ATP-linked Oxygen Consumption Research Articles

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.

Bioenergetic function is decreased in peripheral blood mononuclear cells of veterans with Gulf War Illness.

Gulf War Illness (GWI) is a major health problem for approximately 250,000 Gulf War (GW) veterans, but the etiology of GWI is unclear. We hypothesized that mitochondrial dysfunction is an important contributor to GWI, based on the similarity of some GWI symptoms to those occurring in some mitochondrial diseases; the plausibility that certain pollutants to which GW veterans were exposed affect mitochondria; mitochondrial effects observed in studies in laboratory models of GWI; and previous evidence of mitochondrial outcomes in studies in GW veterans. A primary role of mitochondria is generation of energy via oxidative phosphorylation. However, direct assessment of mitochondrial respiration, reflecting oxidative phosphorylation, has not been carried out in veterans with GWI. In this case-control observational study, we tested multiple measures of mitochondrial function and integrity in a cohort of 114 GW veterans, 80 with and 34 without GWI as assessed by the Kansas definition. In circulating white blood cells, we analyzed multiple measures of mitochondrial respiration and extracellular acidification, a proxy for non-aerobic energy generation; mitochondrial DNA (mtDNA) copy number; mtDNA damage; and nuclear DNA damage. We also collected detailed survey data on demographics; deployment; self-reported exposure to pesticides, pyridostigmine bromide, and chemical and biological warfare agents; and current biometrics, health and activity levels. We observed a 9% increase in mtDNA content in blood in veterans with GWI, but did not detect differences in DNA damage. Basal and ATP-linked oxygen consumption were respectively 42% and 47% higher in veterans without GWI, after adjustment for mtDNA amount. We did not find evidence for a compensatory increase in anaerobic energy generation: extracellular acidification was also lower in GWI (12% lower at baseline). A subset of 27 and 26 veterans returned for second and third visits, allowing us to measure stability of mitochondrial parameters over time. mtDNA CN, mtDNA damage, ATP-linked OCR, and spare respiratory capacity were moderately replicable over time, with intraclass correlation coefficients of 0.43, 0.44, 0.50, and 0.57, respectively. Other measures showed higher visit-to-visit variability. Many measurements showed lower replicability over time among veterans with GWI compared to veterans without GWI. Finally, we found a strong association between recalled exposure to pesticides, pyridostigmine bromide, and chemical and biological warfare agents and GWI (p < 0.01, p < 0.01, and p < 0.0001, respectively). Our results demonstrate decreased mitochondrial respiratory function as well as decreased glycolytic activity, both of which are consistent with decreased energy availability, in peripheral blood mononuclear cells in veterans with GWI.

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.

Abstract 485: Mitochondrial Complex I Promotes Smooth Muscle Cell Plasticity And Vascular Remodeling

Remodeling of the mature vasculature occurs in response to diseases such as atherosclerosis or after vascular injury due to angioplasty or stent placement. Smooth muscle cell (SMC) plasticity is a key factor in this process, but the significance of cellular metabolic and mitochondrial activities in control of SMC plasticity is not well understood. We hypothesized an essential role for mitochondrial respiratory complex I (CI) in SMC proliferation and migration and tested this idea by inactivating the CI subunit NDUFS4 or by using pharmacologic CI inhibitors. In cultured Ndufs4 -/- SMCs, we found lower overall CI expression and activity (P=0.0027), plus decreased respirasome levels, ATP-linked oxygen consumption (P&lt;0.0001), respiratory capacity, NAD + /NADH ratio (P=0.0035), aspartate, and cell proliferation (P&lt;0.0001). NDUFS4 loss increased mitochondrial fragmentation (P&lt;0.0001); interestingly, inhibition of mitochondrial fission restored proliferation of NDUFS4-deficient mouse SMCs and CI inhibitor-treated human SMCs. In addition, loss of NDUFS4 or CI inhibitor treatment limited migration of mouse (P=0.0012) and human (P=0.0022) SMCs, respectively. In vivo , NDUFS4 was highly expressed in the arterial wall during embryogenesis and in cells forming the neointima after arterial injury in adulthood. Mice with embryonic inactivation of SMC Ndufs4 were born in Mendelian ratios, reached adulthood, and lacked an obvious phenotype up to 20 weeks of age; by electron microscopy, however, SMCs in the aortic media revealed altered mitochondrial ultrastructure. To evaluate how SMC CI contributes to arterial injury response in the adult, we inactivated Ndufs4 conditionally at age 6 weeks (NDUFS4 iSMKO) and performed carotid artery ligation; these studies showed that loss of SMC NDUFS4 reduced neointima formation (57% lower intima/media ratio; control, n=13; NDUFS4 iSMKO, n=8; P=0.02). In conclusion, NDUFS4 and thus proper levels of SMC CI are essential for proliferation, migration, normal mitochondrial ultrastructure, and vascular remodeling in adulthood. Mechanistic studies suggest that CI promotes SMC plasticity by supporting biosynthesis of aspartate and ATP, maintaining NAD + /NADH balance, and limiting mitochondrial fragmentation.

