Effects of glycogen availability on human skeletal muscle molecular responses to exercise

Ten healthy and recreationally active males aged between 18 – 30 years old with a body mass index (BMI) of 18.5 – 27.0 kg/m2 will be recruited to participate in this study to investigate the effects of skeletal muscle glycogen availability on molecular responses to exercise. Preliminary testing (week -2) VISIT 1 (~30 mins): At the first visit, participants will meet the trial coordinator, go through the participant information letter and have an opportunity to ask any questions they may have about the study. 1. Prior to any measurement participants will sign the study consent and muscle biopsy consent forms. 2. The Exercise and Sports Science Australia (ESSA) pre-exercise screening tool will be completed with trial coordinator. 3. Three-day food record explained: Participants will be asked to record ALL food and fluid consumed over a three-day period prior to the experimental period to establish habitual dietary intake using the ‘Easy Diet Diary’ or ‘My Fitness Pal’ phone application. To assist with compliance and reduce participant burden, household measures will be recorded. 4. Anthropometric measurements: Height will be measured using a wall mounted stadiometer. Weight will be measured using digital scales. Hip, waist and neck circumferences will be measured with a metal tape measure. All measures will be taken at the Australian Catholic University (ACU) lab in duplicate. VISIT 2 (~2 h; week -1): At the second visit, participants will participate in the following measures: 1. Body composition assessment: A dual-energy x-ray absorptiometry scan (DXA) is a specialised x-ray technique used to provide a measure of body composition (i.e. total body mass, fat mass and lean mass). Participants are required to lay supine on the scanning bed based at ACU for the duration of the scan, which is approximately 15 min and will be one to two scans depending on body shape. The machine uses small doses (<1% of the yearly radiation dose) of radiation to estimate tissue density. This test requires participants to be fasted with no food, fluid or exercise/activity prior to the test and requires participants to wear light clothing with no metal items (i.e. zips, domes, clips, underwire etc.) and remove all jewellery. Measures will be obtained by trained researchers who hold radiation licenses with Victorian Government and comply to the Code of Practice set out by the Australian Radiation Protection and Nuclear Safety Agency. 2. Maximum effort cycling exercise test [VO2 peak]: An experienced exercise physiologist will assess aerobic capacity at the ACU lab by measuring the maximum volume of oxygen (VO2) participants can breathe in and transport to the working muscles while exercising at maximal intensity. After a light 10 min warm-up on a cycling ergometer (exercise bike), participants will pedal at a fixed intensity (watts) that will increase every 1-2.5 min until volitional fatigue. During this test participants will wear a mouthpiece and headpiece that is connected to a metabolic cart for the collection of expired air. Following the warm-up, the test will last for 10-15 min where the last few minutes should be perceived as exhaustive, with recovery at low intensity for 2-3 min. VISIT 3 (~2 h; > 72 h post Visit 2): At the third visit, participants will participate in the following protocol: 1. One-legged glycogen depletion and two legged high intensity cycling exercise familiarization: Subjects will arrive at the ACU lab mid-afternoon to be familiarised with both one-legged cycling (to be used during glycogen depletion session) and two-legged interval cycling (to be used for exercise trial day) as practice runs for the glycogen depletion and exercise experimental trial day. Between each exercise protocol familiarisation performed with an experienced exercise physiologist, participants will rest for 15-30 min. VISIT 4 (~3 h; > 72 h post Visit 3): At the fourth visit, participants will participate in the following measures: 1. Continuous glucose monitoring (CGM): Participants will be provided with a continuous glucose monitor to assess circulating glucose levels before the glycogen depletion protocol, and this monitor will remain on the participant until the next morning following the experimental exercise session. These monitors enable detection of blood glucose changes associated with glycogen depletion and diet to ensure the participant has not consumed any food other than what was provided for their evening standardised meal. 2. One-legged muscle glycogen depletion exercise protocol: The exercise glycogen depletion protocol will be performed with an experienced exercise physiologist at the ACU lab and consist of repeated 10-min work bouts with 2 min recovery between work bouts. The time spent completing these work bouts will be ~100 min. This work-to-rest ratio will continue until volitional fatigue at which time power output will be decreased by 10W. Subjects then