Bidirectional modulation of TCA cycle metabolites and anaplerosis by metformin and its combination with SGLT2i.

BACKGROUND: Metformin and sodium-glucose-cotransporter-2 inhibitors (SGLT2i) are cornerstone therapies for managing hyperglycemia in diabetes. However, their detailed impacts on metabolic processes, particularly within the citric acid (TCA) cycle and its anaplerotic pathways, remain unclear. This study investigates the tissue-specific metabolic effects of metformin, both as a monotherapy and in combination with SGLT2i, on the TCA cycle and associated anaplerotic reactions in both mice and humans. METHODS: Metformin-specific metabolic changes were initially identified by comparing metformin-treated diabetic mice (MET) with vehicle-treated db/db mice (VG). These findings were then assessed in two human cohorts (KORA and QBB) and a longitudinal KORA study of metformin-naïve patients with Type 2 Diabetes (T2D). We also compared MET with db/db mice on combination therapy (SGLT2i + MET). Metabolic profiling analyzed 716 metabolites from plasma, liver, and kidney tissues post-treatment, using linear regression and Bonferroni correction for statistical analysis, complemented by pathway analyses to explore the pathophysiological implications. RESULTS: Metformin monotherapy significantly upregulated TCA cycle intermediates such as malate, fumarate, and α-ketoglutarate (α-KG) in plasma, and anaplerotic substrates including hepatic glutamate and renal 2-hydroxyglutarate (2-HG) in diabetic mice. Downregulated hepatic taurine was also observed. The addition of SGLT2i, however, reversed these effects, such as downregulating circulating malate and α-KG, and hepatic glutamate and renal 2-HG, but upregulated hepatic taurine. In human T2D patients on metformin therapy, significant systemic alterations in metabolites were observed, including increased malate but decreased citrulline. The bidirectional modulation of TCA cycle intermediates in mice influenced key anaplerotic pathways linked to glutaminolysis, tumorigenesis, immune regulation, and antioxidative responses. CONCLUSION: This study elucidates the specific metabolic consequences of metformin and SGLT2i on the TCA cycle, reflecting potential impacts on the immune system. Metformin shows promise for its anti-inflammatory properties, while the addition of SGLT2i may provide liver protection in conditions like metabolic dysfunction-associated steatotic liver disease (MASLD). These observations underscore the importance of personalized treatment strategies.

The tricarboxylic acid cycle is inhibited under acute stress from carbonate alkalinity in the gills of Eriocheir sinensis.

Owing to population growth and environmental pollution, freshwater aquaculture has been rapidly shrinking in recent years. Aquaculture in saline-alkaline waters is a crucial strategy to meet the increasing demand for aquatic products. The Chinese mitten crab is an important economic food in China, but the molecular mechanism by which it tolerates carbonate alkalinity (CA) in water remains unclear. Here, we found that enzyme activities of the tricarboxylic acid (TCA) cycle in the gills, such as citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase, were markedly reduced under CA stress induced by 40 mM NaHCO3. Secondly, the TCA cycle in the gills is inhibited under acute CA stress, according to proteomic and metabolomic analyses. The expressions of six enzymes, namely aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, dihydrolipoyl dehydrogenase, succinate-CoA ligase, and malate dehydrogenase, were downregulated, resulting in the accumulation of phosphoenolpyruvic acid, citric acid, cis-aconitate, and α-ketoglutaric acid. Finally, we testified that if the TCA cycle is disturbed by malonate, the survival rate increases in CA water. To our knowledge, this is the first study to show that the TCA cycle in the gills is inhibited under CA stress. Overall, the results provide new insights into the molecular mechanism of tolerance to saline-alkaline water in crabs, which helped us expand the area for freshwater aquaculture and comprehensively understand the physiological characteristics of crab migration.

Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenic Escherichia coli.

Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after the completion of successful antibiotic treatment. Many clinical isolates, including the well-characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex integration host factor and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two-hybrid assays. We report direct interactions between the succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions may enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility. IMPORTANCE: Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections (UTIs). Upon invasion into bladder epithelial cells, UPEC establish quiescent intracellular reservoirs that may lead to antibiotic tolerance and recurrent UTIs. Here, we demonstrate using an in vitro system that quiescent UPEC cells are tolerant to ampicillin and have decreased metabolism characterized by succinyl-CoA limitation. We identify the global regulator integration host factor complex and the cell division protein ZapE as critical modifiers of quiescence and antibiotic tolerance. Finally, we show that ZapE interacts with components of both the cell division machinery and the tricarboxylic acid cycle, and this interaction is conserved in non-pathogenic E. coli, establishing a novel link between cell division and metabolism.

