Reduced Oxygen Consumption Research Articles

ATP-binding cassette A1 deficiency causes cardiolipin-driven mitochondrial dysfunction in podocytes.

Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1fl/fl) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1fl/fl mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.

Prefrontal cortex activity triggered by affective faces exposure and its relationship with neuroticism

The emotional processing of affective faces is an essential element of social relationships. Individual differences in personality traits such as neuroticism can influence how we manage these interactions. The objective of this study was to analyze changes in prefrontal cortex (PFC) activity in response to visual exposure to affective faces, and to ascertain whether changes in PFC activity were related to scores in neuroticism, including depression, anxiety, low self-esteem and dependence facets. Fifty-two healthy undergraduate female students participated in the present study. Results showed significant differences depending on face valence in the left and right ventrolateral PFC. We found a reduction in oxygen consumption in reaction to neutral and happy faces, and a small increase in oxygenation in reaction to angry faces both in the left and the right PFC. There were significant positive correlations in the left ventrolateral PFC between oxygenation changes during exposure to neutral and happy faces and the neuroticism factor. Anxiety and depression facets showed positive significant correlations with oxygenation changes for all Time blocks. Notice that participants with high neuroticism scores did not show differences in ventrolateral PFC activity depending on face valence. We suggest that PFC would play a protective role in response to emotional stimuli. The reduced regulatory control of PFC over the amygdala could explain vulnerability to emotional disorders in subjects with high neuroticism.

Fatty Acid Profile of Pacific Oyster, Crassostrea gigas, Fed Different Ratios of Dietary Seaweed and Microalgae during Broodstock Conditioning.

The fatty acid (FA) profile of oysters generally reflects the dietary FA composition. Moreover, incorporation of FA into tissues is modulated by various metabolic factors, and final composition will depend upon the dietary sources, cumulative intake, and oysters' development stage. Thus, the aim of this study was to assess the impact of dietary incorporation of seaweed (SW) Ulva rigida, in replacement of traditional microalgae diet, on the FA composition of Pacific oysters Crassostrea gigas, during broodstock conditioning. The dietary conditioning consisted of direct replacement of microalgae (33% Tisochrysis lutea, 50.25% Skeletonema costatum, and 16.75% Chaetoceros calcitrans) by SW at four different substitution levels (0%, 25%, 50%, and 100% diet). The dietary docosahexaenoic acid (DHA) (22:6n-3) and eicosapentaenoic acid (EPA) (20:5n-3) contents showed a positive correlation with the dietary microalgae level. During the trial, oysters fed with higher percentages of microalgae revealed a depletion of DHA and accumulation of EPA. The 100% SW caused a significant reduction in oxygen consumption and, consequently, in the standard metabolic rate. Based on these results, a partial substitution of up to 25% of dietary microalgae seems to be a suitable alternative, because it elicited similar results to the commercial 100% microalgae diet.

Maternal fructose–induced oxidative stress occurs via Tfam and Ucp5 epigenetic regulation in offspring hippocampi

Fructose consumption is rising globally, but maternal high fructose intake might adversely affect offspring. Our previous report demonstrated that excess maternal fructose intake impairs hippocampal function in offspring, indicating that the hippocampi of offspring are highly sensitive to maternal fructose. Here, we examined the effect of maternal high fructose on mitochondrial physiology and uncoupling protein (UCP) expression. Rat dams received a 20% fructose solution during gestation and lactation. Immediately after weaning, offspring hippocampi were isolated. Maternal high fructose consumption attenuated the mitochondrial O2 consumption rate and stimulated lipid hydroperoxide production in the hippocampi of offspring. Reduced Ucp5 and mitochondrial transcription factor A (Tfam) mRNA levels were also observed after maternal exposure to fructose. We assessed the promoter regions of both genes and found that this treatment enhanced DNA methylation levels. In addition, luciferase assays showed that this DNA methylation could reduce the transcription of both genes. Chromatin immunoprecipitation analysis demonstrated that specificity protein 1 binding to the Ucp5 promoter regions was reduced by DNA methylation. In addition, Ucp5 knockdown induced the up-regulation of reactive oxygen species levels in a rat brain glioma cell line, whereas reduced O2 consumption was observed with Tfam knockdown. Maternal high fructose intake thus induces reduced O2 oxygen consumption and increases oxidative stress in offspring, at least partly through epigenetic mechanisms involving Ucp5 and Tfam.-Yamada, H., Munetsuna, E., Yamazaki, M., Mizuno, G., Sadamoto, N., Ando, Y., Fujii, R., Shiogama, K., Ishikawa, H., Suzuki, K., Shimono, Y., Ohashi, K., Hashimoto, S. Maternal fructose-induced oxidative stress occurs via Tfam and Ucp5 epigenetic regulation in offspring hippocampi.

