Mitochondrial Respiration Rate Research Articles

Abstract 10422: Sesn2 Mediates Metformin Rescued Age-Related Cardiac Dysfunctions of Cardiorenal Syndrome

Introduction: Cardiorenal syndrome (CRS) consists of disorders of the kidneys and heart, whereby dysfunction of one organ may induce dysfunction of the other. Acute kidney injury (AKI) leads to a reduction in blood flow and energy supply to the heart. AMP-activated protein kinase (AMPK) as an energy sensor regulates energy metabolism via interacting with a scaffold protein Sesn2. The anti-diabetes drug metformin is an AMPK agonist, and it has been reported to regulate substrate metabolism under physiological and pathological conditions. But the beneficial role of metformin in CRS remains unknown. Hypothesis: Age-related impaired AMPK signaling contributes to cardiac intolerance to AKI-induced insults in the elderly. Metformin treatment ameliorates CRS through triggering Sens2/AMPK activation. Methods: Young (3-5 months) and aged (21-23 months) C57BL/6J wild type (WT), cardiomyocyte specific knock out (cSesn2 -/- ) and littermates (Sesn2 f/f ) C57BL/6J mice were subjected to AKI with a laparotomy and the renal artery and vein were clamped for 15 min followed by 72 hr of reperfusion (IRI). Seahorse XF Analyzer for mitochondrial respirational functions. Results: The higher levels of serum creatinine were observed after IRI-induced AKI in the aged versus young WT and cSens2 -/- versus Sesn2 f/f mice. Interestingly, administration of metformin can significantly reduce serum creatinine levels by IRI-induced AKI in young/aged WT and Sesn2 f/f but not cSesn2 -/- mice. Renal histological analysis showed higher incidence of tubular necrosis in the aged versus young AKI mice, the tubular necrosis was significantly reduced in the metformin treated young and aged AKI mice. Echocardiography data showed that AKI caused cardiac systolic dysfunctions in all experimental groups, while metformin treatment improved cardiac functions of young/aged WT and Sesn2 f/f but not cSesn2 -/- mice. Moreover, metformin treatment triggered AMPK activation and improved mitochondrial respiration rate and ATP production of cardiomyocytes from young/aged WT and Sesn2 f/f but not cSesn2 -/- hearts. Conclusions: Aging augments cardiac vulnerability to IRI-induced AKI. Metformin can rescue cardiac dysfunctions and renal injury caused by AKI through Sesn2/AMPK signaling pathway.

Metabolic rate increases with acclimation temperature and is associated with mitochondrial function in some tissues of threespine stickleback.

The metabolic rate (ṀO2) of eurythermal fishes changes in response to temperature, yet it is unclear how changes in mitochondrial function contribute to changes in ṀO2. We hypothesized that ṀO2would increase with acclimation temperature in the threespine stickleback (Gasterosteus aculeatus) in parallel with metabolic remodeling at the cellular level but that changes in metabolism in some tissues, such as liver, would contribute more to changes in ṀO2than others. Threespine stickleback were acclimated to 5, 12 and 20°C for 7 to 21 weeks. At each temperature, standard and maximum metabolic rate (SMR and MMR, respectively), and absolute aerobic scope (AAS) were quantified, along with mitochondrial respiration rates in liver, oxidative skeletal and cardiac muscles, and the maximal activity of citrate synthase (CS) and lactate dehydrogenase (LDH) in liver, and oxidative and glycolytic skeletal muscles. SMR, MMR and AAS increased with acclimation temperature, along with rates of mitochondrial phosphorylating respiration in all tissues. Low SMR and MMR at 5°C were associated with low or undetectable rates of mitochondrial complex II activity and a greater reliance on complex I activity in liver, oxidative skeletal muscle and heart. SMR was positively correlated with cytochrome c oxidase (CCO) activity in liver and oxidative muscle, but not mitochondrial proton leak, whereas MMR was positively correlated with CCO activity in liver. Overall, the results suggest that changes in ṀO2in response to temperature are driven by changes in some aspects of mitochondrial function in some, but not all, tissues of threespine stickleback.

