bims-celmim 2025-05-04 papers

bims-celmim

Biomed News

on Cellular and mitochondrial metabolism

Issue of 2025–05–04
24 papers selected by
Marc Segarra Mondejar

Mitochondrial metabolism in laryngeal cancer: Therapeutic mechanisms and prospects.
Intracellular metabolic gradients dictate dependence on exogenous pyruvate.
Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis.
Mitochondrial CCN1 drives ferroptosis via fatty acid β-oxidation.
Coenzyme Q10 protects keratinocytes against oxidation-induced energy stress as revealed by spatiotemporal analysis of cell energetics.
Advances in research on mitochondrial dysfunction in neurodegenerative diseases.
Lactate Provides Metabolic Substrate Support and Attenuates Ischemic Brain Injury in Mice, Revealed by 1H-13C Nuclear Magnetic Resonance Metabolic Technique.
Untargeted metabolomics reveals key metabolic alterations in pediatric epilepsy with insights into Tryptophan metabolism and the gut-brain axis.
Mitochondria - the CEO of the cell.
Time-resolved mitochondrial screen identifies regulatory components of oxidative metabolism.
Large-Scale Metabolite Imaging Gallery of Mouse Organ Tissues to Study Spatial Metabolism.
STING and metabolism-related diseases: Roles, mechanisms, and applications.
Integrating metagenomics and metabolomics to study the gut microbiome and host relationships in sports across different energy systems.
Label-Free Visualization and Segmentation of Endothelial Cell Mitochondria Using Holotomographic Microscopy and U-Net.
LRRC8 channel complexes counterbalance KATP channels to mediate swell-secretion coupling in mouse pancreatic β-cells.
Blocking the TCA Cycle in Cancer Cells Potentiates CD36+ T-cell-Mediated Antitumor Immunity by Suppressing ER Stress-Associated THBS2 Signaling.
The role of protein lactylation in brain health and disease: current advances and future directions.
Epigenetic Activation of PTCD3 Promotes CRC Glutamine Metabolism and Metastasis via IGF2BP2-Mediated SLC38A2 m6A Modification.
Protocol for in vivo CRISPR knockout screening of autophagy genes to identify breast cancer metastasis suppressors.
NAD depletion in skeletal muscle does not compromise muscle function or accelerate aging.
Glia phagocytose neuronal sphingolipids to infiltrate developing synapses.
Attenuated PINK1 autophosphorylation play neuroprotective and anti-seizure roles in neonatal hypoxia.
Signaling switches: Metabolism regulates gastruloid self-organization.
Quantifying small GTPase activation status using a novel fluorescence HPLC-based assay.

Biochim Biophys Acta Rev Cancer. 2025 Apr 29. pii: S0304-419X(25)00077-0. [Epub ahead of print] 189335

Mitochondrial metabolism in laryngeal cancer: Therapeutic mechanisms and prospects.

Yun-Jing Hou, Xin-Xin Yang, Hong-Xue Meng.

  Tumours reprogram pathways that regulate nutrient uptake and metabolism to meet the biosynthetic, bioenergetic, and redox requirements of cancer cells. This phenomenon is known as metabolic reprogramming and is edited by the deletion of oncogenes and the activation of proto-oncogenes. This article highlights the pathological mechanisms associated with metabolic reprogramming in laryngeal cancer (LC), including enhanced glycolysis, tricarboxylic acid cycle, nucleotide synthesis, lipid synthesis and metabolism, and amino acid metabolism, with a special emphasis on glutamine, tryptophan, and arginine metabolism. All these changes are regulated by HPV infection, hypoxia, and metabolic mediators in the tumour microenvironment. We analyzed the function of metabolic reprogramming in the development of drug resistance during standard LC treatment, which is challenging. In addition, we revealed recent advances in targeting metabolic strategies, assessing the strengths and weaknesses of clinical trials and treatment programs to attack resistance. This review summarises some currently important biomarkers and lays the foundation for therapeutic pathways in LC.

Keywords:  Laryngeal cancer (LC); Metabolic reprogramming; Mitochondria; Pathological mechanisms; Tumour microenvironment

DOI:  https://doi.org/10.1016/j.bbcan.2025.189335
Nat Metab. 2025 Apr 28.

Intracellular metabolic gradients dictate dependence on exogenous pyruvate.

Benjamin T Jackson, Angela M Montero, Sangita Chakraborty, Julia S Brunner, Paige K Arnold, Anna E Bridgeman, Pavlina K Todorova, Katrina I Paras, Lydia W S Finley.

During developmental transitions, cells frequently remodel metabolic networks, including changing reliance on metabolites such as glucose and glutamine to fuel intracellular metabolic pathways. Here we used embryonic stem (ES) cells as a model system to understand how changes in intracellular metabolic networks that characterize cell state transitions affect reliance on exogenous nutrients. We find that ES cells in the naive ground state of pluripotency increase uptake and reliance on exogenous pyruvate through the monocarboxylate transporter MCT1. Naive ES cells, but not their more committed counterparts, rely on exogenous pyruvate even when other sources of pyruvate (glucose, lactate) are abundant. Pyruvate dependence in naive ES cells is a consequence of their elevated mitochondrial pyruvate consumption at the expense of cytosolic NAD+ regeneration. Indeed, across a range of cell types, increased mitochondrial pyruvate consumption is sufficient to drive demand for extracellular pyruvate. Accordingly, restoring cytosolic NAD+ regeneration allows naive ES cells to tolerate pyruvate depletion in diverse nutrient microenvironments. Together, these data demonstrate that intracellular metabolic gradients dictate uptake and reliance on exogenous pyruvate and highlight mitochondrial pyruvate metabolism as a metabolic vulnerability of naive ES cells.