Abstract 2720: Mitochondrial ClpP-mediated proteolysis induces selective cancer cell lethality

Abstract ClpP is a mitochondrial protease and a major protein quality control mediator that primarily interacts with metabolic enzymes in mitochondria. Here, we demonstrate that activation of this protease results in prominent anti-cancer activity, and propose ClpP activation as a novel therapeutic strategy for cancer and hematologic malignancies. We used genetic and chemical tools to activate ClpP. In a genetic approach, we tested the anti-cancer effects of ClpP activation by expressing a constitutively active ClpP mutant. Indeed, induction of the active ClpP mutant induced apoptosis in vitro and inhibited tumor progression in vivo. To further explore the antineoplastic effects of ClpP activation, we then performed a chemical screen of an in-house library of on-patent and off-patent drugs and identified imipridones (ONC201 and ONC212) as potent ClpP agonists. Imipridones are first-in-class antineoplastic agents and have shown preclinical efficacy in various malignancies in vitro and in vivo and are currently being evaluated in clinical trials in a diverse spectrum of cancers. Importantly, we and others have shown that their activity is agnostic to TP53 mutational status. Of note, molecular targets of imipridones that bind the drugs and are functionally important for their cytotoxicity have never been identified. Through extensive chemical investigations, including analysis of binding mechanism of the compounds to ClpP in cell free (ITC) and cell based assays (CETSA) as well as molecular analysis of the crystal structure, we demonstrate that these molecules bind ClpP non-covalently, and activate the protease by stabilizing the ClpP 14-mer, enlarging the axial pores of the complex, and inducing structural changes in the residues surrounding and including the catalytic triad. In leukemia, lymphoma and colon cancer cells including primary acute myeloid leukemia (AML) cells, both compounds displayed potent ClpP-dependent cytotoxicity with IC50s in low micro- or nanomolar ranges. Importantly, in primary AML samples, pretreatment ClpP levels correlated with response to imipridones. In lymphoma and AML xenograft models, both genetic and chemical activation of ClpP resulted in antitumor effects, while expression of inactive D190A ClpP mutant induced resistance. Mechanistically, ClpP activation leads to increased degradation of substrates of the enzyme including respiratory chain complex subunits and mitochondrial translation system. The resultant impaired mitochondrial structure and reduction in oxygen consumption is selectively cytotoxic to malignant cells that rely highly on mitochondrial energy production for their survival, whereas normal cells are not affected. In conclusion, ClpP activation is an entirely novel therapeutic strategy for malignant tumors. Our findings also suggest a general concept of inducing TP53-independent cancer cell lethality through activation of mitochondrial proteolysis. Citation Format: Jo Ishizawa, Sarah F. Zarabi, R Eric Davis, Ondrej Halgas, Takenobu Nii, Yulia Jitkova, Ran Zhao, Jonathan St-Germain, Lauren E. Heese, Grace Egan, Vivian R. Ruvolo, Samir H. Barghout, Yuki Nishida, Rose Hurren, Wencai Ma, Marcela Gronda, Todd Link, Keith Wong, Mark Mabanglo, Kensuke Kojima, Gautam Borthakur, Neil MacLean, John Man Chun Ma, Andrew B. Leber, Mark D. Minden, Walid Houry, Hagop Kantarjian, Martin Stogniew, Brian Raught, Emil F. Pai, Aaron D. Schimmer, Michael Andreeff. Mitochondrial ClpP-mediated proteolysis induces selective cancer cell lethality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2720.

Abstract 2716: Autophagy modulates lipid metabolism in liver kinase B1 (LKB1)-deficient Kras-driven lung tumorigenesis