Synergistic Anti-Tumor Effect of Palmitoylcarnitine and Dasatinib in Liver Cancer

Hepatocellular carcinoma (HCC) is the third major cause of cancer-related death worldwide and responds positively to tyrosine kinase inhibitors (TKIs). Dasatinib (Das) is an Src/Abl family kinase and has been successfully utilized in the treatment of various cancers. Cancer cells are known to limit their oxidative phosphorylation to minimize oxidative stress. Palmitoylcarnitine (Pcar) incubation triggers mitochondria-mediated apoptosis in cancer cells by increasing the mitochondrial respiration rate. It stimulates the H2O2 production in cancer cells and thus induces oxidative stress. Thus, considering the above observations, the combined effect of Pcar and Das on HepG2, liver cancer cells has been evaluated in the present study. Results demonstrated that combined exposure to Pcar and dasatinib inhibited cell growth, proliferation, and invasion efficiency of cancerous cells more than single-drug treatment. Further, cells undergo membrane depolarization and caspase-dependent apoptosis upon exposure to combined treatment. In addition, in vivo study showed that Pcar and dasatinib treatment reduced the tumor size in mice more significantly than single-drug treatment. Thus, considering the above remarks, combined therapy of Pcar and dasatinib may serve as a potential candidate in the treatment of liver cancer in human and animal tissues.

Alternative or cytochrome? Respiratory pathways in traps of aquatic carnivorous bladderwort Utricularia reflexa

ABSTRACT Carnivorous plants of the genus Utricularia (bladderwort) form modified leaves into suction bladder traps. The bladders are metabolically active plant tissue with high rates of mitochondrial respiration (RD). In general, plants possess two mitochondrial electron transport pathways to reduce oxygen to water: cytochrome and an alternative. Due to the high metabolic rate in the bladders, it is tempting to assume that the bladders prefer the cytochrome c oxidative pathway. Surprisingly, we revealed that alternative oxidase (AOX), which yields only a little ATP, is much more abundant in the bladders of Utricularia reflexa in comparison with the shoots. This pattern is similar to the carnivorous plants with passive pitcher traps (e.g. Sarracenia, Nepenthes) and seems to be widespread across many carnivorous taxa. The exact role of AOX in the traps of carnivorous plants remains to be investigated.

Upregulation of Succinate Dehydrogenase (SDHA) Contributes to Enhanced Bioenergetics of Ovarian Cancer Cells and Higher Sensitivity to Anti-Metabolic Agent Shikonin.

Simple SummaryIn recent years, targeting tumor specific metabolism has gained an interest as a promising therapeutic strategy. We discovered that the overexpression of mitochondrial enzyme succinate dehydrogenase (SDHA) is highly prevalent in ovarian carcinoma patients and contributes to elevated mitochondrial metabolism in ovarian tumor models. The SDHA overexpressing tumor cells are highly metabolically active, relying on both glycolysis and oxidative phosphorylation in mitochondria to meet their energy requirements. Further, we found that those cells are particularly vulnerable to deprivation of essential nutrients such as glucose and glutamine, which led to a substantial reduction of ATP yield. Lastly, we identified an anti-metabolic compound shikonin, which demonstrated a potent anti-tumor efficacy against SDHA overexpressing ovarian cancer cells. Overall, we strive to advance scientific knowledge by uncovering the previously unappreciated role of SDHA in reprogramming of ovarian cancer metabolism, which holds untapped opportunities for therapeutic intervention.We discovered that the overexpression of mitochondrial enzyme succinate dehydrogenase (SDHA) is particularly prevalent in ovarian carcinoma and promotes highly metabolically active phenotype. Succinate dehydrogenase deficiency has been previously studied in some rare disorders. However, the role of SDHA upregulation and its impact on ovarian cancer metabolism has never been investigated, emphasizing the need for further research. We investigated the functional consequences of SDHA overexpression in ovarian cancer. Using proteomics approaches and biological assays, we interrogated protein content of metabolic pathways, cell proliferation, anchorage-independent growth, mitochondrial respiration, glycolytic function, and ATP production rates in those cells. Lastly, we performed a drug screening to identify agents specifically targeting the SDHA overexpressing tumor cells. We showed that SDHA overexpressing cells are characterized by enhanced energy metabolism, relying on both glycolysis and oxidative phosphorylation to meet their energy needs. In addition, SDHA-high phenotype was associated with cell vulnerability to glucose and glutamine deprivation, which led to a substantial reduction of ATP yield. We also identified an anti-metabolic compound shikonin with a potent efficacy against SDHA overexpressing ovarian cancer cells. Our data underline the unappreciated role of SDHA in reprogramming of ovarian cancer metabolism, which represents a new opportunity for therapeutic intervention.

