The role of mitochondria linked fatty-acid uptake-driven adipogenesis in Graves’ Orbitopathy

Context Depot-specific expansion of orbital-adipose-tissue (OAT) in Graves’ Orbitopathy (GO, an autoimmune condition producing proptosis, visual impairment and reduced quality of life) is associated with fatty-acid (FA) uptake-driven adipogenesis in preadipocytes/fibroblasts (PFs). Objective A role for mitochondria in OAT-adipogenesis in GO. Design, Setting, Participants Confluent PFs from healthy OAT (OAT-H), OAT from GO (OAT-GO) and white-adipose-tissue in culture-medium compared with culture-medium containing a mixed hormonal-cocktail as adipogenic-medium (ADM); or culture-medium containing FA-supplementation, oleate:palmitate:linoleate (45:30:25%) with/without different concentration of mitochondrial bio-substrate ADP/GDP, AICAR (adenosine-analog) or inhibitor oligomycin-A for 17 days. Main outcome measures Oil-Red-O staining and foci-count of differentiated adipocytes for in-vitro adipogenesis; flow-cytometry, relative-QPCR, MTS-assay/10 6 cells, total cellular-ATP detection kit and Seahorse-XFe96-Analyzer for mitochondria and OXPHOS/Glycolysis-ATP production analysis. Results During early adipogenesis before adipocyte formation (day-0,4&7), we observed OAT-specific cellular ATP-production via mitochondrial-OXPHOS in PFs from both OAT-H/OAT-GO, and substantially disrupted OXPHOS-ATP/Glycolysis-ATP production in PFs from OAT-GO, e.g. 40% reduction in OXPHOS-ATP and trend-increased Glycolysis-ATP production on day-4&7 compared with day-0, which contrasted with the stable levels in OAT-H.FA-supplementation in culture-medium triggered adipogenesis in PFs from both OAT-H/OAT-GO, which was substantially enhanced by 1mM GDP reaching 7-18% of ADM-adipogenesis. The FA-uptake-driven adipogenesis was diminished by oligomycin-A but unaffected by treatment with ADP or AICAR. Furthermore, we observed significant positive correlation between FA-uptake-driven adipogenesis by GDP and the ratios of OXPHOS-ATP/Glycolysis-ATP through adipogenesis of PFs from OAT-GO. Conclusions Our study confirmed that FA-uptake can drive OAT-adipogenesis and revealed a fundamental role for mitochondria-OXPHOS in GO development, which provides potential for therapeutic interventions.

Targeting the ATP Synthase in Staphylococcus aureus Small Colony Variants, Streptococcus pyogenes and Pathogenic Fungi

The ATP synthase has been validated as a druggable target with the approval of the ATP synthase inhibitor, bedaquiline, for treatment of drug-resistant Mycobacterium tuberculosis, a bacterial species in which the ATP synthase is essential for viability. Gene inactivation studies have also shown that the ATP synthase is essential among Streptococci, and some studies even suggest that inhibition of the ATP synthase is a strategy for the elimination of Staphylococcus aureus small colony variants with deficiencies in the electron transport chain, as well as pathogenic fungi, such as Candida albicans. Here we investigated five structurally diverse ATP synthase inhibitors, namely N,N′-dicyclohexylcarbodiimide (DCCD), oligomycin A, tomatidine, resveratrol and piceatannol, for their growth inhibitory activity against the bacterial strains Streptococcus pyogenes, S. aureus and two isogenic small colony variants, as well as the pathogenic fungal species, C. albicans and Aspergillus niger. DCCD showed broad-spectrum inhibitory activity against all the strains (minimum inhibitory concentration (MIC) 2–16 µg/mL), except for S. aureus, where the ATP synthase is dispensable for growth. Contrarily, oligomycin A selectively inhibited the fungal strains (MIC 1–8 µg/mL), while tomatidine showed very potent, but selective, activity against small colony variants of S. aureus with compromised electron transport chain activity (MIC 0.0625 µg/mL). Small colony variants of S. aureus were also more sensitive to resveratrol and piceatannol than the wild-type strain, and piceatannol inhibited S. pyogenes at 16–32 µg/mL. We previously showed that transposon inactivation of the ATP synthase sensitizes S. aureus towards polymyxin B and colistin, and here we demonstrate that treatment with structurally diverse ATP synthase inhibitors sensitized S. aureus towards polymyxin B. Collectively, our data show that ATP synthase inhibitors can have selective inhibitory activity against pathogenic microorganisms in which the ATP synthase is essential. The data also show that the inhibition of the ATP synthase in Streptococcus pyogenes may be a new strategy for development of a narrow-spectrum antibiotic class. In other major bacterial pathogens, such as S. aureus and potentially Escherichia coli, where the ATP synthase is dispensable, the ATP synthase inhibitors may be applied in combination with antimicrobial peptides to provide new therapeutic options.

