Depolarization Of Membrane Potential Research Articles

A role of ROS-dependent defects in mitochondrial dynamic and autophagy in carbon black nanoparticle-mediated myocardial cell damage

Carbon black nanoparticles (CBNPs) are widely distributed in the environment and are increasingly recognized as a contributor in the development of cardiovascular disease.A variety of cardiac injuries and diseases result from structural and functional damage to cardiomyocytes. This study explored the mechanisms of CBNPs-mediated myocardial toxicity. CBNPs were given to mice through intra-tracheal instillation and it was demonstrated that the particles can be taken up into the cardiac tissue. Exposure to CBNPs induced cardiomyocyte inflammation and apoptosis. In combination with in vitro experiments, we showed that CBNPs increased the ROS and induced mitochondria fragmentation. Functionally, CBNPs-exposed cardiomyocyte exhibited depolarization of the mitochondrial membrane potential, release of cytochrome c, and activation of pro-apoptotic BAX, thereby initiating programmed cell death. On the other hand, CBNPs impaired autophagy, leading to the inadequate removal of dysfunctional mitochondria. The excess accumulation of damaged mitochondria further stimulated NF-κB activation and triggered the NLRP3 inflammasome pathway. Both the antioxidant N-acetylcysteine and the autophagy activator rapamycin were effective to attenuate the damage of CBNPs on cardiomyocytes. Taken together, this study elucidated the potential mechanism underlying CBNPs-induced myocardial injury and provided a scientific reference for the evaluation and prevention of the CBNPs-related heart risk.

Moringin, an isothiocyanate modulates multiple cellular signalling molecules in breast cancer cells

Prohibitin (PHB) is a pleiotropic molecule with a variety of known functions and subcellular locations. PHB's function in breast cancer is poorly understood. Herein, we report that PHB is expressed in cancer types of diverse origin including breast cancer. The cancer patients with changes in PHB were reported to have significantly reduced ‘overall survival’ in comparison to the cases without alterations in PHB. The expression of PHB was increased by H2O2 and also by Moringin (MG), which is an isothiocyanate derived from the seeds of Moringa oleifera. MG interacted with PHB, DRP1, and SLP2 and inhibited the growth of MCF-7 and MDAMB-231 cells. The isothiocyanate triggered apoptosis in breast cancer cells as revealed by AO/PI assay, phosphatidylserine externalization, cell cycle analysis and DAPI staining. MG induced proapoptotic proteins expression such as cytochrome c, p53, and cleaved caspase-7. Further, cell survival proteins such as survivin, Bcl-2, and Bcl-xL were suppressed. A depolarization of membrane potential suggested that the apoptosis was triggered through mitochondria. The isothiocyanate suppressed the cancer cell migration and interacted with NF-κB subunits. MG suppressed p65 nuclear translocation induced by TNF-α. The reactive oxygen species generation was also induced by the isothiocyanate in breast cancer cells. MG also modulated the expression of lncRNAs. Collectively, the functions of PHB in breast cancer growth is evident from this study. The activities of MG against breast cancer might result from its ability to modulate multiple cancer-related targets.

Membrane potential dynamics of C5a-stimulated neutrophil granulocytes

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3—4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

A tonically active master neuron modulates mutually exclusive motor states at two timescales.

Continuity of behaviors requires animals to make smooth transitions between mutually exclusive behavioral states. Neural principles that govern these transitions are not well understood. Caenorhabditis elegans spontaneously switch between two opposite motor states, forward and backward movement, a phenomenon thought to reflect the reciprocal inhibition between interneurons AVB and AVA. Here, we report that spontaneous locomotion and their corresponding motor circuits are not separately controlled. AVA and AVB are neither functionally equivalent nor strictly reciprocally inhibitory. AVA, but not AVB, maintains a depolarized membrane potential. While AVA phasically inhibits the forward promoting interneuron AVB at a fast timescale, it maintains a tonic, extrasynaptic excitation on AVB over the longer timescale. We propose that AVA, with tonic and phasic activity of opposite polarities on different timescales, acts as a master neuron to break the symmetry between the underlying forward and backward motor circuits. This master neuron model offers a parsimonious solution for sustained locomotion consisted of mutually exclusive motor states.

