Nanoplastics Penetrate Human Bronchial Smooth Muscle and Small Airway Epithelial Cells and Affect Mitochondrial Metabolism.

Micro- and nanoplastic particles, including common forms like polyethylene and polystyrene, have been identified as relevant pollutants, potentially causing health problems in living organisms. The mechanisms at the cellular level largely remain to be elucidated. This study aims to visualize nanoplastics in bronchial smooth muscle (BSMC) and small airway epithelial cells (SAEC), and to assess the impact on mitochondrial metabolism. Healthy and asthmatic human BSMC and SAEC in vitro cultures were stimulated with polystyrene nanoplastics (PS-NPs) of 25 or 50 nm size, for 1 or 24 h. Live cell, label-free imaging by holotomography microscopy and mitochondrial respiration and glycolysis assessment were performed. Furthermore, 25 and 50 nm NPs were shown to penetrate SAEC, along with healthy and diseased BSMC, and they impaired bioenergetics and induce mitochondrial dysfunction compared to cells not treated with NPs, including changes in oxygen consumption rate and extracellular acidification rate. NPs pose a serious threat to human health by penetrating airway tissues and cells, and affecting both oxidative and glycolytic metabolism.

Calculation of ATP production rates using the Seahorse XF Analyzer.

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.

Dissecting Rubella Placental Infection in an In Vitro Trophoblast Model.

Vertical transmission of rubella virus (RuV) occurs at a high rate during the first trimester of pregnancy. The modes of vertical transmission including the response of trophoblasts to RuV are not well understood. Here, RuV-trophoblast interaction was studied in the BeWo trophoblast cell line. Analysis included early and late time-point kinetics of virus infection rate and the antiviral innate immune response at mRNA and protein level. BeWo characteristics were addressed through metabolic activity by extracellular flux analysis and syncytiotrophoblast formation through incubation with forskolin. We found that RuV infection of BeWo led to profuse type III interferon (IFN) production. Transfecting trophoblast cells with dsRNA analog induced an increase in the production of type I IFN-ß and type III IFNs; however, this did not occur in RuV-infected BeWo trophoblasts. IFN-ß and to a lesser extent type III IFN-λ1 were inhibitory to RuV. While no significant metabolic alteration was detected, RuV infection reduced the cell number in the monolayer culture in comparison to the mock control and resulted in detached and floating cells. Syncytia formation restricted RuV infection. The use of BeWo as a relevant cell culture model for infection of trophoblasts highlights cytopathogenicity in the absence of a type I IFN response as a pathogenic alteration by RuV.

The Inhibition of Glycolysis in T Cells by a Jak Inhibitor Ameliorates the Pathogenesis of Allergic Contact Dermatitis in Mice.

Allergic contact dermatitis (ACD) and atopic dermatitis develop through delayed-type hypersensitivity reactions mediated by T cells. The development of immunomodulatory drugs, such as Jak inhibitors, would be useful for the long-term management of these diseases owing to their profile of favorable adverse effects. However, the efficacy of Jak inhibitors for ACD treatment has not been fully determined under a variety of settings. Therefore, we evaluated the effects of ruxolitinib, a Jak inhibitor for Jak1 and Jak2, using a mouse ACD model. As a result, the lower numbers of immune cells, including CD4+ T cells, CD8+ T cells, neutrophils, and possibly macrophages, as well as milder pathophysiological aspects have been observed in the inflamed skin of ACD with the administration of ruxolitinib. In addition, the treatment of differentiating T cells with ruxolitinib downregulated the level of IL-2-mediated glycolysis in vitro. Furthermore, symptoms of ACD did not develop in T-cell-specific Pgam1-deficient mice whose T cells had no glycolytic capacity. Taken together, our data suggest that the downregulation of glycolysis in T cells by ruxolitinib could be an important factor in the suppression of ACD development in mice.

Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF, and Temozolomide: Impact on the Mitochondrial Metabolism, Clonogenic Potential, and Migration of Human U251 Glioma Cells.

Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 µM), TMZ (155 µM), and NGF (7.55 × 10-3 µM) inhibited 43.9%-60.3%, 73.5%-81.3%, 66.2% the clonogenicity of glioma U251 cells for 1-2 days, respectively. LL-37 (4 µM), and NGF (7.55 × 10-3 µM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1-3 days. In contrast, PG-1 (16 µM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.

Glycolysis, monocarboxylate transport, and purinergic signaling are key events in Eimeria bovis-induced NETosis.

