Altered Bioenergetics Research Articles

Ultraviolet radiation inhibits mitochondrial bioenergetics activity.

Mitochondria play an important role in cellular function, not only as a major site of adenosine triphosphate (ATP) production but also by regulating energy expenditure, apoptosis signaling, control of the cell cycle, cellular growth, cell differentiation, transportation of metabolites, and production of reactive oxygen species. Interaction with electromagnetic waves can lead to dysregulation or alterations in the patterns of energy activities in the mitochondria. Ultraviolet light (UV) can be found in sunlight and artificial sources, such as lamps. UV radiation can cause damage to DNA, proteins, and lipids. Besides that, UV radiation is largely used in microorganism disinfection. To establish possible alterations in mitochondrial bioenergetics, this study proposes to investigate the UV (at two distinct intervals) effects on isolated mitochondria from mice liver to obtain direct responses and selective permeability of the internal membrane information. UVA-371 and UVC-255 nm lamps were used to irradiate, at different doses varying from 22.5 to 756 mJ/cm2, isolated mitochondria samples. Mitochondrial respiration pathways were investigated by high-resolution respirometry, and possible mitochondrial membrane damages were evaluated by mitochondrial swelling by spectrophotometer analysis. UVC irradiation results (in the higher dose) indicate decrease in 75% of mitochondrial bioenergetics capacity, such as limitation of oxidative phosphorylation in 60% and increased energy dissipation in 30%. Mitochondrial swelling experiments (spectrophotometer) indicated inner membrane damage, and consequently a loss of selective permeability. Direct correlation between irradiation and effect responses was observed, mitochondrial bioenergetics is severely affected by UVC radiation, but (UVA) radiation did not present bioenergetic alterations. These alterations can contribute to improving the knowledge behind the cell death mechanism in disinfection UV light and UV therapy such as phototherapy.

The bacterial quorum sensing signal 2'-aminoacetophenone rewires immune cell bioenergetics through the Ppargc1a/Esrra axis to mediate tolerance to infection.

How bacterial pathogens exploit host metabolism to promote immune tolerance and persist in infected hosts remains elusive. To achieve this, we show that Pseudomonas aeruginosa (PA), a recalcitrant pathogen, utilizes the quorum sensing (QS) signal 2'-aminoacetophenone (2-AA). Here, we unveil how 2-AA-driven immune tolerization causes distinct metabolic perturbations in murine macrophages' mitochondrial respiration and bioenergetics. We present evidence indicating that these effects stem from decreased pyruvate transport into mitochondria. This reduction is attributed to decreased expression of the mitochondrial pyruvate carrier (Mpc1), which is mediated by diminished expression and nuclear presence of its transcriptional regulator, estrogen-related nuclear receptor alpha (Esrra). Consequently, Esrra exhibits weakened binding to the Mpc1 promoter. This outcome arises from the impaired interaction between Esrra and the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a). Ultimately, this cascade results in diminished pyruvate influx into mitochondria and, consequently reduced ATP production in tolerized murine and human macrophages. Exogenously added ATP in infected macrophages restores the transcript levels of Mpc1 and Esrra and enhances cytokine production and intracellular bacterial clearance. Consistent with the in vitro findings, murine infection studies corroborate the 2-AA-mediated long-lasting decrease in ATP and acetyl-CoA and its association with PA persistence, further supporting this QS signaling molecule as the culprit of the host bioenergetic alterations and PA persistence. These findings unveil 2-AA as a modulator of cellular immunometabolism and reveal an unprecedented mechanism of host tolerance to infection involving the Ppargc1a/Esrra axis in its influence on Mpc1/OXPHOS-dependent energy production and PA clearance. These paradigmatic findings pave the way for developing treatments to bolster host resilience to pathogen-induced damage. Given that QS is a common characteristic of prokaryotes, it is likely that 2-AA-like molecules with similar functions may be present in other pathogens.

