Development and validation of novel fluorescent probes to advance research into glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP1) receptor localisation and activity

Thesis

Diabetes and obesity are chronic conditions which cause significant impact upon the individual, healthcare services and society. Medications agonising glucagon-like peptide-1 receptor (GLP1R) and dual agonists targeting GLP1R and glucose-dependent insulinotropic polypeptide receptor (GIPR) are revolutionising treatment for type 2 diabetes and obesity and showing promise in other conditions such as MASLD and IIH. Dual agonists demonstrate superior efficacy compared to GLP1R agonists for glycaemic control and weight loss. However, the mechanisms of their superiority remain unclear as confirmation of GIPR presence and dual agonist cellular targets remains challenging. In this thesis, we present the development and validation of novel GIPR (sGIPs) and dual GLP1R/GIPR (daLUXendins) fluorescent agonist probes and apply them to relevant models to visualise cellular targets.

The sGIP probes demonstrate specificity in over- and endogenously expressed systems and suggest that GLP1R may be upregulated in GIPRKO models.

With the daLUXendin probes, we have generated non-lipidated (daLUXendin) and lipidated (daLUXendin+) forms. We show that the non-lipidated probes advantageously show greater selectivity for mouse GIPR over mouse GLP1R and label all major endocrine cell types in mouse islets and human stem-cell derived islet-like structures. Such labelling shows greatest intensity with ß cells > α cells = δ cells. Peripheral administration of daLUXendins labels circumventricular organs in a similar manner to single GLP1R agonists and antagonists, suggesting that depth of brain penetration is not a contributor to superior dual agonist efficacy. Central daLUXendin administration shows labelling of tanycytes. daLUXendins are also suitable for single-molecule imaging and enhance nanodomain formation compared to single agonist/antagonist binding.

Together, this thesis presents the novel sGIP and daLUXendin probes which are specific for their cognate receptors and versatile for use in a range of super-resolution imaging modalities. We have used them to confirm dual agonist targets in the pancreas and brain and presented interrogable avenues to investigate the superior efficacy of dual agonists compared to single agonists.

Authors: De Bray, AY

• DOI: 10.5287/ora-gpg5vjayz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Dual GLP1R/GIPR agonist; GIPR; GLP1R; fluorescent probe
• Subjects: Glucose-dependent insulinotropic polypeptide; Fluorescent labeling; Glucagon-like peptide 1; Obesity; Diabetes