Congenital Hypermetabolism and Uncoupled Oxidative Phosphorylation

SummaryWe describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B, which encodes the β subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism. (Funded by the National Institutes of Health.)

Astaxanthin Is Able to Prevent Alcohol-Induced Dysfunction of Liver Mitochondria.

The search for new targets for the pathological action of ethanol remains an urgent task of biomedicine. Since degenerative changes in the liver are associated with the development of oxidative stress, antioxidants are promising agents for the treatment of alcohol-related diseases. In this work, we studied the ability of the carotenoid antioxidant, astaxanthin (AX), to prevent ethanol-induced changes in the liver of rats. It was shown that AX is able to protect the structure of mitochondria from degenerative changes caused by ethanol to improve mitochondrial functions. AX positively influences the activity and expression of proteins of the mitochondrial respiratory chain complexes and ATPase. In addition, a protective effect of AX on the rate and activity of mitochondrial respiration was demonstrated in the work. Thus, studies have shown that AX is involved in protective mechanisms in response to ethanol-induced mitochondrial dysfunction.

Different Effects of Quercetin Glycosides and Quercetin on Kidney Mitochondrial Function-Uncoupling, Cytochrome C Reducing and Antioxidant Activity.

Flavonols are found in plants as aglycones and as glycosides. Antioxidant activity of flavonols may occur via several mechanisms within the cell, and mitochondria as a target may play an important role. There is a lack of information about the influence of the sugar moiety on biological activity of flavonoid glycosides. The aims of study were to investigate the effects of quercetin and its glycosides on mitochondrial respiration rates at various metabolic states, and to evaluate their antioxidant potential using chemical and biological approaches. Mitochondrial function was measured using an oxygraphic method, cytochrome c reduction spectrophotometrically, H2O2 generation in mitochondria fluorimetrically, and antioxidant activity of flavonoids using an HPLC-post column system. Our data revealed that quercetin and its glycosides isoquercitrin, rutin, and hyperoside uncouple kidney mitochondrial respiration (increasing the State 2 respiration rate) and significantly reduce cytochrome c. Moreover, quercetin, and its glycosides decrease the production of mitochondrial H2O2 and possess radical scavenging and ferric reducing capacities. The highest activity was characteristic for quercetin, showing that the sugar moiety significantly diminishes its activity. In conclusion, our results show the efficient radical scavenging, ferric and cytochrome c reducing capacities, and uncoupling properties of quercetin and its glycosides, as well as the importance of the sugar residue and its structure in the regulation of kidney mitochondrial function.

The aquatic macrophyte Salvinia molesta mitigates herbicides (glyphosate and aminomethylphosphonic acid) effects to aquatic invertebrates.

We evaluated the individual and combined effects of different environmentally representative concentrations of glyphosate (0, 25, 50, 75, and 100µgl-1) and aminomethylphosphonic acid (AMPA; 0, 12.5, 25, 37.5, and 50µgl-1) on the physiology of Aedes aegypti larvae, as well as the capacity of the aquatic macrophyte Salvinia molesta to attenuate those compounds' toxicological effects. Larvae of Ae. aegypti (between the third and fourth larval stages) were exposed for 48h to glyphosate and/or AMPA in the presence or absence of S. molesta. Glyphosate and AMPA induced sublethal responses in Ae. aegypti larvae during acute exposures. Plants removed up to 49% of the glyphosate and 25% of AMPA from the water, resulting in the exposure of larvae to lower concentration of those compounds in relation to media without plants. As a result, lesser effects of glyphosate and/or AMPA were observed on larval acetylcholinesterase, P450 reductase, superoxide dismutase, mitochondrial electron transport chain enzymes, respiration rates, and lipid peroxidation. In addition to evidence of deleterious effects by media contamination with glyphosate and AMPA on aquatic invertebrates, our results attest to the ability of S. molesta plants to mitigate the toxicological impacts of those contaminants.