Sex differences in cardiac mitochondrial respiration and reactive oxygen species production may predispose Scn1a−/+ mice to cardiac arrhythmias and Sudden Unexpected Death in Epilepsy

Dravet Syndrome (DS) is a pediatric-onset epilepsy with an elevated risk of Sudden Unexpected Death in Epilepsy (SUDEP). Most individuals with DS possess mutations in the voltage-gated sodium channel gene Scn1a, expressed in both the brain and heart. Previously, mutations in Scn1a have been linked to arrhythmia. We used a Scn1a−/+ DS mouse model to investigate changes to cardiac mitochondrial function that may underlie arrhythmias and SUDEP. We detected significant alterations in mitochondrial bioenergetics that were sex-specific. Mitochondria from male Scn1a−/+ hearts had deficits in maximal (p = 0.02) and Complex II-linked respiration (p = 0.03). Male Scn1a−/+ mice were also more susceptible to cardiac arrhythmias under increased workload. When isolated cardiomyocytes were subjected to diamide, cardiomyocytes from male Scn1a−/+ hearts were less resistant to thiol oxidation. They had decreased survivability compared to Scn1a+/+ (p = 0.02) despite no whole-heart differences. Lastly, there were no changes in mitochondrial ROS production between DS and wild-type mitochondria at basal conditions, but Scn1a−/+ mitochondria accumulated more ROS during hypoxia/reperfusion. This study determines novel sex-linked differences in mitochondrial and antioxidant function in Scn1a-linked DS. Importantly, we found that male Scn1a−/+ mice are more susceptible to cardiac arrhythmias than female Scn1a−/+ mice. When developing new therapeutics to address SUDEP risk in DS, sex should be considered.

Placental Bioenergetics and Antioxidant Homeostasis in Maternal Obesity and Gestational Diabetes.

Maternal obesity has been associated with short- and long-term risks of pregnancy-perinatal adverse events, possibly due to alterations of placental mitochondrial bioenergetics. However, several detrimental mechanisms occurring in the placentas of women with obesity still need to be clarified. Here, we analyzed placental mitochondrial features and oxidative environment of 46 pregnancies in relation to pre-pregnancy BMI. Seventeen Caucasian normal-weight (NW) and twenty-nine women who were obese (OB) were enrolled. The protein expression of mitochondrial CypD and electron transfer chain complexes (C) I-V were measured, as well as ATP production and oxygen consumption rates (OCRs). The protein levels of the pro/anti-oxidant enzymes TXNIP, SOD2, and PON2 were also analyzed. Despite no differences in CypD expression, OCRs were significantly lower in OB vs. NW women. Accordingly, ATP synthase (CV) levels and ATP content were decreased in OB women, positively correlating with placental efficiency, suggesting a link between ATP deficiency and placental dysfunction. SOD2 expression negatively correlated with maternal BMI, indicating a possible impairment of antioxidant defenses with increasing BMI. These changes were worsened in 10 OB women presenting with gestational diabetes mellitus. Overall, these results suggest alterations of placental bioenergetics in pregnancies of women with obesity, possibly leading to placental dysfunction and altered fetal development and programming.

METABOLIC AND BIOENERGETIC ALTERATIONS ARE ASSOCIATED WITH INFECTION SUSCEPTIBILITY IN SURVIVORS OF SEVERE TRAUMA: AN EXPLORATORY STUDY.

Background : Trauma and blood loss are frequently associated with organ failure, immune dysfunction, and a high risk of secondary bacterial lung infections. We aim to test if plasma metabolomic flux and monocyte bioenergetics are altered in association with trauma and related secondary infections. Methods : Plasma samples were collected from trauma patients at three time points: days 0, 3, and 7 postadmission. Metabolites (140) were measured in plasma from trauma survivors ( n = 24) and healthy control individuals (HC, n = 10). Further analysis within the trauma cohort included subsets of trauma/infection-negative (TIneg, n = 12) and trauma/infection-positive patients (TIpos, n = 12). The bioenergetic profile in monocytes was determined using mitochondrial and glycolytic stress tests. Results : In the trauma cohort, significant alterations were observed in 29 metabolites directly affecting 11 major metabolic pathways, while 34 metabolite alterations affected 8 pathways in 9, versus TIneg patients. The most altered metabolic pathways included protein synthesis, the urea cycle/arginine metabolism, phenylalanine, tyrosine, tryptophan biosynthesis, and carnitine compound family. In monocytes from trauma patients, reduced mitochondrial indices and loss of glycolytic plasticity were consistent with an altered profile of plasma metabolites in the tricarboxylic acid cycle and glycolysis. Conclusions : Our study highlights that the metabolic profile is significantly and persistently affected by trauma and related infections. Among trauma survivors, metabolic alterations in plasma were associated with reduced monocyte bioenergetics. These exploratory findings establish a groundwork for future clinical studies aimed at enhancing our understanding of the interplay between metabolic/bioenergetic alterations associated with trauma and secondary bacterial infections.

