Decrease In Oxidative Phosphorylation Research Articles

Single-cell profiling reveals transcriptome dynamics during bovine oocyte growth

BackgroundMammalian follicle development is characterized by extensive changes in morphology, endocrine responsiveness, and function, providing the optimum environment for oocyte growth, development, and resumption of meiosis. In cattle, the first signs of transcription activation in the oocyte are observed in the secondary follicle, later than during mouse and human oogenesis. While many studies have generated extensive datasets characterizing gene expression in bovine oocytes, they are mostly limited to the analysis of fully grown and matured oocytes. The aim of the present study was to apply single-cell RNA sequencing to interrogate the transcriptome of the growing bovine oocyte from the secondary follicle stage through to the mid-antral follicle stage.ResultsSingle-cell RNA-seq libraries were generated from oocytes of known diameters (< 60 to > 120 μm), and datasets were binned into non-overlapping size groups for downstream analysis. Combining the results of weighted gene co-expression network and Trendy analyses, and differently expressed genes (DEGs) between size groups, we identified a decrease in oxidative phosphorylation and an increase in maternal -genes and transcription regulators across the bovine oocyte growth phase. In addition, around 5,000 genes did not change in expression, revealing a cohort of stable genes. An interesting switch in gene expression profile was noted in oocytes greater than 100 μm in diameter, when the expression of genes related to cytoplasmic activities was replaced by genes related to nuclear activities (e.g., chromosome segregation). The highest number of DEGs were detected in the comparison of oocytes 100–109 versus 110–119 μm in diameter, revealing a profound change in the molecular profile of oocytes at the end of their growth phase.ConclusionsThe current study provides a unique dataset of the key genes and pathways characteristic of each stage of oocyte development, contributing an important resource for a greater understanding of bovine oogenesis.

Abstract 5119: Exploring the relationship between metabolism and the epigenome in MYCN-amplified neuroblastoma

Abstract Background: Neuroblastoma (NB) is the most common cancer of infancy and is responsible for the most cancer-related deaths in children under the age of five. The transcription factor MYCN is the most studied oncogene in NB and automatically confers high-risk. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. NB has also been demonstrated to be a highly methylated cancer, with DNA methyltransferases (DNMTs) largely responsible for these modifications. Epigenetics and metabolism are closely connected, with oncogene-driven metabolic rewiring modifying the epigenetic landscape through increases of metabolites that are critical for the activities of epigenetic enzymes, and conversely the enhanced epigenetic activity further regulating the activity of metabolic enzymes. Aims: As metabolism is believed to be a key regulator of the epigenome, the objective of this proposal is to understand the connection between MYCN-DNMT1-driven epigenetic alterations and dysregulated metabolism in MYCN-amplified NB. Methods: RNA-seq and mass spectrometry analyses were performed to explore the MYCN-driven metabolome in NB and changes that occur with DNMT1 inhibition. The efficacy of DNMT1 inhibition in MYCN-amplified NB was tested in vivo. Results: Our data demonstrate that sensitivity to DNMT1 inhibition is linked to MYCN status in NB. We found that MYCN indeed dysregulates metabolism in NB, and DNMT1 inhibition led to a global decrease in oxidative phosphorylation and exacerbated mitochondrial dysfunction in MYCN-amplified NB. Expression of MYCN was linked to an increase in methionine and subsequently SAM, the universal methyl donor, further supporting reliance on DNMT1. Most importantly, we have found that treatment with DNMT1 inhibitors in vivo inhibited tumor growth, even after treatment had stopped. Conclusion: We have found a correlation between MYCN and metabolism that supports DNMT1 activity. This relationship allows for a potential treatment for a high-risk disease in which there is few clinically actionable targets. Citation Format: Krista M. Dalton, Timothy L. Lochmann, Colin Coon, Marissa L. Calbert, Mikhail Dozmorov, Lisa Shock, Jennifer Koblinski, Anthony C. Faber. Exploring the relationship between metabolism and the epigenome in MYCN-amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5119.

Chronic stress hinders sensory axon regeneration via impairing mitochondrial cristae and OXPHOS.

Spinal cord injury (SCI) often leads to physical limitations, persistent pain, and major lifestyle shifts, enhancing the likelihood of prolonged psychological stress and associated disorders such as anxiety and depression. The mechanisms linking stress with regeneration remain elusive, despite understanding the detrimental impact of chronic stress on SCI recovery. In this study, we investigated the effect of chronic stress on primary sensory axon regeneration using a preconditioning lesions mouse model. Our data revealed that chronic stress-induced mitochondrial cristae loss and a decrease in oxidative phosphorylation (OXPHOS) within primary sensory neurons, impeding central axon regrowth. Corticosterone, a stress hormone, emerged as a pivotal player in this process, affecting satellite glial cells by reducing Kir4.1 expression. This led to increased neuronal hyperactivity and reactive oxygen species levels, which, in turn, deformed mitochondrial cristae and impaired OXPHOS, crucial for axonal regeneration. Our study underscores the need to manage psychological stress in patients with SCI for effective sensory-motor rehabilitation.

