Intracellular Metabolite Levels Research Articles

Elucidating the pivotal role of TSPO in porphyrin-related cellular processes, in Bacillus cereus

A structural homolog of the mammalian TSPO has been identified in the human pathogen Bacillus cereus. BcTSPO, in its recombinant form, has previously been shown to bind and degrade porphyrins. In this study, we generated a ΔtspO mutant strain in B. cereus ATCC 14579 and assessed the impact of the absence of BcTSPO on cellular proteomics and physiological characteristics. The proteomic analysis revealed correlations between the lack of BcTSPO and the observed growth defects, increased oxygen consumption, ATP deficiency, heightened tryptophan catabolism, reduced motility, and impaired biofilm formation in the ΔtspO mutant strain. Our results also suggested that BcTSPO plays a crucial role in regulating intracellular levels of metabolites from the coproporphyrin-dependent branch of the heme biosynthetic pathway. This regulation potentially underlies alterations in the metabolic landscape, emphasizing the pivotal role of BcTSPO in B. cereus aerobic metabolism. Notably, our study unveils, for the first time, the involvement of TSPO in tryptophan metabolism. These findings underscore the multifaceted role of TSPO, not only in metabolic pathways but also potentially in the microorganism's virulence mechanisms.

CD8+ T Cells Keep Their (K+)urrency for Function.

CD8+ T-cell responses are influenced by ion abundance, which can widely vary within the tumor microenvironment. In this issue, Collier and colleagues investigated how intracellular versus extracellular potassium ion (K+) regulates intratumoral CD8+ T cells. They show that, while excessive extracellular K+ induces exhaustion, intracellular K+ is needed for protection from dysfunction. This work shows additional evidence that the regulation of CD8+ T-cell responses depends on a fine balance between intracellular and extracellular metabolite levels. See related article by Collier et al., p. 36 (2) .

Disruption of trehalose periplasmic recycling dysregulates cAMP-CRP signaling in Escherichia coli during stationary phase.

Survival during starvation hinges on the ability to manage intracellular energy reserves and to initiate appropriate metabolic responses to perturbations of such reserves. How Escherichia coli manage carbon storage systems under starvation stress, as well as transpose changes in intracellular metabolite levels into regulatory signals, is not well understood. Endogenous trehalose metabolism may be at the center of these processes, coupling carbon storage with carbon starvation responses. The coupled transport to the periplasm and subsequent hydrolysis of trehalose back to glucose for transport to the cytoplasm may function as a crucial metabolic signaling pathway. Although trehalose has been characterized as a stress protectant in E. coli, the disaccharide also functions as both an energy storage compound and a regulator of carbohydrate metabolism in fungi, plants, and other bacteria. Our research explores the metabolic regulatory properties of trehalose in E. coli and a potential mechanism by which the intracellular carbon pool is interconnected with regulatory circuits, enabling long-term survival.

Genetic dissection of regulation by a repressing and novel activating corrinoid riboswitch enables engineering of synthetic riboswitches.

In addition to proteins, microbes can use structured RNAs such as riboswitches for the important task of regulating gene expression. Riboswitches control gene expression by changing their structure in response to binding a small molecule and are widespread among bacteria. Here we determine the mechanism of regulation in a riboswitch that responds to corrinoids-a family of coenzymes related to vitamin B12. We report the alternative RNA secondary structures that couple corrinoid sensing with response in a repressing and novel activating corrinoid riboswitch. We then applied this knowledge to flipping the regulatory sign by constructing synthetic riboswitches that activate expression to a higher level than the natural one. In the process, we observed patterns in which sequence, in addition to structure, impacts function in paired RNA regions. The synthetic riboswitches we describe here have potential applications as biosensors.

Fundamental Neurochemistry Review: Microglial immunometabolism in traumatic brain injury.