Protective effect of low-dose radiation on doxorubicin-induced brain injury in mice

BackgroundTo investigate the protective effect of low-dose radiation (LDR) on brain injury in mice induced by doxorubicin (DOX). MethodsSixty female BALB/C mice were randomly divided into the control (CTR) group, low-dose radiation (LDR) group, doxorubicin treatment (DOX) group and low-dose radiation before doxorubicin treatment (COM) group. After 72 h of exposure to 75 mGy, the mice were intraperitoneally injected with 7.5 mg/kg of doxorubicin and sacrificed 5 days later. Neuron-specific enolase (NSE), lactate dehydrogenase (LDH), adenosine triphosphate (ATP), neurotransmitters, inflammatory mediators, apoptosis- and oxidative stress-related mediators as well as mitochondrial dysfunction were examined. ResultsCompared to the DOX group, the concentrations of DA, 5-HT, EPI and GABA in the COM group were significantly decreased, and the number of TUNEL-positive cells was decreased. In addition, the expression of proapoptotic proteins was downregulated in the COM group compared to the DOX group. Low-dose radiation in advance reduced reactive oxygen species and activated the SOD antioxidant defense system as indicated by significantly reduced GSH expression, increased GSSG expression, increased GPx expression and activation of the Nrf2 redox pathway. After low-dose radiation, the expression levels of ATP5f1, NDUFV1 and CYC1 were close to normal, and the mitochondrial respiratory control rate (RCR) and activity of respiratory chain complex enzymes also tended to be normal. Low-dose radiation upregulated the expression levels of IL-2 and IL-4 but downregulated the expression levels of IL-10 and TGF-β. ConclusionLDR has a protective effect on brain injury in mice treated with DOX. The mechanism is related to LDR alleviating mitochondrial dysfunction and oxidative stress, which promotes the production of antioxidant damage proteins, thus exerting an adaptive protective effect on cells.

Experimental study of mitochondrion-targeted small molecule IR-61 ameliorated exhaustive exercise-induced cardiac injury in rats

To investigate the effects of mitochondrion-targeted cyanine fluorescent small molecule IR-61 on cardiac injury induced by exhaustive exercise in rats. Thirty-six adult male SD rats were randomly divided into 3 groups(n=12),control group (Ctrl), exhaustive exercise group (EE) and IR-61+ exhaustive exercise group (IR-61+EE). IR-61+EE group were intraperitoneally injected with 2 mg/kg IR-61 at the same time on day 1, 4 and 7. One hour after the end of the last drug administration, the two exhaustive exercise groups were subjected to exhaustive exercise modeling. The rats were placed on an animal treadmill with a slope of 0° at a speed of 10~15 m/min to coordinate their limbs running posture, and then ran at a speed of 25~30 m/min until exhaustion about 15 minutes later. After the animal models established, ECG was recorded by physiological recorder, myocardial injury was observed by light microscope, mitochondrial injury was observed by transmission electron microscope, myocardial cell apoptosis was detected by TUNEL method, markers of myocardial injury were detected by ELISA, and myocardial mitochondrial respiration rate was measured by high-resolution Oxygraph-2K mitochondrial instrument. ① Compared with Ctrl group, heart rate was increased, PR interval was shortened, QRS interval was prolonged, QTc was prolonged and ST segment was depressed significantly in EE group (P＜0.05). In EE group, myocardial fiber fracture and mitochondrial inner chamber swelling were obvious, mitochondrial crest was fuzzy, mitochondrial outer membrane was incomplete, and a large number of mitochondrial rupture and fusion were visible. In EE group, TUNEL staining cells were abundant, chromatin concentration and marginalization, nuclear membrane lysis, chromatin fragmentation into massive apoptotic bodies, apoptosis score increased (P＜0.05). The levels of creatine kinase isoenzyme-MB (CK-MB), cardiac troponin I(cTn-I) and N-terminal B-type natriuretic peptide (NT-proBNP) were increased in EE group (P＜0.05). Basal respiration rate, oxidative respiration rate of fatty acids and respiration rate of complex Ⅰ, Ⅱ and Ⅳ were all decreased (P＜ 0.05). ② Compared with EE group, the heart rate in IR-61+EE group was increased, PR interval was prolonged, QRS interval was shortened, QTc was shortened, ST segment was not significantly depressed (P＜0.05). In IR-61+EE group, myocardial fiber arrangement was loose, no obvious fracture was observed, mitochondrial inner ventricle was swelling, mitochondrial outer membrane was intact, TUNEL stained cells and unstained cells were observed, the overall morphology was more similar to Ctrl group. Apoptosis index was decreased (P＜0.05), the levels of CK-MB and cTn-I were decreased in IR-61+EE group (P＜0.05). The oxidative respiration rate of fatty acids and the respiration rate of complex Ⅱ and Ⅳ were increased (P＜0.05). Mitochondrion-targeted cyanine fluorescent small molecule IR-61 can improve cardiac electrical activity, reduce myocardial cell injury and mitochondrial injury, reduce myocardial cell apoptosis, and improve the myocardial mitochondrial energy metabolism condition in exhausted rats.

