Altered Bioenergetics Research Articles

Guanosine protects against behavioural and mitochondrial bioenergetic alterations after mild traumatic brain injury

Traumatic brain injury (TBI) constitutes a heterogeneous cerebral insult induced by traumatic biomechanical forces. Mitochondria play a critical role in brain bioenergetics, and TBI induces several consequences related with oxidative stress and excitotoxicity clearly demonstrated in different experimental model involving TBI. Mitochondrial bioenergetics alterations can present several targets for therapeutics which could help reduce secondary brain lesions such as neuropsychiatric problems, including memory loss and motor impairment. Guanosine (GUO), an endogenous neuroprotective nucleoside, affords the long-term benefits of controlling brain neurodegeneration, mainly due to its capacity to activate the antioxidant defense system and maintenance of the redox system. However, little is known about the exact protective mechanism exerted by GUO on mitochondrial bioenergetics disruption induced by TBI. Thus, the aim of this study was to investigate the effects of GUO in brain cortical and hippocampal mitochondrial bioenergetics in the mild TBI model. Additionally, we aimed to assess whether mitochondrial damage induced by TBI may be related to behavioral alterations in rats. Our findings showed that 24 h post-TBI, GUO treatment promotes an adaptive response of mitochondrial respiratory chain increasing oxygen flux which it was able to protect against the uncoupling of oxidative phosphorylation (OXPHOS) induced by TBI, restored the respiratory electron transfer system (ETS) established with an uncoupler. Guanosine treatment also increased respiratory control ratio (RCR), an indicator of the state of mitochondrial coupling, which is related to the mitochondrial functionality. In addition, mitochondrial bioenergetics failure was closely related with locomotor, exploratory and memory impairments. The present study suggests GUO treatment post mild TBI could increase GDP endogenous levels and consequently increasing ATP levels promotes an increase of RCR increasing OXPHOS and in substantial improve mitochondrial respiration in different brain regions, which, in turn, could promote an improvement in behavioral parameters associated to the mild TBI. These findings may contribute to the development of future therapies with a target on failure energetic metabolism induced by TBI.

COX5B-Mediated Bioenergetic Alteration Regulates Tumor Growth and Migration by Modulating AMPK-UHMK1-ERK Cascade in Hepatoma.

The oxidative phosphorylation machinery in mitochondria, which generates the main bioenergy pool in cells, includes four enzyme complexes for electron transport and ATP synthase. Among them, the cytochrome c oxidase (COX), which constitutes the fourth complex, has been suggested as the major regulatory site. Recently, abnormalities in COX were linked to tumor progression in several cancers. However, it remains unclear whether COX and its subunits play a role in tumor progression of hepatoma. To search for the key regulatory factor(s) in COX for hepatoma development, in silico analysis using public transcriptomic database followed by validation for postoperative outcome associations using independent in-house patient cohorts was performed. In which, COX5B was highly expressed in hepatoma and associated with unfavorable postoperative prognosis. In addressing the role of COX5B in hepatoma, the loss- and gain-of-function experiments for COX5B were conducted. Consequently, COX5B expression was associated with increased hepatoma cell proliferation, migration and xenograft growth. Downstream effectors searched by cDNA microarray analysis identified UHMK1, an oncogenic protein, which manifested a positively correlated expression level of COX5B. The COX5B-mediated regulatory event on UHMK1 expression was subsequently demonstrated as bioenergetic alteration-dependent activation of AMPK in hepatoma cells. Phosphoproteomic analysis uncovered activation of ERK- and stathmin-mediated pathways downstream of UHMK1. Finally, comprehensive phenotypic assays supported the impacts of COX5B-UHMK1-ERK axis on hepatoma cell growth and migration.

Psychological distress and lack of PINK1 promote bioenergetics alterations in peripheral blood mononuclear cells

Psychological distress induces oxidative stress and alters mitochondrial metabolism in the nervous and immune systems. Psychological distress promotes alterations in brain metabolism and neurochemistry in wild-type (WT) rats in a similar manner as in Parkinsonian rats lacking endogenous PTEN-induced kinase 1 (PINK1), a serine/threonine kinase mutated in a recessive forms of Parkinson’s disease. PINK1 has been extensively studied in the brain, but its physiological role in peripheral tissues and the extent to which it intersects with the neuroimmune axis is not clear. We surmised that PINK1 modulates the bioenergetics of peripheral blood mononuclear cells (PBMCs) under basal conditions or in situations that promote oxidative stress as psychological distress. By using an XF metabolic bioanalyzer, PINK1-KO-PBMCs showed significantly increased oxidative phosphorylation and basal glycolysis compared to WT cells and correlated with motor dysfunction. In addition, psychological distress enhanced the glycolytic capacity in PINK1-KO-PBMCs but not in WT-PBMCs. The level of antioxidant markers and brain-derived neurotrophic factor were altered in PINK1-KO-PBMCs and by psychological distress. In summary, our data suggest that PINK1 is critical for modulating the bioenergetics and antioxidant responses in PBMCs whereas lack of PINK1 upregulates compensatory glycolysis in response to oxidative stress induced by psychological distress.