The unique interplay of mitochondrial oxidative phosphorylation (OXPHOS) and immunity and its potential implication for the sex- and age-related morbidity of severe COVID-19 patients.

Aged male patients are more vulnerable to severe or critical symptoms of COVID-19, but the underlying mechanism remains elusive. In this study, we analyzed previously published scRNA-seq data from a large cohort of COVID-19 patients, castrated and regenerated mice, and bulk RNA-seq of a RNAi library of 400 genes, and revealed that both immunity and OXPHOS displayed cell-type-, sex-, and age-related variation in the severe or critical COVID-19 patients during disease progression, with a more prominent increase in immunity and decrease in OXPHOS in myeloid cells in the males relative to the females (60-69 years old). Male severe or critical patients above 70 years old were an exception in that the compromised negative correlation between OXPHOS and immunity in these patients was associated with its disordered transcriptional regulation. Finally, the expression levels of OXPHOS and androgens were revealed to be positively correlated, and the responses of macrophages to android fluctuation were more striking than other types of detected immune cells in the castrated mice model. Therefore, the interplay of OXPHOS and immunity displayed a cell-type-specific, age-related, and sex-biased pattern, and the underlying potential regulatory role of the hormonal milieu should not be neglected.

Abstract P3072: Effects Of Antiviral Innate Immune Activation On Cytokine Secretion And Function Of Human Ipsc-derived Cardiomyocytes

Introduction: Acute and long-term cardiovascular complications of COVID-19 have been reported, ranging from arrhythmias, ischemic heart disease, and heart failure to cerebrovascular and cardiometabolic disorders, yet the underlying molecular mechanisms are not well understood. Here, we examine the effects of stimulating the double-stranded RNA (dsRNA) pattern recognition receptor pathway, which is strongly induced to activate interferon-stimulated gene (ISG) expression in the heart during COVID-19, on excitation-contraction coupling and mitochondrial function. While providing antiviral protection, we hypothesize that, even in the absence of cardiac viral replication, this pathway might contribute to COVID-19-related cardiac dysfunction. Results: We used the viral dsRNA mimetic polyinosinic: polycytidylic (PIC) to induce a type I interferon response in iPSC-CM or A549 (lung epithelial) cells. In iPSC-CM, PIC induced robust protein expression of STAT1/pSTAT1, OAS2, OAS3, IRF7, pIRF7, IRF9 and MX1. A marked increase in cytokine protein secretion was also observed, with significant increases in CXCL-10 (~160 fold), IL6, CCL5 (~20-fold) IL8 (15-fold), and CXCL1 (~10-fold), and a 2-5-fold increase in ~30 other cytokines. Negative effects on excitation-contraction coupling were observed after 72 hours of PIC treatment, manifested as a 25% reduction in iPSC-CM Ca2+ transient amplitude. Microelectrode array analysis of iPSC-CM monolayers revealed electrophysiological dysfunction: PIC treatment increased cardiac field potential duration and spike amplitude, but reduced beat periods. A549 cells also showed increases in interferon pathway genes (STAT1, pSTAT1, IRF3, pIRF3, OAS1,2,3 and IFNb1), along with increased rates of RNA degradation after PIC treatment, consistent with activation of the OAS/RNAseL dsRNA sensor pathway. PIC-treated A549 cells also showed a large decrease in oxidative phosphorylation (maximal respiration and spare respiratory capacity), which was prevented by the mitochondrial antioxidant, mitoTEMPO. Conclusions: Activation of the viral dsRNA innate immune sensor pathway, even in the absence of active cardiac viral replication, might contribute to mitochondrial and cardiac dysfunction in COVID-19.

