Increase In Oxygen Consumption Rate Research Articles

Mitochondria dysfunction is one of the causes of diclofenac toxicity in the green alga Chlamydomonas reinhardtii.

Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), form a significant group of environmental contaminants. When the toxic effects of DCF on plants are analyzed, authors often focus on photosynthesis, while mitochondrial respiration is usually overlooked. Therefore, an in vivo investigation of plant mitochondria functioning under DCF treatment is needed. In the present work, we decided to use the green alga Chlamydomonas reinhardtii as a model organism. Synchronous cultures of Chlamydomonas reinhardtii strain CC-1690 were treated with DCF at a concentration of 135.5 mg × L-1, corresponding to the toxicological value EC50/24. To assess the effects of short-term exposure to DCF on mitochondrial activity, oxygen consumption rate, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) production were analyzed. To inhibit cytochrome c oxidase or alternative oxidase activity, potassium cyanide (KCN) or salicylhydroxamic acid (SHAM) were used, respectively. Moreover, the cell's structure organization was analyzed using confocal microscopy and transmission electron microscopy. The results indicate that short-term exposure to DCF leads to an increase in oxygen consumption rate, accompanied by low MMP and reduced mtROS production by the cells in the treated populations as compared to control ones. These observations suggest an uncoupling of oxidative phosphorylation due to the disruption of mitochondrial membranes, which is consistent with the malformations in mitochondrial structures observed in electron micrographs, such as elongation, irregular forms, and degraded cristae, potentially indicating mitochondrial swelling or hyper-fission. The assumption about non-specific DCF action is further supported by comparing mitochondrial parameters in DCF-treated cells to the same parameters in cells treated with selective respiratory inhibitors: no similarities were found between the experimental variants. The results obtained in this work suggest that DCF strongly affects cells that experience mild metabolic or developmental disorders, not revealed under control conditions, while more vital cells are affected only slightly, as it was already indicated in literature. In the cells suffering from DCF treatment, the drug influence on mitochondria functioning in a non-specific way, destroying the structure of mitochondrial membranes. This primary effect probably led to the mitochondrial inner membrane permeability transition and the uncoupling of oxidative phosphorylation. It can be assumed that mitochondrial dysfunction is an important factor in DCF phytotoxicity. Because studies of the effects of NSAIDs on the functioning of plant mitochondria are relatively scarce, the present work is an important contribution to the elucidation of the mechanism of NSAID toxicity toward non-target plant organisms.

Flavopiridol inhibits adipogenesis and improves metabolic homeostasis by ameliorating adipose tissue inflammation in a diet-induced obesity model

Repositioning of FDA approved/clinical phase drugs has recently opened a new opportunity for rapid approval of drugs, as it shortens the overall process of drug discovery and development. In previous studies, we predicted the possibility of better activity profiles of flavopiridol, the FDA approved orphan drug with better fit value 2.79 using a common feature pharmacophore model for anti-adipogenic compounds (CFMPA). The present study aimed to investigate the effect of flavopiridol on adipocyte differentiation and to determine the underlying mechanism. Flavopiridol inhibited adipocyte differentiation in different cell models like 3T3-L1, C3H10T1/2, and hMSCs at 150 nM. Flavopiridol was around 135 times more potent than its parent molecule rohitukine. The effect was mediated through down-regulation of key transcription factors of adipogenesis i.e. Peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and their downstream targets, including adipocyte protein −2 (aP2) and fatty acid synthase (FAS). Further, results revealed that flavopiridol arrested the cell cycle in G1/S phase during mitotic clonal expansion by suppressing cell cycle regulatory proteins i.e. Cyclins and CDKs. Flavopiridol inhibited insulin-stimulated signalling in the early phase of adipocyte differentiation by downregulation of AKT/mTOR pathway. In addition, flavopiridol improved mitochondrial function in terms of increased oxygen consumption rate (OCR) in mature adipocytes. In the mouse model of diet-induced obesity, flavopiridol attenuated obesity-associated adipose tissue inflammation and improved serum lipid profile, glucose tolerance as well as insulin sensitivity. In conclusion, the FDA approved drug flavopiridol could be placed as a potential drug candidate for the treatment of cancer and obesity comorbid patients.

