Maximal Mitochondrial Respiration Research Articles

Respiratory and Metabolic Responses of CD4+ T Cells to Acute Exercise and their Association with Cardiorespiratory Fitness.

The study aimed to investigate to what extent acute endurance exercise, especially eccentric exercise and cardiorespiratory fitness affect the metabolic profile of CD4+ cells. 15 male, healthy adults aged between 20 and 33 years with a maximal oxygen uptake (VO2max) between 44 and 63 ml/kg/min performed a downhill run (DR) and a level run (LR) for 45 minutes at 70% of their VO2max on a treadmill in a cross-over design. Blood samples were taken before (T0), directly after (T1), 3 hours after (T3), and 24 hours (T24) after each exercise for analyzing leukocyte numbers and cytokine levels. Isolated CD4+ cells were incubated for 4 hours in autologous resting versus 3 hours after exercise serum (T3 DR and T3 LR), and subsequently, cellular respiration, transcriptomic, and metabolomics profiles were measured. The systemic immune inflammation index increased significantly after DR and LR at T1 and T3 (p < .001). In contrast, the transcriptomic and metabolic profile of CD4+ cells showed no significant alterations after incubation in T3 exercise serum. However, cardiorespiratory fitness positively correlated with the maximal mitochondrial respiration in CD4+ cells after incubation with T3 LR serum (r = .617, p = .033) and with gene expression of oxidative phosphorylation and levels of different metabolites. Similarly, VO2max was associated with an anti-inflammatory profile on RNA level. Lower lactate, methylmalonic acid, and D-gluconic acid levels were found in CD4+ cells of participants with a high VO2max (p < .001). Acute exercise leads to a mild pro-inflammatory milieu with only small changes in the metabolic homeostasis of CD4+ cells. High cardiorespiratory fitness is associated with a metabolic shift to oxidative phosphorylation in CD4+ cells. Functional relevance of this metabolic shift needs to be further investigated.

Association of poly(rC)-binding protein-2 with sideroflexin-3 through TOM20 as an iron entry pathway to mitochondria

Iron is essential for all the lives and mitochondria integrate iron into heme and Fe-S clusters for diverse use as cofactors. Here, we screened mitochondrial proteins in KU812 human chronic myelogenous leukemia cells by glutathione S-transferase pulldown assay with PCBP2 to identify mitochondrial receptors for PCBP2, a major cytosolic Fe(II) chaperone. LC–MS analyses identified TOM20, sideroflexin-3 (SFXN3), SFXN1 and TOM70 in the affinity-score sequence. Stimulated emission depletion microscopy and proteinase-K digestion of mitochondria in HeLa cells revealed that TOM20 is located in the outer membrane of mitochondria whereas SFXN3 is located in the inner membrane. Although direct association was not observed between PCBP2 and SFXN3 with co-immunoprecipitation, proximity ligation assay demonstrated proximal localization of PCBP2 with TOM20 and there was a direct binding between TOM20 and SFXN3. Single knockdown either of PCBP2 and SFXN3 in K562 leukemia cells significantly decreased mitochondrial catalytic Fe(II) and mitochondrial maximal respiration. SFXN3 but not MFRN1 knockout (KO) in mouse embryonic fibroblasts decreased FBXL5 and heme oxygenase-1 (HO-1) but increased transferrin uptake and induced ferritin, indicating that mitochondrial iron entry through SFXN3 is distinct. MFRN1 KO revealed more intense mitochondrial Fe(II) deficiency than SFXN3 KO. Insufficient mitochondrial heme synthesis was evident under iron overload both with SFXN3 and MFRN KO, which was partially reversed by HO-1 inhibitor. Conversely, SFXN3 overexpression caused cytosolic iron deficiency with mitochondrial excess Fe(II), which further sensitized HeLa cells to RSL3-induced ferroptosis. In conclusion, we discovered a novel pathway of iron entry into mitochondria from cytosol through PCBP2-TOM20-SFXN3 axis.

