Extracellular Flux Analyzer Research Articles

Metabolomics, Lipidomics, and Immunometabolism.

Metabolomics, lipidomics, and the study of cellular metabolism are gaining increasing interest particularly in the field of immunology, since the activation and effector functions of immune cells are profoundly controlled by changes in cellular metabolic asset. Among the different techniques that can be used for the evaluation of cellular metabolism, the Seahorse Extracellular Flux Analyzer allows the real time measurement of both glycolytic and mitochondrial respiration pathways in cells of interest, through the assessment of extracellular acidification and oxygen consumption rate. Metabolomics, on the other hand, is the high-throughput analysis of metabolites, i.e., the substrates, intermediates, and products of cellular metabolism, starting from biofluids, cells or tissues. The metabolome does not include lipids as their properties are different from water-soluble metabolites and are classified under the lipidome. Lipidomics analysis allows the identification and quantification of lipid species. Metabolomics and lipidomics are currently performed with mass-spectrometry coupled with liquid or gas chromatography (LC-MS or GC-MS) and/or nuclear-magnetic resonance (NMR). Here we describe the protocol for the evaluation of metabolic rate, metabolomics, and lipidomics in T cells, examining the detailed experimental approaches.

Bioenergetics of Islet Preparations in a Pilot Clinical Trial of Peri-Transplant Hydroxychloroquine for Autologous Islet Transplantation

The inflammatory response is an obstacle to success in both allogeneic and autologous islet transplantation. In autologous islet transplantation (AIT), however, the recipient is also the donor, permitting pretreatment of donor/recipient for a controlled duration prior to transplantation. We sought to exploit this feature of (AIT) by pretreating donor/recipients with chronic pancreatitis undergoing total pancreatectomy and autologous islet transplantation (TPAIT) to test the hypothesis that peri-transplant treatment with the FDA-approved anti-inflammatory hydroxychloroquine (HCQ) improves graft function. In this randomized placebo-controlled pilot clinical study, patients (n = 6) were treated with oral HCQ for 30 days prior to and 90 days after TPAIT. In vivo islet function was assessed via Mixed Meal Tolerance Testing before HCQ treatment, 6- and 12-months after surgery. In vitro islet bioenergetics were assessed at the time of transplantation via extracellular flux analysis of islet preparation samples from the clinical trial cohort and six additional patients (n = 12). Our study shows that HCQ did not alter clinical endpoints, but HCQ-treated patients showed greater spare respiratory capacity (SRC) compared to samples from control patients (P=0.028). Glycolytic metabolism of islet preparations directly correlated with stimulated C-peptide secretion both before and after TPAIT (P=0.01, R2=0.489 and P=0.03, R2=0.674, respectively), and predicted in vivo islet function better than mitochondrial metabolism of islet preps or islet equivalents infused. Overnight culture of islet preparations altered bioenergetic function, significantly decreasing SRC and maximal respiration (P<0.001). In conclusion, while HCQ did not alter clinical outcomes, it was associated with significantly increased SRC in islet preparations. Bioenergetic analyses of islet preparations suggests that culture should be avoided and that glycolysis may be a more sensitive indicator of in vivo islet function than current metrics, including islet oxygen consumption and islet equivalents infused.

CPNE1 Enhances Colorectal Cancer Cell Growth, Glycolysis, and Drug Resistance Through Regulating the AKT-GLUT1/HK2 Pathway

IntroductionColorectal cancer (CRC) is a major cause of cancer-related mortality worldwide. Copines-1 (CPNE1) has been shown to be overexpressed in various cancers; however, the role of CPNE1 in CRC remains unknown. Therefore, it is of great importance to elucidate the role of CPNE1 in CRC and its underlying mechanism of action.MethodsCPNE1 expression in CRC tissues was measured by quantitative real-time PCR and immunohistochemical (IHC) staining. CPNE1 was knocked down (KD) or overexpressed using small inferring RNAs or lentiviral transduction in CRC cells. The proliferation, apoptosis, glycolysis, and mitochondrial respiration of CRC cells were assessed by cell counting kit-8, flow cytometry, and Xfe24 extracellular flux analyzer assays, respectively. The role of CPNE1 in tumor growth and chemoresistance was further confirmed in xenograft and patient-derived tumor xenograft models, respectively.ResultsCPNE1 mRNA and protein were upregulated in CRC tissues. CPNE1 promoted proliferation, inhibited apoptosis, increased mitochondrial respiration, enhanced aerobic glycolysis by activating AKT signaling, upregulated glucose transporter 1 (GLUT1) and hexokinase 2 (HK2), and downregulated the production of cleaved Caspase-3 (c-Caspase 3). CPNE1 also contributed to chemoresistance in CRC cells. CPNE1 KD inhibited tumor growth and increased the sensitivity of tumors to oxaliplatin in vivo.ConclusionCPNE1 promotes CRC progression by activating the AKT-GLUT1/HK2 cascade and enhances chemoresistance.

