Cell type-selective secretome profiling in vivo.

Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Ion Channels in Drug Discovery and Safety Pharmacology.

Ion channels are membrane proteins involved in almost all physiological processes, including neurotransmission, muscle contraction, pace-making activity, secretion, electrolyte and water balance, immune response, and cell proliferation. Due to their broad distribution in human body and physiological roles, ion channels are attractive targets for drug discovery and safety pharmacology. Over the years ion channels have been associated to many genetic diseases ("channelopathies"). For most of these diseases the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a number of patients. The search for the development of new and more specific therapeutic approaches is therefore strongly pursued. At the same time acquired channelopathies or dangerous side effects (such as proarrhythmic risk) can develop as a consequence of drugs unexpectedly targeting ion channels. Several noncardiovascular drugs are known to block cardiac ion channels, leading to potentially fatal delayed ventricular repolarization. Thus, the search of reliable preclinical cardiac safety testing in early stage of drug discovery is mandatory. To fulfill these needs, both ion channels drug discovery and toxicology strategies are evolving toward comprehensive research approaches integrating ad hoc designed in silico predictions and experimental studies for a more reliable and quick translation of results to the clinic side.Here we discuss two examples of how the combination of in silico methods and patch clamp experiments can help addressing drug discovery and safety issues regarding ion channels.

Antidiabetic potential of phytochemicals isolated from the stem bark of Myristica fatua Houtt. var. magnifica (Bedd.) Sinclair.

Phytochemical investigation of the stem bark of Myristica fatua Houtt. led to the isolation of a new compound 1 (3-tridecanoylbenzoic acid), along with six known acylphenols (2-7). All the compounds displayed moderate inhibitory activity on α-amylase and significant activity on α-glucosidase; however malabaricone B (6) and C (7) were identified as potent α-glucosidase inhibitors with IC50 values of 63.70 ±â€¯0.546, and 43.61 ±â€¯0.620 µM respectively. Acylphenols (compounds 3-7) also showed significant antiglycation property. The molecular docking and dynamics simulation studies confirmed the efficient binding of malabaricone C with C-terminus of human maltase-glucoamylase (2QMJ). Malabaricone B also enhanced the 2-NBDG [2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy glucose] uptake in L6 myotubes. These findings demonstrate that acylphenols isolated from Myristica fatua Houtt. can be considered as a lead scaffold for the treatment of type II diabetes mellitus.

Bioreactor model of neuromuscular junction with electrical stimulation for pharmacological potency testing.

In vitro models of the neuromuscular junction (NMJ) are emerging as a valuable tool to study synaptogenesis, synaptic maintenance, and pathogenesis of neurodegenerative diseases. Many models have previously been developed using a variety of cell sources for skeletal muscle and motoneurons. These models can advanced by integrating beneficial features of the native developmental milieu of the NMJ. We created a functional in vitro model of NMJ by bioreactor cultivation of transdifferentiated myocytes and stem cell-derived motoneurons, in the presence of electrical stimulation. In conjunction with a coculture medium, electrical stimulation resulted in improved maturation and function of motoneurons and myocytes, as evidenced by mature cellular structures, increased expression of neuronal and muscular genes, clusterization of acetylcholine receptors (AChRs) in the vicinity of motoneurons, and the response to glutamate stimulation. To validate the model and demonstrate its utility for pharmacological testing, we documented the potency of drugs that affect key pathways during NMJ signal transduction: (i) acetylcholine (ACh) synthesis, (ii) ACh vesicular storage, (iii) ACh synaptic release, (iv) AChR activation, and (v) ACh inactivation in the synaptic cleft. The model properly responded to the drugs in a concentration-dependent manner. We thus propose that this in vitro model of NMJ could be used as a platform in pharmacological screening and controlled studies of neuromuscular diseases.

[Effects of Electroacupuncture on Apoptosis-related Protein Expression of Gastrocnemius Muscle Cells in Rats with Denervated Sciatic Nerve].