Abstract Tumor suppressor liver kinase B1 (LKB1) activates 5’-adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non-small cell lung cancer (NSCLC). Compared to p53 mutation, mutations in LKB1 causes more aggressive tumor growth and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1-deficient NSCLC treatment is urgently needed. Autophagy degrades and recycles macromolecules for cells to survive starvation. Using genetically engineered mouse models for NSCLC with Kras activation and LKB1 loss (KL), we found that autophagy (Atg7) deficiency prevents tumor initiation, reduced tumor growth and extended the mouse lifespan compared to mice bearing Atg7 wild-type (WT) tumors. Most importantly, tumor growth inhibition by Atg7 deficiency was much more dramatic in KL than Kras/p53 (KP) lung tumors. Furthermore, we generated mouse tumor-derived cell lines (TDCLs) and found that Atg7 null TDCLs were more sensitive to starvation-induced cell death than Atg7 WT TDCLs, which can be rescued by palmitate supplementation, indicating that autophagy is required to maintain free fatty acid (FA) level for cells to survive starvation. We further performed 13C glucose tracing study and found that Atg7 deletion increased de novo FA synthesis and reduced FAs elongation. Fasnall, a selective FA synthase inhibitor, inhibited Atg7 null cell proliferation. Treatment with Orlistat, a lipase inhibitor, did not impact cell proliferation in nutrient-rich conditions but accelerated starvation-induced Atg7 null cell death, indicating that lipase-medicated lipid degradation compensates for autophagy loss to provide free FAs in starvation. A carnitine palmitoyltransferase I inhibitor, Etomoxir treatment, accelerated the starvation-induced Atg7 null cell death and reduced oxygen consumption rate (OCR) of Atg7 null cells in starvation, compared with Atg7 WT cells, indicating that autophagy-deficient cells rely more on FA oxidation for energy production in nutrient deprivation. To probe the significance of autophagy in human lung cancer cell lines with LKB1 mutation, we knocked down ATG7 using siRNA. ATG7 knockdown (KD) inhibited clonogenic survival of LKB1 mutant cells in starvation, but had no effect in LKB1 WT cells. Supplementation of glutamine or palmitate rescued the survival of LKB1 mutant cells with ATG7 KD in starvation and maintained OCR, demonstrating that KRAS-driven human lung cancer cells with mutant LKB1 rely on autophagy to maintain mitochondrial metabolism to survive energy crises. Taken together, our results demonstrate that autophagy modulates lipid metabolism for KL cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to specifically treat LKB1-deficient NSCLC. Citation Format: Vrushank D. Bhatt, Khoosheh Khayati, Zhixian Hu, Xiaoyang Su, Jessie Yanxiang Guo. Autophagy modulates lipid metabolism in liver kinase B1 (LKB1)-deficient Kras-driven lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2716.

Abstract LB-023: CYP monooxygenases regulate nuclear localization of ERá and mTORC1 component RagC in ER+HER2- breast cancer cells

Abstract Introduction: Cancer cell-intrinsic CYP monooxygenases promote tumor progression. In ER+ breast cancer cells, CYP3A4 is required for tumor growth and localizes to mitochondria where it promotes electron transport chain and respiration, while suppressing autophagy, in part, through epoxyeicosatrienoic acid (EET) biosynthesis (1). The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 arachidonic acid epoxygenase activity, reduced oxygen consumption rates and suppressed growth of breast cancer cell lines (IC50=3-30 μM) and an established ER+ mammary tumor model. HBB inhibited activation of mTOR and suppressed S6Kinase phosphorylation. HBB also suppressed intratumoral mTOR. Recently, nuclear transit of RagC has been shown to promote mTORC1 function, a process sensitive to metformin (2), which inhibits the nuclear pore complex (NPC). Therefore, we hypothesized that HBB as a more potent biguanide may inhibit mTOR through reduction of nuclear RagC by regulating NPC. HBB may also regulate the localization of other nuclear proteins such as estrogen receptor alpha (ERα). Method: ER+HER2- breast cancer cells (MCF-7 and T47D) were seeded on chamber slides and treated with vehicle or HBB for varied periods of time. Cells were fixed, permeabilized, and incubated with specific antibodies against RagC and ERα. After incubation with fluorescence tagged secondary antibody, slides were stained with DAPI and analyzed by confocal microscopy. Levels of nuclear proteins of interest were normalized against DAPI staining. Results: CYP3A4 shRNA knockdown reduced nuclear RagC in MCF-7 cells by 61.2±12.3 % (n=3, p<0.05) relative to a non-targeted shRNA control line. HBB treatment for 6 hours (20 μM) reduced nuclear RagC by 66.1±15.4% (n=4, p<0.001) in MCF-7 cells and by 32.4±5.1% (n=3, p<0.001) in T47D cells. HBB treatment for 6 hours (20 μM) also reduced nuclear ERα by 36.8±11.1% (n=6, p<0.001) in MCF-7 cells and by 32.2±8.1% (n=3, p<0.01) in T47D cells grown in complete media. HBB treatment for 1 hour (20 μM) reduced passive transport of 70 kDa FITC-dextran through the NPC into the nuclear compartment by 17.5±3.6% (n=3, p<0.01) in MCF-7 cells and by 28.3±3.2% (n=3, p<0.01) in T47D cells. Immunofluorescence study results of HBB-mediated reduction of nuclear RagC and ERα were confirmed by Western-blotting analysis of nuclear extraction of T47D cells treated with HBB. Conclusion: These data suggest that the biguanide sensitive enzyme CYP3A4 may play a role in nuclear localization of ERα and RagC, which work in tandem to promote the growth of ER+HER2- breast cancer cells. 1. Cell Chemical Biology. 2017 Oct 19; 24(10) 1259 – 1275. 2. Cell. 2016 Dec 15;167(7):1705-1718 Citation Format: Zhijun Guo, Shaoping Wu, Julissa Molina-Vega, Rafael Castillo, Jaime Barrera, Veronica Johnson, Carol Lange, David Potter. CYP monooxygenases regulate nuclear localization of ERá and mTORC1 component RagC in ER+HER2- breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-023.