DOI: https://doi.org/10.1038/s42255-025-01289-8
Nat Metab. 2025 May 02.

Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis.

Kyle M Flickinger, Carlos A Mellado Fritz, Kimberly S Huggler, Gina M Wade, Gavin R Chang, Kathryn C Fox, Judith A Simcox, Jason R Cantor.

Nicotinamide adenine dinucleotide kinase (NADK) catalyses the phosphorylation of NAD+ to produce NAD phosphate, the oxidized form of NADPH, a cofactor that serves a critical role in driving reductive metabolism. Cancer cells co-express two distinct NAD kinases that differ by localization (NADK, cytosol; NADK2, mitochondria). CRISPR screens performed across hundreds of cancer cell lines indicate that both are dispensable for growth in conventional culture media. By contrast, NADK deletion impaired cell growth in human plasma-like medium. Here we trace this conditional NADK dependence to the availability of folic acid. NADPH is the preferred cofactor of dihydrofolate reductase (DHFR), the enzyme that mediates metabolic activation of folic acid. We find that NADK is required for enabling cytosolic NADPH-driven DHFR activity sufficient to maintain folate-dependent nucleotide synthesis under low folic acid conditions. Our results reveal a basis for conditional NADK essentiality and suggest that folate availability determines whether DHFR activity can be sustained by alternative electron donors such as NADH.

DOI: https://doi.org/10.1038/s42255-025-01272-3
Dev Cell. 2025 Apr 18. pii: S1534-5807(25)00206-0. [Epub ahead of print]

Mitochondrial CCN1 drives ferroptosis via fatty acid β-oxidation.

Wanxin Guo, Congcong Zhang, Qianjun Zhou, Tianxiang Chen, Xin Xu, Jianfeng Zhang, Xuewen Yu, Han Wu, Xiao Zhang, Lifang Ma, Kun Qian, Daniel J Klionsky, Rui Kang, Guido Kroemer, Yongchun Yu, Daolin Tang, Jiayi Wang.

  Ferroptosis is a type of oxidative cell death, although its key metabolic processes remain incompletely understood. Here, we employ a comprehensive multiomics screening approach that identified cellular communication network factor 1 (CCN1) as a metabolic catalyst of ferroptosis. Upon ferroptosis induction, CCN1 relocates to mitochondrial complexes, facilitating electron transfer flavoprotein subunit alpha (ETFA)-dependent fatty acid β-oxidation. Compared with a traditional carnitine O-palmitoyltransferase 2 (CPT2)-ETFA pathway, the CCN1-ETFA pathway provides additional substrates for mitochondrial reactive oxygen species production, thereby stimulating ferroptosis through lipid peroxidation. A high-fat diet can enhance the anticancer efficacy of ferroptosis in lung cancer mouse models, depending on CCN1. Furthermore, primary lung cancer cells derived from patients with hypertriglyceridemia or high CCN1 expression demonstrate increased susceptibility to ferroptosis in vitro and in vivo. These findings do not only identify the metabolic role of mitochondrial CCN1 but also establish a strategy for enhancing ferroptosis-based anticancer therapies.

Keywords:  CCN1; cell death; mitochondria

DOI:  https://doi.org/10.1016/j.devcel.2025.04.004
Sci Rep. 2025 Apr 25. 15(1): 14501

Coenzyme Q10 protects keratinocytes against oxidation-induced energy stress as revealed by spatiotemporal analysis of cell energetics.

Roland Abi Nahed, Ali Hussein, Cécile Cottet-Rousselle, Alexandra Vogelsang, Francesco Aulicino, Imre Berger, Thomas Blatt, Julia M Weise, Uwe Schlattner.

  Coenzyme Q10 (Q10) plays a critical role in cellular energy conversion within the mitochondrial respiratory chain and offers protective effects against oxidative and metabolic stress. In this study, we investigated the impact of Q10 on the spatio-temporal patterns of cellular energetics in keratinocyte-derived HaCaT cells, utilizing the genetically-encoded FRET sensor AMPfret. Engineered from the AMP-activated protein kinase (AMPK), this sensor leverages endogenous affinities of the kinase that evolved to detect energy stress, specifically decreases in ATP/ADP and ATP/AMP ratios that pose a threat to cell survival. We successfully established HaCaT cells stably expressing AMPfret, validated their functionality by inducing energy stress with 2-deoxy-D-glucose, and demonstrated that Q10, together with high glucose conditions in culture, can enhance cellular energetics compared to low glucose controls. We then employed AMPfret to analyze the spatio-temporal response of HaCaT keratinocytes to Luperox (tert-butyl peroxide), a potent organic prooxidant, in the presence of varying intracellular levels of Q10. Preloading cells with Q10 was protective, slowing the speed and reducing the extend of the energy stress response. In contrast, preincubation with Simvastatin, an inhibitor of the mevalonate Q10 biosynthesis pathway, depleted cellular Q10 levels, accelerated the onset of energy stress, and led to early cell death as compared to controls. Under all conditions, AMPfret revealed cell-to-cell heterogeneity in energy stress at baseline and in the response to Luperox. Overall, tracking changes in energy state in time and at single-cell level allows further insights into the beneficial role of Q10 in enhancing cellular bioenergetics in skin cells, and a potential role of AMPK in mediating responses to altered Q10 levels.

