Publications by authors named "Makoto Kanzaki"

72 Publications

Skeletal muscle-specific Keap1 disruption modulates fatty acid utilization and enhances exercise capacity in female mice.

Redox Biol 2021 Apr 5;43:101966. Epub 2021 Apr 5.

Department of Gene Expression Regulation, IDAC, Tohoku University, Sendai, 980-8575, Japan. Electronic address:

Skeletal muscle health is important for the prevention of various age-related diseases. The loss of skeletal muscle mass, which is known as sarcopenia, underlies physical disability, poor quality of life and chronic diseases in elderly people. The transcription factor NRF2 plays important roles in the regulation of the cellular defense against oxidative stress, as well as the metabolism and mitochondrial activity. To determine the contribution of skeletal muscle NRF2 to exercise capacity, we conducted skeletal muscle-specific inhibition of KEAP1, which is a negative regulator of NRF2, and examined the cell-autonomous and non-cell-autonomous effects of NRF2 pathway activation in skeletal muscles. We found that NRF2 activation in skeletal muscles increased slow oxidative muscle fiber type and improved exercise endurance capacity in female mice. We also observed that female mice with NRF2 pathway activation in their skeletal muscles exhibited enhanced exercise-induced mobilization and β-oxidation of fatty acids. These results indicate that NRF2 activation in skeletal muscles promotes communication with adipose tissues via humoral and/or neuronal signaling and facilitates the utilization of fatty acids as an energy source, resulting in increased mitochondrial activity and efficient energy production during exercise, which leads to improved exercise endurance.
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http://dx.doi.org/10.1016/j.redox.2021.101966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050939PMC
April 2021

Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5.

PLoS One 2020 2;15(12):e0231064. Epub 2020 Dec 2.

Division of Nephrology, Endocrinology and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.

Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and several reports have suggested that mitochondrial abnormalities are involved in its etiology. We recruited 9 sIBM patients and found significant histological changes and an elevation of growth differential factor 15 (GDF15), a marker of mitochondrial disease, strongly suggesting the involvement of mitochondrial dysfunction. Bioenergetic analysis of sIBM patient myoblasts revealed impaired mitochondrial function. Decreased ATP production, reduced mitochondrial size and reduced mitochondrial dynamics were also observed in sIBM myoblasts. Cell vulnerability to oxidative stress also suggested the existence of mitochondrial dysfunction. Mitochonic acid-5 (MA-5) increased the cellular ATP level, reduced mitochondrial ROS, and provided protection against sIBM myoblast death. MA-5 also improved the survival of sIBM skin fibroblasts as well as mitochondrial morphology and dynamics in these cells. The reduction in the gene expression levels of Opa1 and Drp1 was also reversed by MA-5, suggesting the modification of the fusion/fission process. These data suggest that MA-5 may provide an alternative therapeutic strategy for treating not only mitochondrial diseases but also sIBM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231064PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710105PMC
January 2021

LRRK2 Inhibition Ameliorates Dexamethasone-Induced Glucose Intolerance via Prevents Impairment in GLUT4 Membrane Translocation in Adipocytes.

Biol Pharm Bull 2020 ;43(11):1660-1668

Department of Regulation Biochemistry, Graduate School of Medical Sciences, Kitasato University.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson's disease. LRRK2 is a large protein with multiple functional domains, including a guanosine 5'-triphosphate (GTP)-binding domain and a protein kinase domain. Recent studies indicated that the members of the Rab GTPase family, Rab8a and Rab10, which are involved in the membrane transport of the glucose transporter type 4 (GLUT4) during insulin-dependent glucose uptake, are phosphorylated by LRRK2. However, the physiological role of LRRK2 in the regulation of glucose metabolism is largely unknown. In the present study, we investigated the role of LRRK2 using dexamethasone (DEX)-induced glucose intolerance in mice. LRRK2 knockout (KO) mice exhibited suppressed glucose intolerance, even after treatment with DEX. The phosphorylation of LRRK2, Rab8a and Rab10 was increased in the adipose tissues of DEX-treated wild-type mice. In addition, inhibition of the LRRK2 kinase activity prevented the DEX-induced inhibition of GLUT4 membrane translocation and glucose uptake in cultured 3T3-L1 adipocytes. These results suggest that LRRK2 plays an important role in glucose metabolism in adipose tissues.
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http://dx.doi.org/10.1248/bpb.b20-00377DOI Listing
January 2020

Imaging of muscle activity-induced morphometric changes in fibril network of myofascia by two-photon microscopy.

J Anat 2021 Mar 19;238(3):515-526. Epub 2020 Oct 19.

Department of Nursing, Tohoku Fukushi University, Sendai, Japan.

