Non-mitochondrial Oxygen Consumption Research Articles

ALS-associated VRK1 R321C mutation causes proteostatic imbalance and mitochondrial defects in iPSC-derived motor neurons

Vaccinia-related kinase 1 (VRK1) is a gene which has been implicated in the pathological process of a broad range of neurodevelopmental disorders as well as neuropathies, such as Amyotrophic Lateral Sclerosis (ALS). Here we report a family presenting ALS in an autosomal recessive mode of inheritance, segregating with a homozygous missense mutation located in VRK1 gene (p.R321C; Arg321Cys). Proteomic analyses from iPSC-derived motor neurons identified 720 proteins eligible for subsequent investigation, and our exploration of protein profiles revealed significant enrichments in pathways such as mTOR signaling, E2F, MYC targets, DNA repair response, cell proliferation and energetic metabolism. Functional studies further validated such alterations, showing that affected motor neurons presented decreased levels of global protein output, ER stress and downregulation of mTOR signaling. Mitochondrial alterations also pointed to decreased reserve capacity and increased non-mitochondrial oxygen consumption. Taken together, our results present the main pathological alterations associated with VRK1 mutation in ALS.

Adipocyte-derived inflammatory molecules induce senescent B cells through metabolic pathways.

The aim of this study was to demonstrate that an adipocyte tissue-derived conditioned medium (ACM) contains inflammatory molecules that induce senescence in B cells. We incubated blood-derived B cells from lean donors with ACM obtained from the adipose tissue of adult female donors with obesity undergoing weight reduction surgery or with medium as control. After 24 h, cells were harvested, and the expression of transcripts for proinflammatory cytokines (TNF/IL-6), chemokines (IL-8), and for markers of the senescence-associated secretory phenotype (SASP) was measured by quantitative polymerase chain reaction. B cells were also stained with the marker of immunosenescence β-galactosidase, and their metabolic status was evaluated in Seahorse using a Mito Stress Test. We show that the incubation of B cells from lean donors with ACM induces the expression of transcripts for inflammatory and SASP transcripts, increases the amount of β-galactosidase staining, and induces a metabolic phenotype characterized by higher basal and maximal oxygen consumption, spare respiratory capacity (difference between maximal and basal respiration), nonmitochondrial oxygen consumption, ATP production, and proton leak. These results demonstrate that B cells from lean individuals, after incubation with ACM, become inflammatory and senescent, and this occurs through metabolic pathways needed to support their secretory phenotype.

Platelet Bioenergetics and Associations With Delirium and Coma in Patients With Sepsis: A Prospective Cohort Study

BackgroundAcute brain dysfunction during sepsis, which manifests as delirium or coma, is common and associated with multiple adverse outcomes including longer periods of mechanical ventilation, prolonged hospital stays, and increased mortality. Delirium and coma during sepsis may be manifestations of alteration in systemic metabolism. Since access to brain mitochondrial is a limiting factor, measurement of peripheral platelet bioenergetics offers a potential opportunity to understand metabolic changes associated with acute brain dysfunction during sepsis. Research QuestionAre altered platelet mitochondrial bioenergetics associated with acute brain dysfunction during sepsis? Study Design and MethodsWe assessed participants with critical illness in the ICU for the presence of delirium or coma via validated assessment measures. Blood samples were collected and processed to isolate and measure platelet mitochondrial oxygen consumption. We used Seahorse extracellular flux to directly measure baseline, proton leak, maximal oxygen consumption rate, and extracellular acidification rate. We calculated ATP-linked, spare respiratory capacity, and non-mitochondrial oxygen consumption rate from the measured values. ResultsMaximal oxygen consumption was highest in patients with coma, as was spare respiratory capacity and ECAR in unadjusted analysis. After adjusting for age, sedation, modified Sequential Organ Failure Assessment (SOFA) without the neurological component, and pre-existing cognitive function, increased spare respiratory capacity remained associated with coma. Delirium was not associated with any platelet mitochondrial bioenergetics. InterpretationIn this single-center, exploratory, prospective cohort study, we found increased platelet mitochondrial spare respiratory capacity was associated with coma in patients with sepsis. Future studies powered to determine any relationship between delirium and mitochondrial respiration bioenergetics are needed.