Abstract 4466: Omega-3 fatty acid-induced ferroptosis in 4T1 breast cancer through CD36/FABP5 axis

Abstract Mammalian cells utilize energy from many different sources, and fatty acids are one of these sources. Dietary fats contain various structures of fatty acids, each impacting cell lipid metabolism in unique ways. Omega-3 fatty acids are known to induce breast cancer cell death through ferroptosis. However, the specific molecular mechanisms by which omega-3 fatty acids trigger ferroptosis have yet to be fully elucidated. Fatty acid binding proteins (FABPs) constitute a family of evolutionarily conserved lipid chaperones, facilitating the transport and utilization of fatty acids within cells. Notably, FABP5 is highly expressed in breast cancer, especially in the most aggressive form, triple-negative breast cancer. We hypothesized that FABP5 acts as a lipid sensor, mediating the ferroptosis induced by omega-3 fatty acids in triple-negative breast cancer cells. To test this, we created Fabp5 stable knockout cell lines in 4T1 and MDA-MB-231, two commonly used triple-negative breast cancer cell lines, using CRISPR/Cas9 technology. When treated with docosapentaenoic acid (DPA), these Fabp5-knockout cells exhibited reduced ferroptosis compared to their wild-type counterparts. Interestingly, blocking the surface protein CD36, either with inhibitors or antibodies, also reduced DPA-induced ferroptosis. Furthermore, mutations in Fabp5 decreased maximal mitochondrial respiration in both 4T1 and MDA-MB-231 cells, suggesting a crucial role for FABP5 in mediating oxygen consumption and reactive oxygen species (ROS) production during DPA-induced ferroptosis. Additionally, mice fed a high-fat diet rich in omega-3 fatty acids showed reduced tumor size and lung metastasis. Collectively, our data indicate that FABP5 is a key molecular sensor mediating oxygen consumption and ROS production, thus contributing to ferroptosis induced by omega-3 fatty acids in triple-negative breast cancer. Citation Format: Anthony Avellino, Jiaqing Hao, Matthew Yorek, Jian yu, Bing Li. Omega-3 fatty acid-induced ferroptosis in 4T1 breast cancer through CD36/FABP5 axis [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 4466.

BDE-99 stimulates generation of aberrant brown/beige adipocytes

Adipose tissue compromises one of the principal depots where brominated flame retardants (BFR) accumulate in vivo, yet whether BFR disturb thermogenic brown/beige adipocytes is still not referred to date. Herein, effects of BDE-99, a major congener of polybrominated diphenyl ethers (PBDEs) detected in humans, on brown/beige adipocytes were explored for the first time, aiming to provide new knowledge evaluating the obesogenic and metabolic disrupting effects of BFR. Our results firstly demonstrated that exposure to BDE-99 during the lineage commitment period significantly promoted C3H10T1/2 MSCs differentiating into brown/beige adipocytes, evidenced by the increase of brown/beige adipocyte marker UCP1, Cidea as well as mitochondrial membrane potential and basal respiration rate, which was similar to pharmacological PPARγ agonist rosiglitazone. Unexpectedly, the mitochondrial maximal respiration rate of BDE-99 stimulated brown/beige adipocytes was not synchronously enhanced and resulted in a significant reduction of mitochondrial spare respiration capacity (SRC) compared to control or rosiglitazone stimulated adipocytes, indicating a deficient energy-dissipating capacity of BDE-99 stimulated thermogenic adipocytes. Consistently with compromised mitochondrial SRC, lipidomic analysis further revealed that the lipids profile of mitochondria derived from BDE-99 stimulated brown/beige adipocytes were quite different from control or rosiglitazone stimulated cells. In detail, BDE-99 group contains more free fatty acid (FFA) and lyso-PE in mitochondria. In addition to energy metabolism, our results also demonstrated that BDE-99 stimulated brown/beige adipocytes were deficient in endocrine, which secreted more adverse adipokine named resistin, coinciding with comparable beneficial adipokine adiponectin compared with that of rosiglitazone. Taken together, our results showed for the first time that BDE-99 stimulated brown/beige adipocytes were aberrant in energy metabolism and endocrine, which strongly suggests that BDE-99 accumulated in human adipose tissue could interfere with brown/beige adipocytes to contribute to the occurrence of obesity and relevant metabolic disorders.

Fatty Acids Play a Critical Role in Mitochondrial Oxidative Phosphorylation in Effector T Cells in Graft-versus-Host Disease.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.