Resistance training prevents dynamics and mitochondrial respiratory dysfunction in vastus lateralis muscle of ovariectomized rats

To investigate whether ovariectomy affects mitochondrial respiratory function, gene expression of the biogenesis markers and mitochondrial dynamics of the vastus lateralis muscle, female Wistar rats divided into ovariectomized (OVX) and intact (INT) groups were kept sedentary (SED) or submitted to resistance training (RT) performed for thirteen weeks on a vertical ladder in which animals climbed with a workload apparatus. RT sessions were performed with four climbs with 65, 85, 95, and 100 % of the rat's previous maximum workload. Mitochondrial Respiratory Function data were obtained by High-resolution respirometry. Gene expression of FIS1, MFN1 and PGC1-α was evaluated by real-time PCR. There was a decrease on oxidative phosphorylation capacity in OVX-SED compared to other groups. Trained groups presented increase on oxidative phosphorylation capacity when compared to sedentary groups. For respiratory control ratio (RCR), OVX-SED presented lower values when compared to INT-SED and to trained groups. Trained groups presented RCR values higher compared to INT-SED. Exercise increased the values of FIS1, MFN1 and PGC1-α expression compared to OVX-SED. Our results demonstrated that in the absence of ovarian hormones, there is a great decrease in oxidative phosphorylation and electron transfer system capacities of sedentary animals. RT was able to increase the expression of genes related to mitochondrial dynamics markers, reversing the condition determined by ovariectomy.

Ribitol alters multiple metabolic pathways of central carbon metabolism with enhanced glycolysis: A metabolomics and transcriptomics profiling of breast cancer.

Breast cancer is heterogenous in development and cell population with prognoses being highly dependent on numerous factors from driving mutations, biomarker expression and variation in extracellular environment, all affecting response to therapies. Recently, much attention has been given to the role of metabolic alteration in cancers, expanding from the Warburg effect to highlight unique patterns in different cancer cell populations for improving diagnostic and therapeutic approaches. We recently reported on modulation of mannosylation of α-dystroglycan with the metabolite ribitol in breast cancer lines. Here we investigate the effects of pentose sugars ribitol, ribose, and xylitol media supplementation in breast cancer cells by metabolomics and differential gene expression profiling. This combined approach revealed distinctive patterns of alterations in metabolic pathways by ribitol, contrasted with the closely related pentose ribose and pentitol xylitol. Significantly, ribitol supplementation enhances utilization of glucose by glycolysis, whereas ribose improves oxidative phosphorylation and fatty acid synthesis. Ribitol supplementation also increased levels of reduced glutathione (associated with a decrease in oxidative phosphorylation, gluconeogenesis), where ribose supplementation elevated levels of oxidized glutathione (GSSG) indicating an increase in oxidative stress. Treatment with ribitol also enhanced nucleotide biosynthesis. The apparent TCA cycle dysregulation, with distinctive pattern in response to the individual pentitol and pentose, such as ribitol increasing succinate and fumarate while decreasing citrate, demonstrate the adaptive capability of cancer cells to nutritional environment. This metabolic reprogramming presents new avenues for developing targeted therapies to cancers with metabolites, especially in combination with other drug treatments.

Simultaneous Inhibition of SIRT3 and Cholesterol Homeostasis Targets AML Stem Cells By Perturbing Fatty Acid β-Oxidation and Inducing Lipotoxicity