Versatile Fluorescence Lifetime-Based Copper Probe to Quantify Mitochondrial Membrane Potential and Reveal Its Interaction with Protein Aggregation.

Mitochondria play a crucial role in maintaining cellular homeostasis, and the depolarization of mitochondrial membrane potential (MMP) is an important signal of apoptosis. Additionally, protein misfolding and aggregation are closely related to diseases including neurodegenerative diseases, diabetes, and cancers. However, the interaction between MMP changes and disease-related protein aggregation was rarely studied. Herein, we report a novel "turn-on" fluorescent probe MitoRhB that specifically targets to mitochondria for Cu2+ detection in situ. The fluorescence lifetime (τ) of MitoRhB exhibits a positive correlation with MMP changes, allowing us to quantitatively determine the relative MMP during SOD1 (A4 V) protein aggregation. Finally, we found that (1) the increasing concentrations of copper will accelerate the depolarization of mitochondria and reduce MMP; (2) the depolarization of mitochondria can intensify the degree of protein aggregation, suggesting a new routine of copper-induced cell death mediated through abnormal MMP depolarization and protein aggregation.

L-Ornithine-l-Aspartate (LOLA) Normalizes Metabolic Parameters in Models of Steatosis, Insulin Resistance and Metabolic Syndrome.

l-Ornithine- l-aspartate (LOLA) reduces toxic ammonium (NH3) plasma levels in hepatic encephalopathy. NH3 detoxification/excretion is achieved by its incorporation into urea and glutamine via activation of carbamoyl phosphate synthetase 1 (CSP1) by l-ornithine and stimulation of arginase by l-aspartate. We aimed at identifying additional molecular targets of LOLA as a potential treatment option for non-alcoholic fatty liver disease (NAFLD). In primary hepatocytes from NAFLD patients, urea cycle enzymes CSP1 and ornithine transcarbamylase (OTC) increase, while the catabolism of branched-chain amino acids (BCAAs) decreases with disease severity. In contrast, LOLA increased the expression rates of the BCAA enzyme transcripts bcat2, bckdha, and bckdk. In untreated HepG2 hepatoblastoma cells and HepG2-based models of steatosis, insulin resistance, and metabolic syndrome (the latter for the first time established herein), LOLA reduced the release of NH3; beneficially modulated the expression of genes related to fatty acid import/transport (cd36, cpt1), synthesis (fasn, scd1, ACC1), and regulation (srbf1); reduced cellular ATP and acetyl-CoA; and favorably modulated the expression of master regulators/genes of energy balance/mitochondrial biogenesis (AMPK-α, pgc1α). Moreover, LOLA reconstituted the depolarized mitochondrial membrane potential, while retaining mitochondrial integrity and avoiding induction of superoxide production. Most effects were concentration-dependent at ≤40 mM LOLA. We demonstrate for l-ornithine-l-aspartate a broad range of reconstituting effects on metabolic carriers and targets of catabolism/energy metabolism impaired in NAFLD. These findings strongly advocate further investigations to establish LOLA as a safe, efficacious, and cost-effective basic medication for preventing and/or alleviating NAFLD.

Isobavachalcone induces hepatotoxicity in zebrafish embryos and HepG2 cells via the System Xc--GSH-GPX4 signaling pathway in ferroptosis response.

