title: Quantification of substrate metabolism in endurance athletes using microscopy techniques.
Creators: name: Jevons, E
Publisher: name: Liverpool John Moores University
Year: 2021

description: 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.