Mechanisms associated with cuproptosis and implications for ovarian cancer.

Ovarian cancer, a profoundly fatal gynecologic neoplasm, exerts a substantial economic strain on nations globally. The formidable challenge of its frequent relapse necessitates the exploration of novel cytotoxic agents, efficacious antineoplastic medications with minimal adverse effects, and strategies to surmount resistance to primary chemotherapeutic agents. These endeavors aim to supplement extant pharmacological interventions and elucidate molecular mechanisms underlying induced cytotoxicity, distinct from conventional therapeutic modalities. Recent scientific research has unveiled a novel form of cellular demise, known as copper-death, which is contingent upon the intracellular concentration of copper. Diverging from conventional mechanisms of cellular demise, copper-death exhibits a pronounced reliance on mitochondrial respiration, particularly the tricarboxylic acid (TCA) cycle. Tumor cells manifest distinctive metabolic profiles and elevated copper levels in comparison to their normal counterparts. The advent of copper-death presents alluring possibilities for targeted therapeutic interventions within the realm of cancer treatment. Hence, the primary objective of this review is to present an overview of the proteins and intricate mechanisms associated with copper-induced cell death, while providing a comprehensive summary of the knowledge acquired regarding potential therapeutic approaches for ovarian cancer. These findings will serve as valuable references to facilitate the advancement of customized therapeutic interventions for ovarian cancer.

Itaconate reduces proliferation and migration of fibroblast-like synoviocytes and ameliorates arthritis models.

Fibroblast-like synoviocytes (FLS) play critical roles in rheumatoid arthritis (RA). Itaconate (ITA), an endogenous metabolite derived from the tricarboxylic acid (TCA) cycle, has attracted attention because of its anti-inflammatory, antiviral, and antimicrobial effects. This study evaluated the effect of ITA on FLS and its potential to treat RA. ITA significantly decreased FLS proliferation and migration in vitro, as well as mitochondrial oxidative phosphorylation and glycolysis measured by an extracellular flux analyzer. ITA accumulates metabolites including succinate and citrate in the TCA cycle. In rats with type II collagen-induced arthritis (CIA), intra-articular injection of ITA reduced arthritis and bone erosion. Irg1-deficient mice lacking the ability to produce ITA had more severe arthritis than control mice in the collagen antibody-induced arthritis. ITA ameliorated CIA by inhibiting FLS proliferation and migration. Thus, ITA may be a novel therapeutic agent for RA.

Quercetin induces itaconic acid-mediated M1/M2 alveolar macrophages polarization in respiratory syncytial virus infection.

BACKGROUND: Quercetin has received extensive attention for its therapeutic potential treating respiratory syncytial virus (RSV) infection diseases. Recent studies have highlighted quercetin's ability of suppressing alveolar macrophages (AMs)-derived lung inflammation. However, the anti-inflammatory mechanism of quercetin against RSV infection still remains elusive. PURPOSE: This study aims to elucidate the mechanism about quercetin anti-inflammatory effect on RSV infection. METHODS: BALB/c mice were intranasally infected with RSV and received quercetin (30, 60, 120 mg/kg/d) orally for 3 days. Additionally, an in vitro infection model utilizing mouse alveolar macrophages (MH-S cells) was employed to validate the proposed mechanism. RESULTS: Quercetin exhibited a downregulatory effect on glycolysis and tricarboxylic acid (TCA) cycle metabolism in RSV-infected AMs. However, it increased itaconic acid production, a metabolite derived from citrate through activating immune responsive gene 1 (IRG1), and further inhibiting succinate dehydrogenase (SDH) activity. While the suppression of SDH activity orchestrated a cascading downregulation of Hif-1α/NLRP3 signaling, ultimately causing AMs polarization from M1 to M2 phenotypes. CONCLUSION: Our study demonstrated quercetin stimulated IRG1-mediated itaconic acid anabolism and further inhibited SDH/Hif-1α/NLRP3 signaling pathway, which led to M1 to M2 polarization of AMs so as to ameliorate RSV-induced lung inflammation.

Elevated Na is a dynamic and reversible modulator of mitochondrial metabolism in the heart.

