Increase In Mitochondrial Activity Research Articles

P-024 Revitalizing male fertility: Crocin’s dual role in antioxidant mastery and mitochondrial activation to enhance sperm motility

Abstract Study question The study aims to investigate the potential effect of crocin, a major constituent of Crocus sativus (saffron), on improving sperm motility in patients with asthenozoospermia. Summary answer In vitro crocin treatment enhances sperm motility, reduces ROS, improves mitochondrial membrane potential, and boosts mitochondrial activity, suggesting the potential role of enhancing sperm motility. What is known already Male infertility can result from various factors, with excessive production of reactive oxygen species (ROS) adversely affecting sperm motility. Crocin, known for its antioxidant properties in scavenging ROS, has led to increasing interest in natural compounds to enhance sperm quality. Study design, size, duration The current study involved the evaluation of 95 patients at the Service of Reproductive Biology at the Maternity and Neonatology Center of Monastir. Sperm samples were collected and incubated for 3 hours at 37 °C with increasing concentrations of Crocin (0, 0.2, 0.5, 1, 1.5 mM). Participants/materials, setting, methods This study included asthenozoospermic patients, categorized into two groups: G1 with progressive motility (PM) between 20% and 30%, and G2 with PM between 10% and 15%. Sperm samples underwent treatment with Crocin. Evaluation encompassed sperm motility, considering the distance covered and velocity via video-microscopy. Additionally, intracellular ROS levels were assessed using DCFH-DA fluorochrome, mitochondrial membrane potential was measured with rh123, and mitochondrial activity was determined through succinate dehydrogenase activity via the MTT assay. Main results and the role of chance The results demonstrate that sperm motility is significantly enhanced after in vitro treatment with 1mM Crocin for 3 hours, with 1.9-fold (p < 0.001) and 1.7-fold (p < 0.001) increases in sperm velocity and distance traveled, respectively. Additionally, Crocin reduces intracellular ROS levels, exhibiting a reduction of 1.5-fold (p < 0.001) in G1 and 2.4-fold (p < 0.001) in G2. Fluorescence microscopy of spermatozoa from a patient with severe asthenozoospermia showed intense ROS-induced fluorescence, which was attenuated after incubation with 1 mM Crocin for 3 hours. Furthermore, Crocin demonstrates a noteworthy improvement in mitochondrial membrane potential, with 1.7-fold (p < 0.001) for G1 and 1.9-fold (p < 0.001) for G2, suggesting its capability to prevent the decline of mitochondrial potential. Microscopic fluorescence revealed a marked increase in fluorescence, particularly in the midpiece after Crocin treatment, confirming the protective effect of Crocin on mitochondrial membrane potential. Moreover, there is a substantial increase in mitochondrial activity with Crocin, particularly in G1, showing a 2.14-fold (p < 0.001) increase compared to the G2 group’s 1.85-fold (p < 0.001) increase. These findings collectively highlight the multifaceted benefits of Crocin, encompassing enhanced sperm motility, reduced ROS levels, preserved mitochondrial membrane potential, and heightened mitochondrial activity, underscoring its potential as a therapeutic agent for male infertility, particularly in cases of asthenozoospermia. Limitations, reasons for caution While promising, this study’s limitations include the in vitro treatment, and not fully replicating complex in vivo conditions. Focused on asthenozoospermia, generalizing findings to other male infertility forms necessitates further investigation. Validation of Crocin’s therapeutic potential in male infertility requires additional clinical trials. Wider implications of the findings Our findings suggest that in vitro enhancement of sperm quality with crocin may serve as a promising solution to improve assisted reproductive techniques, mitigating exogenous oxidative stress during laboratory procedures. This insight holds potential for advancing reproductive interventions and warrants further exploration in clinical settings. Trial registration number not applicable

Aberrant expression of thyroidal hormone receptor α exasperating mitochondrial dysfunction induced sarcopenia in aged mice.

