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.

Vaspin promotes insulin sensitivity in elderly muscle and is upregulated in obesity

“…However, it should be noted that primary human myotubes in culture elicit relatively low insulin-stimulated glucose uptake (Montell et al 2001, Al-Khalili et al 2003, Aas et al 2004), compared to skeletal muscle tissue. This could be due to low basal expression of GLUT4, which has been attributed to denervation (Chowdhury et al 2005, Jensen et al 2009) and the subsequent absence of contraction. Therefore, in our model system, treatment of myotubes with vaspin may only be increasing the pool of bioavailable GLUT4 towards physiological levels.…”

Are cultured human myotubes far from home?

“…It is also important to note that denervation of skeletal muscles reduces GLUT4 expression and causes insulin resistance ). Indeed, innervation of human myotubes is possible in vitro by coculturing with rat motor neurons, and such innervation increases GLUT4 expression (Chowdhury, et al, 2005). However, more convenient methodologies are required.…”

Interactions of Antibodies to the Gram-Negative Gastric Bacterium Helicobacter pylori with the Synaptic Calcium Sensor Synaptotagmin 5, Correlate to Impaired Vesicle Recycling in SiMa Human Neuroblastoma Cells

“…Effects of α-HPy and α-CJe on acetylcholine(ACh)-elicited exocytosis and subsequent vesicle recycling by endocytosis were visualized by incubation with the fluorescent dye FM1-43 (Chowdhury et al 2005;Gaffield and Betz 2006). Cells with or without a 12-h pretreatment with 10 μg/ml of a given antibacterial antiserum were transferred to a recording chamber, continuously superfused with a standard bath solution (at a rate of 5 ml/ min).…”