MITOCHONDRIAL ANTIVIRAL PATHWAYS CONTROL ANTI-HIV RESPONSES AND ISCHEMIC STROKE OUTCOMES VIA THE RIG-1 SIGNALING AND INNATE IMMUNITY MECHANISMS.

Occludin (ocln) is one of the main regulatory cells of the blood-brain barrier (BBB). Ocln silencing resulted in alterations of the gene expression signatures of a variety of genes of the innate immunity system, including IFN-stimulated genes (ISGs) and the antiviral retinoic acid-inducible gene-1 (RIG-1) signaling pathway, which functions as a regulator of the cytoplasmic sensors upstream of the mitochondrial antiviral signaling protein (MAVS). Indeed, we observed dysfunctional mitochondrial bioenergetics, dynamics, and autophagy in our system. Alterations of mitochondrial bioenergetics and innate immune protection translated into worsened ischemic stroke outcomes in EcoHIV-infected ocln deficient mice. Overall, these results allow for a better understanding of the molecular mechanisms of viral infection in the brain and describe a previously unrecognized role of ocln as a key factor in the control of innate immune responses and mitochondrial dynamics, which affect cerebral vascular diseases such as ischemic stroke.

Distinct metabolic responses to heme in inflammatory human and mouse macrophages – Role of nitric oxide

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

Acyl-CoA synthetase 4 modulates mitochondrial function in breast cancer cells

Mitochondria are dynamic organelles that respond to cellular stress through changes in global mass, interconnection, and subcellular location. As mitochondria play an important role in tumor development and progression, alterations in energy metabolism allow tumor cells to survive and spread even in challenging conditions. Alterations in mitochondrial bioenergetics have been recently proposed as a hallmark of cancer, and positive regulation of lipid metabolism constitutes one of the most common metabolic changes observed in tumor cells. Acyl-CoA synthetase 4 (ACSL4) is an enzyme catalyzing the activation of long chain polyunsaturated fatty acids with a strong substrate preference for arachidonic acid (AA). High ACSL4 expression has been related to aggressive cancer phenotypes, including breast cancer, and its overexpression has been shown to positively regulate the mammalian Target of Rapamycin (mTOR) pathway, involved in the regulation of mitochondrial metabolism genes. However, little is known about the role of ACSL4 in the regulation of mitochondrial function and metabolism in cancer cells. In this context, our objective was to study whether mitochondrial function and metabolism, processes usually altered in tumors, are modulated by ACSL4 in breast cancer cells. Using ACSL4 overexpression in MCF-7 cells, we demonstrate that this enzyme can increase the mRNA and protein levels of essential mitochondrial regulatory proteins such as nuclear respiratory factor 1 (NRF-1), voltage-dependent anion channel 1 (VDAC1) and respiratory chain Complex III. Furthermore, respiratory parameters analysis revealed an increase in oxygen consumption rate (OCR) and in spare respiratory capacity (SRC), among others. ACSL4 knockdown in MDA-MB-231 cells led to the decrease in OCR and in SCR, supporting the role of ACSL4 in the regulation of mitochondrial bioenergetics. Moreover, ACSL4 overexpression induced an increase in glycolytic function, in keeping with an increase in mitochondrial respiratory activity. Finally, there was a decrease in mitochondrial mass detected in cells that overexpressed ACSL4, while the knockdown of ACSL4 expression in MDA-MB-231 cells showed the opposite effect. Altogether, these results unveil the role of ACSL4 in mitochondrial function and metabolism and expand the knowledge of ACSL4 participation in pathological processes such as breast cancer.

Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Primary mitochondrial diseases: The intertwined pathophysiology of bioenergetic dysregulation, oxidative stress and neuroinflammation.