Keywords:  Cellular bioenergetics; Coenzyme Q10; FRET sensor; HaCaT cells; Keratinocytes; Oxidative stress

DOI:  https://doi.org/10.1038/s41598-025-98793-4
J Neurol. 2025 Apr 28. 272(5): 364

Advances in research on mitochondrial dysfunction in neurodegenerative diseases.

Yao Zhang, Xiao-Wen Li, Yuan Zhang, Xing Li.

  Given the high energy demand of the nervous system, mitochondrial dysfunction is a key factor in the pathogenesis of neurodegenerative diseases. Thus, a comprehensive understanding of its mechanisms and potential therapeutic targets is essential. This review discusses the roles of mitochondrial oxidative stress, mitochondrial dynamics alterations, and mtDNA damage in Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and multiple sclerosis (MS). In addition, it summarizes the contributions of novel technological approaches in detecting mitochondrial dysfunction, which assist in disease diagnosis. We also emphasize emerging therapeutic strategies and drugs aimed at enhancing mitochondrial quality control and reducing oxidative stress, thereby laying the groundwork for innovative therapeutic approaches in neurodegenerative disease treatment.

Keywords:  Detection technology; Mitochondrial dysfunction; Neurodegenerative diseases; Therapeutic targets

DOI:  https://doi.org/10.1007/s00415-025-13101-4
Biomedicines. 2025 Mar 24. pii: 789. [Epub ahead of print]13(4):

Lactate Provides Metabolic Substrate Support and Attenuates Ischemic Brain Injury in Mice, Revealed by 1H-13C Nuclear Magnetic Resonance Metabolic Technique.

Kefan Wu, Yajing Liu, Yuxuan Wang, Jiabao Hou, Meng Jiang, Shaoqin Lei, Bo Zhao, Zhongyuan Xia.

  Background/Objectives: Lactate, classically considered a metabolic byproduct of anaerobic glycolysis, is implicated in ischemic acidosis and neuronal injury. The recent evidence highlights its potential role in sustaining metabolic networks and neuroprotection. This study investigates lactate's compensatory mechanisms in ischemic brain injury by analyzing post-ischemic metabolic enrichments and inter-regional metabolite correlations. Methods: Dynamic metabolic profiling was conducted using 13C-labeled glucose combined with 1H-13C NMR spectroscopy to quantify the metabolite enrichment changes in a murine cerebral ischemia model (n = 8). In vivo validation included intracerebroventricular pH-neutral lactate infusion in ischemic mice to assess the behavioral, electrophysiological, and mitochondrial outcomes. In vitro, HT22 hippocampal neurons underwent oxygen-glucose deprivation (OGD) with pH-controlled lactate supplementation (1 mM), followed by the evaluation of neuronal survival, mitochondrial membrane potential, and glycolytic enzyme expression. Results: NMR spectroscopy revealed a 30-50% reduction in most cerebral metabolites post-ischemia (p < 0.05), while the quantities of lactate and the related three-carbon intermediates remained stable or increased. Correlation analyses demonstrated significantly diminished inter-metabolite coordination post-ischemia, yet lactate and glutamate maintained high metabolic activity levels (r > 0.80, p < 0.01). Lactate exhibited superior cross-regional metabolic mobility compared to those of the other three-carbon intermediates. In vivo, lactate infusion improved the behavioral/electrophysiological outcomes and reduced mitochondrial damage. In the OGD-treated neurons, pH-neutral lactate (7.4) reduced mortality (p < 0.05), preserved the mitochondrial membrane potential (p < 0.05), and downregulated the glycolytic enzymes (HK, PFK, and PKM; p < 0.01), thereby attenuating H+ production. Conclusions: Under ischemic metabolic crisis, lactate and the three-carbon intermediates stabilize as critical substrates, compensating for global metabolite depletion. pH-neutral lactate restores energy flux, modulates the glycolytic pathways, and provides neuroprotection by mitigating acidotoxicity.

Keywords:  NMR; cerebral ischemia; glycolytic; lactate

DOI:  https://doi.org/10.3390/biomedicines13040789
Sci Rep. 2025 Apr 30. 15(1): 15262

Untargeted metabolomics reveals key metabolic alterations in pediatric epilepsy with insights into Tryptophan metabolism and the gut-brain axis.

Karol Chojnowski, Mikołaj Opiełka, Krzysztof Urbanowicz, Marta Zawadzka, Karolina Wangin, Ryszard Tomasz Smoleński, Maria Mazurkiewicz-Bełdzińska.