Traumatic brain injury (TBI) is a devastating neurological disorder caused by a physical impact to the brain that promotes diffuse damage and chronic neurodegeneration. Key mechanisms believed to support secondary brain injury include mitochondrial dysfunction and chronic neuroinflammation. Microglia and brain-infiltrating macrophages are responsible for neuroinflammatory cytokine and reactive oxygen species (ROS) production after TBI. Their production is associated with loss of homeostatic microglial functions such as immunosurveillance, phagocytosis, and immune resolution. Beyond providing energy support, mitochondrial metabolic pathways reprogram the pro- and anti-inflammatory machinery in immune cells, providing a critical immunometabolic axis capable of regulating immunologic response to noxious stimuli. In the brain, the capacity to adapt to different environmental stimuli derives, in part, from microglia's ability to recognize and respond to changes in extracellular and intracellular metabolite levels. This capacity is met by an equally plastic metabolism, capable of altering immune function. Microglial pro-inflammatory activation is associated with decreased mitochondrial respiration, whereas anti-inflammatory microglial polarization is supported by increased oxidative metabolism. These metabolic adaptations contribute to neuroimmune responses, placing mitochondria as a central regulator of post-traumatic neuroinflammation. Although it is established that profound neurometabolic changes occur following TBI, key questions related to metabolic shifts in microglia remain unresolved. These include (a) the nature of microglial mitochondrial dysfunction after TBI, (b) the hierarchical positions of different metabolic pathways such as glycolysis, pentose phosphate pathway, glutaminolysis, and lipid oxidation during secondary injury and recovery, and (c) how immunometabolism alters microglial phenotypes, culminating in chronic non-resolving neuroinflammation. In this basic neurochemistry review article, we describe the contributions of immunometabolism to TBI, detail primary evidence of mitochondrial dysfunction and metabolic impairments in microglia and macrophages, discuss how major metabolic pathways contribute to post-traumatic neuroinflammation, and set out future directions toward advancing immunometabolic phenotyping in TBI.

Assays for profiling changes in T cell metabolism

Abstract Immune cell therapy, including engineered T cells, engineered NK cells, and treatments to enhance native T cell response, is a rapidly advancing field of research with great promise to treat multiple diseases. However, multiple challenges, from isolating the correct cell type, determining its mode of action, manufacturing and establishing appropriate quality control criteria, must be overcome to reach its potential. In recent years, cellular energy metabolism has emerged as an important consideration for efficacy and persistence when developing cell therapies. Here we evaluate a set of bioluminescent cell-based assays for measuring rate of metabolite uptake, monitoring activity for key metabolic pathways and determining changes in intracellular metabolite levels during T cell transition from naïve to activated to memory phenotypes. Metabolite levels measured include ATP, glucose uptake, malate, branched chain amino acids, and glycogen levels. Depending on the activation state of the cells, the cells respond differently to glycolysis and oxidative phosphorylation inhibitors, as demonstrated by changes to metabolite levels. Energy storage as measured by intracellular glycogen levels also differs depending on the T cell phenotype (naïve, activated, or memory). Understanding the processes governing dynamic metabolic changes during cell modification and expansion is required for next-generation cell therapy development. The developed bioluminescent cell-based assays provide the sensitivity, throughput and robustness required for rapid evaluation of such changes.

Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors.

Quantification of the concentration of particular cellular metabolites reports on the actual utilization of metabolic pathways in physiological and pathological conditions. Metabolite concentration also constitutes the readout for screening cell factories in metabolic engineering. However, there are no direct approaches that allow for real-time assessment of the levels of intracellular metabolites in single cells. In recent years, the modular architecture of natural bacterial RNA riboswitches has inspired the design of genetically encoded synthetic RNA devices that convert the intracellular concentration of a metabolite into a quantitative fluorescent signal. These so-called RNA-based sensors are composed of a metabolite-binding RNA aptamer as the sensor domain, connected through an actuator segment to a signal-generating reporter domain. However, at present, the variety of available RNA-based sensors for intracellular metabolites is still very limited. Here, we go through natural mechanisms for metabolite sensing and regulation in cells across all kingdoms, focusing on those mediated by riboswitches. We review the design principles underlying currently developed RNA-based sensors and discuss the challenges that hindered the development of novel sensors and recent strategies to address them. We finish by introducing the current and potential applicability of synthetic RNA-based sensors for intracellular metabolites.