N6-Methyladenosine Demethylase Fto Promotes Growth and Metastasis of Gastric Cancer &lt;i&gt;via&lt;/i&gt; M &lt;sup&gt;6&lt;/sup&gt;A Modification of Caveolin-1 and Metabolic Regulation of Mitochondrial Dynamics

Background: Gastric cancer (GC) is the fifth most common tumor and the third most deadly cancer worldwide. N6-methyladenosine (m6A) modification has been reported to play a regulatory role in human cancers. However, the exact role of m6A in GC remains largely unknown, and the dysregulation of m6A on mitochondrial metabolism has never been studied. Method: The expression level of FTO in the GC tissue was analyzed by real-time quantitative PCR, Western blotting, IF and IHC. Colorimetric m6A RNA methylation assay kit and Dot blotting were used to evaluate the level of m6A methylated RNA. In vitro assays, including cell viability, colony formation assay, wound healing assay and invasion assay were performed to detect the effects of FTO and caveolin-1 manipulation on the biological functions of GC cells. Xenografts models were constructed to evaluate the effects of FTO and caveolin-1 manipulation on the biological function of GC cell in vivo. HiSeq RNA-Seq was used to screen the potential targets of FTO. Real-time integrated cellular oxygen consumption rate (OCR) was measured using the Seahorse XF24 Extracellular Flux Analyzer. Finding: We demonstrated that FTO, a key demethylase for RNA m6A modification, was up-regulated in GC tissues, especially in tissues with liver metastasis. Functionally, FTO acted as a promoter for the proliferation and metastasis in GC. Moreover, FTO enhanced the degradation of caveolin-1 mRNA via its demethylation, which regulated the mitochondrial fission/fusion and metabolism. Interpretation: Our current findings provided some valuable insights into FTO-mediated m6A demethylation modification and could be used as a new strategy for more careful surveillance and aggressive therapeutic intervention. Funding: This project was supported by grants from the National Key R&D Plan (2018YFC1313400), National Natural Science Foundation of China (31701111), Young Talent Development Plan of Changzhou Health Commission (CZQM2020008, CZQM2020018) and Youth Talent Science and Technology Project of Changzhou Health Commission (QN202014). Declaration of Interest: None to declare. Ethical Approval: All animal experiments were approved by the Animal Care Committee of the Third Affiliated Hospital of Soochow University. All patients have provided written informed consent, and our study was preformed after formal approval by the Institutional Review Board of the Third Affiliated Hospital of Soochow University.

Extracellular Flux Analysis to Investigate the Impact of NF-κB on Mitochondrial Respiration in Colorectal Carcinoma (CRC).

The reprogramming of cell metabolism is a hallmark of cancer. NF-κB transcription factors coordinate the host defense responses to stress, injury, and infection. They also play a central role in oncogenesis, at least in part by regulating cell metabolism and the adaptation to energy stress conditions in various types of cancer, such as colorectal carcinoma (CRC). Here, we describe the XF Cell Mito Stress Test methodology aimed at characterizing the metabolic and bioenergetic profile of CRC cells following the silencing of the essential NF-κB subunit, RelA. This methodology may also be applied to other cancers to reveal novel core vulnerabilities of malignant cells.

Considerations for using isolated cell systems to understand cardiac metabolism and biology

Changes in myocardial metabolic activity are fundamentally linked to cardiac health and remodeling. Primary cardiomyocytes, induced pluripotent stem cell-derived cardiomyocytes, and transformed cardiomyocyte cell lines are common models used to understand how (patho)physiological conditions or stimuli contribute to changes in cardiac metabolism. These cell models are helpful also for defining metabolic mechanisms of cardiac dysfunction and remodeling. Although technical advances have improved our capacity to measure cardiomyocyte metabolism, there is often heterogeneity in metabolic assay protocols and cell models, which could hinder data interpretation and discernment of the mechanisms of cardiac (patho)physiology. In this review, we discuss considerations for integrating cardiomyocyte cell models with techniques that have become relatively common in the field, such as respirometry and extracellular flux analysis. Furthermore, we provide overviews of metabolic assays that complement XF analyses and that provide information on not only catabolic pathway activity, but biosynthetic pathway activity and redox status as well. Cultivating a more widespread understanding of the advantages and limitations of metabolic measurements in cardiomyocyte cell models will continue to be essential for the development of coherent metabolic mechanisms of cardiac health and pathophysiology.