The biochemical processes of childhood-onset epilepsy remain unclear, with no reliable biomarkers for prognosis or management. Untargeted plasma metabolomics offers a valuable approach to uncover underlying pathomechanisms and identify actionable biomarkers. In this study, plasma samples from 18 pediatric patients with epilepsy and 11 age-matched healthy controls were analyzed using liquid chromatography-mass spectrometry. Data were analyzed using univariate and multivariate statistical methods and pathway enrichment analysis. Multivariate analyses demonstrated separation between the patient and control groups. A total of 19 endogenous metabolites (VIP > 1, adjusted p < 0.05) emerged as key differentiators. Compared with controls, patients exhibited significant reductions in tryptophan (Trp), 5-Hydroxyindoleacetic acid (5-HIAA), several gut microbiota-derived metabolites, including indole, indoxyl sulfate, and p-cresyl sulfate, as well as in niacin metabolism end-products - N1-Methyl-2-pyridone-5-carboxamide (Met2PY) and N1-Methyl-4-pyridone-3-carboxamide (Met4PY). In addition, patients showed decreased levels of tricarboxylic acid (TCA) cycle intermediates, concomitant with an increase in fatty acid derivatives and N-acetylneuraminic acid (Neu5Ac). The most substantially altered metabolic pathways in epilepsy patients involved the TCA cycle, vitamin A and C metabolism, prostaglandin synthesis, and D4/E4-neuroprostane formation. Observed alterations in tryptophan and microbiota-derived metabolites suggest gut dysbiosis may contribute to epilepsy development through the gut-brain axis. Moreover, the circulatory metabolic markers indicating an energy deficit and oxidative stress underscore the systemic impact of seizure activity.

DOI: https://doi.org/10.1038/s41598-025-99805-z
J Cell Sci. 2025 May 01. pii: jcs263403. [Epub ahead of print]138(9):

Mitochondria - the CEO of the cell.

Laurie P Lee-Glover, Martin Picard, Timothy E Shutt.

  As we have learned more about mitochondria over the past decades, including about their essential cellular roles and how altered mitochondrial biology results in disease, it has become apparent that they are not just powerplants pumping out ATP at the whim of the cell. Rather, mitochondria are dynamic information and energy processors that play crucial roles in directing dozens of cellular processes and behaviors. They provide instructions to enact programs that regulate various cellular operations, such as complex metabolic networks, signaling and innate immunity, and even control cell fate, dictating when cells should divide, differentiate or die. To help current and future generations of cell biologists incorporate the dynamic, multifaceted nature of mitochondria and assimilate modern discoveries into their scientific framework, mitochondria need a 21st century 'rebranding'. In this Opinion article, we argue that mitochondria should be considered as the 'Chief Executive Organelle' - the CEO - of the cell.

Keywords:  Mitochondria; Organelle; mtDNA

DOI:  https://doi.org/10.1242/jcs.263403
EMBO Rep. 2025 Apr 29.

Time-resolved mitochondrial screen identifies regulatory components of oxidative metabolism.

Marcos Zamora-Dorta, Sara Laine-Menéndez, David Abia, Pilar González-García, Luis C López, Paula Fernández-Montes, Enrique Calvo, Jesús Vázquez, José Antonio Enríquez, Eduardo Balsa.

  Defects in mitochondrial oxidative metabolism underlie many genetic disorders with limited treatment options. The incomplete annotation of mitochondrial proteins highlights the need for a comprehensive gene inventory, particularly for Oxidative Phosphorylation (OXPHOS). To address this, we developed a CRISPR/Cas9 loss-of-function library targeting nuclear-encoded mitochondrial genes and conducted galactose-based screenings to identify novel regulators of mitochondrial function. Our study generates a gene catalog essential for mitochondrial metabolism and maps a dynamic network of mitochondrial pathways, focusing on OXPHOS complexes. Computational analysis identifies RTN4IP1 and ECHS1 as key OXPHOS genes linked to mitochondrial diseases in humans. RTN4IP1 is found to be crucial for mitochondrial respiration, with complexome profiling revealing its role as an assembly factor required for the complete assembly of complex I. Furthermore, we discovered that ECHS1 controls oxidative metabolism independently of its canonical function in fatty acid oxidation. Its deletion impairs branched-chain amino acids (BCAA) catabolism, disrupting lipoic acid-dependent enzymes such as pyruvate dehydrogenase (PDH). This deleterious phenotype can be rescued by restricting valine intake or catabolism in ECHS1-deficient cells.

Keywords:  CRISPR Screening; ECHS1; Mitochondria; OXPHOS; RTN4IP1

DOI:  https://doi.org/10.1038/s44319-025-00459-9
J Proteome Res. 2025 Apr 28.

Large-Scale Metabolite Imaging Gallery of Mouse Organ Tissues to Study Spatial Metabolism.

Karl W Smith, Antonia Fecke, Siva Swapna Kasarla, Prasad Phapale.

  Mass spectrometry imaging (MSI) has revolutionized the study of tissue metabolism by enabling the visualization of small molecule metabolites (SMMs) with high spatial resolution. However, comprehensive SMM imaging databases for different organ tissues are lacking, hindering our understanding of spatial organ metabolism. To address this resource gap, we present a large-scale SMM imaging gallery for mouse brain, kidney, and liver, capturing SMMs spanning eight chemical super classes and encompassing over 40 metabolic pathways. Manual curation and display of these imaging data sets unveil spatial patterns of metabolites that are less documented in the reported organs. Specifically, we identify 65 SMMs in brain coronal sections and 71 in sagittal tissue sections, including spatial patterns for neurotransmitters. Furthermore, we map 98 SMMs in kidneys and 66 SMMs in liver, providing insights into their amino acid and glutathione metabolism. Our insightful SMM imaging gallery serves as a critical resource for the spatial metabolism research community, filling a significant resource gap. This resource is freely available for download and can be accessed through the BioImage Archive and METASPACE repositories, providing high-quality annotated images for potential future computational models and advancing our understanding of tissue metabolism at the spatial level.

