T1D is a classic example of an organ-specific autoimmune disease. It has two stages. The first is an occult phase, termed insulitis, in which a mixed population of leukocytes invades the islets of Langerhans of the pancreas. This, in turn, eventually provokes specific destruction of the insulin-producing beta cells. The second stage is the overt phase (diabetes), in which the bulk of beta cells have been destroyed, and insulin production is no longer sufficient to regulate blood-glucose levels. This eventually results in hyperglycemia. To date, a major hindrance to both research on and treatment of T1D, has been the inability to directly visualize beta cells during disease progression. Our goal has therefore been to develop robust, non-invasive methods to image BCM (both experimentally in mouse models and clinically). Thus far, we have tested most of the published fluorescent probes in vivo in MIP-GFP mice, but with little or modest success. Based on profiling studies, we also investigated a number of novel targets including carboxypeptidase E (CPE), proprotein convertase subtilisin/kexin 2 (PCSK2) or islet amyloid polypeptide (IAPP), among others. This study now reports on the first selective fluorophore-based probe for in vivo imaging of pancreatic beta cells, and we confirm its potency in biochemical assays, in in vitro assays, by histology and by in vivo imaging.
The islets of Langerhans, encompassing the endocrine pancreas, represent approximately 1–2% of the pancreatic mass. They are also scattered throughout the pancreas, which is likely the underlying reason for the previous difficulties in visualizing BCM with macroscopic imaging techniques (5
). Within each islet, there are five major endocrine cell types: insulin-producing beta cells (60–80% of the islet), glucagon-producing alpha cells (15–20% of the islet), somatostatin-producing delta cells (5–10% of the islet), and pancreatic-polypeptide-producing (PP) cells (15–20% of the islet), and ghrelin-producing epsilon-cells (< 1%).
The adult mouse pancreas contains about 3,000 islets, with about 2×106
beta cells (27
). The human pancreas contains about 106
islets. These pancreatic islets measure 50–300 μm in diameter and are highly vascularized, with 1–5 arterioles branching into numerous capillaries. Capillaries are fenestrated and are therefore highly permeable. It has been estimated that islets receive 10–20% of pancreatic blood flow, which is disproportionally large for an area that assumes 1 to 2% of pancreatic volume and < 0.005% of body weight. This feature, however, creates a favorable environment for the delivery of imaging agents. In our studies, we used intravital confocal microscopy to study islet microvascularity and were able to show rapid delivery of the E4K12
-Fl agent to the pancreatic islets with high spatial overlap with EGFP labeled beta cells.
In the pancreas of mice, rats, and humans, the GLP-1R is highly expressed within the islets and to a lesser extent within some ducts. Within the islets the GLP-1R protein almost exclusively co-localizes with insulin and is highly restricted to pancreatic beta cells (28
). This distribution makes the GLP-1R a good target for measuring BCM. Glucagon-like peptide-1 (GLP-1) is secreted by intestinal L cells as a gut hormone and its secretion is dependent on the presence of nutrients in the small intestine. The physiological functions of GLP-1 include potentiation of insulin secretion in a glucose dependent manner as well as influencing a number of gastric functions (slowing gastric emptying and inhibiting acid secretion). GLP-1 analogues are a new class of drugs for the treatment of diabetes as they have a low risk of causing hypoglycemia (29
). Exenatide (Byetta), the first FDA approved GLP-1 analog, is a synthetic version of exendin-4, a hormone found in the saliva of the Gila monster. It has 53% sequence homology with GLP-1 (30
). A recent crystal structure shows that exendin-4 binds to the extracellular domain of GLP-1R (30
). From this crystal structure, it follows that the amino acid K12 is not involved in GLP-1R binding () and explains its efficacy as an imaging agent. Conversely, other modifications with fluorochromes do alter binding.
In this study, we focused on the exendin-4 analog E4K12
-Fl. There are, however, several other pharmaceutical analogues of GLP-1 that are currently in development or in clinical use, including liraglutide, albiglutide and taspoglutide, each of which have variable substitutions and modifications that alter their pharmacokinetics (21
). It is entirely possible that we will be able to further optimize probe development by using some of these newer GLP-1 analogues as platforms. Another potential avenue for probe development would be to use the peptide as a targeting ligand for the delivery of multimodal, nanoparticle based constructs for imaging (31
) and or therapeutics (32
). We believe that the featured E4K12
-Fl, and possibly similar derivatives, will prove useful agents for the selective visualization of beta cells. Finally, it appears reasonable that more immediately one should be able to use the described agent in the imaging of often difficult to localize insulinomas, possibly via positron emission tomography (PET) tracers, via magnetic resonance imaging (MRI) with agents based on this peptide, or by intraoperative imaging of fluorescent compounds as described.