Long-term, high-fat feeding exacerbates short-term increases in adipose mitochondrial reactive oxygen species, without impairing mitochondrial respiration.

White adipose tissue (WAT) dysfunction in obesity is implicated in the onset of whole body insulin resistance. Alterations in mitochondrial bioenergetics, namely impaired mitochondrial respiration and increased mitochondrial reactive oxygen species (mtROS) production, have been suggested to contribute to this metabolic dysregulation. However, techniques investigating mitochondrial function are classically normalized to tissue weight, which may be confounding when considering obesity-related adipocyte hypertrophy. Furthermore, the effect of long-term high-fat diet (HFD) on mtROS in WAT has yet to be elucidated. Therefore, we sought to determine the HFD-mediated temporal changes in mitochondrial respiration and mtROS emission in WAT. C57BL/6N mice received low-fat diet or HFD for 1 or 8 wk and changes in inguinal WAT (iWAT) and epididymal WAT (eWAT) were assessed. While tissue weight-normalized mitochondrial respiration was reduced in iWAT following 8-wk HFD-feeding, this effect was mitigated when adipocyte cell size and/or number were considered. These data suggest HFD does not impair mitochondrial respiratory capacity per adipocyte within WAT. In support of this assertion, within eWAT compensatory increases in lipid-supported and maximal succinate-supported respiration occurred at 8 wk despite cell hypertrophy and increases in WAT inflammation. Although these data suggest impairments in mitochondrial respiration do not contribute to HFD-mediated WAT phenotype, lipid-supported mtROS emission increased following 1-wk HFD in eWAT, while both lipid and carbohydrate-supported mtROS were increased at 8 wk in both depots. Combined, these data establish that while HFD does not impair adipocyte mitochondrial respiratory capacity, increased mtROS is an enduring physiological occurrence within WAT in HFD-induced obesity.

Abstract A076: Mechanistic pathways and novel prognostic indicators of lifestyle intervention outcomes for African American breast cancer survivors

Abstract Background: For African American (AA) women in the US, breast cancer (BC) is the leading cancer diagnosis and the second leading cause of cancer death. The rising prevalence of obesity and related comorbidities are important contributors to widening racial disparities in BC outcomes. 82% of AA women are obese or overweight, compared to 64% of Non-Hispanic White (NHW) women. BC mortality rates are 40% higher in AA women, and incidence rates surpassed NHW women in 2013. Obesity is linked to poorer BC outcomes through upregulation of leptin, cytokines, and hormone expression. Conversely, weight loss and/or increased physical activity decrease inflammation and estrogen expression, decrease cellular growth factor signaling and angiogenesis, increase antitumor immunity, and reduce overall BC risk. Methods: Between 2011 and 2014, 246 AA breast cancer survivors were recruited and randomized to either the Moving Forward interventionist-guided or self-guided weight loss program. Results showed significant weight loss, improved diet, increased physical activity, and increased social support. In addition to collecting anthropometric and behavioral data, the study team banked serum samples for the majority of participants at baseline, six months (post-intervention) and twelve months (follow-up). For all samples, weight loss outcomes were recorded, and potential biomarkers were explored between groups. Results: Interventionist-guided study participants exhibited greater improvements than self-guided control participants for weight loss (-3.49kgs vs -1.27kgs, p = <0.0001) and % weight loss (3.6% vs 1.4%, p = 0.001). Among all participants, those losing >5% of their body weight showed significant improvements in leptin (65.6 ± 28.3 vs. 45.0 ± 23.3 at baseline and 6 months, respectively, p = <0.0001) and c-peptide (805.3 ± 470.6 vs. 632.3 ± 453.4 at baseline and 6 months, respectively, p = 0.0001). Planned studies will utilize RNA sequencing approaches to fully elucidate the role of non-coding RNAs in lifestyle intervention outcomes. Conclusions: Early changes in triglycerides, leptin, and HbA1c are detectable in participant serum samples and are likely linked to alterations in body composition, inflammatory cytokines and mitochondrial bioenergetics. Our future directions for this study are completely novel in their use of next generation sequencing to examine noncoding RNA-mediated mechanisms of reduced breast cancer risk, and the first to focus specifically on outcomes of lifestyle intervention in AA women. Citation Format: Jessica Olson, Patricia Sheehan, Giamila Fantuzzi, Lisa Sharp, Melinda Stolley. Mechanistic pathways and novel prognostic indicators of lifestyle intervention outcomes for African American breast cancer survivors [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A076.