Keywords:  MALDI-MSI; imaging database; mass spectrometry imaging; metabolite imaging; organ metabolism; small molecules; spatial metabolomics; tissue metabolism

DOI:  https://doi.org/10.1021/acs.jproteome.4c00594
Cell Signal. 2025 Apr 26. pii: S0898-6568(25)00246-3. [Epub ahead of print]132 111833

STING and metabolism-related diseases: Roles, mechanisms, and applications.

Zhengyang Zhang, Xirui Wang, Chuangchuang Zhao, Haitao Zhu, Xiang Liao, Hsiang-I Tsai.

  The stimulator of interferon genes (STING) pathway plays a critical role in innate immunity, acting as a central mediator that links cytosolic DNA sensing to inflammatory signaling. STING not only responds to cellular metabolic states but also actively regulates key metabolic processes, including glycolysis, lipid metabolism, and redox balance. This bidirectional interaction underscores the existence of a dynamic feedback mechanism between STING signaling and metabolic pathways, which is essential for maintaining cellular homeostasis. This review provides a comprehensive analysis, beginning with an in-depth overview of the classical STING signaling pathway, followed by a detailed examination of its reciprocal regulation of various metabolic pathways. Additionally, it explores the role and mechanisms of STING signaling in metabolic disorders, including obesity, diabetes, and atherosclerosis. By integrating these insights into the mutual regulation between STING and its metabolism, novel therapeutic strategies targeting this pathway in metabolic diseases have been proposed.

Keywords:  Cell metabolism; Metabolic disease; cGAS-STING pathway

DOI:  https://doi.org/10.1016/j.cellsig.2025.111833
Sci Rep. 2025 May 02. 15(1): 15356

Integrating metagenomics and metabolomics to study the gut microbiome and host relationships in sports across different energy systems.

Viviana Aya, Daniel Pardo-Rodriguez, Laura Camila Vega, Mónica P Cala, Juan David Ramírez.

  The gut microbiome plays a critical role in modulating host metabolism, influencing energy production, nutrient utilization, and overall physiological adaptation. In athletes, these microbial functions may be further specialized to meet the unique metabolic demands of different sports disciplines. This study explored the role of the gut microbiome in modulating host metabolism among Colombian athletes by comparing elite weightlifters (n = 16) and cyclists (n = 13) through integrative omics analysis. Fecal and plasma samples collected one month before an international event underwent metagenomic, metabolomic, and lipidomic profiling. Metagenomic analysis revealed significant microbial pathways, including L-arginine biosynthesis III and fatty acid biosynthesis initiation. Key metabolic pathways, such as phenylalanine, tyrosine, and tryptophan biosynthesis; arginine biosynthesis; and folate biosynthesis, were enriched in both athlete groups. Plasma metabolomics and lipidomics revealed distinct metabolic profiles and a separation between athlete types through multivariate models, with lipid-related pathways such as lipid droplet formation and glycolipid synthesis driving the differences. Notably, elevated carnitine, amino acid, and glycerolipid levels in weightlifters suggest energy system-specific metabolic adaptations. These findings underscore the complex relationship between the gut microbiota composition and metabolic responses tailored to athletic demands, laying the groundwork for personalized strategies to optimize performance. This research highlights the potential for targeted modulation of the gut microbiota as a basis for tailored interventions to support specific energy demands in athletic disciplines.

Keywords:  Athletes; Gut microbiome; Lipidomics; Metabolomics; Metagenomics; Sports

DOI:  https://doi.org/10.1038/s41598-025-98973-2
Chem Biomed Imaging. 2025 Apr 28. 3(4): 225-231

Label-Free Visualization and Segmentation of Endothelial Cell Mitochondria Using Holotomographic Microscopy and U-Net.

Raul Michael, Tallah Modirzadeh, Tahir Bachar Issa, Patrick Jurney.

Understanding the physiological processes underlying cardiovascular disease (CVD) requires examination of endothelial cell (EC) mitochondrial networks, because mitochondrial function and adenosine triphosphate production are crucial in EC metabolism, and consequently influence CVD progression. Although current biochemical assays and immunofluorescence microscopy can reveal how mitochondrial function influences cellular metabolism, they cannot achieve live observation and tracking changes in mitochondrial networks through fusion and fission events. Holotomographic microscopy (HTM) has emerged as a promising technique for real-time, label-free visualization of ECs and their organelles, such as mitochondria. This nondestructive, noninterfering live cell imaging method offers unprecedented opportunities to observe mitochondrial network dynamics. However, because existing image processing tools based on immunofluorescence microscopy techniques are incompatible with HTM images, a machine-learning model is required. Here, we developed a model using a U-net learner with a Resnet18 encoder to identify four classes within HTM images: mitochondrial networks, cell borders, ECs, and background. This method accurately identifies mitochondrial structures and positions. With high accuracy and similarity metrics, the output image successfully provides visualization of mitochondrial networks within HTM images of ECs. This approach enables the study of mitochondrial networks and their effects, and holds promise in advancing understanding of CVD mechanisms.

DOI: https://doi.org/10.1021/cbmi.4c00100
JCI Insight. 2025 Apr 29. pii: e188020. [Epub ahead of print]

LRRC8 channel complexes counterbalance KATP channels to mediate swell-secretion coupling in mouse pancreatic β-cells.