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.

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&lt;0.05; n =3-4) and PGJ 2 (Sed 92.63±16.08 vs Exe 227.90±30.23 pg/mL; P&lt;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&lt;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&lt;0.05; n =3-4), maximum (Sed 9.20±0.30 vs Exe 12.02±0.82; P&lt;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&lt;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&lt;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&lt;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&lt;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&lt;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&lt;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.

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.

Novel mitochondria-targeting compounds selectively kill human leukemia cells.

Acute myeloid leukemia (AML) is a heterogeneous group of aggressive hematological malignancies commonly associated with treatment resistance, high risk of relapse, and mitochondrial dysregulation. We identified six mitochondria-affecting compounds (PS compounds) that exhibit selective cytotoxicity against AML cells in vitro. Structure-activity relationship studies identified six analogs from two original scaffolds that had over an order of magnitude difference between LD50 in AML and healthy peripheral blood mononuclear cells. Mechanistically, all hit compounds reduced ATP and selectively impaired both basal and ATP-linked oxygen consumption in leukemic cells. Compounds derived from PS127 significantly upregulated production of reactive oxygen species (ROS) in AML cells and triggered ferroptotic, necroptotic, and/or apoptotic cell death in AML cell lines and refractory/relapsed AML primary samples. These compounds exhibited synergy with several anti-leukemia agents in AML, acute lymphoblastic leukemia (ALL), or chronic myelogenous leukemia (CML). Pilot in vivo efficacy studies indicate anti-leukemic efficacy in a MOLM14/GFP/LUC xenograft model, including extended survival in mice injected with leukemic cells pre-treated with PS127B or PS127E and in mice treated with PS127E at a dose of 5 mg/kg. These compounds are promising leads for development of future combinatorial therapeutic approaches for mitochondria-driven hematologic malignancies such as AML, ALL, and CML.

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 &lt; 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.

Oils Rich in α-Linolenic Acid or Docosahexaenoic Acid Have Distinct Effects on Plasma Oxylipin and Adiponectin Concentrations and on Monocyte Bioenergetics in Women with Obesity

BackgroundOmega-3 fatty acids, including DHA and α-linolenic acid (ALA), are proposed to improve metabolic health by reducing obesity-associated inflammation. Their effects are mediated in part by conversion to oxylipins. ALA is relatively understudied, and direct comparisons to other omega-3 fatty acids are limited. ObjectivesWe compared the effects of equal doses of ALA and DHA on plasma oxylipins and markers of metabolic health in women with obesity. MethodsWe carried out a randomized, double-blind, crossover clinical trial where women aged 20–51 with a BMI of 30–51 kg/m2 were supplemented with 4 g/day of ALA or DHA for 4 weeks in the form of ALA-rich flaxseed oil or DHA-rich fish oil. The primary outcome, the plasma oxylipin profile, was assessed at Days 0 and 28 of each phase by HPLC-MS/MS. Plasma fatty acids, inflammatory markers, and the monocyte glucose metabolism were key secondary outcomes. Data were analyzed using a mixed model. ResultsCompared to the baseline visit, there were higher plasma levels of nearly all oxylipins derived from DHA (3.8-fold overall; P < 0.001) and EPA (2.7-fold overall; P < 0.05) after 28 days of fish-oil supplementation, while there were no changes to oxylipins after flaxseed-oil supplementation. Neither supplement altered plasma cytokines; however, adiponectin was increased (1.1-fold; P < 0.05) at the end of the fish-oil phase. Compared to the baseline visit, 28 days of flaxseed-oil supplementation reduced ATP-linked oxygen consumption (0.75-fold; P < 0.05) and increased spare respiratory capacity (1.4-fold; P < 0.05) in monocytes, and countered the shift in oxygen consumption induced by LPS. ConclusionsFlaxseed oil and fish oil each had unique effects on metabolic parameters in women with obesity. The supplementation regimens were insufficient to reduce inflammatory markers but adequate to elicit increases in omega-3 oxylipins and adiponectin in response to fish oil and to alter monocyte bioenergetics in response to flaxseed oil. This trial was registered at clinicaltrials.gov as NCT03583281.