Myofascia, deep fascia enveloping skeletal muscles, consists of abundant collagen and elastin fibres that play a key role in the transmission of muscular forces. However, understanding of biomechanical dynamics in myofascia remains very limited due to less quantitative and relevant approaches for in vivo examination. The purpose of this study was to evaluate the myofascial fibril structure by means of a quantitative approach using two-photon microscopy (TPM) imaging in combination with intravital staining of Evans blue dye (EBD), a far-red fluorescence dye, which potentially labels elastin. With focus on myofascia of the tibial anterior (TA) muscle, the fibril structure intravitally stained with EBD was observed at the depth level of collagen fibrous membrane above the muscle belly. The EBD-labelled fibril structure and orientation in myofascia indicated biomechanical responses to muscle activity and ageing. The orientation histograms of EBD-labelled fibrils were significantly modified depending upon the intensity of muscle activity and ageing. Moreover, the density of EBD-labelled fibrils in myofascia decreased with habitual exercise but increased with muscle immobilization or ageing. In particular, the diameter of EBD-labelled fibrils in aged mice was significantly higher. The orientation histograms of EBD-labelled fibrils after habitual exercise, muscle immobilization and ageing showed significant differences compared to control. Indeed, the histograms in bilateral TA myofascia of exercise mice made simple waveforms without multiple sharp peaks, whilst muscular immobilization or ageing significantly shifted a histogram with sustaining multiple sharp peaks. Therefore, the dynamics of fibre network with EBD fluorescence in response to the biomechanical environment possibly indicate functional tissue adaptation in myofascia. Furthermore, on the basis of the knowledge that neutrophil recruitment occurs locally in working muscles, we suggested the unique reconstruction mechanism involving neutrophilic elastase in the myofascial fibril structure. In addition to the elastolytic susceptibility of EBD-labelled fibrils, distinct immunoreactivities and activities of neutrophil elastase in the myofascia were observed after electric pulse stimulation-induced muscle contraction for 15 min. Our findings of EBD-labelled fibril dynamics in myofascia through quantitative approach using TPM imaging and intravital fluorescence labelling potentially brings new insights to examine muscle physiology and pathology.
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http://dx.doi.org/10.1111/joa.13339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855069PMC
March 2021

TRPA1 and TRPV1 channels participate in atmospheric-pressure plasma-induced [Ca] response.

Sci Rep 2020 06 16;10(1):9687. Epub 2020 Jun 16.

Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.

Despite successful clinical application of non-equilibrium atmospheric pressure plasma (APP), the details of the molecular mechanisms underlying APP-inducible biological responses remain ill-defined. We previously reported that exposure of 3T3L1 cells to APP-irradiated buffer raised the cytoplasmic free Ca ([Ca]) concentration by eliciting Ca influx in a manner sensitive to transient receptor potential (TRP) channel inhibitors. However, the precise identity of the APP-responsive channel molecule(s) remains unclear. In the present study, we aimed to clarify channel molecule(s) responsible for indirect APP-responsive [Ca] rises. siRNA-mediated silencing experiments revealed that TRPA1 and TRPV1 serve as the major APP-responsive Ca channels in 3T3L1 cells. Conversely, ectopic expression of either TRPA1 or TRPV1 in APP-unresponsive C2C12 cells actually triggered [Ca] elevation in response to indirect APP exposure. Desensitization experiments using 3T3L1 cells revealed APP responsiveness to be markedly suppressed after pretreatment with allyl isothiocyanate or capsaicin, TRPA1 and TRPV1 agonists, respectively. APP exposure also desensitized the cells to these chemical agonists, indicating the existence of a bi-directional heterologous desensitization property of APP-responsive [Ca] transients mediated through these TRP channels. Mutational analyses of key cysteine residues in TRPA1 (Cys421, Cys621, Cys641, and Cys665) and in TRPV1 (Cys258, Cys363, and Cys742) have suggested that multiple reactive oxygen and nitrogen species are intricately involved in activation of the channels via a broad range of modifications involving these cysteine residues. Taken together, these observations allow us to conclude that both TRPA1 and TRPV1 channels play a pivotal role in evoking indirect APP-dependent [Ca] responses.
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http://dx.doi.org/10.1038/s41598-020-66510-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297720PMC
June 2020

Biomechanics of C2C12 Cells Observed with Cellular Resolution Scanning Acoustic Microscope Combined with Optical Microscope

Annu Int Conf IEEE Eng Med Biol Soc 2019 Jul;2019:4828-4831

Biomechanics of the cell indicates the inner structure and viability of the cell. Mechanical properties are represented by acoustic properties such as speed of sound (SOS) or acoustic impedance. In the present study, cellular resolution scanning acoustic microscope combined with optical microscope (OptSAM) is developed to observe the change of mechanical properties in cell differentiation. Main part of the OptSAM was consisted of 350 MHz ultrasound transducer mechanically scanned by a piezo-actuator. Thickness, SOS, acoustic impedance, density and elastic bulk modulus of the cell were deduced by the ultrasound responses in both time domain and frequency domain. C2C12 cell changing its form from myoblast to myotube was observed by OptSAM. The value of bulk modulus slightly increased in response to differentiation process. OptSAM non-invasively provides important information on biomechanics of cells without contact or staining.
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http://dx.doi.org/10.1109/EMBC.2019.8857008DOI Listing
July 2019

Exercise-evoked intramuscular neutrophil-endothelial interactions support muscle performance and GLUT4 translocation: a mouse gnawing model study.

J Physiol 2020 01 13;598(1):101-122. Epub 2019 Dec 13.

Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.

Key Points: Fractalkine receptor antagonist inhibited neutrophil recruitment to masseter muscles and exacerbated fatigability during masticatory activity. Fractalkine-mediated neutrophil recruitment is required for both upregulation of myokines (CXCL1, interleukin-6) and enhanced GLUT4 translocation in response to masticatory activity. Fractalkine and intercellular adhesion molecule-1 expression in endothelial cells increased in response to masticatory activity. In vitro experiments demonstrated that contracting myotubes lack the ability to upregulate fractalkine but revealed that endothelial fractalkine upregulation is induced using a conditioned medium of contracting myotubes.

