Increase In Oxygen Consumption Research Articles

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.

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.

Modulation of mitochondrial function by extracellular acidosis in tumor cells and normal fibroblasts: Role of signaling pathways

In many tumors pronounced extracellular acidosis resulting from glycolytic metabolism is found. Since several environmental stress factors affect the mitochondrial activity the aim of the study was to analyze the impact of acidosis on cellular oxygen consumption and which signaling pathways may be involved in the regulation.In two tumor cell lines and normal fibroblasts cellular oxygen consumption rate (OCR) and mitochondrial function were measured after 3 h at pH 6.6. Besides the activation of ERK1/2, p38 and PI3K signaling in the cytosolic and mitochondrial compartment, the mitochondrial structure and proteins related to mitochondria fission were analyzed.The acidic extracellular environment increased OCR in tumor cells but not in fibroblasts. In parallel, the mitochondrial membrane potential increased at low pH. In both tumor lines (but not in fibroblasts), the phosphorylation of ERK1/2 and PI3K/Akt was significantly increased, and both cascades were involved in OCR modulation. The activation of signaling pathways was located predominantly in the mitochondrial compartment of the cells. At low pH, the mitochondrial structure in tumor cells showed structural changes related to elongation whereas mitochondria fragmentation was reduced indicating mitochondria fusion. However, these morphological changes were not related to ERK1/2 or PI3K signaling.Acidic stress seems to induce an increased oxygen consumption, which might further aggravate tumor hypoxia. Low pH also induces mitochondria fusion that is not mediated by ERK1/2 or PI3K signaling. The mechanism by which these signaling cascades modulate the respiratory activity of tumor cells needs further investigation.

Defecation after magnesium supplementation enhances cognitive performance in triathletes

Constipation is correlated with diminished cognitive function, revealing a possible rectum-brain connection. In this counter-balanced crossover trial, 13 elite triathletes underwent a Stroop test to assess cognitive function and executive control. The Stroop test was conducted both with and without magnesium oxide intake, with a 1-week washout period between sessions. Oxygenation and blood distribution during the cognitive challenge were measured using Near-Infrared Spectroscopy (NIRS). Measurements were taken in both the prefrontal brain and the sub-navel region, where the highest glucose uptake was detected under the 18F-fluorodeoxyglucose Positron Emission Tomography (PET) scan. A significant reduction in completion time for the Stroop test was observed after defecation compared to the non-defecated condition (non-defecation: [27.1 ​± ​1.1] s; non-magnesium defecation: [24.4 ​± ​0.9] s; magnesium defecation: [23.4 ​± ​0.8] s, p ​< ​0.05). Stroop test performance was improved in all (100%, 13/13) of the participants after magnesium-induced defecation and most (69%, 9/13) of the participants after non-magnesium-induced defecation. While no alterations in oxygenation and blood distribution were observed in the prefrontal brain during the Stroop test, decreased oxygenation levels were observed in the sub-navel region under both defecated conditions, without significant changes in blood distribution (p ​< ​0.05). This data suggests an acute increase in oxygen consumption at this specific region. The result of this study suggests an unexplored causal link between the state of the rectum and cognitive performance. Magnesium supplementation to improved rectal emptying presents a novel application for optimizing cognitive function in athletes navigating intricate racing conditions.

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.

Folate induces stemness and increases oxygen consumption under glucose deprivation by notch-1 pathway activation in colorectal cancer cell.

Evidence for folate's protective effects on neural tube defects led the USA and Chile to start mandatory folic acid (FA) fortification programs, decreasing up to 50%. However, ∼30% of the population consuming fortified foods reach supraphysiologic serum levels. Although controversial, several epidemiological and clinical observations suggest that folate increases cancer risk, giving concern about the risks of FA supplementation. The Cancer stem cells (CSCs) model has been used to explain survival to anticancer therapies. The Notch-1 pathway plays a role in several cancers and is associated with the stemness process. Different studies show that modulation of metabolic pathways regulates stemness capacity in cancer. Supraphysiologic concentrations of FA increase the proliferation of HT-29 cells by Notch-1 activation. However, whether folate can induce a stemness-like phenotype in cancer is not known. We hypothesized that FA protects from glucose deprivation-induced cell death through Notch-1 activation. HT-29 cells were challenged with glucose deprivation at basal (20 nM) and supraphysiological (400 nM) FA and 5-MTHF concentrations. We analyzed changes in stemness-like gene expression, cell death and different energetic metabolic functions. Supraphysiological concentrations of FA increased stemness-like genes, and improved survival and oxygen consumption, inducing AMPK phosphorylation and HSP-70 protein expression. We evaluated the Notch-1 pathway using the DAPT and siRNA as inhibitors, decreasing the stemness-like gene expression and preventing the FA protection against glucose deprivation-induced cell death. Moreover, they decreased oxygen consumption and AMPK phosphorylation. These results suggest that FA protects against glucose deprivation. These effects were associated with AMPK activation, a critical metabolic mediator in nutrient consumption and availability that activates the Notch-1 pathway.