Disrupted mitochondrial dynamics and mitophagy contribute to functional deterioration of skeletal muscle (SM) during aging, but the regulatory mechanisms are poorly understood. Our previous study demonstrated that the expression of thyroid hormone receptor α (TRα) decreased significantly in aged mice, suggesting that the alteration of thyroidal elements, especially the decreased TRα, might attenuate local THs action thus to cause the degeneration of SM with aging, while the underlying mechanism remains to be further explored. In this study, decreased expression of myogenic regulators Myf5, MyoD1, mitophagy markers Pink1, LC3II/I, p62, as well as mitochondrial dynamic factors Mfn1 and Opa1, accompanied by increased reactive oxygen species (ROS), showed concomitant changes with reduced TRα expression in aged mice. Further TRα loss- and gain-of-function studies in C2C12 revealed that silencing of TRα not only down-regulated the expression of above-mentioned myogenic regulators, mitophagy markers and mitochondrial dynamic factors, but also led to a significant decrease in mitochondrial activity and maximum respiratory capacity, as well as more mitochondrial ROS and damaged mitochondria. Notedly, overexpression of TRα could up-regulate the expression of those myogenic regulators, mitophagy markers and mitochondrial dynamic factors, meanwhile also led to an increase in mitochondrial activity and number. These results confirmed that TRα could concertedly regulate mitochondrial dynamics, autophagy, and activity, and myogenic regulators rhythmically altered with TRα expression. Summarily, these results suggested that the decline of TRα might cause the degeneration of SM with aging by regulating mitochondrial dynamics, mitophagy and myogenesis.

BI-3231, an enzymatic inhibitor of HSD17B13, reduces lipotoxic effects induced by palmitic acid in murine and human hepatocytes.

17-β-hydroxysteroid dehydrogenase 13 (HSD17B13), a lipid droplet-associated enzyme, is primarily expressed in the liver and plays an important role in lipid metabolism. Targeted inhibition of enzymatic function is a potential therapeutic strategy for treating steatotic liver disease (SLD). The present study is aimed at investigating the effects of the first selective HSD17B13 inhibitor, BI-3231, in a model of hepatocellular lipotoxicity using human cell lines and primary mouse hepatocytes in vitro. Lipotoxicity was induced with palmitic acid in HepG2 cells and freshly isolated mouse hepatocytes and the cells were coincubated with BI-3231 to assess the protective effects. Under lipotoxic stress, triglyceride (TG) accumulation was significantly decreased in the BI-3231-treated cells compared with that of the control untreated human and mouse hepatocytes. In addition, treatment with BI-3231 led to considerable improvement in hepatocyte proliferation, cell differentiation, and lipid homeostasis. Mechanistically, BI-3231 increased the mitochondrial respiratory function without affecting β-oxidation. BI-3231 inhibited the lipotoxic effects of palmitic acid in hepatocytes, highlighting the potential of targeting HSD17B13 as a specific therapeutic approach in steatotic liver disease.NEW & NOTEWORTHY 17-β-Hydroxysteroid dehydrogenase 13 (HSD17B13) is a lipid droplet protein primarily expressed in the liver hepatocytes. HSD17B13 is associated with the clinical outcome of chronic liver diseases and is therefore a target for the development of drugs. Here, we demonstrate the promising therapeutic effect of BI-3231 as a potent inhibitor of HSD17B13 based on its ability to inhibit triglyceride accumulation in lipid droplets (LDs), restore lipid metabolism and homeostasis, and increase mitochondrial activity in vitro.

The Relationship between Clock Genes, Sirtuin 1, and Mitochondrial Activity in Head and Neck Squamous Cell Cancer: Effects of Melatonin Treatment.

The circadian clock is a regulatory system, with a periodicity of approximately 24 h, which generates rhythmic changes in many physiological processes, including mitochondrial activity. Increasing evidence links chronodisruption with aberrant functionality in clock gene expression, resulting in multiple diseases such as cancer. Melatonin, whose production and secretion oscillates according to the light-dark cycle, is the principal regulator of clock gene expression. In addition, the oncostatic effects of melatonin correlate with an increase in mitochondrial activity. However, the direct links between circadian clock gene expression, mitochondrial activity, and the antiproliferative effects of melatonin in cancers, including head and neck squamous cell carcinoma (HNSCC), remain largely unknown. In this study, we analyzed the effects of melatonin on HNSCC cell lines (Cal-27 and SCC9), which were treated with 500 and 1000 µM melatonin. We found that the antiproliferative effect of melatonin is not mediated by the Bmal1 clock gene. Additionally, high doses of melatonin were observed to result in resynchronization of oscillatory circadian rhythm genes (Per2 and Sirt1). Surprisingly, the resynchronizing effect of melatonin on Per2 and Sirt1 did not produce alterations in the oscillation of mitochondrial respiratory activity. These results increase our understanding of the possible antiproliferative mechanisms in melatonin in the treatment of head and neck squamous cell carcinoma and suggest that its antiproliferative effects are independent of clock genes but are directly related to mitochondrial activity.

