p66Shc signaling and autophagy impact on C2C12 myoblast differentiation during senescence.

During aging, muscle regenerative capacities decline, which is concomitant with the loss of satellite cells that enter in a state of irreversible senescence. However, what mechanisms are involved in myogenic senescence and differentiation are largely unknown. Here, we showed that early-passage or "young" C2C12 myoblasts activated the redox-sensitive p66Shc signaling pathway, exhibited a strong antioxidant protection and a bioenergetic profile relying predominantly on OXPHOS, responses that decrease progressively during differentiation. Furthermore, autophagy was increased in myotubes. Otherwise, late-passage or "senescent" myoblasts led to a highly metabolic profile, relying on both OXPHOS and glycolysis, that may be influenced by the loss of SQSTM1/p62 which tightly regulates the metabolic shift from aerobic glycolysis to OXPHOS. Furthermore, during differentiation of late-passage C2C12 cells, both p66Shc signaling and autophagy were impaired and this coincides with reduced myogenic capacity. Our findings recognized that the lack of p66Shc compromises the proliferation and the onset of the differentiation of C2C12 myoblasts. Moreover, the Atg7 silencing favored myoblasts growth, whereas interfered in the viability of differentiated myotubes. Then, our work demonstrates that the p66Shc signaling pathway, which highly influences cellular metabolic status and oxidative environment, is critical for the myogenic commitment and differentiation of C2C12 cells. Our findings also support that autophagy is essential for the metabolic switch observed during the differentiation of C2C12 myoblasts, confirming how its regulation determines cell fate. The regulatory roles of p66Shc and autophagy mechanisms on myogenesis require future attention as possible tools that could predict and measure the aging-related state of frailty and disability.

Oestradiol and osteoclast differentiation: Effects on p53 and mitochondrial metabolism.

BACKGROUND: Oestrogen deficiency increases bone resorption, contributing to osteoporosis development. Yet, the mechanisms mediating the effects of oestrogen on osteoclasts remain unclear. This study aimed to elucidate the early metabolic alteration induced by RANKL, the essential cytokine in osteoclastogenesis and 17-beta-oestradiol (E2) on osteoclast progenitor cells, using RAW 264.7 macrophage cell line and primary bone marrow-derived macrophages as biological models. RESULTS: This research demonstrated that, in osteoclast precursors, RANKL stimulates complex I activity, oxidative phosphorylation (OXPHOS) and mitochondria-derived ATP production as early as 3 h of exposure. This effect on mitochondrial bioenergetics is associated with an increased capacity to oxidize TCA cycle substrates, fatty acids and amino acids. E2 inhibited all effects of RANKL on mitochondria metabolism. In the presence of RANKL, E2 also decreased cell number and stimulated the mitochondrial-mediated apoptotic pathway, detected as early as 3 h. Further, the pro-apoptotic effects of E2 during osteoclast differentiation were associated with an accumulation of p392S-p53 in mitochondria. CONCLUSIONS: These findings elucidate the early effects of RANKL on osteoclast progenitor metabolism and suggest novel p53-mediated mechanisms that contribute to postmenopausal osteoporosis.

Age-dependent energy metabolism and transcriptome changes in urine-derived stem cells.

The global population over 60 years old is projected to reach 1.5 billion by 2050. Understanding age-related disorders and gender-specificities is crucial for a healthy aging. Reliable age-related biomarkers are needed, preferentially obtained through non-invasive methods. Urine-derived stem cells (UDSCs) can be easily obtained, although a detailed bioenergetic characterization, according to the donor aging, remain unexplored. UDSCs were isolated from young and elderly adult women (22-35 and 70-94 years old, respectively). Surprisingly, UDSCs from elderly subjects exhibited significantly higher maximal oxygen consumption and bioenergetic health index than those from younger individuals, evaluated through oxygen consumption rate. Exploratory data analysis methods were applied to engineer a minimal subset of features for the classification and stratification of UDSCs. Additionally, RNAseq of UDSCs was performed to identify age-related transcriptional changes. Transcriptional analysis revealed downregulation of genes related to glucuronidation and estrogen metabolism, and upregulation of inflammation-related genes in UDSCs from elderly individuals. This study demonstrates unexpected differences in the UDSCs' OCR between young and elderly individuals, revealing improved bioenergetics in concurrent with an aged-like transcriptome signature. UDSCs offer a non-invasive model for studying age-related changes, holding promise for aging research and therapeutic studies.