Abstract 4439: Decoding the metabolic symphony TP53 mutation-driven rewiring of cellular metabolism in non-small cell lung carcinoma

Abstract TP53 is a critical regulator of major metabolic pathways. Metabolic reprogramming is one of the "hallmarks of cancer" and drives tumorigenesis. Frequent p53mutations not only eradicate tumour suppressor capacities but also confer various activities that impact the alteration of metabolic pathways now regarded as central for tumour development and progression. We selected three hotspot mutants (R175H, R273H and R249S) and wtp53 to evaluate the effects in the human non-small cell lung carcinoma cell line (NCI-H1299). Differential expressions of p53 mutants were observed in normal and glucose/glutamine starvation. However, WTp53 expression was not found to be affected. Under both the glucose and glutamine starvation, inhibition of the cell growth and migratory potential of mutant cells was observed which was more prominent in glutamine starvation. Basal respiration and ATP production in p53-/- R273H and R249S significantly increased under glucose starvation. However, Non-mitochondrial oxygen consumption and maximal respiration were found to be decreased in all the cell lines. Importantly, the cells harbouring mutation R175H increase the glycolysis and TCA cycle. The metabolic intermediates of the urea cycle were found to be increased by nearly fourfold in R273H cells. Indicating increased utilization of the urea cycle instead of the TCA cycle, which has been known to be fueled by glutamine for anaplerosis. This study demonstrates that mutp53 influences several metabolic processes and has an influence on cancer cell aggression under starvation of main carbon sources (Glucose and Glutamine). In starved conditions, cells with p53 mutations enhance mitochondrial activity and use alternative pathways in non-small cell lung cancer cells. This might provide a foundation for the development of more effective targeted therapeutics/pharmacological approaches toward variants of mutant p53. Citation Format: Jiyauddin Khan, Asad Ur Rehman, Ankit Mathur, Naveen Kumar Bhatrajuc, Anannya Tulid, Divya Rajput, Mohammad Askandar Iqbal, Daman Saluja. Decoding the metabolic symphony TP53 mutation-driven rewiring of cellular metabolism in non-small cell lung 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 4439.

Exploring mitochondrial metabolism of wild-type and diabetic mice skin explants using the Seahorse technology.

Skin wound healing is a complex mechanism which requires a lot of energy, mainly provided by mitochondrial respiration. However, little is known about the mitochondrial bioenergetics of mice skin. We sought to develop a microplate-based assay to directly measure oxygen consumption in whole mice skin with the goal of identifying mitochondrial dysfunction in diabetic skin using an extracellular flux. Different parameters were optimized to efficiently measure the oxygen consumption rate (OCR). First, the most pertinent skin side of wild-type mice was first determined. Then, concentrations of mitochondrial inhibitors were then optimized to get the best efficacy. Finally, punch sizes were modulated to get the best OCR profile. Dermis had the best metabolic activity side of the skin. Unlike the increased concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone/antimycin A, which showed no improvement of these drugs' effects, varying the skin punch size was successful. Finally, type II diabetic (T2D) skin produced less ATP through mitochondrial metabolism and had a greater non-mitochondrial oxygen consumption than wild-type or type I diabetic (T1D) skin. Here we designed, for the first time, a reliable protocol to measure mitochondria function in whole mouse skin. Our optimized protocol was valuable in assessing alterations associated with diabetes and could be applied to future studies of pathological human skin metabolism.

Alcohol Impairs Bioenergetics and Differentiation Capacity of Myoblasts from Simian Immunodeficiency Virus-Infected Female Macaques.

