Increase In Oxygen Consumption Research Articles

#1762 Reduction in intradialytic glycemia and increased oxygen consumption as potential indicators of intradialytic parenchymal stress

Abstract Background and Aims Patients undergoing chronic hemodialysis (HD) face heightened all-cause and cardiovascular mortality due to various risk factors, with intradialytic stress playing a fundamental role. Recently, the assessment of intradialytic oxygen trends has emerged as a potential parameter for evaluating parenchymal stress. Specifically, an intradialytic increase in the oxygen extraction ratio (OER), derived from the ratio of peripherical oxygen saturation to central venous oxygen saturation, identifies patients at a greater risk of intradialytic events and increased clinical risk. In addition to OER, intradialytic consumption of glucose indicates higher tissue metabolic demand, likely indicative of parenchymal stress. We hypothesize that OER and intradialytic glucose consumption may be phenomena related to increased intradialytic parenchymal stress and, therefore, may be correlated. This study aimed to assess the potential correlation between intradialytic glycemia trends and oxygen extraction (OER). Method Patients in chronic HD treatment with a central venous catheter (CVC) for HD were enrolled from January 2019 to January 2020. The exclusion criteria were: ongoing acute pathology, diabetes mellitus, and undergoing hemodialysis for less than six months. OER and glycemia were measured during three consecutive HD sessions at baseline, 15, 30, 60, and 120 minutes into treatment, and post-HD. For statistical analysis, average values for each patient across the three dialysis sessions were considered. Results Twenty patients with a permanent CVC (10 males, 10 females; age 74 ± 13 years; HD vintage 46 ± 34 months) were enrolled. The pre-HD mean OER in the initial three HD sessions was 34 ± 7, increasing to 46 ± 9 post-HD, with a mean percentage delta OER of 39 ± 20%. Evaluating OER trends in the entire population revealed an increase in OER (∆OER) from the first 15 minutes of treatment (∆OER% 15’: 14 ± 7; ∆OER% 30’: 18 ± 22, p < 0.01). Patients' baseline glycemia was 120 ± 31 mg/dl at the start of the HD session and 111 ± 21 at the end, with a delta of −11 ± 21 mg/dl and a glucose delta percentage of 4%. Patients with higher oxygen consumption after 15 and 30 minutes of treatment experienced a greater decrease in glycemia. Specifically, ∆OER at 15’ negatively correlated with glycemia values at 15’ (r: −0.494, p < 0.05), 30’ (r: −0.521, p < 0.05), 60’ (r: −0.644, p < 0.01, Fig. 1), and 120’ (r: −0.714, p < 0.01), and post-HD (r: −0.634, p < 0.05). Patients were then divided based on the magnitude of mean O2 consumption measured post-HD into two groups: ΔOER ≤ mean and ΔOER > mean (threshold 40%). Patients with a higher ∆OER showed a more significant decrease in glycemia from the first 15 minutes of HD, confirmed at the end of HD (ΔOER ≤ 40%: Δglucose post-HD −2 ± 40; ΔOER > 40%: −22 ± 28, p < 0.05, Fig. 2). Conclusion The data confirm that oxygen extraction during hemodialysis progressively increases from the first 30 minutes of treatment. Changes in OER are associated with a reduction in glycemia, particularly patients with higher ΔOER have a more pronounced decrease in glycemia. This association between OER and glycemia supports the hypothesis that these two parameters are linked to the same pathophysiological phenomenon, such as increased intradialytic cellular metabolic activity. Studies with larger populations will help confirm this association and propose glycemia delta as an indicator of intradialytic parenchymal stress.

COMPARATIVE ANALYSIS OF ERGOSPYROMETRIC VARIABLES BETWEEN SEDENTARY AND NON-SEDENTARY PATIENTS WITH METABOLIC SYNDROME AND ESTABLISHED CARDIOVASCULAR DISEASE

Objective: To analyze the ergospirometric prognostic variables of VO2MAX, OUES, SLOPE VE/VCO2, and ventilatory class, between sedentary and non-sedentary patients with metabolic syndrome all suffering from any kind of established cardiovascular disease. Design and method: From November 2022 to September 2023, 99 patients, with an average age of 70 years, 51.5% women and 48.5% men, underwent an ergospirometry study. They were divided into two groups: sedentary (SP) and non-sedentary (NSP) according to the responses obtained from the IPAQ survey. Subsequently, the ergospirometric variables that measure cardiorespiratory and cardiovascular capacity related to maximum exercise capacity O2 metabolism (VO2MAX), the increase in oxygen consumption according to minute ventilation (OUES) and the response to ventilation according to CO2 production (slope VE/VCO2) were analyzed in both groups. For statistical analysis, the Student's t test and the Fisher exact test were used. The R v4.0.3 software was used. Results: The level of exercise was low in 56 (56.6%) and moderate or high in 43 patients. (43.4%). The NSP group presented higher levels of maximum oxygen consumption (18.3 vs 12.7, p=0.001), higher mean percentage of OUES (92 vs 63.9, p=0.001) and lower mean VE/VCO2 SLOPE ratio (26 vs 31, p=0.001). Among the NSP, 14% were found in ventilatory class 2 and 86% in class 3 and in the SP, 25% were found in class 1, 21.4% in class 2, and 48.2% in class 3. Although that a high percentage of NSP were in functional class 3 (86%) the same presented better values than those SP with better ventilatory class. We did not obtain statistical differences between both groups with respect to their underlying cardiovascular pathologies. Conclusions: A sedentary lifestyle is associated with higher erogospirometric variables risk in patients with metabolic syndrome and established cardiovascular disease. The level of physical activity is a factor that modifies this cardiovascular risk. Ergospirometry is an effective tool to measure cardiovascular prognosis.

