Thermogenesis must be precisely controlled to avoid wasting energy. In this issue of Nature Metabolism, Tang et al. reveal that macrophage-derived itaconate acts as a paracrine metabolic brake that blocks MCT1-dependent succinate uptake and restrains brown adipose tissue thermogenesis.

Particulate matter exposure, especially diesel exhaust particles (DEP), can exacerbate neutrophilic airway inflammation which presents corticosteroid insensitivity, resulting in the loss of asthma control. The underlying biological mechanisms remain poorly understood, thereby impeding the development of innovative therapeutic strategies. Itaconate (ITA) is an anti-inflammatory metabolite that suppresses excessive immune activation in multiple pathological conditions. In this study, we identified that neutrophil acted as an essential regulator in DEP-induced corticosteroid-resistant asthma mouse models. Multi-omics and single-cell sequencing analysis found that aconitate decarboxylase 1 (ACOD1)/ITA was significantly elevated in neutrophils via the NF-κB signaling pathway in DEP-exacerbated asthma. Knockout of Acod1 exacerbated asthma pathogenesis, while treatment with exogenous ITA or 4-octyl itaconate (4-OI) conferred protection against airway inflammation and reversed corticosteroid resistance in asthma mouse models. Mechanistically, neutrophil-derived ITA helped maintain immune homeostasis by reducing the formation of neutrophil extracellular traps (NETs), which further inhibited Th17 cell differentiation in DEP-exacerbated asthma. Our results delineate the dual immunoregulatory function of neutrophils in DEP-induced corticosteroid-resistant asthma, wherein they simultaneously propagate inflammation through NETosis and Th17 activation while restraining immune hyperactivation via ITA-mediated metabolic regulation. ITA serves as a negative regulator of airway inflammation and corticosteroid resistance, highlighting its promising therapeutic potential in asthma.

AIMS:Inherited ventricular arrhythmias (VAs) frequently occur in the absence of pathogenic variants in canonical ion channel genes, suggesting alternative mechanisms of electrical instability. DENND3 is a guanine nucleotide exchange factor that regulates Rab GTPase-mediated trafficking, but its role in cardiac excitation-contraction coupling and membrane microdomain organization remains undefined. METHODS AND RESULTS:We studied induced pluripotent stem cell-derived cardiomyocytes generated from a CRISPR/CAS9-engineered ultra-rare DENND3-p.R534S variant-inserted line (previously identified in an idiopathic ventricular fibrillation pedigree) and matched isogenic controls. Multielectrode array recordings, live-cell calcium imaging, super-resolution imaging using expansion microscopy, and biochemical analyses were used to assess electrical activity, calcium handling, membrane architecture, and calcium release unit organization. Potentially therapeutic studies were performed using genetic and pharmacologic inhibition of Rab11b. DENND3-p.R534S induced pluripotent stem cell-derived cardiomyocytes exhibited multicellular electrical instability characterized by increased beat-to-beat variability, arrhythmic activity, conduction slowing, and prolonged excitation-contraction delay. These abnormalities were accompanied by heterogeneous and dyssynchronous calcium cycling despite preserved expression of major calcium-handling proteins. Super-resolution imaging revealed disruption of BIN1-dependent membrane architecture and nanoscale uncoupling of Cav1.2 and RyR2. Inhibition of Rab11b restored BIN1 organization, re-established dyadic coupling, normalized calcium cycling, and improved electrical stability. CONCLUSION:These findings support a model in which altered trafficking balance contributes to disruption of membrane microdomain organization, leading to dyadic uncoupling, calcium instability, and electrical dysfunction. Modulation of the Rab11b-mediated trafficking pathway restored structural and functional abnormalities, supporting the trafficking-associated pathway as a potential therapeutic target in DENND3-associated ventricular arrhythmia.