Novel Role of CETP in Macrophages: Reduction of Mitochondrial Oxidants Production and Modulation of Cell Immune-Metabolic Profile.

Plasma cholesteryl ester transfer protein (CETP) activity diminishes HDL-cholesterol levels and thus may increase atherosclerosis risk. Experimental evidence suggests CETP may also exhibit anti-inflammatory properties, but local tissue-specific functions of CETP have not yet been clarified. Since oxidative stress and inflammation are major features of atherogenesis, we investigated whether CETP modulates macrophage oxidant production, inflammatory and metabolic profiles. Comparing macrophages from CETP-expressing transgenic mice and non-expressing littermates, we observed that CETP expression reduced mitochondrial superoxide anion production and H2O2 release, increased maximal mitochondrial respiration rates, and induced elongation of the mitochondrial network and expression of fusion-related genes (mitofusin-2 and OPA1). The expression of pro-inflammatory genes and phagocytic activity were diminished in CETP-expressing macrophages. In addition, CETP-expressing macrophages had less unesterified cholesterol under basal conditions and after exposure to oxidized LDL, as well as increased HDL-mediated cholesterol efflux. CETP knockdown in human THP1 cells increased unesterified cholesterol and abolished the effects on mitofusin-2 and TNFα. In summary, the expression of CETP in macrophages modulates mitochondrial structure and function to promote an intracellular antioxidant state and oxidative metabolism, attenuation of pro-inflammatory gene expression, reduced cholesterol accumulation, and phagocytosis. These localized functions of CETP may be relevant for the prevention of atherosclerosis and other inflammatory diseases.

Standardization and validation of novel ex-vivo method for mitochondrial bioenergetics using mitochondrial modulators

Mitochondria is an essential organelle; it produces 95% of the adenine triphosphate (ATP) of cells, their dysfunction is related to the pathogenesis of multiple diseases, such as diabetes mellitus, cardiovascular and neurological disorders. Various pharmacologic agents are known to target mitochondrial function. Moreover, the toxic side effects of multiple drugs used to treat diseases are related to the impairment of mitochondrial function. Thus, there is a need to develop a method to evaluate the effect of pharmacologic agents for their potential and side effects to identify effective mitochondrial-modulating agents. Therefore, the objective of this study was to develop and validate an ex-vivo method for studying the effect of pharmacologic agents on mitochondrial function and rescue of dysfunction. Dimethyl sulfoxide (DMSO) concentrations that drugs were soluble in and maintained mitochondrial function were determined. Metformin (MET) is a known mitochondrial complex-1 inhibitor tested for its ability to compromise mitochondrion function. Coenzyme Q10 (Q10) and Resveratrol (RSV), which are known to enhance mitochondrial function, were added alone and dose-dependent, tested for the ability to rescue metformin-induced mitochondrial dysfunction. Ex-vivo liver and brain mitochondrial function was assessed using an oxytherm Clark-type oxygen electrode. DMSO was found to be toxic above 10% and drugs insoluble below 5%. The addition of 0.5 mg/ml MET decreased liver and brain mitochondrial respiratory control rate (RCR). At the same time, Q10 improved RCR in normal mitochondria and a concentration-dependent manner in MET-induced dysfunctional mitochondria. RSV was added in the last step of the experiment to confirm that compromised function is due to MET. Hence this method can be used to screen pharmacological agents for their potential therapeutics or toxic effect on mitochondria.

Pharmacological activation of PPARβ/δ preserves mitochondrial respiratory function in ischemia/reperfusion via stimulation of fatty acid oxidation-linked respiration and PGC-1α/NRF-1 signaling.

