Electron Transport Chain Activity Research Articles

Fecal microbiota transplantation and replenishment of short-chain fatty acids protect against chronic cerebral hypoperfusion-induced colonic dysfunction by regulating gut microbiota, differentiation of Th17 cells, and mitochondrial energy metabolism

BackgroundLittle is known about the association between gut microbiota and intestinal injury under a state of chronic cerebral hypoperfusion (CCH). Here, the effects of gut microbiota and short-chain fatty acids (SCFAs), as important metabolic products, on intestinal function and potential mechanisms after CCH were investigated.MethodsRats were subjected to bilateral common carotid artery occlusion (BCCAo) to induce CCH. The gut microbiota and metabolites of SCFAs were assessed by 16S rRNA sequencing and targeted metabolomics, respectively. Transcriptomic analysis of colon tissues was also conducted. Subsequently, potential molecular pathways and differentially expressed genes were verified by western blot, immunoprecipitation, and immunofluorescence analyses. Furthermore, the integrity of the colonic barrier was evaluated by hematoxylin and eosin and mucin 2 staining and expression levels of tight junction proteins. Besides, colonic inflammation was further assessed by flow cytometry and expression levels of inflammatory cytokines. In addition, colonic mitochondrial dysfunction was analyzed via membrane potential, reactive oxygen species, electron transport chain (ETC) activities, adenosine triphosphate content, and mitochondrial ultrastructure.ResultsCCH modified gut microbial composition and microbial metabolism of SCFAs, which may be associated with inhibition of mitochondrial ETC activities and oxidative phosphorylation, leading to dysregulation of mitochondrial energy metabolism. Furthermore, CCH induced differentiation of pathogenic Th17 cells, promoted the formation of complexes of interferon regulatory factor 4 and signal transducer and activator of transcription 3 (STAT3), and increased the phosphorylation of STAT3. This was associated with an impairment of colonic barrier function and chronic colonic inflammation. In contrast, FMT and SCFA replenishment ameliorated CCH-induced gut microbial dysbiosis by increasing the intestinal content of Ruminococcus_sp_N15_MGS_57 and modulating microbial metabolism of SCFAs by increasing acetic acid contents associated with an improvment of the balance between Tregs and Th17 cells, mitochondrial ETC activities, and oxidative phosphorylation to prevent colonic inflammation and dysregulation of mitochondrial energy metabolism.ConclusionThese findings indicate that FMT and SCFA replenishment present a promising therapeutic strategy against colonic dysfunction under a state of chronic cerebral ischemia.

Antidepressants that increase mitochondrial energetics may elevate risk of treatment-emergent mania

Preclinical evidence suggests that antidepressants (ADs) may differentially influence mitochondrial energetics. This study was conducted to investigate the relationship between mitochondrial function and illness vulnerability in bipolar disorder (BD), specifically risk of treatment-emergent mania (TEM). Participants with BD already clinically phenotyped as TEM+ (n = 176) or TEM− (n = 516) were further classified whether the TEM associated AD, based on preclinical studies, increased (Mito+, n = 600) or decreased (Mito−, n = 289) mitochondrial electron transport chain (ETC) activity. Comparison of TEM+ rates between Mito+ and Mito− ADs was performed using generalized estimating equations to account for participants exposed to multiple ADs while adjusting for sex, age at time of enrollment into the biobank and BD type (BD-I/schizoaffective vs. BD-II). A total of 692 subjects (62.7% female, 91.4% White, mean age 43.0 ± 14.0 years) including 176 cases (25.3%) of TEM+ and 516 cases (74.7%) of TEM- with previous exposure to Mito+ and/or Mito- antidepressants were identified. Adjusting for age, sex and BD subtype, TEM+ was more frequent with antidepressants that increased (24.7%), versus decreased (13.5%) mitochondrial energetics (OR = 2.21; p = 0.000009). Our preliminary retrospective data suggests there may be merit in reconceptualizing AD classification, not solely based on monoaminergic conventional drug mechanism of action, but additionally based on mitochondrial energetics. Future prospective clinical studies on specific antidepressants and mitochondrial activity are encouraged. Recognizing pharmacogenomic investigation of drug response may extend or overlap to genomics of disease risk, future studies should investigate potential interactions between mitochondrial mechanisms of disease risk and drug response.