Mitochondria dysfunction induced by decyl-TPP mitochondriotropic antioxidant based on caffeic acid AntiOxCIN6 sensitizes cisplatin lung anticancer therapy due to a remodeling of energy metabolism.

The pharmacological interest in mitochondria is very relevant since these crucial organelles are involved in the pathogenesis of multiple diseases, such as cancer. In order to modulate cellular redox/oxidative balance and enhance mitochondrial function, numerous polyphenolic derivatives targeting mitochondria have been developed. Still, due to the drug resistance emergence in several cancer therapies, significant efforts are being made to develop drugs that combine the induction of mitochondrial metabolic reprogramming with the ability to generate reactive oxygen species, taking into consideration the varying metabolic profiles of different cell types. We previously developed a mitochondria-targeted antioxidant (AntiOxCIN6) by linking caffeic acid to lipophilic triphenylphosphonium cation through a 10-carbon aliphatic chain. The antioxidant activity of AntiOxCIN6 has been documented but how the mitochondriotropic compound impact energy metabolism of both normal and cancer cells remains unknown. We demonstrated that AntiOxCIN6 increased antioxidant defense system in HepG2 cells, although ROS clearance was ineffective. Consequently, AntiOxCIN6 significantly decreased mitochondrial function and morphology, culminating in a decreased capacity in complex I-driven ATP production without affecting cell viability. These alterations were accompanied by an increase in glycolytic fluxes. Additionally, we demonstrate that AntiOxCIN6 sensitized A549 adenocarcinoma cells for CIS-induced apoptotic cell death, while AntiOxCIN6 appears to cause metabolic changes or a redox pre-conditioning on lung MRC-5 fibroblasts, conferring protection against cisplatin. We propose that length and hydrophobicity of the C10-TPP+ alkyl linker play a significant role in inducing mitochondrial and cellular toxicity, while the presence of the antioxidant caffeic acid appears to be responsible for activating cytoprotective pathways.

ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens.

Introduction: Estrogens inhibit bone resorption and preserve bone mass, at least in part, via direct effects on osteoclasts. The binding of RANKL, the critical cytokine for osteoclast differentiation, to its receptor in osteoclast precursor cells of the monocyte lineage recruits the adaptor protein TRAF6 and activates multiple signaling pathways. Early effects of RANKL include stimulation of mitochondria. 17ß-estradiol (E2) prevents the effects of RANKL on mitochondria and promotes mitochondria mediated apoptotic cell death. However, the molecular mechanisms responsible for the actions of RANKL and estrogens on mitochondria remain unknown. Evolutionarily Conserved Signaling Intermediate in Toll Pathway (ECSIT) is a complex I-associated protein that regulates immune responses in macrophages following the engagement of Toll-like receptors, which also recruit TRAF6. Here, we examined whether ECSIT could be implicated in the rapid effects of RANKL and E2 on osteoclast progenitors. Methods: Bone marrow-derived macrophages (BMMs) from C57BL/6 mice were cultured with RANKL (30 ng/ml) with or without E2 (10-8 M). ECSIT-TRAF6 interaction was evaluated by co-immunoprecipitation and ECSIT levels in mitochondria and cytosolic fractions by Western blot. ShRNA lentivirus particles were used to knockdown ECSIT. Osteoclasts were enumerated after tartrate-resistant acid phosphatase staining. Oxygen consumption and extracellular acidification rates were measured with Seahorse XFe96 Analyzer. ATP, lactate, and NAD/NADH were measured with commercial assay kits. NADH oxidation to NAD was used to evaluate Complex I activity. Total and mitochondrial ROS, and mitochondrial membrane potential were measured with H2DCFDA, MitoSOX, and TMRM probes, respectively. Degradation of DEVD-AFC was used to measure Caspase-3 activity. Results: We found that RANKL promoted ECSIT-TRAF6 interaction and increased the levels of ECSIT in mitochondria. E2 abrogated these effects of RANKL. Silencing of ECSIT decreased osteoclast differentiation and abrogated the inhibitory effects of E2 on osteoclastogenesis. Loss of ECSIT decreased complex I activity, oxygen consumption, NAD+/NADH redox ratio, and ATP production and increased mitochondrial ROS. In the absence of ECSIT, the stimulatory actions of RANKL on complex I activity and all other markers of oxidative phosphorylation, as well as their inhibition by E2, were prevented. Instead, RANKL stimulated apoptosis of osteoclast progenitors. Discussion: These findings suggest that dysregulated mitochondria cause a switch in RANKL signaling from pro-survival to pro-apoptotic. In addition, our results indicate that ECSIT represents a central node for the early effects of RANKL on mitochondria and that inhibition of ECSIT-mediated mitochondria stimulation might contribute to the bone protective actions of estrogens.