Elevated intracellular sodium Nai adversely affects mitochondrial metabolism and is a common feature of heart failure. The reversibility of acute Na induced metabolic changes is evaluated in Langendorff perfused rat hearts using the Na/K ATPase inhibitor ouabain and the myosin-uncoupler para-aminoblebbistatin to maintain constant energetic demand. Elevated Nai decreases Gibb's free energy of ATP hydrolysis, increases the TCA cycle intermediates succinate and fumarate, decreases ETC activity at Complexes I, II and III, and causes a redox shift of CoQ to CoQH2, which are all reversed on lowering Nai to baseline levels. Pseudo hypoxia and stabilization of HIF-1α is observed despite normal tissue oxygenation. Inhibition of mitochondrial Na/Ca-exchange with CGP-37517 or treatment with the mitochondrial ROS scavenger MitoQ prevents the metabolic alterations during Nai elevation. Elevated Nai plays a reversible role in the metabolic and functional changes and is a novel therapeutic target to correct metabolic dysfunction in heart failure.

DRP1 inhibition-mediated mitochondrial elongation abolishes cancer stemness, enhances glutaminolysis, and drives ferroptosis in oral squamous cell carcinoma.

BACKGROUND: Mitochondrial dynamics play a fundamental role in determining stem cell fate. However, the underlying mechanisms of mitochondrial dynamics in the stemness acquisition of cancer cells are incompletely understood. METHODS: Metabolomic profiling of cells were analyzed by MS/MS. The genomic distribution of H3K27me3 was measured by CUT&Tag. Oral squamous cell carcinoma (OSCC) cells depended on glucose or glutamine fueling TCA cycle were monitored by 13C-isotope tracing. Organoids and tumors from patients and mice were treated with DRP1 inhibitors mdivi-1, ferroptosis inducer erastin, or combination with mdivi-1 and erastin to evaluate treatment effects. RESULTS: Mitochondria of OSCC stem cells own fragment mitochondrial network and DRP1 is required for maintenance of their globular morphology. Imbalanced mitochondrial dynamics induced by DRP1 knockdown suppressed stemness of OSCC cells. Elongated mitochondria increased α-ketoglutarate levels and enhanced glutaminolysis to fuel the TCA cycle by increasing glutamine transporter ASCT2 expression. α-KG promoted the demethylation of histone H3K27me3, resulting in downregulation of SNAI2 associated with stemness and EMT. Significantly, suppressing DRP1 enhanced the anticancer effects of ferroptosis. CONCLUSION: Our study reveals a novel mechanism underlying mitochondrial dynamics mediated cancer stemness acquisition and highlights the therapeutic potential of mitochondria elongation to increase the susceptibility of cancer cells to ferroptosis.

Wild-type IDH2 is a therapeutic target for triple-negative breast cancer.

Mutations in isocitrate dehydrogenases (IDH) are oncogenic events due to the generation of oncogenic metabolite 2-hydroxyglutarate. However, the role of wild-type IDH in cancer development remains elusive. Here we show that wild-type IDH2 is highly expressed in triple negative breast cancer (TNBC) cells and promotes their proliferation in vitro and tumor growth in vivo. Genetic silencing or pharmacological inhibition of wt-IDH2 causes a significant increase in α-ketoglutarate (α-KG), indicating a suppression of reductive tricarboxylic acid (TCA) cycle. The aberrant accumulation of α-KG due to IDH2 abrogation inhibits mitochondrial ATP synthesis and promotes HIF-1α degradation, leading to suppression of glycolysis. Such metabolic double-hit results in ATP depletion and suppression of tumor growth, and renders TNBC cells more sensitive to doxorubicin treatment. Our study reveals a metabolic property of TNBC cells with active utilization of glutamine via reductive TCA metabolism, and suggests that wild-type IDH2 plays an important role in this metabolic process and could be a potential therapeutic target for TNBC.

Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids.

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.

Activity-based protein profiling technology reveals malate dehydrogenase as the target protein of cinnamaldehyde against Aspergillus niger.