Alcohol misuse and HIV independently induce myopathy. We previously showed that chronic binge alcohol (CBA) administration, with or without simian immunodeficiency virus (SIV), decreases differentiation capacity of male rhesus macaque myoblasts. We hypothesized that short-term alcohol and CBA/SIV would synergistically decrease differentiation capacity and impair bioenergetic parameters in female macaque myoblasts. Myoblasts from naïve (CBA-/SIV-), vehicle [VEH]/SIV, and CBA/SIV (N = 4-6/group) groups were proliferated (3 days) and differentiated (5 days) with 0 or 50 mM ethanol (short-term). CBA/SIV decreased differentiation and increased non-mitochondrial oxygen consumption rate (OCR) versus naïve and/or VEH/SIV. Short-term alcohol decreased differentiation; increased maximal and non-mitochondrial OCR, mitochondrial reactive oxygen species (ROS) production, and aldolase activity; and decreased glycolytic measures, ATP production, mitochondrial membrane potential (ΔΨm), and pyruvate kinase activity. Mitochondrial ROS production was closely associated with mitochondrial network volume, and differentiation indices were closely associated with key bioenergetic health and function parameters. Results indicate that short-term alcohol and CBA non-synergistically decrease myoblast differentiation capacity. Short-term alcohol impaired myoblast glycolytic function, driving the bioenergetic deficit. Results suggest potentially differing mechanisms underlying decreased differentiation capacity with short-term alcohol and CBA, highlighting the need to elucidate the impact of different alcohol use patterns on myopathy.

Inversion of a paradigm: The positive roles of plant phenolics in dairy goat nutrition

The paradigm prevailing in the nineties, based on studies with goats fed tannin-rich browse as sole feed, was that dietary tannins are associated with impaired feed intake, increased fecal loss of N, and impaired NDF digestibility, hence, impaired productivity, with much research aiming at alleviating these negative effects. However, dairy goats fed plant phenolic-rich diets, and supplemented with concentrates will voluntarily ingest diets comprising more than 4% of tannins, without impairment of total feed intake and milk yield. Also, supplementing them with Poly (ethylene-glycol) MW 4000 is not needed. All plant phenolics, including condensed tannins, are partly digested, inferring that phenolic derivatives are absorbed and may reach tissues. In some studies, diets rich in plant phenolics are associated with the production of milk richer in protein and fat, reduced urea content, and improved curdling performance. Feeding goats with plant phenolics-rich diets is generally associated with inhibited bio-hydrogenation (BH) of fatty acids (FA) in the rumen, resulting in decreased saturated FA, increased polyunsaturated FA (PUFA) and, in particular C18:3–n3 but also a modified composition of de novo synthesized FA in goat milk that suggest direct action of phenolics on the mammary gland. Knowledge of the absorption of plant phenolics is still scarce for goats but even modest daily intakes seem to be associated with increased phenolic derivatives and anti-oxidative activity in plasma and milk. Dietary phenolic compounds may induce a local effect in the mammary gland and its production units (the mammary epithelium cells) encompassing increased cellular triglyceride and casein contents, and increased ATP production and non-mitochondrial oxygen consumption. Plant phenolics and their derivatives divert energy to production of milk fat, protein and lactose, with less energy directed to cellular damage control. Dietary plant phenolics are associated with improved goat health and production of healthier milk. In addition, dietary plant phenolics have anti-parasitic properties and are environment-friendly as they decrease gut methane emission and they increase fecal N at the expense of rapidly decomposable urinary N. Therefore, the attitude of scientists towards feeding plant phenolics has evoluted from mostly negative to mostly positive.

Vitamin D Modulates the Response of Patient-Derived Metastatic Melanoma Cells to Anticancer Drugs.

Melanoma is considered a lethal and treatment-resistant skin cancer with a high risk of recurrence, making it a major clinical challenge. Our earlier studies documented that 1,25(OH)2D3 and its low-calcaemic analogues potentiate the effectiveness of dacarbazine and cediranib, a pan-VEGFR inhibitor. In the current study, a set of patient-derived melanoma cultures was established and characterised as a preclinical model of human melanoma. Thus, patient-derived cells were preconditioned with 1,25(OH)2D3 and treated with cediranib or vemurafenib, a BRAF inhibitor, depending on the BRAF mutation status of the patients enrolled in the study. 1,25(OH)2D3 preconditioning exacerbated the inhibition of patient-derived melanoma cell growth and motility in comparison to monotherapy with cediranib. A significant decrease in mitochondrial respiration parameters, such as non-mitochondrial oxygen consumption, basal respiration and ATP-linked respiration, was observed. It seems that 1,25(OH)2D3 preconditioning enhanced cediranib efficacy via the modulation of mitochondrial bioenergetics. Additionally, 1,25(OH)2D3 also decreased the viability and mobility of the BRAF+ patient-derived cells treated with vemurafenib. Interestingly, regardless of the strict selection, cancer-derived fibroblasts (CAFs) became the major fraction of cultured cells over time, suggesting that melanoma growth is dependent on CAFs. In conclusion, the results of our study strongly emphasise that the active form of vitamin D, 1,25(OH)2D3, might be considered as an adjuvant agent in the treatment of malignant melanoma.