Isobavachalcone (IBC) is a flavonoid component of the traditional Chinese medicine Psoraleae Fructus, with a range of pharmacological properties. However, IBC causes some hepatotoxicity, and the mechanism of toxicity is unclear. The purpose of this paper was to investigate the possible mechanism of toxicity of IBC on HepG2 cells and zebrafish embryos. The results showed that exposure to IBC increased zebrafish embryo mortality and decreased hatchability. Meanwhile, IBC induced liver injury and increased expression of ALT and AST activity. Further studies showed that IBC caused the increase of ROS and MDA the decrease of CAT, GSH, and GSH-Px; the increase of Fe2+ content; and the changes of ferroptosis related genes (acsl4, gpx4, and xct) and iron storage related genes (tf, fth, and fpn) in zebrafish embryos. Through in vitro verification, it was found that IBC also caused oxidative stress and increased Fe2+ content in HepG2 cells. IBC caused depolarization of mitochondrial membrane potential (MMP) and reduction of mitochondrial ATP, as well as altered expression of ACSl4, SLC7A11, GPX4, and FTH1 proteins. Treatment of HepG2 cells with ferrostatin-1 could reverse the effect of IBC. Targeting the System Xc--GSH-GPX4 pathway of ferroptosis and preventing oxidative stress damage might offer a theoretical foundation for practical therapy and prevention of IBC-induced hepatotoxicity.

5-Hydroxytryptamine Enhances the Pacemaker Activity of Interstitial Cells of Cajal in Mouse Colon.

We examined the localization of the 5-hydroxytryptamine (5-HT) receptor and its effects on mouse colonic interstitial cells of Cajal (ICCs) using electrophysiological techniques. Treatment with 5-HT increased the pacemaker activity in colonic ICCs with depolarization of membrane potentials in a dose-dependent manner. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers blocked pacemaker activity and 5-HT-induced effects. Moreover, an adenylate cyclase inhibitor inhibited 5-HT-induced effects, and cell-permeable 8-bromo-cAMP increased the pacemaker activity. Various agonists of the 5-HT receptor subtype were working in colonic ICCs, including the 5-HT4 receptor. In small intestinal ICCs, 5-HT depolarized the membrane potentials transiently. Adenylate cyclase inhibitors or HCN blockers did not show any influence on 5-HT-induced effects. Anoctamin-1 (ANO1) or T-type Ca2+ channel blockers inhibited the pacemaker activity of colonic ICCs and blocked 5-HT-induced effects. A tyrosine protein kinase inhibitor inhibited pacemaker activity in colonic ICCs under controlled conditions but did not show any influence on 5-HT-induced effects. Among mitogen-activated protein kinase (MAPK) inhibitors, a p38 MAPK inhibitor inhibited 5-HT-induced effects on colonic ICCs. Thus, 5-HT's effect on pacemaker activity in small intestinal and colonic ICCs has excitatory but variable patterns. ANO1, T-type Ca2+, and HCN channels are involved in 5-HT-induced effects, and MAPKs are involved in 5-HT effects in colonic ICCs.

Phaseolus vulgaris STP13.1 is an H+-coupled monosaccharide transporter, present in source leaves and seed coats, with higher substrate affinity at depolarized potentials.

Sugar transport proteins (STPs) are high-affinity H+-coupled hexose symporters. Recently, the contribution of STP13 to bacterial and fungal pathogen resistance across multiple plant species has garnered significant interest. Quantitative PCR analysis of source leaves, developing embryos, and seed coats of Phaseolus vulgaris L. (common bean) revealed that PvSTP13.1 was expressed in source leaves and seed coats throughout seed development. In contrast, PvSTP13.1 transcripts were detected at exceedingly low levels in developing embryos. To characterize the transport mechanism, PvSTP13.1 was expressed in Xenopus laevis oocytes, and inward-directed currents were analyzed using two-electrode voltage clamping. PvSTP13.1 was shown to function as an H+-coupled monosaccharide symporter exhibiting a unique high affinity for hexoses and aldopentoses at depolarized membrane potentials. Specifically, of the 31 assessed substrates, which included aldohexoses, deoxyhexoses, fructose, 3-O-methyl-D-glucose, aldopentoses, polyols, glycosides, disaccharides, trisaccharides, and glucuronic acid, PvSTP13.1 displayed the highest affinity (K 0.5) for glucose (43 μM), mannose (92 μM), galactose (145 μM), fructose (224 μM), xylose (1.0mM), and fucose (3.7mM) at pH5.6 at a depolarized membrane potential of -40 mV. The results presented here suggest PvSTP13.1 contributes to retrieval of hexoses from the apoplasmic space in source leaves and coats of developing seeds.