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapies to fully eradicate disease initiating leukemia stem cells (LSCs) (Shlush et al., 2017). Oxidative phosphorylation (OXPHOS) is a highly targetable and essential process for LSC energy production and survival (Lagadinou et al., 2013). Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation (Finley et al., 2016), has been specifically shown to regulate OXPHOS in cancer models (Li et al., 2019, Xu et al., 2019). Thus, we sought to identify if SIRT3 is essential for LSC function. To determine if SIRT3 is essential for LSCs, we measured cell viability, colony forming potential, and engraftment potential of primary AML specimens upon perturbation of SIRT3 function using both RNAi and a small molecule inhibitor (YC8-02) (Li et al., 2019). SIRT3 reduction significantly decreased viability, colony forming potential and engraftment of LSCs (Fig. 1A), suggesting that SIRT3 inhibition decreases LSC function. Importantly, neither SIRT3 knockdown nor inhibition impacted the viability, colony forming potential, or engraftment potential of normal hematopoietic stem and progenitor cells (HSPCs). Altogether, these data suggest that SIRT3 is essential for LSCs and not HSPCs. To determine the molecular mechanisms by which SIRT3 is essential for LSC function, we performed proximity-dependent biotin labeling (BioID) in 293 T-Rex cells to map the interactome of SIRT3. Strikingly, our BioID analysis identified several fatty acid oxidation (FAO) enzymes as proximity interactors including CPT2, ACADVL, ACADM, ECHI, HADH, and ACAA2. To corroborate these findings in LSCs, we performed RNA-sequencing upon SIRT3 inhibition in primary LSCs derived from three primary AML specimens. Gene set enrichment analysis revealed a significant decrease in OXPHOS and FAO gene signatures upon SIRT3 inhibition. Importantly, FAO is known to support OXPHOS which suggests that SIRT3 may regulate OXPHOS through mediation of FAO. To determine if protein interactions and gene expression changes result in functional changes in mitochondrial biology, we measured OXPHOS and mitochondrial ATP production in LSCs upon SIRT3 perturbation in LSCs using a seahorse assay. This analysis revealed that SIRT3 perturbation resulted in decreased OXPHOS and ATP production. To determine if SIRT3 regulates OXPHOS via FAO mediation, we performed mass spectrometry-based lipidomic and metabolic interrogation on LSCs from three AML samples treated with YC8-02, which showed an accumulation of fatty acid upon SIRT3 inhibition. To interrogate fatty acid metabolism further, we performed stable isotope tracing analysis using 13C16-palmitate on LSC enriched from three patient specimens upon SIRT3 inhibition. Tracing studies revealed a reduction in 13C enrichment in tricarboxylic acid (TCA) cycle metabolites in the YC8-02-treated LSCs (Fig. 1B), suggesting an impairment to fatty acid utilization upon SIRT3 inhibition. Decreasing availability of TCA products impairs OXPHOS and ultimately causes cell death. Importantly, viability of LSCs was partially rescued through addition of the TCA intermediate α-ketoglutarate, demonstrating reduced FAO to be an essential part of the mechanism by which SIRT3 targets LSCs. In addition to decreasing OXPHOS, the accumulation of fatty acids should induce cell death by causing lipotoxicity. However, phenocopy experiments revealed that LSCs are protected from lipotoxicity unlike AML blasts or AML cell lines. Lipidomic analysis suggested that fatty acids may be converted to cholesterol esters, which can then be stored or excreted from cells, specifically in LSCs and not in AML cell lines. Therefore, we postulated that targeting cholesterol homeostasis using dipyridamole would enhance the effect of SIRT3 inhibition (Esquejo et al., 2021). The combination treatment significantly reduced the viability of LSCs compared to individual agents, indicating that co-inhibition of SIRT3 and cholesterol homeostasis enhances the toxic effects of FAO inhibition on LSCs. In all, we identify SIRT3 as a key regulator of LSC survival, through the mediation of lipid metabolism. We further demonstrate that LSCs are tolerant to FAO inhibition which can be overcome by concurrent inhibition of FAO and cholesterol metabolism, which has the potential to lead to improved treatment response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison of metabolic effects of mitochondrial dysfunctions in the context of vulnerability to fatigue: computer simulation study

In recent years, the accumulation of phosphate ions and the increase in acidity have been described as crucial metabolic fatigue-leading factors that disturb muscle fiber contractions. This fact is especially important in the context of mitochondrial dysfunctions in which excessive fatigue is one of the possible symptoms. However, little is known about the precise fatigue-inducing thresholds of work intensity in mitochondrial dysfunctions of various types and at various stages of their severity. Possible interactions of additional factors such as disturbances in electrolyte concentrations (i.e. magnesium ions) were also not precisely defined. One of the best-suited tools for this kind of problem is systems biology, which enables modeling of metabolic pathways. In this research, a computer model of working skeletal muscle was adapted. The relationship between the decrease in oxidative phosphorylation and the workload shows a linear dependence for dysfunctions that evenly disturb the activity of each element of the pathway (which is equivalent to the decrease in mitochondrial mass). In case of dysfunctions that disrupt only one element of the pathway, the relationship between fatigue threshold and exercise intensity is exponential, but with higher threshold deficiency values. Muscle phosphate levels were the most vulnerable to disruptions of complex III and ATP synthase. Surprisingly, disruptions of the ATP/ADP exchanger emerged as equally disruptive and capable of significantly increasing phosphate concentrations also in the rest state, whereas the impact of the impairment of the phosphate transporter was negligible. Perturbations in magnesium concentration also did not show a significant effect in any of these models.

Ym155 localizes to the mitochondria leading to mitochondria dysfunction and activation of AMPK that inhibits BMP signaling in lung cancer cells

The imidazolium compound Ym155 was first reported to be a survivin inhibitor. Ym155 potently induces cell death of many types of cancer cells in preclinical studies. However, in phase II clinical trials Ym155 failed to demonstrate a significant benefit. Studies have suggested that the cytotoxic effects of Ym155 in cancer cells are not mediated by the inhibition of survivin. Understanding the mechanism by which Ym155 induces cell death would provide important insight how to improve its efficacy as a cancer therapeutic. We demonstrate a novel mechanism by which Ym155 induces cell death by localizing to the mitochondria causing mitochondrial dysfunction. Our studies suggest that Ym155 binds mitochondrial DNA leading to a decrease in oxidative phosphorylation, decrease in TCA cycle intermediates, and an increase in mitochondrial permeability. Furthermore, we show that mitochondrial stress induced by Ym155 and other mitochondrial inhibitors activates AMP-activated kinase leading to the downregulation to bone morphogenetic protein (BMP) signaling. We provide first evidence that Ym155 initiates cell death by disrupting mitochondrial function.