Abstract: Physical exercise stimulates neutrophil recruitment within working skeletal muscle, although its underlying mechanisms remain ill-defined. By employing a masticatory behaviour (gnawing) model, we demonstrate the importance of intramuscular paracrine and autocrine systems that are triggered by muscle contractile activity and reliant upon fractalkine/CX3CL1-mediated signals. These signals were revealed to be required for achieving proper GLUT4 translocation and glucose uptake to meet the glucose demands for fatigue alleviation. Specifically, fractalkine expression and neutrophil recruitment both increased in the masseter muscle tissues upon masticatory activity. Importantly, a fractalkine antagonist inhibited neutrophil accumulation and exacerbated fatigability during masticatory activity. We found that fractalkine-dependent neutrophil recruitment is required for both upregulation of myokines (i.e. CXCL1 and interleukin-6) and enhanced GLUT4 translocation in response to gnawing activity. Immunofluorescence analysis of masseter muscles demonstrated that fractalkine and intercellular adhesion molecule-1 expression are both upregulated in endothelial cells but not in myofibres. The in vitro exercise model further revealed that contractile activity failed to stimulate fractalkine upregulation in myotubes, implying that fractalkine is not a myokine (myofibre-derived factor). Nevertheless, endothelial fractalkine expression was markedly stimulated by a conditioned medium from the contracting myotubes. Moreover, intercellular adhesion molecule-1, a key adhesion molecule for neutrophils, was upregulated in endothelial cells by fractalkine. Taken together, our findings strongly suggest that endothelial fractalkine serves as a key factor for organizing a physiologically beneficial intramuscular microenvironment by recruiting neutrophils in response to relatively mild exercise (i.e. masticatory muscle activity).
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http://dx.doi.org/10.1113/JP278564DOI Listing
January 2020

In vitro exercise model using contractile human and mouse hybrid myotubes.

Sci Rep 2019 08 15;9(1):11914. Epub 2019 Aug 15.

Graduate School of Biomedical Engineering, Tohoku University, 980-8579, 6-6-04 Aoba, Aramaki, Aoba-ku, Sendai, Japan.

Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses (i.e. myokine secretion). We herein describe an "in vitro exercise model", using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the "in vitro exercise model".
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http://dx.doi.org/10.1038/s41598-019-48316-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695424PMC
August 2019

Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis.

FASEB J 2019 09 18;33(9):10240-10256. Epub 2019 Jun 18.

Division of Neurology, Department of Neuroscience and Sensory Organs, Tohoku University Graduate School of Medicine, Sendai, Japan.

The neuropathological hallmarks of Parkinson's disease (PD) include the appearance of α-synuclein (α-SYN)-positive Lewy bodies (LBs) and the loss of catecholaminergic neurons. Thus, a potential mechanism promoting the uptake of extracellular α-SYN may exist in susceptible neurons. Of the various differentially expressed proteins, we are interested in flotillin (FLOT)-1 because this protein is highly expressed in the brainstem catecholaminergic neurons and is strikingly up-regulated in PD brains. In this study, we found that extracellular monomeric and fibrillar α-SYN can potentiate FLOT1-dopamine transporter (DAT) binding and pre-endocytic clustering of DAT on the cell surface, thereby facilitating DAT endocytosis and down-regulating its transporter activity. Moreover, we demonstrated that α-SYN itself exploited the DAT endocytic process to enter dopaminergic neuron-like cells, and both FLOT1 and DAT were found to be the components of LBs. Altogether, these findings revealed a novel role of extracellular α-SYN on cellular trafficking of DAT and may provide a rationale for the cell type-specific, functional, and pathologic alterations in PD.-Kobayashi, J., Hasegawa, T., Sugeno, N., Yoshida, S., Akiyama, T., Fujimori, K., Hatakeyama, H., Miki, Y., Tomiyama, A., Kawata, Y., Fukuda, M., Kawahata, I., Yamakuni, T., Ezura, M., Kikuchi, A., Baba, T., Takeda, A., Kanzaki, M., Wakabayashi, K., Okano, H., Aoki, M. Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis.
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http://dx.doi.org/10.1096/fj.201802051RDOI Listing
September 2019

Sparc, an EPS-induced gene, modulates the extracellular matrix and mitochondrial function via ILK/AMPK pathways in C2C12 cells.

Life Sci 2019 Jul 28;229:277-287. Epub 2019 May 28.

CREMI, CHU de Québec Research Center, Quebec, Quebec G1V 4G2, Canada; Department of Molecular Medicine, Faculty of Medicine, Laval University, Quebec, Quebec G1V 4G2, Canada. Electronic address:

Aims: Secreted protein acidic and rich in cysteine, (SPARC), is a matricellular protein implicated in the modulation of the extracellular matrix (ECM) and mitochondrial proteins expression.

Main Methods: To study the mechanism through which SPARC is involved in the possible link between ECM and mitochondria, C2C12 myoblasts were cultured with/without the exogenous addition/inhibition of SPARC as well as activation/inhibition of adenosine monophosphate-activated protein kinase (AMPK). Electrical pulse stimulation (EPS), was applied for 2 days in myotubes.

Key Findings: The expressions of ECM-related (integrin-linked kinase (ILK), glycogen synthase kinase-3 beta (GSK-3ß), phosphorylated-GSK-3ß (p-GSK-3ß) and collagen 1a1), mitochondrial-related (AMPK, phosphorylated-AMPK (p-AMPK), succinate dehydrogenase (SDHB) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α)) and SPARC proteins and/or genes were measured after modulation of SPARC and/or AMPK as well as with or without EPS. The addition of SPARC in C2C12 myoblast increased the expression of ILK, p-GSK-3ß and p-AMPK whereas anti-SPARC antibody decreased them at different incubation times (0, 10, and 30 min, and 6 h). The AMPK activation increased SPARC, collagen 1a1, p-AMPK and SDHB proteins level, however, AMPK inhibition blunted the effects. EPS induced Sparc and Pgc1a genes expression.

