Oxygen Consumption Rate Research Articles

Lipid Variability Induces Endothelial Dysfunction by Increasing Inflammation and Oxidative Stress.

This study investigates the impact of fluctuating lipid levels on endothelial dysfunction. Human aortic and umbilical vein endothelial cells were cultured under varying palmitic acid (PA) concentrations: 0, 50, and 100 μM, and in a variability group alternating between 0 and 100 μM PA every 8 hours for 48 hours. In the lipid variability group, cells were exposed to 100 μM PA during the final 8 hours before analysis. We assessed inflammation using real-time polymerase chain reaction, Western blot, and cytokine enzyme-linked immunosorbent assay (ELISA); reactive oxygen species (ROS) levels with dichlorofluorescin diacetate assay; mitochondrial function through oxygen consumption rates via XF24 flux analyzer; and endothelial cell functionality via wound healing and cell adhesion assays. Cell viability was evaluated using the MTT assay. Variable PA levels significantly upregulated inflammatory genes and adhesion molecules (Il6, Mcp1, Icam, Vcam, E-selectin, iNos) at both transcriptomic and protein levels in human endothelial cells. Oscillating lipid levels reduced basal respiration, adenosine triphosphate synthesis, and maximal respiration, indicating mitochondrial dysfunction. This lipid variability also elevated ROS levels, contributing to a chronic inflammatory state. Functionally, these changes impaired cell migration and increased monocyte adhesion, and induced endothelial apoptosis, evidenced by reduced cell viability, increased BAX, and decreased BCL2 expression. Lipid variability induce endothelial dysfunction by elevating inflammation and oxidative stress, providing mechanistic insights into how lipid variability increases cardiovascular risk.

Ginsenoside Rh4 prevents endothelial dysfunction as a novel AMPK activator.

The AMP-activated protein kinase (AMPK) signalling pathway is a desirable target for various cardiovascular diseases (CVD), while the involvement of AMPK-mediated specific downstream pathways and effective interventions in hyperlipidaemia-induced endothelial dysfunction remain largely unknown. Herein, we aim to identify an effective AMPK activator and to explore its efficacy and mechanism against endothelial dysfunction. Molecular docking technique was adopted to screen for the potent AMPK activator among 11 most common rare ginsenosides. In vivo, poloxamer 407 (P407) was used to induce acute hyperlipidaemia in C57BL/6J mice. In vitro, palmitic acid (PA) was used to induce lipid toxicity in HAEC cells. We discovered the strongest binding of ginsenoside Rh4 to AMPKα1 and confirmed the action of Rh4 on AMPK activation. Rh4 effectively attenuated hyperlipidaemia-related endothelial injury and oxidative stress both in vivo and in vitro and restored cell viability, mitochondrial membrane potential and mitochondrial oxygen consumption rate in HAEC cells. Mechanistically, Rh4 bound to AMPKα1 and simultaneously up-regulated AKT/eNOS-mediated NO release, promoted PGC-1α-mediated mitochondrial biogenesis and inhibited P38 MAPK/NFκB-mediated inflammatory responses in both P407-treated mice and PA-treated HAEC cells. The AMPK inhibitor Compound C treatment completely abrogated the regulation of Rh4 on the above pathways and weakened the lowering effect of Rh4 on endothelial impairment markers, suggesting that the beneficial effects of Rh4 are AMPK dependent. Rh4 may serve as a novel AMPK activator to protect against hyperlipidaemia-induced endothelial dysfunction, providing new insights into the prevention and treatment of endothelial injury-associated CVD.

Killer whale respiration rates.

