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The process of glycolysis breaks down glycogen stored in muscles, producing lactate through pyruvate to generate energy. Excess lactate is then released into the bloodstream. When lactate reaches the liver, it is converted to glucose, which muscles utilize as a substrate to generate ATP. Although the biochemical study of lactate metabolism in hepatocytes and skeletal muscle cells has been extensive, the spatial and temporal dynamics of this metabolism in live cells are still unknown. We observed the dynamics of metabolism-related molecules in primary cultured hepatocytes and a skeletal muscle cell line upon lactate overload. Our observations revealed an increase in cytoplasmic pyruvate concentration in hepatocytes, which led to glucose release. Skeletal muscle cells exhibited elevated levels of lactate and pyruvate levels in both the cytoplasm and mitochondrial matrix. However, mitochondrial ATP levels remained unaffected, indicating that the increased lactate can be converted to pyruvate but is unlikely to be utilized for ATP production. The findings suggest that excess lactate in skeletal muscle cells is taken up into mitochondria with little contribution to ATP production. Meanwhile, lactate released into the bloodstream can be converted to glucose in hepatocytes for subsequent utilization in skeletal muscle cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: The use of multi-electrode arrays (MEA) in combination with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provides a promising method to predict comprehensive cardiotoxicity, including drug-induced QT prolongation and arrhythmia. We previously demonstrated that MEA in combination with hiPSC-CMs could provide a generalizable platform by using 7 reference drugs at 10 testing facilities. Using this approach, we evaluated responses to reference drugs that modulate a range of cardiac ion currents and have a range of arrhythmogenic effects., Methods: We used the MEA system (MED64) and commercially available hiPSC-CMs (iCell cardiomyocytes) to evaluate drug effects on the beat rate, field potential duration (FPD), FPD corrected by Fridericia's formula (FPDc), and the incidence of arrhythmia-like waveforms., Results: This assay detected the repolarization effects of Bay K8644, mibefradil, NS1643, levcromakalim, and ouabain; and the chronotropic effects of isoproterenol, ZD7288, and BaCl 2 . Chronotropy was also affected by K + and Ca 2+ current modulation. This system detected repolarization delays and the arrhythmogenic effects of quinidine, cisapride, thioridazine, astemizole, bepridil, and pimozide more sensitively than the established guinea pig papillary muscle action potential assay. It also predicted clinical QT prolongation by drugs with multiple ion channel effects (fluoxetine, amiodarone, tolterodine, vanoxerine, alfuzosin, and ranolazine)., Discussion: MEA in combination with hiPSC-CMs may provide a powerful method to detect various cardiac electrophysiological effects, QT prolongation, and arrhythmia during drug discovery. However, the data require careful interpretation to predict chronotropic effects and arrhythmogenic effects of candidate drugs with multiple ion channel effects., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Recent increasing evidence suggests that the currently-used platforms in vitro I Kr and APD, and/or in vivo QT assays are not fully predictive for TdP, and do not address potential arrhythmia (VT and/or VF) induced by diverse mechanisms of action. In addition, other cardiac safety liabilities such as functional dysfunction of excitation-contraction coupling (contractility) and structural damage (morphological damage to cardiomyocytes) are also major causes of drug attrition, but current in vitro assays do not cover all these liabilities. We organized the Consortium for Safety Assessment using Human iPS cells (CSAHi; http://csahi.org/en/), based on the Japan Pharmaceutical Manufacturers Association (JPMA), to verify the application of human iPS/ES cell-derived cardiomyocytes in drug safety evaluation. The main goal of the CSAHi HEART team has been to propose comprehensive screening strategies to predict a diverse range of cardiotoxicities by using recently introduced platforms (multi-electrode array (MEA), patch clamp, cellular impedance, motion field imaging [MFI], and Ca transient systems) while identifying the strengths and weaknesses of each. Our study shows that hiPS-CMs used in these platforms have pharmacological responses more relevant to humans in comparison with existent hERG, APD