Molecular mechanisms of metabolic dysregulation in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM), one of the most serious complications of diabetes mellitus, has become recognized as a cardiometabolic disease. In normoxic conditions, the majority of the ATP production (>95%) required for heart beating comes from mitochondrial oxidative phosphorylation of fatty acids (FAs) and glucose, with the remaining portion coming from a variety of sources, including fructose, lactate, ketone bodies (KB) and branched chain amino acids (BCAA). Increased FA intake and decreased utilization of glucose and lactic acid were observed in the diabetic hearts of animal models and diabetic patients. Moreover, the polyol pathway is activated, and fructose metabolism is enhanced. The use of ketones as energy sources in human diabetic hearts also increases significantly. Furthermore, elevated BCAA levels and impaired BCAA metabolism were observed in the hearts of diabetic mice and patients. The shift in energy substrate preference in diabetic hearts results in increased oxygen consumption and impaired oxidative phosphorylation, leading to diabetic cardiomyopathy. However, the precise mechanisms by which impaired myocardial metabolic alterations result in diabetes mellitus cardiac disease are not fully understood. Therefore, this review focuses on the molecular mechanisms involved in alterations of myocardial energy metabolism. It not only adds more molecular targets for the diagnosis and treatment, but also provides an experimental foundation for screening novel therapeutic agents for diabetic cardiomyopathy.

Abstract 45: Driving AMPK signaling in CD4+ CART cells enhances metabolic capacity, memory formation, and anti-leukemia activity

Abstract Background: While Chimeric Antigen Receptor (CAR)T cell therapy has seen great success in pediatric and young adult acute lymphoblastic leukemia, up to 50% of patients will relapse following treatment. Increased memory formation and mitochondrial capacity, particularly in the CD4+ subset, have been linked to improved CART cell function and enhanced in vivo persistence, leading to long-term relapse-free survival. AMP-activated protein kinase (AMPK) is a cellular energy sensor responsible for promoting metabolic efficiency and T cell memory formation. We have previously demonstrated AMPK activity can be increased in human T cells via overexpression of the AMPKγ2 domain. We hypothesized that overexpression of AMPKγ2 in CART cells would enhance their metabolic capacity and improve their anti-leukemia function. Methods: Human CART cells bearing a CD19-CAR utilizing a 41BB costimulatory domain were generated from healthy human donors via lentiviral transduction and co-transduced with AMPKγ2 (AMPK) or an empty vector (EV) control. Flow sorting for the co-transduced populations yielded &amp;gt;95% CD4+ CART cells. CARTs were expanded in IL-2 for approximately 2 weeks, followed by overnight stimulation and subsequent metabolic analysis using the Seahorse Metabolic Analyzer. Memory status was assessed by CD62L and CCR7 co-expression. In vitro CART function was assessed using Zs-green+ NALM6 targets, with loss of green fluorescence over time measured on the Incucyte analyzer. Where indicated, tumor conditioned media was harvested from 72-hour NALM6 cultures. For in vivo experiments, immunodeficient NOD-scid IL2Rγnull (NSG) mice were injected with luciferase+ NALM6 targets, followed one week later by AMPK- or EV-CARTs. Leukemia burden, via IVIS imaging, and survival were followed over time. Results: CD4+ CARTs co-transduced with AMPKγ2 (AMPK-CARTs) increased oxygen consumption, maximal respiration, and spare respiratory capacity following overnight stimulation by ~20% compared to CARTs co-transduced with the EV (EV-CARTs) (p&amp;lt;0.05). Following expansion, CD62L+CCR7+ central memory cells were 4.5-fold more abundant in the AMPK-CART group (2.4-6.2-fold, p&amp;lt;0.01). In vitro, while AMPK-CARTs demonstrated similar cytotoxicity against NALM6 targets as EV-CARTs in standard media, they mediated faster killing of NALM6 targets when challenged in tumor conditioned media (p&amp;lt;0.001). In vivo, recipients of AMPK-CARTs showed a consistent decrease in tumor burden as measured by median radiance values, translating to a significant survival advantage (p&amp;lt;0.05). Conclusions: Increasing AMPK signaling in CD4+ CARTs via overexpression of AMPKγ2 improved metabolic profile, increased memory generation, and enhanced toxicity against leukemia targets both in vitro and in vivo. Together, these features make elevating AMPK signaling an attractive method to increase the efficacy of CART therapy. Citation Format: Erica Braverman, Herbert Schuler, Mengtao Qin, Craig Byersdorfer. Driving AMPK signaling in CD4+ CART cells enhances metabolic capacity, memory formation, and anti-leukemia activity [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 45.