The protozoan parasite Eimeria bovis is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis-an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)-is elicited by E. bovis stages. So far, the metabolic requirements of E. bovis-triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in E. bovis sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied via chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished via PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in E. bovis sporozoite-induced NETosis.

mTORC1 and mTORC2 Complexes Regulate the Untargeted Metabolomics and Amino Acid Metabolites Profile through Mitochondrial Bioenergetic Functions in Pancreatic Beta Cells.

Background: Pancreatic beta cells regulate bioenergetics efficiency and secret insulin in response to glucose and nutrient availability. The mechanistic Target of Rapamycin (mTOR) network orchestrates pancreatic progenitor cell growth and metabolism by nucleating two complexes, mTORC1 and mTORC2. Objective: To determine the impact of mTORC1/mTORC2 inhibition on amino acid metabolism in mouse pancreatic beta cells (Beta-TC-6 cells, ATCC-CRL-11506) using high-resolution metabolomics (HRM) and live-mitochondrial functions. Methods: Pancreatic beta TC-6 cells were incubated for 24 h with either: RapaLink-1 (RL); Torin-2 (T); rapamycin (R); metformin (M); a combination of RapaLink-1 and metformin (RLM); Torin-2 and metformin (TM); compared to the control. We applied high-resolution mass spectrometry (HRMS) LC-MS/MS untargeted metabolomics to compare the twenty natural amino acid profiles to the control. In addition, we quantified the bioenergetics dynamics and cellular metabolism by live-cell imaging and the MitoStress Test XF24 (Agilent, Seahorse). The real-time, live-cell approach simultaneously measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine cellular respiration and metabolism. Statistical significance was assessed using ANOVA on Ranks and post-hoc Welch t-Tests. Results: RapaLink-1, Torin-2, and rapamycin decreased L-aspartate levels compared to the control (p = 0.006). Metformin alone did not affect L-aspartate levels. However, L-asparagine levels decreased with all treatment groups compared to the control (p = 0.03). On the contrary, L-glutamate and glycine levels were reduced only by mTORC1/mTORC2 inhibitors RapaLink-1 and Torin-2, but not by rapamycin or metformin. The metabolic activity network model predicted that L-aspartate and AMP interact within the same activity network. Live-cell bioenergetics revealed that ATP production was significantly reduced in RapaLink-1 (122.23 ± 33.19), Torin-2 (72.37 ± 17.33) treated cells, compared to rapamycin (250.45 ± 9.41) and the vehicle control (274.23 ± 38.17), p < 0.01. However, non-mitochondrial oxygen consumption was not statistically different between RapaLink-1 (67.17 ± 3.52), Torin-2 (55.93 ± 8.76), or rapamycin (80.01 ± 4.36, p = 0.006). Conclusions: Dual mTORC1/mTORC2 inhibition by RapaLink-1 and Torin-2 differentially altered the amino acid profile and decreased mitochondrial respiration compared to rapamycin treatment which only blocks the FRB domain on mTOR. Third-generation mTOR inhibitors may alter the mitochondrial dynamics and reveal a bioenergetics profile that could be targeted to reduce mitochondrial stress.

Multiplexing Seahorse XFe24 and ImageXpress® Nano Platforms for Comprehensive Evaluation of Mitochondrial Bioenergetic Profile and Neuronal Morphology.

The measurement of mitochondrial function has become imperative to understand and characterize diseases characterized by bioenergetic alterations. The advancement of automation and application of high-throughput technologies has propelled our understanding of biological complexity and facilitated drug discovery. Seahorse extracellular flux (XFe) technology measures changes in dissolved oxygen and proton concentration in cell culture media, providing kinetic measurements of oxidative phosphorylation and glycolytic metabolism. ImageXpress® Nano is an automated fluorescent microscope with the ability to perform high-content, fast, and robust imaging in multi-well formats. In this chapter, we present a comprehensive protocol to multiplex the Seahorse XFe24 analyzer with the ImageXpress® Nano high content imaging microscope to provide a comprehensive yet rigorous profile of bioenergetics and its correlation to neuronal function and morphology.

Mitochondrial bioenergetics in ocular fibroblasts of two myasthenia gravis cases.