Significance: Sparc, an EPS-induced gene, may be involved in the link between ECM remodeling and mitochondrial function in muscle via its interaction with ILK/AMPK.
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http://dx.doi.org/10.1016/j.lfs.2019.05.070DOI Listing
July 2019

Cooperative actions of Tbc1d1 and AS160/Tbc1d4 in GLUT4-trafficking activities.

J Biol Chem 2019 01 27;294(4):1161-1172. Epub 2018 Nov 27.

Graduate School of Biomedical Engineering, Sendai 980-8579, Japan; Department of Information and Intelligent Systems, Tohoku University, Sendai 980-8579, Japan. Electronic address:

AS160 and Tbc1d1 are key Rab GTPase-activating proteins (RabGAPs) that mediate release of static GLUT4 in response to insulin or exercise-mimetic stimuli, respectively, but their cooperative regulation and its underlying mechanisms remain unclear. By employing GLUT4 nanometry with cell-based reconstitution models, we herein analyzed the functional cooperative activities of the RabGAPs. When both RabGAPs are present, Tbc1d1 functionally dominates AS160, and stimuli-inducible GLUT4 release relies on Tbc1d1-evoking proximal stimuli, such as AICAR and intracellular Ca Detailed functional assessments with varying expression ratios revealed that AS160 modulates sensitivity to external stimuli in Tbc1d1-mediated GLUT4 release. For example, Tbc1d1-governed GLUT4 release triggered by Ca plus insulin occurred more efficiently than that in cells with little or no AS160. Series of mutational analyses revealed that these synergizing actions rely on the phosphotyrosine-binding 1 (PTB1) and calmodulin-binding domains of Tbc1d1 as well as key phosphorylation sites of both AS160 (Thr) and Tbc1d1 (Ser and Thr). Thus, the emerging cooperative governance relying on the multiple regulatory nodes of both Tbc1d1 and AS160, functioning together, plays a key role in properly deciphering biochemical signals into a physical GLUT4 release process in response to insulin, exercise, and the two in combination.
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http://dx.doi.org/10.1074/jbc.RA118.004614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349102PMC
January 2019

Effects of Acute Exercise Combined With Calorie Restriction Initiated Late-in-Life on Insulin Signaling, Lipids, and Glucose Uptake in Skeletal Muscle From Old Rats.

J Gerontol A Biol Sci Med Sci 2020 01;75(2):207-217

School of Kinesiology, University of Michigan, Ann Arbor.

We evaluated effects of calorie restriction (CR: consuming 60-65% of ad libitum [AL] intake) initiated late-in-life with or without acute exercise on insulin-stimulated glucose uptake (ISGU) of skeletal muscle by studying four groups of 26-month-old rats: sedentary-AL, sedentary-CR (8-week duration), 3 hours post-exercise (3hPEX)-AL and 3hPEX-CR. ISGU was determined in isolated epitrochlearis muscles incubated ± insulin. Muscles were assessed for signaling proteins (immunoblotting) and lipids (mass spectrometry). ISGU from sedentary-CR and 3hPEX-AL exceeded sedentary-AL; 3hPEX-CR exceeded all other groups. Akt (Ser473, Thr308) and Akt substrate of 160 kDa (AS160; Ser588, Thr642, Ser704) phosphorylation levels tracked with ISGU. Among the 477 lipids detected, 114 were altered by CR (including reductions in 15 of 25 acylcarnitines), and 27 were altered by exercise (including reductions in 18 of 22 lysophosphatidylcholines) with only six lipids overlapping between CR and exercise. ISGU significantly correlated with 23 lipids, including: acylcarnitine 20:1 (r = .683), lysophosphatidylethanolamine19:0 (r = -.662), acylcarnitine 24:0 (r = .611), and plasmenyl-phosphatidylethanolamine 37:5 (r = -.603). Muscle levels of ceramides (a lipid class previously linked to insulin resistance) were not altered by CR and/or exercise nor significantly correlated with ISGU, implicating other mechanisms (which potentially involve other lipids identified in this study) for greater ISGU and Akt and AS160 phosphorylation with these interventions.
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http://dx.doi.org/10.1093/gerona/gly222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176061PMC
January 2020

Three-Dimensional Tracking of Quantum Dot-Conjugated Molecules in Living Cells.

Methods Mol Biol 2018 ;1814:425-448

LENS-European Laboratory for Non-linear Spectroscopy, Sesto Fiorentino, Italy.

Here, we describe protocols for three-dimensional tracking of single quantum dot-conjugated molecules with nanometer accuracy in living cells using conventional fluorescence microscopy. The technique exploits out-of-focus images of single emitters combined with an automated pattern-recognition open-source software that fits the images with proper model functions to extract the emitter coordinates. We describe protocols for targeting quantum dots to both membrane components and cytosolic proteins.
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http://dx.doi.org/10.1007/978-1-4939-8591-3_26DOI Listing
March 2019

Neutrophils Provide a Favorable IL-1-Mediated Immunometabolic Niche that Primes GLUT4 Translocation and Performance in Skeletal Muscles.