Oxidative stress and mitochondrial dysfunction of retinal ganglion cells injury exposures in long-term blue light.

To investigate the phototoxic effect of long-term excessive narrow-band blue light in staurosporine-induced differentiated retinal ganglion cells-5 (SSRGC-5). SSRGC-5 cells were divided into two groups, blue light group (BL group) and control group. Cell viability was assessed by using CCK-8 assay. Metabolic profile analysis was performed by using Seahorse extracellular flux analyzer. Mitochondria ultrastructure were studied via transmission electron microscope (TEM). Mitochondria contents and oxidative stress was evaluated by flow cytometry. Western blotting was performed to monitor the changes in mitogen-activated protein kinases (MAPK) pathway and PI3K/AKT pathway. Blue light caused morphological changes of SSRGC-5 cells. The cell viability was significantly decreased from 3h in BL group. Intercellular ROS and mitochondrial superoxide levels were increased following blue light exposure. Metabolic profiling identified blue light induced SSRGC-5 cells to have severely compromised mitochondrial function. This was accompanied by impaired mitochondrial ultrastructure and remodeling, increased expression of the mitochondrial related proteins, and increased glycolysis as compensation. Moreover, the results showed that blue light induced higher expression of p-p38, p38, p-JNK, p-ERK, p-c-Jun, c-Jun, and p-AKT. These findings indicate that excessive narrow-band blue light induces oxidative stress and mitochondrial metabolic remodeling dysregulate in SSRGC-5 cells. Activated MAPK and AKT signaling pathways are involved in this process.

Palmitic acid methyl ester inhibits cardiac arrest-induced neuroinflammation and mitochondrial dysfunction

We previously discovered that palmitic acid methyl ester (PAME) is a potent vasodilator released from the sympathetic ganglion with vasoactive properties. Post-treatment with PAME can enhance cortical cerebral blood flow and functional learning and memory, while inhibiting neuronal cell death in the CA1 region of the hippocampus under pathological conditions (i.e. cerebral ischemia). Since mechanisms underlying PAME-mediated neuroprotection remain unclear, we investigated the possible neuroprotective mechanisms of PAME after 6 min of asphyxial cardiac arrest (ACA, an animal model of global cerebral ischemia). Our results from capillary-based immunoassay (for the detection of proteins) and cytokine array suggest that PAME (0.02 mg/kg) can decrease neuroinflammatory markers, such as ionized calcium binding adaptor molecule 1 (Iba1, a specific marker for microglia/macrophage activation) and inflammatory cytokines after cardiopulmonary resuscitation. Additionally, the mitochondrial oxygen consumption rate (OCR) and respiratory function in the hippocampal slices were restored following ACA (via Seahorse XF24 Extracellular Flux Analyzer) suggesting that PAME can ameliorate mitochondrial dysfunction. Finally, hippocampal protein arginine methyltransferase 1 (PRMT1) and PRMT8 are enhanced in the presence of PAME to suggest a possible pathway of methylated fatty acids to modulate arginine-based enzymatic methylation. Altogether, our findings suggest that PAME can provide neuroprotection in the presence of ACA to alleviate neuroinflammation and ameliorate mitochondrial dysfunction.

Metabolic Profiling of Female Tg2576 Mouse Brains Provides Novel Evidence Supporting Intranasal Low-Dose Pioglitazone for Long-Term Treatment at an Early Stage of Alzheimer's Disease.