Mitochondrial proteome analysis reveals that an augmented cytochrome c oxidase assembly and activity potentiates respiratory capacity in sarcoma

Mitochondrial oxidative phosphorylation (OXPHOS) is an obligatory process in sarcoma. Despite that, the metabolic programming of sarcoma mitochondria is still unknown. To obtain a comprehensive metabolic insight of mitochondria, we developed a mouse fibrosarcoma model by injecting 3-methylcholanthrene and compared mitochondrial proteomes between sarcoma and its contralateral normal muscle using mass spectrometry. Our study identified ∼449 proteins listed in the SwissProt databases, and all the data sets are available via ProteomeXchange with the identifier PXD044903. In sarcoma, 49 mitochondrial proteins were found differentially expressed, including 36 proteins up-regulated and 13 proteins down-regulated, with the significance of p-value <0.05 and the log2[fold change] > 1 and < −1 as compared to normal muscle. Our data revealed that various anaplerotic reactions actively replenish the TCA cycle in sarcoma. The comparative expression profile and Western blotting analysis of OXPHOS subunits showed that complex-IV subunits, MT-CO3 and COX6A1, were significantly up-regulated in sarcoma vs. normal muscle. Further, biochemical and physiological assays confirmed enhanced complex-IV specific enzymatic and supercomplex activities with a concomitant increase of oxygen consumption rate in sarcoma mitochondria compared to normal muscle. Validation with human post-operative sarcoma tissues also confirms an increased MT-CO3 expression compared to normal tissue counterparts. Thus, our data comprehensively analyses the mitochondrial proteome and identifies augmented complex-IV assembly and activity in sarcoma.

Acyl-CoA synthetase 4 modulates mitochondrial function in breast cancer cells

Mitochondria are dynamic organelles that respond to cellular stress through changes in global mass, interconnection, and subcellular location. As mitochondria play an important role in tumor development and progression, alterations in energy metabolism allow tumor cells to survive and spread even in challenging conditions. Alterations in mitochondrial bioenergetics have been recently proposed as a hallmark of cancer, and positive regulation of lipid metabolism constitutes one of the most common metabolic changes observed in tumor cells. Acyl-CoA synthetase 4 (ACSL4) is an enzyme catalyzing the activation of long chain polyunsaturated fatty acids with a strong substrate preference for arachidonic acid (AA). High ACSL4 expression has been related to aggressive cancer phenotypes, including breast cancer, and its overexpression has been shown to positively regulate the mammalian Target of Rapamycin (mTOR) pathway, involved in the regulation of mitochondrial metabolism genes. However, little is known about the role of ACSL4 in the regulation of mitochondrial function and metabolism in cancer cells. In this context, our objective was to study whether mitochondrial function and metabolism, processes usually altered in tumors, are modulated by ACSL4 in breast cancer cells. Using ACSL4 overexpression in MCF-7 cells, we demonstrate that this enzyme can increase the mRNA and protein levels of essential mitochondrial regulatory proteins such as nuclear respiratory factor 1 (NRF-1), voltage-dependent anion channel 1 (VDAC1) and respiratory chain Complex III. Furthermore, respiratory parameters analysis revealed an increase in oxygen consumption rate (OCR) and in spare respiratory capacity (SRC), among others. ACSL4 knockdown in MDA-MB-231 cells led to the decrease in OCR and in SCR, supporting the role of ACSL4 in the regulation of mitochondrial bioenergetics. Moreover, ACSL4 overexpression induced an increase in glycolytic function, in keeping with an increase in mitochondrial respiratory activity. Finally, there was a decrease in mitochondrial mass detected in cells that overexpressed ACSL4, while the knockdown of ACSL4 expression in MDA-MB-231 cells showed the opposite effect. Altogether, these results unveil the role of ACSL4 in mitochondrial function and metabolism and expand the knowledge of ACSL4 participation in pathological processes such as breast cancer.

Abstract 1126: Thiamine Derivative Enhances Mitochondrial Function To Reduce Vascular Calcification

Introduction/Background: Vascular calcification significantly impacts cardiovascular disease and mortality. It's exacerbated by mitochondrial dysfunction in vascular smooth muscle cells (VSMCs), influenced by factors such as calcium and phosphate. This study examines the role of thiamine derivatives in improving mitochondrial function, potentially reducing vascular calcification. Research Question/Hypothesis: We hypothesize that thiamine derivatives enhance mitochondrial function, thereby attenuating vascular calcification. Goals/Aims: The aim is to assess the efficacy of thiamine derivatives in enhancing mitochondrial integrity and function to mitigate vascular calcification. Methods/Approach: VSMCs were treated in vitro with phosphate and calcium chloride to induce calcification, along with TTFD (Thiamine tetrahydrofurfuryl disulfide) treatment. Similarly, C57BL/6J mice were administered thiamine derivatives in vivo before inducing aortic calcification. Analysis included calcium deposition, apoptosis, and mitochondrial function assessment. Results/Data: TTFD significantly reduced vascular calcium deposition in VSMCs, both in vitro and in vivo. Mitochondrial function improvement was evidenced by increased oxygen consumption rates and JC-1 aggregate levels. Additionally, TTFD mitigated mitochondrial fission by inhibiting Drp1 phosphorylation at serine 616, leading to reduced apoptosis and osteogenic gene expression. Conclusion: Thiamine derivatives, particularly TTFD, demonstrate potential in mitigating vascular calcification through enhanced mitochondrial function and inhibition of mitochondrial fission. These findings highlight the therapeutic potential of thiamine derivatives in treating vascular calcification-related conditions.

