The bisphenol S contamination level observed in human follicular fluid affects the development of porcine oocytes

Bisphenol S (BPS), the main replacement for bisphenol A (BPA), is thought to be toxic, but limited information is available on the effects of Bisphenol S on ovarian follicles. In our study, we demonstrated the presence of Bisphenol S in the follicular fluid of women at a concentration of 22.4 nM. The effect of such concentrations of Bisphenol S on oocyte maturation and subsequent embryo development is still unknown. Therefore, we focused on the effect of Bisphenol S on in vitro oocyte maturation, fertilization, and embryo development. As a model, we used porcine oocytes, which show many physiological similarities to human oocytes. Oocytes were exposed to Bisphenol S concentrations similar to those detected in female patients in the ART clinic. We found a decreased ability of oocytes to successfully complete meiotic maturation. Mature oocytes showed an increased frequency of meiotic spindle abnormalities and chromosome misalignment. Alarming associations of oocyte Bisphenol S exposure with the occurrence of aneuploidy and changes in the distribution of mitochondria and mitochondrial proteins were demonstrated for the first time. However, the number and quality of blastocysts derived from oocytes that successfully completed meiotic maturation under the influence of Bisphenol S was not affected

The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction

Glucose restriction (GR) impairs muscle cell differentiation and evokes myotube atrophy. Resveratrol treatment in skeletal muscle cells improves inflammatory-induced reductions in skeletal muscle cell differentiation. We therefore hypothesised that resveratrol treatment would improve muscle cell differentiation and myotube hypertrophy in differentiating C2C12 myoblasts and mature myotubes during GR. Glucose restriction at 0.6 g/L (3.3 mM) blocked differentiation and myotube hypertrophy versus high-glucose (4.5 g/L or 25 mM) differentiation media (DM) conditions universally used for myoblast culture. Resveratrol (10 μM) treatment increased SIRT1 phosphorylation in DM conditions, yet did not improve differentiation when administered to differentiating myoblasts in GR conditions. Resveratrol did evoke increases in hypertrophy of mature myotubes under DM conditions with corresponding elevated Igf-I and Myhc7 gene expression, coding for the ‘slow’ type I MYHC protein isoform. Inhibition of SIRT1 via EX-527 administration (100 nM) also reduced myotube diameter and area in DM conditions and resulted in lower gene expression of Myhc 1, 2 and 4 coding for ‘intermediate’ and ‘faster’ IIx, IIa and IIb protein isoforms, respectively. Resveratrol treatment did not appear to modulate phosphorylation of energy-sensing protein AMPK or protein translation initiator P70S6K. Importantly, in mature myotubes, resveratrol treatment was able to ameliorate reduced myotube growth in GR conditions over an acute 24-h period, but not over 48–72 h. Overall, resveratrol evoked myotube hypertrophy in DM conditions while favouring ‘slower’ Myhc gene expression and acutely ameliorated impaired myotube growth observed during glucose restriction

Energetický metabolismus kosterního svalu

Skeletal muscle is the largest tissue in the body and plays a marked role in the homeostasis of the body metabolic state. Mitochondria have been proven to contribute to the pathophysiology of various metabolic diseases, either due to defects in their bioenergetic properties or the production of reactive oxygen species. In this work murine myoblasts C2C12 were used as a model of skeletal muscle in vitro, and rat muscle was used to prepare homogenate enriched in the mitochondrial fraction. This work investigates the changes in respiratory parameters in models where mitochondrial oxidative phosphorylation is induced by changing the available consumable substrates in the culture media, such as replacing glucose by galactose, and the effect of treating the cells with high glucose concentration during the process of differentiation on mitochondrial performance. It also investigates the changes in bioenergetic profiles in samples treated with inactive derivatives of the widely used triphenylphosphonium (TPP+) salts to target mitochondria by various probes and antioxidants. The methods used in this study included evaluating mitochondrial parameters in intact and permeabilized cells by real time measurement of the oxygen consumption rate using the extracellular flux analyzer, measuring the enzymatic...Souhrn Kosterní svalovina, jakožto nejobjemnější tkáň v těle, má nezastupitelnou úlohu při udržování metabolické homeostázy. Bylo dokázáno, že mitochondrie přispívají k patofyziologii nejrůznějších metabolických onemocnění, ať již kvůli poškození bioenergetických vlastností nebo produkcí reaktivních forem kyslíku. V této práci byly jako in vitro model kosterního svalu použity myší myoblasty C2C12 a sval krysy, ze kterého byl připraven homogenát obohacený o mitochon- driální frakci. Cílem práce bylo stanovit změny v mitochondriálních respiračních parame- trech daných dostupností využitelných substrátů v kultivačním mediu, např. nahrazením glukózy galaktózou, a také zjistit vliv vysokých koncentrací glukózy na mitochondriální aktivitu během diferenciace. Dalším cílem bylo objasnit efekt inaktivních derivátů trifenyl fosfoniových (TPP+) solí, hojně využívaných pro doručení různých prób a antioxidantů do mitochondrií, na bioenergetický profil buněk. Data byla získána pomocí metod umožňující měření spotřeby kyslíku v reál- ném čase na extracelulárním flux analyzátoru a to jak v buňkách s neporušenou membránou, tak v permeabilizovaných buňkách, dále pomocí spektrofotometrick- ého měření enzymatické aktivity Krebsova cyklu a elektronového transportního řetězce, a nakonec pomocí fluorometrické detekce změn...Department of Internal Medicine 3FM CU and UHKVInterní klinika 3. LF UK a FNKV3. lékařská fakultaThird Faculty of Medicin