Myocardial ischemia/reperfusion (I/R) injury leads to significant impairment of cardiac function and remains the leading cause of morbidity and mortality worldwide. Activation of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) confers cardioprotection via pleiotropic effects including antioxidant and anti-inflammatory actions; however, the underlying mechanisms are not yet fully elucidated. The aim of this study was to investigate the effect of PPARβ/δ activation on myocardial mitochondrial respiratory function and link this effect with cardioprotection after ischemia/reperfusion (I/R). For this purpose, rats were treated with the PPARβ/δ agonist GW0742 and/or antagonist GSK0660 in vivo. Mitochondrial respiration and ROS production rates were determined using high-resolution fluororespirometry. Activation of PPARβ/δ did not alter mitochondrial respiratory function in the healthy heart, however, inhibition of PPARβ/δ reduced fatty acid oxidation (FAO) and complex II-linked mitochondrial respiration and shifted the substrate dependence away from succinate-related energy production and towards NADH. Activation of PPARβ/δ reduced mitochondrial stress during in vitro anoxia/reoxygenation. Furthermore, it preserved FAO-dependent mitochondrial respiration and lowered ROS production at oxidative phosphorylation (OXPHOS)-dependent state during ex vivo I/R. PPARβ/δ activation was also followed by increased mRNA expression of components of FAO -linked respiration and of transcription factors governing mitochondrial homeostasis (carnitine palmitoyl transferase 1b and 2-CPT-1b and CPT-2, electron transfer flavoprotein dehydrogenase -ETFDH, peroxisome proliferator-activated receptor gamma co-activator 1 alpha- PGC-1α and nuclear respiratory factor 1-NRF-1). In conclusion, activation of PPARβ/δ stimulated both FAO-linked respiration and PGC-1α/NRF -1 signaling and preserved mitochondrial respiratory function during I/R. These effects are associated with reduced infarct size.

Antibiotic therapy does not alter mitochondrial bioenergetics in lymphocytes of patients with septic shock – A prospective cohort study

Antibiotics may trigger alterations in mitochondrial function, which has been explored in cells culture, and in animal model of sepsis. This study sought to evaluate whether antibiotic therapy affects mitochondrial bioenergetics in a 68-patients clinical study. We studied mitochondrial respiratory rates at two time points: the first day of antibiotic administration and three days after. The Δbasal, ΔCI, ΔCII respiration, and ΔBCE respiratory rates were not different between patients administered with polymyxin, vancomycin, amoxicillin-clavulanate, and azithromycin compared to those who were not administered. Specific beta-lactams are associated with specific modifications in mitochondrial respiratory endpoints – patients who used meropenem had higher delta C2 values compared to those who did not (p = 0.03). Patients who used piperacillin-tazobactam had lower delta C1 (p = 0.03) values than those who did not, but higher delta C2 values (p = 0.02). These mitochondrial metabolic signatures in isolated lymphocytes challenges the proposed effects of antibiotics in mitochondrial bioenergetics of cell cultures, but at current status have an uncertain clinical significance.

Obesity elicits a unique metabolomic signature in human airway smooth muscle cells.

Obesity can aggravate asthma by enhancing airway hyperresponsiveness (AHR) and attenuating response to treatment. However, the precise mechanisms linking obesity and asthma remain unknown. Human airway smooth muscle (HASM) cells exhibit amplified excitation-contraction (EC) coupling and force generation in obesity. Therefore, we posit that airway smooth muscle (ASM) cells obtained from obese donors manifest a metabolomic phenotype distinct from that of nonobese donor cells and that a differential metabolic phenotype, at least in part, drives enhanced ASM cell EC coupling. HASM cells derived from age-, sex-, and race-matched nonobese [body mass index (BMI) ≤ 24.9 kg·m-2] and obese (BMI ≥ 29.9 kg·m-2) lung donors were subjected to unbiased metabolomic screening. The unbiased metabolomic screening identified differentially altered metabolites linked to glycolysis and citric acid cycle in obese donor-derived cells compared with nonobese donor cells. The Seahorse assay measured the bioenergetic profile based on glycolysis, mitochondrial respiration, palmitate oxidation, and glutamine oxidation rates in HASM cells. Glycolytic rate and capacity were elevated in obese donor-derived HASM cells, whereas mitochondrial respiration, palmitate oxidation, and glutamine oxidation rates were comparable between obese and nonobese groups. PFKFB3 mRNA and protein expression levels were also elevated in obese donor-derived HASM cells. Furthermore, pharmacological inhibition of PFKFB3 attenuated agonist-induced myosin light chain (MLC) phosphorylation in HASM cells derived from obese and nonobese donors. Our findings identify elevated glycolysis as a signature metabolic phenotype of obesity and inhibition of glycolysis attenuates MLC phosphorylation in HASM cells. These findings identify novel therapeutic targets to mitigate AHR in obesity-associated asthma.

Flying on empty: reduced mitochondrial function and flight capacity in food-deprived monarch butterflies.