Importance of Per2 in Cardiac Mitochondrial Function during Stress

Background and Hypothesis: Ischemic heart disease is the worldwide leading cause of death. Cardiac cellular damage from ischemia is mainly inflicted in the form of mitochondrial dysfunction by inflammatory cytokines and reactive oxidative species (ROS). Mitochondria are critical for metabolic function to maintain cardiac activity. Interventions against inflammatory cytokines and ROS are therefore cardioprotective during ischemic damage. Period Circadian Regulator 2 (Per2) is a circadian rhythm protein involved in metabolic regulation as a time-responsive gene in cardiomyocytes during ischemic damage. Overexpression of Per2 has been shown to decrease infarct size following myocardial infarction. In this study, we hypothesize that Per2 protein plays a regulatory role in the mitochondrial response to inflammatory cytokine TNFα and oxidative stressor H2O2 in human cardiomyocytes. &#x0D; Project Methods: AC16 Human Cardiomyocytes (HCM) transfected with Per2 or control siRNA were subjected to stress treatment of 100ng/mL TNFa or 100μM H2O2. RT-PCR and Western blot were used to detect Per2 expression. After two hours of treatment, mitochondrial membrane potential ([Equation]M) was detected using JC1 fluorescence probe and mitochondrial respiration capacity was evaluated via Seahorse Mito Stress Test. After four hours of treatment, cell death was measured using Annexin V and propidium iodide (PI) apoptosis kit via flow cytometry. &#x0D; Results: Per2 siRNA significantly reduced Per2 mRNA and protein levels in HCM. Increased cell death and decreased [Equation]M were observed in HCM treated with TNFa or H2O2. Knockdown of Per2 potentiated TNFa-induced cell death, TNFa- or H2O2 -disrupted [Equation]M, and TNFa- or H2O2- impaired mitochondrial maximal respiration. &#x0D; Conclusion and Potential Impact: Per2 knockdown increases apoptotic susceptibility and mitochondrial dysfunction in human cardiomyocytes exposed to TNFa or H2O2. Delivery of Per2 may serve as a promising therapeutic strategy to protect cardiomyocyte mitochondrial function during periods of stress, such as myocardial infarction, organ transplantation, and cardiac surgery.

Allopregnanolone pleiotropic action in neurons and astrocytes: calcium signaling as a unifying mechanism.

Allopregnanolone (Allo) is a neurosteroid with pleiotropic action in the brain that includes neurogenesis, oligogenesis, human and rodent neural stem cell regeneration, increased glucose metabolism, mitochondrial respiration and biogenesis, improved cognitive function, and reduction of both inflammation and Alzheimer's disease (AD) pathology. Because the breadth of Allo-induced responses requires activation of multiple systems of biology in the absence of an Allo-specific nuclear receptor, analyses were conducted in both neurons and astrocytes to identify unifying systems and signaling pathways. Mechanisms of Allo action were investigated in embryonic hippocampal neurons and astrocytes cultured in an Aging Model (AM) media. Cellular morphology, mitochondrial function, and transcriptomics were investigated followed by mechanistic pathway analyses. In hippocampal neurons, Allo significantly increased neurite outgrowth and synaptic protein expression, which were paralleled by upregulated synaptogenesis and long-term potentiation gene expression profiles. Mechanistically, Allo induced Ca2+/CREB signaling cascades. In parallel, Allo significantly increased maximal mitochondrial respiration, mitochondrial membrane potential, and Complex IV activity while reducing oxidative stress, which required both the GABAA and L-type Ca2+ channels. In astrocytes, Allo increased ATP generation, mitochondrial function and dynamics while reducing oxidative stress, inflammasome indicators, and apoptotic signaling. Mechanistically, Allo regulation of astrocytic mitochondrial function required both the GABAA and L-type Ca2+ channels. Furthermore, Allo activated NRF1-TFAM signaling and increased the DRP1/OPA1 protein ratio, which led to increased mitochondrial biogenesis and dynamics. Collectively, the cellular, mitochondrial, transcriptional, and pharmacological profiles provide evidence in support of calcium signaling as a unifying mechanism for Allo pleiotropic actions in the brain.