Opa1 and Drp1 reciprocally regulate cristae morphology, ETC function, and NAD+ regeneration in KRas-mutant lung adenocarcinoma

Oncogenic KRas activates mitochondrial fission through Erk-mediated phosphorylation of the mitochondrial fission GTPase Drp1. Drp1 deletion inhibits tumorigenesis of KRas-driven pancreatic cancer, but the role of mitochondrial dynamics in other Ras-driven malignancies is poorly defined. Here we show that invitro and invivo growth of KRas-driven lung adenocarcinoma is unaffected by deletion of Drp1 but is inhibited by deletion of Opa1, the GTPase that regulates inner membrane fusion and proper cristae morphology. Mechanistically, Opa1 knockout disrupts cristae morphology and inhibits electron transport chain (ETC) assembly and activity, which inhibits tumor cell proliferation through loss of NAD+ regeneration. Simultaneous inactivation of Drp1 and Opa1 restores cristae morphology, ETC activity, and cell proliferation indicating that mitochondrial fission activity drives ETC dysfunction induced by Opa1 knockout. Our results support a model in which mitochondrial fission events disrupt cristae structure, and tumor cells with hyperactive fission activity require Opa1 activity to maintain ETC function.

Energy substrate metabolism and oxidative stress in metabolic cardiomyopathy.

Metabolic cardiomyopathy is an emerging cause of heart failure in patients with obesity, insulin resistance, and diabetes. It is characterized by impaired myocardial metabolic flexibility, intramyocardial triglyceride accumulation, and lipotoxic damage in association with structural and functional alterations of the heart, unrelated to hypertension, coronary artery disease, and other cardiovascular diseases. Oxidative stress plays an important role in the development and progression of metabolic cardiomyopathy. Mitochondria are the most significant sources of reactive oxygen species (ROS) in cardiomyocytes. Disturbances in myocardial substrate metabolism induce mitochondrial adaptation and dysfunction, manifested as a mismatch between mitochondrial fatty acid oxidation and the electron transport chain (ETC) activity, which facilitates ROS production within the ETC components. In addition, non-ETC sources of mitochondrial ROS, such as β-oxidation of fatty acids, may also produce a considerable quantity of ROS in metabolic cardiomyopathy. Augmented ROS production in cardiomyocytes can induce a variety of effects, including the programming of myocardial energy substrate metabolism, modulation of metabolic inflammation, redox modification of ion channels and transporters, and cardiomyocyte apoptosis, ultimately leading to the structural and functional alterations of the heart. Based on the above mechanistic views, the present review summarizes the current understanding of the mechanisms underlying metabolic cardiomyopathy, focusing on the role of oxidative stress.

Abstract 12168: Pi3kγ is a Key Modulator of Cardiac Metabolism Rewiring Occurring in Doxorubicin-Treated Mice

Introduction: Doxorubicin (DOX) is a potent chemotherapeutic drug, whose use is hampered by the onset of long-term cardiotoxicity and lack of effective therapeutic approaches. Recently, metabolic perturbations have emerged as an early predictor factor of cardiac dysfunction, but how DOX affects cardiac metabolism is still unknown. We have previously demonstrated that phosphoinositide 3-kinase γ (PI3Kγ) is a major driver of DOX-evoked cardiotoxicity through mitophagy inhibition and accumulation of dysfunctional mitochondria. Hypothesis: Here we sought to explore the role of PI3Kγ in modulating cardiac metabolism upon DOX administration. Methods: Metabolomic analysis, glycolysis and oxidative phosphorylation were measured in whole hearts from wild-type (WT) and knock-in mice expressing a kinase-inactive PI3Kγ (kinase-dead; KD) treated with DOX (4 mg/kg on day 0, 7 and 14). Similar analyses were performed in WT and KD hearts infected with AAV9-shATG7 to explore the role of autophagy. Moreover, glucose uptake and GLUT-4 expression on the plasma membrane were measured in WT and KD neonatal mouse cardiomyocytes (NMCs) exposed to 1μM DOX. Results: Metabolomic analysis revealed an increased level of succinate and malic acid in DOX-treated hearts, suggesting defects in the TCA cycle. In agreement, DOX promotes the accumulation of metabolites normally catabolised in the TCA cycle, such as aminoacids and pyruvate, indicating a severe mitochondria dysfunction. Accordingly, fatty acid oxidation and electron transport chain activity were impaired in DOX-treated hearts at day 3 and exacerbated at day 42 after DOX. At the same time, glycolysis was upregulated. These changes were prevented in KD animals. Interestingly, upon autophagy inhibition, KD mice lost their cardioprotection at the mitochondrial level, but they still failed to upregulate glycolysis. This suggests a new autophagy-independent mechanism through which PI3Kγ regulates glucose utilisation. In agreement with the pharmacological and genetic inhibition of PI3Kγ abrogates glucose uptake in NMCs and normalises the plasma membrane exposure of GLUT-4. Conclusions: Overall, these results promote PI3Kγ as a master regulator of metabolic rewiring occurring in DOX-treated cardiomyocytes.