Primary mitochondrial diseases (PMDs) are rare genetic disorders resulting from mutations in genes crucial for effective oxidative phosphorylation (OXPHOS) that can affect mitochondrial function. In this review, we examine the bioenergetic alterations and oxidative stress observed in cellular models of primary mitochondrial diseases (PMDs), shedding light on the intricate complexity between mitochondrial dysfunction and cellular pathology. We explore the diverse cellular models utilized to study PMDs, including patient-derived fibroblasts, induced pluripotent stem cells (iPSCs) and cybrids. Moreover, we also emphasize the connection between oxidative stress and neuroinflammation. The central nervous system (CNS) is particularly vulnerable to mitochondrial dysfunction due to its dependence on aerobic metabolism and the correct functioning of OXPHOS. Similar to other neurodegenerative diseases affecting the CNS, individuals with PMDs exhibit several neuroinflammatory hallmarks alongside neurodegeneration, a pattern also extensively observed in mouse models of mitochondrial diseases. Based on histopathological analysis of postmortem human brain tissue and findings in mouse models of PMDs, we posit that neuroinflammation is not merely a consequence of neurodegeneration but a potential pathogenic mechanism for disease progression that deserves further investigation. This recognition may pave the way for novel therapeutic strategies for this group of devastating diseases that currently lack effective treatments. In summary, this review provides a comprehensive overview of bioenergetic alterations and redox imbalance in cellular models of PMDs while underscoring the significance of neuroinflammation as a potential driver in disease progression.

Platelet bioenergetic profiling uncovers a metabolic pattern of high dependency on mitochondrial fatty acid oxidation in type 2 diabetic patients who developed in-stent restenosis

Although platelet bioenergetic dysfunction is evident early in the pathogenesis of diabetic macrovascular complications, the bioenergetic characteristics in type 2 diabetic patients who developed coronary in-stent restenosis (ISR) and their effects on platelet function remain unclear. Here, we performed platelet bioenergetic profiling to characterize the bioenergetic alterations in 28 type 2 diabetic patients with ISR compared with 28 type 2 diabetic patients without ISR (non-ISR) and 28 healthy individuals. Generally, platelets from type 2 diabetic patients with ISR exhibited a specific bioenergetic alteration characterized by high dependency on fatty acid (FA) oxidation, which subsequently induced complex III deficiency, causing decreased mitochondrial respiration, increased mitochondrial oxidant production, and low efficiency of mitochondrial ATP generation. This pattern of bioenergetic dysfunction showed close relationships with both α-granule and dense granule secretion as measured by surface P-selectin expression, ATP release, and profiles of granule cargo proteins in platelet releasates. Importantly, ex vivo reproduction of high dependency on FA oxidation by exposing non-ISR platelets to its agonist mimicked the bioenergetic dysfunction observed in ISR platelets and enhanced platelet secretion, whereas pharmaceutical inhibition of FA oxidation normalized the respiratory and redox states of ISR platelets and diminished platelet secretion. Further, causal mediation analyses identified a strong association between high dependency on FA oxidation and increased angiographical severity of ISR, which was significantly mediated by the status of platelet secretion. Our findings, for the first time, uncover a pattern of bioenergetic dysfunction in ISR and enhance current understanding of the mechanistic link of high dependency on FA oxidation to platelet abnormalities in the context of diabetes.

Abstract 2037: Enhanced chemosensitivity to DNA-damaging agents in human high-grade glioma through 2-DG treatment

