ATP-linked Oxygen Consumption Rate Research Articles

Comprehensive characterization of mitochondrial bioenergetics at different larval stages reveals novel insights about the developmental metabolism of Caenorhabditis elegans.

Mitochondrial bioenergetic processes are fundamental to development, stress responses, and health. Caenorhabditis elegans is widely used to study developmental biology, mitochondrial disease, and mitochondrial toxicity. Oxidative phosphorylation generally increases during development in many species, and genetic and environmental factors may alter this normal trajectory. Altered mitochondrial function during development can lead to both drastic, short-term responses including arrested development and death, and subtle consequences that may persist throughout life and into subsequent generations. Understanding normal and altered developmental mitochondrial biology in C. elegans is currently constrained by incomplete and conflicting reports on how mitochondrial bioenergetic parameters change during development in this species. We used a Seahorse XFe24 Extracellular Flux (XF) Analyzer to carry out a comprehensive analysis of mitochondrial and non-mitochondrial oxygen consumption rates (OCR) throughout larval development in C. elegans. We optimized and describe conditions for analysis of basal OCR, basal mitochondrial OCR, ATP-linked OCR, spare and maximal respiratory capacity, proton leak, and non-mitochondrial OCR. A key consideration is normalization, and we present and discuss results as normalized per individual worm, protein content, worm volume, mitochondrial DNA (mtDNA) count, nuclear DNA (ncDNA) count, and mtDNA:ncDNA ratio. Which normalization process is best depends on the question being asked, and differences in normalization explain some of the discrepancies in previously reported developmental changes in OCR in C. elegans. Broadly, when normalized to worm number, our results agree with previous reports in showing dramatic increases in OCR throughout development. However, when normalized to total protein, worm volume, or ncDNA or mtDNA count, after a significant 2-3-fold increase from L1 to L2 stages, we found small or no changes in most OCR parameters from the L2 to the L4 stage, other than a marginal increase at L3 in spare and maximal respiratory capacity. Overall, our results indicate an earlier cellular shift to oxidative metabolism than suggested in most previous literature.

HSP47 complements cholesterol synthesis-independent HMGCoA-reductase pathway-mediated fibroblast bioenergetics

Fibrosis, characterized by excessive extracellular matrix (ECM) components deposition, is a common pathological process underlying numerous chronic diseases. Differentiating fibroblasts into myofibroblasts leads to the deposition of fibrous proteins like collagen in the ECM. Heat shock protein 47 (HSP47) is a molecular chaperone protein that assists in the folding of collagen. Recent studies show that inhibition of cholesterol synthesis-independent HMGCoA-reductase pathway enzyme geranylgeranyl pyrophosphate synthase 1 (GGPS1) decreases fibroblast differentiation and its bioenergetics. However, the role of HSP47 in this pathway is unknown, although recent observations show that HSP47 is decreased when GGPS1 is inhibited. Hence, we tested the hypothesis that HSP47 promotes cellular respiration in myofibroblasts. Seahorse assays were performed on different fibroblasts/myofibroblasts repeatedly and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in the presence and absence of HSP47 inhibitor (HY124817). Complex V of the electron transport chain was measured by immunoblotting ATP-5A. HY124817 tends to decrease the OCR, basal respiration rate, ATP-linked, and spare respiratory capacity. Co-incubation with HY124817 and digeranyl bisphosphonate (DGBP), an inhibitor of GGPS1, significantly (P = 0.043) inhibited further the ATP-linked OCR compared to the TGF group. Immunoblotting showed that Complex V did not change significantly across treatments. These results suggest that HSP47 is involved in fibroblast bioenergetics related to ATP-linked OCR in both differentiated myofibroblasts and normal fibroblasts. Inhibition of HSP47 complements the decrease in myofibroblast cellular respiration caused by the GGPS1 inhibition. This finding is novel in that HSP47 plays a role in fibroblast bioenergetics regulated by the cholesterol synthesis-independent HMGCoA-reductase pathway. Further research is necessary to identify what HSP47 targets and regulatory elements involved in this process. This would provide more insight into the mechanisms that underlie critical fibrosis conditions like myocardial fibrosis and potential therapeutics. This experience was supported by the Medical College of Wisconsin's Advancing Student Potential for Inclusion with Research Experiences (ASPIRE) program. The program is funded by a federal grant from the National Heart, Lung, and Blood Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modulation of Endothelial Cell Mitochondrial Phenotype and Angiogenesis by TRPV4