Abstract 1830: Role of heat shock factor 1 (HSF1) on metabolic reprogramming of T -cells

Abstract Heat shock transcription factor 1 (HSF1) is normally activated in response to a variety of environmental stressors. Higher levels of HSF1 expression has been shown in many tumor types and this is associated with poor prognosis. Previous data from our laboratory indicate that genetic inactivation of HSF1 significantly delays ERBB2-induced mammary tumorigenesis and hepatocellular carcinoma induced by chemical carcinogen in mouse models. We have also shown that HSF1 is involved in controlling cellular bioenergetics. CD8-positive T cells plays an important role in anti-tumor immunity response. Our preliminary results show that genetic inactivation of HSF1 in T-cells leads to reduced oxygen consumption rate (OCR) as well as Extracellular Acidification Rate (ECAR). We have also observed that deletion of HSF1 from T cells delayed in-vitro activation of purified CD8-positive T cells upon stimulation with anti-CD3 and anti-CD28. Above characteristics corresponds to reduced cellular glycolysis and T cell proliferation. Further studies will be directed towards exploring the effects of HSF1 deletion in T cell activation induced anti-tumor immunity. This study is supported by the NCI grants; NCICA062130 and NCICA132640. Citation Format: Bhaumik D. Pandya, Nahid M. Mivechi, Dimitrios Moskophidis. Role of heat shock factor 1 (HSF1) on metabolic reprogramming of T -cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1830.

Oxygen-enhanced MRI Is Feasible, Repeatable, and Detects Radiotherapy-induced Change in Hypoxia in Xenograft Models and in Patients with Non-small Cell Lung Cancer.

Hypoxia is associated with poor prognosis and is predictive of poor response to cancer treatments, including radiotherapy. Developing noninvasive biomarkers that both detect hypoxia prior to treatment and track change in tumor hypoxia following treatment is required urgently. We evaluated the ability of oxygen-enhanced MRI (OE-MRI) to map and quantify therapy-induced changes in tumor hypoxia by measuring oxygen-refractory signals in perfused tissue (perfused Oxy-R). Clinical first-in-human study in patients with non-small cell lung cancer (NSCLC) was performed alongside preclinical experiments in two xenograft tumors (Calu6 NSCLC model and U87 glioma model). MRI perfused Oxy-R tumor fraction measurement of hypoxia was validated with ex vivo tissue pathology in both xenograft models. Calu6 and U87 experiments showed that MRI perfused Oxy-R tumor volume was reduced relative to control following single fraction 10-Gy radiation and fractionated chemoradiotherapy (P < 0.001) due to both improved perfusion and reduced oxygen consumption rate. Next, evaluation of 23 patients with NSCLC showed that OE-MRI was clinically feasible and that tumor perfused Oxy-R volume is repeatable [interclass correlation coefficient: 0.961 (95% CI, 0.858-0.990); coefficient of variation: 25.880%]. Group-wise perfused Oxy-R volume was reduced at 14 days following start of radiotherapy (P = 0.015). OE-MRI detected between-subject variation in hypoxia modification in both xenograft and patient tumors. These findings support applying OE-MRI biomarkers to monitor hypoxia modification, to stratify patients in clinical trials of hypoxia-modifying therapies, to identify patients with hypoxic tumors that may fail treatment with immunotherapy, and to guide adaptive radiotherapy by mapping regional hypoxia.

Understanding ‘ride’ perception in running using reliable testers: ankle joint mechanics matter

Improved dynamic comfort has been associated with reduced oxygen consumption (Luo, Stergiou, Worobets, Nigg, & Stefanyshyn, 2009). As a matter of fact, improving comfort during running is important...

From DNA to ecological performance: Effects of anthropogenic noise on a reef-building mussel

Responses of marine invertebrates to anthropogenic noise are insufficiently known, impeding our understanding of ecosystemic impacts of noise and the development of mitigation strategies. We show that the blue mussel, Mytilus edulis, is negatively affected by ship-noise playbacks across different levels of biological organization. We take a novel mechanistic multi-method approach testing and employing established ecotoxicological techniques (i.e. Comet Assay and oxidative stress tests) in combination with behavioral and physiological biomarkers. We evidence, for the first time in marine species, noise-induced changes in DNA integrity (six-fold higher DNA single strand-breaks in haemocytes and gill epithelial cells) and oxidative stress (68% increased TBARS in gill cells). We further identify physiological and behavioral changes (12% reduced oxygen consumption, 60% increase in valve gape, 84% reduced filtration rate) in noise-exposed mussels. By employing established ecotoxicological techniques we highlight impacts not only on the organismal level, but also on ecological performance. When investigating species that produce little visually obvious responses to anthropogenic noise, the above mentioned endpoints are key to revealing sublethal effects of noise and thus enable a better understanding of how this emerging, but often overlooked stressor, affects animals without complex behaviors. Our integrated approach to noise research can be used as a model for other invertebrate species and faunal groups, and inform the development of effective methods for assessing and monitoring noise impacts. Given the observed negative effects, noise should be considered a potential confounding factor in studies involving other stressors.