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.

Chronic DDE Exposure Modifies Mitochondrial Respiration during Differentiation of Human Adipose-Derived Mesenchymal Stem Cells into Mature Adipocytes.

The contribution of environmental pollutants to the obesity pandemic is still not yet fully recognized. Elucidating possible cellular and molecular mechanisms of their effects is of high importance. Our study aimed to evaluate the effect of chronic, 21-day-long, 2,2-bis (4-chlorophenyl)-1,1-dichlorethylenedichlorodiphenyldichloroethylene (p,p´-DDE) exposure of human adipose-derived mesenchymal stem cells committed to adipogenesis on mitochondrial oxygen consumption on days 4, 10, and 21. In addition, the mitochondrial membrane potential (MMP), the quality of the mitochondrial network, and lipid accumulation in maturing cells were evaluated. Compared to control differentiating adipocytes, exposure to p,p´-DDE at 1 μM concentration significantly increased basal (routine) mitochondrial respiration, ATP-linked oxygen consumption and MMP of intact cells on day 21 of adipogenesis. In contrast, higher pollutant concentration seemed to slow down the gradual increase in ATP-linked oxygen consumption typical for normal adipogenesis. Organochlorine p,p´-DDE did not alter citrate synthase activity. In conclusion, in vitro 1 μM p,p´-DDE corresponding to human exposure is able to increase the mitochondrial respiration per individual mitochondrion at the end of adipocyte maturation. Our data reveal that long-lasting exposure to p,p´-DDE could interfere with the metabolic programming of mature adipocytes.

Role of IRE1\u03b1 in podocyte proteostasis and mitochondrial health

Glomerular epithelial cell (GEC)/podocyte proteostasis is dysregulated in glomerular diseases. The unfolded protein response (UPR) is an adaptive pathway in the endoplasmic reticulum (ER) that upregulates proteostasis resources. This study characterizes mechanisms by which inositol requiring enzyme-1α (IRE1α), a UPR transducer, regulates proteostasis in GECs. Mice with podocyte-specific deletion of IRE1α (IRE1α KO) were produced and nephrosis was induced with adriamycin. Compared with control, IRE1α KO mice had greater albuminuria. Adriamycin increased glomerular ER chaperones in control mice, but this upregulation was impaired in IRE1α KO mice. Likewise, autophagy was blunted in adriamycin-treated IRE1α KO animals, evidenced by reduced LC3-II and increased p62. Mitochondrial ultrastructure was markedly disrupted in podocytes of adriamycin-treated IRE1α KO mice. To pursue mechanistic studies, GECs were cultured from glomeruli of IRE1α flox/flox mice and IRE1α was deleted by Cre–lox recombination. In GECs incubated with tunicamycin, deletion of IRE1α attenuated upregulation of ER chaperones, LC3 lipidation, and LC3 transcription, compared with control GECs. Deletion of IRE1α decreased maximal and ATP-linked oxygen consumption, as well as mitochondrial membrane potential. In summary, stress-induced chaperone production, autophagy, and mitochondrial health are compromised by deletion of IRE1α. The IRE1α pathway is cytoprotective in glomerular disease associated with podocyte injury and ER stress.

Mitochondrial DNA Haplogroup M7 Confers Disability in a Chinese Aging Population.