BACKGROUND:H3-altered gliomas, now recognized by WHO CNS5 as grade 4 neoplasms driven by histone H3 mutations (notably H3K27M and H3G34R/V), exhibit marked molecular heterogeneity, dismal survival, and resistance to standard therapies. Despite their classification as WHO grade 4 neoplasms, current stratification fails to capture their clinical and epigenetic diversity. METHODS:We performed genome-wide DNA methylation profiling on 49 representative cases from a clinical cohort of 375 H3-altered gliomas and validated subtype classifications using TCGA data. Single-cell RNA sequencing, ChIP-seq, and spatial transcriptomic analyses were employed to elucidate tumor-intrinsic programs and microenvironmental niches across subtypes. RESULTS:Unsupervised clustering identified four robust DNA methylation-defined subtypes: DMG_K27M, GBM_RTK, GBM_G34, and IDH/H3_comut, a novel subtype harboring co-occurring IDH and H3 mutations. The IDH/H3_comut subtype exhibited global DNA hypermethylation and significantly improved survival. In contrast, GBM_RTK tumors showed hypomethylation of cell-cycle enhancers and aggressive phenotypes, while GBM_G34 and DMG_K27M displayed subtype-specific epigenetic features. Single-cell transcriptomics revealed distinct lineage compositions and microenvironments, with IDH/H3_comut tumors enriched in NPC-like cells and GBM_RTK in astrocyte-like and vascular mimicry programs. Clonal and spatial analyses uncovered a compartmentalized coexistence of IDH- and H3-mutant cells, suggesting cooperative rather than exclusive evolution. CONCLUSIONS:Our study redefines H3-altered gliomas through methylation-based taxonomy, identifies a novel IDH/H3_comut subtype with favorable prognosis, and reveals subtype-specific therapeutic vulnerabilities, including potential responsiveness to hypomethylating agents or CDK4/6 inhibitors.

BACKGROUND AND AIMS:Type 1 short QT syndrome (SQT1) is a genetic channelopathy caused by gain-of-function variants in KCNH2, resulting in shortened cardiac repolarization and QT intervals, which predispose patients to ventricular arrhythmias and sudden cardiac death. This study aimed to investigate the therapeutic efficacy of KCNH2-specific suppression-and-replacement (KCNH2-SupRep) gene therapy in a transgenic rabbit model of SQT1. METHODS:KCNH2-SupRep was developed by combining a KCNH2-shRNA with its corresponding shRNA-immune KCNH2-cDNA into an AAV9 vector, delivered directly into the aortic root (1 × 1010 vg/kg). Therapeutic efficacy was evaluated in vivo by electrocardiogram, ex vivo by optical mapping, and at cellular levels by patch-clamp, calcium imaging, and qPCR in ventricular cardiomyocytes (VCMs). RESULTS:In vivo, KCNH2-SupRep normalized the heart rate-corrected QT interval (QTc) in SQT1 rabbits, without affecting repolarization heterogeneity. Ex vivo, KCNH2-SupRep corrected the action potential duration (APD90) and resolved the increased apicobasal APD90 heterogeneity observed in untreated (UT)-SQT1 hearts, supporting an antiarrhythmic effect, which was further validated by reduced re-entry formation in silico. At cellular levels, KCNH2-SupRep prolonged APD90 in VCMs from SupRep-SQT1 rabbits closer to wildtype levels compared with UT- and sham-SQT1. Additionally, KCNH2-SupRep restored the cellular surrogate of the electro-mechanical window and normalized IKr in nearly 50% of VCMs, in line with a 50%-60% suppression of the mutant KCNH2 transcript. CONCLUSIONS:This proof-of-concept study is the first to demonstrate the efficacy of gene therapy for SQT1 in a medium-sized animal model. KCNH2-SupRep gene therapy successfully corrected the pathologic phenotype in vivo, ex vivo, and at cellular levels in transgenic SQT1 rabbits.