OBJECTIVE: To observe the anti-apoptosis effect of electroacupuncture (EA) on gastrocnemius muscle cells in rats with denervated sciatic nerve, so as to explore its possible mechanisms underlying delaying atrophy of skeletal muscle. METHODS: Sixty-three male Sprague-Dawley rats were randomly divided into sham operation group, model group, and EA group, and then further divided into 3 subgroups in each group(n=7/subgroup). Gastrocnemius muscle atrophy model was established by transecting the sciatic nerve of rat. EA (5 Hz, 1.5 mA) was applied to "Zusanli" (ST 36) and "Chengshan" (BL 57) acupoints at the affected side for 10 min, once a day for 1, 2, 3 weeks, respectively. The gastrocnemius muscles were sampled on the 7th, 14th, 21th d after modeling, separately. The apoptotic cells were detected by TUNEL staining. Bcl-2, Bax, Cyt-C and Caspase-3 protein expressions were determined by Western blot. RT-PCR was used to check Caspase-3 gene expression. RESULTS: Compared with the sham operation group, the cell apoptotic index in gastrocnemius was markedly higher, gastrocnemius Bcl-2 protein expression was markedly down-regulated, and gastrocnemius Bax, Cyt-C and Caspase-3 protein expressions were considerably up-regulated in the model group at all time-points (P<0.05). Compared with the model group, the cell apoptotic indexes in gastrocnemius were significantly lower in the three EA subgroups (P<0.05). Bcl-2 protein expressions were markedly up-regulated while Bax, Cyt-C and Caspase-3 protein expressions were significantly down-regulated in the EA group 14, 21 days after modeling compared with the corresponding model subgroups (P<0.05). The changes of Caspase-3 mRNA expression levels of gastrocnemius in all groups were similar to those of Caspase-3 protein expressions. CONCLUSIONS: Electroacupuncture intervention can effectively increase Bcl-2 expression and decrease Bax, Cyt-C and Caspase-3 expression in gastrocnemius muscle, and consequently reduce the apoptosis of muscle cell, which may contribute to its effect in delaying the skeletal muscle atrophy of denervated sciatic nerve.

Panduratin A Prevents Tumor Necrosis Factor-Alpha-Induced Muscle Atrophy in L6 Rat Skeletal Muscle Cells.

Panduratin A, a prenylated chalcone compound, has anti-obesity, anti-bacterial, anti-cancer, anti-inflammatory, and anti-oxidative activities. However, its preventive effect on muscle atrophy has not been studied. The purpose of this study was to evaluate the inhibitory effect of panduratin A on muscle atrophy and to investigate its molecular mechanisms in tumor necrosis factor-alpha (TNF-α)-treated L6 rat skeletal muscle cells. Panduratin A restored the myotube diameter reduced by TNF-α. At the molecular level, panduratin A elevated phosphatidylinositol 3 kinase/Akt/mammalian target of rapamycin pathway and stimulated the MyoD and myogenin mRNA expression decreased by TNF-α. However, panduratin A attenuated the mRNA expression of E3 ubiquitin ligase and autophagy-related genes. Moreover, panduratin A significantly inhibited reactive oxygen species production by increasing the mRNA expression of catalase and superoxide dismutase. Overall, panduratin A might be a useful agent for the treatment of muscle atrophy.

Creatine Supplementation and Skeletal Muscle Metabolism for Building Muscle Mass- Review of the Potential Mechanisms of Action.

Creatine, a very popular supplement among athletic populations, is of growing interest for clinical applications. Since over 90% of creatine is stored in skeletal muscle, the effect of creatine supplementation on muscle metabolism is a widely studied area. While numerous studies over the past few decades have shown that creatine supplementation has many favorable effects on skeletal muscle physiology and metabolism, including enhancing muscle mass (growth/hypertrophy); the underlying mechanisms are poorly understood. This report reviews studies addressing the mechanisms of action of creatine supplementation on skeletal muscle growth/hypertrophy. Early research proposed that the osmotic effect of creatine supplementation serves as a cellular stressor (osmosensing) that acts as an anabolic stimulus for protein synthesis signal pathways. Other reports indicated that creatine directly affects muscle protein synthesis via modulations of components in the mammalian target of rapamycin (mTOR) pathway. Creatine may also directly affect the myogenic process (formation of muscle tissue), by altering secretions of myokines, such as myostatin and insulin-like growth factor-1, and expressions of myogenic regulatory factors, resulting in enhanced satellite cells mitotic activities and differentiation into myofiber. Overall, there is still no clear understanding of the mechanisms of action regarding how creatine affects muscle mass/growth, but current evidence suggests it may exert its effects through multiple approaches, with converging impacts on protein synthesis and myogenesis.