Alterations of mitochondrial bioenergetics, dynamics, and morphology support the theory of oxidative damage involvement in autism spectrum disorder.

Autism spectrum disorder (ASD) has been hypothesized to be a result of the interplay between genetic predisposition and increased vulnerability to early environmental insults. Mitochondrial dysfunctions appear also involved in ASD pathophysiology, but the mechanisms by which such alterations develop are not completely understood. Here, we analyzed ASD primary fibroblasts by measuring mitochondrial bioenergetics, ultrastructural and dynamic parameters to investigate the hypothesis that defects in these pathways could be interconnected phenomena responsible or consequence for the redox imbalance observed in ASD. High levels of 4-hydroxynonenal protein adducts together with increased NADPH (nicotinamide adenine dinucleotide phosphateoxidase) activity and mitochondrial superoxide production coupled with a compromised antioxidant response guided by a defective Nuclear Factor Erythroid 2-Related Factor 2 pathway confirmed an unbalanced redox homeostasis in ASD. Moreover, ASD fibroblasts showed overactive mitochondrial bioenergetics associated with atypical morphology and altered expression of mitochondrial electron transport chain complexes and dynamics-regulating factors. We suggest that many of the changes observed in mitochondria could represent compensatory mechanisms by which ASD cells try to adapt to altered energy demand, possibly resulting from a chronic oxinflammatory status.

Effects of Atrial Natriuretic Peptide knockout on renal mitochondrial bioenergetics in salt‐sensitive hypertension

BackgroundAtrial Natriuretic Peptide (ANP) encoded by Nppa, is a hormone known to promote salt excretion and BP reduction; there are clinical data implicating inherently low levels of ANP in the development of salt‐sensitive (SS) hypertension. In the heart and fat tissue, ANP is known to affect lipid peroxidation, mitochondrial bioenergetics and biogenesis. However, there is a gap in knowledge regarding the effects of ANP on mitochondria in the kidney. We hypothesized here that in SS hypertension ANP deficiency causes renal mitochondrial dysfunction.MethodsSS hypertension was induced in SSNPPA−/− (ANP knockout in Dahl SS background) and SSWT (wild type Dahl SS) rats by placing them on a high salt diet (HS, 4% NaCl) or a normal salt diet (control, NS, 0.4% NaCl) for 21 days. A combination of in vivo techniques with studies performed on isolated renal mitochondria (seahorse respiration and spectrofluorimetry assays) were used to test the role of ANP knockout in the mitochondria bioenergetics.ResultsOur previous studies demonstrated that during a HS challenge SSNPPA−/− rats exhibit exacerbated salt‐sensitivity of blood pressure, reduced sodium excretion, decreased urine flow, and aggravated kidney injury. In addition, there is a significant reduction in renal cortical cGMP production in the HS fed SSNPPA−/− rats vs SSWT. cGMP is known to exert protective effects in mitochondria; in order to test mitochondrial function in our model, we measured membrane potential and levels of superoxide and hydrogen peroxide (H2O2) in mitochondria isolated from kidney cortex. Specifically, TMRM, Amplex Red and MCLA were used to detect mitochondrial membrane potential, H2O2 and superoxide, respectively. We report a decrease in mitochondrial membrane potential in the SSNPPA−/− rats vs SSWT (both on NS and HS diets), and an increase in mitochondrial H2O2 and superoxide levels in the same groups. Swelling assay revealed a trend for an increased mitochondria swelling rate in the SSNPPA−/− rats. Pilot calcium uptake studies implicated calcium influx via MCU (mitochondrial calcium uniporter) and mitochondrial permeability transition pore (mPTP) opening in this process. Interestingly, seahorse respirometry performed on cortical mitochondria revealed elevation of the oxygen consumption rate (OCR) in the knockout rats on HS, while in SSWT rats HS diet feeding reduced OCR (both vs NS). In addition, we observed activation of the antioxidant system in the SSNPPA−/− rats as opposed to SSWT (on HS diet, assessed in a Trolox‐based assay).ConclusionsTaken together, our data indicate that lack of ANP, via disruption of protective cGMP‐mediated pathways, results in significant alterations in mitochondrial bioenergetics in the renal tissue. By establishing the mechanistic link between ANP and mitochondrial function, further studies will advance the understanding of the mitochondria‐mediated mechanisms affecting renal disease progression.Support or Funding InformationThe study was supported by NIH R00 DK105160 and Dialysis Clinic Inc Reserve Funds.