Nickel induces mitochondrial damage in renal cells in vitro and in vivo through its effects on mitochondrial biogenesis, fusion, and fission

Nickel (Ni) and its compounds are common, widely distributed components of hazardous waste in the chemical industry. Excessive exposure to Ni can cause kidney damage in humans and animals. We investigated the impact of Ni on renal mitochondria using in vivo and in vitro models of Ni nephrotoxicity, and explored the Ni nephrotoxic mechanism. We showed that nickel chloride (NiCl2) damaged the renal mitochondria, causing mitochondrial swelling, breakage of the mitochondrial cristae, increased levels of mitochondrial reactive oxygen species (mt-ROS), and depolarization of the mitochondrial membrane potential (MMP). The levels of the mitochondrial respiratory chain complexes I–IV were reduced in the kidneys of mice treated with NiCl2. In addition, NiCl2 treatment inhibited mitochondrial biogenesis in renal cells by down-regulating mRNA and the protein expression of TFAM, PGC-1α, and NRF1. Moreover, NiCl2 reduced the levels of the proteins involved in mitochondrial fusion, including Mfn1 and Mfn2, while significantly augmenting the levels of the proteins Fis1 and Drip1 involved in mitochondrial fission in renal cells. Taken together, these results suggested that NiCl2 inhibited mitochondrial biogenesis, suppressed mitochondrial fusion, and promoted mitochondrial fission, resulting in mitochondrial dysfunction in renal cells, ultimately causing renal injury. This study provided novel insights into the mechanisms of nephrotoxicity of Ni and new ideas for the development of targeted treatments for Ni-induced kidney injury.

13-oxyingenol dodecanoate derivatives induce mitophagy and ferroptosis through targeting TMBIM6 as potential anti-NSCLC agents

Ingenol diterpenoids continue to attract the attention for their extensive biological activity and novel structural features. To further explore this type of compound as anti-tumor agent, 13-oxyingenol dodecanoate (13-OD) was prepared by a standard chemical transformation from an Euphorbia kansui extract, and 29 derivatives were synthesized through parent 13-OD. Their inhibition activities against different types of cancer were screened and some derivatives showed superior anti-non-small cell lung cancer (NSCLC) cells cytotoxic potencies than oxaliplatin. In addition, TMBIM6 was identified as a crucial cellular target of 13-OD using ABPP target angling technique, and subsequently was verified by pull down, siRNA interference, BLI and CETSA assays. With modulating the function of TMBIM6 protein by 13-OD and its derivatives, Ca2+ release function was affected, causing mitochondrial Ca2+ overload, depolarisation of membrane potential. Remarkably, 13-OD, B6, A2, and A10–2 induced mitophagy and ferroptosis. In summary, our results reveal that 13-OD, B6, A2, and A10–2 holds great potential in developing anti-tumor agents for targeting TMBIM6.

Unveil the mechanism for EHMT -- A novel triterpenoid inhibits proliferation and induces apoptosis in colon cancer through ROS-mediated JNK signaling pathway

Colon cancer ranks among the most prevalent malignancies worldwide, trailing only lung and breast cancer in incidence. Despite the availability of numerous therapeutic strategies, the burden of new cases and fatalities remains high in countries undergoing socioeconomic transitions. Natural products offer promising avenues for developing more effective and less toxic anticancer agents, expanding the clinical arsenal. In this investigation, we isolated a triterpenoid, (21 S,23 R,24 R)-21,23-epoxy-24-hydroxy-21-methoxytirucalla-7,25-dien-3-one (EHMT), from the fruits of Melia azedarach L., which exhibited significant inhibitory activity against colon cancer cells while sparing normal cells. EHMT effectively curtailed colony formation and induced apoptosis and cell cycle arrest in the HCT116 cell line. Furthermore, EHMT prompted the generation of reactive oxygen species (ROS) and the depolarization of mitochondrial membrane potential. Notably, EHMT treatment triggered ROS-mediated cell apoptosis via activation of the JNK signaling pathway in HCT116 cells. Additionally, our findings extended to Caenorhabditis elegans, where EHMT induced ROS accumulation and apoptosis. Collectively, these findings position EHMT as a promising candidate for the development of anticancer agents in the treatment of colon cancer, offering new hope in the battle against this formidable disease.