Transient receptor potential vanilloid type 4 (TRPV4) is a mechanically activated ion channel implicated in the regulation of endothelial cell (EC) proliferation, migration, and angiogenesis. However, the molecular mechanism(s) by which TRPV4 regulates EC functions remains underexplored. Here, we investigated if TRPV4 channels mediate endothelial function via modulation of mitochondria. Confocal microscopy showed a peri-nuclear localization of mitochondria in normal EC (NEC), while they were localized throughout the cell in TRPV4 knockout EC (KOEC). Furthermore, transmission electron microscopy confirmed clear round mitochondria in NEC compared to elongated mitochondria with distinct cristae in KOEC. Importantly, we found increased distribution of mitochondria in KOEC with increasing stiffness, when cultured on extracellular matrix (ECM) gels of varying stiffness that mimic the stiffness of matrix in pathophysiological conditions such as tumor or heart failure (0.2, 8, and 50 kPa). Mechanistically, western blot analysis showed increased fusion/fission protein ratio (Optic Atrophy 1 (OPA1)/mitochondrial fission factor (MFF)), in KOEC compared to NEC. Seahorse flux analyzer analysis demonstrated increased basal oxygen consumption rate (OCR), maximal OCR, ATP-linked OCR, and spare capacity in KOEC compared to NEC, which were significantly attenuated by a small molecule inhibitor of OPA1, MYLS22. Finally, MYLS22 normalized TRPV4-knockout mediated abnormal proliferation, migration, and angiogenesis ex vivo. Taken together, these findings indicate that mechanosensitive TRPV4 channels regulate mitochondrial phenotype and function during angiogenesis through OPA1. This work was supported by the National Institutes of Health (and R01HL148585 and AHA-TPA-971237) to CKT. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Platinum iodido drugs show potential anti-tumor activity, affecting cancer cell metabolism and inducing ROS and senescence in gastrointestinal cancer cells

Cisplatin-based chemotherapy has associated clinical disadvantages, such as high toxicity and resistance. Thus, the development of new antitumor metallodrugs able to overcome different clinical barriers is a public healthcare priority. Here, we studied the mechanism of action of the isomers trans and cis-[PtI2(isopropylamine)2] (I5 and I6, respectively) against gastrointestinal cancer cells. We demonstrate that I5 and I6 modulate mitochondrial metabolism, decreasing OXPHOS activity and negatively affecting ATP-linked oxygen consumption rate. Consequently, I5 and I6 generated Reactive Oxygen Species (ROS), provoking oxidative damage and eventually the induction of senescence. Thus, herein we propose a loop with three interconnected processes modulated by these iodido agents: (i) mitochondrial dysfunction and metabolic disruptions; (ii) ROS generation and oxidative damage; and (iii) cellular senescence. Functionally, I5 reduces cancer cell clonogenicity and tumor growth in a pancreatic xenograft model without systemic toxicity, highlighting a potential anticancer complex that warrants additional pre-clinical studies.

A Thia-Analogous Indirubin N-Glycoside Disrupts Mitochondrial Function and Causes the Death of Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells.

Skin cancer is the most common malignant disease worldwide and, therefore, also poses a challenge from a pharmacotherapeutic perspective. Derivatives of indirubin are an interesting option in this context. In the present study, the effects of 3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(β-d-glucopyranosyl)-oxindole (KD87), a thia-analogous indirubin N-glycoside, on the viability and mitochondrial properties of melanoma (A375) and squamous cell carcinoma cells (A431) of the skin were investigated. In both cell lines, KD87 caused decreased viability, the activation of caspases-3 and -7, and the inhibition of colony formation. At the mitochondrial level, a concentration-dependent decrease in both the basal and ATP-linked oxygen consumption rate and in the reserve capacity of oxidative respiration were registered in the presence of KD87. These changes were accompanied by morphological alterations in the mitochondria, a release of mitochondrial cytochrome c into the cytosol and significant reductions in succinate dehydrogenase complex subunit B (SDHB, subunit of complex II) in A375 and A431 cells and NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8, subunit of complex I) in A375 cells. The effect of KD87 was accompanied by a significant upregulation of the enzyme heme oxygenase-1, whose inhibition led to a partial but significant reduction in the metabolic-activity-reducing effect of KD87. In summary, our data show a mitochondria-targeting effect of KD87 as part of the cytotoxic effect of this compound on skin cancer cells, which should be considered in future studies with this class of compounds.