Cell Rep 2018 05;23(8):2354-2364

Graduate School of Biomedical Engineering, Tohoku University, Sendai, Miyagi, Japan. Electronic address:

Metabolic immunomodulation involving IL-1 has been investigated for unfavorable metabolic effects, including obesity, but a potentially favorable role for IL-1 remains unclear. Here, we find mechanistic interactions between working skeletal muscles and locally recruited neutrophils expressing IL-1β, which supports muscle performance through priming exercise-dependent GLUT4 translocation. Thus, during exercise, both IL-1α/β-deficient and neutrophil-depleted mice similarly exhibit increased fatigability associated with impaired muscle glucose homeostasis due to GLUT4 dysregulation. Deficiency of IL-1-producing neutrophils results in intrinsic abnormalities represented by aberrant Rac1 signaling and irregular GLUT4-storage vesicles, suggesting that these properties are maintained by local IL-1 produced by recruited neutrophils upon exercise, possibly on a daily basis. We propose that neutrophils are highly engaged in skeletal muscle performance via IL-1 regulation, which coordinates favorable inflammatory microenvironments supporting muscle glucose metabolism.
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http://dx.doi.org/10.1016/j.celrep.2018.04.067DOI Listing
May 2018

Prior treatment with the AMPK activator AICAR induces subsequently enhanced glucose uptake in isolated skeletal muscles from 24-month-old rats.

Appl Physiol Nutr Metab 2018 Aug 8;43(8):795-805. Epub 2018 Mar 8.

a Muscle Biology Laboratory, School of Kinesiology, University of Michigan, Ann Arbor, MI 48109, USA.

5' AMP-activated protein kinase (AMPK) activation may be part of the exercise-induced process that enhances insulin sensitivity. Independent of exercise, acute prior treatment of skeletal muscles isolated from young rats with a pharmacological AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), causes subsequently improved insulin-stimulated glucose uptake (GU). However, efficacy of a single prior AICAR exposure on insulin-stimulated GU in muscles from old animals has not been studied. The purpose of this study was to determine whether brief, prior exposure to AICAR (3.5 h before GU assessment) leads to subsequently increased GU in insulin-stimulated skeletal muscles from old rats. Epitrochlearis muscles from 24-month-old male rats were isolated and initially incubated ±AICAR (60 min), followed by incubation without AICAR (3 h), then incubation ±insulin (50 min). Muscles were assessed for GU (via 3-O-methyl-[H]-glucose accumulation) and site-specific phosphorylation of key proteins involved in enhanced GU, including AMPK, Akt, and Akt substrate of 160 kDa (AS160), via Western blotting. Prior ex vivo AICAR treatment resulted in greater GU by insulin-stimulated muscles from 24-month-old rats. Prior AICAR treatment also resulted in greater phosphorylation of AMPK (T172) and AS160 (S588, T642, and S704). Glucose transporter type 4 (GLUT4) protein abundance was unaffected by prior AICAR and/or insulin treatment. These findings demonstrate that skeletal muscles from older rats are susceptible to enhanced insulin-stimulated GU after brief activation of AMPK by prior AICAR. Consistent with earlier research using muscles from young rodents, increased phosphorylation of AS160 is implicated in this effect, which was not attributable to altered GLUT4 glucose transporter protein abundance.
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http://dx.doi.org/10.1139/apnm-2017-0858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066428PMC
August 2018

Roles of IL-1α/β in regeneration of cardiotoxin-injured muscle and satellite cell function.

Am J Physiol Regul Integr Comp Physiol 2018 07 7;315(1):R90-R103. Epub 2018 Mar 7.

Tohoku University Graduate School of Biomedical Engineering , Sendai , Japan.

Skeletal muscle regeneration after injury is a complex process involving interactions between inflammatory microenvironments and satellite cells. Interleukin (IL)-1 is a key mediator of inflammatory responses and exerts pleiotropic impacts on various cell types. Thus, we aimed to investigate the role of IL-1 during skeletal muscle regeneration. We herein show that IL-1α/β-double knockout (IL-1KO) mice exhibit delayed muscle regeneration after cardiotoxin (CTX) injection, characterized by delayed infiltrations of immune cells accompanied by suppressed local production of proinflammatory factors including IL-6 and delayed increase of paired box 7 (PAX7)-positive satellite cells postinjury compared with those of wild-type (WT) mice. A series of in vitro experiments using satellite cells obtained from the IL-1KO mice unexpectedly revealed that IL-1KO myoblasts have impairments in terms of both proliferation and differentiation, both of which were reversed by exogenous IL-1β administration in culture. Intriguingly, the delay in myogenesis was not attributable to the myogenic transcriptional program since MyoD and myogenin were highly upregulated in IL-1KO cells, instead appearing, at least in part, to be due to dysregulation of cellular fusion events, possibly resulting from aberrant actin regulatory systems. We conclude that IL-1 plays a positive role in muscle regeneration by coordinating the initial interactions among inflammatory microenvironments and satellite cells. Our findings also provide compelling evidence that IL-1 is intimately engaged in regulating the fundamental function of myocytes.
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http://dx.doi.org/10.1152/ajpregu.00310.2017DOI Listing
July 2018

Contractile Skeletal Muscle Cells Cultured with a Conducting Soft Wire for Effective, Selective Stimulation.

Sci Rep 2018 02 2;8(1):2253. Epub 2018 Feb 2.

Department of Finemechanics, Graduate School of Engineering, Tohoku University, 6-6-01 Aramaki, Aoba-ku, Sendai, 980-8579, Japan.