Influence of mudstone on coal spontaneous combustion characteristics and oxidation kinetics analysis

To explore the spontaneous combustion characteristics and hazards of the low-temperature oxidation (LTO) stage in the process of spontaneous combustion of coal and mudstone, the pore structure, spontaneous combustion characteristic parameters, and exothermic characteristics of coal and mudstone were tested and studied, and the oxidation kinetic parameters were calculated. The results show that mudstone has a larger specific surface area and pore volume than coal. From the fractal characteristics, the pore structure of mudstone is more complex than that of coal. According to the comparison of theoretical and actual gas generation and oxygen consumption rate curves, it is found that there is an interaction between coal and mudstone in the LTO process. With the increase of mudstone mass ratio, gas production, and its oxygen consumption rate increase. Among them, CM-4 (Coal:Mudstone = 1:1) has the highest exothermic intensity and the exothermic factor (A) and fire coefficient (K) increase with the increase of mudstone content. The apparent activation energy of the mudstone sample is lower than that of the raw coal, indicating that the sample after adding mudstone is more likely to have spontaneous combustion in the LTO stage.

Glutamine supplementation improves the activity and immunosuppressive action of induced regulatory T cells in vitro and in vivo

BackgroundGlutamine is crucial for the activation and efficacy of T cells, and may play a role in regulating the immune environment. This study aimed to investigate the potential role of glutamine in the activation and proliferation of induced regulatory T cells (iTregs). MethodsCD4+CD45RA+T cells were sorted from peripheral blood mononuclear cells and cultured to analyze iTreg differentiation. Glutamine was then added to the culture system to evaluate the effects of glutamine on iTregs by determining oxidative phosphorylation (OXPHOS), apoptosis, and cytokine secretion. Additionally, a humanized murine graft-versus-host disease (GVHD) model was constructed to confirm the efficacy of glutamine-treated iTregs in vivo. ResultsAfter being cultured in vitro, glutamine significantly enhanced the levels of Foxp3, CTLA-4, CD39, CD69, IL-10, TGF-β, and Ki67 (CTLA-4, IL-10, TGF-β are immunosuppressive markers of iTregs) compared with that of the control iTregs (P < 0.05). Furthermore, the growth curve showed that the proliferative ability of glutamine-treated iTregs was better than that of the control iTregs (P < 0.01). Compared with the control iTregs, glutamine supplementation significantly increased oxygen consumption rates and ATP production (P < 0.05), significantly downregulated Annexin V and Caspase 3, and upregulated BCL2 (P < 0.05). However, GPNA significantly reversed the effects of glutamine (P < 0.05). Finally, a xeno-GVHD mouse model was successfully established to confirm that glutamine-treated iTregs increased the mice survival rate, delayed weight loss, and alleviated colon injury. ConclusionGlutamine supplementation can improve the activity and immunosuppressive action of iTregs, and the possible mechanisms by which this occurs are related to cell proliferation, apoptosis, and OXPHOS.

SIRT3 suppression resulting from the enhanced β-catenin signaling drives glycolysis and promotes hypoxia-induced cell growth in hepatocellular carcinoma cells

ABSTRACT The precise mechanisms underlying the inhibitory effects of SIRT3, a mitochondrial sirtuin protein, on hepatocellular carcinoma (HCC) development, as well as its impact on mitochondrial respiration, remain poorly understood. We assessed sirtuins 3 (SIRT3) levels in HCC tissues and Huh7 cells cultured under hypoxic condition. We investigated the effects of SIRT3 on cell proliferation, glycolytic metabolism, mitochondrial respiration, mitophagy, and mitochondrial biogenesis in Huh7 cells. Besides, we explored the potential mechanisms regulating SIRT3 expression in hypoxically cultured Huh7 cells. Gradual reduction in SIRT3 expressions were observed in both adjacent tumor tissues and tumor tissues. Similarly, SIRT3 expressions were diminished in Huh7 cells cultured under hypoxic condition. Forced expression of SIRT3 attenuated the growth of hypoxically cultured Huh7 cells. SIRT3 overexpression led to a decrease in extracellular acidification rate while increasing oxygen consumption rate. SIRT3 downregulated the levels of hexokinase 2 and pyruvate kinase M2. Moreover, SIRT3 enhanced mitophagy signaling, as indicated by mtKeima, and upregulated key proteins involved in various mitophagic pathways while reducing intracellular reactive oxygen species levels. Furthermore, SIRT3 increased proxisome proliferator-activated receptor-gamma coactivator 1α levels and the amount of mitochondrial DNA in Huh7 cells. Notably, β-catenin expressions were elevated in Huh7 cells cultured under hypoxic condition. Antagonists and agonists of β-catenin respectively upregulated and downregulated SIRT3 expressions in hypoxically cultured Huh7 cells. The modulationsof glycolysis and mitochondrial respiration represent the primary mechanism through which SIRT3, suppressed by β-catenin, inhibits HCC cell proliferation.