Engineered allele substitution at PPARGC1A rs8192678 alters human white adipocyte differentiation, lipogenesis, and PGC-1α content and turnover

Aims/hypothesisPPARGC1A encodes peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), a central regulator of energy metabolism and mitochondrial function. A common polymorphism in PPARGC1A (rs8192678, C/T, Gly482Ser) has been associated with obesity and related metabolic disorders, but no published functional studies have investigated direct allele-specific effects in adipocyte biology. We examined whether rs8192678 is a causal variant and reveal its biological function in human white adipose cells.MethodsWe used CRISPR-Cas9 genome editing to perform an allelic switch (C-to-T or T-to-C) at rs8192678 in an isogenic human pre-adipocyte white adipose tissue (hWAs) cell line. Allele-edited single-cell clones were expanded and screened to obtain homozygous T/T (Ser482Ser), C/C (Gly482Gly) and heterozygous C/T (Gly482Ser) isogenic cell populations, followed by functional studies of the allele-dependent effects on white adipocyte differentiation and mitochondrial function.ResultsAfter differentiation, the C/C adipocytes were visibly less BODIPY-positive than T/T and C/T adipocytes, and had significantly lower triacylglycerol content. The C allele presented a dose-dependent lowering effect on lipogenesis, as well as lower expression of genes critical for adipogenesis, lipid catabolism, lipogenesis and lipolysis. Moreover, C/C adipocytes had decreased oxygen consumption rate (OCR) at basal and maximal respiration, and lower ATP-linked OCR. We determined that these effects were a consequence of a C-allele-driven dysregulation of PGC-1α protein content, turnover rate and transcriptional coactivator activity.Conclusions/interpretationOur data show allele-specific causal effects of the rs8192678 variant on adipogenic differentiation. The C allele confers lower levels of PPARGC1A mRNA and PGC-1α protein, as well as disrupted dynamics of PGC-1α turnover and activity, with downstream effects on cellular differentiation and mitochondrial function. Our study provides the first experimentally deduced insights on the effects of rs8192678 on adipocyte phenotype.Graphical

Regulation of endothelial cell mitochondrial phenotype by mechanosensitive ion channel TRPV4

Transient receptor potential vanilloid type 4 (TRPV4) is a mechanosensor that regulates endothelial cell (EC) proliferation, migration, and angiogenesis. However, the molecular mechanisms by which TRPV4 regulates EC functions are not well understood. In this study, we investigated if TRPV4 regulates EC function via modulation of mitochondria by employing three types of EC expressing different levels of TRPV4, normal (NEC), TRPV4-deficienct (TEC), and TRPV4 knockout (KOEC). First, we confirmed the functional expression of TRPV4 in these EC using qPCR and calcium imaging. Confocal images upon MitoTracker staining revealed spherical and/or round shaped mitochondria with a perinuclear localization in NEC while in TEC and KOEC the mitochondrial network was elongated and rod shaped with a whole cell distribution. Furthermore, Transmission Electron Microscopy confirmed the presence of clear round mitochondria in NEC compared to elongated mitochondria with distinct cristae in TEC and KOEC. These results indicate increased fusion in TRPV4 deficiency or deletion. When cultured on ECM gels of varying stiffness that mimic stiffness of matrix in pathophysiological conditions such as tumor or heart failure (0.2, 8, and 50 kPa), we found increased distribution of mitochondria in EC with increasing stiffness. Moreover, western blot analysis showed increased expression of a fusion/fission protein ratio (Optic Atrophy 1 (OPA1)/mitochondrial fission factor (MFF)), in TEC and KOEC. Further, uncoupling mitochondrial function by mitochondrial stressors (Oligomycin, FCCP, and Rot/AA) significantly increased basal oxygen consumption rate (OCR), maximal OCR, ATP-linked OCR, and spare capacity in TEC and KOEC than NEC. Finally, a small molecule inhibitor of OPA1, MYLS22, attenuated/normalized the mitochondrial energy metabolism to that of NEC but did not affect NEC. These findings suggest that mechanosensitive TRPV4 channels regulate EC mitochondrial morphology and function through increased OPA1. National Institutes of Health (R15CA202847, R01HL119705, and R01HL148585) to CKT 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.

Effects of PFOS, F-53B and OBS on locomotor behaviour, the dopaminergic system and mitochondrial function in developing zebrafish (Danio rerio)