1,8-Cineole alleviates OGD/R-induced oxidative damage and restores mitochondrial function by promoting the Nrf2 pathway.

This study examined the effects of 1,8-cineole on reducing oxidative stress injury and restoring mitochondrial function in oxygen-glucose deprivation and reoxygenation (OGD/R) HT22 cells via the nuclear factor erythrocyte 2 related factor 2 (Nrf2) pathway. The optimal concentration of 1,8-cineole to reduce OGD/R injury was screened via cell morphology, cell survival rate, and lactate dehydrogenase (LDH) leakage rate. Oxidative damage was observed by measuring superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activities, and reactive oxygen species (ROS), glutathione (GSH), protein carbonyl, malondialdehyde (MDA), lipid peroxidation (LPO) content, and 8-hydroxy-2 deoxyguanosine (8-OHDG) expression. Mitochondrial function was observed by mitochondrial membrane potential (MMP) and ATPase activity. Nrf2 pathways were observed by the expression levels of total Nrf2, nucleus Nrf2, NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), the mRNA levels of HO-1 and NQO1. Among different concentrations of 1,8-cineole for promoting HT22 cell proliferation and attenuated OGD/R injury, 10 μmol/L 1,8-cineole was the best. After 1,8-cineole treatment, SOD, GSH-PX, and CAT activities and GSH content increased, while ROS, MDA, LPO, protein carbonyl, and 8-OHDG levels decreased. 1,8-Cineole could restore MMP and increase mitochondrial enzyme activity. It could also increase the total Nrf2, nucleus Nrf2, NQO1, and HO-1, and Nrf2 inhibitor brusatol reduced the effect of 1,8-cineole. Immunofluorescence assay showed that 1,8-cineole could facilitate the transfer of Nrf2 into the nucleus. 1,8-cineole increased the mRNA levels of NQO1 and HO-1. The above results showed that 1,8-cineole could alleviate OGD/R-induced oxidative damage and restores mitochondrial function by activating the Nrf2 signal pathway.

Nicotinamide Riboside Improves Stemness of Human Adipose-Derived Stem Cells and Inhibits Terminal Adipocyte Differentiation.

Adipose tissue plays a crucial role in maintaining metabolic homeostasis by serving as a storage site for excess fat and protecting other organs from the detrimental effects of lipotoxicity. However, the aging process is accompanied by a redistribution of fat, characterized by a decrease in insulin-sensitive subcutaneous adipose depot and an increase in insulin-resistant visceral adipose depot. This age-related alteration in adipose tissue distribution has implications for metabolic health. Adipose-derived stem cells (ASCs) play a vital role in the regeneration of adipose tissue. However, aging negatively impacts the stemness and regenerative potential of ASCs. The accumulation of oxidative stress and mitochondrial dysfunction-associated cellular damage contributes to the decline in stemness observed in aged ASCs. Nicotinamide adenine dinucleotide (NAD+) is a crucial metabolite that is involved in maintaining cellular homeostasis and stemness. The dysregulation of NAD+ levels with age has been associated with metabolic disorders and the loss of stemness. In this study, we aimed to investigate the effects of nicotinamide riboside (NR), a precursor of NAD+, on the stemness of human ASCs in cell culture. Our findings reveal that adipogenesis is accompanied by an increase in mitochondrial activity and the production of reactive oxygen species (ROS). However, treatment with NR leads to a reduction in mitochondrial activity and ROS production in ASCs. Furthermore, NR administration improves the stemness-related genes expression in ASCs and mitigates their propensity for adipocyte differentiation. These results suggest that NR treatment holds promise as a potential strategy to rejuvenate the stemness of aged ASCs. Further investigations, including in vivo evaluations using animal models and human studies, will be necessary to validate these findings and establish the clinical potential of this well-established drug for enhancing the stemness of aged stem cells.