Counteracting Colon Cancer by Inhibiting Mitochondrial Respiration and Glycolysis with a Selective PKCδ Activator.

Metabolic reprogramming is a central hub in tumor development and progression. Therefore, several efforts have been developed to find improved therapeutic approaches targeting cancer cell metabolism. Recently, we identified the 7α-acetoxy-6ß-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz) as a PKCδ-selective activator with potent anti-proliferative activity in colon cancer by stimulating a PKCδ-dependent mitochondrial apoptotic pathway. Herein, we investigated whether the antitumor activity of Roy-Bz, in colon cancer, could be related to glucose metabolism interference. The results showed that Roy-Bz decreased the mitochondrial respiration in human colon HCT116 cancer cells, by reducing electron transfer chain complexes I/III. Consistently, this effect was associated with downregulation of the mitochondrial markers cytochrome c oxidase subunit 4 (COX4), voltage-dependent anion channel (VDAC) and mitochondrial import receptor subunit TOM20 homolog (TOM20), and upregulation of synthesis of cytochrome c oxidase 2 (SCO2). Roy-Bz also dropped glycolysis, decreasing the expression of critical glycolytic markers directly implicated in glucose metabolism such as glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and monocarboxylate transporter 4 (MCT4), and increasing TP53-induced glycolysis and apoptosis regulator (TIGAR) protein levels. These results were further corroborated in tumor xenografts of colon cancer. Altogether, using a PKCδ-selective activator, this work evidenced a potential dual role of PKCδ in tumor cell metabolism, resulting from the inhibition of both mitochondrial respiration and glycolysis. Additionally, it reinforces the antitumor therapeutic potential of Roy-Bz in colon cancer by targeting glucose metabolism.

Brassica oleracea Var italica by-Products Prevent Lipid Accumulation and Cell Death in a Liver Cell Model of Lipid Toxicity.

Obesity, a rising concern in the Eastern world, encompasses several co-morbidities, namely non-alcoholic fatty liver disease (NAFLD). Potential natural-based interventions to decrease the burden of obesity complications are being investigated. Many of the edible parts of plants are not sold for consumption and end up as massive waste, losing nutritional potential. In fact, a sizeable amount of waste is generated within the different steps of the food supply chain, representing a massive loss of both plant material and natural resources. A good example is Brassica by-products (BBPs). The objective of this work was to investigate the effect of three different extracts from broccoli (Brassica oleracea var italica) by-products in an in vitro model of free fatty acid (FFA)-induced lipotoxicity using human hepatoma HepG2 cells. Broccoli leaf, stalk, and inflorescence extracts induced a dose-dependent decrease in the cell viability of HepG2 cells. However, the maximal non-lethal concentrations of leaves, stalks, and inflorescences (10 µg/mL) did not compromise mitochondrial function or neutral lipid accumulation in HepG2 cells. The extracts significantly decreased FFA-induced lipid accumulation in HepG2 cells either in a co-incubation or pre-incubation strategy. The broccoli extracts' capacity to prevent the FFA-induced decrease in catalase activity in HepG2 may explain the observed effects.

1,8-Cineole ameliorates right ventricle dysfunction associated with pulmonary arterial hypertension by restoring connexin43 and mitochondrial homeostasis.