Contractile skeletal muscle cells were cultured so as to wrap around an electrode wire to enable their selective stimulation even when they were co-cultured with other electrically-excitable cells. Since the electrode wire was composed of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) and polyurethane (PU), which is soft and highly capacitive (~10 mF cm), non-faradaic electrical stimulation with charge/discharge currents could be applied to the surrounding cells without causing significant damage even for longer periods (more than a week). The advantage of this new culture system was demonstrated in the study of chemotactic interaction of monocytes and skeletal muscle cells via myokines.
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http://dx.doi.org/10.1038/s41598-018-20729-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797109PMC
February 2018

Olfactory receptors are expressed in pancreatic β-cells and promote glucose-stimulated insulin secretion.

Sci Rep 2018 01 24;8(1):1499. Epub 2018 Jan 24.

Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan.

Olfactory receptors (ORs) mediate olfactory chemo-sensation in OR neurons. Herein, we have demonstrated that the OR chemo-sensing machinery functions in pancreatic β-cells and modulates insulin secretion. First, we found several OR isoforms, including OLFR15 and OLFR821, to be expressed in pancreatic islets and a β-cell line, MIN6. Immunostaining revealed OLFR15 and OLFR821 to be uniformly expressed in pancreatic β-cells. In addition, mRNAs of Olfr15 and Olfr821 were detected in single MIN6 cells. These results indicate that multiple ORs are simultaneously expressed in individual β-cells. Octanoic acid, which is a medium-chain fatty acid contained in food and reportedly interacts with OLFR15, potentiated glucose-stimulated insulin secretion (GSIS), thereby improving glucose tolerance in vivo. GSIS potentiation by octanoic acid was confirmed in isolated pancreatic islets and MIN6 cells and was blocked by OLFR15 knockdown. While Gα expression was not detectable in β-cells, experiments using inhibitors and siRNA revealed that the pathway dependent on phospholipase C-inositol triphosphate, rather than cAMP-protein kinase A, mediates GSIS potentiation via OLFR15. These findings suggest that the OR system in pancreatic β-cells has a chemo-sensor function allowing recognition of environmental substances obtained from food, and potentiates insulin secretion in a cell-autonomous manner, thereby modulating systemic glucose metabolism.
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http://dx.doi.org/10.1038/s41598-018-19765-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5784078PMC
January 2018

Involvement of neutrophils and interleukin-18 in nociception in a mouse model of muscle pain.

Mol Pain 2018 Jan-Dec;14:1744806918757286. Epub 2018 Jan 21.

1 Department of Orthopaedic Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan.

Muscle pain is a common condition that relates to various pathologies. Muscle overuse induces muscle pain, and neutrophils are key players in pain production. Neutrophils also play a central role in chronic pain by secreting interleukin (IL)-18. The aim of this study was to investigate the involvement of neutrophils and IL-18 in a mouse model of muscle pain. The right hind leg muscles of BALB/c mice were stimulated electrically to induce excessive muscle contraction. The left hind leg muscles were not stimulated. The pressure pain threshold, number of neutrophils, and IL-18 levels were investigated. Furthermore, the effects of the IL-18-binding protein and Brilliant Blue G on pain were investigated. In stimulated muscles, pressure pain thresholds decreased, and neutrophil and IL-18 levels increased compared with that in non-stimulated muscles. The administration of IL-18-binding protein and Brilliant Blue G attenuated hyperalgesia caused by excessive muscle contraction. These results suggest that increased IL-18 secretion from larger numbers of neutrophils elicits mechanical hyperalgesia.
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http://dx.doi.org/10.1177/1744806918757286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802617PMC
October 2018

Retained Myogenic Potency of Human Satellite Cells from Torn Rotator Cuff Muscles Despite Fatty Infiltration.

Tohoku J Exp Med 2018 01;244(1):15-24

Department of Orthopaedic Surgery, Tohoku University Graduate School of Medicine.

Rotator cuff tears (RCTs) are a common shoulder problem in the elderly that can lead to both muscle atrophy and fatty infiltration due to less physical load. Satellite cells, quiescent cells under the basal lamina of skeletal muscle fibers, play a major role in muscle regeneration. However, the myogenic potency of human satellite cells in muscles with fatty infiltration is unclear due to the difficulty in isolating from small samples, and the mechanism of the progression of fatty infiltration has not been elucidated. The purpose of this study was to analyze the population of myogenic and adipogenic cells in disused supraspinatus (SSP) and intact subscapularis (SSC) muscles of the RCTs from the same patients using fluorescence-activated cell sorting. The microstructure of the muscle with fatty infiltration was observed as a whole mount condition under multi-photon microscopy. Myogenic differentiation potential and gene expression were evaluated in satellite cells. The results showed that the SSP muscle with greater fatty infiltration surrounded by collagen fibers compared with the SSC muscle under multi-photon microscopy. A positive correlation was observed between the ratio of muscle volume to fat volume and the ratio of myogenic precursor to adipogenic precursor. Although no difference was observed in the myogenic potential between the two groups in cell culture, satellite cells in the disused SSP muscle showed higher intrinsic myogenic gene expression than those in the intact SSC muscle. Our results indicate that satellite cells from the disused SSP retain sufficient potential of muscle growth despite the fatty infiltration.
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http://dx.doi.org/10.1620/tjem.244.15DOI Listing
January 2018

Neuronal signals regulate obesity induced β-cell proliferation by FoxM1 dependent mechanism.

Nat Commun 2017 12 5;8(1):1930. Epub 2017 Dec 5.

Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.