Metabolic signals critically shape innate immune responses. Through pharmacological screening of metabolic pathways, we identified aspartate metabolism as a key regulator of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling. Genetically or aminooxyacetic acid-mediated (AOA-mediated) pharmacologically reducing aspartate levels markedly potentiated the cGAS-STING pathway, leading to stronger upregulation of type I interferons and interferon-stimulated genes. Mechanistically, disruption of de novo pyrimidine synthesis, a major downstream pathway of aspartate, induced mtDNA replication stress and increased mtDNA double-strand breaks, promoting mtDNA release into the cytosol. Cytosolic mtDNA synergized with cGAS-STING agonists to upregulate Z-DNA binding protein 1 (ZBP1), which recruits RIPK1/3 to sustain IRF3 phosphorylation, forming a positive feedback loop that amplifies innate immune signaling. In immunocompetent mouse models, AOA enhanced the antitumor efficacy of STING agonists, chemotherapy, or radiotherapy, whereas aspartate supplementation abrogated these effects. Consistently, aspartate levels negatively correlated with antitumor immunity in colorectal cancer patient samples. Together, our study identifies aspartate-pyrimidine metabolism as a critical metabolic checkpoint that licenses STING signaling by enabling mtDNA stress to cooperate with agonist stimulation, driving type I interferon-dependent ZBP1 induction and feed-forward amplification of STING signaling, thus offering a promising strategy to enhance antitumor immunity.

Isocitrate dehydrogenase-mutant (IDH-MUT) gliomas exhibit distinct metabolic profile marked by 2-hydroxyglutarate (2-HG) accumulation at the expense of α-ketoglutarate. How these tumors maintain the tricarboxylic acid (TCA) cycle, however, remained unclear. We conducted comprehensive metabolomic profiling using clinical cohorts, cell lines, and patient-derived organoids (PDOs). The metabolic dynamics of TCA and urea cycles were interrogated using stable isotope tracing with 13C-aspartate, U5-13C-15N-aspartate, 15NH4Cl and 15N-glutamate. The functional role of carbamoyl-phosphate synthase 1 (CPS1), the key enzyme of the urea cycle, was validated through inhibition experiments in vitro, in vivo and in PDO model, followed by seahorse respirometry and electron microscopy. Metabolomic profiling of two glioma cohorts consistently showed elevated urea cycle metabolites in IDH-MUT tumors. We identified CPS1 as a metabolic checkpoint sustaining TCA cycle through half urea cycle (from ammonia to arginine). Of note, CPS1 upregulation drove fumarate anaplerosis to sustain TCA flux in IDH-MUT gliomas. Thus, CPS1 inhibition not only reduced fumarate levels but also decreased oncometabolite 2-HG both in vitro and in vivo. Consistently, CPS1 inhibition impaired mitochondrial respiration and suppressed tumor growth in vitro, in vivo and in PDOs. Taken together, metabolic checkpoint CPS1 orchestrates half urea cycle to replenish the TCA cycle in IDH-MUT gliomas. Targeting CPS1 represents a promising metabolic therapeutic target for this glioma subtype.

Bacillus Calmette-Guerin (BCG)-induced trained immunity in the macrophages is characterized by exaggerated inflammatory cytokine production with favourable effects on disease controls. However, how BCG-trained macrophages exert anti-tumor effects and underlying mechanisms remain to be clarified. Pan-anti-tumor activity induced by BCG training was evaluated in mouse models with grafted tumors. The proportion and function of tumor infiltrating macrophages (TAMs) and CD8+T cells, as well as the level of reactive oxygen species (ROS), were detected by flow cytometry. The secretion of IL-1β and TNF-α were detected by Enzyme-Linked Immunosorbent Assay (ELISA). The expression and activation of NOX2 complex and NF-κB were detected by qRT-PCR, immunoblotting and immunofluorescence. The methylation modification level and chromatin opening level were detected by CUT RUN and ATAC sequencing. RNA sequencing was used to detect the transcriptome of macrophages and bladder cancer tissues. BCG-trained mice exhibited pan-anti-tumor activity where TAMs originated from newly bone marrow hematopoiesis were the predominant effectors. The anti-tumor effects of BCG-trained TAMs were mediated by ROS production in the tumor microenvironment (TME), resulting from the overactivation of NADPH oxidase 2 (NOX2) complex. Epigenetic rewiring of NOX2 complex occurred both in the myeloid progenitor of BCG-trained mice as well as in BCG-trained macrophages marked by increased deposition of H3K4me3 at the promoter regions of NOX2 complex genes, which in turn facilitated the accessibility of transcription factor such as NF-kB and enhanced transcriptional activation. Clinically, NOX2 gene signatures correlated with a favourable prognosis in bladder cancer patients receiving BCG intravesical instillation. Our findings reveal that BCG training reprograms TAMs to overproduce ROS through epigenetic rewiring of NOX2-ROS axis. Systemic BCG training becomes an effective and promising strategy to remodel the TME with enhanced pan-anti-tumor activity of infiltrating macrophages.