Exendin-4 attenuates atherosclerosis progression via controlling hematopoietic stem/progenitor cell proliferation.

Beyond glycemic control, applications of glucagon-like peptide-1 receptor (GLP-1r) agonists (GLP-1 RAs) inhibit inflammation and plaque development in murine atherosclerotic models. However, whether they modulate hematopoietic stem/progenitor cells (HSPCs) to prohibit skewed myelopoiesis in hypercholesteremia remains unknown. In this study, GLP-1r expression in fluorescence-activated cell sorting (FACS)-sorted wild-type HSPCs was determined by capillary western blotting. Bone marrow cells (BMCs) of wild-type or GLP-1r-/- mice were transplanted into lethally irradiated low-density lipoprotein receptor deficient (LDLr-/-) recipients followed by high-fat diet (HFD) for chimerism analysis by FACS. In parallel, LDLr-/- mice were placed on HFD for 6 weeks and then treated with saline or Exendin-4 (Ex-4) for another 6 weeks. HSPC frequency and cell cycle were analyzed by FACS, and intracellular metabolite levels were assessed by targeted metabolomics. The results demonstrated that HSPCs expressed GLP-1r and transplantation of GLP-1r-/- BMCs resulted in skewed myelopoiesis in hypercholesterolemic LDLr-/- recipients. In vitro, Ex-4 treatment of FACS-purified HSPCs suppressed cell expansion and granulocyte production induced by LDL. In vivo, Ex-4 treatment inhibited plaque progression, suppressed HSPC proliferation, and modified glycolytic and lipid metabolism in HSPCs of hypercholesteremic LDLr-/- mice. In conclusion, Ex-4 could directly inhibit HSPC proliferation induced by hypercholesteremia.

Proteomics and Metabolomics Analysis Reveals the Toxicity of ZnO Quantum Dots on Human SMMC-7721 Cells

ZnO quantum dots (QDs) are composed of less toxic metals than other QDs but have the same interesting photochemical properties. Thus, they have received considerable attention recently. Nevertheless, their toxicity cannot be ignored. In this study, we incubated ZnO QDs with human SMMC-7721 cells for 24 h to assess their nanotoxicity through proteomics (Fold change >1.5 and p-value <0.05) and metabolomics (Fold change ≥ 1.5; VIP ≥ 1; p-value < 0.05) analyses. Both of 174 and 219 significantly changed metabolites were identified in human SMMC-7721 cells treated with 20 and 50 µg/mL ZnO QDs, respectively. ZnO QDs significantly modified metabolic pathways, including purine metabolism, ferroptosis, morphine addiction, alcoholism, cGMP-PKG signaling, and Cushing syndrome. Moreover, we identified 105 and 8 differentially expressed proteins in cells treated with 20 and 50 µg/mL ZnO QDs, and the pathways of alcoholism and Cushing syndrome were enriched. ZnO QDs did not affect cell viability in a CCK8 assay, but disturbed the level of intracellular metabolites and proteins at 20 µg/mL. The KEGG analyses of the metabolomics and proteomics data both enriched the alcoholism and Cushing syndrome pathways. These results provide an experimental basis for future research on the safe use of nanomaterials.

Photoproximity Labeling of Sialylated Glycoproteins (GlycoMap) Reveals Sialylation-Dependent Regulation of Ion Transport.

Sialylation, the addition of sialic acid to glycans, is a crucial post-translational modification of proteins, contributing to neurodevelopment, oncogenesis, and immune response. In cancer, sialylation is dramatically upregulated. Yet, the functional biochemical consequences of sialylation remain mysterious. Here, we establish a μMap proximity labeling platform that utilizes metabolically inserted azidosialic acid to introduce iridium-based photocatalysts on sialylated cell-surface glycoproteins as a means to profile local microenvironments across the sialylated proteome. In comparative experiments between primary cervical cells and a cancerous cell line (HeLa), we identify key differences in both the global sialome and proximal proteins, including solute carrier proteins that regulate metabolite and ion transport. In particular, we show that cell-surface interactions between receptors trafficking ethanolamine and zinc are sialylation-dependent and impact intracellular metabolite levels. These results establish a μMap method for interrogating proteoglycan function and support a role for sialylated glycoproteins in regulating cell-surface transporters.