Astaxanthin Prevents Mitochondrial Impairment Induced by Isoproterenol in Isolated Rat Heart Mitochondria.

Mitochondria are considered to be a power station of the cell. It is known that they play a major role in both normal and pathological heart function. Alterations in mitochondrial bioenergetics are one of the main causes of the origin and progression of heart failure since they have an inhibitory effect on the activity of respiratory complexes in the inner mitochondrial membrane. Astaxanthin (AST) is a xanthophyll carotenoid of mainly marine origin. It has both lipophilic and hydrophilic properties and may prevent mitochondrial dysfunction by permeating the cell membrane and co-localizing within mitochondria. The carotenoid suppresses oxidative stress-induced mitochondrial dysfunction and the development of diseases. In the present study, it was found that the preliminary oral administration of AST upregulated the activity of respiratory chain complexes and ATP synthase and the level of their main subunits, thereby improving the respiration of rat heart mitochondria (RHM) in the heart injured by isoproterenol (ISO). AST decreased the level of cyclophilin D (CyP-D) and increased the level of adenine nucleotide translocase (ANT) in this condition. It was concluded that AST could be considered as a potential mitochondrial-targeted agent in the therapy of pathological conditions associated with oxidative damage and mitochondrial dysfunction. AST, as a dietary supplement, has a potential in the prevention of cardiovascular diseases.

Adaptation of striated muscles to Wolframin deficiency in mice: Alterations in cellular bioenergetics

BackgroundWolfram syndrome (WS), caused by mutations in WFS1 gene, is a multi-targeting disease affecting multiple organ systems. Wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. In this study we aimed to characterize alterations in energy metabolism in the cardiac and in the oxidative and glycolytic skeletal muscles in Wfs1-deficiency. MethodsAlterations in the bioenergetic profiles in the cardiac and skeletal muscles of Wfs1-knock-out (KO) male mice and their wild type male littermates were determined using high resolution respirometry, quantitative RT-PCR, NMR spectroscopy, and immunofluorescence confocal microscopy. ResultsOxygen consumption without ATP synthase activation (leak) was significantly higher in the glycolytic muscles of Wfs1 KO mice compared to wild types. ADP-stimulated respiration with glutamate and malate was reduced in the Wfs1-deficient cardiac as well as oxidative and glycolytic skeletal muscles. ConclusionsWfs1-deficiency in both cardiac and skeletal muscles results in functional alterations of energy transport from mitochondria to ATP-ases. There was a substrate-dependent decrease in the maximal Complex I –linked respiratory capacity of the electron transport system in muscles of Wfs1 KO mice. Moreover, in cardiac and gastrocnemius white muscles a decrease in the function of one pathway were balanced by the increase in the activity of the parallel pathway. General significanceThis work provides new insights to the muscle involvement at early stages of metabolic syndrome like WS as well as developing glucose intolerance.

Pancreatic Acinar Cell Preparation for Oxygen Consumption and Lactate Production Analysis.

Mitochondrial dysfunction is a principal feature of acute pancreatitis (AP) although the underlying mechanisms are still unclear. AP precipitants induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP) in pancreatic acinar cells (PACs), leading to ATP depletion and necrosis. Evaluations of mitochondrial bioenergetics have mainly been performed in isolated PACs using confocal microscopy, with assessment of mitochondrial membrane potential, NADH/FAD+ and ATP levels, coupled with patch-clamp electrophysiology. These studies are technically demanding and time-consuming. Application of Seahorse flux analysis now allows detailed investigations of bioenergetics changes to be performed in cell populations using a multi-well plate-reader format; rates of oxygen consumption (OCR) and extracellular acidification (ECAR) provide important information about cellular respiration and glycolysis, respectively. Parameters such as maximal respiration, ATP-linked capacity and proton leak can be derived from application of a respiratory function "stress" test that involves pharmacological manipulation of the electron transport chain. The use of Seahorse Flux analysis therefore provides a quick, and convenient means to measure detailed cellular bioenergetics and allows results to be coupled with other plate-reader based assays, providing a fuller understanding of the pathophysiological consequences of mitochondrial bioenergetics alterations.

