bims-mepmim 2022-09-25 papers

bims-mepmim

Biomed News

on Metabolites in pathological microenvironments and immunometabolism

Issue of 2022–09–25
forty papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism

"Blocking lipid uptake pathways does not prevent toxicity in adipose triglyceride lipase (ATGL) deficiency".
Formyl-Peptide Receptor 2 Signaling Redirects Glucose and Glutamine into Anabolic Pathways in Metabolic Reprogramming of Lung Cancer Cells.
1-deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells.
Adipose tissue macrophage in obesity-associated metabolic diseases.
Impact of Inhibition of Glutamine and Alanine Transport on Cerebellar Glial and Neuronal Metabolism.
Polarization and β-Glucan Reprogram Immunomodulatory Metabolism in Human Macrophages and Ex Vivo in Human Lung Cancer Tissues.
Bone marrow adipocytes drive the development of tissue invasive Ly6Chigh monocytes during obesity.
Evaluation of Urea Cycle Activity by Metabolic Flux Analysis Using Mass Spectrometry.
Stress-mediated attenuation of translation undermines T-cell activity in cancer.
Targeting the Metabolic Rewiring in Pancreatic Cancer and Its Tumor Microenvironment.
Targeting lipid metabolism reprogramming of immunocytes in response to the tumor microenvironment stressor: A potential approach for tumor therapy.
Isotopic tracing of glucose metabolites in human monocytes to assess changes in inflammatory conditions.
SLC7A1 Overexpression Is Involved in Energy Metabolism Reprogramming to Induce Tumor Progression in Epithelial Ovarian Cancer and Is Associated with Immune-Infiltrating Cells.
Targeted Proteomics for Monitoring One-Carbon Metabolism in Liver Diseases.
The immune landscape of human thymic epithelial tumors.
Inhibition of O-GlcNAcase inhibits hematopoietic and leukemic stem cell self-renewal and drives dendritic cell differentiation via STAT3/5 signaling.
Novel Role of CETP in Macrophages: Reduction of Mitochondrial Oxidants Production and Modulation of Cell Immune-Metabolic Profile.
NAT10: An RNA cytidine transferase regulates fatty acid metabolism in cancer cells.
Protocol to drive human monocyte-to-macrophage polarization in vitro using tumor conditioned media.
Metabolic collateral lethal target identification reveals MTHFD2 paralogue dependency in ovarian cancer.
Liquid chromatography coupled to high-resolution mass spectrometry metabolomics: A useful tool for investigating tumor secretome based on a three-dimensional co-culture model.
Analyses and Localization of Serotonin and L-DOPA in Ocular Tissues by Imaging Mass Spectrometry.
NMR-Based Metabolomic Imprinting Elucidates Macrophage Polarization of THP-1 Cell Lines Stimulated by Zinc Oxide Nanoparticles.
Crosstalk between Hypoxia and Extracellular Matrix in the Tumor Microenvironment in Breast Cancer.
A Mitofusin 2 - Hif1α axis sets a maturation checkpoint in regenerating skeletal muscle.
Emerging Roles of Lipophagy in Cancer Metastasis.
Nitric Oxide, Nitric Oxide Formers and Their Physiological Impacts in Bacteria.
Altered intraperitoneal immune microenvironment in patients with peritoneal metastases from gastric cancer.
Tandem Mass Spectrometry for the Analysis of Plasma/Serum Acylcarnitines for the Diagnosis of Certain Organic Acidurias and Fatty Acid Oxidation Disorders.
A cellular hierarchy in melanoma uncouples growth and metastasis.
Flow pattern-dependent mitochondrial dynamics regulates the metabolic profile and inflammatory state of endothelial cells.
COX-2 Expression in Hepatocytes Improves Mitochondrial Function after Hepatic Ischemia-Reperfusion Injury.
The contribution of astrocytes to obesity-associated metabolic disturbances.
Farnesysltransferase Inhibitor Prevents Burn Injury-Induced Metabolome Changes in Muscle.
Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and type I interferon to promote anti-tumor T cell responses.
Oxygen regulation of breathing is abolished in mitochondrial complex III-deficient arterial chemoreceptors.
Transwell migration assay to interrogate human CAR-T cell chemotaxis.
Recent Development of the Molecular and Cellular Mechanisms of Hydrogen Sulfide Gasotransmitter.
There is no direct competition between arginase and nitric oxide synthase for the common substrate l-arginine.
Activated Hepatic Stellate Cells Promote the M1 to M2 Macrophage Transformation and Liver Fibrosis by Elevating the Histone Acetylation Level.

J Lipid Res. 2022 Sep 14. pii: S0022-2275(22)00107-9. [Epub ahead of print] 100274

"Blocking lipid uptake pathways does not prevent toxicity in adipose triglyceride lipase (ATGL) deficiency".

Jide Oluwadare, Ainara G Cabodevilla, Ni-Huiping Son, Yunying Hu, Adam E Mullick, Michael Verano, Jose O Alemán, Ravichandran Ramasamy, Ira J Goldberg.

  Lipid accumulation in non-adipose tissues can cause lipotoxicity, leading to cell death and severe organ dysfunction. Adipose triglyceride lipase (ATGL) deficiency causes human Neutral Lipid Storage Disease and leads to cardiomyopathy; ATGL deficiency has no current treatment. One possible approach to alleviate this disorder has been to alter the diet and reduce the supply of dietary lipids and, hence, myocardial lipid uptake. However, in this study, when we supplied cardiac Atgl knockout mice a low- or high-fat diet, we found heart lipid accumulation, heart dysfunction, and death were not altered. We next deleted lipid uptake pathways in the ATGL-deficient mice through the generation of double knockout mice also deficient in either cardiac lipoprotein lipase (LpL) or cluster of differentiation (CD) 36, which is involved in an LpL-independent pathway for fatty acid uptake in the heart. We show neither deletion ameliorated ATGL-deficient heart dysfunction. Similarly, we determined non-lipid-containing media did not prevent lipid accumulation by cultured myocytes; rather, the cells switched to increased de novo fatty acid synthesis. Thus, we conclude pathological storage of lipids in ATGL deficiency cannot be corrected by reducing heart lipid uptake.

Keywords:  CD36; LpL; dietary fat; fatty acid synthesis; heart failure; lipid accumulation; lipid droplets; lipotoxicity; myocardial lipid uptake; storage diseases

DOI:  https://doi.org/10.1016/j.jlr.2022.100274
Antioxidants (Basel). 2022 Aug 29. pii: 1692. [Epub ahead of print]11(9):

Formyl-Peptide Receptor 2 Signaling Redirects Glucose and Glutamine into Anabolic Pathways in Metabolic Reprogramming of Lung Cancer Cells.

Tiziana Pecchillo Cimmino, Ester Pagano, Mariano Stornaiuolo, Gabriella Esposito, Rosario Ammendola, Fabio Cattaneo.

  Glucose and glutamine play a crucial role in the metabolic reprogramming of cancer cells. Proliferating cells metabolize glucose in the aerobic glycolysis for energy supply, and glucose and glutamine represent the primary sources of carbon atoms for the biosynthesis of nucleotides, amino acids, and lipids. Glutamine is also an important nitrogen donor for the production of nucleotides, amino acids, and nicotinamide. Several membrane receptors strictly control metabolic reprogramming in cancer cells and are considered new potential therapeutic targets. Formyl-peptide receptor 2 (FPR2) belongs to a small family of GPCRs and is implicated in many physiopathological processes. Its stimulation induces, among other things, NADPH oxidase-dependent ROS generation that, in turn, contributes to intracellular signaling. Previously, by phosphoproteomic analysis, we observed that numerous proteins involved in energetic metabolism are uniquely phosphorylated upon FPR2 stimulation. Herein, we investigated the role of FPR2 in cell metabolism, and we observed that the concentrations of several metabolites associated with the pentose phosphate pathway (PPP), tricarboxylic acid cycle, nucleotide synthesis, and glutamine metabolism, were significantly enhanced in FPR2-stimulated cells. In particular, we found that the binding of specific FPR2 agonists: (i) promotes NADPH production; (ii) activates the non-oxidative phase of PPP; (iii) induces the expression of the ASCT2 glutamine transporter; (iv) regulates oxidative phosphorylation; and (v) induces the de novo synthesis of pyrimidine nucleotides, which requires FPR2-dependent ROS generation.

Keywords:  NADPH oxidase; formyl-peptide receptors; glucose metabolism; glutamine transporter; metabolic reprogramming; synthesis of pyrimidine nucleotides

DOI:  https://doi.org/10.3390/antiox11091692
J Lipid Res. 2022 Sep 14. pii: S0022-2275(22)00114-6. [Epub ahead of print] 100281

1-deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells.

Thekla Cordes, Ramya S Kuna, Grace H McGregor, Sanika V Khare, Jivani Gengatharan, Thangaselvam Muthusamy, Christian M Metallo.

  Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I (HSAN1), resulting in the synthesis and accumulation of 1-deoxysphingolipids. These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxysphingolipid synthesis, they impact numerous other metabolic pathways important for cancer cells. Here we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxysphingolipid toxicity in cancer cells. We determined that both alanine treatment and SPTLC1C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxysphingolipid synthesis was induced via SPTLC1C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxysphingolipid synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.

Keywords:  1-deoxy(dihydro)ceramide; 1-deoxysphingolipid accumulation; Metabolism; RAB5; SPT promiscuity; alanine; mitochondrial stress; serine; serine palmitoyltransferase; soft agar

DOI:  https://doi.org/10.1016/j.jlr.2022.100281
Front Immunol. 2022 ;13 977485

Adipose tissue macrophage in obesity-associated metabolic diseases.

Jingfei Yao, Dongmei Wu, Yifu Qiu.

  Adipose tissue macrophage (ATM) has been appreciated for its critical contribution to obesity-associated metabolic diseases in recent years. Here, we discuss the regulation of ATM on both metabolic homeostatsis and dysfunction. In particular, the macrophage polarization and recruitment as well as the crosstalk between ATM and adipocyte in thermogenesis, obesity, insulin resistance and adipose tissue fibrosis have been reviewed. A better understanding of how ATM regulates adipose tissue remodeling may provide novel therapeutic strategies against obesity and associated metabolic diseases.

Keywords:  adaptive thermogenesis; adipokines; adipose tissue macrophage; fibrosis; insulin resistance; obesity

DOI:  https://doi.org/10.3389/fimmu.2022.977485
Biomolecules. 2022 Aug 27. pii: 1189. [Epub ahead of print]12(9):

Impact of Inhibition of Glutamine and Alanine Transport on Cerebellar Glial and Neuronal Metabolism.

Abhijit Das, Gregory Gauthier-Coles, Stefan Bröer, Caroline D Rae.

  The cerebellum, or "little brain", is often overlooked in studies of brain metabolism in favour of the cortex. Despite this, anomalies in cerebellar amino acid homeostasis in a range of disorders have been reported. Amino acid homeostasis is central to metabolism, providing recycling of carbon backbones and ammonia between cell types. Here, we examined the role of cerebellar amino acid transporters in the cycling of glutamine and alanine in guinea pig cerebellar slices by inhibiting amino acid transporters and examining the resultant metabolism of [1-13C]d-glucose and [1,2-13C]acetate by NMR spectroscopy and LCMS. While the lack of specific inhibitors of each transporter makes interpretation difficult, by viewing results from experiments with multiple inhibitors we can draw inferences about the major cell types and transporters involved. In cerebellum, glutamine and alanine transfer is dominated by system A, blockade of which has maximum effect on metabolism, with contributions from System N. Inhibition of neural system A isoform SNAT1 by MeAIB resulted in greatly decreased metabolite pools and reduced net fluxes but showed little effect on fluxes from [1,2-13C]acetate unlike inhibition of SNAT3 and other glutamine transporters by histidine where net fluxes from [1,2-13C]acetate are reduced by ~50%. We interpret the data as further evidence of not one but several glutamate/glutamine exchange pools. The impact of amino acid transport inhibition demonstrates that the cerebellum has tightly coupled cells and that glutamate/glutamine, as well as alanine cycling, play a major role in that part of the brain.

Keywords:  alanine; amino acid transporters; glutamine

DOI:  https://doi.org/10.3390/biom12091189
J Immunol. 2022 Sep 23. pii: ji2200178. [Epub ahead of print]

Polarization and β-Glucan Reprogram Immunomodulatory Metabolism in Human Macrophages and Ex Vivo in Human Lung Cancer Tissues.

Teresa W-M Fan, Saeed Daneshmandi, Teresa A Cassel, Mohammad B Uddin, James Sledziona, Patrick T Thompson, Penghui Lin, Richard M Higashi, Andrew N Lane.

Immunomodulatory (IM) metabolic reprogramming in macrophages (Mϕs) is fundamental to immune function. However, limited information is available for human Mϕs, particularly in response plasticity, which is critical to understanding the variable efficacy of immunotherapies in cancer patients. We carried out an in-depth analysis by combining multiplex stable isotope-resolved metabolomics with reversed phase protein array to map the dynamic changes of the IM metabolic network and key protein regulators in four human donors' Mϕs in response to differential polarization and M1 repolarizer β-glucan (whole glucan particles [WGPs]). These responses were compared with those of WGP-treated ex vivo organotypic tissue cultures (OTCs) of human non-small cell lung cancer. We found consistently enhanced tryptophan catabolism with blocked NAD+ and UTP synthesis in M1-type Mϕs (M1-Mϕs), which was associated with immune activation evidenced by increased release of IL-1β/CXCL10/IFN-γ/TNF-α and reduced phagocytosis. In M2a-Mϕs, WGP treatment of M2a-Mϕs robustly increased glucose utilization via the glycolysis/oxidative branch of the pentose phosphate pathway while enhancing UDP-N-acetyl-glucosamine turnover and glutamine-fueled gluconeogenesis, which was accompanied by the release of proinflammatory IL-1β/TNF-α to above M1-Mϕ's levels, anti-inflammatory IL-10 to above M2a-Mϕ's levels, and attenuated phagocytosis. These IM metabolic responses could underlie the opposing effects of WGP, i.e., reverting M2- to M1-type immune functions but also boosting anti-inflammation. Variable reprogrammed Krebs cycle and glutamine-fueled synthesis of UTP in WGP-treated OTCs of human non-small cell lung cancer were observed, reflecting variable M1 repolarization of tumor-associated Mϕs. This was supported by correlation with IL-1β/TNF-α release and compromised tumor status, making patient-derived OTCs unique models for studying variable immunotherapeutic efficacy in cancer patients.

DOI: https://doi.org/10.4049/jimmunol.2200178
Elife. 2022 Sep 20. pii: e65553. [Epub ahead of print]11

Bone marrow adipocytes drive the development of tissue invasive Ly6Chigh monocytes during obesity.

Parastoo Boroumand, David C Prescott, Tapas Mukherjee, Philip J Bilan, Michael Wong, Jeff Shen, Ivan Tattoli, Yuhuan Zhou, Angela Li, Tharini Sivasubramaniyam, Nan Shi, Lucie Y Zhu, Zhi Liu, Clinton Robbins, Dana J Philpott, Stephen E Girardin, Amira Klip.

  During obesity and high fat-diet (HFD) feeding in mice, sustained low-grade inflammation includes not only increased pro-inflammatory macrophages in the expanding adipose tissue, but also bone marrow (BM) production of invasive Ly6Chigh monocytes. As BM adiposity also accrues with HFD, we explored the relationship between the gains in BM white adipocytes and invasive Ly6Chigh monocytes in vivo and through ex vivo paradigms. We find a temporal and causal link between BM adipocyte whitening and the Ly6Chigh monocyte surge, preceding the adipose tissue macrophage rise during HFD. Phenocopying this, ex vivo treatment of BM cells with conditioned media from BM adipocytes or from bona fide white adipocytes favoured Ly6Chigh monocyte preponderance. Notably, Ly6Chigh skewing was preceded by monocyte metabolic reprogramming towards glycolysis, reduced oxidative potential and increased mitochondrial fission. In sum, short-term HFD changes BM cellularity, resulting in local adipocyte whitening driving a gradual increase and activation of invasive Ly6Chigh monocytes.

Keywords:  immunology; inflammation; medicine; mouse

DOI:  https://doi.org/10.7554/eLife.65553
Methods Mol Biol. 2022 ;2544 129-144

Evaluation of Urea Cycle Activity by Metabolic Flux Analysis Using Mass Spectrometry.

Yasuharu Ueno, Takuji Maeda, Satoshi Okamoto, Hideki Taniguchi.

  Hepatocytes play an important role in maintaining homeostasis in living organisms by carrying out various metabolic functions. The urea cycle, one of the metabolic pathways taking place in hepatocytes, is an important metabolic pathway that converts toxic ammonia to nontoxic urea. Performing quantitative assessments of individual metabolite levels using a mass spectrometer is useful for assessing the metabolic state of the urea cycle in hepatocytes. In addition, metabolic flux analysis using stable isotopes and a mass spectrometer is a new technique for measuring the metabolic state. It enables conducting specific, objective, and quantitative measurement of the activated state of the target metabolic pathway regardless of external disturbing factors. This section describes the technical background and methodology of performing metabolic flux analysis of the urea cycle by mass spectrometry.

Keywords:  Hepatocytes; Metabolic flux analysis; Urea cycle

DOI:  https://doi.org/10.1007/978-1-0716-2557-6_9
Cancer Res. 2022 Sep 20. pii: CAN-22-1744. [Epub ahead of print]

Stress-mediated attenuation of translation undermines T-cell activity in cancer.

Brian P Riesenberg, Elizabeth G Hunt, Megan D Tennant, Katie E Hurst, Alex M Andrews, Lee R Leddy, David M Neskey, Elizabeth G Hill, Guillermo O Rangel Rivera, Chrystal M Paulos, Peng Gao, Jessica E Thaxton.

