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We examined the effects of carbohydrate (CHO) feeding on lipid droplet (LD) morphology, muscle glycogen utilisation and exercise‐induced skeletal muscle cell signalling. After a 36 h CHO loading protocol and pre‐exercise meal (12 and 2 g kg–1, respectively), eight trained males ingested 0, 45 or 90 g CHO h–1 during 180 min cycling at lactate threshold followed by an exercise capacity test (150% lactate threshold). Muscle biopsies were obtained pre‐ and post‐completion of submaximal exercise. Exercise decreased (P < 0.01) glycogen concentration to comparable levels (∼700 to 250 mmol kg–1 DW), though utilisation was greater in type I (∼40%) versus type II fibres (∼10%) (P < 0.01). LD content decreased in type I (∼50%) and type IIa fibres (∼30%) (P < 0.01), with greater utilisation in type I fibres (P < 0.01). CHO feeding did not affect glycogen or IMTG utilisation in type I or II fibres (all P > 0.05). Exercise decreased LD number within central and peripheral regions of both type I and IIa fibres, though reduced LD size was exclusive to type I fibres. Exercise induced (all P < 0.05) comparable AMPKThr172 (∼4‐fold), p53Ser15 (∼2‐fold) and CaMKIIThr268 phosphorylation (∼2‐fold) with no effects of CHO feeding (all P > 0.05). CHO increased exercise capacity where 90 g h–1 (233 ± 133 s) > 45 g h–1 (156 ± 66 s; P = 0.06) > 0 g h–1 (108 ± 54 s; P = 0.03). In conditions of high pre‐exercise CHO availability, we conclude CHO feeding does not influence exercise‐induced changes in LD morphology, glycogen utilisation or cell signalling pathways with regulatory roles in mitochondrial biogenesis.

This study aimed to quantify net glycogen utilization in the vastus lateralis (VL) and gastrocnemius (G) of male (n = 11) and female (n = 10) recreationally active runners during three outdoor training sessions.After 2-d standardization of carbohydrate intakes (6 g·kg body mass per day), glycogen was assessed before and after 1) a 10-mile road run (10-mile) at lactate threshold, 2) 8 × 800-m track intervals (8 × 800 m) at velocity at V˙O2max, and 3) 3 × 10-min track intervals (3 × 10 min) at lactate turnpoint.Resting glycogen concentration was lower in the G of female compared with males (P0.001) runners, although no sex differences were apparent in the VL (P = 0.40). Within the G and VL of male runners, net glycogen utilization differed between training sessions where 10 miles was greater than both track sessions (all comparisons, P0.05). In contrast, net glycogen utilization in female runners was not different between training sessions in either muscle (all comparisons, P0.05). Net glycogen utilization was greater in male than in female runners in both VL (P = 0.02) and G (P = 0.07) during the 10-mile road run. With the exception of male runners during the 3 × 10-min protocol (P = 0.28), greater absolute glycogen utilization was observed in the G versus the VL muscle in both male and female runners and during all training protocols (all comparisons, P0.05).Data demonstrate that 1) prolonged steady-state running necessitates a greater glycogen requirement than shorter but higher-intensity track running sessions, 2) female participants display evidence of reduced resting muscle glycogen concentration and net muscle glycogen utilization when compared with male participants, and 3) net glycogen utilization is higher in the G muscle compared with the VL.

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.

Intramuscular triglyceride (IMTG) only accounts for a small proportion of total lipid in the human body. However, over the past two decades there has been significant interest in the area of IMTG metabolism, largely due to the contribution that IMTG makes to ATP production during moderate-intensity exercise, and the proposed role of IMTG in the development of skeletal muscle insulin resistance. This article is protected by copyright. All rights reserved

Carbohydrate and lipid are important substrates during exercise, and their relative contribution is heavily dependent on exercise intensity and duration. Not only this, but it now appears that the utilisation of muscle glycogen and lipid (intramuscular triglyceride; IMTG) is specific to muscle fibre type and/or the subcellular location of these substrates. However, to date, our understanding of muscle substrate use stems largely from lab-based research. The first aim of this thesis was, therefore, to investigate muscle glycogen and IMTG utilisation on a fibre type and subcellular- specific basis in exercise undertaken in the field. The exercise-induced decrease in muscle glycogen and IMTG necessitates a replenishment of these substrates in the post-exercise period, where traditionally carbohydrate ingestion is the focus. However, the mechanisms regulating post-exercise IMTG resynthesis, especially in the face of carbohydrate ingestion is not well understood. Therefore, a second aim of this thesis was to examine the influence of post-exercise carbohydrate consumption on IMTG resynthesis and the muscle mechanisms regulating this. Study 1 aimed to quantify glycogen and lipid utilisation in the vastus lateralis and gastrocnemius muscle of 11 recreationally active male runners during either steady state endurance exercise (10-mile trial) or high intensity interval exercise (8x800m trial) in a field-based training environment. Using transmission electron microscopy (TEM), this study identified 1) a preferential use of intramyofibrillar glycogen during exercise, independent of exercise trial, and 2) lipid utilisation occurred primarily in the intermyofibrillar region of type I fibres during steady state exercise. These data highlight a novel pattern of muscle glycogen and lipid utilisation during field-based exercise which is dependent on exercise intensity and duration. Study 2 aimed to examine if immunofluorescence microscopy could be used to investigate changes in IMTG on a subcellular level, and additionally compared the agreement between immunofluorescence microscopy with TEM. This study continued the field-based ‘theme’ examining IMTG utilization during a competitive endurance event. ~1 hour of cross-country skiing in a field-based environment reduced IMTG i content in both type I and type IIa fibres, and in both the peripheral and central subcellular regions. When assessing the agreement between the two microscopy methods, it appears that immunofluorescence microscopy provides a suitable method to quantify IMTG content, but not LD morphology. Therefore, these data have important implications for future studies aiming to understand the mechanisms regulating subcellular LD pools. As such, this directly influenced the methodology for study 3 of this thesis. Finally, study 3 aimed to investigate the hypothesis that post-exercise IMTG resynthesis would be accelerated under conditions of acute CHO restriction in elite endurance athletes, with a secondary aim to investigate changes in the distribution of perilipin proteins relative to LDs during exercise and in the post-exercise period. IMTG resynthesis was shown to occur rapidly by 4 h post-exercise in the central region of type I fibres following prolonged exercise in highly-trained individuals, independent of CHO availability. Further, increases in IMTG content following exercise preceded an increase in the association of LDs with regulatory perilipin proteins. These data, therefore, suggest that the perilipin proteins do not play a key role in the mechanism of IMTG synthesis, as originally believed. Overall, this thesis expands our knowledge on the effect of exercise intensity and duration on substrate utilisation by investigating this on a fibre and subcellular basis, whilst also providing an insight into the field-based demands of training and competition on substrate utilization. Furthermore, the work deepens our understanding of the proposed role of the LD regulatory perilipin proteins during IMTG breakdown and resynthesis by providing key insights into the mechanisms underpinning IMTG resynthesis. From a research perspective, it is hoped that this thesis will elicit further field-based studies examining substrate utilisation on a fibre and region-specific basis.

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 P