Mechanism of Porphyra Yezoensis Polysaccharides in Inhibiting Hyperoxalate-Induced Renal Injury and Crystal Deposition.

Oxidative damage to the kidneys is a primary factor in the occurrence of kidney stones. This study explores the inhibitory effect of Porphyra yezoensis polysaccharides (PYP) on oxalate-induced renal injury by detecting levels of oxidative damage, expression of adhesion molecules, and damage to intracellular organelles and revealed the molecular mechanism by molecular biology methods. Additionally, we validated the role of PYP in vivo using a crystallization model of hyperoxalate-induced rats. PYP effectively scavenged the overproduction of reactive oxygen species (ROS) in HK-2 cells, inhibited the adhesion of calcium oxalate (CaOx) crystals on the cell surface, unblocked the cell cycle, restored the depolarization of the mitochondrial membrane potential, and inhibited cell death. PYP upregulated the expression of antioxidant proteins, including Nrf2, HO-1, SOD, and CAT, while decreasing the expression of Keap-1, thereby activating the Keap1/Nrf2 signaling pathway. PYP inhibited CaOx deposition in renal tubules in the rat crystallization model, significantly reduced high oxalate-induced renal injury, decreased the levels of the cell surface adhesion proteins, improved renal function in rats, and ultimately inhibited the formation of kidney stones. Therefore, PYP, which has crystallization inhibition and antioxidant properties, may be a therapeutic option for the treatment of kidney stones.

Synthesis and biological evaluation of folic acid-rotenol conjugate as a potent targeted anticancer prodrug

Rotenone, a plant-based agricultural insecticide, has been shown to have anti-tumor activity through targeting mitochondrial complex I in cancer cells. However, off-target toxic side effect on nervous systems have greatly restricted the application of rotenone as anticancer drugs. Here, a folic acid-rotenol (FA-rotenol) conjugate was prepared by covalent coupling of the tumor-targeting ligand folic acid with rotenone derivative—rotenol to enhance its accumulation at tumor site. FA-rotenol conjugates present high in vitro cytotoxicties against several cell lines by inducing mitochondrial membrane potential depolarization and increasing the level of intracellular reactive oxygen species (ROS) to activate the mitochondrial pathway of apoptosis and enhance the G2/M cell cycle arrest. Because of the high affinity with over-expressed folate receptors, FA-rotenol conjugate demonstrated more effective in vivo therapeutic outcomes in 4T1 tumor-bearing mice than rotenone and rotenol. In addition, FA-rotenol conjugate can markedly inhibit the cell migration and invasion of HepG-2 cells. These studies confirm the feasibility of tumor-targeted ligand conjugated rotenone derivatives for targeted antitumor therapy; likewise, they lay the foundations for the development of other rotenol-conjugates with antitumor potential.

Mitochondrial flashes are interlinked with adaptive thermogenesis in brown adipocytes

Mitochondrial flash (mitoflash) is the electrochemical excitation of a single mitochondrion and plays diverse signaling roles in a variety of cells. From the viewpoint of bioenergetics, it represents the transient uncoupling of mitochondrial respiration from ATP synthesis. Brown adipose tissue (BAT) produces heat, primarily via mitochondrial uncoupled respiration. Here, we aim to illustrate the mitoflash activity in BAT thermogenesis. We show that BAT mitochondria undergo stochastic and intermittent mitoflashes at the levels of isolated mitochondria, cultured brown adipocytes, and even intact BAT. A BAT mitoflash contains multiple signals, including transient depolarization of mitochondrial membrane potential, transient matrix alkalization, and bursting production of reactive oxygen species. Importantly, thermogenic activation by β3-adrenergic stimulation dramatically augments mitoflash incidence, with mild effects on its unitary characteristics. Ablation of the thermogenic uncoupling protein 1 (UCP1) showed little effect on mitoflash biogenesis during heat production. Collectively, our results suggest that mitoflash might constitute an elemental signaling activity of mitochondria to regulate BAT thermogenesis.