Under insulin-resistant conditions such as obesity, pancreatic β-cells proliferate to prevent blood glucose elevations. A liver-brain-pancreas neuronal relay plays an important role in this process. Here, we show the molecular mechanism underlying this compensatory β-cell proliferation. We identify FoxM1 activation in islets from neuronal relay-stimulated mice. Blockade of this relay, including vagotomy, inhibits obesity-induced activation of the β-cell FoxM1 pathway and suppresses β-cell expansion. Inducible β-cell-specific FoxM1 deficiency also blocks compensatory β-cell proliferation. In isolated islets, carbachol and PACAP/VIP synergistically promote β-cell proliferation through a FoxM1-dependent mechanism. These findings indicate that vagal nerves that release several neurotransmitters may allow simultaneous activation of multiple pathways in β-cells selectively, thereby efficiently promoting β-cell proliferation and maintaining glucose homeostasis during obesity development. This neuronal signal-mediated mechanism holds potential for developing novel approaches to regenerating pancreatic β-cells.
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http://dx.doi.org/10.1038/s41467-017-01869-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717276PMC
December 2017

3D electrochemical and ion current imaging using scanning electrochemical-scanning ion conductance microscopy.

Phys Chem Chem Phys 2017 Oct;19(39):26728-26733

WPI-Advanced Institute for Materials Research, Tohoku University, 980-8577, Japan.

Local cell-membrane permeability and ionic strength are important factors for maintaining the functions of cells. Here, we measured the spatial electrochemical and ion concentration profile near the sample surface with nanoscale resolution using scanning electrochemical microscopy (SECM) combined with scanning ion-conductance microscopy (SICM). The ion current feedback system is an effective way to control probe-sample distance without contact and monitor the kinetic effect of mediator regeneration and the chemical concentration profile. For demonstrating 3D electrochemical and ion concentration mapping, we evaluated the reaction rate of electrochemical mediator regeneration on an unbiased conductor and visualized inhomogeneous permeability and the ion concentration 3D profile on a single fixed adipocyte cell surface.
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http://dx.doi.org/10.1039/c7cp05157cDOI Listing
October 2017

Heterotypic endosomal fusion as an initial trigger for insulin-induced glucose transporter 4 (GLUT4) translocation in skeletal muscle.

J Physiol 2017 08 10;595(16):5603-5621. Epub 2017 Jul 10.

Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.

Key Points: Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted. Quantum dot-based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving 'static retention' and 'stimulus-dependent liberation'. Vital imaging analyses and super-resolution microscopy-based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4-containing vesicles in skeletal myofibres. Prior exposure to exercise-mimetic stimuli potentiated this insulin-responsive endomembrane fusion event involving GLUT4-containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise.

Abstract: Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletal myofibres, we show that one of the initial triggers of insulin-induced GLUT4 translocation is heterotypic endomembrane fusion arising from very small static GLUT4-containing vesicles with a subset of transferrin receptor-containing endosomes. Importantly, pretreatment with exercise-mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe-like localization close to sarcomeric α-actinin and that insulin induced a reduction of the stripe-like localization accompanying changes in its detergent solubility. The results of the present study thus provide a conceptual framework indicating that GLUT4 protein trafficking via heterotypic fusion is a critical feature of GLUT4 translocation in skeletal muscles and also suggest that the efficacy of the endomembranous fusion process in response to insulin is involved in the benefits of exercise.
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http://dx.doi.org/10.1113/JP273985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556175PMC
August 2017

Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization.

J Clin Biochem Nutr 2017 Jan 17;60(1):3-11. Epub 2016 Dec 17.

Department of Biomedical Engineering, Tohoku University, 6-6-05 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan.

Gas-liquid interfacial atmospheric-pressure plasma jets (GLI-APPJ) are used medically for plasma-induced cell-membrane permeabilization. In an attempt to identify the dominant factors induced by GLI-APPJ responsible for enhancing cell-membrane permeability, the concentration and distribution of plasma-produced reactive species in the gas and liquid phase regions are measured. These reactive species are classified in terms of their life-span: long-lived (e.g., HO), short-lived (e.g., O), and extremely-short-lived (e.g., OH). The concentration of plasma-produced OH in the liquid phase region decreases with an increase in solution thickness (<1 mm), and plasma-induced cell-membrane permeabilization is found to decay markedly as the thickness of the solution increases. Furthermore, the horizontally center-localized distribution of OH, resulting from the center-peaked distribution of OH in the gas phase region, corresponds with the distribution of the permeabilized cells upon APPJ irradiation, whereas the overall plasma-produced oxidizing species such as HO in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that OH is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization.
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http://dx.doi.org/10.3164/jcbn.16-73DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5281536PMC
January 2017

Live-cell single-molecule labeling and analysis of myosin motors with quantum dots.

Mol Biol Cell 2017 01 9;28(1):173-181. Epub 2016 Nov 9.

Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8579, Japan.

Quantum dots (QDs) are a powerful tool for quantitatively analyzing dynamic cellular processes by single-particle tracking. However, tracking of intracellular molecules with QDs is limited by their inability to penetrate the plasma membrane and bind to specific molecules of interest. Although several techniques for overcoming these problems have been proposed, they are either complicated or inconvenient. To address this issue, in this study, we developed a simple, convenient, and nontoxic method for labeling intracellular molecules in cells using HaloTag technology and electroporation. We labeled intracellular myosin motors with this approach and tracked their movement within cells. By simultaneously imaging myosin movement and F-actin architecture, we observed that F-actin serves not only as a rail but also as a barrier for myosin movement. We analyzed the effect of insulin on the movement of several myosin motors, which have been suggested to regulate intracellular trafficking of the insulin-responsive glucose transporter GLUT4, but found no significant enhancement in myosin motor motility as a result of insulin treatment. Our approach expands the repertoire of proteins for which intracellular dynamics can be analyzed at the single-molecule level.
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http://dx.doi.org/10.1091/mbc.E16-06-0413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5221621PMC
January 2017

The proneurotrophin receptor sortilin is required for Mycobacterium tuberculosis control by macrophages.