Abstract 15660: Impaired Mitochondrial Quality Control Drives the Development of Cardiomyopathy in Anderson-Fabry Disease

Introduction: Anderson-Fabry disease (AFD) is a systemic disorder caused by impaired alpha-galactosidase (GLA) activity, leading to a progressive cardiomyopathy, with hypertrophy and diastolic dysfunction. However, the molecular mechanisms underlying these features are unknown. Aim: To explore the role of mitochondria as a pathogenic mechanism of cardiac dysfunction in AFD. Methods: We tested a transgenic mouse that lacks endogenous GLA and expresses human R301Q-GLA [Tg/GLA knockout]. We performed serial ecochardiographic, histological, and biochemical analyses on cardiac tissue and single cell contractility on isolated adult cardiomyocytes (CM). Moreover, we assessed mitochondrial morphology/function in mouse embryonic fibroblasts (MEFs). Results: AFD-MEFs display an impaired mitochondrial respiration and reduced ATP synthesis. Increased mitochondrial levels of DRP1, LC3II, and MFN-ubiquitination along with accumulation of fragmented mitochondria and autophagosomes indicate the presence of mitophagy-targeted mitochondria that were not removed. Reduced PGC-1a and mitochondrial-DNA synthesis in AFD-MEFs indicate impaired mitochondrial biogenesis as well. Altogether, our data indicate impaired mitophagy and quality control of AFD-mitochondria. At echocardiography, AFD mice showed diastolic dysfunction and hypertrophy starting from 9 months. Isolated AFD-CMs exhibit reduced inotropic response to isoproterenol. Remarkably, alterations of cardiac mitochondrial function were observed starting from 3 months, including increased levels of MFN2-ubiquitination, DRP1 and LC3II, decreased levels of PGC-1a along with accumulation of disarranged mitochondria and autophagosomes. Frank impairment of mitochondrial respiration in AFD-CMs and reduced myocardial ATP-content was detected from 6 months, together with increased release of Cytochrome-C in the cytosol. Strikingly, mitochondrial dysfunction was also detected in fibroblasts isolated from AFD patients, which have a significantly reduced electron transport chain activity. Conclusions: In AFD, energetic alterations associated with impaired autophagy/mitophagy, are evident at 3 months and progressively lead to cardiac hypertrophy and dysfunction.

Abstract 12624: Smooth Muscle Cell Alpha-Actin Pathogenic Variant That Predisposes to Moyamoya-Like Cerebrovascular Disease Alters Smooth Muscle Cell Phenotype and Metabolism