Apoptotic Potential and Molecular Docking of 3,4-Dihydro-lactucin, a Compound With Anticancer Properties Derived from Microbispora rosea AL22.

Tetrazolium-based cell proliferation assays using MDA-MB-231 and HeLa cells revealed that 3,4-dihydro-lactucin (3,4-DHL), a compound isolated from Microbispora rosea AL22, possesses anticancer properties. Apoptotic cell death was observed in 3,4-DHL-treated cells. Lactucopicrin, a related compound, reportedly exerts anticancer activity against different cancer types. However, data on the anticancer mechanism of lactucins are limited. This study aimed to investigate apoptosis induction in MDA-MB-231 cells treated with 3,4-DHL. Morphological changes, changes in mitochondrial membrane potential, and apoptosis induction in MDA-MB-231 cells treated with 3,4-DHL were investigated. Furthermore, molecular docking and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis of anti-apoptotic proteins were performed to determine the effector mechanism of 3,4-DHL. 3,4-DHL induced cytotoxicity at a half-maximal inhibitory concentration of 37.62 μg/ml, along with various morphological alterations in apoptotic and viable cells. Furthermore, 3,4-DHL-treated cells showed mitochondrial membrane potential depolarization, intense annexin V-fluorescein isothiocyanate staining, and increased caspase 3 and 8 activities. Molecular-docking studies demonstrated that 3,4-DHL should bind to the active site of various anti-apoptotic proteins, forming stable complexes. Our findings revealed that 3,4-DHL has great potential to be used as an apoptosis-inducing agent in cancer therapy. However, further in-vivo confirmation is required in evaluation of 3,4-DHL as an anticancer agent in cancer chemotherapy.

Quercetin protects cardiomyoblasts against hypertonic cytotoxicity by abolishing intracellular Ca2+ elevations and mitochondrial depolarisation

Background and AimOsmotic changes represent a burden for the body and their limitation would be beneficial. We hypothesized that ubiquitous natural compounds could guard against cytotoxic effects of osmotic stress. We evaluated the anti-hypertonic mechanism of quercetin and 2,3-dehydrosilybin in H9c2 cells in vitro. Experimental procedureProtective effect of both compounds was determined by neutral red assay, cell apoptosis was estimated by measuring caspase-3 activity and verified by western blot and annexin V assay. Phosphorylation level of selected proteins was also detected. Mitochondrial membrane potential was evaluated using dye JC-1. Ca2+ signals were evaluated using genetically encoded fluorescent Ca2+ biosensor GCaMP7f. Formation of reactive oxygen species was measured using an oxidant-sensing probe dihydrofluorescein diacetate. Key resultsQuercetin protected H9c2 cells against hypertonic stress-induced cell death. We observed a significant increase in intracellular Ca2+ levels ([Ca2+]cyto) when cells originally placed in a hypertonic solution were returned to a normotonic environment. Quercetin was found to prevent this increase in [Ca2+]cyto and also the depolarization of mitochondrial membrane potential. Conclusions and implicationsQuercetin, but not 2,3-dehydrosilybin, reduced adverse effects of osmotic stress mainly by dampening the elevation of [Ca2+]cyto and mitochondrial Ca2+ overload. This may consequently prevent MPTP pore opening and activation of apoptosis.

Development and prenatal exposure to androgens alter potassium currents in gonadotropin-releasing hormone neurons from female mice.

