Stimulated Glucagon as a Biomarker of Hypoglycemic Risk in Type 1 Diabetes

Blood glucose levels are normally controlled by insulin, a hormone that lowers high blood glucose levels, and glucagon which is released into the blood to raise glucose when levels are low. For people with Type 1 diabetes (T1D), taking insulin medication to lower glucose levels can sometimes result in dangerously low blood sugar levels (hypoglycaemia). The ability to release glucagon and correct blood glucose can vary, particularly in people who have had T1D for years. The study aims to find out how blood glucagon levels after a stimulus compare to a person's own experience of hypoglycaemia, and hypoglycaemia. This research will establish whether glucagon measured after a meal could be used as a blood marker of hypoglycaemia risk and help to identify individuals at high risk.

Hypothesis: Low post-mixed meal (MM) stimulated glucagon secretion predicts hypoglycaemia.

Prior to consent, all potential participants will be provided with detailed written information about the study (that complies with the UK General Data Protection Regulation (GDPR) and Data Protection Act 2018) and an opportunity to discuss it with one of the research team. All participants will be informed of their right to withdraw from the study at any time without prejudice or jeopardy to any future clinical care.

A unique study ID will be allocated to the participant to link all participant study information and samples. The participant's clinical characteristics and sample results will be recorded on the participant's Case Report Form and entered on the study database.

All participants will undergo core data collection, measurements and provide fasted and stimulated blood samples via a cannula at the baseline MMTT and AST visits 1 and 2 and again at the 6 months 'Light MMTT' / repeat AST visit. The order of MMTT and AST tests in visits 1 and 2 will be randomised to check for an order effect. Test choice for Visit 3 depends on the peak glucagon results from Visits 1 and 2. At Visits 1 and 3, participants will be asked to complete the hypoglycaemia questionnaire data (Clarke and Gold scores) to assess awareness of hypoglycaemia and quality of life measures which may relate to the MM-glucagon. Participants will be given an Abbot FreeStyle Libre Pro continuous blood glucose monitor (CGM) and asked to perform a 2-week continuous measure of blood glucose (CGM). Clinical diabetes care will not be changed and participants with T1D will use their own insulin regimes and self-monitoring (even if CGM or Abbot FreeStyle Libre Pro) during the study.

Fasted samples will be obtained for baseline measures: routine glucose and HbA1c, plasma and serum for storage and future batched analysis including measures of glucose, glucagon, insulin (C-peptide) and other markers of interest, plus sodium heparin and PAXgenesRNA samples for T1D immune function analysis.

Stimulated samples (see test descriptions below): insulin (C-peptide), glucose and glucagon will be measured at all time points.

At the end of each test period, the participant's blood glucose will be checked and appropriate advice will be given relating to insulin dosage adjustments and a light meal will be provided.

1. Mixed Meal Tolerance Test (MMTT) at Visit 1 or 2 (approx 2½ hours) Rationale: The MMTT will provide confirmatory analysis of post-mixed meal glucagon stored in BD P800 and EDTA plasma samples at a range of standard MMTT time points (0, 30, 60, 90 and 120 min) in 75 people. Glucose will be measured in Fluoride Oxalate samples at the same time points.

Participants will:

• be monitored closely throughout their visit for any discomfort or problems and appropriate advice/support will be given if required.
• be given Ensure HP or Fortisip (6ml/kg to a max of 360mls)
• have blood samples collected, to measure insulin, C-peptide, glucose, and glucagon, at specific intervals: -10, 0, 30, 60, 90, 120 minutes post meal.

1. Arginine Stimulation Test (AST) Visit 1 or 2, and Visit 3 (approx 1 hour) Rationale. An MMTT (despite being convenient because it avoids the need for an IV stimulus) has limitations, including differences in gastric emptying, uptake and effects on enteroendocrine secretion. Therefore, responses will be compared during the MMTT with those obtained during an intravenous arginine challenge, Arginine Stimulation Test (AST), (5 g Arginine during 1 min) at a separate visit from the MMTT visit. Glucose, plasma glucagon and C-peptide will be measured at baseline and specific intervals following the Arginine bolus injection. This test is designed primarily to test the maximum insulin secretion capacity of a participant's beta cells.

The AST test involves:

• Set up. Intravenous catheters will be inserted into antecubital veins in both arms. One arm will be used for infusion of Arginine (amino acid), and the other arm for intermittent sampling.
• Baseline samples will be taken at -10 and 0 minutes. A maximally stimulating dose of Arginine Hydrochloride (5 g) will be injected intravenously for 45s. Samples will be collected at 2, 5, 10, 15 and 30 minute intervals following the Arginine bolus.

1. Home Finger Prick Sample (optional) - collected at home the day after each visit and returned to the research team, using pre-paid packaging provided.