Mitochondrial function is fundamental to organismal performance, health and fitness - especially during energetically challenging events, such as migration. With this investigation, we evaluated mitochondrial sensitivity to ecologically relevant stressors. We focused on an iconic migrant, the North American monarch butterfly (Danaus plexippus), and examined the effects of two stressors: 7days of food deprivation and infection by the protozoan parasite Ophryocystis elektroscirrha (known to reduce survival and flight performance). We measured whole-animal resting metabolic rate (RMR) and peak flight metabolic rate, and mitochondrial respiration of isolated mitochondria from the flight muscles. Food deprivation reduced mass-independent RMR and peak flight metabolic rate, whereas infection did not. Fed monarchs used mainly lipids in flight (respiratory quotient 0.73), but the respiratory quotient dropped in food-deprived individuals, possibly indicating switching to alternative energy sources, such as ketone bodies. Food deprivation decreased mitochondrial maximum oxygen consumption but not basal respiration, resulting in lower respiratory control ratio (RCR). Furthermore, food deprivation decreased mitochondrial complex III activity, but increased complex IV activity. Infection did not result in any changes in these mitochondrial variables. Mitochondrial maximum respiration rate correlated positively with mass-independent RMR and flight metabolic rate, suggesting a link between mitochondria and whole-animal performance. In conclusion, low food availability negatively affects mitochondrial function and flight performance, with potential implications for migration, fitness and population dynamics. Although previous studies have reported poor flight performance in infected monarchs, we found no differences in physiological performance, suggesting that reduced flight capacity may be due to structural differences or low energy stores.

MITOCHONDRIA RESPIRATION IN RAT BRAIN NEURONS UNDER CEREBRAL ISCHEMIA OF VARYING SEVERITY

The knowledge of the mechanisms of energy deficiency development in ischemic lesions is necessary to specify the pathogenesis and assess the damage/compensation ratio.
 The aim of the paper is to study respiration indices of mitochondria of rat brain homogenates in total and subtotal cerebral ischemia.
 Materials and Methods. The experiments were carried out on 88 male outbred white rats weighing 260±20 grams in compliance with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.
 Results. In 1-hour subtotal cerebral ischemia, V2 increased by 24 (18; 27) % (p<0.05), in the presence of malate/glutamate, if compared with the control, while the acceptor control coefficient and the phosphorylation coefficient decreased by 25 (17; 29) % (p<0.05). Other indices (V1, V3, V4, respiratory control coefficient) did not change (p>0.05). In the presence of malate/glutamate under 1-hour SCI, mitochondrial respiration rates V1, V2, V3, and V4 were higher than under 1-hour TCI 89 (82; 93), 58 (55; 63), 24 (21; 29) and 32 (27; 37) % respectively (p<0.05). Decrease in V1, V2, and V3 indices under 24-hour SCI is a consequence of the decrease in oxygen content for mitochondrial respiration. The inhibition of energy processes is more pronounced than under 1-hour SCI, which reflects the extremely low phosphorylation coefficient. Changes in V1, V2, and V3 indices under 1-hour SCI and 1-hour TCI are multidirectional. Their increase under SCI is associated with uncoupling between oxidation and phosphorylation, while their decrease under TCI is associated with a lack of substrates for mitochondrial respiration.
 Conclusion. The most pronounced decrease in respiration indices of the mitochondrial fraction of brain homogenates occurs under total cerebral ischemia due to the complete cessation of neuron blood supply.

P422: OMIPALISIB, A DUAL PI3K/MTOR INHIBITOR, TARGETS MITOCHONDRIA AND IMPAIRS OXIDATIVE PHOSPHORYLATION IN ACUTE MYELOID LEUKEMIA