Accumulating evidence suggests that disruptions in brain energy metabolism may be a key player in the pathogenesis of Alzheimer’s disease (AD). Pioglitazone (PIO) has been found to exert beneficial effects on metabolic dysfunction in many AD preclinical studies. However, limited success in clinical trials remains an obstacle to its development for the treatment of AD. PIO’s poor brain penetration was often cited as a contributing factor to the lack of clinical benefit. In this study, we prepared PIO-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles and administered them as suspended nanoparticles via nebulization. Preliminary investigation of drug distribution to the brain revealed comparatively reduced systemic exposure after administering PIO nanoparticles via the intranasal route. In vitro, extracellular flux analysis showed significantly raised spare respiratory capacity when cells were treated with low-dose PIO nanoparticles. Tg2576 transgenic mice treated with low-dose PIO nanoparticles over four months exhibited an overall trend of reduced hyperactivity in open field tests but did not show any visible effect on alternation rates in the Y-maze task. Subsequent 1H NMR-based metabolic profiling of their plasma and different brain regions revealed differences in metabolic profiles in the cerebellum, cortex, and hippocampus of Tg2576 mice after long-term PIO treatment, but not in their midbrain and plasma. In particular, the specificity of PIO’s treatment effects on perturbed amino acid metabolism was observed in the cortex of transgenic mice with increases in alanine and N-acetylaspartate levels, supporting the notion that PIO treatment exerts beneficial effects on impaired energy metabolism associated with AD. In conclusion, inhalation exposure to PIO nanoparticles presents an exciting opportunity that this drug could be administered intranasally at a much lower dose while achieving a sufficient level in the brain to elicit metabolic benefits at an early stage of AD but with reduced systemic exposure.

Mitochondria as a Platform for Dictating the Cell Fate of Cultured Human Corneal Endothelial Cells.

PurposeAiming to clarify the role of mitochondria in cell fate decision of cultured human corneal endothelial cell (cHCEC) subpopulations.MethodsThe mitochondrial respiratory ability were examined with Mito stress and Mito fuel flex test assays using an extracellular flux analyzer (XFe24; Agilent Technologies; Santa Clara, CA) for human corneal endothelium tissues, mature cHCECs and a variety of cell state transitioned cHCECs. Tricarboxylic acid cycle and acetyl-coenzyme A–related enzymes was analyzed by proteomics for cell lysates using liquid chromatography–tandem mass spectrometry for cHCEC subpopulations.ResultsThe maximum oxygen consumption rate was found to become stable depending on the maturation of cHCECs. In the Mito stress tests, culture supplements, epidermal growth factor, SB203580, and SB431543 significantly repressed oxygen consumption rate, whereas a Rho-associated protein kinase inhibitor Y-27632 increased. Tricarboxylic acid cycle and mitochondria acetyl-coenzyme A–related enzymes were selectively upregulated in mature cHCECs, but not in cell state transitioned cHCECs. The maximum oxygen consumption rate was found to be higher in healthy human corneal endothelium tissues than those with deeply reduced cell density. An upregulated tricarboxylic acid cycle was linked with metabolic rewiring converting cHCECs to acquire the mitochondria-dependent oxidative phenotype.ConclusionsMitochondrial metabolic intermediates and energy metabolism are tightly linked to the endothelial cell fate and function. These findings will help us to standardize a protocol for endothelial cell injection.

Maneb alters central carbon metabolism and thiol redox status in a toxicant model of Parkinson's disease

The dithiocarbamate fungicide maneb (MB) has attracted interest due to increasing concern of the negative health effects of pesticides, as well as its association with Parkinson's disease (PD). Our laboratory has previously reported distinct phenotypic changes of neuroblastoma cells exposed to acute, sub-toxic levels of MB, including decreased mitochondrial respiration, altered lactate dynamics, and metabolic stress. In this study, we aimed to further define the specific molecular mechanisms of MB toxicity through the comparison of several thiol-containing compounds and their effects on cellular energy metabolism and thiol redox nodes. Extracellular flux analyses and stable isotope labeled tracer metabolomics were employed to evaluate alterations in energy metabolism of SK-N-AS human neuroblastoma cells after acute exposure of an array of compounds, including dithiocarbamates (maneb, nabam, zineb) and other thiol-containing small molecules (glutathione, N-acetylcysteine). These studies revealed MB and its methylated form (MeDTC) as unique toxicants with significant alterations to mitochondrial respiration, proliferation, and glycolysis. We observed MB to significantly impact cellular thiol redox status by oxidizing cellular glutathione and altering the thiol redox status of peroxiredoxin 3 (Prx3, mitochondrial) after acute exposure. Redox Western blotting revealed a MB-specific modification of cellular Prx3, strengthening the argument that MB can preferentially target mitochondrial enzymes containing reactive cysteine thiols. Further, stable isotope tracer metabolomics confirmed our energetics assessments, and demonstrated that MB exposure results in acute derangement of central carbon metabolism. Specifically, we observed shunting of cellular glucose into the pentose-phosphate pathway and reduction of TCA intermediates derived from glucose and glutamine. Also, we report novel lactate utilization for TCA enrichment and glutathione synthesis after MB exposure. In summary, our results further confirm that MB exerts its toxic effects via thiol modification, and significantly transforms central carbon metabolism.