Mitochondria-targeted esculetin and metformin delay endothelial senescence by promoting fatty acid β-oxidation: Relevance in age-associated atherosclerosis

Impaired mitochondrial fatty acid β-oxidation (FAO) plays a role in the onset of several age-associated diseases, including atherosclerosis. In the current work, we investigated the efficacies of mitochondria-targeted esculetin (Mito-Esc) and metformin in enhancing FAO in human aortic endothelial cells (HAECs), and its relevance in the delay of cellular senescence and age-associated atherosclerotic plaque formation in Apoe-/- mice. Chronic culturing of HAECs with either Mito-Esc or metformin increased oxygen consumption rates (OCR), and caused delay in senescence features. Conversely, etomoxir (CPT1 inhibitor) reversed Mito-Esc- and metformin-induced OCR, and caused premature endothelial senescence. Interestingly, Mito-Esc, unlike metformin, in the presence of etomoxir failed to preserve OCR. Thereby, underscoring Mito-Esc’s exclusive reliance on FAO as an energy source. Mechanistically, chronic culturing of HAECs with either Mito-Esc or metformin led to AMPK activation, increased CPT1 activity, and acetyl-CoA levels along with a concomitant reduction in malonyl-CoA levels, and lipid accumulation. Similar results were observed in Apoe-/- mice aorta and liver tissue with a parallel reduction in age-associated atherosclerotic plaque formation and degeneration of liver with either Mito-Esc or metformin administration. Together, Mito-Esc and metformin by potentiating FAO, may have a role in the delay of cellular senescence by modulating mitochondrial function.

Abstract 284: Crosstalk with postmenopausal obese mammary adipocytes impacts breast cancer cell metabolism and promotes tumorigenesis

Abstract Introduction: Obesity is associated with an increased risk and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related PM BC are not clearly understood. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the breast tumor microenvironment (TME) promotes the acquisition of an invasive BC cell phenotype and markedly accelerates tumor proliferation and progression. Methods: We asked whether cancer-associated mammary adipocytes (CA-MAd) differ from normal mammary adipocytes (N-MAd) in their ability to promote tumorigenesis of dormant BC cells and if so, what the underlying mechanism(s) might be. Using a 2D transwell model, BC cells (ER- and ER+), were cocultured with obese PM CA-MAd isolated from breast cancer patients or obese mammary N-MAds from PM disease-free women and analyzed for migration, proliferation, phenotypic, signaling and metabolic alterations. Results: Compared to monoculture (MC) conditions, BC cells cocultured (CC) with CA-MAd displayed significantly increased migration and proliferation. Quantitative phase imaging indicated that CA-MAd secretome-treated BC cells displayed increased motility, speed, distance covered and cell size/area compared to N-MAd. Consistent with these results, mesenchymal markers such as fibronectin, vimentin and SNAIL were upregulated whereas the epithelial marker E-cadherin was downregulated in CC vs. MC conditions suggesting that Ads in the TME promote EMT in BC cells. Interestingly, mature Ads CC with BC cells underwent delipidation and dedifferentiation as indicated by reduced Oil Red staining, decreased lipid content and reduced expression of Ad maturation markers such as FABP4, ADIPOQ and PLIN1. Consistently, we determined that CC BC cells exhibited increased lipid uptake and BODIPY staining and upregulated pAMPK, pACC and CPT1A signaling when compared to MC BC cells. Short term treatment of BC cells with both N-MAd and CA-MAd secretomes resulted in increased oxygen consumption rate (OCR), basal respiration, ATP-linked respiration and proton-linked respiration compared to control and as determined by the Seahorse extracellular flux assay. CC with MAds significantly downregulated OCR and all respiration parameters in BC cells suggesting decreased glucose uptake and glycolytic flux. Conclusions: Compared to MC, CC with obese M-Ads promoted functional, phenotypic and metabolic changes in BC cells. Taken together, our results indicate that crosstalk between BC cells and obese MAds promotes delipidation of MAds and a concomitant lipid uptake in BC cells and a switch to lipid metabolism signaling pathways that may, in turn, drive a metabolic shift in BC cells and promote tumor growth. [Supported by NIH RO1CA185530, the Breast Cancer Research Foundation, the Karp Family Foundation and the Nile Albright Research Foundation] Citation Format: Roopali Roy, Rama Aldakhlallah, Stephen Cobbs, Emily Man, Caleb Hallinan, Kwonmoo Lee, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Crosstalk with postmenopausal obese mammary adipocytes impacts breast cancer cell metabolism and promotes tumorigenesis [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 284.

