Abstract
Despite over 50 years of research, a comprehensive understanding of how intramuscular triglyceride (IMTG) is stored in skeletal muscle and its contribution as a fuel during exercise is lacking. Immunohistochemical techniques provide information on IMTG content and lipid droplet (LD) morphology on a fibre type and subcellular-specific basis, and the lipid dye Oil Red O (ORO) is commonly used to achieve this. BODIPY 493/503 (BODIPY) is an alternative lipid dye with lower background staining and narrower emission spectra. Here we provide the first quantitative comparison of BODIPY and ORO for investigating exercise-induced changes in IMTG content and LD morphology on a fibre type and subcellular-specific basis. Estimates of IMTG content were greater when using BODIPY, which was predominantly due to BODIPY detecting a larger number of LDs, compared to ORO. The subcellular distribution of intramuscular lipid was also dependent on the lipid dye used; ORO detects a greater proportion of IMTG in the periphery (5 μm below cell membrane) of the fibre, whereas IMTG content was higher in the central region using BODIPY. In response to 60 min moderate-intensity cycling exercise, IMTG content was reduced in both the peripheral (− 24%) and central region (− 29%) of type I fibres (P < 0.05) using BODIPY, whereas using ORO, IMTG content was only reduced in the peripheral region of type I fibres (− 31%; P < 0.05). As well as highlighting some methodological considerations herein, our investigation demonstrates that important differences exist between BODIPY and ORO for detecting and quantifying IMTG on a fibre type and subcellular-specific basis.
Highlights
Lipid is predominantly stored in adipose tissue in humans, a small amount (~ 200 g) is stored in skeletal muscle in the form of intramuscular triglyceride (IMTG)
BODIPY detected more lipid droplet (LD) compared to Oil red O (ORO) that were ≥ 400 nm2 in type I fibres (P < 0.01), and ≥ 300 nm2 in type IIa and type IIx fibres (P < 0.01)
Given that in most acute and chronic training interventions the changes expected in IMTG content will exceed 10% (Gemmink et al 2016; Shepherd et al 2012, 2013, 2017; van Loon et al 2003), which outweighs our reported coefficient of variation for BODIPY or ORO, we propose imaging of at least 13 fibres in order to generate a reliable estimate of IMTG content
Summary
Lipid is predominantly stored in adipose tissue in humans, a small amount (~ 200 g) is stored in skeletal muscle in the form of intramuscular triglyceride (IMTG). Histochemistry and Cell Biology (2020) 154:369–382 determine net changes in IMTG content. The discrepant findings can be attributed to a large between-biopsy variability, driven by the presence of extramyocellular lipids which are located between the muscle fibres but which cannot be excluded from analysis of mixed muscle homogenates (van Loon 2004). Using immunofluorescence microscopy in conjunction with a lipid dye removes the confounding effect of extramyocellular lipids and permits IMTG content to be determined in a fibre type-specific manner (Koopman et al 2001). Oil red O (ORO) is the most commonly used lipid dye to investigate lipid metabolism in skeletal muscle, and has been employed to demonstrate that IMTG content and use during exercise is greatest in type I fibres (Shepherd et al 2013; van Loon et al 2003). BODIPY may provide the most accurate tool to identify LDs and quantify IMTG content in skeletal muscle, but a quantitative comparison between BODIPY and ORO is yet to be made
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