Measuring breathing rates is a means by which oxygen intake and metabolic rates can be estimated to determine food requirements and energy expenditure of killer whales (Orcinus orca) and other cetaceans. This relatively simple measure also allows the energetic consequences of environmental stressors to cetaceans to be understood but requires knowing respiration rates while they are engaged in different behaviours such as resting, travelling and foraging. We calculated respiration rates for different behavioural states of southern and northern resident killer whales using video from UAV drones and concurrent biologging data from animal-borne tags. Behavioural states of dive tracks were predicted using hierarchical hidden Markov models (HHMM) parameterized with time-depth data and with labeled tracks of drone-identified behavioural states (from drone footage that overlapped with the time-depth data). Dive tracks were sequences of dives and surface intervals lasting ≥ 10 minutes cumulative duration. We calculated respiration rates and estimated oxygen consumption rates for the predicted behavioural states of the tracks. We found that juvenile killer whales breathed at a higher rate when travelling (1.6 breaths min-1) compared to resting (1.2) and foraging (1.5)-and that adult males breathed at a higher rate when travelling (1.8) compared to both foraging (1.7) and resting (1.3). The juveniles in our study were estimated to consume 2.5-18.3 L O2 min-1 compared with 14.3-59.8 L O2 min-1 for adult males across all behaviours based on estimates of mass-specific tidal volume and oxygen extraction. Our findings confirm that killer whales take single breaths between dives and indicate that energy expenditure derived from respirations requires using sex, age, and behavioural-specific respiration rates. These findings can be applied to bioenergetics models on a behavioural-specific basis, and contribute towards obtaining better predictions of dive behaviours, energy expenditure and the food requirements of apex predators.

A Bayesian hierarchical model for predicting rates of oxygen consumption in mechanically ventilated intensive care patients

A Bayesian hierarchical model for predicting rates of oxygen consumption in mechanically ventilated intensive care patients

The AMPK activator ATX-304 alters cellular metabolism to protect against cisplatin-induced acute kidney injury

Acute kidney injury (AKI) disrupts energy metabolism. Targeting metabolism through AMP-activated protein kinase (AMPK) may alleviate AKI. ATX-304, a pan-AMPK activator, was evaluated in C57Bl/6 mice and tubular epithelial cell (TEC) cultures. Mice received ATX-304 (1 mg/g) or control chow for 7 days before cisplatin-induced AKI (CI-AKI). Primary cultures of tubular epithelial cells (TECs) were pre-treated with ATX-304 (20 µM, 4 h) prior to exposure to cisplatin (20 µM, 23 h). ATX-304 increased acetyl-CoA carboxylase phosphorylation, indicating AMPK activation. It protected against CI-AKI measured by serum creatinine (control 0.05 + 0.03 mM vs ATX-304 0.02 + 0.01 mM, P = 0.03), western blot for neutrophil gelatinase-associated lipocalin (NGAL) (control 3.3 + 1.8-fold vs ATX-304 1.2 + 0.55-fold, P = 0.002), and histological injury (control 3.5 + 0.59 vs ATX-304 2.7 + 0.74, P = 0.03). In TECs, pre-treatment with ATX-304 protected against cisplatin-mediated injury, as measured by lactate dehydrogenase release, MTS cell viability, and cleaved caspase 3 expression. ATX-304 protection against cisplatin was lost in AMPK-null murine embryonic fibroblasts. Metabolomic analysis in TECs revealed that ATX-304 (20 µM, 4 h) altered 66/126 metabolites, including fatty acids, tricarboxylic acid cycle metabolites, and amino acids. Metabolic studies of live cells using the XFe96 Seahorse analyzer revealed that ATX-304 increased the basal TEC oxygen consumption rate by 38%, whereas maximal respiration was unchanged. Thus, ATX-304 protects against cisplatin-mediated kidney injury via AMPK-dependent metabolic reprogramming, revealing a promising therapeutic strategy for AKI.

Overlooked shelf sediment reductive sinks of dissolved rhenium and uranium in the modern ocean

Rhenium (Re) and uranium (U) are essential proxies in reconstructing past oceanic oxygenation evolution. However, their removal in continental shelf sediments, hotspots of early diagenesis, were previously treated as quantitatively unimportant sinks in the ocean. Here we examine the sedimentary reductive removal of Re and U and their coupling with organic carbon decomposition, utilizing the 224Ra/228Th disequilibria within the East China Sea shelf. We identified positive correlations between their removal fluxes and the rates of sediment oxygen consumption or organic carbon decomposition. These correlations enable an evaluation of global shelf reductive sinks that are comparable to (for Re) or higher than (~4-fold for U) previously established suboxic/anoxic sinks. These findings suggest potential imbalances in the modern budgets of Re and U, or perhaps a substantial underestimation of their sources. Our study thus highlights shelf sedimentary reductive removal as critical yet overlooked sinks for Re and U in the modern ocean.