Abstract 991: Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology

Abstract Introduction: Pancreatic cancer has the lowest 5-year survival rates among all cancers because of the challenges with early detection, its aggressive nature, and resistance to chemotherapy; this makes prevention a promising approach for reducing related mortalities. The disease is predominantly diagnosed in individuals over 60, suggesting that age-related physiological decline impacts its onset; therefore, delaying aging hallmarks may delay its onset. Nicotinamide dinucleotide (NAD+) precursors may hold a unique advantage over other geroprotectors because they target multiple aging hallmarks. Heron, we investigated how NAD+ supplementation impacts normal human pancreatic ductal epithelial cells (HPDE) under stress as a surrogate for cancer prevention in vitro. We also examined the paradoxical impact of NAD+ precursor supplementation on pancreatic cancer growth during chemotherapy. Methods: In vitro, we mimicked age-related NAD+ decline in the media. We stressed HPDE cells under these conditions and evaluated the effect of supplementation with the NAD+ precursor, Nicotinamide mononucleotide (NMN). We assessed cell viability using the PicoGreen assay, mitochondrial function, and reactive oxygen species (ROS) using the Seahorse XF Analyzer and the mitoSox kit, respectively, DNA repair and DNA damage using a western blot for Poly ADP ribose (PAR) and gamma H2AX, respectively, and SIRT3 expression was determined using a western blot. To evaluate the impact of NAD+ supplementation on pancreatic cancer cells, we treated pancreatic cancer cells with NMN and oxaliplatin. We then assessed the mitochondrial function, ROS levels, DNA damage, and repair. We implanted MiaPaCa-2 xenografts into athymic nude mice and treated them daily with 300mg/Kg of NMN and biweekly 5mg/kg of Oxaliplatin. Results: Experiments revealed that NMN is not toxic to HPDE cells, even at high concentrations. There was increased cell survival of HPDE cells under stress (nutrient deprivation and alkylating agents) following NMN supplementation. We also noted reduced ROS levels, increased SIRT3 expression, and PARylation. Pancreatic cancer cells were less sensitive to Oxaliplatin following NMN supplementation and showed an increased oxygen consumption rate, increased PARylation, and reduced ROS levels. There was reduced sensitivity to oxaliplatin treatment in the mice with MiaPaCa-2 xenografts treated with NMN. Conclusion: These findings demonstrate a cytoprotective role of NAD+ precursor supplementation in normal pancreatic ductal epithelial cells, with implications for possible cancer prevention. NAD+ supplementation also rescued cancer cells under cytotoxic stress, meaning that geroprotectors like NMN, in conjunction with chemotherapy, may unintentionally promote cancer progression. Future studies will directly test NAD+ precursor supplementation in genetically engineered pancreatic cancer models. Citation Format: Faith Nakazzi, Mehrad Zarei, Hallie J. Graor, Alexander W. Loftus, Omid Hajihassanni, Jordan M. Winter. Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology [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 991.

Abstract 4441: The epigenetic factor BMI1 regulates metabolism and tumorigenesis in human pancreatic cancer cells

Abstract Dysregulation of epigenetic factors is a key component of tumorigenesis. BMI1, a member of the polycomb repressor complex 1 (PRC1), is an oncogenic factor studied in many types of cancer, including pancreatic ductal adenocarcinoma (PDAC). PDAC is the third most common cause of cancer-related death in the United States and investigation of the biology involved in transformation is critical for improving outcomes in this devastating disease. In PDAC, BMI1 is required for initiation of pancreatic cancer in mice and enhances in vitro growth of human and murine tumor cell lines. CRISPR-mediated knock out of BMI1 results in reduced subcutaneous and orthotopic tumor growth in mice and decreased expression of genes related to metabolism, specifically glycolysis, and cell proliferation. BMI1 has also been implicated in regulation of metabolism in other epithelial tumor types, including ovarian cancer. Given these preliminary findings, we hypothesized that loss of BMI1 in multiple human PDAC cell lines would result in altered metabolic activity, reducing the growth and tumorigenic properties of these cells. We demonstrated that BMI1 expression is higher in PDAC cell lines compared to normal pancreatic epithelial cells and normal human donor pancreatic tissue. Following CRISPR-mediated BMI1 knock out in human PDAC cell lines, we investigated the alterations to metabolism using metabolic flux analysis. Loss of BMI1 resulted in lower basal and compensatory rates of glycolysis and increased oxygen consumption rates compared to wild-type cells. Metabolomic analysis demonstrated reduction in all the enzymes involved in the glycolysis pathway with loss of BMI1. Cell growth was reduced by loss of BMI1 in vitro, and orthotopic injection into mice of human cells with BMI1 knock out resulted in decreased tumor size compared to wild-type, recapitulating these findings in vivo. Together, these data identify a role of BMI1 in promotion of tumor growth through regulation of metabolism in pancreatic cancer. Experiments are ongoing to determine the underlying mechanism of this regulation and implications on therapeutic targeting with BMI1/PRC1 complex inhibitors in conjunction with traditional chemotherapy. Citation Format: Jamie N. Mills, Dominik Awad, Heather K. Schofield, Joyce K. Thompson, Hannah Watkoske, Damien Sutton, Nicholas Nedzesky, Donovan Drouillard, Zeribe Nwosu, Carlos Espinoza, Yaqing Zhang, Annachiara Del Vecchio, Christopher J. Halbrook, Marina Pasca di Magliano, Costas A. Lyssiotis, Filip Bednar. The epigenetic factor BMI1 regulates metabolism and tumorigenesis in human pancreatic cancer cells [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 4441.

