Effects of CX3CR1 and CXCR2 antagonists on running-dependent intramuscular neutrophil recruitments and myokine upregulation.

Muscle contractile activity stimulates intramuscular recruitment of immune cells including neutrophils emerging to serve as a prerequisite for exerting proper muscular performance, although the underlying mechanisms and their contributions to myokine upregulation remain ill-defined. We previously reported that pharmacological inhibition of CX3CR1, a fractalkine receptor, dampens gnawing-dependent neutrophil recruitment into masseter muscles along with compromising their masticatory activity. By using a running exercise model, we herein demonstrated that hindlimb muscles require collaborative actions of both CX3CR1- and CXCR2-mediated signals for achieving neutrophil recruitment, upregulation of myokines including interleukin (IL)-6, enhanced GLUT4 translocation, and adequate endurance capability. Mechanistically, we revealed that a combination of CX3CR1 and CXCR2 antagonists, i.e., AZD8797 and SB2205002, inhibits exercise-inducible ICAM-1 and fractalkine upregulations in the area of the endothelium and muscle-derived CXCL1 upregulation, both of which apparently contribute to the intramuscular neutrophil accumulation in working muscles. Intriguingly, we also observed that 2 h of running results in intramuscular augmentation of innate lymphoid type 2 cells (ILC2s) markers, i.e., Bcl11b mRNA levels and anti-GATA-3-antibody-positive signals, and that these effects are completely abolished by administration of the combination of CX3CR1 and CXCR2 antagonists. Taken together, our findings strongly suggest that the exercise-evoked regional interplay among working myofibers, the adjacent endothelium, and recruited immune cells including neutrophils and possibly ILC2s, mediated through these local factors, plays a key role in the organization of the intramuscular microenvironment supporting the performance of hindlimb muscles during running.NEW & NOTEWORTHY This study provides compelling evidence that running-dependent intramuscular neutrophil recruitment requires both CX3CR1- and CXCR2-mediated signals that prime not only myofiber-derived myokine upregulations but also endothelium ICAM-1 and fractalkine expressions. The results revealed the importance of the exercise-evoked regional interplay among working myofibers, the adjacent endothelium, and recruited immune cells, including neutrophils and possibly ILC2s, which plays a key role in the organization of the intramuscular microenvironment supporting the performance of hindlimb muscles during running.

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

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.

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

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).

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

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 Ca2+ 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 Ca2+ 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 (Thr642) and Tbc1d1 (Ser237 and Thr596). 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.

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

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.

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

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.

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

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.

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

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/ß-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-1ß 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.

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

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.

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

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.

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

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.

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

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., H2O2), short-lived (e.g., O2•-), and extremely-short-lived (e.g., •OH). The concentration of plasma-produced •OHaq 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 •OHaq, 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 H2O2aq in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that •OHaq is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization.

Bilaterally Aligned Electroosmotic Flow Generated by Porous Microneedle Device for Dual-Mode Delivery.

Here, a novel porous microneedle (PMN) device with bilaterally aligned electroosmotic flow (EOF) enabling controllable dual-mode delivery of molecules is developed. The PMNs placed at anode and cathode compartments are modified with anionic poly-2-acrylamido-2-methyl-1-propanesulfonic acid and cationic poly-(3-acrylamidopropyl) trimethylammonium, respectively. The direction of EOF generated by PMN at the cathode compartment is, therefore, reversed from cathode to anode, countering the unwanted cathodal suctioning of interstitial fluid caused by reverse iontophoresis. With the bilateral alignment of EOF, the versatility of the proposed device is evaluated by delivering molecules with different charges and sizes using Franz cell. In addition, a 3D printed probe device is developed to ease practical handling and minimize electrical stimulation by integrating two PMNs in closed proximity. Finally, the performance of the integrated probe device is demonstrated by dual delivery of a variety of molecules (methylene blue, rhodamine B, and fluorescein isothiocyanate-dextran) using pig skin and vaccination using mice with delivered ovalbumin.

Sporadic inclusion body myositis-derived myotube culture revealed muscle cell-autonomous expression profiles.