Analysis of Electric Field Stimulation in Blue Light Stressed 661W Cells

Though electrical stimulation is used as a therapeutic approach to treat retinal and spinal injuries, many protective mechanisms at cellular level have not been elucidated. We performed a detailed analysis of cellular events in blue light (Li) stressed 661W cells, which were subjected to direct current electric field (EF) stimulation. Our findings revealed that EF stimulation induced protective effects in 661W cells from Li-induced stress by multiple defense mechanisms, such as increase in mitochondrial activity, gain in mitochondrial potential, increase in superoxide levels, and the activation of unfolded protein response (UPR) pathways, all leading to an enhanced cell viability and decreased DNA damage. Here, our genetic screen results revealed the UPR pathway to be a promising target to ameliorate Li-induced stress by EF stimulation. Thus, our study is important for a knowledgeable transfer of EF stimulation into clinical application.

In vivo metabolic imaging identifies lipid vulnerability in a preclinical model of Her2+/Neu breast cancer residual disease and recurrence.

Recurrent cancer cells that evade therapy is a leading cause of death in breast cancer patients. This risk is high for women showing an overexpression of human epidermal growth factor receptor 2 (Her2). Cells that persist can rely on different substrates for energy production relative to their primary tumor counterpart. Here, we characterize metabolic reprogramming related to tumor dormancy and recurrence in a doxycycline-induced Her2+/Neu model of breast cancer with varying times to recurrence using longitudinal fluorescence microscopy. Glucose uptake (2-NBDG) and mitochondrial membrane potential (TMRE) imaging metabolically phenotype mammary tumors as they transition to regression, dormancy, and recurrence. “Fast-recurrence” tumors (time to recurrence ~55 days), transition from glycolysis to mitochondrial metabolism during regression and this persists upon recurrence. “Slow-recurrence” tumors (time to recurrence ~100 days) rely on both glycolysis and mitochondrial metabolism during recurrence. The increase in mitochondrial activity in fast-recurrence tumors is attributed to a switch from glucose to fatty acids as the primary energy source for mitochondrial metabolism. Consequently, when fast-recurrence tumors receive treatment with a fatty acid inhibitor, Etomoxir, tumors report an increase in glucose uptake and lipid synthesis during regression. Treatment with Etomoxir ultimately prolongs survival. We show that metabolic reprogramming reports on tumor recurrence characteristics, particularly at time points that are essential for actionable targets. The temporal characteristics of metabolic reprogramming will be critical in determining the use of an appropriate timing for potential therapies; namely, the notion that metabolic-targeted inhibition during regression reports long-term therapeutic benefit.

The neutral amino acid transporter SLC7A10 in adipose tissue, obesity and insulin resistance.

Obesity, insulin resistance and type 2 diabetes represent major global health challenges, and a better mechanistic understanding of the altered metabolism in these conditions may give improved treatment strategies. SLC7A10, a member of the SLC7 subfamily of solute carriers, also named ASC-1 (alanine, serine, cysteine transporter-1), has recently been implicated as an important modulator of core processes in energy- and lipid metabolism, through its particularly high expression in adipocytes. In human cohorts, adipose SLC7A10 mRNA shows strong inverse correlations with insulin resistance, adipocyte size and components of the metabolic syndrome, strong heritability, and an association with type 2 diabetes risk alleles. SLC7A10 has been proposed as a marker of white as opposed to thermogenic beige and brown adipocytes, supported by increased formation of thermogenic beige adipocytes upon loss of Slc7a10 in mouse white preadipocytes. Overexpression of SLC7A10 in mature white adipocytes was found to lower the generation of reactive oxygen species (ROS) and stimulate mitochondrial respiratory capacity, while SLC7A10 inhibition had the opposite effect, indicating that SLC7A10 supports a beneficial increase in mitochondrial activity in white adipocytes. Consistent with these beneficial effects, inhibition of SLC7A10 was in mouse and human white adipocyte cultures found to increase lipid accumulation, likely explained by lowered serine uptake and glutathione production. Additionally, zebrafish with partial global Slc7a10b loss-of-function were found to have greater diet-induced body weight and larger visceral adipocytes compared to controls. However, challenging that SLC7A10 exerts metabolic benefits only in white adipocytes, suppression of SLC7A10 has been reported to decrease mitochondrial respiration and expression of thermogenic genes also in some beige and brown adipocyte cultures. Taken together, the data point to an important but complex role of SLC7A10 in metabolic regulation across different adipose tissue depots and adipocyte subtypes. Further research into SLC7A10 functions in specific adipocyte subtypes may lead to new precision therapeutics for mitigating the risk of insulin resistance and type 2 diabetes.