Perfluorooctanesulfonic acid (PFOS) has widely been reported to persist in the environment and to elicit neurotoxicological effects in wildlife and humans. Following the restriction of PFOS use, 6:2 chlorinated polyfluorinated ether sulfonate (F-53B) and sodium p-perfluorous nonenoxybenzene sulfonate (OBS) have emerged as novel PFOS alternatives and have been detected in the environment. However, knowledge on the toxicological effects of these alternatives remains scarce. Using developing transgenic Tg(dat:eGFP) zebrafish, we evaluated the consequences of exposure to 0, 0.1 and 1 mg/l PFOS, F-53B and OBS on the dopaminergic system, locomotor behaviour and mitochondrial function. All compounds generally reduced locomotor activity under light conditions irrespective of exposure concentration. Exposure to OBS (at all concentrations), as well as PFOS and F-53B (at 1 mg/l), significantly reduced subpallial dopaminergic neuron abundance. PFOS also significantly reduced dat and pink1 expression irrespective of exposure concentration, while F-53B and OBS tended to reduce mitochondrial pink1 and fis1 expression across concentrations without reaching statistical significance. Mitochondrial function, in the form of reduced oxygen consumption rate and marginally inhibited ATP-linked oxygen consumption rate, was affected only in response to 1 mg/l PFOS. Together, PFOS and the emerging contaminants F-53B and OBS inhibit locomotion at similar concentrations, a finding correlated with decreased dopaminergic neuron numbers in the subpallium and decreased expression of pink1. These findings are relevant to wildlife and human health, as they suggest that PFOS as well as replacement compounds affect locomotion likely in part by negatively impacting the dopamine system.

Benzo(a)pyrene-induced mitochondrial respiration and glycolysis disturbance in human neuroblastoma cells.

Mammalian cells generate ATP through mitochondrial respiration and glycolysis. Mitochondria not only play a key role in cell energy metabolism but also in cell cycle regulation. As a neurotoxic pollutant, benzo(a)pyrene (BaP) can trigger neuronal oxidative damage and apoptosis. However, the features of BaP-induced energy metabolism disturbance in SH-SY5Y cells has rarely been addressed. This study aimed to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as indications of respiratory activities and glycolytic. SH-SY5Y cells were treated with BaP to establish a cytotoxicity model, and butylated hydroxy anisole (BHA) was used to alleviate the damages induced by BaP. Using the Seahorse Extracellular Flux analyzer (XFp), we found that BaP significantly reduced basal respiration, ATP-linked OCR in SH-SY5Y cells with dose- and time-dependent. BHA supplementation recovered the mitochondrial respiration, synchronously attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers, then rescued BaP-induced cell apoptosis. But long-term exposure to BaP or exposure to a high dosage of BaP could decrease OCR associated with maximal respiratory, spare capacity, and glycolysis metabolism. At the same time, the damage to cells is also more severe with the rate of apoptosis and mitochondrial membrane potential (ΔΨm) loss rising sharply, which were not entirely reversed by BHA. This study provides energy metabolism-related, indicative biomarkers of cytotoxicity induced by BaP, which might provide information for early prevention and intervention.

Abstract 12910: Exercise-Induced Specialized Proresolving Mediators Stimulate AMPK Phosphorylation to Promote Mitochondrial Respiration in Macrophages

Emerging epidemiological data suggests that exercise imparts its CVD risk-reducing effects in part through improvements in systemic inflammation. The mechanisms underpinning the salutary effects of physical activity, however, remain incompletely understood. We recently reported that exercise enhances the production of specialized proresolving mediators (SPMs), which act as agonist of inflammation resolution through actions on macrophages (Mϕ). In this study, we find that wheel running accelerated the metabolism of proinflammatory lipid mediators LTB 4 and PGD 2 into their respective metabolites 20-OH-LTB 4 (Sed 27.14±5.88 vs Exe 70.40±10.82 pg/mL; P<0.05; n =3-4) and PGJ 2 (Sed 92.63±16.08 vs Exe 227.90±30.23 pg/mL; P<0.05; n =3-4), resulting in an increase in the ratio of proresolving-to-proinflammatory lipid mediator levels (RvE1+RvD1:leukotrienes; Sed 0.19±0.02 vs Exe 0.32±0.90; P<0.05; n =3-4). These findings were associated with enhanced mitochondrial metabolism in Mϕ, as evidenced by increased basal (Sed 0.12±0.06 vs Exe 1.53±0.07; P<0.05; n =3-4), maximum (Sed 9.20±0.30 vs Exe 12.02±0.82; P<0.05; n =3-4), and ATP-linked oxygen consumption rate (Sed 1.25±0.06 vs Exe 1.47±0.09 pmol/min/μg protein; P<0.05; n =3-4). We also find that SPMs RvD1, RvD2, RvE1 or MaR1 stimulate basal (Ctrl 6.11±0.1 vs RvD1 7.76±0.6 vs RvD2 9.48±0.9 vs RvE1 7.20±0.5 vs MaR1 8.73±0.9 pmol/min/μg protein; P<0.05; n =3) and ATP-linked respiration (Ctrl 2.83±0.2 vs RvD1 4.50±0.3 vs RvD2 4.39±0.2 vs RvE1 4.71±0.3 vs MaR1 4.20±0.2 pmol/min/μg protein; P<0.05; n =3). We find that an increase in basal (Ctrl 1.80±0.13 vs RvD1 2.43±0.19 pmol/min/μg protein; P<0.05; n =3-5) and ATP-linked respiration (Ctrl 1.44±0.10 vs RvD1 1.70±0.15 pmol/min/μg protein; P<0.05; n =3-5) with RvD1 treatment occurs concomitantly with AMPK phosphorylation (Ctrl 1.00±0.07 vs RvD1 1.40±0.02 pAMPK/AMPK fold change; P<0.05; n =3) and inhibiting the kinase activity of AMPK with Compound C abrogates SPM-induced mitochondrial respiration. Taken together, these data suggest that exercise-enhanced SPM production stimulates mitochondrial metabolism in Mϕ via AMPK, which may contribute to exercise-induced amelioration of chronic inflammation.