Abstract 7065: AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer, with emerging evidence highlighting the role of cancer stem cells (CSCs) in its progression. AMBRA1 has been implicated to exert both tumor-promoting and tumor-suppressive roles through regulating cell cycle and mitophagy in various solid tumors. However, the functional roles of AMBRA1 in modulating cancer stemness and tumorigenesis in HCC remain elusive. Initial analysis of The Cancer Genome Atlas data revealed a significant inverse correlation between AMBRA1 expression and stemness indices (TSS) in HCC patients, with low AMBRA1 levels associated with an upregulated oxidative phosphorylation (OXPHOS) gene signature. To elucidate the functional consequences of AMBRA1 modulation in liver tumor development and stemness maintenance, we exploited both hydrodynamic mouse models and human HCC cell lines. Our findings indicate that hepatic AMBRA1 deletion enhances cancer stemness and tumorigenesis both in vivo and in vitro. Moreover, HCC cells with reduced AMBRA1 expression demonstrated heightened mitochondrial activity, characterized by increased oxygen consumption, respiration, mitochondrial membrane potential, and intracellular ATP levels. Apart from OXPHOS, we also observed a concomitant up-regulation and enrichment of MYC targets in TSS-high and AMBRA1-low HCC patients, pointing towards a deregulated c-MYC signaling to promote cancer stemness under AMBRA1 regulation in HCC. Collectively, our results demonstrate that AMBRA1 loss enhances mitochondrial function to support hepatic tumorigenesis in HCC. Citation Format: Yan Liu, Yunong Xie, Yimiao He, Stephanie Ma, Man Tong. AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis in hepatocellular carcinoma [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 7065.

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 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 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.

Abstract 2887: Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain

Abstract This study aims to analyze irradiation-induced metabolic alterations in glioblastoma cells and astrocytes to enhance understanding of cellular responses and facilitate the development of more effective radiotherapeutic strategies. Metabolic reprogramming of tumor cells is considered one of the hallmarks of cancer. Studies have shown that abnormal activation of oncogenes and cancer-related signaling pathways, as well as the inactivation of tumor suppressor genes can induce metabolic reprogramming. These metabolic aberrations facilitate rapid proliferation, continuous growth, invasion, metastasis, and immune evasion. Following the application of radiotherapy, the activity of several metabolic pathways significantly changes, potentially leading to the development of radioresistance. However, a differential effect of ionizing radiation on tumor and normal cells is not well understood. Hence, quantifying irradiation-induced metabolic changes in glioblastoma cells and astrocytes is of high importance. We have developed a novel approach to measure dynamic metabolite changes upon irradiation, in vivo, in situ, and in real-time. This tool combines imaging of recently developed fluorescent biosensors via 2-photon microscopy and millimeter-scaled irradiation. Murine glioblastoma cells and astrocytes were generated with stable expression of biosensors for the quantification of intracellular concentrations of various metabolites, including lactate. Cytosolic concentrations of lactate depend on the balance between glycolytic production, mitochondrial consumption of pyruvate and lactate, and the exchange with the extracellular lactate pool via monocarboxylate transporters. Many cancer cells are characterized by an excessive conversion of glucose to lactate even under normoxic conditions. We quantified this so-called Warburg Effect in glioblastoma cells and astrocytes as the ratio between basal lactate production and the lactate increase after OXPHOS inhibition and determined irradiation-induced metabolic changes on the single-cell level. Complementarily, we used metabolomics and Seahorse analyses before and after irradiation. Our data indicate that irradiation increases oxygen consumption in glioblastoma cells but not in astrocytes. Also, we established an orthotopic glioblastoma mouse model via injection of sensor-expressing tumor cells for in vivo measurements of metabolite changes during and after irradiation. These data are expected to significantly advance the understanding of the cellular response to irradiation on a molecular level in situ and in real-time. This knowledge will help to develop combination treatments and increase the efficiency of radiotherapy. Citation Format: Marvin Kreuzer, Pascal Imseng, Bruno Weber, Martin Pruschy. Impact of ionizing radiation on the energy metabolism of normal and tumor cells in the brain [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 2887.