or Langendorff (MAPD/contraction) assays, and not only MEA but also other methods such as impedance, MFI, and Ca transient systems would offer paradigm changes of platforms for predicting drug-induced QT risk and/or arrhythmia or contractile dysfunctions. Furthermore, we propose a potential multi-parametric platform in which field potential (MEA)-Ca transient-contraction (MFI) could be evaluated simultaneously as an ideal novel platform for predicting a diversity of cardiac toxicities, namely whole effects on the excitation-contraction cascade., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: Drug-induced QT prolongation is a major safety issue during drug development because it may lead to lethal ventricular arrhythmias. In this study, we evaluated the utility of multi-electrode arrays (MEA) with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to predict drug-induced QT prolongation and arrhythmia., Methods: Ten facilities evaluated the effects of 7 reference drugs (E-4031, moxifloxacin, flecainide, terfenadine, chromanol 293B, verapamil, and aspirin) using a MED64 MEA system with commercially available hiPS-CMs. Field potential duration (FPD), beat rate, FPD corrected by Fridericia's formula (FPDc), concentration inducing FPDc prolongation by 10% (FPDc10), and incidence of arrhythmia-like waveform were evaluated., Results: The inter-facility variability of absolute values before drug application was similar to the intra-facility variability for FPD, beat rate, and FPDc. The inter-facility variability of FPDc10 for 5 reference drugs ranged from 1.8- to 5.8-fold. At all 10 facilities, E-4031, moxifloxacin, and flecainide prolonged FPDc and induced arrhythmia-like waveforms at concentrations 1.8- to 6.1-fold higher than their FPDc10. Terfenadine prolonged FPDc and induced beating arrest at 8.0 times the FPDc10. The average FPDc10 values for E-4031, moxifloxacin, and terfenadine were comparable to reported plasma concentrations that caused QT prolongation or Torsade de Pointes in humans. Chromanol 293B, a IKs blocker, also prolonged FPDc but did not induce arrhythmia-like waveforms, even at 7.4 times the FPDc10. In contrast, verapamil shortened FPDc and aspirin did not affect FPDc or FP waveforms., Discussion: MEA with hiPS-CMs can be a generalizable method for accurately predicting both QT prolongation and arrhythmogenic liability in humans., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Adrenaline reacts with three types of adrenergic receptors, α1, α2 and β-adrenergic receptors (ARs), inducing many physiological events including exocytosis. Although adrenaline has been shown to induce glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells, the precise molecular mechanism by which adrenaline regulates GLP-1 secretion remains unknown. Here we show by live cell imaging that all types of adrenergic receptors are stimulated by adrenaline in enteroendocrine L cell line GLUTag cells and are involved in GLP-1 exocytosis. We performed RT-PCR analysis and found that α1B-, α2A-, α2B-, and β1-ARs were expressed in GLUTag cells. Application of adrenaline induced a significant increase of intracellular Ca(2+) and cAMP concentration ([Ca(2+)]i and [cAMP]i, respectively), and GLP-1 exocytosis in GLUTag cells. Blockade of α1-AR inhibited adrenaline-induced [Ca(2+)]i increase and exocytosis but not [cAMP]i increase, while blockade of β1-AR inhibited adrenaline-induced [cAMP]i increase and exocytosis but not [Ca(2+)]i increase. Furthermore, overexpression of α2A-AR suppressed the adrenaline-induced [cAMP]i increase and exocytosis. These results suggest that the fine-turning of GLP-1 secretion from enteroendocrine L cells is established by the balance between α1-, α2-, and β-ARs activation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Recent studies have suggested that astrocytes release gliotransmitters (i.e., ATP, L-glutamate, D-serine, and peptide hormones) and participate actively in synaptic functioning. Although ATP release from astrocytes modulates the activity of neurons, the mechanisms regulating the ATP release from astrocytes and the source of ATP in astrocytes are not well understood. Recently a vesicular nucleotide transporter (VNUT)/solute carrier family 17, member 9 (SLC17A9) has been identified as a mediator of the active accumulation of ATP into vesicles. Here we show by immunocytochemical analysis under confocal microscope and live cell imaging under total internal reflection fluorescence microscope that lysosome-associated VNUT is responsible for ATP release in astrocytes. VNUT was expressed in both primary