Hydrogen Peroxide Promotes the Production of Radiation-Derived EVs Containing Mitochondrial Proteins.

In spite of extensive successes, cancer recurrence after radiation treatment (RT) remains one of the significant challenges in the cure of localized prostate cancer (PCa). This study focuses on elucidating a novel adaptive response to RT that could contribute to cancer recurrence. Here, we used PC3 cell line, an adenocarcinoma from a bone metastasis and radio-resistant clone 695 cell line, which survived after total radiation dose of 66 Gy (2 Gy × 33) and subsequently regrew in nude mice after exposure to fractionated radiation at 10 Gy (2 Gy × 5). Clone 695 cells not only showed an increase in surviving fraction post-radiation but also an increase in hydrogen peroxide (H2O2) production when compared to PC3 cells. At the single cell level, confocal microscope images coupled with IMARIS rendering software demonstrate an increase in mitochondrial mass and membrane potential in clone 695 cells. Utilizing the Seahorse XF96 instrument to investigate mitochondrial respiration, clone 695 cells demonstrated a higher basal Oxygen Consumption Rate (OCR), ATP-linked OCR, and proton leak compared to PC3 cells. The elevation of mitochondrial function in clone 695 cells is accompanied by an increase in mitochondrial H2O2 production. These data suggest that H2O2 could reprogram PCa's mitochondrial homeostasis, which allows the cancer to survive and regrow after RT. Upon exposure to RT, in addition to ROS production, we found that RT induces the release of extracellular vesicles (EVs) from PC3 cells (p < 0.05). Importantly, adding H2O2 to PC3 cells promotes EVs production in a dose-dependent manner and pre-treatment with polyethylene glycol-Catalase mitigates H2O2-mediated EV production. Both RT-derived EVs and H2O2-derived EVs carried higher levels of mitochondrial antioxidant proteins including, Peroxiredoxin 3, Glutathione Peroxidase 4 as well as mitochondrial-associated oxidative phosphorylation proteins. Significantly, adding isolated functional mitochondria 24 h prior to RT shows a significant increase in surviving fractions of PC3 cells (p < 0.05). Together, our findings reveal that H2O2 promotes the production of EVs carrying mitochondrial proteins and that functional mitochondria enhance cancer survival after RT.

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.

Violacein switches off low molecular weight tyrosine phosphatase and rewires mitochondria in colorectal cancer cells

In the last decade, emerging evidence has shown that low molecular weight protein tyrosine phosphatase (LMWPTP) not only contributes to the progression of cancer but is associated with prostate low survival rate and colorectal cancer metastasis. We report that LMWPTP favors the glycolytic profile in some tumors. Therefore, the focus of the present study was to identify metabolic enzymes that correlate with LMWPTP expression in patient samples. Exploratory data analysis from RNA-seq, proteomics, and histology staining, confirmed the higher expression of LMWPTP in CRC. Our descriptive statistical analyses indicate a positive expression correlation between LMWPTP and energy metabolism enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In addition, we examine the potential of violacein to reprogram energetic metabolism and LMWPTP activity. Violacein treatment induced a shift of glycolytic to oxidative metabolism associated with alteration in mitochondrial efficiency, as indicated by higher oxygen consumption rate. Particularly, violacein treated cells displayed higher proton leak and ATP-linked oxygen consumption rate (OCR) as an indicator of the OXPHOS preference. Notably, violacein is able to bind and inhibit LMWPTP. Since the LMWPTP acts as a hub of signaling pathways that offer tumor cells invasive advantages, such as survival and the ability to migrate, our findings highlight an unexplored potential of violacein in circumventing the metabolic plasticity of tumor cells.

