Bodipy Is Superior To Oil-red-O For Detecting And Quantifying Intramuscular Triglyceride In Human Skeletal Muscle

Abstract

PURPOSE: To compare the use of bodipy and oil-red-O (ORO) for quantification of intramuscular triglyceride (IMTG) content and use during exercise as well as investigation of lipid droplet (LD) morphology in human skeletal muscle. METHODS: Biopsies were obtained from the m. vastus lateralis of six fasted healthy, lean sedentary males (age:20±1y, BMI:24.8±1.2kg.m2, VO2peak:39.8±2.4ml.min-1.kg-1) prior to and immediately following 60min cycle ergometer exercise at ~60%VO2peak. Cryosections were labelled using antibodies targeting MHCI and MHCIIa. Anti-laminin was used to identify the cell membrane. LDs were labelled with either bodipy or ORO. Images were captured using confocal immunofluorescence microscopy and analysed using Image Pro Plus. RESULTS: IMTG content (% area stained) was greater when using bodipy compared to ORO (P=0.006), and bodipy detected more LDs (P=0.03) with a greater mean LD size (P=0.03) than ORO. Regional distribution was also different for the two dyes with IMTG content with bodipy being greater in the central vs. peripheral region (5μm from plasma membrane) in both type I (central: 3.1±0.5% vs. peripheral:2.8±0.5%) and type IIa fibres (central:1.4±0.3% vs. peripheral:1.3±0.2%; P=0.036). The opposite was true when using ORO, with a greater IMTG content observed in the peripheral vs. the central region in all fibre types (P=0.013). These regional differences in IMTG content for bodipy and ORO were attributed to differences in LD number (P=0.039). Following exercise, using bodipy, IMTG content was decreased in both the peripheral (-58±21%, P=0.014) and central regions (-63±21%, P=0.016) of type I fibres. However, using ORO only a significant decrease in IMTG content in the peripheral region of type I fibres was observed (-85±19%, P=0.021). CONCLUSION: Bodipy is preferable to investigate IMTG in skeletal muscle due to its superior LD detection capabilities and specificity for detecting IMTG avoiding labelling of membrane structures, compared to ORO. We demonstrate the importance of this in the context of regional distribution of IMTG detected using ORO and bodipy and differences in region-specific IMTG utilisation during exercise. Therefore, it is possible that the choice of lipid dye could impact the conclusions drawn in studies in which subcellular IMTG distribution is considered.