The energy sensor AMP-activated protein kinase (AMPK) promotes tumor cell survival under stress but how to prevent AMPK activation to blunt tumor progression remains unclear. Here we show that the metabolite α-ketoglutarate (α-KG) dictates AMPK translation through a TET-YBX1 axis, which can be exploited to sensitize human cancer cells to energy stress. α-KG-deficient cells fail to activate AMPK under glucose starvation, which elicits cytosolic NADPH depletion and disulfidptosis. Mechanistically, α-KG insufficiency inhibits TET-dependent transcription of YBX1, an RNA-binding protein required for human-specific AMPK protein synthesis. Similarly, α-KG competitors including succinate and itaconate inhibit the YBX1-AMPK axis and sensitize cancer cells to glucose deprivation. Lastly, cotargeting oncogenic YBX1 and GLUT1 creates synthetic lethality and blunts tumor growth in vivo. Together, our findings link α-KG to energy sensing through AMPK translation and propose that targeting α-KG-YBX1-dependent AMPK translation can sensitize human cancer cells to energy stress for treatment.

Neutrophil-associated inflammatory markers (NPR, NHR, SII, and SIRI) have been implicated in various metabolic diseases. However, studies on these markers with metabolic dysfunction-associated steatotic liver disease (MASLD) and advanced liver fibrosis (ALF), as well as their impact on all-cause mortality, remain limited. In this historical cohort study, data from 8051 adults aged 20 years and older were analysed. Weighted logistic regression was used to investigate the associations of neutrophil-associated inflammatory markers with MASLD and ALF. Nonlinear associations were described via restricted cubic spline regression. The diagnostic utility was assessed via receiver operating characteristic (ROC) curves. Furthermore, weighted Kaplan‒Meier survival curves and Cox proportional hazards models were employed to assess all-cause mortality risk. Sensitivity analyses were employed to guarantee the robustness of the findings. Following adjustment for confounding factors, there was a significant positive association between the ln-transformed NPR, NHR, SII, and SIRI and the risk of MASLD (P < 0.001). Conversely, an inverse association was noted between the ln-transformed SII, SIRI and ALF (P < 0.05). Nonlinear relationships were identified between ln-transformed NPR, NHR, and SIRI and the risk of MASLD (P < 0.001), as well as between ln-transformed NPR, SII, and SIRI and the risk of ALF (P < 0.001). Furthermore, the ln-transformed NHR (cut-off value: − 2.571) exhibited the highest diagnostic accuracy for MASLD (AUC 0.71, 95

Glioblastoma (GBM) is a highly lethal malignant brain tumor with poor survival rates, and chemoresistance poses a significant challenge to the treatment of patients with GBM. Here, we show that transketolase (TKT), a metabolic enzyme in the pentose phosphate pathway (PPP), attenuates the chemotherapy sensitivity of glioma cells in a manner independent of catalytic activity. Mechanistically, chemotherapeutic drugs can facilitate the translocation of TKT protein from the cytosol into the nucleus, where TKT physically interacts with XRN2 to regulate the resolution and removal of R-loops. Depletion of TKT leads to increased R-loop accumulation and genome instability, increasing the susceptibility of glioma cells to chemotherapy. In conclusion, our study reveals a non-metabolic function of TKT in regulating R-loop dynamics, genome instability, and chemotherapy sensitivity in gliomas.