Pulsatile gonadotropin-releasing hormone (GnRH) release is critical for reproduction. Disruptions to GnRH secretion patterns may contribute to polycystic ovary syndrome (PCOS). Prenatally androgenized (PNA) female mice recapitulate many neuroendocrine abnormalities observed in PCOS patients. PNA and development induce changes in spontaneous GnRH neuron firing rate, response to synaptic input, and the afterhyperpolarization potential of the action potential. We hypothesized potassium currents are altered by PNA treatment and/or development. Whole-cell patch-clamp recordings were made of transient and residual potassium currents of GnRH neurons in brain slices from 3-week-old and adult control and PNA females. At 3 weeks of age, PNA treatment increased transient current density versus controls. Development and PNA altered voltage-dependent activation and inactivation of the transient current. In controls, transient current activation and inactivation were depolarized at 3 weeks of age versus in adulthood. In GnRH neurons from 3-week-old mice, transient current activation and inactivation were more depolarized in control than PNA mice. Development and PNA treatment interacted to shift the time-dependence of inactivation and recovery from inactivation. Notably, in cells from adult PNA females, recovery was prolonged compared to all other groups. Activation of the residual current occurred at more depolarized membrane potentials in 3-week-old than adult controls. PNA depolarized activation of the residual current in adults. These findings demonstrate the properties of GnRH neuron potassium currents change during typical development, potentially contributing to puberty, and further suggest PNA treatment may both alter some typical developmental changes and induce additional modifications, which together may underlie aspects of the PNA phenotype. There was not any clinical trial involved in this work.

Lactate ameliorates palmitate-induced impairment of differentiative capacity in C2C12 cells through the activation of voltage-gated calcium channels.

Palmitic acid (PA), a saturated fatty acid enriched in high-fat diet, has been implicated in the development of skeletal muscle regeneration dysfunction. This study aimed to examine the effects and mechanisms of lactate (Lac) treatment on PA-induced impairment of C2C12 cell differentiation capacity. Furthermore, the involvement of voltage-gated calcium channels in this context was examined. In this study, Lac could improve the PA-induced impairment of differentiative capacity in C2C12 cells by affecting Myf5, MyoD and MyoG. In addition, Lac increases the inward flow of Ca2+, and promotes the depolarization of the cell membrane potential, thereby activating voltage-gated calcium channels during C2C12 cell differentiation. The enchancement of Lac on myoblast differentiative capacity was abolished after the addition of efonidipine (voltage-gated calcium channel inhibitors). Therefore, voltage-gated calcium channels play an important role in improving PA-induced skeletal muscle regeneration disorders by exercising blood Lac. Our study showed that Lac could rescue the PA-induced impairment of differentiative capacity in C2C12 cells by affecting Myf5, MyoD and MyoG through the activation of voltage-gated calcium channels.

Molecular mechanism responsible for sex differences in electrical activity of mouse pancreatic β cells.

In humans, type 2 diabetes mellitus shows a higher prevalence in men compared with women, a phenotype that has been attributed to a lower peripheral insulin sensitivity in men. Whether sex-specific differences in pancreatic β cell function also contribute is largely unknown. Here, we characterized the electrophysiological properties of β cells in intact male and female mouse islets. Elevation of glucose concentration above 5 mM triggered an electrical activity with a similar glucose dependence in β cells of both sexes. However, female β cells had a more depolarized membrane potential and increased firing frequency compared with males. The higher membrane depolarization in female β cells was caused by approximately 50% smaller Kv2.1 K+ currents compared with males but otherwise unchanged KATP, large-conductance and small-conductance Ca2+-activated K+ channels, and background TASK1/TALK1 K+ current densities. In female β cells, the higher depolarization caused a membrane potential-dependent inactivation of the voltage-gated Ca2+ channels (CaV), resulting in reduced Ca2+ entry. Nevertheless, this reduced Ca2+ influx was offset by a higher action potential firing frequency. Because exocytosis of insulin granules does not show a sex-specific difference, we conclude that the higher electrical activity promotes insulin release in females, improving glucose tolerance.