Gut microbiome is dispensable for normal respiratory function and respiratory chemoreflex

Increasing evidence indicates that gut microbiota can influence changes in respiratory homeostasis. Alterations to the gut microbiome have been hypothesized to play roles in autoimmune diseases, social behavioral deficits, and metabolic diseases such as diabetes and cardiovascular disease. We therefore aimed to determine if a model of gut dysbiosis, the absence of a gut microbiome, would result in alterations to respiratory homeostasis. After 5 days of habituation inside the Gnotobiotic animal facility, we assayed 8-week-old C57Bl/6J germ-free (GF, n=24) and specific-pathogen-free (SPF, n=24) control mice under normoxic (21% O2,79% N2) conditions, followed by a hypercapnic challenge (5% CO2, 21% O2, 74% N2) and hypoxic challenge (10% O2, 90% N2). Importantly, this germfree model does not involve treatment with high-dose antibiotics, which have been implicated in neurotoxic sequela. Whole-body flow through plethysmography was used to monitor respiratory function and metabolic demand in conscious and unrestrained animals. Recordings were interrogated using our newly published open-source software, Breathe Easy, for breath cycle duration, respiratory rate (Vf), tidal volume (VT), minute ventilation (VE), oxygen consumption (VO2), and minute ventilation normalized by oxygen consumption (VE/VO2) during room air, initial hypoxic and hypercapnic exposures, and steady-state hypoxic and hypercapnic respiratory responses. A linear mixed effects model with a Tukey post-hoc test was performed to test for significant differences wherein respiratory outcomes were dependent variables, and strain and sex were independent variables. In male mice, there was a significant decrease in breath cycle duration during room air in GF compared to SPF controls. We also saw a significant decrease in ventilatory equivalents of oxygen in the initial hypoxic response in GF mice compared to SPF controls in males, but not in the steady-state of hypoxic exposure. Finally, we note a significant increase in oxygen consumption in females during room air measurements. Of note, female mice showed significant irregularity in all respiratory parameters except oxygen consumption whereas males only showed significant irregularity in breath cycle duration during hypoxic steady-state and the hypercapnic steady-state, and tidal volume in the initial hypoxic response. While there are significant changes, it remains to be determined what the underlying mechanisms are and how they may play a contributory role in pathophysiologies co-morbid with gut microbiome dysregulation. Taken together, these results suggest that the gut microbiome is largely dispensable for normal respiratory function and respiratory chemoreflexes in adult mice and that the lack of a gut microbiome, a model of gut dysbiosis, does not result in broad significant changes in respiratory homeostasis. NIH: 1F32HL160073-01A1, R01HL130249 44617-S4. BCM McNair Scholar Program, March of Dimes Basil O'Connor Research Award, Parker B. Francis Fellowship, CJ Foundation for SIDS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The use of micro-computed tomography to investigate splenic control of resting oxygen consumption in Tenrec ecaudatus

The canonical functions of the spleen e.g. immune function, blood filtering, and iron recycling overshadow its known role as a blood storage organ in mammals. Boreoeutherians or modern mammals, such as race horses and dogs, experience splenic contractions during exercise where a 30-40% increase in red blood cell availability is associated with an increase in oxygen consumption. Early data (pre-1960) suggest that splenic sequestration may be used in hibernators. Tenrec ecaudatus are basoendothermic mammals that possess numerous ancestral features such as a cloaca. We posit that tenrecs may provide evolutionary insight into the ancestral function of the spleen. Tenrecs may experience as much as a 25-fold variation in resting oxygen consumption as well as a 14-fold variation in spleen size. We contend that sequestration of red blood cells to the spleen in tenrecs controls oxygen consumption. Here, we present data that directly tests the association of spleen size and oxygen consumption using micro-computed tomography. We contend the boreoeutherian functions of the spleen may be a derived condition afforded by a heightened level of endothermy/homeothermy and its associated higher level of homeostasis. National Science Foundation, Department of Education. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Skeletal Muscle Mitochondrial Response to Diet Differs by Genotype and Sex in an Alzheimer’s Disease Mouse Model