Acquired neuromuscular weakness In Intensive Care Unit: Review article

A substantial number of patients admitted to the ICU because of an acute illness, complicated surgery, severe trauma, or burn injury will develop a de novo form of muscle weakness during the ICU stay that is referred to as “intensive care unit acquired weakness” (ICUAW). This ICUAW evoked by critical illness can be due to axonal neuropathy, primary myopathy, or both. Underlying pathophysiological mechanisms comprise microvascular, electrical, metabolic, and bioenergetic alterations, interacting in a complex way and culminating in loss of muscle strength and/or muscle atrophy. ICUAW is typically symmetrical and affects predominantly proximal limb muscles and respiratory muscles, whereas facial and ocular muscles are often spared. ICUAW is diagnosed in awake and cooperative patients by bedside manual testing of muscle strength and the severity is scored by the Medical Research Council sum score. In cases of atypical clinical presentation or evolution, additional electrophysiological testing may be required for differential diagnosis. The cornerstones of prevention are aggressive treatment of sepsis, early mobilization, preventing hyperglycemia with insulin, and avoiding the use parenteral nutrition during the first week of critical illness. Future research should focus on new preventive and/or therapeutic strategies for this detrimental complication of critical illness and on clarifying how ICUAW contributes to poor longer-term prognosis.

Altered Bioenergetics of Blood Cell Sub-Populations in Acute Pancreatitis Patients.

Acute pancreatitis (AP) is a debilitating, sometimes fatal disease, marked by local injury and systemic inflammation. Mitochondrial dysfunction is a central feature of pancreatic damage in AP, however, its involvement in circulating blood cell subtypes is unknown. This study compared mitochondrial bioenergetics in circulating leukocytes from AP patients and healthy volunteers: 15 patients with mild to severe AP were compared to 10 healthy controls. Monocytes, lymphocytes and neutrophils were isolated using magnetic activated cell sorting and mitochondrial bioenergetics profiles of the cell populations determined using a Seahorse XF24 flux analyser. Rates of oxygen consumption (OCR) and extracellular acidification (ECAR) under conditions of electron transport chain (ETC) inhibition (“stress” test) informed respiratory and glycolytic parameters, respectively. Phorbol ester stimulation was used to trigger the oxidative burst. Basal OCR in all blood cell subtypes was similar in AP patients and controls. However, maximal respiration and spare respiratory capacity of AP patient lymphocytes were decreased, indicating impairment of functional capacity. A diminished oxidative burst occurred in neutrophils from AP patients, compared to controls, whereas this was enhanced in both monocytes and lymphocytes. The data demonstrate important early alterations of bioenergetics in blood cell sub-populations from AP patients, which imply functional alterations linked to clinical disease progression.

Synergistic Effect of Carfilzomib and Metformin in Vascular Plasticity; The Emerging Role of Autophagy