Selenium-Binding Protein 1 Reduces Oxygen Consumption and Activates AMPK in Prostate Cancer Cells (OR11-04-19)

ObjectivesSelenium (Se) is a non-metallic, essential trace element for many organisms, including humans. Se may have potential in cancer prevention, as evidenced by multiple animal and human epidemiological studies. Selenium-binding protein 1 (SBP1) is a highly-conserved protein that covalently binds Se. Prostate cells exhibit cytoplasmic and nuclear SBP1 localization. Reduced levels of nuclear SBP1 were previously shown to be associated with a higher tumor grade and a greater likelihood of prostate cancer recurrence following prostatectomy. The Krebs cycle of the normal prostate is inhibited in favor of the production of citrate energy, therefore distinguishing the energy metabolism of the normal prostate from that of other organs. This inhibition is generally relieved during cancer progression. The objective of this study was to investigate a contribution of SBP1 in this process of metabolic shift. MethodsHuman PC-3 prostate cancer cells that express very low levels of SBP1 were engineered to express the native, nuclear-targeted, or nuclear-excluded SBP1 by transfection of constitutive- or inducible-SBP1 expression constructs. Oxidative phosphorylation (OXPHOS) was examined by quantifying the oxygen consumption rate (OCR) using a Seahorse XF analyzer (Agilent, Inc.). Western blotting with SBP1 and phospho-AMPK specific antibodies was employed to interrogate the activation of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis. ResultsOverexpressing SBP1 in PC-3 cells increased the activating phosphorylation of AMPK at Thr172 by approximately 70%. In addition, overexpressing the native, nuclear-targeted or nuclear-excluded SBP1 significantly reduced OCR and mitochondrial ATP synthesis, as measures of mitochondrial respiration (32–43% reduction in basal respiration and 40–55% reduction in maximal respiration). These changes in energy metabolism occurred without affecting cellular proliferation. ConclusionsThese results indicate that the loss of SBP1 during prostate cancer development may contribute to disease progression by facilitating the transition to an energy metabolism that favors increased energy production as well as the building blocks required to sustain tumor growth and survival. Funding SourcesNational Institute of Health.

Diminished Cardiovascular Performance That Persists Up To 3 Days After Completion Of Repeated 6-hour Hyperoxic Resting Dives At 1.35 ATA Is Associated With Reduced Oxygen Consumption

Previous measures of cardiovascular endurance after repeated dives on air and 100% oxygen at 1.35 atmospheres absolute (ATA) have shown reductions in performance that persist up to 3 days post-dive for the hyperoxic group only. PURPOSE: Examine ventilatory parameters associated with changes in treadmill endurance immediately and 3 days after completing repeated air and oxygen resting dives. METHODS: 15 and 12 healthy male Navy divers completed 5 consecutive 6-hr dives with 18-hr surface intervals while breathing air and 100% oxygen, respectively, at 1.35 ATA (Air/Oxygen: 30±5/32±7 yrs; VO 2max: 52±7/53±5 ml/kg/min; mean±SD). Treadmill endurance time at 80-85% of VO 2max and associated physiological variables were tested a few days prior to the first dive (BL), 2 hours post-dive day 5 (PD) and 3-days post-dive (PD3) on day 8. Breath-by-breath (Innocor) minute ventilation (VE), end-tidal CO2 (FETCO2), oxygen consumption (VO 2), and respiratory quotient (R) were collected during the 80-85% VO 2max run at 10% grade until exhaustion. PD and PD3 were compared to BL (BL-PD and BL-PD3) using data from the first minute, mid-run, and last minute of the run. RESULTS: PD cardiovascular endurance significantly decreased for Air and Oxygen phases (Air: -34%; Oxygen: -36%; p<0.05), yet only the Oxygen phase remained reduced PD3 (Air: -11.9%; p>0.05; Oxygen: -31%; p<0.05). VE increased significantly during the run, but was not different between Air and Oxygen phases or across testing days. Although FETCO2 decreased across run times and testing days for Air and Oxygen, values for the Oxygen group remained lower than Air throughout (p<0.05). PD VO 2 showed decline in both groups (Air/Oxygen: PD -7%; p<0.05). VO 2 for Air recovered by PD3, whereas Oxygen remained reduced (Air: +1%; Oxygen: -13%; p<0.05). PD R increased overall for both groups, yet Air returned to baseline while Oxygen remained elevated by PD3 (Air: PD: +4%; p<0.05; Oxygen: PD +7%, PD3 +4%; p<0.05). CONCLUSIONS: Treadmill endurance is reduced after long-duration diving and persists longer when exposed to hyperoxic diving conditions. Reduced VO 2 and elevated R has been reported in the literature to correlate with increased lactate production and a greater reliance on anaerobic energy systems making them likely causes of this decrease in aerobic performance.