Effect of Phenolic Compounds from Elderflowers on Glucose- and Fatty Acid Uptake in Human Myotubes and HepG2-Cells.

Type 2 diabetes (T2D) is manifested by progressive metabolic impairments in tissues such as skeletal muscle and liver, and these tissues become less responsive to insulin, leading to hyperglycemia. In the present study, stimulation of glucose and oleic acid uptake by elderflower extracts, constituents and metabolites were tested in vitro using the HepG2 hepatocellular liver carcinoma cell line and human skeletal muscle cells. Among the crude extracts, the 96% EtOH extract showed the highest increase in glucose and oleic acid uptake in human skeletal muscle cells and HepG2-cells. The flavonoids and phenolic acids contained therein were potent stimulators of glucose and fatty acid uptake in a dose-dependent manner. Most of the phenolic constituents and several of the metabolites showed high antioxidant activity and showed considerably higher α-amylase and α-glucosidase inhibition than acarbose. Elderflower might therefore be valuable as a functional food against diabetes.

H2S inhibits angiotensin II-induced atrial Kv1.5 upregulation by attenuating Nox4-mediated ROS generation during atrial fibrillation.

Our previous study demonstrated that angiotensin II (Ang II) upregulates the expression of Kv1.5, a promising target for atrial fibrillation (AF) therapy, by activating ROS-dependent P-Smad2/3 and P-ERK 1/2. A recent study showed that hydrogen sulfide (H2S) may modulate the effects of angiotensin II (Ang II) by inhibiting the NADPH oxidase 4 (Nox4)-ROS signaling in the heart. The present study aimed to determine whether H2S is involved in the regulation of atrial Kv1.5 via ROS-related mechanisms in AF. Cultured neonatal rat atrial myocytes and a beagle model of AF were used for this study. In the neonatal rat atrial myocytes, quantitative PCR and enzyme immunoassays revealed that the mRNA expression levels of angiotensinogen, angiotensin-converting enzyme, and Ang II type I receptor (AT1R) and the Ang II supernatant concentration were significantly increased by hydrogen peroxide (H2O2) incubation, and these H2O2-induced alterations were reversed by diphenyleneiodonium, apocynin and H2S supplementation. Flow cytometry and Western blotting revealed that blockade of H2S biosynthesis using dl-propargylglycine increased ROS production and the expression of Ang II and Kv1.5. Sodium hydrosulfide (an exogenous H2S donor) and Nox4 siRNA inhibited Ang II-induced ROS production and Ang II-induced expression of Kv1.5, P-Smad2/3, P-ERK 1/2. Sodium hydrosulfide suppressed the Ang II-induced upregulation of Nox4. In our beagle AF model, 24 h of rapid atrial pacing (RAP) increased the atrial Ang II concentration, ROS production and the protein expression of Nox4, Kv1.5, P-Smad2/3 and P-ERK 1/2. These RAP-induced changes were inhibited by H2S supplementation and losartan (an AT1R blocker) pretreatment. In conclusion, our study indicates that H2S downregulates Ang II-induced atrial Kv1.5 expression by attenuating Nox4-related ROS-triggered P-Smad2/3 and P-ERK 1/2 activation during AF. H2S supplementation would be beneficial for AF treatment via the suppression of atrial Kv1.5 expression.

In Silico Evaluation of the Potential Antiarrhythmic Effect of Epigallocatechin-3-Gallate on Cardiac Channelopathies.