The Effects of Elevated Sodium on Mitochondrial Function in Peripheral Blood Mononuclear Cells

INTRODUCTIONDiets high in sodium are associated with impaired vascular function independent of blood pressure. Recent data suggests that sodium accumulation from high salt diets reduces mitochondrial respiration in monocytes and macrophages, resembling what occurs during an inflammatory response. A similar response in circulating peripheral blood mononuclear cells (PBMCs) could implicate systemic inflammation as a mechanism contributing to high salt‐induced vascular dysfunction. Therefore, we aimed to test the hypothesis that exposure to a high sodium environment reduces PBMC mitochondrial respiration.METHODSPBMCs were separated from whole blood of ten healthy subjects (7 men/3 women, 18‐30 years, blood pressure ≤ 130/80mmHg; BMI < 30 kg/m2). PBMCs were then seeded at 2x105 cells/well in Poly‐D‐lysine treated cell culture plates. Plated cells were incubated for 24 hours in RPMI 1640 medium with 10% FBS at 137.7 mM sodium (normal sodium, NS) and 180 mM sodium (high sodium, HS). Basal, maximal, and ATP‐linked oxygen consumption rates were assessed post‐incubation by respirometry assay via Seahorse XFp analyzer (Agilent). Spare respiratory capacity was calculated as the difference between maximal and basal oxygen consumption rate.RESULTSBasal oxygen consumption rate was significantly reduced in HS compared to NS (65.0 ± 12.7 vs. 39.6 ± 6.5 pmol/min, p = 0.03), as was maximal oxygen consumption rate (198.3 ± 32.6 vs. 148.4 ± 27.2 pmol/min, p = 0.008). Basal ATP‐linked oxygen consumption rate was also significantly reduced in HS compared to NS (57.3 ± 9.8 vs. 34.4 ± 5.4 pmol/min, p = 0.01). Spare respiratory capacity in HS was significantly reduced compared to NS (133.2 ± 23.4 vs. 108.8 ± 21.2 pmol/min, p = 0.03).CONCLUSIONReduced basal and maximal oxygen consumption rates and spare respiratory capacity provide evidence that elevated sodium exposure reduces PBMC mitochondrial respiration. Additionally, high sodium exposure reduced ATP‐linked respiration potentially indicating alterations in PBMC energetics, resembling what occurs during inflammation. Collectively, our preliminary findings warrant further investigation into the role of sodium induced mitochondrial dysfunction in PBMCs as a potential mechanism contributing to high salt‐induced vascular dysfunction.

The FoxQ1 transcription factor is a novel regulator of electron transport chain complex I subunits in human breast cancer cells.

The FoxQ1 is an oncogenic transcription factor that is overexpressed in basal-like and luminal-type human breast cancers when compared to the normal mammary tissue. The FoxQ1 is implicated in mammary tumor progression. However, the mechanism by which FoxQ1 promotes mammary tumorigenesis is not fully understood. In this study, we present experimental evidence for a novel function of FoxQ1 in the regulation of complex I activity of the electron transport chain. The RNA-seq data from FoxQ1 overexpressing basal-like SUM159 cells revealed a statistically significant increase in the expression of complex I subunits NDUFS1 and NDUFS2 when compared to the empty vector (EV) transfected control cells. Consistent with these results, the basal and ATP-linked oxygen consumption rates were significantly increased by FoxQ1 overexpression in SUM159 and luminal-type MCF-7 cells. The FoxQ1 overexpression in both cell lines resulted in increased intracellular levels of pyruvate, lactate, and ATP that was associated with overexpression of pyruvate dehydrogenase and pyruvate carboxylase proteins. Activity and assembly of complex I were significantly enhanced by FoxQ1 overexpression in SUM159 and MCF-7 cells that correlated with increased mRNA and/or protein levels of complex I subunits NDUFS1, NDUFS2, NDUFV1, and NDUFV2. The chromatin immunoprecipitation assay revealed the recruitment of FoxQ1 at the promoters of both NDUFS1 and NDUFV1. The cell proliferation of SUM159 and MCF-7 cells was increased significantly by overexpression of NDUFS1 as well as NDUFV1 proteins. In conclusion, we propose that increased complex I-linked oxidative phosphorylation is partly responsible for oncogenic role of FoxQ1 at least in human breast cancer cells.

Impaired Mitochondrial Bioenergetics Function in Pediatric Chronic Overlapping Pain Conditions with Functional Gastrointestinal Disorders.

