Skeletal muscle lipid droplets are resynthesized before being coated with perilipin proteins following prolonged exercise in elite male triathletes

Abstract

Intramuscular triglycerides (IMTG) are a key substrate during prolonged exercise, but little is known about the rate of IMTG resynthesis in the postexercise period. We investigated the hypothesis that the distribution of the lipid droplet (LD)-associated perilipin (PLIN) proteins is linked to IMTG storage following exercise. Fourteen elite male triathletes (27 ± 1 yr, 66.5 ± 1.3 mL·kg−1·min−1) completed 4 h of moderate-intensity cycling. During the first 4 h of recovery, subjects received either carbohydrate or H2O, after which both groups received carbohydrate. Muscle biopsies collected pre- and postexercise and 4 and 24 h postexercise were analyzed using confocal immunofluorescence microscopy for fiber type-specific IMTG content and PLIN distribution with LDs. Exercise reduced IMTG content in type I fibers (−53%, P = 0.002), with no change in type IIa fibers. During the first 4 h of recovery, IMTG content increased in type I fibers ( P = 0.014), but was not increased more after 24 h, where it was similar to baseline levels in both conditions. During recovery the number of LDs labeled with PLIN2 (70%), PLIN3 (63%), and PLIN5 (62%; all P < 0.05) all increased in type I fibers. Importantly, the increase in LDs labeled with PLIN proteins only occurred at 24 h postexercise. In conclusion, IMTG resynthesis occurs rapidly in type I fibers following prolonged exercise in highly trained individuals. Furthermore, increases in IMTG content following exercise preceded an increase in the number of LDs labeled with PLIN proteins. These data, therefore, suggest that the PLIN proteins do not play a key role in postexercise IMTG resynthesis.

Divergence exists in the subcellular distribution of intramuscular triglyceride in human skeletal muscle dependent on the choice of lipid dye

“…Using the optimised protocol and analytical procedure, we compared BODIPY and ORO for determining fibre type and subcellular-specific IMTG utilisation during exercise. Irrespective of the lipid dye used, IMTG utilisation during exercise was specific to type I fibres, in line with a number of previous observations (Jevons et al 2020 ; Shepherd et al 2013 ; van Loon et al 2003 ). However, the choice of lipid dye did reveal differences in IMTG utilisation during exercise when investigated on a subcellular-specific basis.…”

“…Whilst we acknowledge that using BODIPY may also not entirely replicate the observations made using high-resolution electron microscopy (because we observed exercise-induced decreases in IMTG content in both subcellular regions), our data suggest subcellular-specific IMTG utilisation during exercise should not be investigated using ORO. Future work that uses BODIPY to detect IMTG should likely employ a smaller peripheral region (e.g., 2 µm as used in Jevons et al 2020 ) since this may be more representative of the subsarcolemmal region detected using high-resolution electron microscopy.…”

“…An alternative neutral lipid stain is BODIPY 493/503 (BODIPY), which emits a bright green fluorescent signal. Methods for using BODIPY in combination with confocal immunofluorescence microscopy have been demonstrated in vitro (Listenberger et al 2016 ), in rodent skeletal muscle (Prats et al 2006 ; Spangenburg et al 2011 ) and human skeletal muscle (Jevons et al 2020 ; Prats et al 2013 ; Whytock et al 2020 ). Importantly, BODIPY is more specific for detecting IMTG-containing LD’s than Nile red in flow cytometry applications (Gocze and Freeman 1994 ), and when imaged using fluorescence emitted between 570 and 650 nm, it has been confirmed that most of the fluorescence is due to detection of IMTG (Prats et al 2013 ).…”

“…First, we investigated fibre type and region (peripheral and central)-specific differences in IMTG content detected using BODIPY and ORO in muscle biopsies obtained after an overnight fast (study 1). In this context, subcellular distribution is an important consideration because IMTG use during exercise occurs primarily in the central (intermyofibrillar) region of type I fibres of the cell (Jevons et al 2020 ; Koh et al 2017 ), and IMTG deposited in the peripheral (subsarcolemmal) region is inversely related to insulin sensitivity (Nielsen et al 2017 ; Nielsen et al 2010 ). We also critically reviewed several steps in the immunohistochemical protocol and analytical procedure that can affect the visualisation and quantification of IMTG content.…”

The Importance of Fatty Acids as Nutrients during Post-Exercise Recovery

“…Another study in trained men showed IMTG content to be lower compared to both pre-and post-exercise values at both 3, 6, and 18 h in recovery from exhaustive endurance exercise [7]. Importantly, the extent to which IMTG is utilized during exercise and recovery seems to depend on the muscle fiber type, since reductions in IMTG content were only obtained in type I and not in type IIa muscle fibers during [62] and 4 h [63] into recovery from a 2-4 h bicycle exercise bout at 56%-75% of VO 2 peak.…”