Mitochondrial DNA (mtDNA) haplogroups have been associated with functional impairments (i.e., decreased gait speed and grip strength, frailty), which are risk factors of disability. However, the association between mtDNA haplogroups and ADL disability is still unclear. In this study, we conducted an investigation of 25 mtSNPs defining 17 major mtDNA haplogroups for ADL disability in an aging Chinese population. We found that mtDNA haplogroup M7 was associated with an increased risk of disability (OR = 3.18 [95% CI = 1.29–7.83], P = 0.012). The survival rate of the M7 haplogroup group (6.1%) was lower than that of the non-M7 haplogroup group (9.5%) after a 6-year follow-up. In cellular studies, cytoplasmic hybrid (cybrid) cells with the M7 haplogroup showed distinct mitochondrial functions from the M8 haplogroup. Specifically, the respiratory chain complex capacity was significantly lower in M7 haplogroup cybrids than in M8 haplogroup cybrids. Furthermore, an obvious decreased mitochondrial membrane potential and 40% reduced ATP-linked oxygen consumption were found in M7 haplogroup cybrids compared to M8 haplogroup cybrids. Notably, M7 haplogroup cybrids generated more reactive oxygen species (ROS) than M8 haplogroup cybrids. Therefore, the M7 haplogroup may contribute to the risk of disability via altering mitochondrial function to some extent, leading to decreased oxygen consumption, but increased ROS production, which may activate mitochondrial retrograde signaling pathways to impair cellular and tissue function.

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.

Abstract P019: Renal Mitochondrial Bioenergetics In Salt-sensitive Hypertension Is Affected By ANP

There are clinical data suggesting that low levels of Atrial Natriuretic Peptide (ANP) aggravate susceptibility to salt-sensitive (SS) hypertension. ANP is known to affect mitochondria in many tissues, however, little is known about the effects of ANP on renal mitochondrial function. According to our earlier studies, Dahl SS rats lacking ANP exhibit increased blood pressure and pronounced kidney injury. We hypothesized that in SS hypertension ANP deficiency affects renal mitochondrial bioenergetics and contributes to renal function impairment. SS hypertension was induced in male SS NPPA-/- ( Nppa knockout in Dahl SS background, KO) and SS WT (wild type, WT) rats by a high salt (HS) 4% NaCl diet administered for 21 days. Age-matched control animals were fed a normal (NS) 0.4% NaCl salt diet. A combination of in vivo studies, molecular biology and tests of mitochondria isolated from renal cortex (seahorse respiration and spectrofluorimetry assays using TMRM, Amplex Red and MCLA) were used to probe the role ANP in mitochondrial function. Data was analyzed with ANOVA followed by Holm-Sidak post hoc. We report a significant decrease in mitochondrial membrane potential in the SS NPPA-/- rats vs SS WT (25 ± 4% decrease in KO vs WT on NS, and a 16 ± 5% decrease in KO on HS). Furthermore, mitochondrial H 2 O 2 (57.7 ± 7.4 (WT) vs 57.6 ± 1.3 au (KO), p&lt;0.0001) and superoxide (36.6± 1.8 au (WT) vs 67.9 ± 5.2 au (KO), p&lt;0.0001) levels were increased in the KO on a HS diet. Next, we antioxidant capacity was elevated in the SS NPPA-/- rats on HS diet in comparison with SS WT , and increased SOD2 levels were observed in the KO animals. Furthermore, SS NPPA-/- rats exhibit higher MCU (mitochondrial calcium uniporter) activity than SS WT , implying an ANP-dependent effect on mitochondrial calcium influx. Seahorse assay revealed dramatic elevation of the basal, ATP-linked, maximal and spare oxygen consumption rate (OCR) in the cortical mitochondria of the knockout rats on HS, while in HS fed SS WT rats these OCR parameters were, as expected, reduced (vs NS). Therefore, deficiency of circulating ANP leads to significant changes of renal mitochondrial bioenergetics, potentially via effects on mitochondrial calcium uptake, which in turn affects respiratory chain activity leading to oxidative stress.

Ellagic Acid and Its Microbial Metabolite Urolithin A Alleviate Diet-Induced Insulin Resistance in Mice.