Protein synthesis supports robust immune responses. Nutrient competition and global cell stressors in the tumor microenvironment (TME) may impact protein translation in T cells and antitumor immunity. Using human and mouse tumors, we demonstrated here that protein translation in T cells is repressed in solid tumors. Reduced glucose availability to T cells in the TME led to activation of the unfolded protein response (UPR) element eIF2a. Genetic mouse models revealed that translation attenuation mediated by activated p-eIF2a undermines the ability of T cells to suppress tumor growth. Reprogramming T cell metabolism was able to alleviate p-eIF2a accumulation and translational attenuation in the TME, allowing for sustained protein translation. Metabolic and pharmacological approaches showed that proteasome activity mitigates induction of p-eIF2a to support optimal antitumor T cell function, protecting from translation attenuation and enabling prolonged cytokine synthesis in solid tumors. Together, these data identify a new therapeutic avenue to fuel the efficacy of tumor immunotherapy.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-1744
Cancers (Basel). 2022 Sep 07. pii: 4351. [Epub ahead of print]14(18):

Targeting the Metabolic Rewiring in Pancreatic Cancer and Its Tumor Microenvironment.

Keisuke Yamamoto, Dosuke Iwadate, Hiroyuki Kato, Yousuke Nakai, Keisuke Tateishi, Mitsuhiro Fujishiro.

  Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with only a few effective therapeutic options. A characteristic feature of PDAC is its unique tumor microenvironment (TME), termed desmoplasia, which shows extensive fibrosis and extracellular matrix deposition, generating highly hypoxic and nutrient-deprived conditions within the tumor. To thrive in this harsh TME, PDAC undergoes extensive metabolic rewiring that includes the altered use of glucose and glutamine, constitutive activation of autophagy-lysosomal pathways, and nutrient acquisition from host cells in the TME. Notably, these properties support PDAC metabolism and mediate therapeutic resistance, including immune suppression. A deeper understanding of the unique metabolic properties of PDAC and its TME may aid in the development of novel therapeutic strategies against this deadly disease.

Keywords:  KRAS; MHC-I; anabolic glucose metabolism; autophagy; glutamine metabolism; glycolysis; hexosamine biosynthesis pathway; immune checkpoint blockade; immune evasion; lysosome; macropinocytosis; metabolic rewiring; pancreatic ductal adenocarcinoma; pentose phosphate pathway; serine biosynthesis pathway; tumor microenvironment

DOI:  https://doi.org/10.3390/cancers14184351
Front Immunol. 2022 ;13 937406

Targeting lipid metabolism reprogramming of immunocytes in response to the tumor microenvironment stressor: A potential approach for tumor therapy.

Ming Zhang, Tingju Wei, Xiaodan Zhang, Danfeng Guo.

  The tumor microenvironment (TME) has become a major research focus in recent years. The TME differs from the normal extracellular environment in parameters such as nutrient supply, pH value, oxygen content, and metabolite abundance. Such changes may promote the initiation, growth, invasion, and metastasis of tumor cells, in addition to causing the malfunction of tumor-infiltrating immunocytes. As the neoplasm develops and nutrients become scarce, tumor cells transform their metabolic patterns by reprogramming glucose, lipid, and amino acid metabolism in response to various environmental stressors. Research on carcinoma metabolism reprogramming suggests that like tumor cells, immunocytes also switch their metabolic pathways, named "immunometabolism", a phenomenon that has drawn increasing attention in the academic community. In this review, we focus on the recent progress in the study of lipid metabolism reprogramming in immunocytes within the TME and highlight the potential target molecules, pathways, and genes implicated. In addition, we discuss hypoxia, one of the vital altered components of the TME that partially contribute to the initiation of abnormal lipid metabolism in immune cells. Finally, we present the current immunotherapies that orchestrate a potent antitumor immune response by mediating the lipid metabolism of immunocytes, highlight the lipid metabolism reprogramming capacity of various immunocytes in the TME, and propose promising new strategies for use in cancer therapy.

Keywords:  immunocyte; immunometabolism; immunotherapy; lipid metabolism reprogramming; tumor microenvironment

DOI:  https://doi.org/10.3389/fimmu.2022.937406
STAR Protoc. 2022 Sep 22. pii: S2666-1667(22)00595-0. [Epub ahead of print]3(4): 101715

Isotopic tracing of glucose metabolites in human monocytes to assess changes in inflammatory conditions.

Ginevra Giacomello, Chotima Böttcher, Maria Kristina Parr.

  Differences in metabolic profiles can link to functional changes of immune cells in disease conditions. Here, we detail a protocol for the detection and quantitation of 19 metabolites in one analytical run. We provide the parameters for chromatographic separation and mass spectrometric analysis of isotopically labeled and unlabeled metabolites. We include steps for incubation and sample preparation of PBMCs and monocytes. This protocol overcomes the chromatographic challenges caused by the chelating properties of some metabolites.

Keywords:  Chemistry; Immunology; Mass spectrometry; Metabolism; Metabolomics

DOI:  https://doi.org/10.1016/j.xpro.2022.101715
J Oncol. 2022 ;2022 5864826

SLC7A1 Overexpression Is Involved in Energy Metabolism Reprogramming to Induce Tumor Progression in Epithelial Ovarian Cancer and Is Associated with Immune-Infiltrating Cells.

Shijing You, Xiaoying Zhu, Yao Yang, XiuZhen Du, Kejuan Song, Qingmei Zheng, Pengjiao Zeng, Qin Yao.

Cationic amino acid transporters (SLC7A1/CAT1) are highly expressed in human ovarian cancer (OC) tissues. However, the specific biological functions and mechanisms involved remain unclear. We used bioinformatics analysis to explore SLC7A1 expression level, prognostic value, and tumor mutation burden (TMB) in ovarian cancer (OC) tissues. We performed in vitro experiments to identify the expression and biological function of SLC7A1 in epithelial ovarian cancer (EOC) tissues and cells. An amino acid autoanalyzer was used to detect the effect of SLC7A1 on amino acid metabolism in EOC cells. Finally, SLC7A1 in OC was evaluated for cell-to-cell signalling and immune infiltration using online databases. We found that increased SLC7A1 expression in EOC cells and tissues was associated with poorer survival outcomes (P < 0.05) but not with tumor stage or grade of OC (P > 0.05). SLC7A1 is involved in the transport of phenylalanine and arginine in EOC cells, and its knockdown reduced the proliferation and migration of EOC cells and the resistance of cells to cisplatin. Furthermore, the TIMER database indicated that SLC7A1 overexpression was significantly positively correlated with levels of CD4+ memory resting cells, CD8+ effector memory cells, M0 macrophages, and cancer-associated fibroblasts (CAFs) in OC (P < 0.05) and significantly negatively correlated with CD4+ memory-activated cells (P < 0.05). Cell immunofluorescence indicated that SLC7A1 overexpression may affect the distribution of immune-infiltrating lymphocytes in tumors by inhibiting the expression of CCL4. Therefore, we concluded that SLC7A1 is involved in the metabolic remodelling of amino acids in EOC to promote tumor development and cisplatin resistance and is related to the tumor-infiltrating immune microenvironment of OC. SLC7A1 is a biomarker for predicting EOC progression and cisplatin resistance and represents a promising target for EOC treatment.

DOI: https://doi.org/10.1155/2022/5864826
Metabolites. 2022 Aug 24. pii: 779. [Epub ahead of print]12(9):

Targeted Proteomics for Monitoring One-Carbon Metabolism in Liver Diseases.

Laura Guerrero, Alberto Paradela, Fernando J Corrales.

  Liver diseases cause approximately 2 million deaths per year worldwide and had an increasing incidence during the last decade. Risk factors for liver diseases include alcohol consumption, obesity, diabetes, the intake of hepatotoxic substances like aflatoxin, viral infection, and genetic determinants. Liver cancer is the sixth most prevalent cancer and the third in mortality (second in males). The low survival rate (less than 20% in 5 years) is partially explained by the late diagnosis, which remarks the need for new early molecular biomarkers. One-carbon metabolism integrates folate and methionine cycles and participates in essential cell processes such as redox homeostasis maintenance and the regulation of methylation reactions through the production of intermediate metabolites such as cysteine and S-Adenosylmethionine. One-carbon metabolism has a tissue specific configuration, and in the liver, the participating enzymes are abundantly expressed-a requirement to maintain hepatocyte differentiation. Targeted proteomics studies have revealed significant differences in hepatocellular carcinoma and cirrhosis, suggesting that monitoring one-carbon metabolism enzymes can be useful for stratification of liver disease patients and to develop precision medicine strategies for their clinical management. Here, reprogramming of one-carbon metabolism in liver diseases is described and the role of mass spectrometry to follow-up these alterations is discussed.

Keywords:  hepatocellular carcinoma; liver disease; one-carbon metabolism; proteomics

DOI:  https://doi.org/10.3390/metabo12090779
Nat Commun. 2022 Sep 17. 13(1): 5463

The immune landscape of human thymic epithelial tumors.