Monocarboxylate transporter 4 deficiency enhances high-intensity interval training-induced metabolic adaptations in skeletal muscle.

High-intensity exercise stimulates glycolysis, subsequently leading to elevated lactate production within skeletal muscle. While lactate produced within the muscle is predominantly released into the circulation via the monocarboxylate transporter 4 (MCT4), recent research underscores lactate's function as an intercellular and intertissue signalling molecule. However, its specific intracellular roles within muscle cells remains less defined. In this study, our objective was to elucidate the effects of increased intramuscular lactate accumulation on skeletal muscle adaptation to training. To achieve this, we developed MCT4 knockout mice and confirmed that a lack of MCT4 indeed results in pronounced lactate accumulation in skeletal muscle during high-intensity exercise. A key finding was the significant enhancement in endurance exercise capacity at high intensities when MCT4 deficiency was paired with high-intensity interval training (HIIT). Furthermore, metabolic adaptations supportive of this enhanced exercise capacity were evident with the combination of MCT4 deficiency and HIIT. Specifically, we observed a substantial uptick in the activity of glycolytic enzymes, notably hexokinase, glycogen phosphorylase and pyruvate kinase. The mitochondria also exhibited heightened pyruvate oxidation capabilities, as evidenced by an increase in oxygen consumption when pyruvate served as the substrate. This mitochondrial adaptation was further substantiated by elevated pyruvate dehydrogenase activity, increased activity of isocitrate dehydrogenase - the rate-limiting enzyme in the TCA cycle - and enhanced function of cytochrome c oxidase, pivotal to the electron transport chain. Our findings provide new insights into the physiological consequences of lactate accumulation in skeletal muscle during high-intensity exercises, deepening our grasp of the molecular intricacies underpinning exercise adaptation. KEY POINTS: We pioneered a unique line of monocarboxylate transporter 4 (MCT4) knockout mice specifically tailored to the ICR strain, an optimal background for high-intensity exercise studies. A deficiency in MCT4 exacerbates the accumulation of lactate in skeletal muscle during high-intensity exercise. Pairing MCT4 deficiency with high-intensity interval training (HIIT) results in a synergistic boost in high-intensity exercise capacity, observable both at the organismal level (via a treadmill running test) and at the muscle tissue level (through an ex vivo muscle contractile function test). Coordinating MCT4 deficiency with HIIT enhances both the glycolytic enzyme activities and mitochondrial capacity to oxidize pyruvate.

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.

THE INFLUENCE OF BIOCOMPATIBILITY OF THE EXTRACORPOREAL OXYGENATOR CIRCUIT ON THE DEGREE OF HEMOLYSIS AND OPTIMIZATION OF OXYGEN STATUS IN MINIMALLY INVASIVE CORONARY ARTERY BYPASS GRAFTING

Introduction. A distinctive feature of cardiopulmonary bypass in minimally invasive coronary artery bypass grafting (CABG) is the significantly longer perfusion time, involving active blood drainage from the vein to the cardiotomy reservoir, which carries the risk of hemolysis development. In this context, mechanical hemolysis disrupts the gas transport function of the blood.&#x0D; The aim of the research. Reducing hemolysis and optimizing oxygen status of patients during minimally invasive coronary artery bypass grafting surgeries.&#x0D; Materials and methods. The study included 60 patients, who underwent minimally invasive coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) under moderate hypothermia. The patients were divided into two groups of 30 individuals each. To improve the biocompatibility of the oxygenator, the circuit in Group 1 (Gr1) was treated with an adaptive composition, while in Group 2 (Gr2), the oxygenator circuit remained untreated. The following parameters were examined: hemoglobin (Hb), hematocrit (Ht), red blood cell count (RBC), oxygen delivery index (DO2I), oxygen consumption index (VO2I), oxygen extraction ratio (O2ER%), oxygen extraction index (O2EI%), venous (SpvO2), and arterial (SpaO2) oxygen saturation, oxygen tension in arterial (PaO2) and venous blood (PvO2), acid-base status of the blood (pH, pCO2,HCO3ˉ, BE), and the degree of hemolysis.&#x0D; Results. Before initiation of cardiopulmonary bypass (CPB), an insignificant increase in O2IE% was observed in Gr1 (26.07±1.57) and Gr2 (27.11±0.81); p=0.875, indicating an increase in tissue oxygen consumption. At the hypothermic stage CPB, both Gr1 and Gr2 demonstrated a statistically significant decrease in Hb, Ht, and RBC levels (p&lt;0.05) due to hemodilution, accompanied by a decrease in IDO2, IVO2, O2IE%, and O2ER% in both groups due to hypothermia. After rewarming, O2ER% in Gr1 (22.91±2.68) and Gr2 (24.59±2.02); p=0.191, and O2EI% in Gr1 (22.92±2.44) and Gr2 (24.61±2.01); p=0.215, were in the normal range. After weaning off cardiopulmonary bypassin Gr2, O2EI% was 27.34±1.97, indicating an increase in oxygen consumption and oxygen extraction. Upon separation from CPB, Gr2 showed a tendency to mild compensated metabolic acidosis, mainly due to HCO3ˉ 21.9±0.3. During the rewarming stage, the hemolysis value was 0.36±0.09in Gr1 and 0.45±0.17 in Gr2 (p&lt;0.001). After CPB, hemolysis was 0.41±0.15 in Gr1 and 0.61±0.22 in Gr2 (p&lt;0.001).&#x0D; Conclusions. Improving the biocompatibility of the extracorporeal oxygenatorcircuit makes it possible to reduce hemolysis and optimise the patient's oxygen status after cardiopulmonary bypass in minimally invasive coronary artery bypass grafting.