Data Collection and Recording: Data will be initially recorded using a study-specific case report form (CRF), with hard copies stored in the Trial Master File (TMF). Anonymised research data will be recorded on a study-specific database and reviewed for discrepancies and missing data prior to analysis.

End of Study Definition: The end of the study is 8 months after the final participant's Visit 3 to allow for final collection of data.

Criteria for premature withdrawal from the study: Where a participant withdraws, data will be collected up to the point of withdrawal in line with the protocol and study SOPs. Where a participant has prematurely withdrawn but not revoked consent, data and samples will remain within the study and included in any analysis. Where consent is withdrawn, study samples and data will be kept/destroyed as per local guidelines.

Samples: A detailed SOP/work instruction will detail the clinical procedure for sample collection, labelling, logging and management. Central laboratory analysis of the study samples will be undertaken at the Exeter Blood Sciences Laboratory.

Stored samples: Surplus samples for storage should be processed, logged and frozen at -80°C within 24 hours of receipt. All saved samples will be stored under the study ID, with the file linking the study code to personal identifiable information held securely by the Principal Investigator, accessible only to personnel with training in data protection who require this information to perform their duties.

Safety, Definitions and Reporting:

Risks: The blood samples will be collected by members of the research team trained in venepuncture. During the MMTT/AST test, there is a possible risk of hyperglycaemia and appropriate advice/support will be given if required.

Benefits: The study may improve understanding of participants' hypoglycaemia risk. In the longer term, this work may provide evidence that the ability of alpha cells to release glucagon after a mixed meal is variable within people with T1D, associated with hypoglycaemia risk, and could be a biomarker for hypoglycaemia risk. Testing a blood spot assay for C-peptide on home finger prick samples may enable regular assessment of beta cell function without the need for patients/research participants to visit a healthcare/research centre which could revolutionise future diabetes care and research.

Definitions and reporting of adverse effects:

The timeframe for recording SAEs will be from the time of consent to one week following the last visit of a study subject. Any reportable adverse effects noted will be reported within 24 hours to the CI and the Sponsor as per standard NHS R\&D protocols.

Insurance: Sponsor's public liability will be provided by the University of Exeter for the design and management of the study. NHS indemnity will apply for the conduct of the study at the site.

Data Handling \& Record Keeping: The CI is responsible for ensuring that participant anonymity is protected and maintained, ensuring that their identities are protected from any unauthorised parties. The CI is the 'Custodian' of the data. All information related to study participants will be kept confidential and managed in accordance with the UK GDPR and Data Protection Act 2018, NHS Caldicott Guardian, UK Policy Framework for Health \& Social Care, and Research Ethics Committee Approval.

A unique Study ID will be allocated to each participant. All study data and samples will be pseudo-anonymised and stored under the Study ID on a secure password-protected study database. Identifiable information will be stored on a separate, secure password-protected database held on an NHS server to enable the research team to undertake the study.

All paper copies of study data will be stored under ID number and kept in locked, access-controlled offices within the research facilities; research data will be held separately to identifiable information. Researchers involved in data analysis will not have access to personal identifiable data, only the anonymised research data. No identifiable data will be included in research publications or progress reports.

Any participant information required to be sent to a third party will adhere to these pseudo-anonymised parameters. No participant identifiable data will be sent outside the EU.

Record Retention and Archiving: When the research study is complete, it is a requirement of the UK Policy Framework for Health \& Social Care and Sponsor Trust Policy that the records are kept for a further 15 years. At the end of the study, anonymised data will be included in the study's final dataset and stored indefinitely on vivli.org or similar sponsor/funder approved data repository. Personal data will be stored where consent is given by the participant to be contacted for follow-up on their future health status, and/or about participating in future studies. Where consent is given by the participant, their samples and data from the project will be gifted to the Peninsula Research Bank (an approved tissue bank, REC ref 19/SW/1059) to be used for future research.

At the end of the study, it will be archived by the CI at the University of Exeter.

Statistical analysis: For a sample size of 75, it should be possible to detect a correlation between stimulated glucagon and hypoglycaemia of r=0.3, with 95% confidence intervals 0.08 to 0.49, plus correlation of MM-induced increases in plasma glucagon to plasma co-peptin.

Compliance: The CI will ensure that the study is conducted in compliance with the principles of the Declaration of Helsinki (2013), the principles of GCP and in accordance with all applicable regulatory requirements including but not limited to the UK Policy Framework for Health \& Social Care and the Medicines for Human Use (Clinical Trial) Regulations 2004, as amended in 2006, 2008 and 2009 and any future relevant or replacement EU Regulations, Trust and R\&D Office policies and procedures and any subsequent amendments.

Publication Policy: Results will be written up and submitted for publication in a peer-reviewed journal(s). Abstracts will be submitted to national and international conferences. Results will be presented to clinical colleagues at regular in-house meetings. A letter/newsletter outlining the key findings of the study will be sent to all participants and will be uploaded to the study website. An in-person/online event may also be arranged.