Myasthenia gravis (MG) is a rare, treatable, antibody-mediated disease characterized by fatigable muscle weakness of extraocular muscles (EOMs) and non-ocular skeletal muscles. The antibodies are directed against muscle-endplate proteins, most frequently the acetylcholine receptor (AChR) alpha-subunit. Although most MG patients respond to immunosuppressive treatment, some individuals, frequently with African-genetic ancestry, develop treatment-resistant ophthalmoplegia (OP-MG). Although the underlying pathogenetic mechanisms of OP-MG remain unknown, experimental rodent models of MG showed upregulation of genes involved in oxidative metabolism in muscles. EOMs are highly dependent on oxidative metabolism. We opportunistically sampled EOM-tendons of two rare OP-MG patients (and non-MG controls) undergoing re-alignment surgery, and established ocular fibroblast cultures. Metabolic assays were performed on these live cells to assess real-time differences in energy metabolism. To study the cellular bioenergetic profiles in the context of MG, we exposed the cultures to homologous 5% MG sera for 24 h, vs. growth media, from two independent MG patients (with circulating AChR-antibodies) and five controls without MG, and estimated the fold change in oxygen consumption rates in response to three compounds which inhibit different mitochondrial chain complexes. Quantitative PCR (qPCR) was performed in cells before and after MG sera exposure, to assess transcript levels of mitochondrial genes, PDK4, ANGPTL4 and UCP3, which were altered in experimental MG. In response to the mitochondrial stressors, basal oxidative metabolism parameters were similar between OP-MG and control fibroblasts (p = 0.81). However, after exposure to MG sera, bioenergetic parameters (oxygen consumption rate as an indicator of oxidative phosphorylation; extracellular acidification rate as an indicator of glycolysis), were induced to higher levels in OP-MG fibroblasts compared to controls (2.6-fold vs 1.5-fold; p = 0.031) without evidence of mitochondrial insufficiency in the OP-MG ocular fibroblasts. In support of the bioenergetic responses to the same MG sera, gene transcripts of PDK4 and ANGPLT4 in ocular fibroblasts also showed significant upregulation (p ≤ 0.041), but similarly in OP-MG and control cases. Taken together we showed similar basal and metabolic adaptive responses after exposure to mitochondrial inhibitors in ocular fibroblasts derived from OP-MG cases and controls, although the OP-MG cells showed greater activation in response to MG conditions. These pilot results in orbital-derived tissues provide support for myasthenic-induced changes in cellular metabolism and evidence that orbital fibroblasts may be useful for dynamic bioenergetic assessments.

Targeting glutamine utilization to block metabolic adaptation of tumor cells under the stress of carboxyamidotriazole-induced nutrients unavailability.

Tumor cells have unique metabolic programming that is biologically distinct from that of corresponding normal cells. Resetting tumor metabolic programming is a promising strategy to ameliorate drug resistance and improve the tumor microenvironment. Here, we show that carboxyamidotriazole (CAI), an anticancer drug, can function as a metabolic modulator that decreases glucose and lipid metabolism and increases the dependency of colon cancer cells on glutamine metabolism. CAI suppressed glucose and lipid metabolism utilization, causing inhibition of mitochondrial respiratory chain complex I, thus producing reactive oxygen species (ROS). In parallel, activation of the aryl hydrocarbon receptor (AhR) increased glutamine uptake via the transporter SLC1A5, which could activate the ROS-scavenging enzyme glutathione peroxidase. As a result, combined use of inhibitors of GLS/GDH1, CAI could effectively restrict colorectal cancer (CRC) energy metabolism. These data illuminate a new antitumor mechanism of CAI, suggesting a new strategy for CRC metabolic reprogramming treatment.

Bicalutamide may enhance kidney injury in diabetes by concomitantly damaging energy production from OXPHOS and glycolysis.

Bicalutamide (Bic), frequently used in androgen-deprivation therapy for treating prostate cancer, was demonstrated to induce multiple apoptosis and fibrosis pathways and mitochondrial dysfunction in renal mesangial cells. Whether Bic also damages the glycolytic pathway has never been cited. To investigate this, we performed an in vitro model study with mesangial cells, and at the same time, collected data from an in vivo experiment. Bic induced hypoxia-inducible factor (HIF)-1 which upregulates phosphorylated-5'-AMP-activated protein kinase (p-AMPK) and severely suppresses the rate of adenosine triphosphate (ATP) production in both the oxidative phosphorylation and glycolysis pathways. Bic suppressed the oxygen consumption rate, extracellular acidification rate, and mitochondrial proton efflux rate, downregulated in vivo but upregulated in vitro glucose transporter (GLUT)-1, reduced glucose uptake, inhibited key glycolytic enzymes, including phosphofructokinase (PFK), pyruvate kinase (PK), and pyruvate dehydrogenase (PDH), and upregulated hexokinase II (HKII) and lactic dehydrogenase A (LDHA). In vivo, Bic downregulated renal cubilin levels, thereby disrupting the glomerular reabsorption function. Conclusively, Bic can damage bioenergenesis from both mitochondria and glycolysis. It was suggested that long-term administration of Bic can initiate renal damage depending on the duration and dose of treatment, which requires cautious follow-up.