Endothelial specific prolyl hydroxylase domain-containing protein 2 deficiency attenuates aging-related obesity and exercise intolerance

Obesity and exercise intolerance greatly reduce the life quality of older people. Prolyl hydroxylase domain-containing protein 2 (PHD2) is an important enzyme in modulating hypoxia-inducible factor-alpha (HIF) protein. Using vascular endothelial cell-specific PHD2 gene knockout (PHD2 ECKO) mice, we investigated the role of endothelial PHD2 in aging-related obesity and exercise capacity. Briefly, PHD2 ECKO mice were obtained by crossing PHD2-floxed mice with VE-Cadherin (Cdh5)-Cre transgenic mice. The effect of PHD2 ECKO on obesity and exercise capacity in PHD2 ECKO mice and control PHD2f/f mice were determined in young mice (6 to 7 months) and aged mice (16–18 months). We found that aged PHD2 ECKO mice, but not young mice, exhibited a lean phenotype, characterized by lower fat mass, and its ratio to lean weight, body weight, or tibial length, while their food uptake was not reduced compared with controls. Moreover, as compared with aged control mice, aged PHD2 ECKO mice exhibited increased oxygen consumption at rest and during exercise, and the maximum rate of oxygen consumption (VO2 max) during exercise. Furthermore, as compared with corresponding control mice, both young and aged PHD2 ECKO mice demonstrated improved glucose tolerance and lower insulin resistance. Together, these data demonstrate that inhibition of vascular endothelial PHD2 signaling significantly attenuates aging-related obesity, exercise intolerance, and glucose intolerance.

Effect of the Physiological Parameters of Different Age Group Workers on Hoe Weeder

Research was done on the influence of physiological characteristics of different age groups on the hoe weeder at an agriculture working field (10 x 10 m2). Physiological indicators such as heart rate, oxygen consumption rate, and energy expenditure rate of workers of various ages (21–24, 25-28, 29–32, and 33–36 years) were measured while working on a hoe weeder in an agricultural field. Age-related increases in heart rate, oxygen consumption, and energy expenditure were seen among agricultural laborers in a 10 × 10 m2 field. The heart rate, oxygen consumption rate, and energy expenditure rate of those aged 21 to 24 were found to be the lowest while working on a hoe or weeder in an agricultural field. The physiological characteristics of the age group operator of 33–36 years were determined to be the same.

Urolithin A Hijacks ERK1/2-ULK1 Cascade to Improve CD8+ T Cell Fitness for Antitumor Immunity.

According to the latest evidence, the microbial metabolite Urolithin A (UA), known for its role in promoting cellular health, modulates CD8+ T cell-mediated antitumor activity. However, the direct target protein of UA and its underlying mechanism remains unclear. Here, this research identifies ERK1/2 as the specific target crucial for UA-mediated CD8+ T cell activation. Even at low doses, UA markedly enhances the persistence and effector functions of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells both in vitro and in vivo. Mechanistically, UA interacts directly with ERK1/2 kinases, enhancing their activation and subsequently facilitating T cell activation by engaging ULK1. The UA-ERK1/2-ULK1 axis promotes autophagic flux in CD8+ CTLs, enhancing cellular metabolism and maintaining reactive oxygen species (ROS) levels, as evidenced by increased oxygen consumption and extracellular acidification rates. UA-treated CD8+ CTLs also display elevated ATP levels and enhanced spare respiratory capacity. Overall, UA activates ERK1/2, inducing autophagy and metabolic adaptation, showcasing its potential in tumor immunotherapy and interventions for diseases involving ERKs.