Small molecule-mediated rapid maturation of human induced pluripotent stem cell derived cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Gravitation Program “Materials Driven Regeneration” by the Netherlands Organization for Scientific Research (RegmedXB #024.003.013) and the Marie Skłodowska-Curie Actions (Grant agreement RESCUE #801540). The EU-funded project BRAV3 (H2020, ID:874827) Background Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature human primary cardiomyocytes: the ability to use fatty acids as an energy source, high mitochondrial mass, increased nuclei polyploidism, synchronized electrical conduction, and forceful contractions. Instead, their phenotype is similar to immature cardiomyocytes in the late fetal stage. This immaturity represents a bottleneck to their application in 1) disease modeling – as most cardiac (genetic) diseases have a middle-age onset – and 2) clinical use, where integration and functional coupling are key. So far, the mainly used methods to enhance iPSC-CM maturation include prolonged time-in-culture, 3D culture, cyclic mechanical stretch, and electrical stimulation with specialized media. However, these protocols are laborious, costly, and not easily scalable. Methods In this study, we developed a simple, low cost, and rapid protocol using two peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A/PGC-1α) activating small molecules: Asiatic Acid (AA) and GW501516 (GW) to promote cardiomyocyte maturity by inducing a metabolic switch to fatty acid utilization and increased mitochondrial biogenesis. Results Monolayers of iPSC-CMs were incubated with AA and GW every other day for 10 days resulting in increased expression of fatty acid-metabolism-related genes (5 and 10-fold increase in CPT1B gene expression, respectively), mitochondria biogenesis (protein expression of ATP5A) and fusion (50 and 100-fold increase in OPA1 gene expression, respectively). In addition, AA treated iPSC-CMs responded in the seahorse mitochondria stress test more rapidly to an artificial increase in mitochondrial activity and showed a higher flexibility in substrate utilization in the seahorse stress test. A more mature electrophysiological functionality was shown by increased ion channel gene expression (KCNA4, SCN5A, GJA1, CACNA1C, and SCN1B) and enhanced synchronous contraction in treated samples. Moreover, maturation was further shown by increased sarcomeric gene expression (5 and 7-fold increase in TNNI3 in AA and GW respectively) and nuclear polyploidism (>4N fold 2.16 and 1.48-fold increase in AA and GW respectively). Conclusions Collectively, these findings show that AA and GW trigger a metabolic switch and induce extensive maturation of iPSC-CMs, providing a rapid and cost-effective method to obtain iPSC-CMs that more closely resemble their adult counterparts.

Nutraceutical Apigenin regulates DC function in a RelB-dependent manner during neuroinflammation.

Abstract Apigenin, a natural flavonoid, found in several plants is known to have anti-oxidant and anti-inflammatory properties indicated by its use for centuries as a medicinal approach to treat inflammatory disorders. However, there are significant gaps in knowledge regarding its effect on dendritic cell (DC) function in maintaining an immune balance in immunospecialized locations like the central nervous system (CNS). In order to establish the potential utility of Apigenin as a therapeutic agent against neuroinflammatory diseases, we tested and found that Apigenin treatment ameliorated severity of disease progression and relapse after onset of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 and SJL mouse models of multiple sclerosis. An increased retention of DCs and other myeloid cells in the periphery correlated with decreased immune cell infiltration and reduced demyelination in the CNS in treated mice. Mechanistically, Apigenin treatment reduced RelB expression in presence of LPS in human peripheral blood DCs, which is central to DC maturation, its antigen presentation capabilities and DC-mediated T cell activation. IL-12A and IL-23, downstream pro-inflammatory targets of RelB were reduced upon Apigenin treatment in these cells. Further, RelB causes a metabolic switch in immune cells upon inflammation, which was seen as a decrease in glucose uptake and lactate production (glycolysis), and an increase in mitochondrial activity when LPS-induced DCs were treated with Apigenin. These results indicate a protective role of Apigenin against DC-regulated neurodegenerative effects through a probable RelB mediated pathway thus implicating a potential therapy for neuroinflammatory disease. Supported by grants from NIH: 1R01NS0971-47

Preventing White Adipocyte Browning during Differentiation In Vitro: The Effect of Differentiation Protocols on Metabolic and Mitochondrial Phenotypes.