LIX1-like protein promotes liver cancer progression via miR-21-3p-mediated inhibition of fructose-1,6-bisphosphatase.

Limb and CNS expressed 1 like (LIX1L) is over-expressed in several types of tumors. However, the function of LIX1L in glucose metabolism and hepatocellular carcinoma (HCC) progression remains elusive. Here we report that LIX1L is over-expressed in human HCC tissues, which predicts unfavorable prognosis. LIX1L deficiency in vivo significantly attenuated liver cancer initiation in mice. Functional studies indicated that LIX1L overexpression elevated proliferation, migratory, invasive capacities of HCC cells in vitro, and promoted liver cancer growth and metastasis in vivo. LIX1L knockdown up-regulated fructose-1,6-bisphosphatase (FBP1) expression to reduce glucose consumption as well as lactate production. Mechanistically, LIX1L increased miR-21-3p expression, which targeted and suppressed FBP1, thereby promoting HCC growth and metastasis. MiR-21-3p inhibitor could abrogate LIX1L induced enhancement of cell migration, invasion, and glucose metabolism. Inhibition of miR-21-3p suppressed tumor growth in an orthotopic tumor model. Our results establish LIX1L as a critical driver of hepatocarcinogenesis and HCC progression, with implications for prognosis and treatment.

Reduced Energy Metabolism Impairs T Cell-Dependent B Cell Responses in Patients With Advanced HBV-Related Cirrhosis.

Background and Aims: Patients with decompensated HBV-related liver cirrhosis (HBV D-LC) showed compromised immune responses, which manifested as a proneness to develop infections and hyporesponsiveness to vaccines, resulting in accelerated disease progression. The alterations in T cell-dependent B cell responses in this pathophysiological process were not well understood. This study aimed to investigate T cell-dependent B cell responses in this process and discuss the mechanism from the perspective of metabolism. Methods: Changes in phenotypes and subsets of peripheral B cells between HBV D-LC patients and healthy controls (HCs) were compared by flow cytometry. Isolated B cells were activated by coculture with circulating T follicular (cTfh) cells. After coculture, the frequencies of plasmablasts and plasma cells and immunoglobin levels were analyzed. Oxidative phosphorylation (OXPHOS) and glycolysis were analyzed by a Seahorse analyzer. Mitochondrial function and the AKT/mTOR pathway were analyzed by flow cytometry. Results: The proliferation and differentiation capacities of B cells after T cell stimulation were impaired in D-LC. Furthermore, we found that B cells from D-LC patients showed reductions in OXPHOS and glycolysis after activation, which may result from reduced glucose uptake, mitochondrial dysfunction and attenuated activation of the AKT/mTOR pathway. Conclusions: B cells from HBV D-LC patients showed dysfunctional energy metabolism after T cell-dependent activation. Understanding the regulations of B cell metabolic pathway and their changes may provide a new direction to rescue B cell hyporesponsiveness in patients with HBV D-LC, preventing these patients be infected and improving sensitivity to vaccines.

High-resolution spatiotemporal pHe and pO2 imaging in head and neck and oesophageal carcinoma cells.

BACKGROUND: pO2 and pH are physiological parameters relevant for different processes in health and disease, including wound healing and cancer progression. Head and neck squamous cell carcinomas (HNSCC) and oesophageal squamous cell carcinomas (ESCC) have a high rate of local recurrence that is partly related to treatment-resistant residual tumour cells. Hence, novel diagnostic tools are required to visualise potential residual tumour cells and thereby improve treatment outcome for HNSCC and ESCC patients. We developed a device to spatiotemporally measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) to distinguish HNSCC and ESCC cells from healthy cells in vitro, exploiting general metabolic differences between cancer cells and healthy cells. METHODS: OCR and ECAR were measured via a newly developed device named STO2p-Q (SpatioTemporal O2 and pH Quantification) using the VisiSens technology based on ratiometric fluorescence imaging, facilitating spatiotemporal resolution. Results were confirmed using extracellular flux analyses (Seahorse technology). RESULTS: STO2p-Q is described and used to measure OCR and ECAR in HNSCC and ESCC cell lines and normal fibroblast and epithelial cells as components of the tumour microenvironment. OCR measurements showed differences amongst HNSCC and ESCC cell lines and between HNSCC/ESCC and normal cells, which on average had lower OCR than HNSCC/ESCC cells. Both OCR and ECAR measurements were independently verified using the Seahorse technology. Additionally, using STO2p-Q, HNSCC/ESCC, and normal cells could be spatially resolved with a resolution in the low millimetre range. CONCLUSIONS: We developed a method to spatiotemporally measure OCR and ECAR of cells, which has many potential in vitro applications and lays the foundation for the development of novel diagnostic tools for the detection of cancerous tissue in HNSCC and ESCC patients in vivo.