LAT1-specific inhibitor ameliorates severe autoimmune arthritis in SKG mouse

L-type amino acid transporter 1 (LAT1, slc7a5) supplies large neutral amino acids to highly proliferative cells. LAT1 is an attractive therapeutic target for treating overactive T cell-mediated immune disorders due to its high expression in activated T cells, but not in resting T cells. Here, we demonstrate that LAT1 plays a crucial role in T helper (Th) 17-mediated autoimmune arthritis in SKG mice, an animal model of human rheumatoid arthritis (RA). Administration of JPH203, a LAT1-specific inhibitor, suppressed mannan-induced joint swelling, synoviocyte proliferation and inflammatory cell infiltration in SKG mice. A diminished metabolic reprogramming, including a decrease in oxidative phosphorylation that regulates Hif-1α expression and subsequent control of glycolysis enzymes, was involved in the downregulation of Th17 differentiation by LAT1 inhibition. Moreover, publicly released database analysis revealed facilitated expression of LAT1 in T cells with cytotoxic features in patients with RA. Our results demonstrate the essential contribution of LAT1 to the development of RA, proposing a potential therapeutic approach targeting amino acid transporters for treating hypersensitive immune diseases.

PD43-03 MCT INHIBITION SYNERGIZES WITH TYROSINE KINASE AND MTOR INHIBITOR TREATMENT IN RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 May 2022PD43-03 MCT INHIBITION SYNERGIZES WITH TYROSINE KINASE AND MTOR INHIBITOR TREATMENT IN RENAL CELL CARCINOMA Bhaghyasree Jambunathan, Abdo Dergham, Sayani Bhattacharjee, Rebecca Wynn, Puneet Sindhwani, Firas Petros, and Nagalakshmi Nadiminty Bhaghyasree JambunathanBhaghyasree Jambunathan More articles by this author , Abdo DerghamAbdo Dergham More articles by this author , Sayani BhattacharjeeSayani Bhattacharjee More articles by this author , Rebecca WynnRebecca Wynn More articles by this author , Puneet SindhwaniPuneet Sindhwani More articles by this author , Firas PetrosFiras Petros More articles by this author , and Nagalakshmi NadimintyNagalakshmi Nadiminty More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002604.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: RCC has been recognized in recent years as a uniquely metabolic disease. All histologic types of RCC exhibit increase in aerobic glycolysis with disease progression. The TCGA-RCC study showed that high grade, high stage, and low survival are associated with a shift to aerobic glycolysis and a decrease in oxidative phosphorylation. Approaches targeting glucose metabolism in RCC such as GLUT1 inhibitors, hexokinase inhibitors, GAPDH inhibitor, and PKM2 inhibitor showed limited clinical benefit. Monocarboxylate transporters (MCTs) play a major role in the export/import of the glycolytic end-product lactate. Different MCT isoforms are differentially expressed in RCC tissues with an increase in the expression of MCT1 and MCT4 in clear-cell RCC and papillary RCC. Here we hypothesized that MCT inhibition may be an attractive therapeutic strategy against RCC cells. METHODS: We analyzed the expression levels of MCTs in RCC tissues using public datasets from Oncomine. We treated normal renal epithelial and renal cell carcinoma cells with varying concentrations of MCT inhibitors AR-C155858, AZD3965, or syrosingopine either singly or in combination with sunitinib or everolimus and assessed cell survival, cell viability, proliferation, clonogenic ability, survival in 3-D models, and glycolytic activity. We treated xenografts of ccRCC cells in mice with MCT inhibitors, sunitinib, or everolimus singly or in combination and assessed tumor growth. RESULTS: We found that the suppression of MCT activity using the MCT antagonists AR-C155858, AZD3965, or syrosingopine not only diminished the proliferation, survival, and clonogenic ability of RCC cells, but also potentiated the response of RCC cells to treatment with sunitinib or everolimus. MCT inhibitors inhibited the metabolic adaptation of ccRCC cells when treated with sunitinib or everolimus. Tumor growth of ccRCC xenografts was suppressed significantly when treated with a combination of MCT inhibitors with sunitinib or everolimus. These findings imply that MCT inhibition may be an effective strategy to develop combinatorial therapies in renal cell carcinoma. CONCLUSIONS: MCT inhibition may improve the anti-tumor effects of tyrosine kinase or mTOR inhibitors in renal cell carcinoma. Source of Funding: DoD-PCRP PC190332 (W81XWH2010794), University of Toledo College of Medicine and Life Sciences, NIH/NCI 1R21CA202404, NIH/NCI 1R03CA198696 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e702 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bhaghyasree Jambunathan More articles by this author Abdo Dergham More articles by this author Sayani Bhattacharjee More articles by this author Rebecca Wynn More articles by this author Puneet Sindhwani More articles by this author Firas Petros More articles by this author Nagalakshmi Nadiminty More articles by this author Expand All Advertisement PDF DownloadLoading ...