Cinnamaldehyde displays strong antifungal activity against fungi such as Aspergillus niger, but its precise molecular mechanisms of antifungal action remain inadequately understood. In this investigation, we applied chemoproteomics and bioinformatic analysis to unveil the target proteins of cinnamaldehyde in Aspergillus niger cells. Additionally, our study encompassed the examination of cinnamaldehyde's effects on cell membranes, mitochondrial malate dehydrogenase activity, and intracellular ATP levels in Aspergillus niger cells. Our findings suggest that malate dehydrogenase could potentially serve as an inhibitory target of cinnamaldehyde in Aspergillus niger cells. By disrupting the activity of malate dehydrogenase, cinnamaldehyde interferes with the mitochondrial tricarboxylic acid (TCA) cycle, leading to a significant decrease in intracellular ATP levels. Following treatment with cinnamaldehyde at a concentration of 1 MIC, the inhibition rate of MDH activity was 74.90 %, accompanied by an 84.5 % decrease in intracellular ATP content. Furthermore, cinnamaldehyde disrupts cell membrane integrity, resulting in the release of cellular contents and subsequent cell demise. This study endeavors to unveil the molecular-level antifungal mechanism of cinnamaldehyde via a chemoproteomics approach, thereby offering valuable insights for further development and utilization of cinnamaldehyde in preventing and mitigating food spoilage.

Nitric oxide-mediated regulation of Aspergillus flavus asexual development by targeting TCA cycle and mitochondrial function.

Nitric oxide (NO) is a signaling molecule with diverse roles in various organisms. However, its role in the opportunistic pathogen Aspergillus flavus remains unclear. This study investigates the potential of NO, mediated by metabolites from A. oryzae (AO), as an antifungal strategy against A. flavus. We demonstrated that AO metabolites effectively suppressed A. flavus asexual development, a critical stage in its lifecycle. Transcriptomic analysis revealed that AO metabolites induced NO synthesis genes, leading to increased intracellular NO levels. Reducing intracellular NO content rescued A. flavus spores from germination inhibition caused by AO metabolites. Furthermore, exogenous NO treatment and dysfunction of flavohemoglobin Fhb1, a key NO detoxification enzyme, significantly impaired A. flavus asexual development. RNA-sequencing and metabolomic analyses revealed significant metabolic disruptions within tricarboxylic acid (TCA) cycle upon AO treatment. NO treatment significantly reduced mitochondrial membrane potential (Δψm) and ATP generation. Additionally, aberrant metabolic flux within the TCA cycle was observed upon NO treatment. Further analysis revealed that NO induced S-nitrosylation of five key TCA cycle enzymes. Genetic analysis demonstrated that the S-nitrosylated Aconitase Acon and one subunit of succinate dehydrogenase Sdh2 played crucial roles in A. flavus development by regulating ATP production. This study highlights the potential of NO as a novel antifungal strategy to control A. flavus by compromising its mitochondrial function and energy metabolism.

L-DOPA ameliorates hippocampus-based mitochondria respiratory dysfunction caused by GCI/R injury.

Mitochondrial dysmorphology/dysfunction follow global cerebral ischemia-reperfusion (GCI/R) injury, leading to neuronal death. Our previous researches demonstrated that Levodopa (L-DOPA) improves learning and memory impairment in GCI/R rats by increasing synaptic plasticity of hippocampal neurons. This study investigates if L-DOPA, used in Parkinson's disease treatment, alleviates GCI/R-induced cell death by enhancing mitochondrial quality. Metabolomics and transcriptomic results showed that GCI/R damage affected the Tricarboxylic acid (TCA) cycle in the hippocampus. The results of this study show that L-DOPA stabilized mitochondrial membrane potential and ultrastructure in hippocampus of GCI/R rats, increased dopamine level in hippocampus, decreased succinic acid level, and stabilized Ca2+ level in CA1 subregion of hippocampus. As a precursor of dopamine, L-DOPA is presumed to improves mitochondrial function in hippocampus of GCI/R rats. However, dopamine cannot cross the blood-brain barrier, so L-DOPA is used in clinical therapy to supplement dopamine. In this investigation, OGD/R models were established in isolated mouse hippocampal neurons (HT22) and primary rat hippocampal neurons. Notably, dopamine exhibited a multifaceted impact, demonstrating inhibition of mitochondrial reactive oxygen species (mitoROS) production, stabilization of mitochondrial membrane potential and Ca2+ level, facilitation of TCA circulation, promotion of aerobic respiratory metabolism, and downregulation of succinic acid-related gene expression. Consistency between in vitro and in vivo results underscores dopamine's significant neuroprotective role in mitigating mitochondrial dysfunction following global cerebral hypoxia and ischemia injury. Supplement dopamine may represent a promising therapy to the cognitive impairment caused by GCI/R injury.

OGDH and Bcl-xL loss causes synthetic lethality in glioblastoma.

Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the α-ketoglutarate dehydrogenase (OGDH) gene, which encodes an enzyme that is part of the tricarboxylic acid (TCA) cycle, as essential for GBM growth. Moreover, by combining transcriptome and metabolite screening analyses, we discovered that loss of function of OGDH by the clinically validated drug compound CPI-613 was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3 mimetic, ABT263) in patient-derived xenografts as well neurosphere GBM cultures. CPI-613-mediated energy deprivation drove an integrated stress response with an upregulation of the BH3-only domain protein, Noxa, in an ATF4-dependent manner, as demonstrated by genetic loss-of-function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with disruption of the TCA cycle might be a treatment strategy for GBM.

Itaconic acid regulation of TFEB-mediated autophagy flux alleviates hyperoxia-induced bronchopulmonary dysplasia.

BACKGROUND: Premature infants often require oxygen supplementation, which can elicit bronchopulmonary dysplasia (BPD) and lead to mitochondrial dysfunction. Mitochondria play important roles in lung development, in both normal metabolism and apoptosis. Enhancing our comprehension of the underlying mechanisms in BPD development can facilitate the effective treatments. METHODS: Plasma samples from BPD and non-BPD infants were collected at 36 weeks post-menstrual age and used for metabolomic analysis. Based on hyperoxia-induced animal and cell models, changes in mitophagy and apoptosis were evaluated following treatment with itaconic acid (ITA). Finally, the mechanism of action of ITA in lung development was comprehensively demonstrated through rescue strategies and administration of corresponding inhibitors. RESULTS: An imbalance in the tricarboxylic acid (TCA) cycle significantly affected lung development, with ITA serving as a significant metabolic marker for the outcomes of lung development. ITA improved the morphological changes in BPD rats, promoted SP-C expression, and inhibited the degree of alveolar type II epithelial cells (AEC II) apoptosis. Mechanistically, ITA mainly promotes the nuclear translocation of transcription factor EB (TFEB) to facilitate dysfunctional mitochondrial clearance and reduces apoptosis in AEC II cells by regulating autophagic flux. CONCLUSION: The metabolic imbalance in the TCA cycle is closely related to lung development. ITA can improve lung development by regulating autophagic flux and promote the nuclear translocation of TFEB, implying its potential therapeutic utility in the treatment of BPD.

Insulin and glycolysis dependency of cardioprotection by nicotinamide riboside.

Decreased nicotinamide adenine dinucleotide (NAD+) levels contribute to various pathologies such as ageing, diabetes, heart failure and ischemia-reperfusion injury (IRI). Nicotinamide riboside (NR) has emerged as a promising therapeutic NAD+ precursor due to efficient NAD+ elevation and was recently shown to be the only agent able to reduce cardiac IRI in models employing clinically relevant anesthesia. However, through which metabolic pathway(s) NR mediates IRI protection remains unknown. Furthermore, the influence of insulin, a known modulator of cardioprotective efficacy, on the protective effects of NR has not been investigated. Here, we used the isolated mouse heart allowing cardiac metabolic control to investigate: (1) whether NR can protect the isolated heart against IRI, (2) the metabolic pathways underlying NR-mediated protection, and (3) whether insulin abrogates NR protection. NR protection against cardiac IRI and effects on metabolic pathways employing metabolomics for determination of changes in metabolic intermediates, and 13C-glucose fluxomics for determination of metabolic pathway activities (glycolysis, pentose phosphate pathway (PPP) and mitochondrial/tricarboxylic acid cycle (TCA cycle) activities), were examined in isolated C57BL/6N mouse hearts perfused with either (a) glucose + fatty acids (FA) ("mild glycolysis group"), (b) lactate + pyruvate + FA ("no glycolysis group"), or (c) glucose + FA + insulin ("high glycolysis group"). NR increased cardiac NAD+ in all three metabolic groups. In glucose + FA perfused hearts, NR reduced IR injury, increased glycolytic intermediate phosphoenolpyruvate (PEP), TCA intermediate succinate and PPP intermediates ribose-5P (R5P) / sedoheptulose-7P (S7P), and was associated with activated glycolysis, without changes in TCA cycle or PPP activities. In the "no glycolysis" hearts, NR protection was lost, whereas NR still increased S7P. In the insulin hearts, glycolysis was largely accelerated, and NR protection abrogated. NR still increased PPP intermediates, with now high 13C-labeling of S7P, but NR was unable to increase metabolic pathway activities, including glycolysis. Protection by NR against IRI is only present in hearts with low glycolysis, and is associated with activation of glycolysis. When activation of glycolysis was prevented, through either examining "no glycolysis" hearts or "high glycolysis" hearts, NR protection was abolished. The data suggest that NR's acute cardioprotective effects are mediated through glycolysis activation and are lost in the presence of insulin because of already elevated glycolysis.