Ion channels are transmembrane proteins that allow the passage of ions according to the direction of their electrochemical gradients. Mutations in more than 30 genes encoding ion channels have been associated with an increasingly wide range of inherited cardiac arrhythmias. In this line, ion channels become one of the most important molecular targets for several classes of drugs, including antiarrhythmics. Nevertheless, antiarrhythmic drugs are usually accompanied by some serious side effects. Thus, developing new approaches could offer added values to prevent and treat the episodes of arrhythmia. In this sense, green tea catechins seem to be a promising alternative because of the significant effect of Epigallocatechin-3-Gallate (E3G) on the electrocardiographic wave forms of guinea pig hearts. Thus, the aim of this study was to evaluate the benefits-risks balance of E3G consumption in the setting of ion channel mutations linked with aberrant cardiac excitability phenotypes. Two gain-of-function mutations, Nav1.5-p.R222Q and Nav1.5-p.I141V, which are linked with cardiac hyperexcitability phenotypes were studied. Computer simulations of action potentials (APs) show that 30 µM E3G reduces and suppresses AP abnormalities characteristics of these phenotypes. These results suggest that E3G may have a beneficial effect in the setting of cardiac sodium channelopathies displaying a hyperexcitability phenotype.

Protamine zinc insulin combined with sodium selenite improves glycometabolism in the diabetic KKAy mice.

Long-term, high dosage protamine zinc insulin (PZI) treatments produce adverse reactions. The trace element selenium (Se) is a candidate for the prevention of diabetes due to anti-oxidative stress activity and the regulation of glycometabolism. In this study, we aimed to investigate the anti-diabetic effects of a combination of PZI and Se on type 2 diabetes. Diabetic KKAy mice were randomized into the following groups: model group and groups that were subcutaneously injected with PZI, Se, high or low dose PZI + Se for 6 weeks. PZI combined with Se decreased the body weight and fasting blood glucose levels. Moreover, this treatment also improved insulin tolerance, as determined by the reduced values from the oral glucose tolerance test and insulin tolerance test, and increased insulin levels and insulin sensitivity index. PZI combined with Se ameliorated skeletal muscle and ß-cell damage and the impaired mitochondrial morphology. Oxidative stress was also reduced. Furthermore, PZI combined with Se upregulated phosphatidylinositol 3-kinase (PI3K) and downregulated protein tyrosine phosphatase 1B (PTP1B). Importantly, the low dosage combination produced effects similar to PZI alone. In conclusion, PZI combined with Se improved glycometabolism and ameliorated the tissue and mitochondrial damage, which might be associated with the PI3K and PTP1B pathways.

Neuronal control of bone and muscle.

The functional interplay between bone and muscle that enables locomotion is a fundamental aspect of daily life. However, other interactions between bone and muscle continue to attract attention as our understanding of the breath and importance of this inter-relationship continues to expand. Of particular interest is the regulatory connection between bone and muscle, which adds a new insight to the coordination of the bone/muscle unit. We have appreciated the importance of neuronal signaling to the control of bone turnover and muscle contraction, but recent data indicate that neuronal inputs control a far wider range of bone and muscle physiology than previously appreciated. This review outlines the role of the sympathetic nervous system and neuronal/neuropeptide inputs upon the regulation of bone and muscle tissue, and the potential for co-regulatory actions, particularly involving the sympathetic nervous system. This article is part of a Special Issue entitled "Muscle Bone Interactions".

Evidence of Notch-Hesr-Nrf2 Axis in Muscle Stem Cells, but Absence of Nrf2 Has No Effect on Their Quiescent and Undifferentiated State.

Nrf2 is a master regulator of oxidative stresses through the induction of anti-oxidative genes. Nrf2 plays roles in maintaining murine hematopoietic stem cells and fly intestinal stem cells. The canonical Notch signaling pathway is also crucial for maintaining several types of adult stem cells including muscle stem cells (satellite cells). Here, we show that Dll1 induced Nrf2 expression in myogenic cells. In addition, primary targets of Notch signaling, Hesr1 and Hesr3, were involved in the up-regulation of Nrf2 mRNA and expression of its target genes. In vitro, Nrf2 had anti-myogenic and anti-proliferative effects on primary myoblasts. In vivo, although Nrf2-knockout mice showed decreased expression of its target genes in muscle stem cells, adult muscle stem cells of Nrf2-knockout mice did not exhibit the phenotype. Taken together, in muscle stem cells, the Notch-Hesr-Nrf2 axis is a pathway potentially inducing anti-oxidative genes, but muscle stem cells either do not require Nrf2-mediated anti-oxidative gene expression or they have a complementary system compensating for the loss of Nrf2.