Background Fatigue is often the primary complaint of children with functional gastrointestinal disorders (FGDI) and other chronic overlapping pain disorders (COPC). The basis for this symptom remains unknown. We evaluated mitochondrial function in the white blood cells of these patients. Methods This prospective Children's Wisconsin IRB approved study recruited subjects aging 10–18 years from pediatric neurogastroenterology clinics and healthy comparison subjects (HC). Environmental and oxidative stressors can damage the mitochondrial respiratory chain. The known low-grade inflammation in COPC could, therefore, impact the respiratory chain and theoretically account for the disabling fatigue so often voiced by patients. Mitochondrial energy generation can be easily measured in peripheral mononuclear cells (PMC) as a general marker by the Seahorse XF96 Extracellular Flux Analyzer. We measured 5 parameters of oxygen consumption using this methodology: basal respiration (BR), ATP linked oxygen consumption (ATP-LC), maximal oxygen consumption rate (max R), spare respiratory capacity (SRC), and extracellular acidification rate (ECAR), which reflect non-electron chain energy generation through glycolysis. In health, we expect high ATP linked respiration, high reserve capacity, low proton leak, and low non-mitochondrial respiration. In disease, the proton leak typically increases, ATP demand increases, and there is decreased reserve capacity with increased non-mitochondrial respiration. Findings and clinical data were compared to healthy control subjects using a Mann–Whitney test for skewed variables, Fisher's exact test for dichotomous variables, and regression tree for association with functional outcome (functional disability inventory, FDI). Results 19 HC and 31 COPC showed no statistically significant difference in age. FGID, orthostatic intolerance, migraine, sleep disturbance, and chronic fatigue were present in the majority of COPC subjects. BR, ECAR, and ATP-LC rates were lower in the COPC group. The low BR and ATP-LC suggest that mitochondria are stressed with decreased ability to produce ATP. Tree analysis selected SRC as the best predictor of functional disability: patients with SRC >150 had a greater FDI (more disability) compared to patients with SRC <=150, p-value = 0.021. Conclusion Subjects with COPC have reduced mitochondrial capacity to produce ATP. Predisposing genetic factors or reversible acquired changes may be responsible. A higher SRC best predicts disability. Since a higher SRC is typically associated with more mitochondrial reserve, the SRC may indicate an underutilized available energy supply related to inactivity, or a “brake” on mitochondrial function. Prospective longitudinal studies can likely discern whether these findings represent deconditioning, true mitochondrial dysfunction, or both.

Protective Effect of Mitochondrial ND2 C5178A Gene Mutation on Cell and Mitochondrial Functions.

Background Mitochondrial NADH dehydrogenase subunit 2 (MT-ND2) m. 5178C>A gene mutation has protective effects against various diseases, but the molecular mechanism is still unclear. In previous study, we found a heteroplasmy level of MT-ND2 m. 5178C>A mutation in normotensive controls. Peripheral blood samples were obtained from essential hypertension individuals carrying the mutation and healthy controls without gene mutation to establish immortalized lymphocyte lines. To investigate the effect of the MT-ND2 m. 5178C>A gene mutation, comparative analyses of the two group cell lines were performed, including measurements of cell proliferation, viability, ATP synthesis, mitochondrial oxidative stress, and oxidative phosphorylation. Results The cell proliferation rate and viability of the MT-ND2 m. 5178C>A mutant lymphocyte line were higher than those of the control group. Mitochondrial functions of the MT-ND2 m. 5178C>A mutant lymphocyte were increased, including increased ATP synthesis, decreased ROS production, increased mitochondrial membrane potential and Bcl-2 gene transcription and protein translation, decreased Caspase 3/7 activity, and decreased early apoptosis and late apoptosis. The oxygen consumption rate (OCR) of the mutant lymphocyte line was higher than that of the control group, including basal OCR, ATP-linked OCR, maximal OCR, proton leak OCR, and reserve OCR, and there was no significant difference in nonmitochondrial OCR. The activity of Mitochondrial Complex I of the mutant group was increased than that of the control group. Conclusions The MT-ND2 m. 5178C>A mutation is a protective mutation that may be related to improvement of mitochondrial functions and decrease in apoptosis.

The Potent Trypanocidal Effect of LQB303, a Novel Redox-Active Phenyl-Tert-Butyl-Nitrone Derivate That Causes Mitochondrial Collapse in Trypanosoma cruzi.