IGFBP-3 Regulates Mitochondrial Hyperfusion and Metabolic Activity in Ocular Surface Epithelia during Hyperosmolar Stress.

In the eye, hyperosmolarity of the precorneal tear film triggers inflammation and the development of dry eye disease (DED), a highly prevalent condition that causes depression and disability in severe forms. A member of the insulin-like growth factor (IGF) family, the IGF binding protein-3 (IGFBP-3), is a pleiotropic protein with known roles in growth downregulation and survival. IGFBP-3 exerts these effects by blocking IGF-1 activation of the type 1 IGF-receptor (IGF-1R). Here, we examined a new IGF-independent role for IGFBP-3 in the regulation of mitochondrial and metabolic activity in ocular surface epithelial cells subject to hyperosmolar stress and in a mouse model of DED. We found that hyperosmolar stress decreased IGFBP-3 expression in vitro and in vivo. Treatment with exogenous IGFBP-3 induced an early, transient shift in IGF-1R to mitochondria, followed by IGFBP-3 nuclear accumulation. IGFBP-3 nuclear accumulation increased protein translation, blocked the hyperosmolar-mediated decrease in oxidative phosphorylation through the induction of mitochondrial hyperfusion, and restored corneal health in vivo. These data indicate that IGFBP-3 acts a stress response protein in ocular surface epithelia subject to hyperosmolar stress. These findings may lead to the development of first-in-class therapeutics to treat eye diseases with underlying mitochondrial dysfunction.

Features of mitochondrial dynamics in monocytes in inflammatory and metabolic disorders

Mitochondria do not exist as separate formations in the cell; they form a homogeneous network in which the processes of division and fusion continuously occur. A shift in this balance, as well as mitochondrial dysfunction, leads to the development of chronic and metabolic disorders. Metabolic changes in mitochondria control the formation and differentiation of monocytes. Pro-inflammatory activation of monocytes/macrophages leads to a decrease in oxidative phosphorylation and an increase in mitochondrial fusion. To date, the molecular mechanisms that regulate mitochondrial dynamics to control life and death in monocytes are not well understood. In addition, there is ample evidence that abnormal mitochondrial metabolism is involved in the pathogenesis of many diseases. Mitochondrial stress and damage contribute to cell death, metabolic disorders, and inflammation. In this review, we consider in detail the involvement of mitochondrial processes in the development of pathologies and discuss how mitochondria can be therapeutically affected. Attention is also drawn to possible diagnostic studies that target mitochondrial dynamics of disorders in monocytes.

Accumulation of cholesterol suppresses oxidative phosphorylation and altered responses to inflammatory stimuli of macrophages

Hypercholesterolemia induces intracellular accumulation of cholesterol in macrophages and other immune cells, causing immunological dysfunctions. On cellular levels, cholesterol enrichment might lead to mitochondrial metabolic reprogramming and change macrophage functions. Additionally, as cholesterol is permeable to the plasma membrane and might integrate into the membranous organelles, such as endoplasmic reticulum or mitochondria, cholesterol enrichment might change the functions or properties of these organelles, and ultimately alters the cellular functions. In this study, we investigate the mitochondrial alterations and intracellular oxidative stress induced by accumulation of cholesterol in the macrophages, and the possible immunological impacts caused by these alterations. Macrophage cells RAW264.7 were treated with cholesterol to induce intracellular accumulation of cholesterol, which further triggered the reduced production of reactive oxygen/nitrogen species, as well as decrease of oxidative phosphorylation. Basal respiration rate, ATP production and non-mitochondrial oxygen consumption are all suppressed. In contrast, glycolysis remained unaltered in this cholesterol-enriched condition. Previous studies demonstrated that metabolic profiles are associated with macrophage polarization. We further verified whether this metabolic reprogramming influences the macrophage responses to pro-inflammatory or anti-inflammatory stimuli. Our results showed the changes of transcriptional regulations in both pro-inflammatory and anti-inflammatory genes, but not specific toward M1 or M2 polarization. Collectively, the accumulation of cholesterol induced mitochondrial metabolic reprogramming and suppressed the production of oxidative stress, and induced the alterations of macrophage functions.

