In the past we identified Vesicular Monoamine Transporter Type 2 (VMAT2), as a biomarker of beta cell mass that is quantifiable in vivo by Positron Emission Tomography (PET). PET is a tomographic imaging technique providing accurate non-invasive dynamic measurements of regional PET tracer uptake and clearance. These measurements are used to calculate VMAT2 density in the pancreas as a surrogate measure of beta cell mass. Immunohistochemical staining of both control and long term T1D pancreata show excellent correlation between VMAT2 and insulin staining. When PET and the radiopharmaceutical 18F-FP-DTBZ are used to non-invasively measure VMAT2 in vivo, we observe that healthy subjects have a significantly greater pancreatic VMAT2 signal than long term T1D patients with documented inability to secret normal amounts of insulin. However there is a significant residual VMAT2 signal in the pancreata of the T1D patients.

The goals of our project are to understand the nature of this background signal. There are three not mutually exclusive explanations of the background signal observed in pancreata believe to be beta cell ablated due T1D; 1) The radioligand 18F-FP-DTBZ is binding specifically to VMAT2 or some other receptors, not present in beta cells (specific and displaceable off-target binding). Specific off target binding is being measured in a clinical PET study currently in progress. We have also quantified, using immunohistochemistry, the expression of VMAT2 laying outside the beta cell subset in human pancreata (e.g. PP cells). The specific off target bind does not seem to account for the background, 2) 18F-FP-DTBZ may also bind non-specifically and non-displaceably to pancreas tissue (off target non-specific binding). The in vivo non-specific binding is currently being measured in clinical PET study in progress. Our in vitro estimates of non-specific binding using cadaveric pancreas tissue supplied by NPOD suggest that a significant portion of the in vivo background signal is due to non-specific binding. However, these in vitro methods may overestimate in vivo binding of the tracer and must be interpreted with caution, 3) The last formal possibility, that our measurements of VMAT2 in the long term T1D pancreas indeed are on target and specific, representing residual VMAT2 possibly associated with beta linage cells is now under consideration. Such a possibility seems to conflict with preexisting immunohistochemistry data, but immunohistochemistry is not the most sensitive technique available for detecting low levels of expressed proteins. The next phase of our project will be to study long term diabetes pancreata for VMAT2 gene expression using in situ hybridization and RT-PCR techniques. While the pancreas is a difficult organ to obtain RNA suitable for such studies, the generally excellent quality of NPOD tissues will improve our chances for success.