Energy metabolism of skeletal muscle

Skeletal muscle is the largest tissue in the body and plays a marked role in the homeostasis of the body metabolic state. Mitochondria have been proven to contribute to the pathophysiology of various metabolic diseases, either due to defects in their bioenergetic properties or the production of reactive oxygen species. In this work murine myoblasts C2C12 were used as a model of skeletal muscle in vitro, and rat muscle was used to prepare homogenate enriched in the mitochondrial fraction. This work investigates the changes in respiratory parameters in models where mitochondrial oxidative phosphorylation is induced by changing the available consumable substrates in the culture media, such as replacing glucose by galactose, and the effect of treating the cells with high glucose concentration during the process of differentiation on mitochondrial performance. It also investigates the changes in bioenergetic profiles in samples treated with inactive derivatives of the widely used triphenylphosphonium (TPP+) salts to target mitochondria by various probes and antioxidants. The methods used in this study included evaluating mitochondrial parameters in intact and permeabilized cells by real time measurement of the oxygen consumption rate using the extracellular flux analyzer, measuring the enzymatic..

Low glucose but not galactose enhances oxidative mitochondrial metabolism in C2C12 myoblasts and myotubes.

BACKGROUND: Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes. METHODOLOGY/PRINCIPAL FINDINGS: We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels. CONCLUSIONS/SIGNIFICANCE: Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes

Lipophilic triphenylphosphonium cations inhibit mitochondrial electron transport chain and induce mitochondrial proton leak.

The lipophilic positively charged moiety of triphenylphosphonium (TPP+) has been used to target a range of biologically active compounds including antioxidants, spin-traps and other probes into mitochondria. The moiety itself, while often considered biologically inert, appears to influence mitochondrial metabolism.We used the Seahorse XF flux analyzer to measure the effect of a range of alkylTPP+ on cellular respiration and further analyzed their effect on mitochondrial membrane potential and the activity of respiratory complexes. We found that the ability of alkylTPP+ to inhibit the respiratory chain and decrease the mitochondrial membrane potential increases with the length of the alkyl chain suggesting that hydrophobicity is an important determinant of toxicity.More hydrophobic TPP+ derivatives can be expected to have a negative impact on mitochondrial membrane potential and respiratory chain activity in addition to the effect of the biologically active moiety attached to them. Using shorter linker chains or adding hydrophilic functional groups may provide a means to decrease this negative effect

C2C12 cells grow more slowly without glucose and fail to consume galactose.

<p><b>A.</b> Growth rate over 3 days. LG, 1 g/l glucose; GAL, 1 g/l galactose, CF, carbohydrate-free. <b>B.</b> Doubling time. <b>C.</b> Changes in galactose concentration in growth media over time. All results are presented as means and 95% CI, (n = 3, each experiment was performed in triplicate).</p

TPP⁺ derivatives decrease mitochondrial membrane potential.

<p>A typical TMRM fluorescence intensity histogram from a flow cytometry experiment with C2C12 cells in the presence of 1 <i>μ</i>M TPP⁺ compounds. Lower fluorescence intensity corresponds to a lower membrane potential (Δ<i>ψ</i>_<i>m</i>).</p

Dose-dependence of the effect of dodecylTPP⁺ on mitochondrial metabolism.

<p><b>A.</b> Proton leak-driven respiration increases substantially with an increasing extracellular concentration of dodecylTPP⁺. <b>B.</b> A simultaneous decrease in maximal respiration due to increasing doses of dodecylTPP⁺. All results are expressed as the percentage of OCR of the DMSO treated control and are presented as means ± 95% CI, n = 3–6.</p