will repeat the same work-to-rest ratio until fatigue at this reduced work rate. Following a 10 min rest, subjects then will complete a series of 90-s one-leg maximal sprints (with 60-s recovery) on a Repco RE7100 Ergo cycle ergometer until volitional fatigue, defined as the inability to maintain 70 revolutions/min. When participants successfully complete the entire duration of this glycogen depletion exercise protocol, they will achieve the final eligibility component of this study and be formally enrolled to complete the experimental day. 3. Standardised meal: Subjects will consume a low-CHO meal with macronutrient targets based on body mass by an Accredited Practicing Dietician (APD) to minimise glycogen re-synthesis in the 'low glycogen' leg after the glycogen depletion exercise. Prior to undertaking the glycogen depletion, subjects will be instructed to refrain from exercise training and vigorous physical activity, and avoid any alcohol, caffeine and/or food consumption until completing the experimental day. Experimental day and blood/biopsy collection VISIT 5 (~5 h; 12 h post Visit 4): At the fifth visit, participants will participate in the following measures: 1. Muscle biopsy sampling (pre-exercise, immediately post-exercise, 3 h post-exercise): A total of 6 muscle biopsies will be taken over the entire study period by an experienced sports medicine physician, with 3 biopsies taken from each leg on this experimental visit. Samples will immediately be placed in liquid nitrogen for freezing and stored at -80 °C. In preparation for a biopsy, a small amount of local anaesthetic is injected under the skin, which may result in a mild burning sensation while the fluid is injected. A small, 4-5 mm incision is then made into the skin to create an opening for the biopsy needle. There is often a small amount of bleeding from the incision; however, this bleeding is generally minimal. The biopsy needle is then inserted through the incision site. Participants may feel the sensation of deep pressure in the biopsy site, and on some occasions this is moderately painful. However, the discomfort very quickly passes and participants are capable of performing exercise and daily activities within minutes. There may also be some minimal bleeding when the needle is removed which may require the application of pressure for a few minutes. After each biopsy, the incision will be closed with sterile tape and wrapped with a bandage. 2. Blood sampling and analysis (pre-exercise, immediately post-exercise, and 3 h post-exercise): A total of 3 blood samples will be collected over the entire study period via an in-dwelling venous cannula inserted by the sports medicine physician: 12ml blood samples will be taken at 3 time points only for the two-legged high intensity exercise protocol on the experimental day. A total volume of 36 mL of blood will be collected for the whole study across, which is minimal compared to the standard 500 mL for a Red Cross donation. Samples will be collected in two 6 mL vacutainers, with one containing EDTA for plasma and one uncoated for serum. Samples will then be centrifuged and aliquots of plasma/serum will be stored at -80 °C. 3. Experimental Exercise Session (20 x 1 min two-legged high intensity cycling exercise, interspersed with 20 x 1 min active recovery) Participants will have previously completed the glycogen depletion exercise during Visit 4, and the following morning they will complete the following experimental two-legged high intensity cycling exercise protocol with an experienced exercise physiologist in Visit 5: 4. Two legged high intensity cycling exercise protocol: Subjects will report to the ACU lab at 0700 h on the morning of an experiment having completed the glycogen depletion protocol the previous evening. Resting biopsies from the vastus lateralis of the L and R legs will be taken under local anaesthesia. To determine the effects of muscle glycogen availability on exercise-regulated protein and gene responses, subjects will then undertake two-legged cycling 20 x 1 min at a maximum of 110-125% PPO interspersed 20 x 1 min active recovery at 50% PPO. This protocol provides sufficient stimulus to activate proteins and genes in both legs and is the maximum intensity that is sustainable in the ‘low’ glycogen limb. Two further biopsies from both legs will be taken: one immediately upon cessation of exercise to examine the acute protein responses and another 3 h later for the subsequent gene responses. 5. Removal of CGM. 6. Follow up care: Shortly after the completion of the final muscle biopsy sampling at 3 h post-exercise, participants will be offered a light snack (e.g. raisin toast, cereal, Up n' Go). A waterproof dressing will be applied to the muscle biopsy sites on each leg by the sports medicine physician. Participants can shower normally, and for those who may be swimming or sweating heavily, additional dressings and instruction can be provided. Wounds from the muscle biopsies normally heal within four days.