Small extracellular vesicles from senescent stem cells trigger adaptive mechanisms in young stem cells by increasing antioxidant enzyme expression

Extracellular vesicles' biogenesis, shedding, and uptake are redox-sensitive. Indeed, oxidative stress conditions influence extracellular vesicles’ release and content, which can modulate the redox status of the receiving cells. In this study, we aimed to assess the effect of extracellular vesicles from human dental pulp stem cells cultured under 21% O2 (senescent stem cells) on human dental pulp stem cells cultured under 3% O2 (young stem cells). Extracellular vesicles were isolated by ultracentrifugation from senescent stem cells and prepared for the treatment of young stem cells at a final concentration of 10 μg/mL. Cells were analyzed for antioxidant gene expression, mitochondrial bioenergetic parameters, ROS production, culture kinetics, and apoptosis. The results show that extracellular vesicles from senescent stem cells induce overexpression of antioxidant genes (MnSOD, CAT, and GPx) in young stem cells, which show an increased non-mitochondrial oxygen consumption, accompanied by reduced maximal respiration and spare respiratory capacity without altering mitochondrial membrane potential. This is accompanied by improved cell proliferation, viability, and migration rates and a reduction of apoptosis. In conclusion, extracellular vesicles from senescent stem cells trigger an adaptive response in young stem cells which improves their antioxidant defenses and their proliferation, migration, and survival rates. This suggests that extracellular vesicles can modulate the cells' microenvironment and the balance between proliferation and senescence.

Impact of Acute High Glucose on Mitochondrial Function in a Model of Endothelial Cells: Role of PDGF-C

An increase in plasma high glucose promotes endothelial dysfunction mainly through increasing mitochondrial ROS production. High glucose ROS-induced has been implicated in the fragmentation of the mitochondrial network, mainly by an unbalance expression of mitochondrial fusion and fission proteins. Mitochondrial dynamics alterations affect cellular bioenergetics. Here, we assessed the effect of PDGF-C on mitochondrial dynamics and glycolytic and mitochondrial metabolism in a model of endothelial dysfunction induced by high glucose. High glucose induced a fragmented mitochondrial phenotype associated with the reduced expression of OPA1 protein, high DRP1pSer616 levels and reduced basal respiration, maximal respiration, spare respiratory capacity, non-mitochondrial oxygen consumption and ATP production, regarding normal glucose. In these conditions, PDGF-C significantly increased the expression of OPA1 fusion protein, diminished DRP1pSer616 levels and restored the mitochondrial network. On mitochondrial function, PDGF-C increased the non-mitochondrial oxygen consumption diminished by high glucose conditions. These results suggest that PDGF-C modulates the damage induced by HG on the mitochondrial network and morphology of human aortic endothelial cells; additionally, it compensates for the alteration in the energetic phenotype induced by HG.

Characterization of mitochondrial and metabolic alterations induced by trisomy 21 during neural differentiation.

Cellular redox state directs differentiation of induced pluripotent stem cells (iPSC) by energy metabolism control and ROS generation. As oxidative stress and mitochondrial dysfunction have been extensively reported in Down syndrome (DS), we evaluated mitochondrial phenotypes and energy metabolism during neural differentiation of DS iPSCs to neural progenitor cells (NPCs). Our results indicate early maturation of mitochondrial networks and elevated NADPH oxidase 4 (NOX4) expression in DS iPSCs. DS cells also fail to transition from glycolysis to oxidative phosphorylation during differentiation. Specifically, DS NPCs show an increased energetic demand that is limited in their mitochondrial and glycolytic response to mitochondrial distress. Additionally, DS iPSC and NPC non-mitochondrial oxygen consumption was significantly impacted by NOX inhibition. Together, these data build upon previous evidence of accelerated neural differentiation in DS that correlates with cellular redox state. We demonstrate the potential for mitochondrial and non-mitochondrial ROS sources to impact differentiation timing in the context of DS, which could contribute to developmental deficits in this condition.