Metabolite-derived protein modifications modulating oncogenic signaling.

Malignant growth is defined by multiple aberrant cellular features, including metabolic rewiring, inactivation of tumor suppressors and the activation of oncogenes. Even though these features have been described as separate hallmarks, many studies have shown an extensive mutual regulatory relationship amongst them. On one hand, the change in expression or activity of tumor suppressors and oncogenes has extensive direct and indirect effects on cellular metabolism, activating metabolic pathways required for malignant growth. On the other hand, the tumor microenvironment and tumor intrinsic metabolic alterations result in changes in intracellular metabolite levels, which directly modulate the protein modification of oncogenes and tumor suppressors at both epigenetic and post-translational levels. In this mini-review, we summarize the crosstalk between tumor suppressors/oncogenes and metabolism-induced protein modifications at both levels and explore the impact of metabolic (micro)environments in shaping these.

Microbial containment device: A platform for comprehensive analysis of microbial metabolism without sample preparation.

Metabolomics is a mainstream strategy for investigating microbial metabolism. One emerging application of metabolomics is the systematic quantification of metabolic boundary fluxes – the rates at which metabolites flow into and out of cultured cells. Metabolic boundary fluxes can capture complex metabolic phenotypes in a rapid assay, allow computational models to be built that predict the behavior of cultured organisms, and are an emerging strategy for clinical diagnostics. One advantage of quantifying metabolic boundary fluxes rather than intracellular metabolite levels is that it requires minimal sample processing. Whereas traditional intracellular analyses require a multi-step process involving extraction, centrifugation, and solvent exchange, boundary fluxes can be measured by simply analyzing the soluble components of the culture medium. To further simplify boundary flux analyses, we developed a custom 96-well sampling system—the Microbial Containment Device (MCD)—that allows water-soluble metabolites to diffuse from a microbial culture well into a bacteria-free analytical well via a semi-permeable membrane. The MCD was designed to be compatible with the autosamplers present in commercial liquid chromatography-mass spectrometry systems, allowing metabolic fluxes to be analyzed with minimal sample handling. Herein, we describe the design, evaluation, and performance testing of the MCD relative to traditional culture methods. We illustrate the utility of this platform, by quantifying the unique boundary fluxes of four bacterial species and demonstrate antibiotic-induced perturbations in their metabolic activity. We propose the use of the MCD for enabling single-step metabolomics sample preparation for microbial identification, antimicrobial susceptibility testing, and other metabolic boundary flux applications where traditional sample preparation methods are impractical.

Sunitinib versus Pazopanib Dilemma in Renal Cell Carcinoma: New Insights into the In Vitro Metabolic Impact, Efficacy, and Safety.

Sunitinib and pazopanib are tyrosine kinase inhibitors (TKIs) used as first-line therapy for metastatic renal cell carcinoma (RCC). Although these TKIs are associated with similar survival outcomes, some differences have been reported in their safety profiles. In this work, traditional toxicological endpoints (cell viability and growth, oxidative stress, and nuclear morphology) and 1H NMR spectroscopy-based metabolomics analysis were used to provide new insights into the cytotoxicity and metabolic mechanisms underlying sunitinib and pazopanib treatments. Tumoral (Caki-1) and non-tumoral (HK-2) human renal cells were exposed to clinically relevant concentrations of sunitinib (2 µM) or pazopanib (50 µM). Sunitinib showed selectivity for cancer cells, inhibiting proliferation, and inducing apoptotic death of Caki-1 cells, whereas pazopanib had a similar cytotoxic effect in both tumoral and non-tumoral cells. 1H-NMR metabolomics unveiled a higher impact of sunitinib on the levels of intracellular metabolites of Caki-1 cells (seven dysregulated metabolites), suggesting dysregulations on amino acid, glutathione and glycerophospholipid metabolisms. In contrast, pazopanib had a higher impact on the levels of extracellular metabolites of Caki-1 cells (seven dysregulated metabolites in culture medium), unveiling alterations on amino acid and energetic metabolisms. In HK-2 cells, sunitinib caused only a minor increase in intracellular isoleucine levels, whereas pazopanib induced several alterations on the intracellular (three dysregulated metabolites) and extracellular (three dysregulated metabolites) compartments suggesting changes on amino acid, glycerophospholipid, and energy metabolisms. Our results demonstrate that these TKIs elicit distinct cellular and metabolic responses, with sunitinib showing better in vitro efficacy against target RCC cells and lesser nephrotoxic potential than pazopanib.