Zhongwei Xin, Mingjie Lin, Zhixing Hao, Di Chen, Yongyuan Chen, Xiaoke Chen, Xia Xu, Jinfan Li, Dang Wu, Ying Chai, Pin Wu.

Human thymic epithelial tumors (TET) are common malignancies in the anterior mediastinum with limited biological understanding. Here we show, by single cell analysis of the immune landscape, that the developmental pattern of intra-tumoral T-cells identify three types within TETs. We characterize the developmental alterations and TCR repertoires of tumor-infiltrating T cells in the context of the distinguishing epithelial tumor cell types. We demonstrate that a subset of tumor cells, featuring medullary thymic epithelial cell (TEC) phenotype and marked by KRT14/GNB3 expression, accumulate in type 1 TETs, while T-cell positive selection is inhibited. Type 2 TETs are dominated by CCL25+ cortical TEC-like cells that appear to promote T-cell positive selection. Interestingly, the CHI3L1+ medullary TEC-like cells that are the characteristic feature of type 3 TETs don't seem to support T-cell development, however, they may induce a tissue-resident CD8+ T cell response. In summary, our work suggests that the molecular subtype of epithelial tumour cells in TETs determine their tumour immune microenvironment, thus GNB3 and CHI3L1 might predict the immunological behavior and hence prognosis of these tumours.

DOI: https://doi.org/10.1038/s41467-022-33170-7
Stem Cells. 2022 Sep 19. pii: sxac068. [Epub ahead of print]

Inhibition of O-GlcNAcase inhibits hematopoietic and leukemic stem cell self-renewal and drives dendritic cell differentiation via STAT3/5 signaling.

Sudjit Luanpitpong, Napachai Rodboon, Parinya Samart, Montira Janan, Phatchanat Klaihmon, Chanchao Lorthongpanich, Yaowalak U-Pratya, Surapol Issaragrisil.

  Myeloid differentiation blockage at immature and self-renewing stages is a common hallmark across all subtypes of acute myeloid leukemia (AML), despite their genetic heterogeneity. Metabolic state is an important regulator of hematopoietic stem cell (HSC) self-renewal and lineage-specific differentiation as well as several aggressive cancers. However, how O-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, contributes to both normal myelopoiesis and AML pathogenesis remains largely unknown. Using small molecule inhibitors and the CRISPR/Cas9 system, we reveal for the first time that inhibition of either OGA or OGT, which subsequently caused an increase or decrease in cellular O-GlcNAcylation, inhibits the self-renewal and maintenance of CD34 + hematopoietic stem/progenitor cells (HSPCs) and leukemic stem/progenitor cells and drives normal and malignant myeloid differentiation. We further unveiled the distinct roles of OGA and OGT inhibition in lineage-specific differentiation. While OGT inhibition induces macrophage differentiation, OGA inhibition promotes the differentiation of both CD34 + HSPCs and AML cells into dendritic cells (DCs), in agreement with an upregulation of a multitude of genes involved in DC development and function and their ability to induce T cell proliferation, via STAT3/5 signaling. Our novel findings provide significant basic knowledge that could be important in understanding AML pathogenesis and overcoming differentiation blockage-agnostic to the genetic background of AML. Additionally, the parallel findings in normal HSPCs may lay the groundwork for future cellular therapy as a means to improve the ex vivo differentiation of normal DCs and macrophages.

Keywords:   O-GlcNAcylation; acute myeloid leukemia; dendritic cell; differentiation therapy; hematopoiesis; hematopoietic stem cells; leukemic stem cells; macrophage; myeloid differentiation

DOI:  https://doi.org/10.1093/stmcls/sxac068
Antioxidants (Basel). 2022 Aug 31. pii: 1734. [Epub ahead of print]11(9):

Novel Role of CETP in Macrophages: Reduction of Mitochondrial Oxidants Production and Modulation of Cell Immune-Metabolic Profile.

Gabriel G Dorighello, Leandro H P Assis, Thiago Rentz, Joseane Morari, Monique F M Santana, Marisa Passarelli, Neale D Ridgway, Anibal E Vercesi, Helena C F Oliveira.

  Plasma cholesteryl ester transfer protein (CETP) activity diminishes HDL-cholesterol levels and thus may increase atherosclerosis risk. Experimental evidence suggests CETP may also exhibit anti-inflammatory properties, but local tissue-specific functions of CETP have not yet been clarified. Since oxidative stress and inflammation are major features of atherogenesis, we investigated whether CETP modulates macrophage oxidant production, inflammatory and metabolic profiles. Comparing macrophages from CETP-expressing transgenic mice and non-expressing littermates, we observed that CETP expression reduced mitochondrial superoxide anion production and H2O2 release, increased maximal mitochondrial respiration rates, and induced elongation of the mitochondrial network and expression of fusion-related genes (mitofusin-2 and OPA1). The expression of pro-inflammatory genes and phagocytic activity were diminished in CETP-expressing macrophages. In addition, CETP-expressing macrophages had less unesterified cholesterol under basal conditions and after exposure to oxidized LDL, as well as increased HDL-mediated cholesterol efflux. CETP knockdown in human THP1 cells increased unesterified cholesterol and abolished the effects on mitofusin-2 and TNFα. In summary, the expression of CETP in macrophages modulates mitochondrial structure and function to promote an intracellular antioxidant state and oxidative metabolism, attenuation of pro-inflammatory gene expression, reduced cholesterol accumulation, and phagocytosis. These localized functions of CETP may be relevant for the prevention of atherosclerosis and other inflammatory diseases.

Keywords:  CETP; cholesterol; inflammation; macrophage; mitochondria; oxidants

DOI:  https://doi.org/10.3390/antiox11091734
Clin Transl Med. 2022 Sep;12(9): e1045

NAT10: An RNA cytidine transferase regulates fatty acid metabolism in cancer cells.

Mahmood Hassan Dalhat, Mohammed Razeeth Shait Mohammed, Hind Ali Alkhatabi, Mohd Rehan, Aamir Ahmad, Hani Choudhry, Mohammad Imran Khan.

   BACKGROUND: N-4 cytidine acetylation (ac4C) is an epitranscriptomics modification catalyzed by N-acetyltransferase 10 (NAT10); important for cellular mRNA stability, rRNA biogenesis, cell proliferation and epithelial to mesenchymal transition (EMT). However, whether other crucial pathways are regulated by NAT10-dependent ac4C modification in cancer cells remains unclear. Therefore, in this study, we explored the impact of NAT10 depletion in cancer cells using unbiased RNA-seq.
METHODS: High-throughput sequencing of knockdown NAT10 in cancer cells was conducted to identify enriched pathways. Acetylated RNA immunoprecipitation-seq (acRIP-seq) and RIP-PCR were used to map and determine ac4C levels of RNA. Exogenous palmitate uptake assay was conducted to assess NAT10 knockdown cancer cells using Oil Red O staining and lipid content analysis. Gas-chromatography-tandem mass spectroscopy (GC/MS) was used to perform untargeted lipidomics.
RESULTS: High-throughput sequencing of NAT10 knockdown in cancer cells revealed fatty acid (FA) metabolism as the top enriched pathway through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in differentially downregulated genes. FA metabolic genes such as ELOLV6, ACSL1, ACSL3, ACSL4, ACADSB and ACAT1 were shown to be stabilised via NAT10-dependent ac4C RNA acetylation. Additionally, NAT10 depletion was shown to significantly reduce the levels of overall lipid content, triglycerides and total cholesterol. Further, NAT10 depletion in palmitate-loaded cancer cells showed decrease in ac4C levels across the RNA transcripts of FA metabolic genes. In untargeted lipidomics, 496 out of 2 279 lipids were statistically significant in NAT10 depleted cancer cells, of which pathways associated with FA metabolism are the most enriched.
CONCLUSIONS: Conclusively, our results provide novel insights into the impact of NAT10-mediated ac4C modification as a crucial regulatory factor during FA metabolism and showed the benefit of targeting NAT10 for cancer treatment.

Keywords:  NAT10; ac4C; cancer; fatty acid metabolism

DOI:  https://doi.org/10.1002/ctm2.1045
STAR Protoc. 2022 Sep 19. pii: S2666-1667(22)00546-9. [Epub ahead of print]3(4): 101666

Protocol to drive human monocyte-to-macrophage polarization in vitro using tumor conditioned media.

Samantha George, Mirella Georgouli, Victoria Sanz-Moreno.

  Tumor-associated macrophages (TAMs) are key contributors to antitumor immunity. Here, we present a protocol to drive human monocyte-macrophage differentiation using tumor-derived conditioned media, followed by phenotypic and functional characterization of TAMs in vitro. We describe CD14+ cell isolation from healthy human blood, and detail the procedure to induce macrophage polarization. Finally, we outline morphological assessment of macrophages, and validation of their functional behaviors with a tumor cell killing assay. This translatable-based approach can be applied to different cancer cell types. For complete details on the use and execution of this protocol, please refer to Georgouli et al. (2019).