Fusion-fission-mitophagy cycling and metabolic reprogramming coordinate nerve growth factor (NGF)-dependent neuronal differentiation.

Neuronal differentiation is regulated by nerve growth factor (NGF) and other neurotrophins. We explored the impact of NGF on mitochondrial dynamics and metabolism through time-lapse imaging, metabolomics profiling, and computer modeling studies. We show that NGF may direct differentiation by stimulating fission, thereby causing selective mitochondrial network fragmentation and mitophagy, ultimately leading to increased mitochondrial quality and respiration. Then, we reconstructed the dynamic fusion-fission-mitophagy cycling of mitochondria in a computer model, integrating these processes into a single network mechanism. Both the computational model and the simulations are able to reproduce the proposed mechanism in terms of mitochondrial dynamics, levels of reactive oxygen species (ROS), mitophagy, and mitochondrial quality, thus providing a computational tool for the interpretation of the experimental data and for future studies aiming to detail further the action of NGF on mitochondrial processes. We also show that changes in these mitochondrial processes are intertwined with a metabolic function of NGF in differentiation: NGF directs a profound metabolic rearrangement involving glycolysis, TCA cycle, and the pentose phosphate pathway, altering the redox balance. This metabolic rewiring may ensure: (a) supply of both energy and building blocks for the anabolic processes needed for morphological reorganization, as well as (b) redox homeostasis.

Freeze-Thaw Microfluidic System Produces "Themis" Nanocomplex for Cleaning Persisters-Infected Macrophages and Enhancing Uninfected Macrophages.

Macrophages are the primary effectors against potential pathogen infections. They can be "parasitized" by intracellular bacteria, serving as "accomplices", protecting intracellular bacteria and even switching them to persisters. Here, using a freeze-thaw strategy-based microfluidic chip, a "Themis" nanocomplex (TNC) is created. The TNC consists of Lactobacillus reuteri-derived membrane vesicles, heme, and vancomycin, which cleaned infected macrophages and enhanced uninfected macrophages. In infected macrophages, TNC releases heme that led to the reconstruction of the respiratory chain complexes of intracellular persisters, forcing them to regrow. The revived bacteria produces virulence factors that destroyed host macrophages (accomplices), thereby being externalized and becoming vulnerable to immune responses. In uninfected macrophages, TNC upregulates the TCA cycle and oxidative phosphorylation (OXPHOS), contributing to immunoenhancement. The combined effect of TNC of cleaning the accomplice (infected macrophages) and reinforcing uninfected macrophages provides a promising strategy for intracellular bacterial therapy.

Reversine ameliorates hallmarks of cellular senescence in human skeletal myoblasts via reactivation of autophagy.

Cellular senescence leads to the depletion of myogenic progenitors and decreased regenerative capacity. We show that the small molecule 2,6-disubstituted purine, reversine, can improve some well-known hallmarks of cellular aging in senescent myoblast cells. Reversine reactivated autophagy and insulin signaling pathway via upregulation of Adenosine Monophosphate-activated protein kinase (AMPK) and Akt2, restoring insulin sensitivity and glucose uptake in senescent cells. Reversine also restored the loss of connectivity of glycolysis to the TCA cycle, thus restoring dysfunctional mitochondria and the impaired myogenic differentiation potential of senescent myoblasts. Altogether, our data suggest that cellular senescence can be reversed by treatment with a single small molecule without employing genetic reprogramming technologies.

Tumor necrosis factor induces pathogenic mitochondrial ROS in tuberculosis through reverse electron transport.

Tumor necrosis factor (TNF) is a critical host resistance factor against tuberculosis. However, excess TNF produces susceptibility by increasing mitochondrial reactive oxygen species (mROS), which initiate a signaling cascade to cause pathogenic necrosis of mycobacterium-infected macrophages. In zebrafish, we identified the mechanism of TNF-induced mROS in tuberculosis. Excess TNF in mycobacterium-infected macrophages elevates mROS production by reverse electron transport (RET) through complex I. TNF-activated cellular glutamine uptake leads to an increased concentration of succinate, a Krebs cycle intermediate. Oxidation of this elevated succinate by complex II drives RET, thereby generating the mROS superoxide at complex I. The complex I inhibitor metformin, a widely used antidiabetic drug, prevents TNF-induced mROS and necrosis of Mycobacterium tuberculosis-infected zebrafish and human macrophages; metformin may therefore be useful in tuberculosis therapy.