Molar Sodium Lactate Attenuates the Severity of Postcardiac Arrest Syndrome: A Preclinical Study.

To determine whether continuous IV infusion of molar sodium lactate would limit cardiac arrest-induced neurologic injury and cardiovascular failure. Randomized blinded study (animal model). University animal research facility. Twenty-four adult male "New Zealand White" rabbits. Anesthetized rabbits underwent 12.5 minutes of asphyxial cardiac arrest and were randomized to receive either normal saline (control group, n = 12) or molar sodium lactate (molar sodium lactate group, n = 12) at a rate of 5 mL/kg/hr during the whole 120-minute reperfusion period. Pupillary reactivity (primary outcome), levels of S100β protein, in vitro brain mitochondria functions, cardiovascular function, and fluid balance were assessed. Molar sodium lactate reduced brain injury, with a higher proportion of animals exhibiting pupillary reactivity to light (83% vs 25% in the CTRL group, p = 0.01) and lower S100β protein levels (189 ± 42 vs 412 ± 63 pg/mL, p < 0.01) at the end of the protocol. Molar sodium lactate significantly prevented cardiac arrest-induced decrease in oxidative phosphorylation and mitochondrial calcium-retention capacity compared with controls. At 120 minutes of reperfusion, survival did not significantly differ between the groups (10/12, 83% in the molar sodium lactate group vs nine of 12, 75% in the control group; p > 0.99), but hemodynamics were significantly improved in the molar sodium lactate group compared with the control group (higher mean arterial pressure [49 ± 2 vs 29 ± 3 mm Hg; p < 0.05], higher cardiac output [108 ± 4 vs 58 ± 9 mL/min; p < 0.05], higher left ventricle surface shortening fraction [38% ± 3% vs 19% ± 3%; p < 0.05], and lower left ventricular end-diastolic pressure [3 ± 1 vs 8 ± 2 mm Hg; p < 0.01]). While fluid intake was similar in both groups, fluid balance was higher in control animals (11 ± 1 mL/kg) than that in molar sodium lactate-treated rabbits (1 ± 3 mL/kg; p < 0.01) due to lower diuresis. Molar sodium lactate was effective in limiting the severity of the postcardiac arrest syndrome. This preclinical study opens up new perspectives for the treatment of cardiac arrest.

Metabolic evolutionary roots of the macrophage immune response in amoeba-bacteria interactions: The conserved role of hypoxia-induced Factor and AMP kinase

The bacteria Legionella, being able to infect both macrophages and protozoans, reduce oxidative phosphorylation and induce glycolysis, which allows pathogens to grow and replicate in these cells. In amoeba-like inflammatory macrophages (M1), the phagocytizing cells of the primary immune defense, an increase in the rate of glycolysis is followed by a decrease of oxidative phosphorylation. The opposite takes place in anti-inflammatory macrophages (M2). They change from glycolysis to oxidative metabolism when AMP-dependent kinase (AMPK) is activated by a high ratio of AMP/ATP. Stimulation of macrophages with anti-inflammatory cytokines causes activation of AMPK. Infection of macrophages with the parasitic flagellate Leishmania infantum induces a switch from an initial glycolytic phase to oxidative phase with the essential role of AMPK in this change. Activated AMPK induces catabolic pathways effectively producing ATP as well as processes requiring the energy supply. AMPK regulates the migration of cells and enhances the phagocytic activity of macrophages. In macrophages, bacterial products activate TLRs and NF-κB signaling, causing an increase of transcription of hypoxia-induced factor HIF-1α (a subunit of HIF-1). This brings about induction of the enzyme and transporter expression essential for glycolysis and the pentose phosphate pathway to proceed and makes biosynthetic processes and ROS production in macrophages possible. Hypoxia augments macrophage phagocytosis in a HIF-1α-dependent manner. Multicellular parasites experience changes in the availability of oxygen in their life cycle. In the nematode Ascaris suum, HIF participates in the pre-adaptation to hypoxic conditions after infection of their hosts. Also, the freshwater and marine invertebrates meet changes of oxygen concentrations. In the anaerobic branch of the respiratory chain of these invertebrates, fumarate serves as the terminal electron acceptor that is reduced to succinate in complex II of the ETC. In mammalian cells, accumulation of succinate under hypoxic conditions suggests that the mammalian complex II may reduce fumarate to succinate, too. The data reviewed here show that the ability to shift the cell metabolism towards glycolysis observed in activated macrophages can be traced back in evolution to metabolic changes characterizing protozoans infected with bacteria. Anabolic needs of multiplying bacteria direct host metabolism to glycolysis that produces, aside from ATP, precursors of the amino acids used by the pathogen for its protein synthesis. M1-activated mammalian macrophages behave in the same way. Regulation of metabolism in M1 and M2 macrophages is further enhanced by HIF-1 and AMPK, respectively. These archaic functions of AMPK and HIF, important also to control phagocytosis and cell migration were extended to embryonic development in multicellular organisms.