Bile acids promote endothelial cell homeostasis via the regulation of cell metabolism and quiescence

Abstract Background Endothelial cell (EC) homeostasis is characterized by a quiescent and low-metabolic state. Obesity leads to EC dysfunction and CVD. We demonstrated that bariatric surgery improved obesity-induced EC dysfunction and increased fasting and post-prandial plasma levels of bile acids (BAs)(in particular, cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)). BAs are emerging as metabolic signaling molecules in several tissues, but their effect on ECs has not been clearly established yet. Purpose We investigate the effect of BA on the maintenance of EC quiescence. Methods Human Aortic Endothelial Cells (HAECs) were treated for 24h with vehicle (0.1% DMSO), CA, CDCA and DCA (50uM each) in 2%FBS EGM-2 media. Percentage of apoptotic cells was assessed with AnnexinV/PI. Seahorse Mitostress test, Mitoplate and glucose uptake with 0.5µCi/ml of 14C-2-deoxy-D-glucose were used to evaluate changes of HAEC metabolism. Immunostaining for Ki67, Crystal Violet staining and scratch assay were performed to assess HAEC proliferation and migration. NO production and endothelial NO synthase (eNOS) activity were measured via DAF-2 probe and Western Blot, respectively. By qPCR genes involved in Notch pathway were evaluated. Mesoscale vascular injury panel, scratch assay, Ki67 immunostaining and glucose uptake were also performed in HAECs pre-treated with 50uM of BAs and stimulated with 0.5ng/mL TNF-α for 24h. Results HAECs treated with BAs increased NO production compared to vehicle and a reduction in both basal and maximal mitochondrial respiration and extracellular acidification rate was shown by Seahorse Mitostress test. Mitoplate assay displayed an overall reduction in the metabolic rate of HAECs treated with CA and DCA and a peculiar metabolic pattern in cells treated with CDCA. Reduction in glucose uptake was also observed after BA treatment, suggesting that BAs are able to decrease EC metabolism. Immunostaining for Ki67 and Crystal Violet highlighted that BAs reduced cell proliferation. The scratch assay also displayed a reduction in EC migration after BA treatment. BAs also reduced EC apoptosis, as significant differences in the percentage of AnnexinV/PI positive cells was observed in BA-treated cells compared to vehicle. qPCR showed increased expression of genes involved in Notch pathway promoting quiescence after BA treatment. Glucose uptake, scratch assay and Ki67 immunostaining performed in TNF-α stimulated HAECs suggested a BA-dependent endothelial protection in conditions of EC pro-inflammatory activation. We did not observed a reduction in pro-inflammatory biomarkers released in the supernatant of HAECs pre-treated with BA and stimulated with TNF-α. Conclusions Our results suggest that BAs may be involved in the maintenance of EC quiescence by regulating EC metabolism and gene expression profile. These findings highlight a potential role of BAs as endothelial-protective molecules.

The mitochondrial respiration signature of the bovine blastocyst reflects both environmental conditions of development as well as embryo quality

The major limitation of the widespread use of IVP derived embryos is their consistent deficiencies in vitality when compared with their ex vivo derived counterparts. Although embryo metabolism is considered a useful metric of embryo quality, research connecting mitochondrial function with the developmental capacity of embryos is still lacking. Therefore, the aim of the present study was to analyse bovine embryo respiration signatures in relation to developmental capacity. This was achieved by taking advantage of two generally accepted metrics for developmental capacity: (I) environmental conditions during development (vivo vs. vitro) and (II) developmental kinetics (day 7 vs. day 8 blastocysts). Our study showed that the developmental environment affected total embryo oxygen consumption while different morphokinetics illustrating the embryo qualities correlate with maximal mitochondrial respiration, mitochondrial spare capacity, ATP-linked respiration as well as efficiency of ATP generation. This respiration fingerprint for high embryo quality is reflected by relatively lower lipid contents and relatively higher ROS contents. In summary, the results of the present study extend the existing knowledge on the relationship between bovine embryo quality and the signature of mitochondrial respiration by considering contrasting developmental environments as well as different embryo morphokinetics.

Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential

BackgroundHuman mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype.MethodshiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer.ResultsReprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential.ConclusionOverall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations.

HBO treatment enhances motor function and modulates pain development after sciatic nerve injury via protection the mitochondrial function