An Observation on Enhanced Extracellular Acidification and Lactate Production Induced by Inhibition of Lactate Dehydrogenase A.

The Warburg effect, representing enhanced glycolysis and lactate production in adequately oxygenated cancer cells, has been widely regarded to cause increased extracellular acidification. Converting pyruvate to lactate by lactate dehydrogenase A (LDHA) is the last step of glycolysis. Here, we report an interesting counterintuitive observation that inhibition of LDHA resulted in enhanced glycolysis in MDA-MB-231 breast cancer cells. The cells were treated with FX11 (7-benzyl-2,3-dihydroxy-6-methyl-4-propylnaphthalene-1-carboxylic acid), a specific LDHA inhibitor. Seahorse assay reported dose-dependent increases in both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Independent biochemical measurements also confirmed the increase of lactate production under FX11 treatment. The reasons and mechanism of these observations of elevated ECAR and lactate production in the MDA-MB-231 breast cancer cells under FX11 treatment remain to be investigated.

The Role of the Pathogen Dose and PI3Kγ in Immunometabolic Reprogramming of Microglia for Innate Immune Memory.

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.

Real-Time Assessment of Mitochondrial Toxicity in HepG2 Cells Using the Seahorse Extracellular Flux Analyzer.

The liver is the primary organ responsible for drug detoxification. Drug-induced liver injury (DILI) is a leading cause of attrition during drug development and is one of the main reasons that drugs are withdrawn from the market. Hence, the prevention of DILI plays a central role in the overall drug-discovery process. Most of the liver's energy supply comes in the form of adenosine triphosphate (ATP), which is largely generated by mitochondria. This article describes the evaluation of drug-induced mitochondrial dysfunction using the Seahorse Extracellular Flux Analyzer (Agilent). The described protocols detail the accurate measurement of ATP production rate in HepG2 cells after exposure to a panel of potentially toxic compounds. This assay measures changes in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indicators of glycolysis and mitochondrial respiration-the two major energy-generating pathways in a cell. This assay provides a useful model to predict mitochondrial dysfunction-mediated DILI. © 2021 Wiley Periodicals LLC. Basic Protocol: Measurement of cellular ECAR, OCR, and ATP production in live HepG2 cells Support Protocol 1: Culturing and maintaining of HepG2 cells Support Protocol 2: Determining optimal cell density per well.

LIX1-like protein promotes liver cancer progression

Limb and CNS expressed 1 like (LIX1L) is over-expressed in several types of tumors. However, the function of LIX1L in glucose metabolism and hepatocellular carcinoma (HCC) progression remains elusive. Here we report that LIX1L is over-expressed in human HCC tissues, which predicts unfavorable prognosis. LIX1L deficiency

Monitoring Glycolysis and Respiration Highlights Metabolic Inflexibility of Cryptococcus neoformans.

Cryptococcus neoformans is a human fungal pathogen that adapts its metabolism to cope with limited oxygen availability, nutrient deprivation and host phagocytes. To gain insight into cryptococcal metabolism, we optimized a protocol for the Seahorse Analyzer, which measures extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indications of glycolytic and respiratory activities. In doing so we achieved effective immobilization of encapsulated cryptococci, established Rotenone/Antimycin A and 2-deoxyglucose as effective inhibitors of mitochondrial respiration and glycolysis, respectively, and optimized a microscopy-based method of data normalization. We applied the protocol to monitor metabolic changes in the pathogen alone and in co-culture with human blood-derived monocytes. We also compared metabolic flux in wild-type C. neoformans, its isogenic 5-PP-IP5/IP7-deficient metabolic mutant kcs1∆, the sister species of C. neoformans, Cryptococcus deuterogattii/VGII, and two other yeasts, Saccharomyces cerevisiae and Candida albicans. Our findings show that in contrast to monocytes and C. albicans, glycolysis and respiration are tightly coupled in C. neoformans and C. deuterogattii, as no compensatory increase in glycolysis occurred following inhibition of respiration. We also demonstrate that kcs1∆ has reduced metabolic activity that correlates with reduced mitochondrial function. Metabolic inflexibility in C. neoformans is therefore consistent with its obligate aerobe status and coincides with phagocyte tolerance of ingested cryptococcal cells.