Abstract Background: Human high-grade gliomas are aggressive brain cancers known for their resistance to treatment. Targeted therapies with single agents have shown limited success, and the presence of tumor heterogeneity complicates treatment. Combining multiple chemotherapeutic agents has emerged as a potential strategy to overcome these challenges. 2-deoxy-D-glucose (2-DG) is a glucose analog known for its ability to disrupt glycolysis, protein folding, and the pentose phosphate pathway. This disruption renders cancer cells more susceptible to further damage from reactive oxygen species and DNA damage. Previous studies have demonstrated synergistic effects when combining 2-DG with chemotherapeutic drugs in various cancers. Our study examines combining 2-DG with DNA-damaging agents for high-grade gliomas to uncover potential synergies, theorizing that this combination enhances cell death and metabolic stress, offering insights for better treatments against these tumors. Methods: The cell proliferation, clonogenic growth, and cell migration assays assessed viability, clonogenic formation, and migration. Additionally, the cell cycle and reactive oxygen species analysis were employed to evaluate cell division, oxidative stress, and antioxidant capacity. The Illumina NovaSeq 6000 platform measured transcript levels, and Liquid Chromatography-High-Resolution Mass Spectrometry analyzed metabolite changes in high-grade glioma cell lines following 2-DG treatment. The Agilent Seahorse Mito Stress Test evaluated alterations in cellular bioenergetics. SYNERGYFINDER 3 quantified synergistic effects of 2-DG with DNA-damaging agents, while the Annexin V-FITC Conjugates and a ROS multiplex assay assessed cell death and metabolic stress. Results: Our findings demonstrate that when used as a single agent, 2-DG induces cell senescence and limits cell migration but is insufficient to trigger cell death. Moreover, 2-DG treatment shifts glioma cells towards oxidative phosphorylation for energy production, elevates ROS levels, and hampers DNA synthesis and repair mechanisms. Our synergy study reveals promising synergistic effects when combining 2-DG with DNA-damaging agents, including 5-FU, Methotrexate, Gemcitabine, and Lomustine. This combined therapy leads to significant ROS generation and increased cell death compared to single-agent treatments. Furthermore, prolonged exposure to 2-DG leads to increased levels of both glucogenic and ketogenic amino acids within glioma cells, potentially serving as an alternative energy source. This finding hints at a prospective therapeutic approach for targeting drug-resistant cells. Conclusions: Our results suggest that the anticancer therapeutic effect of 2-DG is enhanced when used in combination with DNA-damaging agents, indicating this strategy as a potential approach for treating human high-grade glioma. Citation Format: Tracy Miller, Esther Jane, Matthew Halbert, Taylor Gatesman, Stacey Wendell, Dinesh Mohanakrishnan, Sameer Agnihotri, Daniel Premkumar, Ian Pollack. Enhanced chemosensitivity to DNA-damaging agents in human high-grade glioma through 2-DG treatment [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 2037.

Abstract 3071: Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors

Abstract Advances in research have shown that cancer cells have distinctive bioenergetic alterations to meet their energy requirements. It has been established that certain cancers are significantly reliant on mitochondrial respiration, suggesting that modulation of mitochondrial function may have therapeutic value. The Wnt signaling pathway is a potential therapeutic target of cancer, including breast and colorectal cancers. Recent findings suggest that bi-directional crosstalk between Wnt signaling and mitochondrial function plays a crucial role in tumorigenesis, as well as differentiation and self-renewal of stem cells. In this study, 71 agents known to alter Wnt signaling were screened for effects on mitochondrial function in BT474 and Hs578T breast cancer cell lines, which have highly oxidative and highly glycolytic bioenergetic phenotypes, respectively. This screen was performed using the Agilent Seahorse XF Mito Tox assay, which enables sensitive detection and characterization of mitochondrial modulators via a standardized, quantitative parameter, the Mito Tox Index (MTI). The MTI facilitates the comparison of drug efficacy by detecting modes of mitochondrial toxicity, inhibition, and uncoupling. The MTI-based screening of the Wnt modulators identified one inhibitor and two uncouplers as potent agents inducing mitochondrial dysfunction for both BT474 and Hs578T. Despite their distinctive bioenergetic phenotypes, MTI-based dose-response assays revealed no significant differences in the efficacy of these modulators between BT474 and Hs578T cell types. In contrast, significant differential effects on ATP production rates were detected, showing cellular energy depletion induced by these agents. Using the Agilent Seahorse XF Real-Time ATP Rate assay, BT474 cells showed significant metabolic energy crisis in the presence of all 3 compounds, whereas Hs578T cells maintained stable total ATP production. This differential impact on ATP production rate corresponds directly to differences in cell viability, with BT474 showing higher susceptibility to these compounds. These results suggest that the anti-cancer efficacy of mitochondrial modulators can be different depending on the metabolic phenotype of the target cell and should be considered in therapeutic design. Citation Format: Yoonseok Kam, Lisa Winer, George W. Rogers, Natalia Romero. Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors [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 3071.