Introduction: Skeletal muscle mass is reduced in the early stages of cognitive decline. Apolipoprotein ε4 ( APOE4) is the most prevalent genetic risk factor for Alzheimer’s disease (AD) and is associated with faster motor decline in cognitively healthy individuals. APOE4 also increases the risk of insulin resistance and cardiovascular disease, conditions associated with altered bioenergetic function in muscle. However, no studies have examined the role of APOE4 in modulating muscle physiology and the impact this would have on cognitive function. We sought to determine if APOE4 affects skeletal muscle mitochondrial metabolism in response to a high-fat diet (HFD) and if this is reflected in whole-body metabolism. We also examined the role of sex since APOE4 carriers who are female are more likely to develop AD. Methods: Male and female APOE3 (n=32) and APOE4 (n= 29) targeted-replacement mice on a C57BL/6 background purchased from Taconic were fed a standard chow diet until 4 months old. Mice were then switched to a low-fat diet (LFD, 10% kcal fat) or HFD (45% kcal fat) until sacrifice. Glucose tolerance was tested at 7 months by administering an intraperitoneal glucose injection of 1g/kg lean mass. Blood glucose was measured before and 15, 30, 45, 60, 90 and 120 minutes after glucose injection. Resting respiratory exchange ratio (RER) was determined using the Promethion Indirect Calorimetry system at 8 months to determine the relative contribution of carbohydrates versus lipids to whole body metabolism. Mice were sacrificed at 9 months and carbohydrate-driven oxygen consumption was measured at basal, state 3 (ADP), state 3 + glutamate, state 3S (succinate) and uncoupled respiratory states in mitochondria isolated from quadriceps tissue on the Oroboros Oxygraph-2k system. Results: Mice fed a HFD had greater glucose area under the curve (p<0.0001) and reduced RER (p= <0.001) compared to mice fed a LFD, regardless of sex or genotype. At the level of skeletal muscle, APOE4 female mice had increased state 3 (p=0.027) and state 3 + glutamate oxygen consumption (p=0.037) on a HFD compared to a LFD while no diet effect was observed in APOE4 males or any APOE3 mice. Conclusion: We provide evidence that female APOE4 mice are susceptible to diet-induced increases in carbohydrate-driven mitochondrial oxygen consumption in muscle. Future studies are needed to determine the underlying mechanism and consequences of increased muscle mitochondrial activity, which could help identify treatment targets in female APOE4 carriers. BHTP T32 AG078114, P30 AG072973 (Chelsea Johnson). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exposure to 6-PPD quinone causes damage on mitochondrial complex I/II associated with lifespan reduction in Caenorhabditis elegans

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone (6-PPDQ) is an emerging pollutant transformed from 6-PPD. However, the effect of 6-PPDQ exposure on mitochondrion and underlying mechanism remains largely unclear. Using Caenorhabditis elegans as animal model, exposed to 6-PPDQ at 0.1–10 μg/L was performed form L1 larvae to adult day-1. Exposure to 6-PPDQ (1 and 10 μg/L) could increase oxygen consumption rate and decease adenosine 5′-triphosphate (ATP) content, suggesting induction of mitochondrial dysfunction. Activities of NADH dehydrogenase (complex I) and succinate dehydrogenase (complex II) were inhibited, accompanied by a decrease in expressions of gas-1, nuo-1, and mev-1. RNAi of gas-1 and mev-1 enhanced mitochondrial dysfunction and reduced lifespan of 6-PPDQ exposed nematodes. GAS-1 and MEV-1 functioned in parallel to regulate 6-PPDQ toxicity to reduce the lifespan. Insulin peptides and the insulin signaling pathway acted downstream of GAS-1 and MEV-1 to control the 6-PPDQ toxicity on longevity. Moreover, RNAi of sod-2 and sod-3, targeted genes of daf-16, caused susceptibility to 6-PPDQ toxicity in reducing lifespan and in causing reactive oxygen species (ROS) production. Therefore, 6-PPDQ at environmentally relevant concentrations (ERCs) potentially caused mitochondrial dysfunction by affecting mitochondrial complexes I and II, which was associated with lifespan reduction by affecting insulin signaling in organisms.

Cardioprotection via mitochondrial transplantation supports fatty acid metabolism in ischemia-reperfusion injured rat heart.

In addition to cellular damage, ischemia-reperfusion (IR) injury induces substantial damage to the mitochondria and endoplasmic reticulum. In this study, we sought to determine whether impaired mitochondrial function owing to IR could be restored by transplanting mitochondria into the heart under ex vivo IR states. Additionally, we aimed to provide preliminary results to inform therapeutic options for ischemic heart disease (IHD). Healthy mitochondria isolated from autologous gluteus maximus muscle were transplanted into the hearts of Sprague-Dawley rats damaged by IR using the Langendorff system, and the heart rate and oxygen consumption capacity of the mitochondria were measured to confirm whether heart function was restored. In addition, relative expression levels were measured to identify the genes related to IR injury. Mitochondrial oxygen consumption capacity was found to be lower in the IR group than in the group that underwent mitochondrial transplantation after IR injury (p < 0.05), and the control group showed a tendency toward increased oxygen consumption capacity compared with the IR group. Among the genes related to fatty acid metabolism, Cpt1b (p < 0.05) and Fads1 (p < 0.01) showed significant expression in the following order: IR group, IR + transplantation group, and control group. These results suggest that mitochondrial transplantation protects the heart from IR damage and may be feasible as a therapeutic option for IHD.

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.