title: Insulin-induced exocytosis in single, in vitro innervated human muscle fibres: a new approach.
abstract: We describe a new approach for studying insulin-induced exocytosis in individual, well-differentiated, innervated human muscle fibres. We used an in vitro system in which motor axons extending from embryonic rat spinal cord explants functionally innervate co-cultured human muscle fibres. Under such conditions, the human muscle fibres reach a high degree of differentiation that is never observed in non-innervated, cultured human muscle fibres. To monitor insulin-induced membrane dynamics, we used confocal microscopy to measure the fluorescence intensity changes of the styryl dye FM1-43, a marker for membrane area. The fluorescence intensity increased after insulin stimulation. This increase, as well as the intensity of staining for the glucose transporter 4 (GLUT4), was significantly higher in the innervated and contracting fibres than in myoblasts and myotubes. This shows that in vitro innervation of human muscle cells not only enhances the differentiation stage but also improves the insulin response. Our approach allows continuous monitoring and quantitative assessment of insulin-induced increase in cumulative exocytosis in individual human muscle fibres at a differentiation level practically corresponding to that of adult muscle. It is therefore a suitable system for studying various parameters affecting the mechanisms underlying insulin-induced GLUT4 translocation in human skeletal muscle.
authors.affiliation: Slovenia Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Medical School, University of Ljubljana, Zaloska 4, 1000, Ljubljana, Slovenia.
authors.forename: Helena H
authors.initials: HH
authors.lastname: Chowdhury
authors.forename: Marko
authors.initials: M
authors.lastname: Jevsek
authors.forename: Marko
authors.initials: M
authors.lastname: Kreft
authors.forename: Tomaz
authors.initials: T
authors.lastname: Mars
authors.forename: Robert
authors.initials: R
authors.lastname: Zorec
authors.forename: Zoran
authors.initials: Z
authors.lastname: Grubic
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D005456:Fluorescent Dyes; D051275:Glucose Transporter Type 4; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009121:Muscle Denervation; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism; D011247:Pregnancy; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats; D017208:Rats, Wistar
chemical_list: C073804:FM1 43; D005456:Fluorescent Dyes; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C495692:SLC2A4 protein, human; C495694:Slc2a4 protein, rat