Role of macrophage bioenergetics in N-acetylcysteine-mediated mitigation of lung injury and oxidative stress induced by nitrogen mustard

Nitrogen mustard (NM) is a toxic vesicant that causes acute injury to the respiratory tract. This is accompanied by an accumulation of activated macrophages in the lung and oxidative stress which have been implicated in tissue injury. In these studies, we analyzed the effects of N-acetylcysteine (NAC), an inhibitor of oxidative stress and inflammation on NM-induced lung injury, macrophage activation and bioenergetics. Treatment of rats with NAC (150 mg/kg, i.p., daily) beginning 30 min after administration of NM (0.125 mg/kg, i.t.) reduced histopathologic alterations in the lung including alveolar interstitial thickening, blood vessel hemorrhage, fibrin deposition, alveolar inflammation, and bronchiolization of alveolar walls within 3 d of exposure; damage to the alveolar-epithelial barrier, measured by bronchoalveolar lavage fluid protein and cells, was also reduced by NAC, along with oxidative stress as measured by heme oxygenase (HO)-1 and Ym-1 expression in the lung. Treatment of rats with NAC attenuated the accumulation of macrophages in the lung expressing proinflammatory genes including Ptgs2, Nos2, Il-6 and Il-12; macrophages expressing inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)α protein were also reduced in histologic sections. Conversely, NAC had no effect on macrophages expressing the anti-inflammatory proteins arginase-1 or mannose receptor, or on NM-induced increases in matrix metalloproteinase (MMP)-9 or proliferating cell nuclear antigen (PCNA), markers of tissue repair. Following NM exposure, lung macrophage basal and maximal glycolytic activity increased, while basal respiration decreased indicating greater reliance on glycolysis to generate ATP. NAC increased both glycolysis and oxidative phosphorylation. Additionally, in macrophages from both control and NM treated animals, NAC treatment resulted in increased S-nitrosylation of ATP synthase, protecting the enzyme from oxidative damage. Taken together, these data suggest that alterations in NM-induced macrophage activation and bioenergetics contribute to the efficacy of NAC in mitigating lung injury.

Mitochondrial dysfunction affects hepatic immune and metabolic remodeling in patients with hepatitis B virus-related acute-on-chronic liver failure.

Immune dysregulation and metabolic derangement have been recognized as key factors that contribute to the progression of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF). However, the mechanisms underlying immune and metabolic derangement in patients with advanced HBV-ACLF are unclear. To identify the bioenergetic alterations in the liver of patients with HBV-ACLF causing hepatic immune dysregulation and metabolic disorders. Liver samples were collected from 16 healthy donors (HDs) and 17 advanced HBV-ACLF patients who were eligible for liver transplantation. The mitochondrial ultrastructure, metabolic characteristics, and immune microenvironment of the liver were assessed. More focus was given to organic acid metabolism as well as the function and subpopulations of macrophages in patients with HBV-ACLF. Compared with HDs, there was extensive hepatocyte necrosis, immune cell infiltration, and ductular reaction in patients with ACLF. In patients, the liver suffered severe hypoxia, as evidenced by increased expression of hypoxia-inducible factor-1α. Swollen mitochondria and cristae were observed in the liver of patients. The number, length, width, and area of mitochondria were adaptively increased in hepatocytes. Targeted metabolomics analysis revealed that mitochondrial oxidative phosphorylation decreased, while anaerobic glycolysis was enhanced in patients with HBV-ACLF. These findings suggested that, to a greater extent, hepa-tocytes used the extra-mitochondrial glycolytic pathway as an energy source. Patients with HBV-ACLF had elevated levels of chemokine C-C motif ligand 2 in the liver homogenate, which stimulates peripheral monocyte infiltration into the liver. Characterization and functional analysis of macrophage subsets revealed that patients with ACLF had a high abundance of CD68+ HLA-DR+ macrophages and elevated levels of both interleukin-1β and transforming growth factor-β1 in their livers. The abundance of CD206+ CD163+ macrophages and expression of interleukin-10 decreased. The correlation analysis revealed that hepatic organic acid metabolites were closely associated with macrophage-derived cytokines/chemokines. The results indicated that bioenergetic alteration driven by hypoxia and mitochondrial dysfunction affects hepatic immune and metabolic remodeling, leading to advanced HBV-ACLF. These findings highlight a new therapeutic target for improving the treatment of HBV-ACLF.

Saturated fatty acids differently affect mitochondrial function and the intestinal epithelial barrier depending on their chain length in the in vitro model of IPEC-J2 enterocytes.