Experimental study on inhibition of coal spontaneous combustion by carbon dioxide and nitrogen

ABSTRACT Injecting CO2 and N2 to reduce the concentration of O2 in the air is a commonly used measure to prevent coal spontaneous combustion (CSC). To study the difference in inhibitory effects of CO2 and N2 on coal oxidation, experiments in this study were conducted using a setup of self-developed gas-bath coal oxidation for low temperatures. The oxidation process of the coal-oxygen system was then analyzed using reaction kinetics. Two different coal samples were tested in experiments under two inert gas environments, i.e. CO2 and N2 with various O2 concentrations, respectively. The changes in O2 and CO concentrations were measured during the oxidation process. By studying the standard oxygen consumption rate (SOCR) and the apparent activation energy of the coal-oxygen reaction system, inhibitory effects of CO2 and N2 on CSC were analyzed and compared. The results showed that the SOCR of the same coal sample remained consistent and independent of the O2 concentration within the same inert gas environment although under different O2 levels. In both CO2-O2 and N2-O2 environments, the SOCR of the coal sample exhibits exponential growth with temperature. The SOCR was found to have a functional connection with temperature, it can be represented as y = a ⋅ e bT , and the R 2 (determination coefficients) of the fitting formulas are all greater than 0.94. In the correlation, the inert gas environment has minimal impact on coefficient “b,” while in the CO2-O2 environment, reducing the value of coefficient “a” can effectively lower the coal sample’s SOCR. The GBMK sample and YMWK sample have an “a” value that is 20% of the value in the N2-O2 environment when oxidizing in the CO2-O2 environment. The SOCR in the CO2-O2 environment showed a decrement of between 26.0% and 52.1% when compared to that in the N2-O2 environment, whereas the activation energy showed an increment between 1.4% and 18.6%. The findings in this work provide an in-depth understanding of the prevention of CSC in the gob for risk mitigation.

Elesclomol-copper synergizes with imidazole ketone erastin by promoting cuproptosis and ferroptosis in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) encompass a collection of clonal hematopoietic malignancies distinguished by the depletion of peripheral blood cells. The treatment of MDS is hindered by the advanced age of patients, with a restricted repertoire of drugs currently accessible for therapeutic intervention. In this study, we found that ES-Cu strongly inhibited the viability of MDS cell lines and activated cuproptosis in a copper-dependent manner. Importantly, ferroptosis inducer IKE synergistically enhanced ES-Cu-mediated cytotoxicity both in vitro and in vivo. Of note, the combination of IKE and ES-Cu intensively impaired mitochondrial homeostasis with increased mitochondrial ROS, MMP hyperpolarized, down-regulated iron-sulfur proteins and declined oxygen consumption rate. Additionally, ES-Cu/IKE treatment could enhance the lipoylation-dependent oligomerization of the DLAT. To elucidate the specific order of events in the synergistic cell death, inhibitors of ferroptosis and cuproptosis were utilized to further characterize the basis of cell death. Cell viability assays showed that the glutathione and its precursor N-acetylcysteine could significantly rescue the cell death under either mono or combination treatment, demonstrating that GSH acts at the crossing point in the regulation network of cuproptosis and ferroptosis. Significantly, the reconstitution of xCT expression and knockdown of FDX1 cells have been found to contribute to the tolerance of mono treatment but have little recovery impact on the combined treatment. Collectively, these findings suggest that a synergistic interaction leading to the induction of multiple programmed cell death pathways could be a promising approach to enhance the effectiveness of therapy for MDS.

Toxicity of the sunscreen UV filter benzophenone-3 (OBZ) to the microalga Selenastrum capricornutum: An insight into OBZ’s damage to photosynthesis and respiration