BackgroundPeripheral nerve injury can cause neuroinflammation and neuromodulation that lead to mitochondrial dysfunction and neuronal apoptosis in the dorsal root ganglion (DRG) and spinal cord, contributing to neuropathic pain and motor dysfunction. Hyperbaric oxygen therapy (HBOT) has been suggested as a potential therapeutic tool for neuropathic pain and nerve injury. However, the specific cellular and molecular mechanism by which HBOT modulates the development of neuropathic pain and motor dysfunction through mitochondrial protection is still unclear.MethodsMechanical and thermal allodynia and motor function were measured in rats following sciatic nerve crush (SNC). The HBO treatment (2.5 ATA) was performed 4 h after SNC and twice daily (12 h intervals) for seven consecutive days. To assess mitochondrial function in the spinal cord (L2–L6), high-resolution respirometry was measured on day 7 using the OROBOROS-O2k. In addition, RT-PCR and Immunohistochemistry were performed at the end of the experiment to assess neuroinflammation, neuromodulation, and apoptosis in the DRG (L3–L6) and spinal cord (L2–L6).ResultsHBOT during the early phase of the SNC alleviates mechanical and thermal hypersensitivity and motor dysfunction. Moreover, HBOT modulates neuroinflammation, neuromodulation, mitochondrial stress, and apoptosis in the DRG and spinal cord. Thus, we found a significant reduction in the presence of macrophages/microglia and MMP-9 expression, as well as the transcription of pro-inflammatory cytokines (TNFa, IL-6, IL-1b) in the DRG and (IL6) in the spinal cord of the SNC group that was treated with HBOT compared to the untreated group. Notable, the overexpression of the TRPV1 channel, which has a high Ca2+ permeability, was reduced along with the apoptosis marker (cleaved-Caspase3) and mitochondrial stress marker (TSPO) in the DRG and spinal cord of the HBOT group. Additionally, HBOT prevents the reduction in mitochondrial respiration, including non-phosphorylation state, ATP-linked respiration, and maximal mitochondrial respiration in the spinal cord after SNC.ConclusionMitochondrial dysfunction in peripheral neuropathic pain was found to be mediated by neuroinflammation and neuromodulation. Strikingly, our findings indicate that HBOT during the critical period of the nerve injury modulates the transition from acute to chronic pain via reducing neuroinflammation and protecting mitochondrial function, consequently preventing neuronal apoptosis in the DRG and spinal cord.

A modelling approach to disentangle the factors limiting muscle oxygenation in smokers

Cigarette smoking is associated with a lower exercise capacity and lower muscle fatigue resistance. This is at least partly attributable to carboxyhaemoglobin (HbCO) in the blood that via reduction in the oxygen-carrying capacity, and the left-shift of the Hb-dissociation curve would reduce tissue oxygenation. On the other hand, a reduced oxygen uptake due to mitochondrial dysfunction would result in improved oxygenation. We used previously collected capillarisation, myoglobin and estimated cellular maximal muscle oxygen consumption data derived from succinate dehydrogenase-stained sections from the vastus lateralis muscle from six smokers and five non-smokers. These data were fed into an expanded Krogh tissue oxygenation model to assess whether an impaired muscle fatigue resistance in smokers is primarily due to HbCO or impaired mitochondrial respiration. The model showed that in smokers with 6% and 20% HbCO (causing a left-shift of the Hb-dissociation curve) average muscle oxygenation was reduced by 1.9% and 7.2%, respectively. Muscle oxygenation was increased by 13.3% when maximal mitochondrial respiration was reduced by 29%. A combination of a 29% reduction in maximal mitochondrial respiration and 20% HbCO led to no significant difference in muscle oxygenation from that in non-smokers. This indicates that while HbCO may explain the reduced exercise capacity after just one smoking session, in chronic smokers impaired mitochondrial respiration appears more important in reducing oxygen extraction and exercise capacity with only a small contribution of the left-shift of the Hb-dissociation curve.

Interplay of Impaired Cellular Bioenergetics and Autophagy in PMM2-CDG.

Congenital disorders of glycosylation (CDG) and mitochondrial disorders are multisystem disorders with overlapping symptomatology. Pathogenic variants in the PMM2 gene lead to abnormal N-linked glycosylation. This disruption in glycosylation can induce endoplasmic reticulum stress, contributing to the disease pathology. Although impaired mitochondrial dysfunction has been reported in some CDG, cellular bioenergetics has never been evaluated in detail in PMM2-CDG. This prompted us to evaluate mitochondrial function and autophagy/mitophagy in vitro in PMM2 patient-derived fibroblast lines of differing genotypes from our natural history study. We found secondary mitochondrial dysfunction in PMM2-CDG. This dysfunction was evidenced by decreased mitochondrial maximal and ATP-linked respiration, as well as decreased complex I function of the mitochondrial electron transport chain. Our study also revealed altered autophagy in PMM2-CDG patient-derived fibroblast lines. This was marked by an increased abundance of the autophagosome marker LC3-II. Additionally, changes in the abundance and glycosylation of proteins in the autophagy and mitophagy pathways further indicated dysregulation of these cellular processes. Interestingly, serum sorbitol levels (a biomarker of disease severity) and the CDG severity score showed an inverse correlation with the abundance of the autophagosome marker LC3-II. This suggests that autophagy may act as a modulator of biochemical and clinical markers of disease severity in PMM2-CDG. Overall, our research sheds light on the complex interplay between glycosylation, mitochondrial function, and autophagy/mitophagy in PMM2-CDG. Manipulating mitochondrial dysfunction and alterations in autophagy/mitophagy pathways could offer therapeutic benefits when combined with existing treatments for PMM2-CDG.