Ethyl isopropyl amiloride decreases oxidative phosphorylation and increases mitochondrial fusion in clonal untransformed and cancer cells.

Many cancer cells, regardless of their tissue origin or genetic landscape, have increased expression or activity of the plasma membrane Na-H exchanger NHE1 and a higher intracellular pH (pHi) compared with untransformed cells. A current perspective that remains to be validated is that increased NHE1 activity and pHi enable a Warburg-like metabolic reprogramming of increased glycolysis and decreased mitochondrial oxidative phosphorylation. We tested this perspective and find it is not accurate for clonal pancreatic and breast cancer cells. Using the pharmacological reagent ethyl isopropyl amiloride (EIPA) to inhibit NHE1 activity and decrease pHi, we observe no change in glycolysis, as indicated by secreted lactate and intracellular pyruvate, despite confirming increased activity of the glycolytic enzyme phosphofructokinase-1 at higher pH. Also, in contrast to predictions, we find a significant decrease in oxidative phosphorylation with EIPA, as indicated by oxygen consumption rate (OCR). Decreased OCR with EIPA is not associated with changes in pathways that fuel oxidative phosphorylation or with mitochondrial membrane potential but occurs with a change in mitochondrial dynamics that includes a significant increase in elongated mitochondrial networks, suggesting increased fusion. These findings conflict with current paradigms on increased pHi inhibiting oxidative phosphorylation and increased oxidative phosphorylation being associated with mitochondrial fusion. Moreover, these findings raise questions on the suggested use of EIPA-like compounds to limit metabolic reprogramming in cancer cells.

Control of Macrophage Activation by Coenzyme A

Macrophages are innate immune cells responsible for processes as varied as killing pathogens, initiating wound-healing programs, and presenting antigens to T cells. To achieve this broad range of functions, they acquire pro- or anti-inflammatory programs (or ‘polarize’) by integrating intrinsic, extrinsic, and environmental signals. It is now well established that metabolic changes are critical for this process, though it remains unclear precisely which metabolic alterations are essential for (rather than merely being associated with) activation. Pro-inflammatory macrophage polarization with lipopolysaccharide (LPS) collapses mitochondrial ATP production. This profound decrease in oxidative phosphorylation is associated with increases in glycolysis, signaling metabolites from the TCA cycle, and mitochondrially derived redox signals. Conversely, anti-inflammatory activation with interleukin-4 (IL-4) is associated with substantial increases in oxidative phosphorylation and fatty acid oxidation (FAO). Previous work in our laboratory has shown that although FAO and mitochondrial ATP production are strongly associated with the IL-4 response, these are surprisingly dispensable, whereas coenzyme A homeostasis is obligatory [PMCID: PMC6125190]. This presentation will cover our current understanding of the essential metabolic requirements for anti-inflammatory macrophage activation, with a focus on the potential mechanisms by which coenzyme A may control the IL-4 response.

Metabolic Changes in Peripheral Blood Mononuclear Cells Isolated From Patients With End Stage Renal Disease.

As numerous complex pathologies stem from cellular energy dysfunction, we aimed to elucidate mitochondrial function and associated stress pathologies in kidney disease in a cohort of hemodialysis patients with end-stage kidney disease (ESKD). The bioenergetics study was conducted using peripheral blood mononuclear cells (PBMCs) of ESKD patients (n = 29) and healthy controls (no ESKD, n = 10). PBMCs were isolated from whole blood and seeded into assay plates to detect changes in oxidative phosphorylation and glycolysis. The bioenergetics analysis (i.e., mitochondrial stress test) was performed using Seahorse XFe24 flux analyzer. We observed significant reduction in mitochondrial respiration in patient PBMCs in terms of fundamental bioenergetics parameters such as basal respiration, ATP turnover, maximal respiration and spare respiratory capacity. These findings were correlated with the expression levels of proteins coordinating cellular energy status and regulating mitochondrial dynamics. Our data demonstrates an association between mitochondrial oxygen consumption of PBMCs and ESKD. AMPK activity, its downstream effector PGC-1α and mitochondrial fission/fusion proteins are partially responsible for the decrease in oxidative phosphorylation of PBMCs isolated from ESKD patients. We propose a link between mitochondrial dysfunction and ESKD and a role for mitochondria as a potential site for therapeutic interventions.