Oxybenzone (OBZ; benzophenone-3, CAS# 131–57–7), as a new pollutant and ultraviolet absorbent, shows a significant threat to the survival of phytoplankton. This study aims to explore the acute toxic effects of OBZ on the growth of the microalga Selenastrum capricornutum, as well as the mechanisms for its damage to the primary metabolic pathways of photosynthesis and respiration. The results demonstrated that the concentrations for 50 % of maximal effect (EC50) of OBZ for S. capricornutum were 9.07 mg L−1 and 8.54 mg L−1 at 72 h and 96 h, respectively. A dosage of 4.56 mg L−1 OBZ significantly lowered the photosynthetic oxygen evolution rate of S. capricornutum in both light and dark conditions for a duration of 2 h, while it had no effect on the respiratory oxygen consumption rate under darkness. OBZ caused a significant decline in the efficiency of photosynthetic electron transport due to its damage to photosystem II (PSII), thereby decreasing the photosynthetic oxygen evolution rate. Over-accumulated H2O2 was produced under light due to the damage caused by OBZ to the donor and acceptor sides of PSII, resulting in increased peroxidation of cytomembranes and inhibition of algal respiration. OBZ’s damage to photosynthesis and respiration will hinder the conversion and reuse of energy in algal cells, which is an important reason that OBZ has toxic effects on S. capricornutum. The present study indicated that OBZ has an acute toxic effect on the microalga S. capricornutum. In the two most important primary metabolic pathways in algae, photosynthesis is more sensitive to the toxicity of OBZ than respiration, especially in the dark.

Preparation of complex physicochemical synergistic inhibitors and study on inhibition of spontaneous combustion of coal

ABSTRACT To efficiently isolate oxygen and block free radical reactions， sodium alginate (SA), sodium polyacrylate (PAAS), attapulgite (ATP), and melatonin (MLT) were selected to develop an oxygen-insulating physicochemical synergistic inhibitor. The water loss experiment, EPR experiment, and programmed temperature rise experiment were used to characterize the performance of the inhibitor. The results show that the optimal ratio of the oxygen barrier physicochemical synergistic inhibitor is 2.5 wt% SA, 1.5 wt‰ PAAS, 9 wt% ATP, 6 wt% MLT, and 3 wt% CaCl2. In the oxygen-insulating physicochemical synergistic inhibitor, the MLT can react with oxidizing radicals in the coal to slow down the process of the oxidation reaction of the coal, and the SA gel can fill the pore space to prevent oxygen adsorption on the coal surface, the inhibition rate of the inhibitor reaches 70% at 120°C. Compared with the original coal, the CO concentration of the treated coal samples is reduced by 253.9ppm compared with that of the original coal, the generation rate is reduced by 1. 13 × 10−11 mol·cm−3·s−1, and the rate of oxygen consumption is reduced by 90. 65 × 10−11 mol·cm−3·s−1 at 120°C. The above results show that the oxygen-insulating physicochemical synergistic inhibitor developed in this paper can effectively improve the fire extinguishing efficiency is environmentally friendly and pollution-free, and has good application prospects.

Sulforaphane Exposure Prevents Cadmium-Induced Toxicity and Mitochondrial Dysfunction in the Nematode Caenorhabditis elegans by Regulating the Insulin/Insulin-like Growth Factor Signaling (IIS) Pathway

Cadmium (Cd) is a heavy metal that is highly toxic to humans and animals. Its adverse effects have been widely associated with mitochondrial alterations. However, there are not many treatments that target mitochondria. This study aimed to evaluate the impact of sulforaphane (SFN) pre-exposure against cadmium chloride (CdCl2)-induced toxicity and mitochondrial alterations in the nematode Caenorhabditis elegans (C. elegans), by exploring the role of the insulin/insulin-like growth factor signaling pathway (IIS). The results revealed that prior exposure to SFN protected against CdCl2-induced mortality and increased lifespan, body length, and mobility while reducing lipofuscin levels. Furthermore, SFN prevented mitochondrial alterations by increasing mitochondrial membrane potential (Δψm) and restoring mitochondrial oxygen consumption rate, thereby decreasing mitochondrial reactive oxygen species (ROS) production. The improvement in mitochondrial function was associated with increased mitochondrial mass and the involvement of the daf-16 and skn-1c genes of the IIS signaling pathway. In conclusion, exposure to SFN before exposure to CdCl2 mitigates toxic effects and mitochondrial alterations, possibly by increasing mitochondrial mass, which may be related to the regulation of the IIS pathway. These discoveries open new possibilities for developing therapies to reduce the damage caused by Cd toxicity and oxidative stress in biological systems, highlighting antioxidants with mitochondrial action as promising tools.