Cinnamon and its Components Suppress Vascular Smooth Muscle Cell Proliferation by Up-Regulating Cyclin-Dependent Kinase Inhibitors.

Cinnamomum cassia bark has been used in traditional herbal medicine to treat a variety of cardiovascular diseases. However, the antiproliferative effect of cinnamon extract on vascular smooth muscle cells (VSMCs) and the corresponding restenosis has not been explored. Hence, after examining the effect of cinnamon extract on VSMC proliferation, we investigated the possible involvement of signal transduction pathways associated with early signal and cell cycle analysis, including regulatory proteins. Besides, to identify the active components, we investigated the components of cinnamon extract on VSMC proliferation. Cinnamon extract inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and suppressed the PDGF-stimulated early signal transduction. In addition, cinnamon extract arrested the cell cycle and inhibited positive regulatory proteins. Correspondingly, the protein levels of p21 and p27 not only were increased in the presence of cinnamon extract, also the expression of proliferating cell nuclear antigen (PCNA) was inhibited by cinnamon extract. Besides, among the components of cinnamon extract, cinnamic acid (CA), eugenol (EG) and cinnamyl alcohol significantly inhibited the VSMC proliferation. Overall, the present study demonstrates that cinnamon extract inhibited the PDGF-BB-induced proliferation of VSMCs through a G0/G1 arrest, which down-regulated the expression of cell cycle positive regulatory proteins by up-regulating p21 and p27 expression.

miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction.

Identifying the interaction partners of noncoding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA crosslinking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-crosslinking and Argonaute 2 immunopurification followed by streptavidin affinity purification of probe-linked RNAs provided selectivity in the capture of targets, which were identified by deep sequencing. miR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. miR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long noncoding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation, levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a 'sponge' for these miRNAs.

Activation of calcium- and voltage-gated potassium channels of large conductance by leukotriene B4.

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory ß1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 µm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-ß2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK ß1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate ß1 subunit-containing channels when ß1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK ß1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of ß1-specific BK channel activators.

CLA reduces inflammatory mediators from A427 human lung cancer cells and A427 conditioned medium promotes differentiation of C2C12 murine muscle cells.

Conjugated linoleic acid (CLA) is thought to have anti-proliferative and anti-inflammatory properties, but its effect on cancer cachexia is unknown. Two effects were here investigated: that of CLA on inflammatory mediator production in human lung cancer cells, and that of reduced mediators on the myogenic differentiation of murine muscle C2C12 cells. The latter cells were grown in medium conditioned by human lung cancer A427 cells, with or without CLA, to mimic only the effect of molecules released from the tumor "in vivo", excluding the effect of host-produced cachectic factors. The results obtained show that CLA was found to reduce the production of tumor necrosis factor-α, interleukin (IL)-1ß and prostaglandin E2 (PGE2), but had no effect on IL-6 production. The mechanisms underlying the effect of CLA on cytokine or PGE2 release in A427 cells are probably mediated by activation of peroxisome proliferator-activated receptor (PPAR)α, which increased at 24 h CLA treatment. In turn, the reduced content of inflammatory mediators in medium conditioned by A427 cells, in the presence of CLA, allowed muscle cells to proliferate, again by inducing PPAR. The involvement of PPARα was demonstrated by treatment with the antagonist MK-886. The findings demonstrate the anti-inflammatory and myogenic action of CLA and point to its possible application as a novel dietary supplement and therapeutic agent in inflammatory disease states, such as cachexia.

Hyperthermia: from diagnostic and treatments to new discoveries.