cultured cortical astrocytes and glioma cell line C6 cells, and mainly localized on lysosome in the cells. We found that VNUT-associated secretory lysosomes do not fully collapse into the plasma membrane after lysosomal exocytosis. We also found that inhibition of VNUT function by Evans Blue decreased ATP uptake into secretory lysosomes. Depletion and inhibition of endogenous VNUT by small interference RNA and Evans Blue, respectively decreased the amount of ATP release from the cells, whereas overexpression of VNUT increased it. Taken together, these findings indicate that the participation of VNUT in ATP storage in secretory lysosomes during lysosomal exocytosis of ATP from astrocytes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

lysozyme C (lyz), a glycoside hydrolase expressed exclusively in myeloid cells, is involved in the host defense against bacterial infection. We isolated a 2.4kb zebrafish lyz promoter region and established transgenic lines that drive enhanced green fluorescent protein (EGFP) to examine how lyz expression is regulated during myelopoiesis. We found that the 2.4kb lyz promoter is sufficient to drive myeloid-specific expression of EGFP in zebrafish. We identified potential transcriptional regulatory elements including a Runx element (TGTGGT at -1.70kb) and a C/ebp element (TTTGGCAAT at -1.46kb) in the lyz promoter, and showed that they are required for myeloid-specific expression of EGFP. We found that the myeloid-lineage transcription factors C/ebp1, Runx1 and Pu.1 can bind to the 2.4kb lyz promoter. Forced expression of runx1, c/ebp1 and pu.1 together induced ectopic lyz expression in the intermediated cell mass (ICM). Thus, we propose that c/ebp1 and runx1 presumably cooperated with pu.1 in the transcriptional regulation of lyz during zebrafish myelopoiesis.

Platelet glycoprotein (GP) Ib/IX/V complex is a receptor for von Willebrand factor (vWf), which plays a crucial role in primary hemostasis by mediating platelet adhesion to injured blood vessels. We have expressed in CHO cells a fragment of GPIba that retained a vWf-binding function. The recombinant fragment (rGPIba) was incorporated into liposomes and evaluated their functions in vitro. rGPIba on the liposome surface was detectable by flow cytometric analysis. Addition of vWf and ristocetin caused specific agglutination of rGPIbalpha-liposomes, as evaluated by an aggregometer or a fluorescent microscopy. When ristocetin was added to platelet-rich plasma (PRP) pre-mixed with rhodamine-labeled rGPIbalpha-liposomes, platelets aggregated and rhodamine-fluorescence was strongly positive in the platelet thrombi, suggesting that heterologous aggregation (attachment of liposomes to platelets) occurred. Platelet aggregation in PRP at low platelet concentration (20-80 x 10(6)/ml) was enhanced by rGPIbalpha-liposomes in a dose-dependent manner. Thus, rGPIbalpha-liposomes may accumulate on vWf-exposed subendothelial tissues and enhance platelet function in vivo, supporting hemostasis in thrombocytopenic individuals., (Copyright 1999 Academic Press.)

Platelet-type von Willebrand disease (vWD) is a congenital bleeding disorder characterized by heightened ristocetin-induced platelet aggregation caused by abnormally high affinity between the platelet membrane glycoprotein (GP) Ib/IX complex and von Willebrand factor (vWF). Two distinct point mutations, Gly233 to Val and Met239 to Val, have been reported in GPIb alpha. We have constructed a recombinant GPIb alpha fragment containing the latter mutation, Met239 to Val (M239V) and characterized the mutant molecule using two methods, ie, interaction between soluble vWF and immobilized M239V and inhibition of platelet aggregation by purified soluble M239V. Spontaneous binding (ie, binding without any inducers) was observed between 125I-vWF and immobilized M239V but not between 125I-vWF and immobilized wild-type (WT) GPIb alpha. The addition of low concentrations of ristocetin (0.2 mg/mL) induced specific 125I-vWF binding to immobilized M239V, but not to WT GPIb alpha. At high concentrations of ristocetin (1.2 mg/mL), both WT GPIb alpha and M239V specifically bound to 125I-vWF. Thus, M239V reproduced the unique functional abnormality of the GPIb/IX complex in platelet-type vWD. Moreover, the purified soluble M239V inhibited platelet aggregation induced by low concentration of ristocetin (0.3 mg/mL) in platelet-rich plasma from a patient having Met239 to Val mutation, whereas purified WT did not. These results provide direct evidences that the reported point mutation is the responsible molecular basis of this disorder.