Background: Calcium release channel deficiency syndrome (CRCDS) is caused by biogenic or biophysical loss-of-function (LOF) pathogenic variants in the RYR2 -encoded ryanodine receptor (RyR2), a key intracellular Ca 2+ release channel. Previously, we identified a novel homozygous duplication involving the promoter and exons 1-4 of RYR2 , leading to 80% RyR2 protein loss and exertion-related sudden death in the young. Here, we generated a RYR2 knockout (RYR2-KO) induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model to explore intracellular and electrophysiological compensatory mechanisms that overcome this extreme loss of RyR2 protein. Methods: Using CRISPR/Cas9 gene editing, a homozygous c.163delT variant (p.S55Pfs*46) was inserted into a normal iPSC line (isogenic control) to create a homozygous RYR2-KO iPSC line. After re-engineering the lines into ventricular-like cardiomyocytes (iPSC-CMs), intracellular Ca 2+ handling was assessed by Fluo-4 AM cell imaging (0.5 Hz stimulation). Electrical remodeling in the L-type Ca 2+ current (I CaL ) was assessed using the whole-cell patch clamp technique. Results: Significant differences were observed in Ca 2+ transient parameters between RYR2-KO and isogenic control iPSC-CMs. Biogenic RyR2 loss significantly reduced Ca 2+ transient peak amplitude (CTA: 0.16 ± 0.01 ΔF/F0, p<0.0001) and upstroke velocity (CTV: 0.42 ± 0.06 (ΔF/F0)/t, p<0.0001), and prolonged Ca 2+ transient duration (CTD 90 : 1.59 ± 0.03 s, p=0.01) as compared to isogenic control (CTA: 0.48 ± 0.04 ΔF/F0; CTV: 1.30 ± 0.15 (ΔF/F0)/t; CTD 90 : 1.46 ± 0.05 s). Additionally, RyR2 loss abolished SR Ca 2+ leak (0.9 ± 0.9 % versus 35.1 ± 15.5 %, p=0.03). Lastly, a significant reduction in I CaL density was observed in RYR2-KO iPSC-CMs as compared to isogenic control iPSC-CMs (at 0 mV, RYR2-KO: -8.54 ± 1.60 pA/pF, control: -17.45 ± 3.69 pA/pF, p=0.0004). These data indicate that I CaL reduction may contribute to the reduced Ca 2+ transient peak. Conclusions: Complete loss of RyR2 in iPSC-CMs profoundly disrupts intracellular Ca 2+ handling, abolishes Ca 2+ sparks frequency, and secondarily down-regulates the sarcolemmal L-type Ca 2+ channel.

Itaconate, a macrophage-specific anti-inflammatory metabolite, has recently emerged as a critical regulator in rheumatoid arthritis pathogenesis. We found that itaconate is a TNF-α responsive metabolite significantly elevated in the serum and synovial fluid of rheumatoid arthritis patients and we demonstrated that itaconate is primarily produced by inflammatory macrophages rather than osteoclasts or osteoblasts. In TNF-transgenic and Irg1 −/− hybrid mice, a more severe bone destruction phenotype was observed. Administration of itaconate prevents excessive activation of osteoclasts by inhibiting Tet2 enzyme activity. Furthermore, exogenous administration of itaconate or its derivative, 4-octyl-itaconate, inhibits arthritis progression and mitigates bone destruction, offering a potential therapeutic strategy for rheumatoid arthritis. This study elucidates that TNF-α drives macrophage-derived itaconate production to epigenetically suppress osteoclast hyperactivation through Tet2 inhibition, establishing itaconate and its derivative OI as novel therapeutic agents against rheumatoid arthritis -associated bone destruction.