Alcohol induces mitochondrial derangements in alveolar macrophages by upregulating NADPH oxidase 4.

Excessive alcohol users have increased risk of developing respiratory infections in part due to oxidative stress-induced alveolar macrophage (AM) phagocytic dysfunction. Chronic ethanol exposure increases cellular oxidative stress in AMs via upregulation of NADPH oxidase (Nox) 4, and treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) ligand, rosiglitazone, decreases ethanol-induced Nox4. However, the mechanism by which ethanol induces Nox4 expression and the PPARγ ligand reverses this defect has not been elucidated. Since microRNA (miR)-92a has been predicted to target Nox4 for destabilization, we hypothesized that ethanol exposure decreases miR-92a expression and leads to Nox4 upregulation. Previous studies have implicated mitochondrial-derived oxidative stress in AM dysfunction. We further hypothesized that ethanol increases mitochondrial-derived AM oxidative stress and dysfunction via miR-92a, and that treatment with the PPARγ ligand, pioglitazone, could reverse these derangements. To test these hypotheses, a mouse AM cell line, MH-S cells, was exposed to ethanol invitro, and primary AMs were isolated from a mouse model of chronic ethanol consumption to measure Nox4, mitochondrial target mRNA (qRT-PCR) and protein levels (confocal microscopy), mitochondria-derived reactive oxygen species (confocal immunofluorescence), mitochondrial fission (electron microscopy), and mitochondrial bioenergetics (extracellular flux analyzer). Ethanol exposure increased Nox4, enhanced mitochondria-derived oxidative stress, augmented mitochondrial fission, and impaired mitochondrial bioenergetics. Transfection with a miR-92a mimic invitro or pioglitazone treatment invivo diminished Nox4 levels, resulting in improvements in these ethanol-mediated derangements. These findings demonstrate that pioglitazone may provide a novel therapeutic approach to mitigate ethanol-induced AM mitochondrial derangements.

TRIB3 Promotes Lung Adenocarcinoma Progression via an Enhanced Warburg Effect

BackgroundThe pseudokinase Tribbles 3 (TRIB3) is involved in many cellular processes and various cancers. In recent years, the importance of metabolic transformation in the maintenance of malignant tumors has become increasingly prominent. Abnormal metabolism of cancer cells is considered a hallmark of cancer. However, the exact role and molecular mechanism of TRIB3 in lung adenocarcinoma (LUAD) cell reprogramming is largely unknown.MethodsThe oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells were examined with a Seahorse XF Extracellular Flux Analyzer. In vitro and in vivo RT-qPCR, Western blotting, and functional assays were performed to explore the functional roles of TRIB3 in LUAD.ResultsIn the present study, we demonstrated that TRIB3 is remarkably upregulated in LUAD cell lines as well as tissues. TRIB3 knockdown significantly inhibited LUAD cell growth and suppressed LUAD cell invasion, while TRIB3 overexpression conferred the opposite effects. Moreover, silencing TRIB3 suppressed the tumorigenesis and metastatic ability of LUAD cells. Mechanistically, we demonstrated that silencing TRIB3 significantly impaired aerobic glycolysis ability in LUAD cells. Furthermore, our data indicated that TRIB3 knockdown decreased hypoxia-inducible factor (HIF)1α levels and targeted the glycolytic genes regulated by HIF1α.ConclusionTogether, our findings revealed a previously unappreciated function of TRIB3 in cancer cell metabolism and tumor progression, illustrating that TRIB3 could be considered a valuable therapeutic target for LUAD patients.

CircPVT1 Regulates Cell Proliferation, Apoptosis and Glycolysis in Hepatocellular Carcinoma via miR-377/TRIM23 Axis.