Pathogenic variants in ACTA2 , which encodes smooth muscle α-actin ( α-SMA ), predispose to thoracic aortic disease. ACTA2 variants disrupting arginine 179 ( R179 ) also predispose to moyamoya-like ( MMD ) cerebrovascular disease, characterized by occlusion of the distal internal carotid arteries by neointimal cells that stain positive for smooth muscle cell ( SMC ) markers. Mice with conditional knockin of the Acta2 R179C mutation specifically in SMCs were generated ( Acta2 SMC-R179C/+ ). RNA sequencing and 2D gel electrophoresis confirmed presence of the mutation. Acta2 SMC-R179C/+ SMCs are not fully differentiated, maintain stem cell marker expression, and proliferate and migrate faster than wildtype ( WT ) SMCs. Seahorse analyses show increased glycolysis and decreased oxidative respiration ( OXPHOS ) in Acta2 SMC-R179C/+ SMCs, which have reduced mitochondrial mass and complex I activity, suggestive of deficient electron transport chain activity. Nicotinamide riboside ( NR ) augments complex I activity to boost OXPHOS without altering mitochondrial mass in Acta2 SMC-R179C/+ SMCs. NR also increases differentiation and reduces migration in Acta2 SMC-R179C/+ SMCs. With left carotid artery ligation ( LCAL ), 33% of Acta2 SMC-R179C/+ mice die within 4 days due to strokes, and 100% of surviving Acta2 SMC-R179C/+ mice at postop day 21 have occlusive lesions in the injured artery (8% in WT mice) containing α-SMA+ cells and fibrin deposits. Acta2 SMC-R179C/+ mice also exhibit markedly increased left carotid artery diameter and increased number, diameter, and tortuosity of left-sided collateral vessels compared to WT mice at postop day 21. NR treatment reduces deaths of LCAL-injured Acta2 SMC-R179C/+ mice to 8%, completely resolves occlusive lesion formation in 50% of mutant mice, and attenuates collateral formation and remodeling. These data suggest that immature SMCs with the ACTA2 R179C variant proliferate and migrate into the neointima causing MMD-like occlusive disease. Shifting metabolism to OXPHOS with NR drives differentiation and reduces migration, which diminishes arterial lesions in Acta2 SMC-R179C/+ mice. These data support the use of NR as a therapeutic to prevent MMD-like lesions and ischemic strokes in children with ACTA2 p.R179 variants.

TrkA serves as a virulence modulator in Porphyromonas gingivalis by maintaining heme acquisition and pathogenesis.

Periodontitis is an inflammatory disease of the supporting tissues of the teeth, with polymicrobial infection serving as the major pathogenic factor. As a periodontitis-related keystone pathogen, Porphyromonas gingivalis can orchestrate polymicrobial biofilm skewing into dysbiosis. Some metatranscriptomic studies have suggested that modulation of potassium ion uptake might serve as a signal enhancing microbiota nososymbiocity and periodontitis progression. Although the relationship between potassium transport and virulence has been elucidated in some bacteria, less is mentioned about the periodontitis-related pathogen. Herein, we centered on the virulence modulation potential of TrkA, the potassium uptake regulatory protein of P. gingivalis, and uncovered TrkA as the modulator in the heme acquisition process and in maintaining optimal pathogenicity in an experimental murine model of periodontitis. Hemagglutination and hemolytic activities were attenuated in the case of trkA gene loss, and the entire transcriptomic profiling revealed that the trkA gene can control the expression of genes in relation to electron transport chain activity and translation, as well as some transcriptional factors, including cdhR, the regulator of the heme uptake system hmuYR. Collectively, these results link the heme acquisition process to the potassium transporter, providing new insights into the role of potassium ion in P. gingivalis pathogenesis.

Exogenous trehalose protects photosystem II in heat-stressed wheat

Background: Photosystem II (PSII) is susceptible to heat stress. Plants naturally accumulate trehalose to improve stress tolerance. However, the mechanism by which trehalose affects PSII during heat stress is still unknown.
 Questions: How does trehalose affect PSII during heat stress?
 Studied species: Triticum aestivum L.
 Study site and dates: Shanghai, China. 2019-2021.
 Methods: Trehalose -pretreated wheat seedlings suffered from heat stress and their photosynthetic parameters were measured.
 Results: Heat stress caused a reduction in the photochemical efficiency of PSII, the electron transfer rate (ETR(II)), the quantum yield of regulated energy dissipationY(NPQ) and the coefficient of photochemical quenching (qP), but increased the quantum yield of non-regulated energy dissipation of PSII (Y[NO]). The shape of the fast chlorophyll fluorescence induction kinetics (OJIP) curve in the heat-stressed wheat was altered and the primary photochemistry maximum yield of PSII (Fv/Fo) and the PSII performance indicator PIabs were reduced. Accordingly, the activities of PSII and electron transport chain, the amount of ordered α-helix structures and the content of D1 protein also decreased. However, in trehalose-pretreated wheat, D1 protein and protein secondary structures of PSII were both protected, the electron transport activities of PSII and the whole chain were improved and greater fluorescence parameters values were maintained. Lower Y(NO) and more stable OJIP were obtained.
 Conclusions: Exogenous trehalose acted a vital role in the protection of the function of PSII, resulting in higherphotosynthetic capacity under heat stress.

Targeting DNA methylation can reduce cardiac injury associated with ischemia reperfusion: One step closer to clinical translation with blood-borne assessment.

