Project Abstracts - New Investigators

Start Date: May 1, 2023

NIH HIRN Gateway Investigator Award Recipient 

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Abstract

To date, in vivo measurement of beta cell mass (BCM) is an unmet clinical need that, if possible, would allow tracking and monitoring of recent advances in therapies designed to preserve BCM1 and/or monitor transplanted beta cells.2 Beta cell loss occurs in the endocrine pancreas resulting in loss of insulin secretion and subsequent onset of diabetes. Current clinical in vivo measurements of beta cell function (e.g., C-peptide release in response to oral glucose tolerance test (OGTT)) may underrepresent total BCM due to variability in beta cell function in response to glucose. Beta cells and neurons share common cellular transporters and receptors, and with this in mind, we previously screened potential positron emission tomography (PET) neuroradioligands, already approved for use in humans, to image BCM. From this effort, we published two previous studies demonstrating the potential utility of [11C](+)-PHNO (D2/D3 receptor agonist) to differentiate healthy controls and individuals with type 1 diabetes (T1D).3,4 Dopamine is synthesized within beta cells and co-released with insulin and both D2 and D3 receptors have been shown to regulate insulin secretion.5,6 Interestingly, four of the individuals with T1D that had no measurable C-peptide release had measurable proinsulin and uptake of [11C](+)-PHNO,4 possibly suggesting a subset of not fully functional beta cells, as has been shown previously with proinsulin.7–9 In addition, we found a significant positive correlation of age-at-diagnosis and [11C](+)-PHNO uptake. Previously, age-at-diagnosis has been interpreted to reflect severity of disease (e.g. more severe beta cell loss in younger individuals).9–11 We also performed preliminary immunofluorescence in a healthy control and T1D pancreas.4 We found colocalization of both D2 and D3 receptors with insulin in the healthy pancreas while there was no colocalization with glucagon, somatostatin, and polypeptide Y. In the T1D pancreas, no staining of insulin or dopamine receptors was found, suggesting [11C](+)-PHNO radioligand is binding to D2/D3 receptors on only beta cells. In a preliminary analysis, we recently used state-of-the art proteomics to successfully identify 5382 islet proteins derived from tissue of a single healthy human islet donor; of which at least 200 are categorized as known islet proteins. We used pathway analyses in this sample and found multiple beta cell networks and beta cell-unique proteins associated with our proposed radioligand: D2/D3 receptors. Additional ex vivo human pancreas and islet studies are required to further characterize uptake of [11C](+)-PHNO in human beta cells and fits well within the Human Islet Research Network – Human Pancreas Analysis Consortium (HIRN-HPAC) mission. Evidence of both functional and non-functional BCM has been demonstrated in previous immunohistochemistry characterizations of T1D individuals;10,11 however, similar studies, with the addition of dopamine receptor (D2/D3) immunostaining remain to be performed in T1D. In addition, our assumptions regarding radioligand binding characteristics of dopamine receptors in the pancreas are largely based on those of the brain receptors.4,12 Tissue homogenate binding studies to determine the number of receptors on human beta cells (Bmax) and receptor affinities (Kd) are still necessary.13 We hypothesize dopamine receptors are localized to beta cells and [11C](+)-PHNO PET imaging can be used to assess BCM. To address this hypothesis, we propose to perform immunofluorescence and [11C](+)PHNO homogenate binding studies in healthy and T1D human pancreas tissue collected from surgical margins and/or autopsies and in isolated human Islets (Integrated Islet Distribution Program) (Aim 1). As an exploratory arm of aim 1, the effect of inflammation on islet proteome pathways will be explored in control and ‘stressed’ state (e.g, lipopolysaccharide exposure) to determine the effects on dopaminergic networks in the Islet proteome. To further explore the utility of [11C](+)PHNO PET, in combination with clinical measures such as Cpeptide and proinsulin, we propose to perform PET imaging in three distinct populations; 1) age-matched non-diabetic individuals, 2) individuals with recent onset T1D with detectable C-peptide enrolled in trialNET and 3) individuals with longstanding T1D. If successful, these studies will provide the basis to move from cross-sectional studies to longitudinal monitoring of BCM in trialNET participants to understand efficacy of T1D therapy to maintain BCM. SPECIFIC AIM 1: Ex vivo characterization of [11C](+)-PHNO to measure beta cell mass Hypothesis: We hypothesize ex vivo studies using immunofluorescence will show colocalization of insulin (beta cells) and D2/D3 receptors. Homogenate binding studies, using human islets from the IIDP Islet Award Initiative, will be used to determine D2/D3 receptor density (Bmax) and affinities (Kd). Immunofluorescence will be performed to examine colocalization of insulin and other pancreatic endocrine hormones with D2 and D3 receptors to determine their relationship in human beta cells and the greater Islet architecture. SPECIFIC AIM 2: Cross-sectional PET imaging of non-diabetic, recent onset T1D and longstanding T1D cohorts Hypothesis: We will be able to detect a significant linear correlation between [11C](+)-PHNO uptake and proinsulin (1 predictor, n=12, effect size d≥0.82) with 80% power based previous correlations (Figure 3D; R2=0.45). We will increase our understanding of [11C](+)-PHNO uptake by recruiting three distinct populations; 1) age-matched non-diabetics (n=3) 2) a novel cohort of recent onset T1D and detectable C-peptide enrolled in trialNET (n=6, highest expected within group variance, higher enrollment) and 3) longstanding T1D (n=3).