Abstract P3192: Prdm16 Regulates Cardiac Metabolic Genes Through Perm1

The heart is a metabolically demanding organ that requires significant amounts of energy to function properly, and even minor metabolic deficiencies during development can lead to heart failure. Cardiomyopathies are a group of conditions that affect the heart's contractile function, and PRDM16 deficiency is a primary cause of cardiomyopathies associated with 1p36 deletion syndrome in humans. Conditional deletion of Prmd16 in the heart causes a spectrum of cardiac phenotypes including left ventricular non-compaction, late-onset heart failure, and dilated cardiomyopathy. Still, the mechanisms underlying these cardiac phenotypes with loss of Prdm16 are not fully understood. We and others demonstrated that lack of Prdm16 in the heart causes alterations in fatty acid (FA) oxidation and oxidative phosphorylation (OXPHOS) gene expression. Further, we now show that Prdm16 cardiac deletion reduces the expression of key regulators of FA oxidation and OXPHOS genes including Perm1. Loss and gain of Prdm16 function in vitro confirmed a cell-autonomous regulation of Perm1 by Prdm16 in H9c2 rat cardiomyoblasts and neonatal rat ventricular myocytes (NRVMs). Prdm16 ChIP-sequencing analysis of embryonic hearts revealed direct binding of Prdm16 to the promoter of Perm1. Prdm16 binding to Perm1 promoter was further confirmed by ChIP-qPCR in H9c2 cells. Knockdown of Prdm16 in NRVMs reduced maximal mitochondrial respiration, which was rescued by over-expressing Perm1. Further, PERM1 mRNA was diminished in human induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) derived from a patient with a PRDM16 mutation (Q187X). These results are evidence that PRDM16 is an upstream regulator of PERM1 in the heart.

Amyloid-β Pathology-Specific Cytokine Secretion Suppresses Neuronal Mitochondrial Metabolism

IntroductionNeuroinflammation and metabolic dysfunction are early alterations in Alzheimer’s disease (AD) brain that are thought to contribute to disease onset and progression. Glial activation due to protein deposition results in cytokine secretion and shifts in brain metabolism, which have been observed in AD patients. However, the mechanism by which this immunometabolic feedback loop can injure neurons and cause neurodegeneration remains unclear.MethodsWe used Luminex XMAP technology to quantify hippocampal cytokine concentrations in the 5xFAD mouse model of AD at milestone timepoints in disease development. We used partial least squares regression to build cytokine signatures predictive of disease progression, as compared to healthy aging in wild-type littermates. We applied the disease-defining cytokine signature to wild-type primary neuron cultures and measured downstream changes in gene expression using the NanoString nCounter system and mitochondrial function using the Seahorse Extracellular Flux live-cell analyzer.ResultsWe identified a pattern of up-regulated IFNγ, IP-10/CXCL10, and IL-9 as predictive of advanced disease. When healthy neurons were exposed to these cytokines in proportions found in diseased brain, gene expression of mitochondrial electron transport chain complexes, including ATP synthase, was suppressed. In live cells, basal and maximal mitochondrial respiration were impaired following cytokine stimulation.ConclusionsWe identify a pattern of cytokine secretion predictive of progressing amyloid-β pathology in the 5xFAD mouse model of AD that reduces expression of mitochondrial electron transport complexes and impairs mitochondrial respiration in healthy neurons. We establish a mechanistic link between disease-specific immune cues and impaired neuronal metabolism, potentially causing neuronal vulnerability and susceptibility to degeneration in AD.

Novel Iron Chelator SK4 Drives Cytotoxicity through Inhibiting Mitochondrial Metabolism in Ovarian and Triple Negative Breast Cancer Cell Lines.