Decreased mitochondrial respiration associates with frailty in community-dwelling older adults

Aging population has led to an increased prevalence of chronic and degenerative pathologies. A manifestation of unhealthy aging is frailty, a geriatric syndrome that implies a non-specific state of greater vulnerability. Currently, methods for frailty diagnosis are based exclusively on clinical observation. The aim of this study is to determine whether the bioenergetic capacity defined as mitochondrial oxygen consumption rate (OCR) of peripheral circulation mononuclear cells (PBMC) associates with the frailty phenotype in older adults and with their nutritional status. This is a cross-sectional analytic study of 58 participants 70 years and older, 18 frail and 40 non-frail adults, from the ALEXANDROS cohort study, previously described. Participants were characterized through sociodemographic and anthropometric assessments. Frail individuals displayed a higher frequency of osteoporosis and depression. The mean age of the participants was 80.2 ± 5.2 years, similar in both groups of men and women. Regarding the nutritional status defined as the body mass index, most non-frail individuals were normal or overweight, while frail participants were mostly overweight or obese. We observed that OCR was significantly decreased in frail men (p < 0.01). Age was also associated with significant differences in oxygen consumption in frail patients, with lower oxygen consumption being observed in those over 80 years of age. Therefore, the use of PBMC can result in an accessible fingerprint that may identify initial stages of frailty in a minimally invasive way.

Improved CAR-T cell activity associated with increased mitochondrial function primed by galactose.

CD19 CAR-T cells have led to durable remissions in patients with refractory B-cell malignancies; nevertheless, most patients eventually relapse in the long term. Many interventions aimed at improving current products have been reported, with a subset of them focusing on a direct or indirect link to the metabolic state of the CAR-T cells. We assessed clinical products from an ongoing clinical trial utilizing CD19-28z CAR-T cells from patients with acute lymphoblastic leukemia. CAR-T clinical products leading to a complete response had significantly higher mitochondrial function (by oxygen consumption rate) irrespective of mitochondrial content. Next, we replaced the carbon source of the media from glucose to galactose to impact cellular metabolism. Galactose-containing media increased mitochondrial activity in CAR-T cells, and improved in in-vitro efficacy, without any consistent phenotypic change in memory profile. Finally, CAR-T cells produced in galactose-based glucose-free media resulted in increased mitochondrial activity. Using anin-vivo model of Nalm6 injected mice, galactose-primed CAR-T cells significantly improved leukemia-free survival compared to standard glucose-cultured CAR-T cells. Our results prove the significance of mitochondrial metabolism on CAR-T cell efficacy and suggest a translational pathway to improve clinical products.

Physiological Changes in Nickel-exposed Nile Tilapia Oreochromis niloticus during Exposure and Recovery Periods

Heavy metals are a significant problem in aquatic ecosystems as they are toxic and tend to accumulate, immediately affecting fish physiology. The present investigation was carried out to evaluate the sub-lethality of Nickel chloride on fingerlings of Nile tilapia, Oreochromis niloticus on long-term exposure to it is below safe concentrations1/fifth (9.39 ppm) and 1/10th (4.69 ppm). The physiological studies revealed a significant reduction in Oxygen Consumption Rate (OCR), Ammonia-N Excretion Rate (AER), Oxygen Rate (O: R) and Food Consumption Rate (FCR) and behavioural changes during accumulation and depuration phases. Limited recovery was obtained in all the physiological parameters after depuration for 28 days from the end of the accumulation period.

AMPK regulates immature boar Sertoli cell proliferation through affecting CDK4/Cyclin D3 pathway and mitochondrial function