For the first time, the present study unravels a cardiospecific therapeutic approach for Pulmonary Arterial Hypertension (PAH), a disease with a very poor prognosis and high mortality rates due to right ventricle (RV) dysfunction. We first established a new in vitro model of high-pressure-induced hypertrophy that closely resembles heart defects associated with PAH and validated our in vitro findings on a preclinical in vivo model of monocrotaline (MCT)-induced PAH. Our results showed the in vitro antihypertrophic effect of 1,8-cineole, a monoterpene widely found in several essential oils. Also, a decrease in RV hypertrophy and fibrosis, and an improvement in heart function in vivo was observed, when 1,8-cineole was applied topically. Furthermore, 1,8-cineole restored gap junction protein connexin43 distribution at the intercalated disks and mitochondrial functionality, suggesting it may act by preserving cardiac cell-to-cell communication and bioenergetics. Overall, our results point out a promising therapeutic compound that can be easily applied topically, thus paving the way for the development of effective cardiac-specific therapies to greatly improve PAH outcomes.

The advantages of physical exercise as a preventive strategy against NAFLD in postmenopausal women.

BACKGROUND: The prevalence and severity of nonalcoholic fatty liver disease (NAFLD) increase in women after menopause. This narrative review discusses the causes and consequences of NAFLD in postmenopausal women and describes how physical activity can contribute to its prevention. METHODS: The authors followed the narrative review method to perform a critical and objective analysis of the current knowledge on the topic. The Medical Subject Heading keywords 'physical exercise', 'menopause', 'hormone replacement therapy', 'estradiol' and 'NAFLD' were used to establish a conceptual framework. The databases used to collect relevant references included Medline and specialized high-impact journals. RESULTS: Higher visceral adiposity, higher rate of lipolysis in adipose tissue after oestrogen drop and changes in the expression of housekeeping proteins involved in hepatic lipid management are observed in women after menopause, contributing to NAFLD. Excessive liver steatosis leads to hepatic insulin resistance, oxidative stress and inflammation, accelerating NAFLD progression. Physical activity brings beneficial effects against several postmenopausal-associated complications, including NAFLD progression. Aerobic and resistance exercises partially counteract alterations induced by metabolic syndrome in sedentary postmenopausal women, impacting NAFLD progression and severity. CONCLUSIONS: With the increased global obesity epidemic in developing countries, NAFLD is becoming a severe problem with increased prevalence in women after menopause. Evidence shows that physical activity may delay NAFLD development and severity in postmenopausal women, although the prescription of age-appropriate physical activity programmes is advisable to assure the health benefits.

Carbon Monoxide-Neuroglobin Axis Targeting Metabolism Against Inflammation in BV-2 Microglial Cells.

Microglia are the immune competent cell of the central nervous system (CNS), promoting brain homeostasis and regulating inflammatory response against infection and injury. Chronic or exacerbated neuroinflammation is a cause of damage in several brain pathologies. Endogenous carbon monoxide (CO), produced from the degradation of heme, is described as anti-apoptotic and anti-inflammatory in several contexts, including in the CNS. Neuroglobin (Ngb) is a haemoglobin-homologous protein, which upregulation triggers antioxidant defence and prevents neuronal apoptosis. Thus, we hypothesised a crosstalk between CO and Ngb, in particular, that the anti-neuroinflammatory role of CO in microglia depends on Ngb. A novel CO-releasing molecule (ALF826) based on molybdenum was used for delivering CO in microglial culture.BV-2 mouse microglial cell line was challenged with lipopolysaccharide (LPS) for triggering inflammation, and after 6 h ALF826 was added. CO exposure limited inflammation by decreasing inducible nitric oxide synthase (iNOS) expression and the production of nitric oxide (NO) and tumour necrosis factor-α (TNF-α), and by increasing interleukine-10 (IL-10) release. CO-induced Ngb upregulation correlated in time with CO's anti-inflammatory effect. Moreover, knocking down Ngb reversed the anti-inflammatory effect of CO, suggesting that dependents on Ngb expression. CO-induced Ngb upregulation was independent on ROS signalling, but partially dependent on the transcriptional factor SP1. Finally, microglial cell metabolism is also involved in the inflammatory response. In fact, LPS treatment decreased oxygen consumption in microglia, indicating a switch to glycolysis, which is associated with a proinflammatory. While CO treatment increased oxygen consumption, reverting LPS effect and indicating a metabolic shift into a more oxidative metabolism. Moreover, in the absence of Ngb, this phenotype was no longer observed, indicating Ngb is needed for CO's modulation of microglial metabolism. Finally, the metabolic shift induced by CO did not depend on alteration of mitochondrial population. In conclusion, neuroglobin emerges for the first time as a key player for CO signalling against exacerbated inflammation in microglia.