Ferroptosis is a non-apoptotic mode of cell death characterized by iron-dependent accumulation of lipid peroxidation. While lipid radical elimination reaction catalyzed by glutathione peroxidase 4 (GPX4) is a major anti-ferroptosis mechanism, inhibiting this pathway pharmaceutically shows promise as an antitumor strategy. However, certain tumor cells exhibit redundancy in lipid radical elimination pathways, rendering them unresponsive to GPX4 inhibitors. In this study, we conducted screens across different cancer cell lines and Food and Drug Administration-approved drugs, leading to the identification of temsirolimus in combination with the GPX4 inhibitor RSL3 as a potent inducer of ferroptosis in liver cancer cells. Mechanistically, temsirolimus sensitized liver cancer cells to ferroptosis by directly binding to and inhibiting ferroptosis suppressor protein 1 (FSP1) enzyme. Notably, while temsirolimus is recognized as a potent mammalian target of rapamycin (mTOR) inhibitor, its ferroptosis-inducing effect is primarily attributed to the inhibition of FSP1 rather than mTOR activity. By employing in vitro colony formation assays and in vivo tumor xenograft models, we demonstrated that the combination of temsirolimus and RSL3 effectively suppressed liver tumor progression. This tumoricidal effect was associated with increased lipid peroxidation and induction of ferroptosis. In conclusion, our findings underscore the potential of combining multitarget ferroptosis-inducing agents to circumvent the resistance to ferroptosis of liver cancer cells and highlight temsirolimus as a promising FSP1 inhibitor and ferroptosis inducer, which also deserves further investigation in translational medicine.

The incidence of metabolic dysfunction-associated fatty liver disease (MAFLD) is high among U.S. adults, but studies on its occurrence in different ethnic and age groups are limited. The aim of the present study was to assess MAFLD occurrence among the U.S. adults by considering demographic characteristics, physical indices, and lifestyle conditions. This study utilized the National Health and Nutrition Examination Survey (NHANES) data 2009–2018 from 23,546 participants aged ≥ 20 years. Variables such as age, sex, race, body mass index (BMI), waist circumference (WC), blood pressure, sedentary behavior, sleep, and depression were analyzed. Among 9933 participants, 3562 had MAFLD (34.1

Epigenetic enzyme activity is coupled to cellular metabolism through their reliance on metabolic cofactors and substrates. Here, we describe steps for combining biochemical assays and saturation transfer difference (STD) NMR spectroscopy to experimentally validate metabolite binding and assess the effect on TET2 activity. This protocol enables the identification of both TET2 activators and inhibitors, providing a framework for studying the interplay between metabolism and epigenetic regulation. For complete details on the use and execution of this protocol, please refer to Cheng et al.1.

Spatial metabolomics is a rapidly evolving field to map the distribution of metabolites within tissues, organs, and even single cells. This approach provides contextual metabolic information, which is critical for understanding the biochemical heterogeneity of biological systems. Mid-infrared (MIR) imaging and mass spectrometry imaging (MSI) have emerged as powerful approaches for spatial metabolomics, each offering unique and complementary advantages. In this study, we present a workflow for performing MIR imaging and matrix-assisted laser desorption ionization MSI (MALDI-MSI) on the same tissue section, encompassing experimental procedures, imaging co-registration, data integration, and bioinformatics analysis. MIR imaging is employed as the upstream modality, allowing for non-destructive biochemical analysis of tissues. Subsequently, a matrix is deposited in the tissue for MALDI-MSI, guided by the spatial information obtained from MIR imaging. Images are integrated following co-registration, enabling multi-modal spatial metabolomics analysis using advanced bioinformatics tools such as Seurat. The integration of MIR imaging and MSI represents a transformative advancement in spatial metabolomics, offering unprecedented opportunities to explore the spatial and chemical complexity of metabolic processes in both health and disease. This multi-modal approach holds significant potential for driving innovations in biomarker discovery, disease diagnostics, and therapeutic development.