Sporadic inclusion body myositis (sIBM) is a muscle disease in older people and is characterized by inflammatory cell invasion into intact muscle fibers and rimmed vacuoles. The pathomechanism of sIBM is not fully elucidated yet, and controversy exists as to whether sIBM is a primary autoimmune disease or a degenerative muscle disease with secondary inflammation. Previously, we established a method of collecting CD56-positive myoblasts from human skeletal muscle biopsy samples. We hypothesized that the myoblasts derived from these patients are useful to see the cell-autonomous pathomechanism of sIBM. With these resources, myoblasts were differentiated into myotubes, and the expression profiles of cell-autonomous pathology of sIBM were analyzed. Myoblasts from three sIBM cases and six controls were differentiated into myotubes. In the RNA-sequencing analysis of these "myotube" samples, 104 differentially expressed genes (DEGs) were found to be significantly upregulated by more than twofold in sIBM, and 13 DEGs were downregulated by less than twofold. For muscle biopsy samples, a comparative analysis was conducted to determine the extent to which "biopsy" and "myotube" samples differed. Fifty-three DEGs were extracted of which 32 (60%) had opposite directions of expression change (e.g., increased in biopsy vs decreased in myotube). Apolipoprotein E (apoE) and transmembrane protein 8C (TMEM8C or MYMK) were commonly upregulated in muscle biopsies and myotubes from sIBM. ApoE and myogenin protein levels were upregulated in sIBM. Given that enrichment analysis also captured changes in muscle contraction and development, the triggering of muscle atrophy signaling and abnormal muscle differentiation via MYMK or myogenin may be involved in the pathogenesis of sIBM. The presence of DEGs in sIBM suggests that the myotubes formed from sIBM-derived myoblasts revealed the existence of muscle cell-autonomous degeneration in sIBM. The catalog of DEGs will be an important resource for future studies on the pathogenesis of sIBM focusing on primary muscle degeneration.

Molecular basis of insulin-responsive GLUT4 trafficking systems revealed by single molecule imaging.

Development of a 'static retention' property of GLUT4, the insulin-responsive glucose transporter, has emerged as being essential for achieving its maximal insulin-induced surface exposure. Herein, employing quantum-dot-based nanometrology of intracellular GLUT4 behavior, we reveal the molecular basis of its systematization endowed upon adipogenic differentiation of 3T3L1 cells. Specifically, (i) the endosomes-to-trans-Golgi network (TGN) retrieval system specialized for GLUT4 develops in response to sortilin expression, which requires an intricately balanced interplay among retromers, golgin-97 and syntaxin-6, the housekeeping vesicle trafficking machinery. (ii) The Golgin-97-localizing subdomain of the differentiated TGN apparently serves as an intermediate transit route by which GLUT4 can further proceed to the stationary GLUT4 storage compartment. (iii) AS160/Tbc1d4 then renders the 'static retention' property insulin responsive, i.e. insulin liberates GLUT4 from the static state only in the presence of functional AS160/Tbc1d4. (iv) Moreover, sortilin malfunction and the resulting GLUT4 sorting defects along with retarded TGN function might be etiologically related to insulin resistance. Together, these observations provide a conceptual framework for understanding maturation/retardation of the insulin-responsive GLUT4 trafficking system that relies on the specialized subdomain of differentiated TGN.

Characterization of contraction-induced IL-6 up-regulation using contractile C2C12 myotubes.

Muscle contractile activity functions as a potent stimulus for acute interleukin (IL)-6 expression in working skeletal muscles. Recently, we established an "in vitro contraction model" using highly-developed contractile C2C12 myotubes by applying electric pulse stimulation (EPS). Herein, we characterize the effects of EPS-evoked contraction on IL-6 expression in contractile C2C12 myotubes. Both secretion and mRNA expression of IL-6 were significantly up-regulated by EPS in a frequency-dependent manner in contracting myotubes during a 24-h period, and the response was blunted by cyclosporine A, a calcineurin inhibitor. Longer time (~12h) was required for the induction of IL-6 after the initiation of EPS as compared to that of other contraction-inducible CXC chemokines such as CXCL1/KC, which were induced in less than 3 hours. Furthermore, these acute inducible CXC chemokines exhibited no autocrine effect on IL-6 expression. Importantly, contraction-dependent IL-6 up-regulation was markedly suppressed in the presence of high levels of glucose along with increased glycogen accumulations. Experimental manipulation of intracellular glycogen contents by modulating available glucose or pyruvate during a certain EPS period further established the suppressive effect of glycogen accumulations on contraction-induced IL-6 up-regulation, which appeared to be independent of calcineurin activity. We also document that EPS-evoked contractile activity improved insulin-responsiveness in terms of intracellular glycogen accumulations. Taken together, these data provide important insights into the regulation of IL-6 expression in response to contractile activity of muscle cells, which is difficult to examine using in vivo experimental techniques. Our present results thus expand the usefulness of our "in vitro contraction model".