Keywords:  Cancer; Cell culture; Cell differentiation; Cell isolation; Cell-based assays; Health sciences; Immunology

DOI:  https://doi.org/10.1016/j.xpro.2022.101666
Nat Metab. 2022 Sep;4(9): 1119-1137

Metabolic collateral lethal target identification reveals MTHFD2 paralogue dependency in ovarian cancer.

Abhinav Achreja, Tao Yu, Anjali Mittal, Srinadh Choppara, Olamide Animasahun, Minal Nenwani, Fulei Wuchu, Noah Meurs, Aradhana Mohan, Jin Heon Jeon, Itisam Sarangi, Anusha Jayaraman, Sarah Owen, Reva Kulkarni, Michele Cusato, Frank Weinberg, Hye Kyong Kweon, Chitra Subramanian, Max S Wicha, Sofia D Merajver, Sunitha Nagrath, Kathleen R Cho, Analisa DiFeo, Xiongbin Lu, Deepak Nagrath.

Recurrent loss-of-function deletions cause frequent inactivation of tumour suppressor genes but often also involve the collateral deletion of essential genes in chromosomal proximity, engendering dependence on paralogues that maintain similar function. Although these paralogues are attractive anticancer targets, no methodology exists to uncover such collateral lethal genes. Here we report a framework for collateral lethal gene identification via metabolic fluxes, CLIM, and use it to reveal MTHFD2 as a collateral lethal gene in UQCR11-deleted ovarian tumours. We show that MTHFD2 has a non-canonical oxidative function to provide mitochondrial NAD+, and demonstrate the regulation of systemic metabolic activity by the paralogue metabolic pathway maintaining metabolic flux compensation. This UQCR11-MTHFD2 collateral lethality is confirmed in vivo, with MTHFD2 inhibition leading to complete remission of UQCR11-deleted ovarian tumours. Using CLIM's machine learning and genome-scale metabolic flux analysis, we elucidate the broad efficacy of targeting MTHFD2 despite distinct cancer genetic profiles co-occurring with UQCR11 deletion and irrespective of stromal compositions of tumours.

DOI: https://doi.org/10.1038/s42255-022-00636-3
PLoS One. 2022 ;17(9): e0274623

Liquid chromatography coupled to high-resolution mass spectrometry metabolomics: A useful tool for investigating tumor secretome based on a three-dimensional co-culture model.

Andrea C Pelosi, Anna Maria A P Fernandes, Leonardo F Maciel, Alex A R Silva, Giulia C Mendes, Luísa F Bueno, Lívia Maria F Silva, Rafael F Bredariol, Maycon G Santana, Andreia M Porcari, Denise G Priolli.

Three-dimensional (3D) cell culture technologies, which more closely mimic the complex microenvironment of tissue, are being increasingly evaluated as a tool for the preclinical screening of clinically promising new molecules, and studying of tissue metabolism. Studies of metabolites released into the extracellular space (secretome) allow understanding the metabolic dynamics of tissues and changes caused by therapeutic interventions. Although quite advanced in the field of proteomics, studies on the secretome of low molecular weight metabolites (< 1500 Da) are still very scarce. We present an untargeted metabolomic protocol based on the hybrid technique of liquid chromatography coupled with high-resolution mass spectrometry for the analysis of low-molecular-weight metabolites released into the culture medium by 3D cultures and co-culture (secretome model). For that we analyzed HT-29 human colon carcinoma cells and 3T3-L1 preadipocytes in 3D-monoculture and 3D-co-culture. The putative identification of the metabolites indicated a sort of metabolites, among them arachidonic acid, glyceric acid, docosapentaenoic acid and beta-Alanine which are related to cancer and obesity. This protocol represents a possibility to list metabolites released in the extracellular environment in a comprehensive and untargeted manner, opening the way for the generation of metabolic hypotheses that will certainly contribute to the understanding of tissue metabolism, tissue-tissue interactions, and metabolic responses to the most varied interventions. Moreover, it brings the potential to determine novel pathways and accurately identify biomarkers in cancer and other diseases. The metabolites indicated in our study have a close relationship with the tumor microenvironment in accordance with the literature review.

DOI: https://doi.org/10.1371/journal.pone.0274623
Methods Mol Biol. 2023 ;2571 157-168

Analyses and Localization of Serotonin and L-DOPA in Ocular Tissues by Imaging Mass Spectrometry.

Varun Krishnan, Sean Meehan, Colin Hayter, Sanjoy K Bhattacharya.

  Imaging mass spectrometry (IMS) allows for visualization of the spatial distribution of proteins, lipids, and other metabolites in a targeted or untargeted approach. The identification of compounds through mass spectrometry combined with the mapping of compound distribution in the sample establishes IMS as a powerful tool for metabolomics. IMS analysis for serotonin will allow researchers to pinpoint areas of deficiencies or accumulations associated with ocular disorders such as serotonin selective reuptake inhibitor optic neuropathy. Furthermore, L-DOPA has shown great promise as a therapeutic approach for disorders such as age-related macular degeneration, and IMS allows for localization, and relative magnitudes, of L-DOPA in the eye. We describe here an end-to-end approach of IMS from sample preparation to data analysis for serotonin and L-DOPA analysis.

Keywords:  High-resolution mass spectrometry; Imaging mass spectrometry; L-DOPA; Localization; MALDI; Mass spectrometry; Metabolite localization; Metabolite visualization; Metabolomics; Serotonin

DOI:  https://doi.org/10.1007/978-1-0716-2699-3_16
ACS Appl Bio Mater. 2022 Sep 20.

NMR-Based Metabolomic Imprinting Elucidates Macrophage Polarization of THP-1 Cell Lines Stimulated by Zinc Oxide Nanoparticles.

Vani Mishra, Gurudayal Prajapati, Vikas Baranwal, Rohit Kumar Mishra.

  Zinc oxide (ZnO) nanoparticles (NPs) have been widely used in industry, cosmetics, drugs, bioimaging, and drug delivery. ZnO NPs have been found to interact and interfere with cellular physiology via macrophages, thereby resulting in macrophage polarization. The functional reprogramming of the cells is synchronized through cellular metabolic adaptations. The current study, therefore, aims to establish crosstalk between ZnO-NP-induced metabolic alterations and macrophage polarization in PMA-activated THP-1 cell lines. We observed moderate to heightened cytotoxic response in terms of cell viability and proliferation. The results also revealed increased Th1-type cytokine and chemokine expression. In order to characterize the changes in metabolite concentration in treatment groups, we employed multivariate data analysis (principal component analysis and partial least-squares discriminant analysis) of 1H NMR spectra. The results revealed biologically relevant patterns and alterations in many metabolic pathways. These alterations and patterns were found to be in line across the immune-cytotoxic axis. Furthermore, the results also implicate the role of carbon metabolism toward the classical activation of macrophage polarization. The omics approach could identify the markers involved in NP-induced toxicity, thus elaborating our vision of cytotoxicity that is currently limited to end-point and cytokine assays. Also, it could be emphasized that metabolic reconfiguration upon NP stimulation could direct macrophage polarization toward classical activation.

Keywords:  1H NMR spectroscopy; PCA; PLSDA; macrophage polarization; metabolomics; zinc oxide nanoparticles

DOI:  https://doi.org/10.1021/acsabm.2c00603
Genes (Basel). 2022 Sep 03. pii: 1585. [Epub ahead of print]13(9):

Crosstalk between Hypoxia and Extracellular Matrix in the Tumor Microenvironment in Breast Cancer.

Yasmin Dekker, Sylvia E Le Dévédec, Erik H J Danen, Qiuyu Liu.

  Even though breast cancer is the most diagnosed cancer among women, treatments are not always successful in preventing its progression. Recent studies suggest that hypoxia and the extracellular matrix (ECM) are important in altering cell metabolism and tumor metastasis. Therefore, the aim of this review is to study the crosstalk between hypoxia and the ECM and to assess their impact on breast cancer progression. The findings indicate that hypoxic signaling engages multiple mechanisms that directly contribute to ECM remodeling, ultimately increasing breast cancer aggressiveness. Second, hypoxia and the ECM cooperate to alter different aspects of cell metabolism. They mutually enhance aerobic glycolysis through upregulation of glucose transport, glycolytic enzymes, and by regulating intracellular pH. Both alter lipid and amino acid metabolism by stimulating lipid and amino acid uptake and synthesis, thereby providing the tumor with additional energy for growth and metastasis. Third, YAP/TAZ signaling is not merely regulated by the tumor microenvironment and cell metabolism, but it also regulates it primarily through its target c-Myc. Taken together, this review provides a better understanding of the crosstalk between hypoxia and the ECM in breast cancer. Additionally, it points to a role for the YAP/TAZ mechanotransduction pathway as an important link between hypoxia and the ECM in the tumor microenvironment, driving breast cancer progression.

Keywords:  YAP/TAZ; breast cancer; cell metabolism; extracellular matrix; hypoxia; mechanotransduction; tumor microenvironment

DOI:  https://doi.org/10.3390/genes13091585
J Clin Invest. 2022 Sep 20. pii: e161638. [Epub ahead of print]

A Mitofusin 2 - Hif1α axis sets a maturation checkpoint in regenerating skeletal muscle.