Extracellular DEK Treatment Increases Mitochondrial Dysfunction in the Mouse AML Cell line MLL-AF9


 
 
 Acute myeloid leukemia (AML) is the most common kind of acute leukemia and the second most common type of leukemia in adults. Poor outcomes resulting from AML are thought to occur due to the inability to target the small pool of cancer-initiating cells that develop from cells within hematopoietic stem (HSC) and progenitor (HPC) cell compartments in bone marrow. DEK, a nuclear protein that can be secreted under stress conditions, plays a role in regulating HSC and HPC function. Extracellular DEK has been found to improve functional HSC expansion in in-vivo and ex-vivo mouse studies. Moreover, RNAseq experiments suggest that recombinant human DEK treatment causes the upregulation of the antioxidant gene programs. Indeed, DEK treatment reduces total reactive oxygen species (ROS) in human umbilical cord blood HSCs and HPCs. Thus, extracellular DEK enhances normal HSC function through antioxidant programs, but the role of the extracellular DEK in AML is unclear. We hypothesized that recombinant mouse (rm)DEK treatment of the mouse-derived AML cell line MLL-AF9 would affect mitochondrial function since mitochondria are an important source of ROS production. Since ROS production can contribute to mitochondrial dysfunction by causing damage to the organelles, we investigated the effects of DEK signaling on mitochondrial metabolism in MLL-AF9 cells using the Seahorse XF instrument, which can measure changes in metabolic flux. Compared to vehicle-treated control, cells treated with DEK demonstrated a decrease in basal and maximal mitochondrial respiration, proton leak, and non-mitochondrial oxygen consumption. Experiments to explore the effects of DEK treatment on the glycolytic function of MLL-AF9 cells are ongoing. Our data shows DEK treatment of MLL-AF9 cells alters mitochondrial function. In the future, we wish to investigate DEK’s effect on proliferation and colony formation.
 
 

Visceral Adipose Tissue Bioenergetics Varies According to Individuals' Obesity Class.

Obesity is associated with complex adipose tissue energy metabolism remodeling. Whether AT metabolic reprogramming differs according to body mass index (BMI) and across different obesity classes is unknown. This study’s purpose was to evaluate and compare bioenergetics and energy substrate preference of visceral adipose tissue (VAT) pertaining to individuals with obesity class 2 and class 3. VAT obtained from patients with obesity (n = 15) class 2 (n = 7; BMI 37.53 ± 0.58 kg/m2) or class 3 (n = 8; BMI 47.79 ± 1.52 kg/m2) was used to assess oxygen consumption rate (OCR) bioenergetics and mitochondrial substrate preferences. VAT of patients with obesity class 3 presented significantly higher non-mitochondrial oxygen consumption (p < 0.05). In VAT of patients with obesity class 2, inhibition of pyruvate and glutamine metabolism significantly decreased maximal respiration and spare respiratory capacity (p < 0.05), while pyruvate and fatty acid metabolism inhibition, which renders glutamine the only available substrate, increased the proton leak with a protective role against oxidative stress (p < 0.05). In conclusion, VAT bioenergetics of patients with obesity class 2 depicts a greater dependence on glucose/pyruvate and glutamine metabolism, suggesting that patients within this BMI range are more likely to be responsive to interventions based on energetic substrate modulation for obesity treatment.

Compound C Inhibits Ovarian Cancer Progression via PI3K-AKT-mTOR-NFκB Pathway.