Ischemia reperfusion (I/R) injury is one of the main clinical challenges for cardiac surgeons. No effective strategies or therapy targeting the molecular and cellular mechanisms to reduce I/R exists to date, despite altered gene expression and cellular metabolism/physiology. We aimed to identify whether DNA methylation, an unexplored target, can be a potential site to curb I/R-associated cell death by using the left anterior descending artery occlusion model in male Wistar rats. I/R rat heart exhibited global DNA hypermethylation with a corresponding decline in the mitochondrial genes (PGC-1α, TFAM, POLG, ND1, ND3, ND4, Cyt B, COX1, and COX2), antioxidant genes (SOD2, catalase, and Gpx2) and elevation in apoptotic genes (Casp3, Casp7, and Casp9) expression with corresponding changes in their activity, resulting in injury. Targeting global DNA methylation in I/R hearts by using its inhibitor significantly reduced the I/R-associated infarct size by 45% and improved dysferlin levels via modulating the genes involved in cell death apoptotic pathway (Casp3, Casp7, and PARP), inflammation (IL-1β, TLR4, ICAM1, and MyD88), oxidative stress (SOD1, catalase, Gpx2, and NFkB) and mitochondrial function and its regulation (MT-ND1, ND3, COX1, ATP6, PGC1α, and TFAM) in the cardiac tissue. The corresponding improvement in the genes’ function was reflected in the respective hearts via the reduction in apoptotic TUNEL positive cells and ROS levels, thereby improving myocardial architecture (H&E staining), antioxidant enzymes (SOD, catalase activity) and mitochondrial electron transport chain activities and ATP levels. The analysis of blood from the I/R animals in the presence and absence of methylation inhibition exhibited a similar pattern of changes as that observed in the cardiac tissue with respect to global DNA methylation level and its enzymes (DNMT and TET) gene expression, where the blood cardiac injury markers enzymes like LDH and CK-MB were elevated along with declined tissue levels. Based on these observations, we concluded that targeting DNA methylation to reduce the level of DNA hypermethylation can be a promising approach in ameliorating I/R injury. Additionally, the blood-borne changes reflected I/R-associated myocardial tissue alteration, making it suitable to predict I/R-linked pathology.

Comparative analysis of combined phosphorus and drought stress-responses in two winter wheat.

Phosphorus stress and drought stress are common abiotic stresses for wheat. In this study, two winter wheat varieties “Xindong20” and “Xindong23” were cultured in a hydroponic system using Hoagland nutrient solution and treated with drought stress under conventional (CP: 1.0 mmol/L) and low (LP: 0.05 mmol/L) phosphorus levels. Under drought stress, the root growth was better under LP than under CP. Under LP, root phosphorus content was increased by 94.2% in Xindong20 and decreased by 48.9% in Xindong23 at 3 d after re-watering, compared with those at 0 d under drought stress. However, the potassium (K) content was the highest among the four elements studied and the phosphorus (P) and calcium (Ca) content were reduced in the root of the two varieties. Under CP, the zinc (Zn) content was higher than that under LP in Xindong23. The GeneChip analysis showed that a total of 4,577 and 202 differentially expressed genes (DEGs) were detected from the roots of Xindong20 and Xindong23, respectively. Among them, 89.9% of DEGs were involved in organelles and vesicles in Xindong20, and 69.8% were involved in root anatomical structure, respiratory chain, electron transport chain, ion transport, and enzyme activity in Xindong23. Overall, LP was superior to CP in mitigating drought stress on wheat, and the regulatory genes were also different in the two varieties. Xindong20 had higher drought tolerance for more up-regulated genes involved in the responses compared to Xindong23.

Inner-Membrane-Bound Gold Nanoparticles as Efficient Electron Transfer Mediators for Enhanced Mitochondrial Electron Transport Chain Activity.

Electron transfer through the mitochondrial electron transport chain (ETC) can be critically blocked by the dysfunction of protein complexes. Redox-active molecules have been used to mediate the electron transfer in place of the dysfunctional complexes; however, they are limited to replacing complex I and are known to be toxic. Here we report artificial mitochondrial electron transfer pathways that enhance ETC activity by exploiting inner-membrane-bound gold nanoparticles (GNPs) as efficient electron transfer mediators. The hybridization of mitochondria with GNPs, driven by electrostatic interaction, is successfully visualized in real time at the level of a single mitochondrion. By observing quantized quenching dips via plasmon resonance energy transfer, we reveal that the hybridized GNPs are bound to the inner membrane of mitochondria irrespective of the presence of the outer membrane. The ETC activity of mitochondria with GNPs such as membrane potential, oxygen consumption, and ATP production is remarkably increased in vitro.