Xun Wang, Yuemeng Jia, Jiawei Zhao, Nicholas P Lesner, Cameron J Menezes, Spencer D Shelton, Siva Sai Krishna Venigalla, Jian Xu, Chunyu Cai, Prashant Mishra.

  A fundamental issue in regenerative medicine is whether there exist endogenous regulatory mechanisms that limit the speed and efficiency of the repair process. We report the existence of a maturation checkpoint during muscle regeneration which pauses myofibers at a neonatal stage. This checkpoint is regulated by the mitochondrial protein mitofusin 2 (Mfn2), whose expression is activated in response to muscle injury. Mfn2 is required for growth and maturation of regenerating myofibers; in the absence of Mfn2, new myofibers arrested at a neonatal stage, characterized by centrally nucleated myofibers and loss of H3K27me3 repressive marks at the neonatal myosin heavy chain gene. A similar arrest at the neonatal stage was observed in infantile cases of human centronuclear myopathy. Mechanistically, Mfn2 upregulation suppressed expression of Hypoxia-induced Factor 1α (Hif1α), which is induced in the setting of muscle damage. Sustained Hif1α signaling blocked maturation of new myofibers at the neonatal-to-adult fate transition, revealing the existence of a checkpoint that delays muscle regeneration. Correspondingly, inhibition of Hif1α allowed myofibers to bypass the checkpoint, thereby accelerating the repair process. We conclude that skeletal muscle contains a regenerative checkpoint which regulates the speed of myofiber maturation in response to Mitofusin 2 and Hif1α activity.

Keywords:  Epigenetics; Mitochondria; Muscle Biology; Stem cells

DOI:  https://doi.org/10.1172/JCI161638
Cancers (Basel). 2022 Sep 19. pii: 4526. [Epub ahead of print]14(18):

Emerging Roles of Lipophagy in Cancer Metastasis.

Haimeng Yin, Ying Shan, Tian Xia, Yan Ji, Ling Yuan, Yiwen You, Bo You.

  Obesity is a prominent risk factor for certain types of tumor progression. Adipocytes within tumor stroma contribute to reshaping tumor microenvironment (TME) and the metabolism and metastasis of tumors through the production of cytokines and adipokines. However, the crosstalk between adipocytes and tumor cells remains a major gap in this field. Known as a subtype of selective autophagy, lipophagy is thought to contribute to lipid metabolism by breaking down intracellular lipid droplets (LDs) and generating free fatty acids (FAs). The metastatic potential of cancer cells closely correlates with the lipid degradation mechanisms, which are required for energy generation, signal transduction, and biosynthesis of membranes. Here, we discuss the recent advance in the understanding of lipophagy with tumor lipid metabolism and review current studies on the roles of lipoghagy in the metastasis of certain human malignancies. Additionally, the novel candidate drugs targeting lipophagy are integrated for effective treatment strategies.

Keywords:  cancer metastasis; lipid metabolism; lipophagy

DOI:  https://doi.org/10.3390/cancers14184526
Int J Mol Sci. 2022 Sep 15. pii: 10778. [Epub ahead of print]23(18):

Nitric Oxide, Nitric Oxide Formers and Their Physiological Impacts in Bacteria.

Jinghua Chen, Lulu Liu, Weiwei Wang, Haichun Gao.

  Nitric oxide (NO) is an active and critical nitrogen oxide in the microbe-driven nitrogen biogeochemical cycle, and is of great interest to medicine and the biological sciences. As a gas molecule prior to oxygen, NO respiration represents an early form of energy generation via various reactions in prokaryotes. Major enzymes for endogenous NO formation known to date include two types of nitrite reductases in denitrification, hydroxylamine oxidoreductase in ammonia oxidation, and NO synthases (NOSs). While the former two play critical roles in shaping electron transport pathways in bacteria, NOSs are intracellular enzymes catalyzing metabolism of certain amino acids and have been extensively studied in mammals. NO interacts with numerous cellular targets, most of which are redox-active proteins. Doing so, NO plays harmful and beneficial roles by affecting diverse biological processes within bacterial physiology. Here, we discuss recent advances in the field, including NO-forming enzymes, the molecular mechanisms by which these enzymes function, physiological roles of bacterial NOSs, and regulation of NO homeostasis in bacteria.

Keywords:  NO signaling; NO tolerance; hemoproteins; nitric oxide; nitric oxide forming enzymes; nitric oxide synthase

DOI:  https://doi.org/10.3390/ijms231810778
Front Immunol. 2022 ;13 969468

Altered intraperitoneal immune microenvironment in patients with peritoneal metastases from gastric cancer.

Kazuya Takahashi, Kentaro Kurashina, Hironori Yamaguchi, Rihito Kanamaru, Hideyuki Ohzawa, Hideyo Miyato, Shin Saito, Yoshinori Hosoya, Alan Kawarai Lefor, Naohiro Sata, Joji Kitayama.

   Background: The peritoneal cavity contains many site-specific immune cells which constitute a unique immune microenvironment. However, it is unclear how the local immune signature is altered in patients with peritoneal metastases (PM).
Methods: Peritoneal lavage fluid or ascites were obtained from 122 patients with various stages of gastric cancer (GC). Cells recovered from peritoneal fluids were immunostained with mAbs for lymphocyte-, macrophage- and tumor cell-specific antigens and the frequencies of leukocyte subsets and antigen expression levels were evaluated with multi-color flowcytometry.
Results: The proportions of CD8(+) T cells, CD3(+)CD56(+) NKT-like cells, and CD3(-)CD56(+) NK cells to CD45(+) leukocytes were significantly reduced in patients with PM compared to those without PM. In patients with PM, the rates of CD8 (+) T cells and NKT-like cells correlated inversely with the tumor leukocyte ratio (TLR), the relative frequency of CD326(+) tumor cells to CD45(+) leukocytes. In contrast, the proportion of CD19(+) B cells was significantly increased in patients with PM, and their proportion correlated positively with the TLR and peritoneal carcinomatosis index (PCI) score. In patients with PM, CD14(+) macrophages tended to be increased with enhanced expression of CD14, CD16 and a M2-macrophage marker, CD163. In particular, macrophages in patients with high TLR contained many granules with high side scatter and CD14 expression in their flow profile compared to those without PM.
Conclusion: PM are accompanied by a drastic change in phenotypes of lymphocyte and macrophage in the peritoneal cavity, which might be involved in the development and progression of intraperitoneal tumor growth.

Keywords:  flowcytometry (FCM); gastric cancer; peritoneal immunity; peritoneal lymphocytes; peritoneal macrophage; peritoneal metastasis

DOI:  https://doi.org/10.3389/fimmu.2022.969468
Methods Mol Biol. 2022 ;2546 27-34

Tandem Mass Spectrometry for the Analysis of Plasma/Serum Acylcarnitines for the Diagnosis of Certain Organic Acidurias and Fatty Acid Oxidation Disorders.

David Scott, C Clinton Frazee, Bryce Heese, Uttam Garg.

  Acylcarnitines are formed in the mitochondria by esterification between carnitine and acyl-CoAs. This occurs enzymatically via carnitine acyltransferases. Specific acylcarnitines accumulate as a result of various organic acidurias and fatty acid oxidation disorders, and, thus, acylcarnitines profiles are used for the diagnosis of these disorders. Acylcarnitines monitoring can also be used for the follow-up of patients with these disorders. Tandem mass spectrometry (MS/MS) is the most commonly used method for the analysis of acylcarnitines. An MS/MS method for the quantification of a number of acylcarnitines is described. The method involves butylation of acylcarnitines using acidified butanol. Butylated acylcarnitines are analyzed using flow injection and precursor ion scan. Multiple-reaction monitoring (MRM) is used for the analysis of low-molecular-weight acylcarnitines.

Keywords:  Fatty acid oxidation defects; Inborn error of metabolism; Inherited metabolic disorders; Medium-chain acyl-CoA dehydrogenase deficiency; Organic acidemia; Organic acidurias; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-2565-1_3
Nature. 2022 Sep 21.

A cellular hierarchy in melanoma uncouples growth and metastasis.

Panagiotis Karras, Ignacio Bordeu, Joanna Pozniak, Ada Nowosad, Cecilia Pazzi, Nina Van Raemdonck, Ewout Landeloos, Yannick Van Herck, Dennis Pedri, Greet Bervoets, Samira Makhzami, Jia Hui Khoo, Benjamin Pavie, Jochen Lamote, Oskar Marin-Bejar, Michael Dewaele, Han Liang, Xingju Zhang, Yichao Hua, Jasper Wouters, Robin Browaeys, Gabriele Bergers, Yvan Saeys, Francesca Bosisio, Joost van den Oord, Diether Lambrechts, Anil K Rustgi, Oliver Bechter, Cedric Blanpain, Benjamin D Simons, Florian Rambow, Jean-Christophe Marine.