Mitochondrial dysfunction in white adipose tissue is strongly associated with obesity and its metabolic complications, which are important health challenges worldwide. Human adipose-derived stromal/stem cells (hASCs) are a promising tool to investigate the underlying mechanisms of such mitochondrial dysfunction and to subsequently provide knowledge for the development of treatments for obesity-related pathologies. A substantial obstacle in using hASCs is that the key compounds for adipogenic differentiation in vitro increase mitochondrial uncoupling, biogenesis, and activity, which are the signature features of brown adipocytes, thus altering the white adipocyte phenotype towards brown-like cells. Additionally, commonly used protocols for hASC adipogenic differentiation exhibit high variation in their composition of media, and a systematic comparison of their effect on mitochondria is missing. Here, we compared the five widely used adipogenic differentiation protocols for their effect on metabolic and mitochondrial phenotypes to identify a protocol that enables in vitro differentiation of white adipocytes and can more faithfully recapitulate the white adipocyte phenotype observed in human adipose tissue. We developed a workflow that included functional assays and morphological analysis of mitochondria and lipid droplets. We observed that triiodothyronine- or indomethacin-containing media and commercially available adipogenic media induced browning during in vitro differentiation of white adipocytes. However, the differentiation protocol containing 1 μM of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone prevented the browning effect and would be proposed for adipogenic differentiation protocol for hASCs to induce a white adipocyte phenotype. Preserving the white adipocyte phenotype in vitro is a crucial step for the study of obesity and associated metabolic diseases, adipose tissue pathologies, such as lipodystrophies, possible therapeutic compounds, and basic adipose tissue physiology.

Hyperosmolality in CHO cell culture: effects on the proteome

Chinese hamster ovary (CHO) cells are the most commonly used host cell lines for therapeutic protein production. Exposure of these cells to highly concentrated feed solution during fed-batch cultivation can lead to a non-physiological increase in osmolality (> 300 mOsm/kg) that affects cell physiology, morphology, and proteome. As addressed in previous studies (and indeed, as recently addressed in our research), hyperosmolalities of up to 545 mOsm/kg force cells to abort proliferation and gradually increase their volume—almost tripling it. At the same time, CHO cells also show a significant hyperosmolality-dependent increase in mitochondrial activity. To gain deeper insight into the molecular mechanisms that are involved in these processes, as detailed in this paper, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed CHO cells compared with control cells. Our analysis revealed differentially expressed key proteins that mediate mitochondrial activation, oxidative stress amelioration, and cell cycle progression. Our studies also demonstrate a previously unknown effect: the strong regulation of proteins can alter both cell membrane stiffness and permeability. For example, we observed that three types of septins (filamentous proteins that form diffusion barriers in the cell) became strongly up-regulated in response to hyperosmolality in the experimental setup. Overall, these new observations correlate well with recent CHO-based fluxome and transcriptome studies, and reveal additional unknown proteins involved in the response to hyperosmotic pressure by over-concentrated feed in mammalian cells.Key points• First-time comparative proteome analysis of CHO cells exposed to over-concentrated feed.• Discovery of membrane barrier-forming proteins up-regulation under hyperosmolality.• Description of mitochondrial and protein chaperones activation in treated cells.

Adiponectin receptor agonist AdipoRon improves skeletal muscle function in aged mice.

The loss of skeletal muscle function with age, known as sarcopenia, significantly reduces independence and quality of life and can have significant metabolic consequences. Although exercise is effective in treating sarcopenia it is not always a viable option clinically, and currently, there are no pharmacological therapeutic interventions for sarcopenia. Here, we show that chronic treatment with pan-adiponectin receptor agonist AdipoRon improved muscle function in male mice by a mechanism linked to skeletal muscle metabolism and tissue remodeling. In aged mice, 6 weeks of AdipoRon treatment improved skeletal muscle functional measures in vivo and ex vivo. Improvements were linked to changes in fiber type, including an enrichment of oxidative fibers, and an increase in mitochondrial activity. In young mice, 6 weeks of AdipoRon treatment improved contractile force and activated the energy-sensing kinase AMPK and the mitochondrial regulator PGC-1a (peroxisome proliferator-activated receptor gamma coactivator one alpha). In cultured cells, the AdipoRon induced stimulation of AMPK and PGC-1a was associated with increased mitochondrial membrane potential, reorganization of mitochondrial architecture, increased respiration, and increased ATP production. Furthermore, the ability of AdipoRon to stimulate AMPK and PGC1a was conserved in nonhuman primate cultured cells. These data show that AdipoRon is an effective agent for the prevention of sarcopenia in mice and indicate that its effects translate to primates, suggesting it may also be a suitable therapeutic for sarcopenia in clinical application.