Anti-cancer therapy by iron chelation has been shown to inhibit many cellular processes including DNA replication, mitochondrial metabolism and oncogenic signalling pathways (e.g., EGFR). Iron chelator SK4 represents a double pronged approach towards treating cancer. SK4 enters through LAT1, a commonly overexpressed amino acid transporter in tumours, thus targeting iron addiction and LAT1 overexpression. The aim of this study was to characterise the mode of action of SK4 through proteomics, metabolomics, lipidomics and seahorse real-time analysis in ovarian cell line SKOV3 and triple negative breast cancer cell line MDA MB 231. Pathway enrichment of proteomics data showed an overrepresentation of metabolism related pathways. Metabolic change after SK4 exposure have been confirmed in investigations of changes in basal and maximal mitochondrial respiration using seahorse real-time analysis of mitochondrial metabolism. Metabolomics also showed an increase in AMP and glucose-1-phosphate. Interestingly, our lipidomics data show a decrease in phospholipid synthesis in the SKOV3 cells which is in contrast with previous data which showed an upregulation of ceramide driven apoptosis. In summary, our data highlight impairment of energy metabolism as a mechanism of action underlying SK4 apoptosis, but also suggest a potential role of ceramide induction in the phenotypic outcome of the cell model.

Prostanoid FP and EP2 Receptor Agonists Induce Epithelial and Subepithelial Fibrogenetic Changes in Human Conjunctival Fibroblasts in Different Manners.

Purpose: To examine the effects of prostanoid FP and EP2 receptor agonists, PGF2α and Omidenepag (OMD), respectively, on the transforming growth factor beta (TGF-β2) induced conjunctival fibrogenesis. Methods: Two-dimension (2D) and three-dimension (3D) cultures of these fibroblasts were subjected to following analyses: (1) planar proliferation evaluated by transendothelial electron resistance (TEER) measurements, (2) real-time metabolic analyses, (3) subepithelial proliferation evaluated by 3D spheroid' size and stiffness measurements, and (4) the mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: TGF-β2 induced increase in the planar proliferation was significantly decreased or enhanced by PGF2α or OMD, respectively. The proportion of oxygen consumption required to drive ATP synthesis compared with that driving proton leakage was increased by PGF2α and OMD independently with TGF-β2. In contrast, maximal mitochondrial respiration was decreased by PGF2α and OMD, and the OMD-induced effect was further enhanced by the presence of TGF-β2. In addition, the TGF-β2 dependent increase in the glycolytic capacity was cancelled by PGF2α and/or OMD. Alternatively, subepithelial proliferation, as evidenced by the stiffness of the 3D spheroids, was substantially increased by both PGF2α and OMD, and these were differently modulated by TGF-β2. The expression of several related factors as above fluctuated among the conditions for both 2D and 3D and TGF-β2 untreated or treated cultures. Conclusion: The present findings indicate that the prostanoid FP or the EP2 receptor agonist may solely and differently induce the planar and subepithelial proliferation of HconF cells and these were also modulated by TGF-β2.

Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.

Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subjected to single-limb casting (3 or 7days) and consumed drinking water with or without 1mM sodium nitrate. Compared with the contralateral control limb, 3days of immobilization lowered myofibrillar fractional synthesis rates (FSR, P< 0.0001), resulting in muscle atrophy. Although FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared with intermyofibrillar (IMF) mitochondria, immobilization for 3days decreased FSR in both SS (P=0.009) and IMF (P=0.031) mitochondria. Additionally, 3days of immobilization reduced maximal mitochondrial respiration, decreased mitochondrial protein content, and increased maximal mitochondrial reactive oxygen species emission, without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS and IMF). Although nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics after both 3 and 7days of immobilization. However, in contrast to 3days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7days of immobilization. Therefore, although nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, although dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse.

