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Accumulation of fat in muscle tissue as intramyocellular lipids (IMCLs) is closely related to the development of insulin resistance and subsequent type 2 diabetes. Most IMCLs organize into lipid droplets (LDs), the fates of which are regulated by lipid droplet coat proteins. Perilipin 5 (PLIN5) is an LD coating protein, which is strongly linked to lipid storage in muscle tissue. Here we employ a tandem in vitro/ex vivo approach and use chemical imaging by label-free, hyperspectral coherent Raman microscopy to quantify compositional changes in individual LDs upon PLIN5 overexpression. Our results directly show that PLIN5 overexpression in muscle alters individual LD composition and physiology, resulting in larger LDs with higher esterified acyl chain concentration, increased methylene content, and more saturated lipid species. These results suggest that lipotoxic protection afforded by natural PLIN5 upregulation in muscle involves molecular changes in lipid composition within LDs.

Perilipin 5 mediated lipid droplet remodelling revealed by coherent Raman imaging