Sci Rep 2016 07 8;6:29332. Epub 2016 Jul 8.

Host-pathogen interactions in tuberculosis laboratory, The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, London, NW7 1AA, UK.

Sorting of luminal and membrane proteins into phagosomes is critical for the immune function of this organelle. However, little is known about the mechanisms that contribute to the spatiotemporal regulation of this process. Here, we investigated the role of the proneurotrophin receptor sortilin during phagosome maturation and mycobacterial killing. We show that this receptor is acquired by mycobacteria-containing phagosomes via interactions with the adaptor proteins AP-1 and GGAs. Interestingly, the phagosomal association of sortilin is critical for the delivery of acid sphingomyelinase (ASMase) and required for efficient phagosome maturation. Macrophages from Sort1(-/-) mice are less efficient in restricting the growth of Mycobacterium bovis BCG and M. tuberculosis. In vivo, Sort1(-/-) mice showed a substantial increase in cellular infiltration of neutrophils in their lungs and higher bacterial burden after infection with M. tuberculosis. Altogether, sortilin defines a pathway required for optimal intracellular mycobacteria control and lung inflammation in vivo.
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http://dx.doi.org/10.1038/srep29332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937236PMC
July 2016

Calcium influx through TRP channels induced by short-lived reactive species in plasma-irradiated solution.

Sci Rep 2016 05 12;6:25728. Epub 2016 May 12.

Department of Electronic Engineering, Tohoku University, 6-6-05 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan.

Non-equilibrium helium atmospheric-pressure plasma (He-APP), which allows for a strong non-equilibrium chemical reaction of O2 and N2 in ambient air, uniquely produces multiple extremely reactive products, such as reactive oxygen species (ROS), in plasma-irradiated solution. We herein show that relatively short-lived unclassified reactive species (i.e., deactivated within approximately 10 min) generated by the He-APP irradiation can trigger physiologically relevant Ca(2+) influx through ruthenium red- and SKF 96365-sensitive Ca(2+)-permeable channel(s), possibly transient receptor potential channel family member(s). Our results provide novel insight into understanding of the interactions between cells and plasmas and the mechanism by which cells detect plasma-induced chemically reactive species, in addition to facilitating development of plasma applications in medicine.
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http://dx.doi.org/10.1038/srep25728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864414PMC
May 2016

Involvement of IL-1 in the Maintenance of Masseter Muscle Activity and Glucose Homeostasis.

PLoS One 2015 24;10(11):e0143635. Epub 2015 Nov 24.

Division of Oral Molecular Regulation, Tohoku University Graduate School of Dentistry, Sendai, Japan.

Physical exercise reportedly stimulates IL-1 production within working skeletal muscles, but its physiological significance remains unknown due to the existence of two distinct IL-1 isoforms, IL-1α and IL-1β. The regulatory complexities of these two isoforms, in terms of which cells in muscles produce them and their distinct/redundant biological actions, have yet to be elucidated. Taking advantage of our masticatory behavior (Restrained/Gnawing) model, we herein show that IL-1α/1β-double-knockout (IL-1-KO) mice exhibit compromised masseter muscle (MM) activity which is at least partially attributable to abnormalities of glucose handling (rapid glycogen depletion along with impaired glucose uptake) and dysfunction of IL-6 upregulation in working MMs. In wild-type mice, masticatory behavior clearly increased IL-1β mRNA expression but no incremental protein abundance was detectable in whole MM homogenates, whereas immunohistochemical staining analysis revealed that both IL-1α- and IL-1β-immunopositive cells were recruited around blood vessels in the perimysium of MMs after masticatory behavior. In addition to the aforementioned phenotype of IL-1-KO mice, we found the IL-6 mRNA and protein levels in MMs after masticatory behavior to be significantly lower in IL-1-KO than in WT. Thus, our findings confirm that the locally-increased IL-1 elicited by masticatory behavior, although present small in amounts, contributes to supporting MM activity by maintaining normal glucose homeostasis in these muscles. Our data also underscore the importance of IL-1-mediated local interplay between autocrine myokines including IL-6 and paracrine cytokines in active skeletal muscles. This interplay is directly involved in MM performance and fatigability, perhaps mediated through maintaining muscular glucose homeostasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143635PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4658060PMC
June 2016

Improvement of cell membrane permeability using a cell-solution electrode for generating atmospheric-pressure plasma.

Biointerphases 2015 Jun 21;10(2):029521. Epub 2015 Jun 21.

Department of Biomedical Engineering, Tohoku University, 6-6-04 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan.

The cell membrane permeability, which is strongly related to gene transfection, is improved using a cell-solution electrode for generating atmospheric-pressure plasma (APP) just above the solution. In the case of the floating cells, the cell membrane permeability is significantly improved by the cell-solution electrode APP compared with the conventional diffusion type APP, because the distance between the plasma generation area and the cell solution surface becomes short, which could reduce the radial diffusion loss of the plasma irradiated to the cell suspended solution. In the case of the adherent cells, cell membrane permeability is found to be enhanced by the shorter distance between the solution surface and the adherent cells as well as using the cell-solution electrode, which means that the short-lived reactive oxygen species generated at the solution surface are essential for the improvement of cell membrane permeability.
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http://dx.doi.org/10.1116/1.4921278DOI Listing
June 2015