Background: Mitochondria play a central role in cell metabolism. Recent studies explored mitochondrial alterations contributing to metabolic vulnerability in myeloid leukemia cells may considered as therapeutic targets. We previously reported that omipalisib, a PI3K/mTOR dual inhibitor, could significantly inhibit the growth of myeloid leukemia cells and impair their mitochondrial respiration. However, the precise mechanism of the mitochondria dysfunction in response to omipalisib is still not fully studied. Aims: To explore the mechanism of the mitochondria dysfunction in response to omipalisib. Methods: OCI-AML3 and THP-1 myeloid leukemia cell lines were used in this study. Omipalisib (GSK2126458) and another PI3K/mTOR dual inhibitor gedatolisib (PF-05212384) were used. Flow cytometry was used for mitochondrial analysis. RNA-seq was performed by using an Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) between control and omipalisib (or gedatolisib) were identified by EBseq with a threshold of p ≤ 0.05. The mRNA, nuclear and mitochondrial DNA quantification were measured by using QuantStudio 3 Real-Time PCR Systems. The parameters of mitochondrial respiration were analyzed by using the XFe 24 extracellular flux analyzer. Results: We demonstrated the anti-proliferative effect of omipalisib and gedatolisib on a panel of AML cell lines with different genetic backgrounds. OCI-AML3 cell lines had significant response to omipalisib and gedatolisib with IC50 of 17.45 nM and 153.38 nM, respectively. Subsequently, the transcriptome analysis of 50 nM omipalisib or 200 nM gedatolisib-treated OCI-AML3 cells were analyzed. Compared to DMSO-treated, a total of 1199 (595 upregulated and 604 downregulated) and 326 (218 upregulated and 108 downregulated) DEGs were identified in the omipalisib-treated cells and the gedatolisib-treated cells, respectively. Gene set enrichment analysis (GSEA) indicated that both omipalisib and gedatolisib could suppress “E2F targets”, “Myc targets”, and “G2M checkpoint”, and trigger “TNFα signaling via NFκB”, and “Inflammatory response”. Of them, we found that omipalisib had more significant effect to inhibit “Oxidative phosphorylation” (NES: -2.01) than gedatolisib did (NES: -1.40), especially mitochondrial biogenesis and amino acid metabolism-related pathways. Omipalisib had an inhibitory effect on the mitochondrial basal respiration rate and maximal respiration. Further analysis revealed quantitative reverse-transcription PCR analysis revealed that both omipalisib and gedatolisib could potently suppress serine synthesis (PHGDH, PSAT1, PSPH), glycine synthesis (SHMT1/2), and tetrahydrofolate cycle-related genes (MTHFD1/2) at the transcriptional level. On the other hand, rather than gedatolisib, omipalisib could significantly inhibit expression of glycolysis-related genes (GLUT1, HK2, PKM2, and LDHA) as well as mitochondrial biogenesis-related genes (PPARGC1B, TFAM, and NDUFS6). In addition, omipalisib could strongly decrease mtDNA and mitochondrial mass. These results suggest that omipalisib could suppress the mitochondrial biogenesis through alternative mechanisms, including but not limited to PI3K-AKT-mTOR signaling. Summary/Conclusion: Rather than gedatolisib, omipalisib could significantly inhibit mitochondrial biogenesis and suppress the respiration of mitochondria in myeloid leukemia cells. Our results indicate that omipalisib could suppress cell proliferation not only through PI3K-AKT-mTOR signaling but also via impairing mitochondrial biogenesis. This information may be potentially suitable for future clinical applications.

Cystine reduces mitochondrial dysfunction in C2C12 myotubes under moderate oxidative stress induced by H2O2

Moderate oxidative stress induces temporal impairment in mitochondrial ATP production. As glutathione (GSH) content is reduced to eliminate oxidative stress by oxidation–reduction reaction, intracellular GSH content is crucial for maintaining mitochondrial function under oxidative stress. GSH precursors such as N-acetyl cysteine (NAC) and cysteine are known to suppress oxidative stress based on the supply of cysteine residues being rate-limiting for GSH synthesis. However, it remains unclear whether cystine (Cys2) can suppress mitochondrial dysfunction under oxidative stress conditions. Therefore, we examined whether Cys2 could attenuate mitochondrial dysfunction under moderate oxidative stress without scavenging reactive oxygen species (ROS) in the medium. C2C12 myotubes were incubated for 120 min in a Cys2-supplemented medium and subsequently exposed to hydrogen peroxide (H2O2). Heme oxygenase-1 (HO-1) gene expression, intracellular cysteine and GSH content, intracellular ATP level, and maximal mitochondrial respiration were assessed. Cys2 treatment significantly increased GSH content in a dose-dependent manner under oxidative stress. Cys2 treatment significantly decreased HO-1 expression induced by H2O2 exposure. In addition, maximal mitochondrial respiration rate was decreased by H2O2 exposure, but improved by Cys2 treatment. In conclusion, Cys2 treatment mitigates oxidative stress-induced mitochondrial dysfunction by maintaining GSH content under moderate oxidative stress without scavenging ROS in the medium.