Mitochondrial Ca2+ uptake by the MCU facilitates pyramidal neuron excitability and metabolism during action potential firing

Neuronal activation is fundamental to information processing by the brain and requires mitochondrial energy metabolism. Mitochondrial Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) has long been implicated in the control of energy metabolism and intracellular Ca2+ signalling, but its importance to neuronal function in the brain remains unclear. Here, we used in situ electrophysiology and two-photon imaging of mitochondrial Ca2+, cytosolic Ca2+, and NAD(P)H to test the relevance of MCU activation to pyramidal neuron Ca2+ signalling and energy metabolism during action potential firing. We demonstrate that mitochondrial Ca2+ uptake by the MCU is tuned to enhanced firing rate and the strength of this relationship varied between neurons of discrete brain regions. MCU activation promoted electron transport chain activity and chemical reduction of NAD+ to NADH. Moreover, Ca2+ buffering by mitochondria attenuated cytosolic Ca2+ signals and thereby reduced the coupling between activity and the slow afterhyperpolarization, a ubiquitous regulator of excitability. Collectively, we demonstrate that the MCU is engaged by accelerated spike frequency to facilitate neuronal activity through simultaneous control of energy metabolism and excitability. As such, the MCU is situated to promote brain functions associated with high frequency signalling and may represent a target for controlling excessive neuronal activity.

Glutamine Metabolism Mediates Sensitivity to Respiratory Complex II Inhibition in Acute Myeloid Leukemia.

This work highlights the effects of direct CII inhibition in mediating selective AML cell death and provides insights into glutamine anaplerosis as a metabolic adaptation that can be therapeutically targeted.

O-GlcNAcylation suppresses TRAP1 activity and promotes mitochondrial respiration.

The molecular chaperone TNF-receptor-associated protein-1 (TRAP1) controls mitochondrial respiration through regulation of Krebs cycle and electron transport chain activity. Post-translational modification (PTM) of TRAP1 regulates its activity, thereby controlling global metabolic flux. O-GlcNAcylation is one PTM that is known to impact mitochondrial metabolism, however the major effectors of this regulatory PTM remain inadequately resolved. Here we demonstrate that TRAP1-O-GlcNAcylation decreases TRAP1 ATPase activity, leading to increased mitochondrial metabolism. O-GlcNAcylation of TRAP1 occurs following mitochondrial import and provides critical regulatory feedback, as the impact of O-GlcNAcylation on mitochondrial metabolism shows TRAP1-dependence. Mechanistically, loss of TRAP1-O-GlcNAcylation decreased TRAP1 binding to ATP, and interaction with its client protein succinate dehydrogenase (SDHB). Taken together, TRAP1-O-GlcNAcylation serves to regulate mitochondrial metabolism by the reversible attenuation of TRAP1 chaperone activity.

Dual effect of the herbal matcha green tea (Camellia sinensis L. kuntze) supplement in EA.hy926 endothelial cells and Artemia salina

Ethnopharmacological relevanceMatcha green tea (Camellia sinensis) based-supplements have been widely used since they present a greater content of phenolic compounds than traditional green tea, which is popularly used in the treatment of diabetes. However, there are few studies on the effectiveness and safety of matcha supplements. Aim of the studyThis work aimed to evaluate the efficacy and safety of this supplement in endothelial cells (EA.hy926) in the hyperglycemic model and in vivo Artemia salina. Materials and methodsTo assess the effect of Matcha herbal supplement (MHS), EA. hy926 endothelial cells were treated with 20 μg/mL of MHS for 24 h, in a hyperglycemic medium with 35 mM glucose. After treatment, cells were trypsinized and centrifuged at 4 °C and 47×g for 5 min. The pellet was used to determine the reaction products to thiobarbituric acid and the levels of nitric oxide. Electron transport chain activity and ATP levels were also evaluated. Intracellular pH, apoptosis, and mitochondrial membrane depolarization were evaluated by flow cytometry. MHS chemical characterization was performed by HPLC-UV and total phenolic content analysis. The evaluation of the antioxidant capacity of MHS was performed by 2,2-diphenyl-1-picrylhydrazyl radical scavenger assay. To determine the in vivo acute toxicity of MHS, an A. salina assay was conducted, using 0,2 mL of different concentrations of MHS (10, 50, 100, 250, 500, 750 and 1000 μg/mL). The LD50 values were obtained by interpolation of 50% (y = 50) of the dead individuals in the trend curves. ResultsOur data showed that MHS was able to avoid oxidative and nitrosative stress induced by hyperglycemia, demonstrating important antioxidant activity. However, it was observed that MHS reduced up to 90% the activity of the four-electron transport complexes, reducing the ATP production of the endothelial cells. In the toxicity assay performed in Artemia salina, MHS showed mild toxicity (LD50 = 0,4 mg/mL). The major compounds found in MHS were epigallocatechin gallate, epicatechin, rutin, kaempferol, and quercetin. ConclusionsThis data draws attention to the fact that supplements with high content of phenolic compounds, capable of avoiding oxidative and nitrosative stress can have a dual effect and, simultaneously to antioxidant activity, can induce toxicity in different cell types.