Intracellular pyruvate levels positively correlate with cytokine production capacity in tolerant monocytes from patients with pneumonia

BackgroundCommunity-acquired pneumonia (CAP) is responsible for a high morbidity and mortality worldwide. Monocytes are essential for pathogen recognition and the initiation of an innate immune response. Immune cells induce intracellular glycolysis upon activation to support several functions. ObjectiveTo obtain insight in the metabolic profile of blood monocytes during CAP, with a focus on glycolysis and branching metabolic pathways, and to determine a possible association between intracellular metabolite levels and monocyte function. MethodsMonocytes were isolated from blood of patients with CAP within 24 h of hospital admission and from control subjects matched for age, sex and chronic comorbidities. Changes in glycolysis, oxidative phosphorylation (OXPHOS), tricarboxylic acid (TCA) cycle and the pentose phosphate pathway were investigated through RNA sequencing and metabolomics measurements. Monocytes were stimulated ex vivo with lipopolysaccharide (LPS) to determine their capacity to produce tumor necrosis factor (TNF), interleukin (IL)-1β and IL-10. Results50 patients with CAP and 25 non-infectious control subjects were studied. When compared with control monocytes, monocytes from patients showed upregulation of many genes involved in glycolysis, including PKM, the gene encoding pyruvate kinase, the rate limiting enzyme for pyruvate production. Gene set enrichment analysis of OXPHOS, the TCA cycle and the pentose phosphate pathway did not reveal differences between monocytes from patients and controls. Patients' monocytes had elevated intracellular levels of pyruvate and the TCA cycle intermediate α-ketoglutarate. Monocytes from patients were less capable of producing cytokines upon LPS stimulation. Intracellular pyruvate (but not α-ketoglutarate) concentrations positively correlated with IL-1β and IL-10 levels released by patients' (but not control) monocytes upon exposure to LPS. ConclusionThese results suggest that elevated intracellular pyruvate levels may partially maintain cytokine production capacity of hyporesponsive monocytes from patients with CAP.

Is Monitoring of the Intracellular Active Metabolite Levels of Nucleobase and Nucleoside Analogs Ready for Precision Medicine Applications?

Nucleobase and nucleoside analogs (NAs) play important roles in cancer therapy. Although there are obvious individual differences in NA treatments, most NAs lack direct relationships between their plasma concentrationand efficacy or adverse effects. Accumulating evidence suggests that the intracellular active metabolite levels of NAs predict patient outcomes. This article reviewed the relationships between NA intracellular active metabolite levelsand their efficacy or adverse effects. The factors affecting the formation of intracellular active metabolites and combination regimens that elevate intracellular active metabolite levels were also reviewed. Given the mechanism of NA cytotoxicity, NA intracellular active metabolite levels may be predictive of clinical outcomes. Many clinical studies support this hypothesis. Therefore, the monitoring of intracellular active metabolite levels is beneficial for individualized NA treatment. However, to perform clinical monitoring in practice, well-designed studies are needed to explore the optimal threshold or range and the appropriate regimen adjustment strategies based on these parameters.

Acylations in cardiovascular diseases: advances and perspectives.

Acylations in cardiovascular diseases: advances and perspectives.