Hyperthermia is an important approach for the treatment of several diseases. Hyperthermia is also thought to induce hypertrophy of skeletal muscles in vitro and in vivo, and has been used as a therapeutic tool for millennia. In the first part of our work, we revise several relevant patents related to the utilization of hyperthermia for the treatment and diagnostic of human diseases. In the second part, we present exciting new data on the effects of forced and natural overexpression of HSP72, using murine in vitro (muscle cells) and ex vivo (primary skeletal muscles) models. These studies help to demonstrate that hyperthermia effects are orchestrated by tight coupling between gene expression, protein function, and intracellular Ca2+ signaling pathways with a key role for calcium-induced calcium release. We hope that the review of current patents along with previous unknown information on molecular signaling pathways that underlie the hypertrophy response to hyperthermia in skeletal muscles may trigger the curiosity of scientists worldwide to explore new inventions that fully utilize hyperthermia for the treatment of muscle diseases.

A combination of lipoic acid plus coenzyme Q10 induces PGC1α, a master switch of energy metabolism, improves stress response, and increases cellular glutathione levels in cultured C2C12 skeletal muscle cells.

Skeletal muscle function largely depend on intact energy metabolism, stress response, and antioxidant defense mechanisms. In this study, we tested the effect of a combined supplementation of α-lipoic acid (LA) plus coenzyme Q10 (Q10) on PPARγ-coactivator α (PGC1α) activity, expression of glutathione-related phase II enzymes and glutathione (GSH) levels in cultured C2C12 myotubes. Supplementation of myotubes with 250 µmol/L LA plus 100 µmol/L Q10 significantly increased nuclear levels of PGC1α, a master switch of energy metabolism and mitochondrial biogenesis. The increase of nuclear PGC1α was accompanied by an increase in PPARγ transactivation, a downstream target of PGC1α, and an increase in mitochondrial transcription factor A mRNA centrally involved in mitochondrial replication and transcription. Furthermore, supplementation of myotubes with LA plus Q10 resulted in an increase of genes encoding proteins involved in stress response, GSH synthesis, and its recycling. In LA-plus-Q10-treated myotubes a significant 4-fold increase in GSH was evident. This increase in GSH was accompanied by increased nuclear Nrf2 protein levels, partly regulating γGCS and GST gene expression. Present data suggest that the combined supplementation of skeletal muscle cells with LA plus Q10 may improve energy homeostasis, stress response, and antioxidant defense mechanisms.

Antioxidant effect of a phytoestrogen equol on cultured muscle cells of embryonic broilers.

Previous studies have shown that the in ovo injection of equol can markedly improve the water-holding capacity of muscles of broilers chickens at 7 wk of age through promotion of the antioxidant status. We aimed to investigate directly the antioxidant effects of equol on muscle cells in broilers. Muscle cells were separated from leg muscle of embryos on the 11th day of incubation and treated with equol and H(2)O(2), either alone or together. Cells were pretreated with medium containing 1, 10, or 100 µM equol for 1 h prior to the addition of 1 mM H(2)O(2) for a further 1 h. Photomicrographs of cells were obtained. Cell viability, malondialdehyde (MDA) content, and L-lactate dehydrogenase (LDH) activity in the cell supernatant, as well as intracellular total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities were determined. Treatment with 1 mM H(2)O(2) caused serious damage to cells, indicated by comets with no clear head region but a very apparent tail of DNA fragments. Pretreatment with low (1 µM) but not high concentrations of equol (10 µM) inhibited cell damage, while 100 µM equol caused more serious damage than H(2)O(2) alone. Pretreatment with 1 µM equol had no effect on cell viability, while pretreatment with 10 and 100 µM equol significantly decreased cell viability in a dose-dependent manner. Compared with H(2)O(2) alone, pretreatment with low-dosage equol markedly decreased LDH activity and MDA production in the supernatant, significantly increased intracellular T-SOD activity (P < 0.05) and tended to increase intracellular GSH-Px activity (0.05 < P < 0.1). Pretreatment with high-dosage equol (10 and 100 µM) significantly enhanced LDH activity, but had no effect on MDA content, T-SOD or GSH-Px activity induced by H(2)O(2,) except for an obvious increase in GSH-Px activity caused by 10 µM equol. These results indicate that equol at low dosage can prevent skeletal muscle cell damage induced by H(2)O(2), while pretreatment with high-dosage equol shows a synergistic effect with H(2)O(2) in inducing cell damage.