Introduction: Carfilzomib (Cfz) correlates with a risk of reversible cardiotoxicity in 5-10% of multiple myeloma (MM) patients. We have recently shown that metformin (Met) has a prophylactic role against the Cfz-induced cardiotoxicity in vivo, through activation of AMPKα signaling (Blood 2019;133:710-23). However, the impact of Cfz on vascular function is obscure. Therefore, we sought to investigate: i) the acute, ii) the sub-chronic effect of Cfz on the vascular reactivity, iii) the effect of metformin co-administration on the vascular phenotype and iv) the impact of Cfz and Met co-administration on aged Human Aortic Smooth Muscle Cells (HAoSMCs). Methods: Forty male C57Bl/6 mice were assigned as follows: Acute Protocol: i. Normal Saline (N/S 0.9%); ii. Cfz (8 mg/kg); iii. Met (140 mg/kg); iv. Cfz+Met (8 mg/kg; 140 mg/kg respectively). N/S and Cfz were injected intraperitoneally (IP), while Met was administered via oral gavage daily, for 2 days (n=5 per group). Mice were sacrificed 48h after the initiation of treatments. Sub-chronic Protocol: i. N/S 0.9%; ii. Cfz (8 mg/kg); iii. Met (140mg/kg); iv. Cfz+Met (8 mg/kg; 140mg/kg respectively) for 6 days. N/S and Cfz was injected IP, while Met was administered via oral gavage on alternate days for 6 days (n=5 per group). Mice were sacrificed on the 7th day after the initiation of treatments. At the endpoint of the experiments (48h and 7 days respectively), heparinized whole blood (WB) samples and aortic tissues were collected from the experimental animals. WB samples were used to determine leucocytes derived ROS production (oxidative burst) and aorta sections (3 mm) underwent ex vivo vascular studies (relaxants: Achetlyocholine (ACh), nitroglycerin (Gtn) contractant: PDGF2α). The remaining aortic tissue underwent molecular Western blot analysis for the elucidation of the underlying mechanism. For the in vitro experiments HAoSMCs Passage 7-8 were subjected to Cfz (0.1, 0.3μM), Met (10μM, 10mM) treatment and to the combination of the two compounds. Subsequently, cells were treated with the optimal concentrations of the compounds 24h after treatment with Angiotensin II (AngII, 100nM), CoCL2 (150μM) and 48h after Glucose (25μM) to simulate the hypertensive, hypoxic and diabetic stimuli respectively. Proliferation was assessed by MTT; LC3IIB (as a marker of autophagy) expression was analyzed by immunofluorescence (IF) and cellular bioenergetics and ATP production were investigated through Seahorse XF analyzer, facilitating the study of intracellular glycolysis and oxidative phosphorylation. Results: In the acute setting, Met did not ameliorate the ROS production induced by Cfz, while no effect of Cfz on vascular relaxation/contraction was evident. In the sub-chronic setting, Met limited WB macrophage-derived oxidative burst and, while Cfz did not affect vascular reactivity, the co-administration of Cfz and Met increased PDGF2α induced aortic contractility, deducing an increased plasticity of the vessel. Western blot analysis of the aortas revealed that Cfz led to a de-phosphorylation of AMPKα, an upregulation of mTOR and iNOS while induced endoplasmic reticulum (ER) stress as indicated by increased Calnexin, IRE1a and Bip expression. In the Cfz+Met group, Met restored AMPKα phosphorylation and partially abrogated ER stress markers calnexin and Bip. However, the co-administration of the agents led to a synergistic amplification of autophagy as shown by LC3IIB expression. In the aged HAoSMCs, only Met exhibited cytotoxic capacity, while Cfz failed to exhibit any cytotoxicity. Moreover, Met was found to totally abrogate mitochondrial oxidative phosphorylation and shift cellular ATP production to anaerobic glycolysis, while Cfz had no effect on cellular bioenergetics. In terms of cardiovascular risk factors, the co-treatment of Cfz and Met led to an additive cytotoxic effect in presence of CoCL2 and AngII. Finally, in all tested conditions Cfz and Met were found to have a synergistic effect in inducing autophagy, as shown by LC3IIB upregulation. Conclusions: Sub-chronic co-administration of CFz and Met increases vascular plasticity in vivo, through a converging AMPKα- and ER stress-dependent autophagy. In vitro, Cfz does not present any effect on aged cells, probably due to the decreased proteasome activity, while Met synergizes with Cfz to decrease proliferation through alterations in cellular bioenergetics and induction of autophagy. Disclosures Kastritis: Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Genesis: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Medison: Honoraria; Janssen: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Takeda: Honoraria, Other: Travel expenses, Research Funding.

Glucose starvation-induced oxidative stress causes mitochondrial dysfunction and apoptosis via Prohibitin 1 upregulation in human breast cancer cells

In recent years there has been an upsurge in research focusing on reprogramming cancer cells through understanding of their metabolic signatures. Alterations in mitochondrial bioenergetics and impaired mitochondrial function may serve as effective targeting strategies especially in triple-negative breast cancers (TNBCs) where hormone receptors and endocrine therapy are absent. Glucose starvation (GS) of MDA-MB-231 and MCF-7 breast cancer cells showed decrease in mitochondrial Oxygen Consumption Rate (OCR), which was rescuable to control level through addition of exogenous antioxidant N-Acetyl Cysteine (NAC). Mechanistically, GS led to increase in mitochondrial ROS and upregulation of the pleiotropic protein, Prohibitin 1 (PHB1), leading to its dissociation from Dynamin-related protein 1 (DRP1), perturbance of mitochondrial membrane potential (MMP) and triggering of the apoptosis cascade. PHB1 also reduced the invasive and migratory potential of both cell lines. We emphasize that glucose starvation remarkably sensitized the highly glycolytic metastatic TNBC cell line, MDA-MB-231 to apoptosis and decreased its migratory potential. Based on our findings, additional TNBC cell lines can be evaluated and a nutritional paradigm be proposed for anticancer therapy.