The potential of baicalin to enhance neuroprotection and mitochondrial function in a human neuronal cell model.

Baicalin is a flavone glycoside derived from flowering plants belonging to the Scutellaria genus. Previous studies have reported baicalin's anti-inflammatory and neuroprotective properties in rodent models, indicating the potential of baicalin in neuropsychiatric disorders where alterations in numerous processes are observed. However, the extent of baicalin's therapeutic effects remains undetermined in a human cell model, more specifically, neuronal cells to mimic the brain environment in vitro. As a proof of concept, we treated C8-B4 cells (murine cell model) with three different doses of baicalin (0.1, 1 and 5 μM) and vehicle control (DMSO) for 24 h after liposaccharide-induced inflammation and measured the levels of TNF-α in the medium by ELISA. NT2-N cells (human neuronal-like cell model) underwent identical baicalin treatment, followed by RNA extraction, genome-wide mRNA expression profiles and gene set enrichment analysis (GSEA). We also performed neurite outgrowth assays and mitochondrial flux bioanalysis (Seahorse) in NT2-N cells. We found that in C8-B4 cells, baicalin at ≥ 1 μM exhibited anti-inflammatory effects, lowering TNF-α levels in the cell culture media. In NT2-N cells, baicalin positively affected neurite outgrowth and transcriptionally up-regulated genes in the tricarboxylic acid cycle and the glycolysis pathway. Similarly, Seahorse analysis showed increased oxygen consumption rate in baicalin-treated NT2-N cells, an indicator of enhanced mitochondrial function. Together, our findings have confirmed the neuroprotective and mitochondria enhancing effects of baicalin in human-neuronal like cells. Given the increased prominence of mitochondrial mechanisms in diverse neuropsychiatric disorders and the paucity of mitochondrial therapeutics, this suggests the potential therapeutic application of baicalin in human neuropsychiatric disorders where these processes are altered.

Inoculation with a selected strain of &lt;i&gt;Metschnikowia pulcherrima&lt;/i&gt; as a bioprotective alternative to sulphites for preventing browning of white grape must

In this study, the effectiveness of a selected strain of Metschnikowia pulcherrima (MP1) was compared to that of sulphur dioxide for preventing white grape must from browning. Sulphur dioxide was found to drastically reduce oxygen consumption rate, protect hydroxycinnamic acids against oxidation and prevent grape must from browning, even in the presence of laccase. By contrast, supplementation with the selected strain of Metschnikowia pulcherrima (MP1) drastically increased oxygen consumption rate, thus reducing the amount of oxygen available for polyphenol oxidases. In the absence of laccase, this resulted in a decrease in browning and a certain degree of protection of the hydroxycinnamic acids, but in the presence of laccase it was not effective enough. Consequently, the selected strain of Metschnikowia pulcherrima (MP1) can be considered an interesting alternative to sulphur dioxide for preventing browning in white grape must, but only in conditions of healthy grapes.

Glycolytic state of aortic endothelium favors hematopoietic transition during the emergence of definitive hematopoiesis.

The first definitive hematopoietic progenitors emerge through the process of endothelial-to-hematopoietic transition in vertebrate embryos. With molecular regulators for this process worked out, the role of metabolic pathways used remains unclear. Here, we performed nano-LC-MS/MS-based proteomic analysis and predicted a metabolic switch from a glycolytic to oxidative state upon hematopoietic transition. Mitochondrial activity, glucose uptake, and glycolytic flux analysis supported this hypothesis. Systemic inhibition of lactate dehydrogenase A (LDHA) increased oxygen consumption rate in the hemato-endothelial system and inhibited the emergence of intra-aortic hematopoietic clusters. These findings were corroborated using Tie2-Cre-mediated deletion of Ldha that showed similar effects on hematopoietic emergence. Conversely, stabilization of HIF-1α via inhibition of oxygen-sensing pathway led to decreased oxidative flux and promoted hematopoietic emergence in mid-gestation embryos. Thus, cell-intrinsic regulation of metabolic state overrides oxygenated microenvironment in the aorta to promote a glycolytic metabolic state that is crucial for hematopoietic emergence in mammalian embryos.

Bioenergetic profiles of peripheral mononuclear cells and systemic inflammation in women with Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS).