BackgroundRecent studies reported that circular RNAs (circRNAs) exert essential functions in hepatocellular carcinoma (HCC) progression. However, the expression profile and function of circular RNA PVT1 (circPVT1) in HCC are not fully addressed. Thus, we aimed to probe into the function of circPVT1 in HCC development.MethodsThe levels of circPVT1, microRNA-377 (miR-377) and transcripts encoding tripartite motif containing 23 (TRIM23) were determined by qRT-PCR. The stability and localization of circPVT1 were examined by RNase R digestion assay and subcellular fraction assay, respectively. Cell proliferation and apoptosis were evaluated by MTT assay and flow cytometry analysis, respectively. The relationship between miR-377 and circPVT1 or TRIM23 was determined by dual-luciferase reporter assay and RNA immunoprecipitation (RIP). The protein expression of TRIM23 was measured by Western blot. The glycolysis level was assessed by specific kits and Seahorse Extracellular Flux Analyzer XF96. The function of circPVT1 in vivo was investigated in a murine xenograft model.ResultsCircPVT1 and TRIM23 levels were elevated, while miR-377 was decreased in HCC. CircPVT1 knockdown restrained proliferation and glycolysis, but enhanced apoptosis in HCC cells. CircPVT1 could bind to miR-377 and inhibition of miR-377 restored circPVT1 knockdown-mediated effect on HCC cells. TRIM23 was certified as a target of miR-377, and TRIM23 upregulation overturned the influence of miR-377 upregulation or circPVT1 silence on HCC progression. Moreover, circPVT1 knockdown restrained tumor growth in HCC in vivo.ConclusionCircPVT1 aggravated the progression of HCC by upregulating TRIM23 via sponging miR-377.

Anesthesia and surgery induce age‐dependent changes in postoperative delirium behaviors and microbiota

AbstractBackgroundDelirium is the most common postoperative complication in senior patients and is associated with accelerated cognitive decline and Alzheimer's disease and related dementia (ADRD). However, the neuropathogenesis of POD remains mostly unknown and no effective medication to prevent or treat POD. Gut microbiota dysbiosis, a condition of microbiota imbalance or maladaptation inside the gut, which could lead to altered immune functions and associated with disorders of POD. Aging and neuroinflammation may also contribute to the neuropathogenesis of POD. However, while all patients may have neuroinflammation, not all patients develop POD. Thus, neuroinflammation alone (single‐factor model of postoperative delirium) is not sufficient to cause POD. Therefore, the objective of the current study was to test the hypothesis that anesthesia/surgery causes age‐dependent gut microbiota dysbiosis, changes in brain IL‐6 level, and mitochondrial function, leading to postoperative delirium‐like behavior in mice.MethodFemale 9 or 18 months old mice received abdominal surgery under 1.4% isoflurane for two hours. We employed a battery of natural and learned behavioral tests to determine the delirium‐like behavior, assessed the gut microbiota before and after the anesthesia/surgery by gene pyrosequencing of 16S rRNA, and measured levels of brain IL‐6(ELISA), synaptic marker( Western blot ) and mitochondrial function (Seahorse XFp Extracellular Flux Analyzer) as measures of neuroinflammation and cerebral dysfunction. For intervention, intragastric administration of lactobacillus and probiotic were used to mitigate the anesthesia/surgery‐induced above changes.ResultAnesthesia/surgery caused different alterations in gut microbiota, including change rate of reduction in the levels of gut lactobacillus, between the 18 and 9 months old mice. The anesthesia/surgery induced greater postoperative delirium‐like behavior, increased brain IL‐6 levels, decreased PSD‐95 and synaptophysin levels, and mitochondrial dysfunction in 18 than 9 months old mice. Treatments with lactobacillus and probiotic mitigated the anesthesia/surgery‐induced changes.ConclusionWe demonstrated that anesthesia/surgery induces age‐dependent changes in the gut microbiota(reduction in lactobacillus), increases brain levels of IL‐6, decreases levels of synaptic marker, and reduces mitochondrial function while also promoting the development of postoperative delirium‐like behaviors in the mice. Treatment with lactobacillus or probiotic mitigated these detrimental effects, strongly suggesting that abnormalities in the gut microbiota contribute to the development of postoperative delirium.

251 Impacts of different levels of dietary zinc oxide on mitochondrial energy metabolism and oxidative stress conditions in post-weaned piglets