Simple SummaryOvarian cancer is a deadly cancer due to its late diagnosis. Despite aggressive surgery and chemotherapy recurrence of a resistant aggressive disease is common. Thus, there is an unmet need to develop new therapeutics that target cancer cells and prevent recurrence and resistance. In the present study, we used multiple approaches to report and validate a novel therapeutic compound, compound C, that targets cancer cells and renders them more sensitive to standard of care therapy. Our study also reports novel mechanism of action of compound C and warrants its further development in the treatment of ovarian cancer patients.Epithelial Ovarian cancer (OvCa) is the leading cause of death from gynecologic malignancies in the United States, with most patients diagnosed at late stages. High-grade serous cancer (HGSC) is the most common and lethal subtype. Despite aggressive surgical debulking and chemotherapy, recurrence of chemo-resistant disease occurs in ~80% of patients. Thus, developing therapeutics that not only targets OvCa cell survival, but also target their interactions within their unique peritoneal tumor microenvironment (TME) is warranted. Herein, we report therapeutic efficacy of compound C (also known as dorsomorphin) with a novel mechanism of action in OvCa. We found that CC not only inhibited OvCa growth and invasiveness, but also blunted their reciprocal crosstalk with macrophages, and mesothelial cells. Mechanistic studies indicated that compound C exerts its effects on OvCa cells through inhibition of PI3K-AKT-NFκB pathways, whereas in macrophages and mesothelial cells, CC inhibited cancer-cell-induced canonical NFκB activation. We further validated the specificity of the PI3K-AKT-NFκB as targets of compound C by overexpression of constitutively active subunits as well as computational modeling. In addition, real-time monitoring of OvCa cellular bioenergetics revealed that compound C inhibits ATP production, mitochondrial respiration, and non-mitochondrial oxygen consumption. Importantly, compound C significantly decreased tumor burden of OvCa xenografts in nude mice and increased their sensitivity to cisplatin-treatment. Moreover, compound C re-sensitized patient-derived resistant cells to cisplatin. Together, our findings highlight compound C as a potent multi-faceted therapeutic in OvCa.

Inhibition of Complex I of the Respiratory Chain, but Not Complex III, Attenuates Degranulation and Cytokine Secretion in Human Skin Mast Cells

The mechanisms of mast cell (MC) degranulation and MC-driven skin symptoms are well-described. In contrast, data about the role of mitochondrial respiration for immune functions of human skin MCs are lacking. Oxygen consumption rate (OCR) in primary human skin MCs during IgE-mediated activation in the absence of glucose was examined using a metabolic flux analyzer. Effects of the inhibition of mitochondrial complex I (by rotenone A) and III (by myxothiazol) on degranulation and cytokine secretion (IL-4, IL-5, IL-6, IL-13, TNF-α, and GM-CSF) were explored by the β-hexosaminidase release assay and multiplex ELISA. IgE-mediated activation rapidly increased the mitochondrial OCR and extracellular acidification; the contribution of non-mitochondrial oxygen consumption remained unchanged at lower levels. Both myxothiazol and rotenone A reduced OCR, the mitochondrial parameters, and extracellular acidification; however, myxothiazol did not affect degranulation and cytokine secretion. In contrast, degranulation and the secretion of IL-6, IL-13, TNF-α, and GM-CSF were reduced by rotenone A, whereas the secretion of IL-4 and IL-5 was not significantly affected. The inhibitors did not affect cell viability. Our results highlight the important role played by mitochondrial respiration in primary human skin MCs and allow for a conclusion on a hierarchy of their effector functions. Drugs targeting specific pathways in mitochondria may provide future options to control MC-driven skin symptoms.

MTORC1 and mTORC2 Complexes Regulate the Untargeted Metabolomics and Amino Acid Metabolites Profile through Mitochondrial Bioenergetic Functions in Pancreatic Beta Cells.