Three in One: The Potential of Brassica By-Products against Economic Waste, Environmental Hazard, and Metabolic Disruption in Obesity.

A large amount of waste is generated within the different steps of the food supply chain, representing a significant loss of natural resources, plant material, and economic value for producers and consumers. During harvesting and processing, many parts of edible plants are not sold for consumption and end up as massive waste, adding environmental hazards to the list of concerns regarding food wastage. Examples are Brassica oleracea var. Italica (broccoli) by-products, which represent 75% of the plant mass. A growing concern in the Western world is obesity, which results from incorrect lifestyles and comprises an extensive array of co-morbidities. Several studies have linked these co-morbidities to increased oxidative stress; thus, naturally occurring and readily available antioxidant compounds are an attractive way to mitigate metabolic diseases. The idea of by-products selected for their biomedical value is not novel. However, there is innovation underlying the use of Brassica by-products in the context of obesity. For this reason, Brassica by-products are prime candidates to be used in the treatment of obesity due to its bioactive compounds, such as sulforaphane, which possess antioxidant activity. Here, we review the economic and health potential of Brassica bioactive compounds in the context of obesity.

Analysis of Proapoptotic Protein Trafficking to and from Mitochondria.

Mitochondria play a key role in cell death and its regulation. The permeabilization of the outer mitochondrial membrane, which is mainly controlled by proteins of the BCL-2 family, is a key event that can be directly induced by different signaling pathways, including p53-mediated, and results in the release of proapoptotic factors to the cytosol, such as cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor-of-apoptosis (IAP) binding protein with low pI (SMAC/Diablo), Omi serine protease (Omi/HtrA2), apoptosis-inducing factor (AIF), or endonuclease G (Endo-G). Hence, the determination of subcellular localization of these proteins is extremely important to predict cell fate and elucidate the specific mechanism of apoptosis. Here we describe experimental protocols that can be used to study the subcellular location of different proapoptotic proteins to be used in basic cell biology and toxicology studies.

The mitochondrial permeability transition pore: an evolving concept critical for cell life and death.

In this review, we summarize current knowledge of perhaps one of the most intriguing phenomena in cell biology: the mitochondrial permeability transition pore (mPTP). This phenomenon, which was initially observed as a sudden loss of inner mitochondrial membrane impermeability caused by excessive calcium, has been studied for almost 50 years, and still no definitive answer has been provided regarding its mechanisms. From its initial consideration as an in vitro artifact to the current notion that the mPTP is a phenomenon with physiological and pathological implications, a long road has been travelled. We here summarize the role of mitochondria in cytosolic calcium control and the evolving concepts regarding the mitochondrial permeability transition (mPT) and the mPTP. We show how the evolving mPTP models and mechanisms, which involve many proposed mitochondrial protein components, have arisen from methodological advances and more complex biological models. We describe how scientific progress and methodological advances have allowed milestone discoveries on mPTP regulation and composition and its recognition as a valid target for drug development and a critical component of mitochondrial biology.

Afrostyrax lepidophyllus Mildbr. and Monodora myristica (Gaertn.) Dunal Extracts Decrease Doxorubicin Cytotoxicity on H9c2 Cardiomyoblasts.

MATERIALS AND METHODS: Bark extracts of these plants (1 and 25 µg/mL) were added 3 hours before coincubating H9c2 cardiomyoblasts with Dox (0.5 and 1 µM) for 24 hours more. We measured cell mass and metabolic viability, mitochondrial transmembrane potential, superoxide anion content, and activity-like of caspase-3 and caspase-9 following treatment with the extracts and/or Dox. Also, selenium and vitamin C contents were measured in the plant extracts. RESULTS: The results confirmed that Dox treatment decreased cell mass, mitochondrial membrane potential and metabolic viability, increased mitochondrial superoxide anion, and stimulated caspase-3 and caspase-9-like activities. Pretreatment of the cells with the plant extracts significantly inhibited Dox cytotoxicity, with more significant results at the higher concentration. Measurements of selenium and vitamin C in the extracts revealed higher concentration of both when compared with other Cameroonian spices. CONCLUSION: Both extracts of A. lepidophyllus and M. myristica were effective against Dox-induced cytotoxicity, most likely due to their content in antioxidants.