Abstract P3037: Metabolic Transitions In The Developing Mouse Heart

During cardiac development, cellular energy production evolves dramatically. Mechanisms that control these changes and activation of electron transport chain (ETC) remain unclear. Our aim was to provide a comprehensive analysis of mitochondrial biology and bioenergetics in the developing wild type heart for use in experimental models, such as mice, in which the mitochondrial protein, cyclophilin D (CyPD), was deleted. Mouse hearts were harvested from wild type and CyPD null hearts throughout development and examined by electron microscopy, liquid chromatography/mass spectrometry, oxygen consumption and enzyme assays, gel electrophoresis/immunoblotting, and calcium retention capacity (CRC). As the heart develops, myocyte size and mitochondrial number, size, and complexity increases with transitions at the end of the embryonic period and immediately after birth. Metabolomic profiling demonstrated changes in glycolysis, the TCA cycle, and redox state during development and these changes may facilitate proliferation in the neonatal heart. Activation of ETC complexes I, III, IV, and V occurs in the mid-embryonic period, while assembly of ETC supercomplexes begins at the late embryonic period. In contrast, complex II activity is relatively stable during development. CRC assays demonstrated a gradual decrease in the probability of mPTP opening during development, and while CyPD expression increased, its specific enzymatic activity decreased. For most assays tested, these metabolic transitions occurred earlier in development in CyPD null mice. In conclusion, the developing heart undergoes changes in cellular bioenergetics with two transitions at the end of the embryonic period and immediately after birth. Deletion of CyPD causes these transitions to occur earlier in development, suggesting that CyPD and closure of the mPTP controls mitochondrial activation and bioenergetics during cardiac development.

Abstract P3054: Alpha-1a Adrenergic Receptor Activation Increases Electron Transport Chain Activity And Preserves Cardiac Contractility Following Myocardial Infarction

Heart failure features decreased electron transport chain (ETC) activity in cardiomyocyte mitochondria. Previous studies have demonstrated that alpha-1A adrenergic receptor (α1A-AR) activation increases cardiac contractility in the failing heart. However, α1A-AR activation as a method of ETC regulation following cardiac injury has not been studied. Here, we assessed the hypothesis that α1A-AR activation increases ETC activity following myocardial infarction (MI) induced by left coronary artery ligation. We measured cardiac contractility using echocardiography at baseline and 7 days after MI in male CL57Bl/6J mice receiving the selective α1A-AR agonist A61603 (10 ng/kg/d) (n = 8) or vehicle (0.9% saline) (n = 9) via subcutaneous osmotic minipumps (Panel A). We then profiled citrate synthase (CS), a key enzyme in the citric acid cycle and common marker for mitochondrial mass, and ETC Complex I-IV activities in ventricular lysates. ETC enzyme activities were normalized to CS to define ETC capacity on a per mitochondrion basis. On Day 0, there were no differences in fractional shortening between groups (Panel B, 46 ± 3% vs. 49 ± 3%, p = 0.27) (mean ± SEM). On Day 7, fractional shortening was preserved in A6-treated mice compared to vehicle controls (Panel B, 24 ± 3% vs. 14 ± 1%, p = 0.01). A6 decreased CS activity compared to vehicle controls (Panel C, 11,024 ± 1,035 vs. 15,966 ± 1,068 nmol/mg/min, p < 0.01). When normalized to CS activity, A6 increased Complex I (16 ± 2% vs. 7 ± 1%, p < 0.01) (1,584 ± 116 vs. 1,106 ± 154 nmol/min/mg), Complex III (82 ± 8% vs. 56 ± 5%, p = 0.01) (8,435 ± 461 vs. 8,542 ± 327 nmol/min/mg), and Complex IV (121 ± 12% vs. 92 ± 9.%, p = 0.04) (12,523 ± 673 vs. 14,174 ± 1,156) activities (Panel D), suggesting enhanced ETC activity on a per mitochondrion basis. In conclusion, we show that a low dose of a selective α1A-AR agonist enhances cardiac contractility following MI. Increased ETC enzyme activity, particularly Complex I, may contribute to this beneficial effect. Ours is the first study in which selective α1A-AR activation has been linked to enhanced cardiac contractility and mitochondrial metabolism following MI. These findings may outline a novel mechanism through which α1A-AR activation preserves cardiac function in the injured and failing heart.