The transcriptomic responses of the ark shell, Anadara broughtonii, to sulfide and hypoxia exposure.

Sulfide and hypoxia threaten marine organisms in various ways. Anadara broughtonii, a commercial marine bivalve in China which has great potential exposure to sulfide and hypoxia, was selected to test the responses to these stresses. Digital gene expression profile (DGE) analysis was performed on the juveniles' gills after exposed to normal condition (CG group), hypoxia (LO group), and low/high concentration of sulfide (LS/HS group, administered in hypoxia), respectively, using RNA-seq technology. A total of over 30 million clean reads were filtered from each DGE library and over 90% of them were annotated successfully. In total, 774 significant differentially expressed genes (DEGs) were detected and assigned to Gene ontology (GO) classification and KEGG Pathway enrichment analysis. The results show that many of the upregulated DEGs are related to hemoglobin, immunology, and stress responding. In the stressed A. broughtonii, cytochrome P450 and phosphoenolpyruvate carboxykinase may stimulate the glycolysis process to reduce oxygen consumption; Aminoacyl-tRNA synthetases, heat shock protein and protein disulfide isomerase probably help to maintain the genome integrity; Baculoviral IAP repeat-containing protein 2/3, mitogen-activated protein kinase and tumor necrosis factor pathways were probably responsible for protein repair, proteolysis, apoptosis and immune responses to high concentration of sulfide. Combined challenges also induced alternative oxidase and sushi repeat-containing protein, which have indistinct but probably indispensable function in invertebrates. For the first time, comprehensive transcriptome information on A. broughtonii in response to sulfide and hypoxia were provided. Our research offers new insights into the molecular mechanism behind the resistance of shellfish to sulfide and hypoxia.

Elevated Heme Synthesis and Uptake Underpin Intensified Oxidative Metabolism and Tumorigenic Functions in Non–Small Cell Lung Cancer Cells

Tumors of human non-small cell lung cancer (NSCLC) are heterogeneous but exhibit elevated glycolysis and glucose oxidation relative to benign lung tissues. Heme is a central molecule for oxidative metabolism and ATP generation via mitochondrial oxidative phosphorylation (OXPHOS). Here, we showed that levels of heme synthesis and uptake, mitochondrial heme, oxygen-utilizing hemoproteins, oxygen consumption, ATP generation, and key mitochondrial biogenesis regulators were enhanced in NSCLC cells relative to nontumorigenic cells. Likewise, proteins and enzymes relating to heme and mitochondrial functions were upregulated in human NSCLC tissues relative to normal tissues. Engineered heme-sequestering peptides (HSP) reduced heme uptake, intracellular heme levels, and tumorigenic functions of NSCLC cells. Addition of heme largely reversed the effect of HSPs on tumorigenic functions. Furthermore, HSP2 significantly suppressed the growth of human NSCLC xenograft tumors in mice. HSP2-treated tumors exhibited reduced oxygen consumption rates (OCR) and ATP levels. To further verify the importance of heme in promoting tumorigenicity, we generated NSCLC cell lines with increased heme synthesis or uptake by overexpressing either the rate-limiting heme synthesis enzyme ALAS1 or uptake protein SLC48A1, respectively. These cells exhibited enhanced migration and invasion and accelerated tumor growth in mice. Notably, tumors formed by cells with increased heme synthesis or uptake also displayed elevated OCRs and ATP levels. These data show that elevated heme flux and function underlie enhanced OXPHOS and tumorigenicity of NSCLC cells. Targeting heme flux and function offers a potential strategy for developing therapies for lung cancer. SIGNIFICANCE: These findings show that elevated heme availability due to increased heme synthesis and uptake causes intensified oxygen consumption and ATP generation, promoting tumorigenic functions and tumor growth in NSCLC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2511/F1.large.jpg.