Sustaining efficient immune functions with regular physical exercise in the COVID-19 era and beyond.

The new coronavirus (SARS-CoV-2) appearance in Wuhan, China, did rise the new virus disease (COVID-19), which spread globally in a short time, leading the World Health Organization to declare a new global pandemic. To contain and mitigate the spread of SARS-CoV-2, specific public health procedures were implemented in virtually all countries, with a significant impact on society, making it difficult to keep the regular practice of physical activity. It is widely accepted that an active lifestyle contributes to the improvement of general health and preservation of cardiovascular, respiratory, osteo-muscular and immune system capacities. The positive effects of regular physical activity on the immune system have emerged as a pivotal trigger of general health, underlying the beneficial effects of physical activity on multiple physiological systems. Accordingly, recent studies have already pointed out the negative impact of physical inactivity caused by the social isolation imposed by the public sanitary authorities due to COVID-19. Nevertheless, there are still no current narrative reviews evaluating the real impact of COVID-19 on active lifestyle or even discussing the possible beneficial effects of exercise-promoted immune upgrade against the severity or progression of COVID-19. Based on the consensus in the scientific literature, in this review, we discuss how an exercise adherence could adequately improve immune responses in times of the 'COVID-19 Era and beyond'.

Glutaminolysis is a metabolic route essential for survival and growth of prostate cancer cells and a target of 5α-dihydrotestosterone regulation.

PURPOSE: Resistance to androgen-deprivation therapies and progression to so-called castrate-resistant prostate cancer (CRPC) remain challenges in prostate cancer (PCa) management and treatment. Among other alterations, CRPC has been associated with metabolic reprogramming driven by androgens. Here, we investigated the role of androgens in regulating glutaminolysis in PCa cells and determined the relevance of this metabolic route in controlling the survival and growth of androgen-sensitive (LNCaP) and CRPC (DU145 and PC3) cells. METHODS: PCa cells (LNCaP, DU145 and PC3) and 3-month old rats were treated with 5α-dihydrotestosterone (DHT). Alternatively, LNCaP cells were exposed to the glutaminase inhibitor BPTES, alone or in combination with the anti-androgen bicalutamide. Biochemical, Western blot and extracellular flux assays were used to evaluate the viability, proliferation, migration and metabolism of PCa cells in response to DHT treatment or glutaminase inhibition. RESULTS: We found that DHT up-regulated the expression of the glutamine transporter ASCT2 and glutaminase, both in vitro in LNCaP cells and in vivo in rat prostate cells. BPTES diminished the viability and migration of PCa cells, while increasing caspase-3 activity. CRPC cells were found to be more dependent on glutamine and more sensitive to glutaminase inhibition. BPTES and bicalutamide co-treatment had an additive effect on suppressing LNCaP cell viability. Finally, we found that inhibition of glutaminolysis differentially affected glycolysis and lipid metabolism in both androgen-sensitive and CRPC cells. CONCLUSION: Our data reveal glutaminolysis as a central metabolic route controlling PCa cell fate and highlight the relevance of targeting glutaminase for CRPC treatment.

Maternal obesity in sheep impairs foetal hepatic mitochondrial respiratory chain capacity.