Asiatic improves brain mitochondrial function, activates antioxidant response and attenuates cognitive deficits in beta amyloid overexpressing mice

AbstractBackgroundThe plant Centella asiatica can enhance mitochondrial function, promote antioxidant activity and improve cognitive deficits in mouse models of aging and Alzheimer’s disease. Asiatic acid (AA) is one of the constituent triterpene compounds present in the plant. In this study we explore the effects of increasing concentrations of AA on mitochondrial function and antioxidant response in healthy mice and investigate whether high doses of AA can elicit beneficial effects on mitochondrial function, antioxidant response and cognitive function in the 5xFAD mouse model of beta‐amyloid accumulation.MethodsNine month old female CF1 mice were treated with 0, 5mg, 50mg or 1000 mg of AA per 1kg of AIN‐93M diet for four weeks. Toxicity was also monitored. Hippocampal mitochondrial bioenergetics and the expression of mitochondrial and antioxidant response genes was determined. Results were compared to animals treated with 1000mg/kg of a water extract of Centella asiatica (CAW) incorporated into their diet. Additionally, six month old male and female 5xFAD mice were treated with 0 or 1000 mg/kg AA in their diet for four weeks. In the third week mice underwent cognitive testing. Hippocampal mitochondrial bioenergetics and expression of synaptic, mitochondrial and antioxidant response genes was assessed.ResultsIn the healthy mice increasing concentrations of AA resulted in a dose dependent increase in basal and maximal mitochondrial respiration, mitochondrial gene expression and antioxidant gene expression. Results from the 1000mg/kg AA dose were similar to what was observed with CAW. No evidence of toxicity was observed at any concentration of AA. In the 5xFAD mice 1000mg/kg AA attenuated deficits in cognitive and mitochondrial function and induced the expression of antioxidant genes. Interestingly, there was not a consistent effect of AA on mitochondrial or synaptic gene expression.ConclusionThese data show that high concentrations of AA can improve mitochondrial function and antioxidant response in the brains of treated animals. Consistent with the fact that mitochondrial dysfunction and oxidative stress are known to contribute to cognitive impairments this improvement was accompanied by improved cognitive function in the 5xFAD mice. Taken together these results suggest that AA is a promising cognitive enhancing therapy to develop for use in AD.

Two Weeks of Nrf2 Activation Improves Maximal Mitochondrial Respiration in Older Adults with Lower Starting Values

Background: The impact of aging on skeletal muscle mitochondrial function, as assessed by maximal mitochondrial respiration (OXPHOS), is a developing field of research with no clear consensus. Older individuals often display a greater range of OXPHOS, compared to younger individuals, possibly reflecting heterogeneity in the aging process. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a role in the transcription of genes involved in many pathways including the regulation of metabolism, inflammation, oxidative stress, and mitochondrial physiology. Since Nrf2 is downregulated with aging and significantly inactivated with sedentary behaviors, we hypothesized that activating this pathway with the nutraceutical PB125 would improve OXPHOS in older individuals with lower OXPHOS starting values. Methods: OXPHOS (CI&amp;II, State 3) was assessed in permeabilized muscle fibers via high-resolution respirometry (Oroboros, O2k) on samples biopsied from the vastus lateralis in 19 young (26 ± 5 years; 13 female) and 18 older (67 ± 7 years; 11 female) volunteers. Sixteen of the older individuals were then randomized to receive a two-week supplementation of either PB125 (n = 10) or Placebo (n = 6) and OXPHOS was reassessed post-supplementation. Results: Baseline OXPHOS was not different between the young (32 ± 8 pmol/s/mg; Range: 18-44) and old (35 ± 12 pmol/s/mg; Range: 14-60; P = 0.49). The supplement-induced change in OXPHOS was not different between the older groups (PB125: 3 ± 9 vs Placebo: -0 ± 10 pmol/s/mg; P = 0.56) and neither was different from baseline (both, P &gt; 0.05). However, a “threshold-like” relationship between the baseline OXPHOS and the PB125-induced change was identified within the PB125-treated group, whereby older individuals with a relatively lower baseline OXPHOS (&lt;25 pmol/s/mg) had a significant improvement (11 ± 7 pmol/s/mg, n = 4; P = 0.03) compared to baseline, while individuals with a normal baseline OXPHOS had no significant change (-2 ± 2 pmol/s/mg, n = 6; P &gt; 0.05). Conclusion: These findings support previous reports that mitochondrial function, as measured by OXPHOS, is not impaired in older individuals compared to their younger counterparts. However, older individuals with low mitochondrial function exhibited improved OXPHOS following Nrf2 activation via PB125 supplementation. These findings highlight the importance of heterogeneity in aging and provide novel evidence that activating Nrf2 may prove beneficial to improve maximal mitochondrial respiration. NIH: T32HL007576, T32HL139451, R01HL142603; VA: I01CX001999 This is the full abstract presented at the American Physiology Summit 2023 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.