Chagas disease, which is caused by Trypanosoma cruzi, establishes lifelong infections in humans and other mammals that lead to severe cardiac and gastrointestinal complications despite the competent immune response of the hosts. Furthermore, it is a neglected disease that affects 8 million people worldwide. The scenario is even more frustrating since the main chemotherapy is based on benznidazole, a drug that presents severe side effects and low efficacy in the chronic phase of the disease. Thus, the search for new therapeutic alternatives is urgent. In the present study, we investigated the activity of a novel phenyl-tert-butyl-nitrone (PBN) derivate, LQB303, against T. cruzi. LQB303 presented trypanocidal effect against intracellular [IC50/48 h = 2.6 μM] and extracellular amastigotes [IC50/24 h = 3.3 μM] in vitro, leading to parasite lysis; however, it does not present any toxicity to host cells. Despite emerging evidence that mitochondrial metabolism is essential for amastigotes to grow inside mammalian cells, the mechanism of redox-active molecules that target T. cruzi mitochondrion is still poorly explored. Therefore, we investigated if LQB303 trypanocidal activity was related to the impairment of the mitochondrial function of amastigotes. The investigation showed there was a significant decrease compared to the baseline oxygen consumption rate (OCR) of LQB303-treated extracellular amastigotes of T. cruzi, as well as reduction of “proton leak” (the depletion of proton motive force by the inhibition of F1Fo ATP synthase) and “ETS” (maximal oxygen consumption after uncoupling) oxygen consumption rates. Interestingly, the residual respiration (“ROX”) enhanced about three times in LQB303-treated amastigotes. The spare respiratory capacity ratio (SRC: cell ability to meet new energy demands) and the ATP-linked OCR were also impaired by LQB303 treatment, correlating the trypanocidal activity of LQB303 with the impairment of mitochondrial redox metabolism of amastigotes. Flow cytometric analysis demonstrated a significant reduction of the ΔΨm of treated amastigotes. LQB303 had no significant influence on the OCR of treated mammalian cells, evidencing its specificity against T. cruzi mitochondrial metabolism. Our results suggest a promising trypanocidal activity of LQB303, associated with parasite bioenergetic inefficiency, with no influence on the host energy metabolism, a fact that may point to an attractive alternative therapy for Chagas disease.

13C isotope-based metabolic flux analysis revealing cellular landscape of glucose metabolism in human liver cells exposed to perfluorooctanoic acid

Perfluorooctanoic acid (PFOA) is well known to break glucose homeostasis. However, the effects of PFOA on glucose metabolism are difficult to be evaluated because related metabolites may be synthesized from other nutritional substrates. Here, the relative contribution of glucose to metabolites (e.g., pyruvate and citrate) in the PFOA-treated human liver cells (HepG2) was determined using the 13C isotope-based metabolic flux analysis (MFA), i.e., pathway activities. The relative percentage of [U-13C6] glucose-derived pyruvate in cells exposed to PFOA was not significantly different from that in the controls, indicating that the metabolic pattern of glycolysis was not substantially changed by PFOA. The pathway activity of [U-13C6] glucose-driven tricarboxylic acid (TCA) cycle was dramatically inhibited by PFOA. Consequently, mitochondrial respiratory function was phenotypically impaired by PFOA, as observed from the decreasing basal oxygen consumption rate (OCR), ATP-linked OCR and spare respiratory capacity. This study suggests that PFOA may cause the abnormal glucose metabolism via altering the metabolic pattern of TCA cycle instead of glycolysis. The MFA is strongly recommended as a promising and robust tool to address the toxicity mechanisms of contaminants associated with glucose metabolism.

Elevated ROS depress mitochondrial oxygen utilization efficiency in cardiomyocytes during acute hypoxia.

Mitochondria are important sites for the production of ATP and the generation of ROS in cells. However, whether acute hypoxia increases ROS generation in cells or affects ATP production remains unclear, and therefore, monitoring the changes in ATP and ROS in living cells in real time is important. In this study, cardiomyocytes were transfected with RoGFP for ROS detection and MitGO-Ateam2 for ATP detection, whereby ROS and ATP production in cardiomyocytes were respectively monitored in real time. Furthermore, the oxygen consumption rate (OCR) of cardiomyocytes was measured. Similar results were produced for adult and neonatal rat cardiomyocytes. Hypoxia (1% O2) reduced the basal OCR, ATP-linked OCR, and maximal OCR in cardiomyocytes compared with these OCR levels in the cardiomyocytes in the normoxic group (21% O2). However, ATP-linked OCR, normalized to maximal OCR, was increased during hypoxia, indicating that the electron leakage of complex III exacerbated the increase of ATP-linked oxygen consumption during hypoxia and vice versa. Combined with the result that cardiomyocytes expressing MitGO-Ateam2 showed a significant decrease in ATP production during hypoxia compared with that of normoxic group, acute hypoxia might depress the mitochondrial oxygen utilization efficiency of the cardiomyocytes. Moreover, cardiomyocytes expressing Cyto-RoGFP or IMS-RoGFP showed an increase in ROS generation in the cytosol and the mitochondrial intermembrane space (IMS) during hypoxia. All of these results indicate that acute hypoxia generated more ROS in complex III and increased mitochondrial oxygen consumption, leading to less ATP production. In conclusion, acute hypoxia depresses the mitochondrial oxygen utilization efficiency by decreasing ATP production and increasing oxygen consumption as a result of the enhanced ROS generation at mitochondrial complex III.