Background: Pancreatic beta cells regulate bioenergetics efficiency and secret insulin in response to glucose and nutrient availability. The mechanistic Target of Rapamycin (mTOR) network orchestrates pancreatic progenitor cell growth and metabolism by nucleating two complexes, mTORC1 and mTORC2. Objective: To determine the impact of mTORC1/mTORC2 inhibition on amino acid metabolism in mouse pancreatic beta cells (Beta-TC-6 cells, ATCC-CRL-11506) using high-resolution metabolomics (HRM) and live-mitochondrial functions. Methods: Pancreatic beta TC-6 cells were incubated for 24 h with either: RapaLink-1 (RL); Torin-2 (T); rapamycin (R); metformin (M); a combination of RapaLink-1 and metformin (RLM); Torin-2 and metformin (TM); compared to the control. We applied high-resolution mass spectrometry (HRMS) LC-MS/MS untargeted metabolomics to compare the twenty natural amino acid profiles to the control. In addition, we quantified the bioenergetics dynamics and cellular metabolism by live-cell imaging and the MitoStress Test XF24 (Agilent, Seahorse). The real-time, live-cell approach simultaneously measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine cellular respiration and metabolism. Statistical significance was assessed using ANOVA on Ranks and post-hoc Welch t-Tests. Results: RapaLink-1, Torin-2, and rapamycin decreased L-aspartate levels compared to the control (p = 0.006). Metformin alone did not affect L-aspartate levels. However, L-asparagine levels decreased with all treatment groups compared to the control (p = 0.03). On the contrary, L-glutamate and glycine levels were reduced only by mTORC1/mTORC2 inhibitors RapaLink-1 and Torin-2, but not by rapamycin or metformin. The metabolic activity network model predicted that L-aspartate and AMP interact within the same activity network. Live-cell bioenergetics revealed that ATP production was significantly reduced in RapaLink-1 (122.23 ± 33.19), Torin-2 (72.37 ± 17.33) treated cells, compared to rapamycin (250.45 ± 9.41) and the vehicle control (274.23 ± 38.17), p < 0.01. However, non-mitochondrial oxygen consumption was not statistically different between RapaLink-1 (67.17 ± 3.52), Torin-2 (55.93 ± 8.76), or rapamycin (80.01 ± 4.36, p = 0.006). Conclusions: Dual mTORC1/mTORC2 inhibition by RapaLink-1 and Torin-2 differentially altered the amino acid profile and decreased mitochondrial respiration compared to rapamycin treatment which only blocks the FRB domain on mTOR. Third-generation mTOR inhibitors may alter the mitochondrial dynamics and reveal a bioenergetics profile that could be targeted to reduce mitochondrial stress.

Ozone high dose therapy (OHT) improves mitochondrial bioenergetics in peripheral blood mononuclear cells

BackgroundThe worldwide increasing number of people with chronic diseases is pushing conventional therapy to its limits. The so-called Major AutoHaemo Therapy (MAH) has been used in many practices for years. Despite suspicions, especially the 10-passes ozone-high-dosis Therapy (OHT) has shown substantial benefits in chronic ailments. However, knowledge of scientifically based effects of high ozone concentrations are still rare. The present investigation focussed on verifying whether OHT may be linked to a beneficial effect on mitochondrial bioenergetics which can be expressed as a bioenergetic health index (BHI).MethodsWe report on six patients which received OHT for preventive purposes twice within one week. The BHI in peripheral blood mononuclear cells (PBMC) is calculated from parameters of a cellular mitochondrial function assay, which gives insights into different aspects of mitochondrial function: 1) Basal oxygen consumption rate (OCR); 2) ATP-linked OCR and proton leak; 3) Maximal OCR and reserve capacity; 4) Non-mitochondrial OCR.ResultsThe results clearly show that the bioenergetic health index in PBMC improves significantly after just 2 OHT applications over a period of 1 week. The overall improvement of the BHI is based primarily on a significant increase in the reserve capacity and the maximum respiration of the mitochondria. The increase in non-mitochondrial oxygen consumption, which has a negative impact on the BHI value, is indicative for the Nrf-2 dependent activation of antioxidant and detoxifying enzymes activated through OHT.ConclusionThese data demonstrate for the first time the beneficial effect of OHT on mitochondrial parameters. Thus, the results of this study suggest that OHT could be a safe and effective therapeutic option alone or as integrative and complementary support for pharmacological therapy in a variety of chronic and acute diseases where mitochondrial dysfunction plays a central role.

Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures.