Changes in cardiac and hepatic energetic metabolism in gerbils infected by Listeria monocytogenes

Energy metabolism is a sensitive indicator of cellular disorders. Therefore, the objective of this study was to investigate changes in cardiac and hepatic energy metabolism during listeriosis using an experimental model. We divided gerbils into two groups: Control (n = 11) and orally Infected (n = 12) with 5 × 109 CFU/mL of Listeria monocytogenes. Euthanasia and sampling were performed on days 6 and 12 post-infection (PI). Histopathological lesions were not found in the heart; however, the liver showed pyogranuloma. In the hearts of infected animals, cytosolic creatine kinase activity was lower on day 6 and 12 PI; mitochondrial creatine kinase/pyruvate kinase (PK), and sodium potassium pump (Na+/K+-ATPase) activities were lower on day 12 PI. Hepatic PK and Na+/K+-ATPase activities were lower in the infected group on day 12 PI. Lipoperoxidation was higher in the livers and hearts of infected animals on day 12 PI, and antioxidant capacity against peroxyl radicals (ACAP) was also higher in this group. These data suggest that subclinical listeriosis alters hepatic and cardiac energy metabolism, possibly related to decreased activity of phosphotransferases and ATPase. Subsequent antioxidant responses are not sufficient to correct alterations in lipid peroxidation and bioenergetics, possibly leading to important cellular pathological mechanisms.

P3116New role of EPAC1 in Anthracycline-induced cardiotoxicity and anticancer therapy

Abstract Introduction Doxorubicin (Dox) is an anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent induces many side effects such as cardiotoxicity leading to dilated cardiomyopathy (DCM). The cardiotoxicity of Dox has been related to reactive oxygen species generation, DNA intercalation, topoisomerase II inhibition and bioenergetics alterations leading to cardiomyocyte death. Objective Nowadays the challenge is to find new treatment options aiming at reducing Dox cardiotoxicity. Epac (exchange protein directly activated by cAMP) signaling could be worth investigating as Epac activates small G proteins which are known to be involved in Dox-induced cardiotoxicity. Methods We investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/ Knock-out Epac1 mice, iv injections, 12mg/kg cumulative dose) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM, 24h, Dox 1μM). Results In vivo, Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction (increase of Ca2+ waves and Ca2+ leaks). In vitro, as measured by flow cytometry and western blot, Dox (1μM) induced DNA damages and cell death in NRVM. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation and cell size reduction. The inhibition of Epac1 (ESI09, CE3F4) decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. Moreover, in vivo, Epac1 KO mice were protected against Dox-induced cardiotoxicity by unaltered cardiac function (no DCM) and calcium homeostasis at 15 weeks post-treatment. Conclusion Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy. Indeed, preliminary data show also that preventing Dox-induced cardiotoxicity, the inhibition of Epac1 can also potentiate cancerous cells death. Acknowledgement/Funding Labex Lermit (ANR 0033), Torino and Inserm

Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells

Unlike most other tissues, the colon epithelium is exposed to high levels of H2S derived from gut microbial metabolism. H2S is a signaling molecule that modulates various physiological effects. It is also a respiratory toxin that inhibits complex IV in the electron transfer chain (ETC). Colon epithelial cells are adapted to high environmental H2S exposure as they harbor an efficient mitochondrial H2S oxidation pathway, which is dedicated to its disposal. Herein, we report that the sulfide oxidation pathway enzymes are apically localized in human colonic crypts at the host-microbiome interface, but that the normal apical-to-crypt gradient is lost in colorectal cancer epithelium. We found that sulfide quinone oxidoreductase (SQR), which catalyzes the committing step in the mitochondrial sulfide oxidation pathway and couples to complex III, is a critical respiratory shield against H2S poisoning. H2S at concentrations ≤20 μm stimulated the oxygen consumption rate in colon epithelial cells, but, when SQR expression was ablated, H2S concentrations as low as 5 μm poisoned cells. Mitochondrial H2S oxidation altered cellular bioenergetics, inducing a reductive shift in the NAD+/NADH redox couple. The consequent electron acceptor insufficiency caused uridine and aspartate deficiency and enhanced glutamine-dependent reductive carboxylation. The metabolomic signature of this H2S-induced stress response mapped, in part, to redox-sensitive nodes in central carbon metabolism. Colorectal cancer tissues and cell lines appeared to counter the growth-restricting effects of H2S by overexpressing sulfide oxidation pathway enzymes. Our findings reveal an alternative mechanism for H2S signaling, arising from alterations in mitochondrial bioenergetics that drive metabolic reprogramming.