Inflammation is thought to contribute to the etiology of interstitial cystitis/bladder pain syndrome (IC/BPS). It is well-known that disruption in metabolism in immune cells contributes to inflammation in several inflammatory diseases. The purpose of this study was to investigate whether cellular bioenergetics is altered in monocytes and lymphocytes from women with IC/BPS, and if these alterations correlate with systemic inflammatory markers. Age and BMI matched adult healthy women (HS; n = 18) and women with IC/BPS (n = 18) were included in the study. Blood was collected to assess cellular bioenergetics in monocytes and lymphocytes using a Seahorse XF96 Analyzer and plasma cytokine levels were measured using Meso Scale Discovery immunoassays. The correlation between bioenergetic parameters, cytokines, and demographics was determined using Pearson correlation coefficients. Means of the two groups were compared using the two-group t-test. Patients with IC/BPS had reduced monocyte oxygen consumption rates and glycolytic rates compared to healthy subjects. In contrast, lymphocytes from these patients had increased oxygen consumption rates and glycolytic rates. Several cytokines and chemokines including Interferon-gamma (IFN-ɣ), tumor necrosis factor alpha (TNF-ɑ), Interleukin-6 (IL-6), Interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) levels were significantly elevated in the plasma of patients with IC/BPS. However, Transforming growth factor (TGF-β) and Interleukin-10 (IL-10) levels were significantly decreased in IC/BPS patients compared to HS. In addition, Interferon gamma (IFN-ɣ), TNF-ɑ, IL-8, and TGF-β levels correlated with several bioenergetic parameters in monocytes or lymphocytes from healthy subjects. In contrast, TNF-ɑ and IL-8 correlated with bioenergetic parameters in monocytes from IC/BPS patients. Monocyte and lymphocyte cellular bioenergetics and plasma cytokine levels are different in patients with IC/PBS compared to HS. It appears that systemic inflammation is greater in this cohort which may negatively impact immune cell function. The relationship between cellular bioenergetics and inflammation in monocytes and lymphocytes could be important in understanding the pathogenesis of IC/PBS and warrants further investigation.

Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation, and myofibrillar organization.

Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.

Potential role of mitochondrial dysfunction in arrhythmogenesis in coronary artery disease

Introduction. Coronary artery disease (CAD) continues to be the most common pathology in the structure of cardiovascular diseases over the past decades, both in Russia and around the world. In the normal condition, the mitochondria of all body cells have the same function capabilities due to the carriage of the same genome. Therefore, it is possible to assess the respiration activity of cardiomyocyte mitochondria by the respiration of mitochondria from peripheral blood leukocytes.Aim: To compare respiratory activity of mitochondria of peripheral blood leukocytes in patients diagnosed with coronary artery disease and coronary artery disease with developed cardiac rhythm disorders (CRD).Material and methods. The studied groups included 45 patients with CAD without CRD and 39 patients with CAD complicated by CRD. Mitochondria were isolated from peripheral blood leukocytes by differential centrifugation. The rate of oxygen loss in pyruvate-malate and succinate incubation buffers was measured when isolated mitochondria were introduced, as well as when palmitic acid was added to the medium. Oxygen consumption rate for the V3 (active phosphorylating) and V4 (nonphosphorylating) metabolic sates was determined, and on their basis respiratory control coefficient was calculating using the formula V3/V4. Statistical data processing was carried out using STATISTICA 13.0 software.Results. Oxygen consumption rate in mitochondria of patients with uncomplicated CAD and CAD with CRD had no significant differences in either pyruvate-malate or succinate buffers. When palmitic acid was added to the incubation medium, the mitochondria of CAD patients without CRD significantly increased oxygen consumption rate in both incubation media. Mitochondria of CAD patients with CRD did not change oxygen consumption rate in both metabolic states after the addition of palmitic acid in incubation media.Conclusion. On the basis of the data obtained, it can be concluded that the function capabilities of mitochondria in the complicated course of CAD has been exhausted, which manifests itself in the inability to increase ATP synthesis in response to the introduction of additional substrates.

FoxO3a Drives the Metabolic Reprogramming in Tamoxifen-Resistant Breast Cancer Cells Restoring Tamoxifen Sensitivity.

Tamoxifen-resistant breast cancer cells (TamR-BCCs) are characterized by an enhanced metabolic phenotype compared to tamoxifen-sensitive cells. FoxO3a is an important modulator of cell metabolism, and its deregulation has been involved in the acquisition of tamoxifen resistance. Therefore, tetracycline-inducible FoxO3a was overexpressed in TamR-BCCs (TamR/TetOn-AAA), which, together with their control cell line (TamR/TetOn-V), were subjected to seahorse metabolic assays and proteomic analysis. FoxO3a was able to counteract the increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) observed in TamR by reducing their energetic activity and glycolytic rate. FoxO3a caused glucose accumulation, very likely by reducing LDH activity and mitigated TamR biosynthetic needs by reducing G6PDH activity and hindering NADPH production via the pentose phosphate pathway (PPP). Proteomic analysis revealed a FoxO3a-dependent marked decrease in the expression of LDH as well as of several enzymes involved in carbohydrate metabolism (e.g., Aldolase A, LDHA and phosphofructokinase) and the analysis of cBioPortal datasets of BC patients evidenced a significant inverse correlation of these proteins and FoxO3a. Interestingly, FoxO3a also increased mitochondrial biogenesis despite reducing mitochondrial functionality by triggering ROS production. Based on these findings, FoxO3a inducing/activating drugs could represent promising tools to be exploited in the management of patients who are refractory to antiestrogen therapy.