Although melanoma is notorious for its high degree of heterogeneity and plasticity1,2, the origin and magnitude of cell-state diversity remains poorly understood. Equally, it is unclear whether growth and metastatic dissemination are supported by overlapping or distinct melanoma subpopulations. Here, by combining mouse genetics, single-cell and spatial transcriptomics, lineage tracing and quantitative modelling, we provide evidence of a hierarchical model of tumour growth that mirrors the cellular and molecular logic underlying the cell-fate specification and differentiation of the embryonic neural crest. We show that tumorigenic competence is associated with a spatially localized perivascular niche, a phenotype acquired through an intercellular communication pathway established by endothelial cells. Consistent with a model in which only a fraction of cells are fated to fuel growth, temporal single-cell tracing of a population of melanoma cells with a mesenchymal-like state revealed that these cells do not contribute to primary tumour growth but, instead, constitute a pool of metastatic initiating cells that switch cell identity while disseminating to secondary organs. Our data provide a spatially and temporally resolved map of the diversity and trajectories of melanoma cell states and suggest that the ability to support growth and metastasis are limited to distinct pools of cells. The observation that these phenotypic competencies can be dynamically acquired after exposure to specific niche signals warrant the development of therapeutic strategies that interfere with the cancer cell reprogramming activity of such microenvironmental cues.

DOI: https://doi.org/10.1038/s41586-022-05242-7
JCI Insight. 2022 Sep 22. pii: e159286. [Epub ahead of print]7(18):

Flow pattern-dependent mitochondrial dynamics regulates the metabolic profile and inflammatory state of endothelial cells.

Soon-Gook Hong, Junchul Shin, Soo Young Choi, Jeffery C Powers, Benjamin M Meister, Jacqueline Sayoc, Jun Seok Son, Ryan Tierney, Fabio A Recchia, Michael D Brown, Xiaofeng Yang, Joon-Young Park.

  Endothelial mitochondria play a pivotal role in maintaining endothelial cell (EC) homeostasis through constantly altering their size, shape, and intracellular localization. Studies show that the disruption of the basal mitochondrial network in EC, forming excess fragmented mitochondria, implicates cardiovascular disease. However, cellular consequences underlying the morphological changes in the endothelial mitochondria under distinctively different, but physiologically occurring, flow patterns (i.e., unidirectional flow [UF] versus disturbed flow [DF]) are largely unknown. The purpose of this study was to investigate the effect of different flow patterns on mitochondrial morphology and its implications in EC phenotypes. We show that mitochondrial fragmentation is increased at DF-exposed vessel regions, where elongated mitochondria are predominant in the endothelium of UF-exposed regions. DF increased dynamin-related protein 1 (Drp1), mitochondrial reactive oxygen species (mtROS), hypoxia-inducible factor 1, glycolysis, and EC activation. Inhibition of Drp1 significantly attenuated these phenotypes. Carotid artery ligation and microfluidics experiments further validate that the significant induction of mitochondrial fragmentation was associated with EC activation in a Drp1-dependent manner. Contrarily, UF in vitro or voluntary exercise in vivo significantly decreased mitochondrial fragmentation and enhanced fatty acid uptake and OXPHOS. Our data suggest that flow patterns profoundly change mitochondrial fusion/fission events, and this change contributes to the determination of proinflammatory and metabolic states of ECs.

Keywords:  Atherosclerosis; Endothelial cells; Mitochondria; Vascular Biology

DOI:  https://doi.org/10.1172/jci.insight.159286
Antioxidants (Basel). 2022 Aug 30. pii: 1724. [Epub ahead of print]11(9):

COX-2 Expression in Hepatocytes Improves Mitochondrial Function after Hepatic Ischemia-Reperfusion Injury.

Marina Fuertes-Agudo, María Luque-Tévar, Carme Cucarella, Rocío Brea, Lisardo Boscá, Rubén Quintana-Cabrera, Paloma Martín-Sanz, Marta Casado.

  Cyclooxygenase 2 (COX-2) is a key enzyme in prostanoid biosynthesis. The constitutive hepatocyte expression of COX-2 has a protective role in hepatic ischemia-reperfusion (I/R) injury (IRI), decreasing necrosis, reducing reactive oxygen species (ROS) levels, and increasing autophagy and antioxidant and anti-inflammatory response. The physiopathology of IRI directly impacts mitochondrial activity, causing ATP depletion and being the main source of ROS. Using genetically modified mice expressing human COX-2 (h-COX-2 Tg) specifically in hepatocytes, and performing I/R surgery on the liver, we demonstrate that COX-2 expression has a beneficial effect at the mitochondrial level. Mitochondria derived from h-COX-2 Tg mice livers have an increased respiratory rate associated with complex I electron-feeding pathways compared to Wild-type (Wt) littermates, without affecting complex I expression or assembly. Furthermore, Wt-derived mitochondria show a loss of mitochondrial membrane potential (ΔΨm) that correlates to increased proteolysis of fusion-related OPA1 through OMA1 protease activity. All these effects are not observed in h-COX-2 Tg mitochondria, which behave similarly to the Sham condition. These results suggest that COX-2 attenuates IRI at a mitochondrial level, preserving the proteolytic processing of OPA1, in addition to the maintenance of mitochondrial respiration.

Keywords:  COX-2; high-resolution respirometry; ischemia-reperfusion; liver; mitochondrial dynamics; prostaglandins

DOI:  https://doi.org/10.3390/antiox11091724
J Biomed Res. 2022 Aug 28. 1-13

The contribution of astrocytes to obesity-associated metabolic disturbances.

Marta Obara-Michlewska.

  Obesity is a worldwide health, economic and social concern, despite efforts made to counteract the spreading wave of eating and nourishment-associated disorders. The review aims to show how the glial cells, astrocytes, contribute to the central regulation of appetite and energy metabolism. The hypothalamus is the brain center responsible for nutrients and nutritional hormone sensing, signal processing, and execution of metabolic and behavioral responses, directed at sustaining energy homeostasis. The astrocytes are endowed with receptors, transporters and enzymatic machinery responsible for glucose, lactate, fatty acids, ketone bodies, as well as leptin or ghrelin transport and metabolism, and that render them supporters and partners for neurons in governing the brain and body energy intake and expenditure. However, the role of astrocytes associated with brain energy metabolism reaches far beyond simple fuel contingent-they contribute to cognitive performance. The cognitive decline which often accompanies high fat- and/or high-calorie diets and correlates with neuroinflammation and astrogliosis, is a major concern. The last two decades of research enabled us to acknowledge the astroglia in obesity-associated dysfunctions and to investigate astrocytes as contributors to the pathology, as well as targets for therapy.

Keywords:  astrocytes; astrocytosis; brain energy metabolism; hypothalamic inflammation; obesity

DOI:  https://doi.org/10.7555/JBR.36.20200020
Metabolites. 2022 Aug 27. pii: 800. [Epub ahead of print]12(9):

Farnesysltransferase Inhibitor Prevents Burn Injury-Induced Metabolome Changes in Muscle.

Harumasa Nakazawa, Lai Ping Wong, Laura Shelton, Ruslan Sadreyev, Masao Kaneki.

  Burn injury remains a significant public health issue worldwide. Metabolic derangements are a major complication of burn injury and negatively affect the clinical outcomes of severely burned patients. These metabolic aberrations include muscle wasting, hypermetabolism, hyperglycemia, hyperlactatemia, insulin resistance, and mitochondrial dysfunction. However, little is known about the impact of burn injury on the metabolome profile in skeletal muscle. We have previously shown that farnesyltransferase inhibitor (FTI) reverses burn injury-induced insulin resistance, mitochondrial dysfunction, and the Warburg effect in mouse skeletal muscle. To evaluate metabolome composition, targeted quantitative analysis was performed using capillary electrophoresis mass spectrometry in mouse skeletal muscle. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and hierarchical cluster analysis demonstrated that burn injury induced a global change in metabolome composition. FTI treatment almost completely prevented burn injury-induced alterations in metabolite levels. Pathway analysis revealed that the pathways most affected by burn injury were purine, glutathione, β-alanine, glycine, serine, and threonine metabolism. Burn injury induced a suppressed oxidized to reduced nicotinamide adenine dinucleotide (NAD+/NADH) ratio as well as oxidative stress and adenosine triphosphate (ATP) depletion, all of which were reversed by FTI. Moreover, our data raise the possibility that burn injury may lead to increased glutaminolysis and reductive carboxylation in mouse skeletal muscle.

Keywords:  burn injury; farnesyltransferase inhibitor; metabolomics; skeletal muscle

DOI:  https://doi.org/10.3390/metabo12090800
Cancer Cell. 2022 Aug 29. pii: S1535-6108(22)00380-4. [Epub ahead of print]

Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and type I interferon to promote anti-tumor T cell responses.