Pharmacological inhibition of sphingolipid synthesis reduces ferroptosis by stimulating the HIF-1 pathway

SummaryFerroptosis is crucial to the pathology of many neurological diseases. Here, we found pre-treatment with myriocin, an inhibitor of de novo synthesis of sphingolipid, significantly decreased the erastin- or glutamate-induced ferroptosis of HT22 cells without requiring the recovery of intracellular glutathione. The transcriptome analysis of HT22 cells treated with or without myriocin identified the hypoxia-inducible factor 1 (HIF-1) pathway as a prime and novel drug target. Further study validated that HIF1α was required for the cytoprotective effects of myriocin. Myriocin treatment promoted the expression of HIF-1 pathway effectors including PDK1 and BNIP3 and altered the intracellular levels of glucose metabolites. Additionally, myriocin treatment stabilized HIF1α protein by decreasing its ubiquitination and proteasomal degradation. Similar effects of myriocin on HIF1α stabilization were also found in other mammalian cell lines indicating this is a common mechanism for the cytoprotective role of myriocin.

Conversion of Hyperpolarized [1-13C]Pyruvate in Breast Cancer Cells Depends on Their Malignancy, Metabolic Program and Nutrient Microenvironment.

Simple SummaryThe metabolic phenotype of cancer cells depends on their metabolic program and the availability of nutrients in the tumor microenvironment. Metabolic activities in vivo can be characterized by using magnetic resonance spectroscopy (MRS) for analyzing the conversion of hyperpolarized 13C-pyruvate to 13C-lactate. To investigate how the conversion rates (kpl) of exogenous pyruvate to lactate are affected by glucose and glutamine availability, two breast cancer cell lines of different malignancy were analyzed in vitro for the Warburg effect, enzyme activities, and flux of 13C-glucose-derived metabolites. Conversion of 13C-pyruvate correlated with glucose/glutamine-dependent glycolytic activity, but not with the Warburg effect. Unexpectedly, the more malignant cells had lower kpl-values, in spite of having higher lactate production. It is suggested that exogenous pyruvate is converted by LDH associated with the glycolytic micro-compartment. Analyses of pyruvate-to-lactate conversion with MRS thus depend on both the metabolic program and the nutritional state of the tumors.Hyperpolarized magnetic resonance spectroscopy (MRS) is a technology for characterizing tumors in vivo based on their metabolic activities. The conversion rates (kpl) of hyperpolarized [1-13C]pyruvate to [1-13C]lactate depend on monocarboxylate transporters (MCT) and lactate dehydrogenase (LDH); these are also indicators of tumor malignancy. An unresolved issue is how glucose and glutamine availability in the tumor microenvironment affects metabolic characteristics of the cancer and how this relates to kpl-values. Two breast cancer cells of different malignancy (MCF-7, MDA-MB-231) were cultured in media containing defined combinations of low glucose (1 mM; 2.5 mM) and glutamine (0.1 mM; 1 mM) and analyzed for pyruvate uptake, intracellular metabolite levels, LDH and pyruvate kinase activities, and 13C6-glucose-derived metabolomics. The results show variability of kpl with the different glucose/glutamine conditions, congruent with glycolytic activity, but not with LDH activity or the Warburg effect; this suggests metabolic compartmentation. Remarkably, kpl-values were almost two-fold higher in MCF-7 than in the more malignant MDA-MB-231 cells, the latter showing a higher flux of 13C-glucose-derived pyruvate to the TCA-cycle metabolites 13C2-citrate and 13C3-malate, i.e., pyruvate decarboxylation and carboxylation, respectively. Thus, MRS with hyperpolarized [1-13C-pyruvate] is sensitive to both the metabolic program and the nutritional state of cancer cells.

Pulsed-Ultrasound Irradiation Induces the Production of Itaconate and Attenuates Inflammatory Responses in Macrophages.