Introduction: Maintenance of the intestinal barrier mainly relies on the mitochondrial function of intestinal epithelial cells that provide ATP through oxidative phosphorylation (OXPHOS). Dietary fatty acid overload might induce mitochondrial dysfunction of enterocytes and may increase intestinal permeability as indicated by previous in vitro studies with palmitic acid (C16:0). Yet the impact of other dietary saturated fatty acids remains poorly described. Methods: To address this question, the in vitro model of porcine enterocytes IPEC-J2 was treated for 3days with 250µM of lauric (C12:0), myristic (C14:0), palmitic (C16:0) or stearic (C18:0) acids. Results and discussion: Measurement of the transepithelial electrical resistance, reflecting tight junction integrity, revealed that only C16:0 and C18:0 increased epithelial permeability, without modifying the expression of genes encoding tight junction proteins. Bioenergetic measurements indicated that C16:0 and C18:0 were barely β-oxidized by IPEC-J2. However, they rather induced significant OXPHOS uncoupling and reduced ATP production compared to C12:0 and C14:0. These bioenergetic alterations were associated with elevated mitochondrial reactive oxygen species production and mitochondrial fission. Although C12:0 and C14:0 treatment induced significant lipid storage and enhanced fusion of the mitochondrial network, it only mildly decreased ATP production without altering epithelial barrier. These results point out that the longer chain fatty acids C16:0 and C18:0 increased intestinal permeability, contrary to C12:0 and C14:0. In addition, C16:0 and C18:0 induced an important energy deprivation, notably via increased proton leaks, mitochondrial remodeling, and elevated ROS production in enterocytes compared to C12:0 and C14:0.

Fatigue as hallmark of Fabry disease: role of bioenergetic alterations.

Fabry disease (FD) is a lysosomal storage disorder due to the impaired activity of the α-galactosidase A (GLA) enzyme which induces Gb3 deposition and multiorgan dysfunction. Exercise intolerance and fatigue are frequent and early findings in FD patients, representing a self-standing clinical phenotype with a significant impact on the patient's quality of life. Several determinants can trigger fatigability in Fabry patients, including psychological factors, cardiopulmonary dysfunctions, and primary alterations of skeletal muscle. The "metabolic hypothesis" to explain skeletal muscle symptoms and fatigability in Fabry patients is growing acknowledged. In this report, we will focus on the primary alterations of the motor system emphasizing the role of skeletal muscle metabolic disarrangement in determining the altered exercise tolerance in Fabry patients. We will discuss the most recent findings about the metabolic profile associated with Fabry disease offering new insights for diagnosis, management, and therapy.

Targeting Key Inflammatory Mechanisms Underlying Heart Failure: A Comprehensive Review.

Inflammation is a major component of heart failure (HF), causing peripheral vasculopathy and cardiac remodeling. High levels of circulating inflammatory cytokines in HF patients have been well recognized. The hallmark of the inflammatory imbalance is the insufficient production of anti-inflammatory mediators, a condition that leads to dysregulated cytokine activity. The condition progresses because of the pathogenic consequences of the cytokine imbalance, including the impact of endothelial dysfunction and adrenergic responsiveness deterioration, and unfavorable inotropic effects on the myocardium. Hence, to develop possible anti-inflammatory treatment options that will enhance the outcomes of HF patients, it is essential to identify the potential pathophysiological mechanisms of inflammation in HF. Inflammatory mediators, such as cytokines, adhesion molecules, and acute-phase proteins, are elevated during this process, highlighting the complex association between inflammation and HF. Therefore, these inflammatory markers can be used in predicting prognosis of the syndrome. Various immune cells impact on myocardial remodeling and recovery. They lead to stimulation, release of alarmins and risk-related molecule patterns. Targeting key inflammatory mechanisms seems a quite promising therapy strategy in HF. Cytokine modulation is only one of several possible targets in the fight against inflammation, as the potential molecular targets for therapy in HF include immune activation, inflammation, oxidative stress, alterations in mitochondrial bioenergetics, and autophagy.