Jessica K Mandula, Shiun Chang, Eslam Mohamed, Rachel Jimenez, Rosa A Sierra-Mondragon, Darwin C Chang, Alyssa N Obermayer, Carlos M Moran-Segura, Satyajit Das, Julio A Vazquez-Martinez, Karol Prieto, Ann Chen, Keiran S M Smalley, Brian Czerniecki, Peter Forsyth, Richard C Koya, Brian Ruffell, Juan R Cubillos-Ruiz, David H Munn, Timothy I Shaw, Jose R Conejo-Garcia, Paulo C Rodriguez.

  Activation of unfolded protein responses (UPRs) in cancer cells undergoing endoplasmic reticulum (ER) stress promotes survival. However, how UPR in tumor cells impacts anti-tumor immune responses remains poorly described. Here, we investigate the role of the UPR mediator pancreatic ER kinase (PKR)-like ER kinase (PERK) in cancer cells in the modulation of anti-tumor immunity. Deletion of PERK in cancer cells or pharmacological inhibition of PERK in melanoma-bearing mice incites robust activation of anti-tumor T cell immunity and attenuates tumor growth. PERK elimination in ER-stressed malignant cells triggers SEC61β-induced paraptosis, thereby promoting immunogenic cell death (ICD) and systemic anti-tumor responses. ICD induction in PERK-ablated tumors stimulates type I interferon production in dendritic cells (DCs), which primes CCR2-dependent tumor trafficking of common-monocytic precursors and their intra-tumor commitment into monocytic-lineage inflammatory Ly6C+CD103+ DCs. These findings identify how tumor cell-derived PERK promotes immune evasion and highlight the potential of PERK-targeting therapies in cancer immunotherapy.

Keywords:  PERK; immunogenic cell death; tumor immunity; type I IFN; unfolded protein responses

DOI:  https://doi.org/10.1016/j.ccell.2022.08.016
Proc Natl Acad Sci U S A. 2022 Sep 27. 119(39): e2202178119

Oxygen regulation of breathing is abolished in mitochondrial complex III-deficient arterial chemoreceptors.

Daniel Cabello-Rivera, Patricia Ortega-Sáenz, Lin Gao, Ana M Muñoz-Cabello, Victoria Bonilla-Henao, Paul T Schumacker, José López-Barneo.

  Acute oxygen (O2) sensing is essential for adaptation of organisms to hypoxic environments or medical conditions with restricted exchange of gases in the lung. The main acute O2-sensing organ is the carotid body (CB), which contains neurosecretory chemoreceptor (glomus) cells innervated by sensory fibers whose activation by hypoxia elicits hyperventilation and increased cardiac output. Glomus cells have mitochondria with specialized metabolic and electron transport chain (ETC) properties. Reduced mitochondrial complex (MC) IV activity by hypoxia leads to production of signaling molecules (NADH and reactive O2 species) in MCI and MCIII that modulate membrane ion channel activity. We studied mice with conditional genetic ablation of MCIII that disrupts the ETC in the CB and other catecholaminergic tissues. Glomus cells survived MCIII dysfunction but showed selective abolition of responsiveness to hypoxia (increased [Ca2+] and transmitter release) with normal responses to other stimuli. Mitochondrial hypoxic NADH and reactive O2 species signals were also suppressed. MCIII-deficient mice exhibited strong inhibition of the hypoxic ventilatory response and altered acclimatization to sustained hypoxia. These data indicate that a functional ETC, with coupling between MCI and MCIV, is required for acute O2 sensing. O2 regulation of breathing results from the integrated action of mitochondrial ETC complexes in arterial chemoreceptors.

Keywords:  acute O2 sensing; carotid body glomus cell; hypoxia; mitochondrial O2 sensing and signaling; mitochondrial complex III

DOI:  https://doi.org/10.1073/pnas.2202178119
STAR Protoc. 2022 Sep 20. pii: S2666-1667(22)00588-3. [Epub ahead of print]3(4): 101708

Transwell migration assay to interrogate human CAR-T cell chemotaxis.

Arman Oner, Sebastian Kobold.

  A major impediment to effective cellular therapies in solid tumors is the limited access of therapeutic cells to the tumor site. One strategy to overcome this challenge is to endow T cells with chemotactic properties required to access tumor tissue. Here, we present a chimeric antigen receptor (CAR)-modified T cell strategy centered around enhanced T cell trafficking. We outline isolation, activation, and transduction of human T cells, as well as techniques for assessing migratory and cytotoxic capacity of CAR-T cells. For complete details on the use and execution of this protocol, please refer to Lesch et al. (2021).

Keywords:  Biotechnology and bioengineering; Cancer; Cell isolation; Cell-based assays; Immunology

DOI:  https://doi.org/10.1016/j.xpro.2022.101708
Antioxidants (Basel). 2022 Sep 10. pii: 1788. [Epub ahead of print]11(9):

Recent Development of the Molecular and Cellular Mechanisms of Hydrogen Sulfide Gasotransmitter.

Jianyun Liu, Fikir M Mesfin, Chelsea E Hunter, Kenneth R Olson, W Christopher Shelley, John P Brokaw, Krishna Manohar, Troy A Markel.

  Hydrogen sulfide has been recently identified as the third biological gasotransmitter, along with the more well studied nitric oxide (NO) and carbon monoxide (CO). Intensive studies on its potential as a therapeutic agent for cardiovascular, inflammatory, infectious and neuropathological diseases have been undertaken. Here we review the possible direct targets of H2S in mammals. H2S directly interacts with reactive oxygen/nitrogen species and is involved in redox signaling. H2S also reacts with hemeproteins and modulates metal-containing complexes. Once being oxidized, H2S can persulfidate proteins by adding -SSH to the amino acid cysteine. These direct modifications by H2S have significant impact on cell structure and many cellular functions, such as tight junctions, autophagy, apoptosis, vesicle trafficking, cell signaling, epigenetics and inflammasomes. Therefore, we conclude that H2S is involved in many important cellular and physiological processes. Compounds that donate H2S to biological systems can be developed as therapeutics for different diseases.

Keywords:  gasotransmitter; persulfidation; reactive species interactome; reactive sulfur species

DOI:  https://doi.org/10.3390/antiox11091788
Nitric Oxide. 2022 Sep 17. pii: S1089-8603(22)00105-7. [Epub ahead of print]

There is no direct competition between arginase and nitric oxide synthase for the common substrate l-arginine.

Tony Y Momma, Javier I Ottaviani.

   AIMS: Extrahepatic arginases are postulated to be involved in cardiovascular-related pathologies by competing with nitric oxide synthase (NOS) for the common substrate l-arginine, subsequently decreasing nitric oxide production. However, previous models used to study arginase and NOS competition did not account for steady state level of l-arginine pool, which is dependent on conditions of l-arginine supply and utilization pathways. This work aimed at revisiting the concept of NOS and arginase competition while considering different conditions of l-arginine supply and l-arginine utilization pathways.
METHODS AND RESULTS: Mouse macrophage-like RAW cells and human vascular endothelial cells co-expressing NOS and arginase were used to reevaluate the concept of substrate competition between arginase and NOS under conditions of l-arginine supply that mimicked either a continuous (similar to in vivo conditions) or a limited supply (similar to previous in vitro models). Enzyme kinetics simulation models were used to gain mechanistic insight and to evaluate the tenability of a substrate competition between the two enzymes. In addition to arginase and NOS, other l-arginine pathways such as transporters and utilization towards protein synthesis were considered to understand the intricacies of l-arginine metabolism. Our results indicate that when there is a continuous supply of l-arginine, as is the case for most cells in vivo, arginase does not affect NOS activity by a substrate competition. Furthermore, we demonstrate that l-arginine pathways such as transporters and protein synthesis are more likely to affect NOS activity than arginase.
CONCLUSIONS: Arginase does not outcompete NOS for the common substrate l-arginine. Findings from this study should be considered to better understand the role of arginase in certain pathologies and for the interpretation of in vivo studies with arginase inhibitors.

Keywords:  Arginase; Nitric oxide; Nitric oxide synthase; l-arginine metabolism

DOI:  https://doi.org/10.1016/j.niox.2022.09.002
Dis Markers. 2022 ;2022 9883831

Activated Hepatic Stellate Cells Promote the M1 to M2 Macrophage Transformation and Liver Fibrosis by Elevating the Histone Acetylation Level.

Junru Chen, Xueqing Huang, Zhi Huang, Yu Cao.

Liver fibrosis results from the formation of fibrous scars of hepatic stellate cells by various chronic liver diseases. Considering that the liver is the most important metabolic organ in the human body, exploring the metabolic characteristics of liver fibrosis is expected to discover new markers and therapeutic targets. In this study, we first used mouse model to verify that both lactate content and histone acetylation levels were significantly increased in hepatic fibrosis mice. At the same time, it was confirmed that activated hepatic stellate cells (HSCs) cocultured with M1 macrophages can promote their transformation into M2 macrophages in hepatic stellate cell line and primary hepatic stellate cells. In addition, the addition of lactic acid to the medium in which M1 cells are cultured can promote their transformation into M2 macrophages. Therefore, we concluded that activated HSCs can promote the transformation of M1 to M2 macrophages through lactate accumulation, thereby causing liver fibrosis.

DOI: https://doi.org/10.1155/2022/9883831