A traceable nanoplatform for enhanced chemo-photodynamic therapy by reducing oxygen consumption

Tumor hypoxia impedes the efficiencies of oxygen-dependent photodynamic therapy (PDT) and chemotherapy. Herein, we design a traceable nanoplatform (DOX/Met/BSA-HA-CDs) by reducing oxygen (O2) consumption to overcome the hypoxia-caused cancer therapy. Carbon dots (CDs) are used not only as a PDT agent but also applied for in vivo traceable imaging. Metformin (Met), a potent antihyperglycemic agent, to improve tumor oxygenation and enhance the efficiencies of hypoxia-caused cancer therapy. In the hypoxic tumor microenvironment, Met was released more rapidly than DOX, which is advantageous for improving hypoxic cancer to exert a better therapeutic efficiency. Ex vivo immunofluorescence staining revealed that the DOX/Met/BSA-HA-CDs nanoparticles greatly reduce O2 consumption in tumor site. Followed by in vivo synergistic treatment achieved considerably enhanced cancer therapeutic efficiency. This system holds great clinical promise as a traceable imaging approach to guide the improvement of PDT and chemotherapy efficiencies through utilizing a simple, safe method improved hypoxic tumor microenvironment.

Romelt Process Production Indices with Partial Replacement of Coal by Natural Gas

Results are provided for calculation of the material and heat balances of cast iron smelting in a Romelt furnace using coal with a different fixed carbon content with addition of natural gas to the blast of the lower tuyeres. It is shown that under constant heat flow conditions due to gas combustion in the slag bath an increase in natural gas consumption is accompanied by a reduction in coal consumption. In this case saving coal does not depend on its form and is proportional to natural gas consumption. An increase in natural gas consumption is accompanied by a reduction in oxygen consumption in the lower tuyeres and an increase in the upper tuyeres. Saving oxygen in the Romelt process is at a maximum for coal with a low fixed carbon content. It decreases as there is an increase in fixed carbon content and with ≈ 75% Cfix saving oxygen is replaced by a small over-consumption.

Retinal oximetry: Metabolic imaging for diseases of the retina and brain.

Retinal oximetry imaging of retinal blood vessels measures oxygen saturation of hemoglobin. The imaging technology is non-invasive and reproducible with remarkably low variability on test-retest studies and in healthy cohorts. Pathophysiological principles and novel biomarkers in several retinal diseases have been discovered, as well as possible applications for systemic and brain disease. In diabetic retinopathy, retinal venous oxygen saturation is elevated and arteriovenous difference progressively reduced in advanced stages of retinopathy compared with healthy persons. This correlates with pathophysiology of diabetic retinopathy where hypoxia stimulates VEGF production. Laser treatment and vitrectomy both improve retinal oximetry values, which correlate with clinical outcome. The oximetry biomarker may allow automatic measurement of severity of diabetic retinopathy and predict its response to treatment. Central retinal vein occlusion is characterized by retinal hypoxia, which is evident in retinal oximetry. The retinal hypoxia seen on oximetry correlates with the extent of peripheral ischemia, visual acuity and thickness of macular edema. This biomarker may help diagnose and measure severity of vein occlusion and degree of retinal ischemia. Glaucomatous retinal atrophy is associated with reduced oxygen consumption resulting in reduced arteriovenous difference and higher retinal venous saturation. The oximetry findings correlate with worse visual field, thinner nerve fiber layer and smaller optic disc rim. This provides an objective biomarker for glaucomatous damage. In retinitis pigmentosa, an association exists between advanced atrophy, worse visual field and higher retinal venous oxygen saturation, lower arteriovenous difference. This biomarker may allow measurement of severity and progression of retinitis pigmentosa and other atrophic retinal diseases. Retinal oximetry offers visible light imaging of systemic and central nervous system vessels. It senses hypoxia in cardiac and pulmonary diseases. Oximetry biomarkers have been discovered in Alzheimer's disease and multiple sclerosis and oxygen levels in the retina correspond well with brain.

Sex Differences in Type‐2 Diabetes: Implications for Caveolin‐3 Regulated Mitochondrial Function