Tarek Mohamed Abd El-Aziz, Chen Kang, Litao Xie, John D Tranter, Sumit Patel, Rahul Chadda, Maria S Remedi, Rajan Sah.

  Insulin secretion from pancreatic β-cells is initiated by membrane potential depolarization followed by activation of voltage-gated Ca2+ channels to trigger Ca2+-mediated insulin vesicle fusion with the β-cell plasma membrane. Here, we show that β-cell swelling associated with glucose metabolism is sensed by LRRC8 channel complexes and contributes to insulin secretion. Hypertonic perfusate (360-380 mOsm) dose-dependently impairs glucose-stimulated insulin secretion by counteracting β-cell swelling. Hypotonic perfusate alone, independent of glucose stimulation or KATP channel closure, is sufficient to increase β-cell intracellular Ca2+ and trigger insulin secretion. Inhibition of sodium-potassium-chloride cotransporter-1 with bumetanide, which diminishes the intracellular Cl- concentration in β-cells and consequently reduces Cl- efflux via LRRC8 channel complexes, also significantly reduces hypotonic-stimulated insulin secretion. Finally, stimulation of insulin secretion by the glucokinase activator GKA50, which is known to induce β-cell swelling, is entirely suppressed in β-cell-targeted Lrrc8a KO islets. These data support a model wherein the LRRC8 channel complex senses β-cell swelling triggered by glucose metabolism and regulates β-cell insulin secretion through activation of LRRC8-mediated Cl- efflux.

Keywords:  Beta cells; Cell biology; Insulin; Ion channels; Metabolism

DOI:  https://doi.org/10.1172/jci.insight.188020
Cancer Res. 2025 Apr 28. OF1-OF13

Blocking the TCA Cycle in Cancer Cells Potentiates CD36+ T-cell-Mediated Antitumor Immunity by Suppressing ER Stress-Associated THBS2 Signaling.

Jianqiang Yang, Fanghui Chen, Zhenzhen Fu, Fan Yang, Nabil F Saba, Yong Teng.

The tricarboxylic acid (TCA) cycle is often rewired or dysregulated to meet the increased energy and biosynthetic demands of rapidly dividing cancer cells, and targeting the TCA cycle is a potential therapeutic strategy for treating cancer. However, tumor cell metabolism can impact other cells in the tumor microenvironment, and disrupting the TCA cycle in cancer cells could impact the antitumor immune response. In this study, using CPI-613 as a model drug for TCA cycle inhibition, we identified a molecular mechanism by which blocking the TCA cycle enhances T-cell-mediated antitumor immunity in the context of head and neck squamous cell carcinoma (HNSCC). Impairment of mitochondrial metabolism by CPI-613 induced endoplasmic reticulum stress in HNSCC cells, leading to increased expression of spliced X-box-binding protein 1. This, in turn, directly suppressed the transcriptional activity of the thrombospondin-2 gene. Correspondingly, CPI-613 reduced the secretion of thrombospondin-2 from HNSCC cells, enhancing the proliferation and cytotoxic potential of tumor-infiltrating CD36+CD8+ T cells by upregulating AKT-mTOR signaling. This mechanism ultimately enhanced antitumor immunity in a syngeneic mouse model of orthotopic HNSCC following CPI-613 treatment. These findings uncover the immunomodulatory role of the TCA cycle in cancer cells and suggest that targeting it is a promising approach to harness tumor-reactive immune cells. Significance: The immunomodulatory role of the TCA cycle in cancer cells provides a therapeutic opportunity to enhance antitumor immunity by targeting tumor cell metabolism.

DOI: https://doi.org/10.1158/0008-5472.CAN-24-3477
Cell Death Discov. 2025 Apr 30. 11(1): 213

The role of protein lactylation in brain health and disease: current advances and future directions.

Mingrui Han, Wenfeng He, Wengen Zhu, Linjuan Guo.

Lactate, the end product of glycolysis, plays a crucial role in cellular signaling and metabolism. The discovery of lactylation, a novel post-translational modification, has uncovered the role of lactate in regulating diseases, especially in the brain. Lactylation connects genetic encoding with protein function, thereby influencing key biological processes. Increasing evidence supports lactate-mediated lactylation as a critical modulator in neurological disorders. This review offers an overview of lactate metabolism and lactylation, highlighting recent advances in understanding the regulatory enzymes of lactylation and their role in the central nervous system. We investigate the impact of lactylation on brain dysfunctions, including neurodegenerative diseases, cerebrovascular disorders, neuroinflammation, brain tumors, and psychiatric conditions. Moreover, we highlight the therapeutic potential of targeting lactylation in treating brain disorders and outline key research gaps and future directions needed to advance this promising field.

DOI: https://doi.org/10.1038/s41420-025-02408-w
FASEB J. 2025 May 15. 39(9): e70558

Epigenetic Activation of PTCD3 Promotes CRC Glutamine Metabolism and Metastasis via IGF2BP2-Mediated SLC38A2 m6A Modification.

Weihui Peng, Zhijun Zeng.