Fibrillar tau induces blood brain barrier permeability, endothelial cell activation and bioenergetic alterations

AbstractBackgroundAlzheimer’s Disease, the most common form of dementia, is characterized by the accumulation of amyloid‐beta plaques and neurofibrillary tangles formed by hyperphosphorylated tau in the brain, leading to neuronal loss and cognitive decline. The neurovascular unit, a complex system of several cell types surrounding brain vessels, is extremely important for blood flow regulation, amyloid and tau clearance, and proper brain function. Tau aggregates produced by neurons and glial cells reach endothelial cells (EC) at vessel walls by tau spreading. Currently, the mechanisms responsible for the effects of tau on ECs remain to be understood. We hypothesize that aggregated tau fibrils mediate alterations in bioenergetics in these cells, associated to pro‐inflammatory EC activation, BBB permeability and eventually EC death.MethodImmortalized human brain microvascular endothelial cells (D3) and primary human cerebral endothelial cells (hCECs) were challenged with aggregated 1N4R tau fibrils (5nM‐25nM) for 3‐72h. Trans‐endothelial electrical resistance (TEER) was measured by the ECIS Zθ system as an in vitro model of the BBB. Cytokine production and release was assessed using a V‐Plex Neuroinflammatory Panel 1 (MSD). We performed immunocytochemical analysis of junction protein expression and western blotting for EC activation markers. EC bioenergetics were measured by Seahorse Extracellular Flux Analyzer and EC death was determined by Cell Death Elisa and LDH Release (Roche).ResultTEER was reduced following incubation with fibrillar 1N4R tau. VCAM‐1 expression significantly increased with 25nM tau (24h) and 5nM (48h), and ICAM‐1 followed similar trends. Junction protein (ZO‐1, VE‐Cadherin) expression was decreased. Aggregated tau (25nM) caused an increase in glycolysis at 24‐hours, which reverted at further timepoints, whereas mitochondrial ATP production increased (48h) and then decreased (72h). After 72h treatment, 25nM tau induced a significant increase in apoptotic cell death and LDH release, yet no significant changes in cell death were observed at previous timepoints.ConclusionOur results suggest that tau fibrils mediate a stress response with a metabolic shift in ECs, in association with pro‐inflammatory EC activation. These molecular changes can contribute to BBB permeability and eventually cell death, further exacerbating cerebrovascular dysfunction.

Dissociations between APP pathology and metabolic signalling in the neuron‐specific hBACE1 knock‐in PLB4 mouse

AbstractBackgroundPatients with Alzheimer’s Disease (AD) frequently suffer comorbidities such as type 2 diabetes mellitus (T2DM) alongside shared common pathologies e.g. increased inflammation, endoplasmic reticulum (ER) stress, and impaired energy homeostasis (Plucinska et al., 2016, https://doi.org/10.1007/s00125-016-3960-1). The complexity of neurodegenerative and metabolic diseases requires a systems‐based approach that examines the interplay between brain pathology and metabolic regulation. Emerging evidence points towards abnormal cellular energy regulation as a common denominator, investigated here in hBACE1 associated pathologies (Findlay et al., 2015, https://doi.org/10.3389/fncel.2015.00382).MethodGlucose tolerance tests were performed in male PLB4 and PLB‐WT controls (3 – 8 months) to determine diabetic status (Plucinska et al., 2014, https://doi.org/10.1523/JNEUROSCI.0433-14.2014). Protein analysis was employed to characterise beta‐amyloid/APP processing, ER stress, glucose regulatory signalling, and bioenergetic metabolites in brain, liver, muscle or plasma, and gene expression analysis examined hypothalamic alterations in energy regulation.ResultNeuronal hBACE1 expression caused early pathological cleavage of APP, in parallel with increased brain ER stress (p‐eIF2α) and the chaperone binding immunoglobulin protein (BIP). APP processing declined over time, alongside increased ER stress (IRE1α) in brain and liver. Systemic glucose intolerance was evident from 3 months, varied between tissues and ages, and was confined to the periphery. All key markers normalised to control levels at 8 months. Evidence for altered bioenergetics metabolites (LDH‐A and BCKDH) in brain and liver, also affected by ageing, were detected in PLB4 mice. Furthermore, hypothalamus‐specific changes in energy and appetite regulators (NPY and GLP‐1 receptor) were uncovered in the hBACE1 mice.ConclusionhBACE1 resulted in early APP misprocessing alongside ER stress but not post‐insulin receptor signalling changes. Peripheral metabolic and bioenergetic alterations were age‐ and tissue‐specific but did not correlate with APP processing. Further tissue analyses are ongoing to determine cellular consequences of hBACE1 expression on systemic bioenergetics.