Sertoli cell (SC) proliferation plays an important role in sperm production and quality; however, the regulatory mechanism of SC proliferation is not well understood. This study investigated the role of adenosine monophosphate-activated protein kinase (AMPK) in the regulation of immature boar SC activity. Cell counting kit-8, Seahorse XFe96, mitochondrial respiratory enzyme-related assay kits, and transmission electron microscopy were used to detect SC proliferative viability, oxygen consumption rate (OCR), mitochondrial respiratory enzyme activity, and the ultrastructure of primary cultured SCs in vitro from the testes of 21-day-old boars. A dual luciferase reporter assay was performed to determine the miRNA-mRNA target interaction. Western blotting was used to analyze cell proliferation-related protein expression of p38, p21, proliferating cell nuclear antigen (PCNA), Cyclin-dependent kinase 4 (CDK4), Cyclin D3, and phosphorylated retinoblastoma protein (Rb). Each experiment had a completely randomized design, with three replicates in each experiment. The results showed that the AMPK inhibitor (Compound C, 20 μM-24 h) increased cell proliferation viability, ATP production, and maximal respiration of SCs by 0.64-, 0.12-, and 0.08-fold (p < 0.05), respectively; increased the SC protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.13-, 0.09-, 0.88-, and 0.12-fold (p < 0.05), respectively; and decreased the SC protein expression of p38 and p21 by 0.36- and 0.27-fold (p < 0.05), respectively. The AMPK agonist AICAR (2 mM-6 h) significantly inhibited SC ultrastructure, OCR, mitochondrial respiratory enzyme activity, and cell proliferation-related protein levels. AMPK was validated to be a target gene of miR-1285 based on the result in which the miR-1285 mimic inhibited the luciferase activity of wild-type AMPK by 0.54-fold (p < 0.001). MiR-1285 mimic promoted the OCR of SCs, with 0.45-, 0.15-, 0.21-, and 0.30-fold (p < 0.01) increases in ATP production, basal and maximal respiration, and spare capacity, respectively. MiR-1285 mimic increased the mitochondrial respiratory enzyme activity of SCs, with 0.63-, 0.70-, and 0.97-fold (p < 0.01) increases in NADH-Q oxidoreductase, cytochrome c oxidase, and ATP synthase, respectively. Moreover, the miR-1285 mimic increased the protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.24-, 0.30-, 0.22-, and 0.13-fold (p < 0.05), respectively, and reduced the protein expression of p38 and p21 by 0.58- and 0.66-fold (p < 0.001). MiR-1285 inhibitor showed opposite effects on the above indicators and induced numerous autophagosomes and large lipid droplets in SCs. A high dose of estradiol (10 μM-6 h, showed a promotion of AMPK activation in a previous study) significantly inhibited SC ultrastructure, mitochondrial function, and proliferation-related pathways, while these adverse effects were weakened by Compound C treatment or miR-1285 mimic transfection. Our findings suggest that the activation and inhibition of AMPK induced by specific drugs or synthesized targeted miRNA fragments could regulate immature boar SC proliferative activity by influencing the CDK4/Cyclin D3 pathway and mitochondrial function; this helps to provide a basis for the prevention and treatment of male sterility in clinical practice.

Abstract PO5-23-08: Adenylosuccinate lyase is essential for proliferation and mitochondrial function of endocrine therapy resistant breast cancer cells

Abstract Eighty percent of breast cancers express estrogen receptor alpha (ERα, ESR1) at the time of diagnosis. Endocrine therapy (ET) is the standard treatment that either block estrogen mediated ER activation (such as tamoxifen, fulvestrant) or suppress estrogen synthesis (such as exemestane, letrozole, or anastrozole). While ET is initially effective, emergence of ET-resistance is common and possess a major clinical challenge. Hence, there is an ongoing need to develop new treatments for ET-resistant-ERα positive breast cancer. In this study we investigated the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway as a potential target of ET-resistant breast cancer. The protein level of ADSL in two independent ET-resistant cell line model LCC9 and T47D-4HT cells were significantly higher in comparison to its parental ET-sensitive cell lines, MCF7 and T47D cells, respectively. Transient knockdown of ADSL using two independent siRNA in both LCC9 and T47D-4HT cells results in a significant decrease of cell growth, colony, and spheroid formation. Notably, reduced ADSL levels in LCC9 cells prevented the cells from progressing from G1 to S phase of cell cycle with concurrent elevation of cyclin D1/D3, CDK2/4, and cyclin E. On the other hand, in T47D-4HT cells, ADSL reduction caused cells to accumulate in S-phase of cell cycle and showed lower levels of total cyclin D1 protein. Mechanistically, in LCC9 cells (but not in T47D-4HT cells), ADSL depletion induced DNA replication stress which activates (phosphorylate) ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its major downstream effector checkpoint kinase 1 (Chk1) that in turn failed to de-phosphorylate the inhibitory-phospho-groups of cyclin-dependent kinase 2 (CDK2). In addition, ADSL depletion perturbed the mitochondrial function in both LCC9 and T47D-4HT cells. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was lower in both the cells along with the mitochondrial membrane potential. Pertinently, lower concentration of AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), a product of ADSL enzyme function, partially rescued the cell growth and restored mitochondrial membrane potential in ADSL-depleted LCC9 cells. To further understand the global effects of ADSL depletion we performed RNA-sequencing of LCC9 and T47D-4HT cells after ADSL depletion and compared with respective control siRNA transfected cells followed by gene set enrichment analysis. Intriguingly, diverse effect was observed in LCC9 and T47D-4HT cells as very few genes were found to be commonly regulated between these two cell lines. Analysis of clinical data sets revealed high level of ADSL transcript was associated with adverse progression free survival and overall survival in breast cancer patients treated with endocrine therapies. Overall, our findings demonstrate that ADSL-mediated de novo purine synthesis is critical for cellular growth, proliferation, and mitochondrial function of endocrine therapy-resistant breast cancer cells. Therefore, targeting ADSL is a novel potential therapeutic approach for ERα positive ET- resistant breast cancer. Citation Format: Anil Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Adenylosuccinate lyase is essential for proliferation and mitochondrial function of endocrine therapy resistant breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-23-08.