IntroductionFemale diabetic patients have worse cardiovascular disease outcomes when compared to diabetic males. The underlying molecular mechanism responsible for this sex‐specific disease progression is still unclear. Caveolins are ubiquitously expressed membrane localized signaling platforms that regulate cardiac stress responses. Cardiac specific overexpression of caveolin‐3 (Cav3 OE) was shown to protect the heart from stressors such as ischemia or pressure‐overload.HypothesisSince we previously reported beneficial effects of cardiac specific Cav3 OE during cardiac stress adaptation, we tested the rationale that diabetic Cav3 OE female mice may be protected from diabetes‐induced mitochondrial dysfunction.Methods11–14‐month (m)‐old male and female Cav3 OE mice and transgene negative littermates (Tg‐neg) were injected with a single dose of streptozotocin (non‐fasted 75mg/kg i.p.) or citrate buffer and either placed on a 60% kcal high‐fat diet (HFD, TD 6414) to induce diabetes or placed on a 4% kcal low fat diet (LFD, TD 7001) as control. Food intake was monitored weekly and body weight assessed in two‐week intervals. Glucose tolerance testing (GTT) was performed 4 month‐post diabetes induction. 5m post diabetes induction, cardiac function was determined by echocardiography. Animals were sacrificed 6m post HFD and cardiac tissues harvested for ultrastructural analysis and high‐resolution respirometry on left ventricular free wall.ResultsHFD induced similar weight gain in mid‐age male and female Cav3 OE and Tg‐neg mice. In both sexes and genotypes GTT showed a comparable increase in glucose levels 4m post HFD. No differences in cardiac contractility (%EF) or diastolic function (E/A, and E′/A′), were detected between the four groups 5m post HFD. We identified cardiac hypertrophy with preserved systolic and diastolic function in male and female Tg‐neg mice on HFD while male and female Cav3OE mice did not show cardiac hypertrophy. Swollen cristae and diffuse mitochondrial inner and outer membrane structures were found in male and female Tg‐neg mice on HFD, but in Cav3 OE mice mitochondrial ultrastructure was preserved in both sexes. Mitochondrial function was preserved in all male T2DM hearts while reduced oxygen consumption was found in female Tg‐neg T2DM but not in female Cav3 OE T2DM.ConclusionWe show for the first time that mid‐age diabetic female mice present mitochondrial dysfunction earlier than male diabetic animals and Cav3 OE protects females from cardiac hypertrophy and mitochondrial dysfunction. The findings here mimic the clinical phenomenon that female diabetic patients show worsened cardiovascular outcomes. Therefore, Cav3 may present a novel target to protect the female heart from diabetes induced metabolic injury.Support or Funding InformationNational Institutes of Health (HL091071, AG052722) and the Veterans Administration (BX001963) to HHPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autophagy modulates lipid metabolism in Liver Kinase B1 (LKB1)‐deficient Kras‐driven lung tumorigenesis

Tumor suppressor liver kinase B1 (LKB1) activates 5′‐adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non‐small cell lung cancer (NSCLC). Compared to p53 mutation, mutations in LKB1 causes more aggressive tumor growth and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1‐deficient NSCLC treatment is urgently needed. Autophagy degrades and recycles macromolecules for cells to survive starvation. Using genetically engineered mouse models for NSCLC with Kras activation and LKB1 loss (KL), we found that autophagy (Atg7) deficiency prevents tumor initiation, reduced tumor growth and extended the mouse lifespan compared to mice bearing Atg7 wild‐type (WT) tumors. Most importantly, tumor growth inhibition by Atg7 deficiency was much more dramatic in KL than Kras/p53 (KP) lung tumors. Furthermore, we generated mouse tumor‐derived cell lines (TDCLs) and found that Atg7 null TDCLs were more sensitive to starvation‐induced cell death than Atg7 WT TDCLs, which can be rescued by palmitate supplementation, indicating that autophagy is required to maintain free fatty acid (FA) level for cells to survive starvation. We further performed 13C glucose tracing study and found that Atg7 deletion increased de novo FA synthesis and reduced FAs elongation. Fasnall, a selective FA synthase inhibitor, inhibited Atg7 null cell proliferation. Treatment with Orlistat, a lipase inhibitor, did not impact cell proliferation in nutrient‐rich conditions but accelerated starvation‐induced Atg7 null cell death, indicating that lipase‐medicated lipid degradation compensates for autophagy loss to provide free FAs in starvation. A carnitine palmitoyltransferase I inhibitor, Etomoxir treatment, accelerated the starvation‐induced Atg7 null cell death and reduced oxygen consumption rate (OCR) of Atg7 null cells in starvation, compared with Atg7 WT cells, indicating that autophagy‐deficient cells rely more on FA oxidation for energy production in nutrient deprivation. To probe the significance of autophagy in human lung cancer cell lines with LKB1 mutation, we knocked down ATG7 using siRNA. ATG7 knockdown (KD) inhibited clonogenic survival of LKB1 mutant cells in starvation but had no effect in LKB1 WT cells. Supplementation of glutamine or palmitate rescued the survival of LKB1 mutant cells with ATG7 KD in starvation and maintained OCR, demonstrating that KRAS‐driven human lung cancer cells with mutant LKB1 rely on autophagy to maintain mitochondrial metabolism to survive energy crises. Taken together, our results demonstrate that autophagy modulates lipid metabolism for KL cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to specifically treat LKB1‐deficient NSCLC.Support or Funding InformationThis research has been supported by the research funds from K22 CA190521 (NIH) grant, American Cancer Society Early Investigator Pilot Award, Lung Cancer Research Foundation grant and startup funding from Rutgers Cancer Institute of New Jersey.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.