Disrupted and Elevated Circadian Secretion of Glucagon-Like Peptide-1 in a Murine Model of Type 2 Diabetes.

Metabolism and circadian rhythms are intimately linked, with circadian glucagon-like peptide-1 (GLP-1) secretion by the intestinal L-cell entraining rhythmic insulin release. GLP-1 secretion has been explored in the context of obesogenic diets, but never in a rodent model of type 2 diabetes (T2D). There is also considerable disagreement regarding GLP-1 levels in human T2D. Furthermore, recent evidence has demonstrated decreased expression of the β-cell exocytotic protein secretagogin (SCGN) in T2D. To extend these findings to the L-cell, we administered oral glucose tolerance tests at 6 time points in 4-hour intervals to the high-fat diet/streptozotocin (HFD-STZ) mouse model of T2D. This revealed a 10-fold increase in peak GLP-1 secretion with a phase shift of the peak from the normal feeding period into the fasting-phase. This was accompanied by impairments in the rhythms of glucose, glucagon, mucosal clock genes (Arntl and Cry2), and Scgn. Immunostaining revealed that L-cell GLP-1 intensity was increased in the HFD-STZ model, as was the proportion of L-cells that expressed SCGN; however, this was not found in L-cells from humans with T2D, which exhibited decreased GLP-1 staining but maintained their SCGN expression. Gcg expression in isolated L-cells was increased along with pathways relating to GLP-1 secretion and electron transport chain activity in the HFD-STZ condition. Further investigation into the mechanisms responsible for this increase in GLP-1 secretion may give insights into therapies directed toward upregulating endogenous GLP-1 secretion.

Obesity and Fatty Acids Promote Mitochondrial Translocation of STAT3 Through ROS-Dependent Mechanisms.

Obesity promotes the onset and progression of metabolic and inflammatory diseases such as type 2 diabetes. The chronic low-grade inflammation that occurs during obesity triggers multiple signaling mechanisms that negatively affect organismal health. One such mechanism is the persistent activation and mitochondrial translocation of STAT3, which is implicated in inflammatory pathologies and many types of cancers. STAT3 in the mitochondria (mitoSTAT3) alters electron transport chain activity, thereby influencing nutrient metabolism and immune response. PBMCs and CD4+ T cells from obese but normal glucose-tolerant (NGT) middle-aged subjects had higher phosphorylation of STAT3 on residue serine 727 and more mitochondrial accumulation of STAT3 than cells from lean subjects. To evaluate if circulating lipid overabundance in obesity is responsible for age- and sex-matched mitoSTAT3, cells from lean subjects were challenged with physiologically relevant doses of the saturated and monounsaturated fatty acids, palmitate and oleate, respectively. Fatty acid treatment caused robust accumulation of mitoSTAT3 in all cell types, which was independent of palmitate-induced impairments in autophagy. Co-treatment of cells with fatty acid and trehalose prevented STAT3 phosphorylation and mitochondrial accumulation in an autophagy-independent but cellular peroxide–dependent mechanism. Pharmacological blockade of mitoSTAT3 either by a mitochondria-targeted STAT3 inhibitor or ROS scavenging prevented obesity and fatty acid–induced production of proinflammatory cytokines IL-17A and IL-6, thus establishing a mechanistic link between mitoSTAT3 and inflammatory cytokine production.