Abstract Zinc oxide (ZnO) is commonly added to weanling piglet diets for its documented benefits as growth promoting agent and usefulness for preventing diarrhea. We have recently reported the occurrence of mitochondrial energetic deficiencies and oxidative stress conditions in post-weaned piglets. In cells, Zn is known to be primarily located in mitochondria, where it is involved in mitochondrial function. The objective of this study was to determine the impacts of different levels of dietary ZnO on mitochondrial function and oxidative stress. One hundred twenty suckling piglets (5.65 ± 0.68 kg) were selected at d14. After weaning (d21), they were fed a basal diet supplemented with 100 mg/kg (low; L), 1000 mg/kg (medium; M) or 3000 mg/kg (high; H) of ZnO until d42 (n = 40/treatment). Piglets were sacrificed at d21, d23, d35, and d42 of age (n = 10/treatment/d) for sample collection. Mitochondrial respiration was evaluated using an extracellular flux analyzer and our results showed higher oxygen consumption rates in liver mitochondria from H group (P &amp;lt; 0.05) at d42. The cellular energy status was characterized by measuring liver adenosine 5’-triphosphate (ATP) content, and lower levels were reported in H group (P &amp;lt; 0.01) at d42. These observations were concomitant with a decrease in activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in cytosol and mitochondria. Quantitative RT-PCR analyses revealed that expression of genes implicated in oxidative stress response such as glutathione synthase (GSS), heme oxygenase 1 (HO1) and metallothionein 3 (MT3) is increased in H group at d42 (P &amp;lt; 0.05). In plasma, GPx activity and oxidative damage to proteins (carbonyls) was higher in H group (P &amp;lt; 0.05). These findings indicate that ZnO affects mitochondrial function and oxidative stress in post-weaned piglets and provide insights to guide the prophylactic practices using high levels of dietary ZnO.

The Effect of Angiotensin II, Retinoic Acid, EGCG, and Vitamin C on the Cardiomyogenic Differentiation Induction of Human Amniotic Fluid-Derived Mesenchymal Stem Cells.

Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may be potentially applied in cell therapy or regenerative medicine as a new alternative source of stem cells. They could be particularly valuable in restoring cardiac tissue after myocardial infarction or other cardiovascular diseases. We investigated the potential of biologically active compounds, namely, angiotensin II, retinoic acid (RA), epigallocatechin-3-gallate (EGCG), vitamin C alone, and the combinations of RA, EGCG, and vitamin C with angiotensin II to induce cardiomyogenic differentiation of AF-MSCs. We observed that the upregulated expression of cardiac gene markers (NKX2-5, MYH6, TNNT2, and DES) and cardiac ion channel genes (sodium, calcium, the potassium) as well as the increased levels of Connexin 43 and Nkx2.5 proteins. Extracellular flux analysis, applied for the first time on AF-MSCs induced with biologically active compounds, revealed the switch in AF-MSCs energetic phenotype and enhanced utilization of oxidative phosphorylation for energy production. Moreover, we demonstrated changes in epigenetic marks associated with transcriptionally active (H3K4me3, H3K9ac, and H4hyperAc) or repressed (H3K27me3) chromatin. All in all, we demonstrated that explored biomolecules were able to induce alterations in AF-MSCs at the phenotypic, genetic, protein, metabolic, and epigenetic levels, leading to the formation of cardiomyocyte progenitors that may become functional heart cells in vitro or in vivo.

Abstract 16939: Metabolic and Electrophysiological Alterations Underlie Proarrhythmic Properties of Highly Reactive Isolevuglandins in Atrial Cardiomyocytes

Background: Hypertension is one of the most common risk factors for atrial fibrillation (AF), although the precise cellular and molecular mechanism(s) by which hypertension leads to AF are not well understood. Isolevuglandins (IsoLGs) are highly reactive dicarbonyl products of lipid peroxidation responsible for a major component of oxidative stress-related injury. In a mouse model of hypertension, we recently demonstrated that IsoLGs are elevated in hypertensive mouse atria and that an IsoLG scavenger reduced both IsoLG burden and AF susceptibility. Hypothesis: In this study, we hypothesized that IsoLGs can promote AF by inducing proarrhythmic metabolic and electrophysiologic (EP) changes in atrial cardiomyocytes. Methods and Results: Using standard patch clamp methods, we found significant changes in action potential properties of isolated mouse atrial cardiomyocytes exposed to IsoLGs (1μM, n=15 cells), including elevation of resting membrane potential, shortening of APD and reduction of V max . Acute IsoLG treatment led to a reduction of intracellular ATP production in atrial HL-1 cardiomyocytes, as measured by using a luminescence assay. Employing TMRM and Mitotracker Green staining for confocal and high-throughput screening (HTS) live-cell imaging assays, we also found that IsoLGs decreased mitochondrial membrane potential (compared to control, TMRM fluorescence decreased by 23%, 28%, 36% and 42%, respectively, when exposed to 0.01, 0.1, 0.5 and 1μM concentrations of IsoLG) accompanied by increased apoptosis (Cell Event Caspase-3/7 Green Detection Reagent) in a concentration-dependent manner, suggesting a prolonged mitochondrial transition pore opening. Moreover, cell metabolism assays performed using Agilent’s Seahorse XF96 extracellular flux analyzer revealed that IsoLGs exert a concentration dependent decrease in basal oxygen consumption rate and ATP production in HL-1 atrial cardiomyocytes. Conclusion: Together, these findings indicate that IsoLGs promote proarrhythmic EP and mitochondrial effects in atrial cells and thus may provide a novel therapeutic target for AF.

