable immune biomarkers.

DOI: https://doi.org/10.1038/s41467-023-37806-0
J Immunol. 2023 Apr 19. pii: ji2300065. [Epub ahead of print]

IL-18 Binding Protein-Producing Cells Attenuate Anemia in Murine Macrophage Activation Syndrome.

Mathilde Harel, Sébastien Fauteux-Daniel, Emiliana Rodriguez, Gaby Palmer, Cem Gabay.

IL-18 is a pleiotropic immunoregulatory cytokine of the IL-1 family. IL-18 has been identified as a potent IFN-γ inducer in synergy with IL-12 and IL-15 and thus as a powerful Th1 cell-polarizing cytokine. IL-18 activity is regulated by its naturally occurring soluble inhibitor IL-18 binding protein (IL-18BP), the production of which is stimulated by IFN-γ in a negative feedback loop. Circulating levels of IL-18BP are elevated, and unbound bioactive free IL-18 is thus not detectable in the circulation in physiologic conditions. However, emerging evidence indicates that the IL-18/IL-18BP balance could be dysregulated in macrophage activation syndrome (MAS), as mirrored by the presence of free IL-18 in the circulation of patients with MAS. Herein, we sought to identify IL-18BP-producing cells in a murine CpG-induced MAS model using IL-18BP knock-in tdTomato reporter mice. Endothelial cells, tissue-resident macrophages, and neutrophils appeared as major cellular sources of IL-18BP. We also identified extramedullary and medullary early erythroid progenitors as IL-18BP-producing cells in an IFN-γ-dependent manner. This finding suggests a novel regulation of IL-18 activity by erythroid precursors, which are likely involved in the prevention of the negative effects of IL-18 on erythropoiesis. Indeed, coherent in vivo and in vitro results indicate that IL-18 indirectly impairs erythropoiesis while favoring myelopoiesis and thus contributes to anemia associated with MAS and potentially with other IL-18-driven inflammatory diseases. In conclusion, IL-18BP production by endothelial cells, neutrophils, macrophages, and erythroid precursors attenuates the anemia associated with murine CpG-induced MAS.

DOI: https://doi.org/10.4049/jimmunol.2300065
Nat Metab. 2023 Apr 20.

Amino acids downregulate SIRT4 to detoxify ammonia through the urea cycle.

Song-Hua Hu, Yu-Yang Feng, Yuan-Xin Yang, Hui-Da Ma, Shu-Xian Zhou, Ya-Nan Qiao, Kai-Hui Zhang, Lei Zhang, Lin Huang, Yi-Yuan Yuan, Yan Lin, Xin-Yan Zhang, Yao Li, Hai-Tao Li, Jian-Yuan Zhao, Wei Xu, Shi-Min Zhao.

Ammonia production via glutamate dehydrogenase is inhibited by SIRT4, a sirtuin that displays both amidase and non-amidase activities. The processes underlying the regulation of ammonia removal by amino acids remain unclear. Here, we report that SIRT4 acts as a decarbamylase that responds to amino acid sufficiency and regulates ammonia removal. Amino acids promote lysine 307 carbamylation (OTCCP-K307) of ornithine transcarbamylase (OTC), which activates OTC and the urea cycle. Proteomic and interactome screening identified OTC as a substrate of SIRT4. SIRT4 decarbamylates OTCCP-K307 and inactivates OTC in an NAD+-dependent manner. SIRT4 expression was transcriptionally upregulated by the amino acid insufficiency-activated GCN2-eIF2α-ATF4 axis. SIRT4 knockout in cultured cells caused higher OTCCP-K307 levels, activated OTC, elevated urea cycle intermediates and urea production via amino acid catabolism. Sirt4 ablation decreased male mouse blood ammonia levels and ameliorated CCl4-induced hepatic encephalopathy phenotypes. We reveal that SIRT4 safeguards cellular ammonia toxicity during amino acid catabolism.

DOI: https://doi.org/10.1038/s42255-023-00784-0