  Cancer cells undergo metabolic reprogramming, shifting their programs toward aerobic glycolysis and enhanced glutaminolysis to fulfill the requirements of rapid proliferation. Investigating the mechanisms underlying glutaminolysis and its connection with colorectal cancer (CRC) could aid in identifying novel therapeutic targets. PTCD3, a mitochondrial RNA-binding protein, is implicated in cancer progression, and IGF2BP2 regulates mRNA stability and translation. SLC38A2, a key transporter in glutamine metabolism, plays a crucial role in supporting cancer cell growth. This study aims to develop inhibitors of PTCD3 or SLC38A2 to prevent metabolic changes in cancer cells that facilitate rapid growth and metastasis in CRC. RT-qPCR, western blot, IHC, and IF staining assays confirmed the targeted gene and protein expression. Proliferation, migration, and invasion were evaluated using CCK-8 assay, scratch assay, and Transwell assay, respectively. Co-IP, RIP, and dual-luciferase assays were conducted to investigate the interactions among PTCD3, IGF2BP2, and SLC38A2. A CRC xenograft nude mice model was established for additional in vivo validation. PTCD3 was upregulated in CRC and positively correlated with GLS1. PTCD3 knockdown suppressed CRC cell glutaminolysis, thereby inhibiting CRC migration and invasion. PTCD3 promoted SLC38A2 mRNA stability in an IGF2BP2-dependent manner. KAT2A promoted the expression of PTCD3 by increasing H3K27 acetylation. The inhibitory effect of PTCD3 depletion on the glutaminolysis of CRC cells, as well as CRC cell proliferation and migration, was reversed by SLC38A2 overexpression. The in vivo mouse experiments further confirmed that silencing of PTCD3 inhibited CRC tumor growth. In summary, KAT2A upregulates PTCD3 expression by promoting H3K27 acetylation, which promotes glutaminolysis and metastasis in CRC via enhancing SLC38A2 mRNA stability in an IGF2BP2-dependent manner.

Keywords:  H3K27 acetylation; KAT2A; PTCD3; SLC38A2; colorectal cancer; glutaminolysis; m6A modification

DOI:  https://doi.org/10.1096/fj.202401788RR
STAR Protoc. 2025 Apr 30. pii: S2666-1667(25)00204-7. [Epub ahead of print]6(2): 103798

Protocol for in vivo CRISPR knockout screening of autophagy genes to identify breast cancer metastasis suppressors.

Mingang Hao, Peixin Lu, Jun-Lin Guan.

  Metastasis represents the primary cause of mortality among patients with breast cancer. Here, we present a protocol for utilizing an in vivo custom CRISPR-Cas9 knockout library to systematically investigate autophagy regulatory genes implicated in breast cancer metastasis to the lung in mice. We describe steps for library synthesis, cloning, and virus packaging and transfection. We then detail procedures for genome DNA collection and transplantation, followed by analysis of screening data. This protocol enables efficient identification of potential suppressors of breast cancer metastasis in vivo. For complete details on the use and execution of this protocol, please refer to Hao et al.1.

Keywords:  CRISPR; Cancer; Cell Biology

DOI:  https://doi.org/10.1016/j.xpro.2025.103798
Cell Metab. 2025 Apr 24. pii: S1550-4131(25)00212-8. [Epub ahead of print]

NAD depletion in skeletal muscle does not compromise muscle function or accelerate aging.

Sabina Chubanava, Iuliia Karavaeva, Amy M Ehrlich, Roger M Justicia, Astrid L Basse, Ivan Kulik, Emilie Dalbram, Danial Ahwazi, Samuel R Heaselgrave, Kajetan Trošt, Ben Stocks, Ondřej Hodek, Raissa N Rodrigues, Jesper F Havelund, Farina L Schlabs, Steen Larsen, Caio Y Yonamine, Carlos Henriquez-Olguín, Daniela Giustarini, Ranieri Rossi, Zachary Gerhart-Hines, Thomas Moritz, Juleen R Zierath, Kei Sakamoto, Thomas E Jensen, Nils J Færgeman, Gareth G Lavery, Atul S Deshmukh, Jonas T Treebak.

  Nicotinamide adenine dinucleotide (NAD) is a ubiquitous electron carrier essential for energy metabolism and post-translational modification of numerous regulatory proteins. Dysregulations of NAD metabolism are widely regarded as detrimental to health, with NAD depletion commonly implicated in aging. However, the extent to which cellular NAD concentration can decline without adverse consequences remains unclear. To investigate this, we generated a mouse model in which nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ biosynthesis was disrupted in adult skeletal muscle. The intervention resulted in an 85% reduction in muscle NAD+ abundance while maintaining tissue integrity and functionality, as demonstrated by preserved muscle morphology, contractility, and exercise tolerance. This absence of functional impairments was further supported by intact mitochondrial respiratory capacity and unaltered muscle transcriptomic and proteomic profiles. Furthermore, lifelong NAD depletion did not accelerate muscle aging or impair whole-body metabolism. Collectively, these findings suggest that NAD depletion does not contribute to age-related decline in skeletal muscle function.

Keywords:  NAD metabolism; NAD(+) biosynthesis; NAMPT; aging; epigenetic clock; exercise; mitochondrial supercomplexes; nicotinamide; reactive oxygen species; skeletal muscle

DOI:  https://doi.org/10.1016/j.cmet.2025.04.002
bioRxiv. 2025 Apr 22. pii: 2025.04.14.648777. [Epub ahead of print]

Glia phagocytose neuronal sphingolipids to infiltrate developing synapses.

Emma K Theisen, Irma Magaly Rivas-Serna, Ryan J Lee, Taylor R Jay, Govind Kunduri, Tasha T Nguyen, Vera Mazurak, M Thomas Clandinin, Thomas R Clandinin, John P Vaughen.