Evaluation of the effect of hydrocortisone in 2D and 3D HEp-2 cell culture

Introduction: Cancer is one of the diseases with the highest incidence globally and that associated with the patient's emotional state, can act positively or negatively in the treatment. Cortisol is a principal primary stress hormone in the human body. The corticoids can increase cell proliferation and reactive oxygen species that contribute to DNA damage. Prolonged exposure to stress can contribute to tissues becoming insensitive to cortisol, the primary human stress hormone. Objective: This study explores cortisol's influence on tumor cell development, particularly in human cells of carcinoma of the human laryngeal (HEp-2). Methodology: HEp-2 cells were exposed to increasing cortisol (hydrocortisone) concentrations for 24 or 48 hours, and cytotoxicity (MTT assay) proliferation assay (crystal violet assay), and immunolabeled 3D culture for fibronectin and FAK were analyzed. Results: The group treated with hydrocortisone showed a significant increase in mitochondrial activity, as for the evaluation by the violet crystal, the treated group showed similar behavior to the control. The 3D culture showed dispersed cells within 24 hours with reduced FAK labeling; however, no changes were observed within 48 hours. Conclusion: Although some cases favored corticosteroid use in cancer patients, a more detailed analysis is necessary before prescribing them.

Increased mitochondrial activity upon CatSper channel activation is required for mouse sperm capacitation

To fertilize an oocyte, sperm must undergo several biochemical and functional changes known as capacitation. A key event in capacitation is calcium influx through the cation channel of sperm (CatSper). However, the molecular mechanisms of capacitation downstream of this calcium influx are not completely understood. Capacitation is also associated with an increase in mitochondrial oxygen consumption, and several lines of evidence indicate that regulated calcium entry into mitochondria increases the efficiency of oxidative respiration. Thus, we hypothesized that calcium influx through CatSper during capacitation increases mitochondrial calcium concentration and mitochondrial efficiency and thereby contributes to sperm hyperactivation and fertilization capacity. To test this hypothesis, we used high-resolution respirometry to measure mouse sperm mitochondrial activity. We also measured mitochondrial membrane potential, ATP/ADP exchange during capacitation, and mitochondrial calcium concentration in sperm from wild-type and CatSper knockout mice. We show that the increase in mitochondrial activity in capacitated wild-type sperm parallels the increase in mitochondrial calcium concentration. This effect is blunted in sperm from CatSper knockout mice. Importantly, these mechanisms are needed for optimal hyperactivation and fertilization in wild-type mice, as confirmed by using mitochondrial inhibitors. Thus, we describe a novel mechanism of sperm capacitation. This work contributes to our understanding of the role of mitochondria in sperm physiology and opens the possibility of new molecular targets for fertility treatments and male contraception.

Flavonoids and Omega3 Prevent Muscle and Cardiac Damage in Duchenne Muscular Dystrophy Animal Model.

Nutraceutical products possess various anti-inflammatory, antiarrhythmic, cardiotonic, and antioxidant pharmacological activities that could be useful in preventing oxidative damage, mainly induced by reactive oxygen species. Previously published data showed that a mixture of polyphenols and polyunsaturated fatty acids, mediate an antioxidative response in mdx mice, Duchenne muscular dystrophy animal model. Dystrophic muscles are characterized by low regenerative capacity, fibrosis, fiber necrosis, inflammatory process, altered autophagic flux and inadequate anti-oxidant response. FLAVOmega β is a mixture of flavonoids and docosahexaenoic acid. In this study, we evaluated the role of these supplements in the amelioration of the pathological phenotype in dystrophic mice through in vitro and in vivo assays. FLAVOmega β reduced inflammation and fibrosis, dampened reactive oxygen species production, and induced an oxidative metabolic switch of myofibers, with consequent increase of mitochondrial activity, vascularization, and fatigue resistance. Therefore, we propose FLAVOmega β as food supplement suitable for preventing muscle weakness, delaying inflammatory milieu, and sustaining physical health in patients affected from DMD.