This work aims at evaluating the effect of dietary ellagic acid (EA) and its microbial metabolite urolithin A (UA) on glucose metabolism and insulin resistance (IR) in mice with diet-induced IR. DBA2J mice are fed a high fat/high sucrose diet (HF/HS) for 8 weeks to induce IR and then 0.1% EA, UA, or EA and UA (EA+UA) are added to the HF/HS-diet for another 8 weeks. UA significantly decreases fasting glucose and increases adiponectin compared with HF/HS-controls. During intraperitoneal insulin tolerance test, EA+UA significantly improve insulin-mediated glucose lowering effects at 15 and 120min and reduce blood triglycerides compared with HF/HS-controls. Serum free fatty acids are significantly decreased by EA, UA, and EA+UA. Differential expression of genes related to mitochondrial function by EA, UA, and EA+UA in liver and skeletal muscle is observed. Primary hepatocytes from IR-mice have higher proton leak, basal and ATP-linked oxygen consumption rates compared with healthy controls. EA and EA+UA but not UA reduce the proton leak in hepatocytes from IR-mice. EA and UA induce different metabolic benefits in IR mice. The effects of EA and UA on mitochondrial function suggest a potentially novel mechanism modulating metabolism.

Omega-3 fatty acids increase OXPHOS energy for immune therapy of Alzheimer disease patients.

Sporadic late-onset Alzheimer disease (LOAD) preceded by mild cognitive impairment (MCI) is the most common type of dementia. Long-term studies of immunity to pathogenic amyloid-β (Aβ) in LOAD are lacking. Innate immunity of LOAD patients is malfunctioning in phagocytosis and degradation of Aβ and LOAD patients' macrophage transcriptome and metabolome are deregulated. We previously showed omega-3 fatty acid (ω-3)-mediated repair of unfolded protein response and here we show much broader transcriptomic effects. ω-3 treatment in vitro and ω-3 supplementation by the drink Smartfish (SMF) in vivo increased the transcripts of the genes and pathways of immunity, glycolysis, tricarboxylic acid cycle, OX-PHOS, nicotinamide dinucleotide (NAD+ ) synthesis, and reversed the defects in Aβ phagocytosis. In both peripheral blood mononuclear cells (PBMC) and macrophages, ω-3 increased ATP-linked oxygen consumption rate (OCR) and ω-3 with carnitine was superior to ω-3. ω-3 treatment in vitro and supplementation by the ω-3 drink SMF in vivo rescued macrophage phagocytosis when glycolysis or glycosylation were blocked. ω-3 provide flexible energy for immune clearance of the brain throughout the diurnal cycle, even in hypo- or hyper-glycemia. In certain LOAD patients, ω-3 may delay progression to dementia.

β-Hydroxybutyrate is reduced in humans with obesity-related NAFLD and displays a dose-dependent effect on skeletal muscle mitochondrial respiration in vitro.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation and impaired insulin sensitivity. Reduced hepatic ketogenesis may promote these pathologies, but data are inconclusive in humans and the link between NAFLD and reduced insulin sensitivity remains obscure. We investigated individuals with obesity-related NAFLD and hypothesized that β-hydroxybutyrate (βOHB; the predominant ketone species) would be reduced and related to hepatic fat accumulation and insulin sensitivity. Furthermore, we hypothesized that ketones would impact skeletal muscle mitochondrial respiration in vitro. Hepatic fat was assessed by 1H-MRS in 22 participants in a parallel design, case control study [Control: n = 7, age 50 ± 6 yr, body mass index (BMI) 30 ± 1 kg/m2; NAFLD: n = 15, age 57 ± 3 yr, BMI 35 ± 1 kg/m2]. Plasma assessments were conducted in the fasted state. Whole body insulin sensitivity was determined by the gold-standard hyperinsulinemic-euglycemic clamp. The effect of ketone dose (0.5-5.0 mM) on mitochondrial respiration was conducted in human skeletal muscle cell culture. Fasting βOHB, a surrogate measure of hepatic ketogenesis, was reduced in NAFLD (-15.6%, P < 0.01) and correlated negatively with liver fat (r2 = 0.21, P = 0.03) and positively with insulin sensitivity (r2 = 0.30, P = 0.01). Skeletal muscle mitochondrial oxygen consumption increased with low-dose ketones, attributable to increases in basal respiration (135%, P < 0.05) and ATP-linked oxygen consumption (136%, P < 0.05). NAFLD pathophysiology includes impaired hepatic ketogenesis, which is associated with hepatic fat accumulation and impaired insulin sensitivity. This reduced capacity to produce ketones may be a potential link between NAFLD and NAFLD-associated reductions in whole body insulin sensitivity, whereby ketone concentrations impact skeletal muscle mitochondrial respiration.