High shear stress facilitates von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib/IX, causing activation of GPIIb/IIIa to induce platelet aggregation. Here we report that activated GPIIb/IIIa, even occupied by ligands, is not sufficient to mediate platelet aggregation under high shear stress conditions when vWF binding to GPIb/IX is blocked. Platelet rich plasma or washed platelet suspension supplemented with purified human fibrinogen at a concentration of 2 mg/mL were treated with an anti-vWF monoclonal antibody NMC-4 which blocks the binding of vWF to GPIb/IX. After addition of 10 mumol/L ADP, aggregation was continuously monitored under various shear stress conditions (0-108 dyne/cm2) using a cone-plate type aggregometer previously described (Ikeda Y et al J Clin Invest 1991; 87:1234). The extent of maximal aggregation of agonist-stimulated platelets in the presence of NMC-4 correlated inversely with the level of shear stress applied, with the virtual absence of aggregation at 108 dyne/cm2. Once aggregated by 10 mumol/L ADP under low shear stress (12 dyne/cm2), platelets could be disaggregated, in part, by the application of high shear stress (108 dyne/cm2), and reaggregated when shear stress was returned to 12 dyne/cm2. Flow cytometric analysis revealed that platelets stimulated with 10 mumol/L ADP at 108 dyne/cm2 bound fluorescein isothiocyanate (FITC)-labeled fibrinogen, although aggregation was absent in this experimental condition. These results demonstrate the dual effect of shear stress on platelet functions; a pro-aggregating activity that induces vWF-GPIb/IX interaction leading to platelet activation, and an anti-aggregating force to prevent the growth of platelet thrombi. It is suggested that the efficacy of vWF blockade is greater under high shear than low shear stress conditions, and that a selective inhibition of platelet functions can be possible.

The platelet glycoprotein (GP) Ib/IX/V is a hetero-oligomeric receptor complex for von Willebrand factor (vWF) and mediates platelet adhesion and aggregation under high shear stress conditions. It is composed of alpha and beta chain of GP Ib, GP IX, AND and GP V. To establish transgenic mice carrying human GP Ib alpha, we injected into mouse zygotes a 6 kb DNA fragment containing human GP Ib alpha gene that included entire coding sequence and putative promoter region. One hundred and thirteen offsprings were screened, and only one was found to express human GP Ib alpha protein and has passed the human GP Ib alpha gene as well as the expression of the gene to next generation. The expression of human GP Ib alpha in transgenic mice was limited to platelets and megakaryocytes. Glycocalicin, a proteolytic fragment of human GP Ib alpha found in normal human plasma, was not detected in transgenic mouse plasma. Human vWF in the presence of ristocetin supported agglutination of transgenic mouse platelets, but not of control mouse platelets.

We carried out magnetic resonance imaging (MRI) studies on human brains which had been fixed in formalin solution for over 2 years and had been proven neuropathologically to be cases of multiple sclerosis (MS), progressive multifocal leukoencephalopathy (PML), and Balo's concentric sclerosis (Balo). Using spin echo (SE) and inversion recovery (IR) pulse sequences to detect demyelinated lesions in a living person with MS, the demyelinated lesions of the fixed brains in cases of MS, PML and Balo definitely re-appeared, although T1 and T2 in the gray and white matter were reduced following fixation. High signal areas on the SE images corresponded not only to the characteristic distribution of demyelinated lesions in the white matter but also to sparse myelin, gliosis and mild perivascular cuffing in the white matter around the demyelinated foci in cases of the fixed MS, PML and Balo brains. On the IR images, only MS plaques were evident. This MRI study of fixed brains proved useful to elucidate clinicopathological correlations.