Participants and recruitment The present study will be a pilot, cross-sectional trial in individuals with CFRD (n = 15), as well as age- and gender-matched CF controls without diabetes (n = 15) and age- and gender-matched healthy control participants (n = 15). Participants will be recruited from adult and paediatric CF outpatient clinics within the Southampton CF network. The healthy control participants will be recruited from the University of Portsmouth and local area. Individuals with CF will be tested at a laboratory established within the CF unit at the Southampton General Hospital and/or the Department of Sport and Exercise Sciences (University of Portsmouth), depending on participant convenience. All healthy control participants will attend the Department of Sport and Exercise Sciences at the University of Portsmouth.
Visit 1 During this visit, a valid informed consent and GCP trained member of the research team will fully explain the information sheet and study protocol, as well as answer any questions to ensure that all relevant parties are clear about the study requirements. Following this, fully informed written consent will be obtained. Additionally, informed written assent will be obtained from those \< 16 years of age.
Following the consent procedures the participants resting pulmonary function (forced vital capacity, forced expiratory volume in 1 second, mid force expiratory flow and peak expiratory flow) and anthropometric measures (weight, height and body fat percentage) will obtained. The subsequent aim of this visit is then to habituate all participants with exercising on a cycle ergometer in preparation for their CPET in visit 2, in particular maintaining a certain cadence at a given power output. Appropriate adjustments will be made to the ergometer seat and handlebar position, and noted for visit 2. Furthermore, the protocols of obtaining ones ratings of perceived exertion and dyspnoea will be fully explained. Lastly, the participant will be instructed to avoid consuming mouthwash for the entirety of the study period due to evidence suggesting it may influence the uptake of dietary nitrate.
Visit 2 Participants will arrive to the laboratory at 1900 ± 2 hours. They will be instructed to arrive 2 hours postprandial, having avoided caffeine for \> 12 hours, as well as nitrate rich foods, alcohol and exhaustive exercise for \> 24 hours.
Upon arrival, CGM's will be fixed to the interior surface of the upper arm and worn for the subsequent 14 days. In addition to this, hip worn accelerometers, as well as hourly specific physical activity and food diaries will be distributed and completed for 14 days alongside the CGM. The participants resting pulmonary function will be assessed using the spirometry procedures described below, and a resting blood sample will be collected via venepuncture for the analysis of plasma \[NO-(₂)\] and ET-1. Additionally, the cycle ergometer CPET with a supramaximal (Smax) verification phase will be employed to determine ones aerobic exercise function, and a second venous blood sample will be collected immediately following exercise termination for the analysis of plasma \[NO-(₂)\] and ET-1. The total volume of blood collected over this 2 hour visit will be approximately 20 mL.
Visit 3 Participants will be required to arrive to the laboratory ≥ 3 days post visit 1, at 0800 ± 2 hours, following an overnight fast (\> 10 hours). Furthermore, participants will be instructed to avoid nitrate rich foods, caffeine, alcohol and exhaustive exercise for 24 hours prior to arrival.
Upon arrival participants will undergo the acetylcholine (ACh) and insulin iontophoresis protocols described below, including 5 resting blood pressure measurements. Immediately following this, the participant's pulmonary function will be assessed via spirometry. Participants will be asked to rest, and entertainment will be provided, for the following 60 minutes. The iontophoresis procedures will then be repeated to assess the short-term test-retest variability of the iontophoresis procedures. Following this, a cannula will be inserted into a vein by a trained phlebotomist, prior to the 3 hour OGTT and a baseline blood sample will be taken (measuring all the below biomarkers). Venous blood samples will be drawn at 30, 60, 90, 120 and 180 minutes post glucose ingestion for the analysis of glucose, insulin, active glucagon-like peptide-1 (GLP-1), TNF-α, soluble vascular cell adhesion molecule-1 (sVCAM-1), IL-6, \[NO-(₂)\] and ET-1. NT, total glutathione (tGSH) and total cysteine (tCys) will be analysed at 120 minutes post-glucose ingestion. The total volume of blood collected over this 5 hour visit will be approximately 176 mL. Additionally, the iontophoresis procedures will be repeated at 30, 90 and 150 minutes post-glucose ingestion (i.e. hourly measurements of microvascular endothelial function).
Follow-up CGM's, accelerometers and physical activity/food diaries will be completed for 14 days following visit 1. A member of the research team will collect these from the preferred location of the participant.
