Endocrine Abstracts (2019) 65 OC1.3 | DOI: 10.1530/endoabs.65.OC1.3

Metabolic and functional effects of fatty acid overload on in vitro tissue engineered skeletal muscle

Mark Turner1,2, Rowan Rimington1, Neil Martin1, Jacob Fleming1, Andrew Capel1, Leanne Hodson3 & Mark Lewis1,2

1Loughborough University, Loughborough, UK; 2University Hospitals of Leicester NHS Trust, Leicester, UK; 3University of Oxford, Oxford, UK

Introduction: Efficient fuel selection between glucose and fatty acids in insulin-sensitive organs is a key feature which determines metabolic health in humans. In skeletal muscle, lipid storage and utilisation can differ between healthy and diseased individuals, however to date there are limited in vitro models to explore this. Therefore, aim of this work was to investigate the effects of nutrition on engineered skeletal muscle health and metabolism.

Methods: Three-dimensional tissue engineered skeletal muscles were developed by seeding C2C12 myoblasts into Collagen/Matrigel® solution between 3D printed inserts. Initially cultured for 4 days in growth media followed by 10 days in differentiation media (DM), muscles were then exposed to fatty acid-free BSA alone or conjugated to Oleic, Palmitic, Linoleic, and α-Linoleic Acids (OPLA; physiological ratio 45:30:25:1%) at concentrations of 200 µM or 800 µM for 4 days. Constructs were stained and analysed for lipid droplets and functional capacity was measured by electrical field stimulation. Engineered muscles were also analysed for changes in the mRNA expression of lipid metabolism and storage genes, the expression of mitochondrial and insulin signalling proteins, and morphological changes.

Results: Exposure to OPLA increased the presence of lipid droplets in a dose dependent manner. There was a significant increase in Perilipin 2 and a dose dependent increase Pyruvate dehydrogenase lipoamide kinase isozyme 4 mRNA expression. PGC-1α protein increased with increasing OPLA concentration yet citrate synthase expression remained unchanged. In addition, the increase in the presence of lipid droplets resulted in a reduction force generation and altered the insulin signalling response.

Conclusions: Exposure to OPLA induces lipid droplet accumulation in tissue engineered skeletal muscle as well as increase the expression of key lipid storage and oxidation markers. The presence of lipid droplets also impairs maximal contractile force production in this system.

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