BACKGROUND: Changes in the nutritional environment in utero induced by maternal obesity (MO) lead to foetal metabolic dysfunction predisposing offspring to later-life metabolic diseases. Since mitochondria play a crucial role in hepatic metabolism and function, we hypothesized that MO prior to conception and throughout pregnancy programmes foetal sheep liver mitochondrial phenotype. MATERIAL AND METHODS: Ewes ate an obesogenic diet (150% requirements; MO), or 100% requirements (CTR), from 60 days prior to conception. Foetal livers were removed at 0.9 gestation. We measured foetal liver mitochondrial DNA copy number, activity of superoxide dismutase, cathepsins B and D and selected protein content, total phospholipids and cardiolipin and activity of mitochondrial respiratory chain complexes. RESULTS: A significant decrease in activities of mitochondrial complexes I, II-III and IV, but not aconitase, was observed in MO. In the antioxidant machinery, there was a significant increase in activity of total superoxide dismutase (SOD) and SOD2 in MO. However, no differences were found regarding autophagy-related protein content (p62, beclin-I, LC3-I, LC3-II and Lamp2A) and cathepsin B and D activities. A 21.5% decrease in total mitochondrial phospholipid was observed in MO. CONCLUSIONS: The data indicate that MO impairs foetal hepatic mitochondrial oxidative capacity and affects total mitochondrial phospholipid content. In addition, MO affects the regulation of foetal liver redox pathways, indicating metabolic adaptations to the higher foetal lipid environment. Consequences of in utero programming of foetal hepatic metabolism may persist and compromise mitochondrial bioenergetics in later life, and increase susceptibility to metabolic diseases.

P-cadherin induces anoikis-resistance of matrix-detached breast cancer cells by promoting pentose phosphate pathway and decreasing oxidative stress.

Successful metastatic spreading relies on cancer cells with stem-like properties, glycolytic metabolism and increased antioxidant protection, allowing them to escape anoikis and to survive in circulation. The expression of P-cadherin, a poor prognostic factor in breast cancer, is associated with hypoxic, glycolytic and acidosis biomarkers. In agreement, P-cadherin-enriched breast cancer cell populations presents a glycolytic and an acid-resistance phenotype. Our aim was to evaluate whether P-cadherin expression controls the glycolytic and oxidative phosphorylation fluxes of matrix-detached breast cancer cells, acting as an antioxidant and enhancing their survival in anchorage-independent conditions. By using matrix-detached breast cancer cells, we concluded that P-cadherin increases glucose-6-phosphate dehydrogenase expression, up-regulating the carbon flux through the pentose phosphate pathway, while inhibiting pyruvate oxidation to acetyl-coA via pyruvate dehydrogenase kinase-4 (PDK-4) activation. Accordingly, P-cadherin expression conferred increased sensitivity to dichloroacetate (DCA), a PDK inhibitor. P-cadherin expression also regulates oxidative stress in matrix-detached breast cancer cells, through the control of antioxidant systems, such as catalase and superoxide dismutases (SOD)1 and 2, providing these cells with an increased resistance to doxorubicin-induced anoikis. Importantly, this association was validated in primary invasive breast carcinomas, where an enrichment of SOD2 was found in P-cadherin-overexpressing breast carcinomas. In conclusion, we propose that P-cadherin up-regulates carbon flux through the pentose phosphate pathway and decreases oxidative stress in matrix-detached breast cancer cells. These metabolic remodeling and antioxidant roles of P-cadherin can promote the survival of breast cancer cells in circulation and in metastatic sites, being a possible player in breast cancer therapeutic resistance to pro-oxidant-based interventions.

Estrogens decrease osteoclast number by attenuating mitochondria oxidative phosphorylation and ATP production in early osteoclast precursors.

Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through direct actions on cells of the myeloid lineage. Here, we investigated the molecular mechanism of this effect. We report that 17ß-estradiol (E2) decreased osteoclast number by promoting the apoptosis of early osteoclast progenitors, but not mature osteoclasts. This effect was abrogated in cells lacking Bak/Bax-two pro-apoptotic members of the Bcl-2 family of proteins required for mitochondrial apoptotic death. FasL has been previously implicated in the pro-apoptotic actions of E2. However, we show herein that FasL-deficient mice lose bone mass following ovariectomy indistinguishably from FasL-intact controls, indicating that FasL is not a major contributor to the anti-osteoclastogenic actions of estrogens. Instead, using microarray analysis we have elucidated that ERα-mediated estrogen signaling in osteoclast progenitors decreases "oxidative phosphorylation" and the expression of mitochondria complex I genes. Additionally, E2 decreased the activity of complex I and oxygen consumption rate. Similar to E2, the complex I inhibitor Rotenone decreased osteoclastogenesis by promoting osteoclast progenitor apoptosis via Bak/Bax. These findings demonstrate that estrogens decrease osteoclast number by attenuating respiration, and thereby, promoting mitochondrial apoptotic death of early osteoclast progenitors.