The complex morphologies of mature neurons and glia emerge through profound rearrangements of cell membranes during development. Despite being integral components of these membranes, it is unclear whether lipids might actively sculpt these morphogenic processes. By analyzing lipid levels in the developing fruit fly brain, we discover dramatic increases in specific sphingolipids coinciding with neural circuit establishment. Disrupting this sphingolipid bolus via genetic perturbations of sphingolipid biosynthesis and catabolism leads to impaired glial autophagy. Remarkably, glia can obtain sphingolipid precursors needed for autophagy by phagocytosing neurons. These precursors are then converted into specific long-chain ceramide phosphoethanolamines (CPEs), invertebrate analogs of sphingomyelin. These lipids are essential for glia to arborize and infiltrate the brain, a critical step in circuit maturation that when disrupted leads to reduced synapse numbers. Taken together, our results demonstrate how spatiotemporal tuning of sphingolipid metabolism during development plays an instructive role in programming brain architecture.
Highlights: Brain sphingolipids (SLs) remodel to very long-chain species during circuit maturation Glial autophagy requires de novo SL biosynthesis coordinated across neurons and glia Glia evade a biosynthetic blockade by phagolysosomal salvage of neuronal SLsCeramide Phosphoethanolamine is critical for glial infiltration and synapse density.

DOI: https://doi.org/10.1101/2025.04.14.648777
Sci Rep. 2025 Apr 29. 15(1): 15078

Attenuated PINK1 autophosphorylation play neuroprotective and anti-seizure roles in neonatal hypoxia.

Yi Yuan, Xiaoqian Wang, Yaru Cui, Hua Zhou, Wenna Li, Qian Teng, Hongjin Wang, Bohan Sun, Qiaoyun Wang, Hongliu Sun, Jianhua Tang.

  This study investigated the roles and mechanisms of PINK1 activity in neonatal hypoxia-induced seizures with shRNA intervention targeting translocase outer mitochondrial membrane 7 (TOM7), the positive regulator of PINK1 autophosphorylation, or overlapping with the m-AAA protease 1 homolog (OMA1), the negative regulator of PINK1 autophosphorylation. Studies have suggested that in hypoxia-induced neonatal seizures, the phosphorylation level of PINK1 is significantly increased and the mitophagic pathway is activated, accompanied by neuronal damage and learning-memory deficits. Inhibiting PINK1 phosphorylation by reducing TOM7 expression alleviated mitophagy, mitochondrial oxidative stress, neuronal damage and seizures. In contrast, the inhibition of OMA1 expression resulted in a further increase in PINK1 phosphorylation and aggravated hypoxia-induced seizures and neuronal injury. This study implicated PINK1 activity in neonatal hypoxia and suggest that attenuated PINK1 autophosphorylation may have neuroprotective and anti-seizure effects in neonatal hypoxia.

Keywords:  Mitochondrial oxidative stress; Mitophagy; Neuronal injury; PINK1; Seizure

DOI:  https://doi.org/10.1038/s41598-025-99915-8
Cell Stem Cell. 2025 May 01. pii: S1934-5909(25)00140-7. [Epub ahead of print]32(5): 673-675

Signaling switches: Metabolism regulates gastruloid self-organization.

María J Rodríguez Colman, Katharina F Sonnen.

Metabolic regulation of embryonic development is increasingly recognized. Villaronga-Luque et al.1 and Stopornwongkul et al.2 show that metabolic activity influences gastruloid formation from mouse embryonic stem cells, revealing that the balance between glycolysis and oxidative phosphorylation regulates cell fate decisions during gastruloid self-organization.

DOI: https://doi.org/10.1016/j.stem.2025.04.005
J Biol Chem. 2025 Apr 24. pii: S0021-9258(25)00394-1. [Epub ahead of print] 108545

Quantifying small GTPase activation status using a novel fluorescence HPLC-based assay.

Makoto Araki, Yukika Kasuya, Kaho Yoshimoto, Toshiaki Katada, Kenji Kontani.

  Small GTPases play crucial roles in cellular signaling pathways, with their activation states tightly regulated between GDP-bound inactive and GTP-bound active forms. Dysregulation of these nucleotide-binding states, such as in oncogenic RAS, is implicated in diseases like cancer. Accurately quantifying these states in cells is thus crucial for deciphering their functional roles and regulatory mechanisms. However, current methods do not fully meet the necessary sensitivity and versatility, limiting their effectiveness in small GTPase analysis. Here, we present a highly sensitive HPLC-based assay with fluorescence detection (Fluor-HPLC), enabling precise quantification of guanine nucleotide-binding states in small GTPases. Applying this technique, we successfully quantified the guanine nucleotide-binding states of small GTPases at their endogenous expression levels. We demonstrated the utility of Fluor-HPLC by elucidating RHEB and HRAS activation in response to extracellular stimuli. Furthermore, integration of Fluor-HPLC with syngeneic mouse models provided insights into KRAS activation dynamics in tumor tissues and evaluated the effectiveness of targeted therapeutics. Overall, this versatile method paves the way for investigating activation states and regulatory mechanisms of various small GTPases, potentially accelerating our understanding of their roles in cellular processes and disease pathogenesis.

Keywords:  HRAS; KRAS; RHEB; anticancer drug; high‐performance liquid chromatography (HPLC); small GTPase

DOI:  https://doi.org/10.1016/j.jbc.2025.108545