Alterations in platelet bioenergetics in Group 2 PH-HFpEF patients.

BackgroundPulmonary hypertension (PH) is characterized by elevated pulmonary artery pressure but classified into subgroups based on disease etiology. It is established that systemic bioenergetic dysfunction contributes to the pathogenesis of pulmonary arterial hypertension classified as World Health Organization (WHO) Group 1. Consistent with this, we previously showed that platelets from Group 1 PH patients demonstrate increased glycolysis and enhanced maximal capacity for oxidative phosphorylation, which is due to increased fatty acid oxidation (FAO). However, it remains unclear whether identical mitochondrial alterations contribute to the pathology of other PH subgroups. The most prevalent subgroup of PH is WHO Group 2, which encompasses pulmonary venous hypertension secondary to left heart disease. Here, we hypothesized that platelets from Group 2 subjects show bioenergetic alteration compared to controls, and that these changes were similar to Group 1 PH patients.Method and resultsWe isolated platelets from subjects with Group 2 PH and controls (n = 20) and measured platelet bioenergetics as well as hemodynamic parameters. We demonstrate that Group 2 PH platelets do not show a change in glycolytic rate but do demonstrate enhanced maximal capacity of respiration due at least partially to increased FAO. Moreover, this enhanced maximal capacity correlates negatively with right ventricular stroke work index and is not changed by administration of inhaled nitrite, a modulator of pulmonary hemodynamics.ConclusionsThese data demonstrate that Group 2 PH subjects have altered bioenergetic function though this alteration is not identical to that of Group 1 PH. The implications of this alteration for disease pathogenesis will be discussed.

Platelet bioenergetics correlate with muscle energetics and are altered in older adults.

Physical function decreases with age, and though bioenergetic alterations contribute to this decline, the mechanisms by which mitochondrial function changes with age remains unclear. This is partially because human mitochondrial studies require highly invasive procedures, such as muscle biopsies, to obtain live tissue with functional mitochondria. However, recent studies demonstrate that circulating blood cells are potentially informative in identifying systemic bioenergetic changes. Here, we hypothesize that human platelet bioenergetics reflect bioenergetics measured in muscle biopsies. We demonstrate that maximal and ATP-linked respiratory rate measured in isolated platelets from older adults (86-93 years) correlates significantly with maximal respiration (r = 0.595; P = 0.003) measured by muscle biopsy respirometry and maximal ATP production (r = 0.643; P = 0.004) measured by 31P-MRS respectively, in the same individuals. Comparison of platelet bioenergetics in this aged cohort to platelets from younger adults (18-35 years) shows aged adults demonstrate lower basal and ATP-linked respiration. Platelets from older adults also show enhanced proton leak, which is likely due to increased protein levels of uncoupling protein 2, and correlates with increased gate speed in this cohort (r = 0.58; P = 0.0019). While no significant difference in glycolysis was observed in older adults compared to younger adults, platelet glycolytic rate correlated with fatigability (r = 0.44; P = 0.016). These data advance the mechanistic understanding of age-related changes in mitochondrial function. Further, they suggest that measuring platelet bioenergetics provides a potential supplement or surrogate for muscle biopsy measurement and may be a valuable tool to study mitochondrial involvement in age-related decline of physical function.

Adult stem cell deficits drive Slc29a3 disorders in mice

Mutations exclusively in equilibrative nucleoside transporter 3 (ENT3), the only intracellular nucleoside transporter within the solute carrier 29 (SLC29) gene family, cause an expanding spectrum of human genetic disorders (e.g., H syndrome, PHID syndrome, and SHML/RDD syndrome). Here, we identify adult stem cell deficits that drive ENT3-related abnormalities in mice. ENT3 deficiency alters hematopoietic and mesenchymal stem cell fates; the former leads to stem cell exhaustion, and the latter leads to breaches of mesodermal tissue integrity. The molecular pathogenesis stems from the loss of lysosomal adenosine transport, which impedes autophagy-regulated stem cell differentiation programs via misregulation of the AMPK-mTOR-ULK axis. Furthermore, mass spectrometry-based metabolomics and bioenergetics studies identify defects in fatty acid utilization, and alterations in mitochondrial bioenergetics can additionally propel stem cell deficits. Genetic, pharmacologic and stem cell interventions ameliorate ENT3-disease pathologies and extend the lifespan of ENT3-deficient mice. These findings delineate a primary pathogenic basis for the development of ENT3 spectrum disorders and offer critical mechanistic insights into treating human ENT3-related disorders.