Abstract PO5-23-09: Metabolic alterations in Estrogen Receptor-positive breast cancer contributing to CDK4/6 resistance

Abstract Background CDK4/6 inhibitors (CDK4/6i) are potent FDA-approved agents for the treatment of metastatic and for high-risk early estrogen receptor positive (ER+) breast cancer. However, significant proportion of patients continue to develop disease progression. Response does not correlate well with expression of CDK4/6 or key cell cycle proteins. Ki67, a proliferation marker, is prognostic, but NOT predictive of response. Resistance to CDK4/6i is mediated by multiple mechanisms including metabolic alterations. Herein, we explore the metabolic alterations associated with development of CDK4/6i resistance. Methods To understand the key metabolic alterations associated with resistance, we first developed abemaciclib-resistant ER+ cell lines and compared their metabolic profile with their parental/sensitive counterparts. Seahorse metabolic profiles were performed on parental cells (MCF-7, LCC2, LCC9, T47D and ZR-75.1) and their resistant derivatives. In addition, spatial proteomics analysis was performed using multiplex cyclic immunofluorescence (CycIF) assay for 27 markers including 14 metabolic regulators (MCT1, GLUT1, G6PD, VDAC1/3, LDHA, GAPDH, PGC1A, HK2, ATP5A, GLUD12, CPT1A, CS, C6 Ceramide, COXIV), 9 signaling (CD36, CCND1, CCNE1, pRB, MKI67, pERK, CDH1, GPNMB and MET) and 4 segmentation markers (pan-cytokeratin for epithelial cells, concanavalin A for endoplasmic reticulum, phalloidin for actin, and wheat germ agglutinin (WGA) for Golgi and plasma membrane) in sensitive and resistant cells. Following the image registration (hiPlex) and cell segmentation (CellProfiler), multiplex protein images were clustered for cell phenotypes (HistoCAT). Results Cells resistant to abemaciclib showed G1 phase arrest, but a dramatic increase in invasive capacity. The Seahorse XFp Cell Energy Phenotype Assay demonstrated altered metabolic profiles including decreased extracellular acidification rate (ECAR) and oxygen consumption rate (OCAR) indicative of glycolysis, and mitochondrial respiration, respectively, in resistant cells. Multiplex CycIF utilized a panel of 11 cycles resulting in a total of 27 markers. We demonstrated that three metabolic enzymes, mitochondrial voltage-dependent anion channel protein (VDAC1/3), lactate Dehydrogenase A (LDHA) and PPARγ coactivator 1α (PGC1α) are significantly overexpressed in CDK4/6 resistant cells compared to the control parental sensitive cells. Further analyses are ongoing to better characterize these changes in human samples treated with CDK4/6i. Conclusion Our findings highlight the importance of metabolic pathways in development of resistance to CDK4/6i treatment. Understanding the mechanisms by which these pathways contribute to therapeutic failure will further provide with novel intervention strategies and new avenues for prevent recurrence/resistance in ER+ breast cancer. Citation Format: Mayar Allam, Ahmet Coskun, Thomas Hu, Yuan Gu, Sunil Badve, Yesim Gokmen-Polar. Metabolic alterations in Estrogen Receptor-positive breast cancer contributing to CDK4/6 resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-23-09.