Red LED Light Acts on the Mitochondrial Electron Chain of Donkey Sperm and Its Effects Depend on the Time of Exposure to Light

This work aimed to investigate how stimulation of donkey sperm with red LED light affects mitochondrial function. For this purpose, freshly diluted donkey semen was stimulated with red light for 1, 5, and 10 min, in the presence or absence of oligomycin A (Omy A), a specific inhibitor of mitochondrial ATP synthase, or FCCP, a specific disruptor of mitochondrial electron chain. The results obtained in the present study indicated that the effects of red LED light on fresh donkey sperm function are related to changes in mitochondria function. In effect, irradiation of donkey sperm resulted in an increase in mitochondrial membrane potential (MMP), the activity of cytochrome C oxidase and the rate of oxygen consumption. In addition, in the absence of oligomycin A and FCCP, light-stimulation augmented the average path velocity (VAP) and modified the structure of motile sperm subpopulations, increasing the fastest and most linear subpopulation. In contrast, the presence of either Omy A or FCCP abolished the aforementioned effects. Interestingly, our results also showed that the effects of red light depend on the exposure time applied, as indicated by the observed differences between irradiation protocols. In conclusion, our results suggest that exposing fresh donkey sperm to red light modulates the function of their mitochondria through affecting the activity of the electron chain. However, the extent of this effect depends on the irradiation pattern and does not exclude the existence of other mechanisms, such as those related to thermotaxis.

Red LED Light Acts on the Mitochondrial Electron Chain of Mammalian Sperm via Light-Time Exposure-Dependent Mechanisms

This work analyzes the effects of red LED light on mammalian sperm mitochondrial function, using the pig as an animal model. Liquid-stored pig semen was stimulated with red-light for 1, 5 and 10 min in the presence or absence of oligomycin A, a specific inhibitor of mitochondrial ATP synthase, or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a specific disruptor of mitochondrial electron chain. Whereas exposure for 1 and 5 min significantly (p < 0.05) decreased total motility and intracellular ATP levels, irradiation for 10 min induced the opposite effect. Oligomycin A abolished the light-effects on intracellular ATP levels, O2 consumption and mitochondrial membrane potential, whereas compared to non-irradiated samples, FCCP significantly (p < 0.05) increased O2 consumption when sperm were irradiated for 1 min. Both oligomycin A and FCCP significantly (p < 0.05) decreased total motility. Red-light increased cytochrome c oxidase activity with a maximal effect after 5 min of irradiation, which was abolished by both oligomycin A and FCCP. In conclusion, red-light modulates sperm mitochondrial function via electron chain activity in an exposition, time-dependent manner.

Homo-Tris-Nitrones Derived from α-Phenyl-N-tert-butylnitrone: Synthesis, Neuroprotection and Antioxidant Properties

Herein we report the synthesis, antioxidant and neuroprotective power of homo-tris-nitrones (HTN) 1-3, designed on the hypothesis that the incorporation of a third nitrone motif into our previously identified homo-bis-nitrone 6 (HBN6) would result in an improved and stronger neuroprotection. The neuroprotection of HTNs 1-3, measured against oligomycin A/rotenone, showed that HTN2 was the best neuroprotective agent at a lower dose (EC50 = 51.63 ± 4.32 μM), being similar in EC50 and maximal activity to α-phenyl-N-tert-butylnitrone (PBN) and less potent than any of HBNs 4-6. The results of neuroprotection in an in vitro oxygen glucose deprivation model showed that HTN2 was the most powerful (EC50 = 87.57 ± 3.87 μM), at lower dose, but 50-fold higher than its analogous HBN5, and ≈1.7-fold less potent than PBN. HTN3 had a very good antinecrotic (IC50 = 3.47 ± 0.57 μM), antiapoptotic, and antioxidant (EC50 = 6.77 ± 1.35 μM) profile, very similar to that of its analogous HBN6. In spite of these results, and still being attractive neuroprotective agents, HTNs 2 and 3 do not have better neuroprotective properties than HBN6, but clearly exceed that of PBN.