EVALUATION OF DOSE-DEPENDENT ACTIVITY OF BISPHENOL F ON VIABILITY PARAMETERS AND STEROIDOGENESIS IN H295R CELLS

Increasing concern over bisphenol A (BPA) as an endocrine-disrupting chemical and recent imposition of restriction on the use of BPA paved the way for entry of its analogues in the market. Bisphenol F (BPF) is one of the major analogues of commercial value. Thus, its increasing production and application make it vulnerable to human exposure. The aim of our in vitro study was to assess the potential effect of BPF on H295R cells mitochondrial activity, metabolic activity, membrane integrity, lysosomal function, and testosterone synthesis. Adrenocortical carcinoma cells were cultivated during 24 h in the presence of BPF (0.1, 0.5, 1, 10, 25, 50, 75, 100, 300, 500 μM). Exposure doses of BPF caused a significant decrease of mitochondrial activity starting from 1 μM, we observed a slight increase in mitochondrial activity at the lowest concentration (0.1 μM). Metabolic activity decreased with increasing dose of BPF - from 10 to 500 μM. A significant increase in metabolic activity was observed after cultivation with 0.1 μM BPF and a slight increase was observed after cultivation with 0.5 μM BPF. We observed a slight increase in lysosomal function and membrane integrity after cultivation with 0.1 and 1 μM, although higher exposure doses (25 - 500 μM) caused significant decrease in membrane integrity and lysosomal function. Lowest exposure dose of BPF (0.1 μM) caused a significant increase in testosterone synthesis, on the other hand, higher exposure doses (50 - 500 μM) caused significant decrease of testosterone production. The obtained results confirmed that BPF at higher concentrations caused cytotoxicity and possibly have endocrine-disrupting potential.

Increased mitochondrial calcium uptake and concomitant mitochondrial activity by presenilin loss promotes mTORC1 signaling to drive neurodegeneration

Metabolic dysfunction and protein aggregation are common characteristics that occur in age‐related neurodegenerative disease. However, the mechanisms underlying these abnormalities remain poorly understood. We have found that mutations in the gene encoding presenilin in Caenorhabditis elegans, sel‐12, results in elevated mitochondrial activity that drives oxidative stress and neuronal dysfunction. Mutations in the human presenilin genes are the primary cause of familial Alzheimer's disease. Here, we demonstrate that loss of SEL‐12/presenilin results in the hyperactivation of the mTORC1 pathway. This hyperactivation is caused by elevated mitochondrial calcium influx and, likely, the associated increase in mitochondrial activity. Reducing mTORC1 activity improves proteostasis defects and neurodegenerative phenotypes associated with loss of SEL‐12 function. Consistent with high mTORC1 activity, we find that SEL‐12 loss reduces autophagosome formation, and this reduction is prevented by limiting mitochondrial calcium uptake. Moreover, the improvements of proteostasis and neuronal defects in sel‐12 mutants due to mTORC1 inhibition require the induction of autophagy. These results indicate that mTORC1 hyperactivation exacerbates the defects in proteostasis and neuronal function in sel‐12 mutants and demonstrate a critical role of presenilin in promoting neuronal health.

Anti-Aging Activity of Xylocarpus Granatum Phytoextracts and Xyloccensins K Compound

Cellular aging is promoted by the deleterious effect of free radicals. This can be lowered by antioxidant treatments. Xylocarpus granatum and its compound, Xyloccensins K have been reported to have antioxidant activity but there have been no reports of antioxidant and anti-aging activities at the cellular level. Thus, the aim of this study to investigate the antioxidant and anti-aging properties of X. granatum-derived extract and Xyloccensins K at a cellular level in yeast Schizosaccharomyces pombe. Four vegetative and three generative parts of X. granatum organs including root, stem, leaf, twig, seed, flesh of fruit, and peel of fruit were extracted using 70% ethanol by the maceration method. Whereas, Xyloccensins K was obtained from seed of X. granatum. The samples tested, other than peel of fruit, prolonged cell longevity in lower concentration as compared to that without phytoextracts treatment. Also, our data indicate that all samples could promote oxidative stress tolerance phenotype, as yeast was capable of dealing with H2O2-induced oxidative stress treatment at 1, 2, and 3 mM H2O2 with the best phenotypes by the administration of twig extracts. Most of the phytoextracts showed an increase in mitochondrial activity, except that of seed extract. The result showed the administration of Xyloccensins K compound did not increase the expression of transcriptional factors of oxidative stress response gene cluster, sty1 and pap1. We suggest that the Xyloccensins K compound acts as direct Reactive Oxygen Species (ROS) scavenger. Thus further study in elucidating the phenomenon of longevity-induced X. granatum extract is required.