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.

Postmortem mitochondria function in longissimus lumborum of Angus and Brahman steers

Mitochondria function and integrity may impact postmortem metabolism and meat quality development. Adaptations in heat tolerant Brahman may persist to limit cellular stress postmortem. Our objective was to evaluate glycolysis, pH decline, and mitochondria function in longissimus lumborum (LL) from Angus and Brahman steers (N = 28) early postmortem (1 to 6 h) and after rigor (24 h). We evaluated metabolites of anaerobic glycolysis, ATP, pH, and temperature, and determined mitochondria oxygen consumption rate (OCR) in permeabilized fibers. The main effects of breed (b) and time (t) and the interaction were tested. Brahman LL contained greater ATP during the first 6 h postmortem; Brahman also tended to exhibit a slower pH decline (b × t, P = 0.07) and more rapid temperature decline (b × t, P < 0.001), but metabolites of anaerobic glycolysis were not different. Mitochondria in Brahman and Angus LL were well-coupled and respired at 1 h postmortem. However, outer membrane integrity became increasingly compromised postmortem (t, P < 0.001). Brahman tended to exhibit greater electron transport system capacity (b, P < 0.1) and had greater capacity for oxidative phosphorylation (complex I and II substrates) at 6 h compared with Angus (P < 0.001). In totality, greater ATP, slower pH decline, and enhanced mitochondria capacity indicate that Brahman possess mitochondrial properties or cellular adaptations that help protect the cell during energy stress postmortem. Slower pH and more rapid temperature decline in LL from Brahman may also help preserve mitochondria function postmortem.

Characterization of an in vitro model to study CD4+ T cells metabolism in dairy cows

Dairy cows are susceptible to several health disorders throughout their lactation. Objectives were to characterize an in vitro model to study bioenergetic measures in CD4+ T lymphocytes in dairy cows. Twenty-four healthy mid-lactating multiparous Holstein dairy cows were enrolled at a mean (±standard deviation) of 234 ± 22 d in milk (DIM). Cows were blocked according to DIM and blood was collected to isolate peripheral blood mononuclear cells followed by magnetic separation of CD4+ T lymphocytes using bovine specific monoclonal antibodies. The isolated CD4+ T lymphocytes from each cow were split into 2 tubes and randomly assigned to incubate in an assay medium as control (CON) or with a combination of phorbol myristate acetate and ionomycin (PMA+IMY) to evaluate metabolic function under resting and activated state. Mitochondrial and glycolytic functional kinetics were recorded in CD4+ T lymphocytes based on real-time measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) under basal conditions and in response to complex V inhibitor (oligomycin), a protonophore uncoupler (BAM 15), and complex I and complex III inhibitors (rotenone and antimycin A). The mean (±SD) viability and purity of CD4+ T lymphocytes was 92.5 ± 2.9% and 95.2 ± 2.9%, respectively. The basal OCR in CD4+ T lymphocytes treated with PMA+IMY was greater than CON; nevertheless, the maximal respiration rate (CON = 58.0 vs. PMA+IMY = 47.3 ± 5.7 pmol/min) and sparing respiratory capacity rate (CON = 42.0 vs. PMA+IMY = 28.7 ± 4.2 pmol/min) were decreased in activated CD4+ T lymphocytes. The ECAR in CD4+ T lymphocytes increased progressively over time in PMA+IMY compared with CON, which indicated an increase in aerobic glycolysis in PMA+IMY compared with CON (CON = 46.9 vs. PMA+IMY = 86.4 ± 7.0 pmol/min). Activated CD4+ T lymphocytes exhibit a metabolic switch from oxidative phosphorylation to aerobic glycolysis, which may support rapid cell proliferation. The results observed in this experiment demonstrate the sensitivity of the technique to detect changes in metabolic function under different cellular conditions, providing a robust framework to study immuno-metabolism in dairy cattle.