BackgroundItaconate is a key metabolite in the innate immune system and exerts strong anti-inflammatory effects in macrophages. For the production of itaconate in macrophages, immune-responsive gene 1 (IRG1) is an imperative enzyme, and activating the IRG1-itaconate pathway is reported to alleviate inflammatory diseases by upregulating nuclear factor-erythroid 2-related factor 2 (NRF2). However, there are very few reports on strategies to increase itaconate production. Ultrasound therapy is a widely used intervention for anti-inflammatory and soft-tissue regeneration purposes. Here we show the effect of ultrasound irradiation on the production of itaconate in macrophages.MethodsMurine bone marrow-derived macrophages (BMDMs) were exposed to pulsed ultrasound (3.0 W/cm2) for 5 minutes. Three hours after irradiation, the intracellular levels of metabolites and mRNA expression levels of Irg1 and Nrf2 were measured using CE/MS and qPCR, respectively. To evaluate macrophage inflammation status, 3 h after irradiation, the cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) for 1.5 h and the mRNA expression levels of pro-inflammatory factors (Il-1β, Il-6, and Tnf-α) were measured. Student’s t-test, one-way ANOVA and Tukey’s multiple comparison test were used for statistical processing, and the significance level was set to less than 5%.ResultsUltrasound irradiation significantly increased the intracellular itaconate level and the expression levels of Irg1 and Nrf2 in BMDMs. Upregulation of Il-1β, Il-6, and Tnf-α by LPS was significantly suppressed in BMDMs treated with ultrasound. Ultrasound irradiation did not affect cell viability and apoptosis.ConclusionUltrasound irradiation induces the production of itaconate by upregulating Irg1 expression and attenuates inflammatory responses in macrophages via Nrf2. These results suggest that ultrasound is a potentially useful method to increase itaconate production in macrophages.

Degradation of HIF-1α induced by curcumol blocks glutaminolysis and inhibits epithelial-mesenchymal transition and invasion in colorectal cancer cells.

Colorectal cancer (CRC) has high morbidity and mortality. Epithelial-mesenchymal transition (EMT) is associated with CRC progression and metastasis. Glutaminolysis is essential for malignancy of cancer cells. Here, we examined the effects of curcumol on CRC EMT. We observed that curcumol suppressed invasion and migration in human CRC cells associated with upregulation of epithelial markers E-cadherin and Zonula occludens 1 and downregulation of mesenchymal markers N-cadherin and Vimentin as well as EMT-related transcription factors Snail and Twist. Curcumol increased intracellular levels of glutamine but decreased intracellular levels of glutamate, α-ketoglutarate, ATP, glutathione, and tricarboxylic acid cycle metabolites, suggesting interruption of glutaminolysis. Next, curcumol repressed glutaminase 1 (Gls1) mRNA and protein expression, and overexpression of Gls1 promoted EMT and abolished curcumol effects on CRC cell EMT. Molecular examinations showed that curcumol stimulated protein degradation of hypoxia-inducible factor-1α (HIF-1α) and prevented its nuclear accumulation in CRC cells. HIF-1α agonist deferoxamine (DFO) promoted HIF-1α binding to Gls1 promoter and increased Gls1 expression but abolished curcumol's inhibitory effects on Gls1 expression. DFO also enhanced EMT and invasion and migration in CRC cells and eliminated curcumol effects. Furthermore, mouse CRC models were established with in vivo overexpression of HIF-1α and Gls1. Curcumol effectively inhibited CRC growth, metastasis, and EMT in mice, which was abrogated by overexpression of HIF-1α or Gls1. Altogether, stimulation of HIF-1α degradation was required for curcumol to disrupt EMT and repress invasion and migration in CRC cells through inhibiting Gls1-mediated glutaminolysis. Curcumol could be a promising candidate for intervention of CRC metastasis. • Curcumol inhibits EMT and blocks glutaminolysis in CRC cells. • Inhibition of Gls1 is required for curcumol blockade of glutaminolysis and EMT. • Curcumol induces HIF-1α degradation leading to inhibition of Gls1 and blockade of glutaminolysis and EMT. • Curcumol suppresses CRC growth and metastasis via inhibiting HIF-1α, glutaminolysis and EMT in mice.