Undaria pinnatifida (Wakame) Intake Ameliorates High-Fat Diet-Induced Glucose Intolerance via Promoting GLUT4 Expression and Membrane Translocation in Muscle.

Type 2 diabetes mellitus (T2DM), a lifestyle-related disease, is developed due to eating habits and decreased physical activity. Diabetes also increases the risk of cancer and major neurodegenerative diseases; controlling the onset of diabetes helps prevent various illnesses. Eating seaweed, such as Undaria pinnatifida (wakame), is a part of the Asian food culture. Therefore, we analyzed the antidiabetic effect of wakame intake using the high-fat diet-induced diabetes mouse model. Furthermore, we analyzed the effect of wakame extract on the cell membrane translocation of glucose transporter-4 (GLUT4) and activation of insulin signal molecules, such as AKT and AMPK, in insulin-sensitive tissues. Differentiated C2C12 cells were incubated with wakame components. The membrane translocation of GLUT4 and phosphorylation of AKT and AMPK were investigated with immunofluorescence staining and Western blotting, respectively. Also, male C57BL/6J mice were fed the normal diet (ND), high-fat diet (HFD), ND with 1% wakame powder (ND + W), or HFD with 1% wakame powder (HFD + W). We evaluated the effect of wakame intake on high-fat diet-induced glucose intolerance using an oral glucose tolerance test. Moreover, we analyzed insulin signaling molecules, such as GLUT4, AKT, and AMPK, in muscle using Western blotting. GLUT4 membrane translocation was promoted by wakame components. Also, GLUT4 levels and AKT and AMPK phosphorylation were significantly elevated by wakame components in C2C12 cells. In addition, the area under the curve (AUC) of the HFD + W group was significantly smaller than that of the HFD group. Furthermore, the level of GLUT4 in the muscle was increased in the wakame intake group. This study revealed that various wakame components exerted antidiabetic effects on the mice on a high-fat diet by promoting glucose uptake in the skeletal muscle, enhancing GLUT4 levels, and activating AKT and AMPK.

LRRK2 negatively regulates glucose tolerance via regulation of membrane translocation of GLUT4 in adipocytes.

Epidemiological studies have shown that abnormalities of glucose metabolism are involved in leucine-rich repeat kinase 2 (LRRK2)-associated Parkinson's disease (PD). However, the physiological significance of this association is unclear. In the present study, we investigated the effect of LRRK2 on high-fat diet (HFD)-induced glucose intolerance using Lrrk2-knockout (KO) mice. We found for the first time that HFD-fed KO mice display improved glucose tolerance compared with their wild-type (WT) counterparts. In addition, high serum insulin and leptin, as well as low serum adiponectin resulting from HFD in WT mice were improved in KO mice. Using western blotting, we found that Lrrk2 is highly expressed in adipose tissues compared with other insulin-related tissues that are thought to be important in glucose tolerance, including skeletal muscle, liver, and pancreas. Lrrk2 expression and phosphorylation of its kinase substrates Rab8a and Rab10 were significantly elevated after HFD treatment in WT mice. In cell culture experiments, treatment with a LRRK2 kinase inhibitor stimulated insulin-dependent membrane translocation of glucose transporter 4 (Glut4) and glucose uptake in mouse 3T3-L1 adipocytes. We conclude that increased LRRK2 kinase activity in adipose tissue exacerbates glucose tolerance by suppressing Rab8- and Rab10-mediated GLUT4 membrane translocation.

Protocol for preparing sensor molecules and analyzing heterotypic endomembrane fusion in insulin-responsive cells using live-cell imaging.

Heterotypic endomembrane fusion between static GLUT4-containing vesicles and traveling transferrin receptor-containing endosomes triggers insulin-responsive translocation of the GLUT4 glucose transporter. Here, we provide a protocol for preparing BODIPY-based fluorescent sensor molecules allowing detection of heterotypic endomembrane fusion through dequenching via streptavidin-biotin binding and ratiometrically analyzing insulin-responsive events with live-cell imaging. Although this protocol is for evaluating specific fusion processes relating GLUT4 translocation, it is also applicable to assessing other processes so long as sensor molecules can properly label target molecules. For complete details on the use and execution of this protocol, please refer to Hatakeyama et al. (2022).