Metabolism-Related Features Identify the Combination Metformin Plus NAMPT Inhibitors As a Selective Treatment Strategy in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease, which remains difficult to treat due to its heterogeneous nature. Recently, metabolic adaptation has been recognized as a trademark of leukemogenesis. Notably, altered function of mitochondria (mt) in leukemic stem cells (LSCs) has been linked to chemoresistance in some patients, causing relapse of disease. Thus, targeting the rewired energy metabolism of leukemic cells has gained a lot of attention in recent years. Here, using a large cohort of adult de novo AML samples (n=502 patients, all intensively treated with 3+7 scheme), we identified a subset of patients with increased mitochondrial DNA content (mtDNAcn, n=163) compared to age- and sex-matched healthy controls (n=308). mtDNAcn was significantly higher in patients harboring FLT3-ITD (31%) and IDH1 mutations (50%). Patients with high mtDNAcn were associated with worse clinical outcomes regardless of European Leukemia Net (ELN) risk stratification, sex, and age. In contrast, high mtDNAcn was not a predictive survival marker in a cohort of adult AML patients treated with venetoclax (VEN) + hypomethylating agents (HMA), indicating that these metabolic features are specifically linked to the resistance to intensive chemotherapy. Using label-free quantitative proteome analysis (11272 proteins) combined with metabolomic screening (173 metabolites) on sorted CD34 +/CD117 + AML blasts (n=26), we found that AMLs with high mtDNAcn were enriched for signatures like “GMP-like” and “mt complex I activity”, being metabolically enriched for long-chain fatty acids, suggesting that these cells rely more on oxidative phosphorylation (OXPHOS) metabolism. Functionally, AML blasts with high mtDNAcn displayed increased levels of BCL2, mt mass, and mt membrane potential, which was associated with increased oxygen consumption rate (OCR) measured by extracellular flux analysis. Considering the impact of mtDNAcn on AML survival in patients treated with intensive chemotherapy, which was not observed in the context of VEN+HMA, and considering the increased reliance of LSC on mtOXPHOS, we studied the impact of mtDNAcn on chemotherapy response. Ex vivo analysis of primary AML samples with high mtDNAcn indicated increased resistance to AraC-induced apoptosis, which was reverted upon knock-down of the DNA polymerase gamma gene (POLG) encoding the catalytic subunit of the mtDNA polymerase. Due to the link between mtDNAcn and mt complex I activity, we investigated if the treatment with metformin, known to inhibit complex I, would sensitize AML patients with high mtDNAcn to the standard of care AML drugs, such as Cytarabine (AraC), FLT3-inhibitors and VEN. Treatment with low dose of metformin (1 mM) effectively reduced the mtDNAcn in AML cells (n=40), resulting in decreased OCR, while glycolysis was increased, suggesting metabolic rewiring. Furthermore, the combination of metformin with AraC, VEN and FLT3-inhibitors synergistically increased drug-induced apoptosis in AML cells with high mtDNAcn and helped to overcome VEN resistance in AML cells. In vivo, combination of metformin with VEN prolonged the survival of mice transplanted with K562 cells (VEN resistant), resulting in decreased leukocyte counts and blood chimerism. Since treatment with metformin resulted in metabolic rewiring, with increased glycolysis and NAD + consumption, we wondered if the combination of metformin with glycolytic (2-DG/DAP) and NAMPT (KPT-9274) inhibitors would impede the metabolic rewiring, enhancing cytotoxicity. Among the different treatment combinations, we find that the combination of metformin and KPT-9274 gave the best therapeutic window by significantly increasing drug-induced apoptosis, with more pronounced effects in patients with high mtDNAcn, while sparing healthy CD34 + cells. Concomitantly, OXPHOS and glycolysis were diminished upon KPT-9274+metformin, suggesting that the combination was able to bypass the metabolic rewiring of the AML cells. In conclusion, we uncover a subgroup of patients with high mtDNAcn linked to increased mtOXPHOS that is resistant to chemotherapy-induced apoptosis and is characterized by poor clinical outcomes, which can be overcome by the inhibition of the mt complex I. Moreover, the quantification of mtDNAcn can be easily incorporated into clinical practice as a simple and cost-effective metabolic readout of AML patients to identify metabolic vulnerabilities.