PurposePrimary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O2 concentrations [O2] than what is normally present in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O2] (2.5%; [O2]2.5) and room air ([O2]A).MethodsPrimary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O2]2.5 and [O2]A. Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O2] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates.ResultsHCEnCs grown at [O2]A and [O2]2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O2]2.5 than at [O2]A. HIF-1α was undetectable. Cells displayed greater viability at [O2]2.5 than at [O2]A. HCEnCs showed significantly greater proton leak (P < 0.01), nonmitochondrial oxygen consumption (P < 0.01), and spare capacity (P < 0.05) for oxygen consumption rates, and greater basal glycolysis (P < 0.05) with a decreased glycolytic reserve capacity (P < 0.05) for extracellular acidification rates.ConclusionsPrimary HCEnCs show unique metabolic characteristics at physiologic [O2]. The effect of [O2] for optimization of HCEnC culture conditions should be considered.Translational RelevanceWith the advance of cell-based therapeutics for corneal endothelial diseases, [O2] should be considered an important variable in the optimization of HCEnC culture conditions.

Ketone body oxidation increases cardiac endothelial cell proliferation.

Blood vessel formation is dependent on metabolic adaption in endothelial cells. Glucose and fatty acids are essential substrates for ATP and biomass production; however, the metabolism of other substrates remains poorly understood. Ketone bodies are important nutrients for cardiomyocytes during starvation or consumption of carbohydrate‐restrictive diets. This raises the question whether cardiac endothelial cells would not only transport ketone bodies but also consume some of these to achieve their metabolic needs. Here, we report that cardiac endothelial cells are able to oxidize ketone bodies and that this enhances cell proliferation, migration, and vessel sprouting. Mechanistically, this requires succinyl‐CoA:3‐oxoacid‐CoA transferase, a key enzyme of ketone body oxidation. Targeted metabolite profiling revealed that carbon from ketone bodies got incorporated into tricarboxylic acid cycle intermediates as well as other metabolites fueling biomass production. Elevation of ketone body levels by a high‐fat, low‐carbohydrate ketogenic diet transiently increased endothelial cell proliferation in mouse hearts. Notably, in a mouse model of heart hypertrophy, ketogenic diet prevented blood vessel rarefication. This suggests a potential beneficial role of dietary intervention in heart diseases.

Abstract 13136: Immune Cell Mitochondrial Function and Diastolic Function in Patients With Heart Failure With Preserved Ejection Fraction

Background: Systemic, multimorbidity-driven inflammation may promote diastolic dysfunction in HFpEF. In this regard, anti-inflammatory therapies have been an attractive treatment strategy in clinical trials of HFpEF. Metabolic reprogramming of immune cells that alter the mitochondrial bioenergetic profile may be an upstream mediator of inflammation. Our aim was to investigate the relationship between the immune cell bioenergetic profile and diastolic function in HFpEF. Methods: Nine female patients with a diagnosis of HFpEF (4 Black, 5 White; Median [IQR]: Age 60 [46-67] yrs; BMI 37 [34-42] kg/m 2 ) were enrolled. The mitochondrial bioenergetic profile was assessed in isolated peripheral blood mononuclear cells by respirometry coupled with modulators of cellular respiration to obtain basal respiration, ATP-linked respiration, maximal respiration and non-mitochondrial respiration. Transthoracic echocardiographic Doppler measures of diastolic function were performed and included mitral inflow velocities (E wave) and early diastolic mitral annular velocity (e’). Results: Diastolic function, represented by E/e’ (Median [IQR]: 10 [8-10] was inversely associated with basal respiration ( r =-0.78, p=0.01), maximal respiration ( r =-0.87, p =0.002), ATP linked respiration (54 [40-105] pmol/min, r =-0.75, p =0.02), non-mitochondrial oxygen consumption (24 [20-35] pmol/min, r =-0.82, p =0.007) and spare respiratory capacity (r=-0.82, p=0.007) as shown in Figure 1. Conclusion: There is a strong negative association between the immune cell mitochondrial bioenergetic profile and diastolic function in HFpEF. Patients with greater impairment in diastolic function, as shown by higher E/e’ ratios, have lower immune cell respiration and reduced respiratory reserve. The relationship between immune cell mitochondrial function, inflammation and diastolic function in HFpEF patients warrants further investigation.