Defective immunometabolism pathways in cystic fibrosis macrophages

BackgroundMitochondria play a key role in immune defense pathways, particularly for macrophages. We and others have previously demonstrated that cystic fibrosis (CF) macrophages exhibit weak autophagy activity and exacerbated inflammatory responses. Previous studies have revealed that mitochondria are defective in CF epithelial cells, but to date, the connection between defective mitochondrial function and CF macrophage immune dysregulation has not been fully elucidated. Here, we present a characterization of mitochondrial dysfunction in CF macrophages. MethodsMitochondrial function in wild-type (WT) and CF F508del/F508del murine macrophages was measured using the Seahorse Extracellular Flux analyzer. Mitochondrial morphology was investigated using transmission electron and confocal microscopy. Mitochondrial membrane potential (MMP) as well as mitochondrial reactive oxygen species (mROS) were measured using TMRM and MitoSOX Red fluorescent dyes, respectively. All assays were performed at baseline and following infection by Burkholderia cenocepacia, a multi-drug resistant bacterium that causes detrimental infections in CF patients. ResultsWe have identified impaired oxygen consumption in CF macrophages without and with B. cenocepacia infection. We also observed increased mitochondrial fragmentation in CF macrophages following infection. Lastly, we observed increased MMP and impaired mROS production in CF macrophages following infection with B. cenocepacia. ConclusionsThe mitochondrial defects identified are key components of the macrophage response to infection. Their presence suggests that mitochondrial dysfunction contributes to impaired bacterial killing in CF macrophages. Our current study will enhance our understanding of the pathobiology of CF and lead to the identification of novel mitochondrial therapeutic targets for CF.

TAMI-33. AURKA INHIBITION REPROGRAMS METABOLISM AND IS SYNTHETICALLY LETHAL WITH FATTY ACID OXIDATION INHIBITION IN GLIOBLASTOMA

Abstract Aurora kinase A (AURKA) has emerged as a viable drug target for glioblastoma (GBM), the most common malignant primary brain tumor in adults with a life expectancy of 12-15 months. However, resistance to therapy remains a critical issue, which partially may be driven by reprogramming of metabolism. By integration of transcriptome, chromatin immunoprecipitation with sequencing (CHIP-seq.), assay for transposase-accessible chromatin with sequencing (ATAC-seq.), proteomic and metabolite screening followed by carbon tracing (U-13C-Glucose, U-13C-Glutamine and U-13C-Palmitic acid) and extracellular flux analysis we provided evidence that genetic (shRNA and CRISPR/Cas9) and pharmacological (Alisertib) AURKA inhibition elicited substantial metabolic reprogramming mediated in part by inhibition of MYC targets and concomitant activation of PPARA (e.g. PGC1A) signaling. While glycolysis was suppressed by AURKA inhibition, we noted a compensatory increase in oxygen consumption rate (OCR) fueled by fatty acid oxidation (FAO). Whereas interference with AURKA elicited a suppression of c-Myc, we detected an upregulation of PGC1A, a master regulator of oxidative metabolism, upon AURKA inhibition. Silencing of PGC1A reversed the increase in OCR and sensitized GBM cells to AURKA inhibition mediated reduction in cellular viability. CHIP experiments confirmed binding of c-Myc to the promoter region of PGC1A, which is abrogated by AURKA inhibition and in turn unleashed PGC1A expression. ATAC-seq. confirmed higher accessibility of the MYC binding region within the PGC1A promoter. Forced expression of c-Myc blocked AURKA inhibition mediated increase of PGC1A, suggesting that c-Myc acted as a repressor. To interfere with this oxidative metabolic reprogramming, we combined AURKA inhibitors with blockers of FAO (etomoxir), which elicited substantial synergistic growth inhibition and extension of overall survival in orthotopic patient derived xenografts of GBM in mice without induction of toxicity in normal tissue. Taken together, these data support that simultaneous targeting of oxidative metabolism and AURKA inhibition might be a potential novel therapy against GBM.