Quantification Of Beta Cell Mass Research Articles

3D quantification of changes in pancreatic islets in mouse models of diabetes type I and II.

ABSTRACTDiabetes is characterized by rising levels of blood glucose and is often associated with a progressive loss of insulin-producing beta cells. Recent studies have demonstrated that it is possible to regenerate new beta cells through proliferation of existing beta cells or trans-differentiation of other cell types into beta cells, raising hope that diabetes can be cured through restoration of functional beta cell mass. Efficient quantification of beta cell mass and islet characteristics is needed to enhance drug discovery for diabetes. Here, we report a 3D quantitative imaging platform for unbiased evaluation of changes in islets in mouse models of type I and II diabetes. To determine whether the method can detect pharmacologically induced changes in beta cell volume, mice were treated for 14 days with either vehicle or the insulin receptor antagonist S961 (2.4 nmol/day) using osmotic minipumps. Mice treated with S961 displayed increased blood glucose and insulin levels. Light-sheet imaging of insulin and Ki67 (also known as Mki67)-immunostained pancreata revealed a 43% increase in beta cell volume and 21% increase in islet number. S961 treatment resulted in an increase in islets positive for the cell proliferation marker Ki67, suggesting that proliferation of existing beta cells underlies the expansion of total beta cell volume. Using light-sheet imaging of a non-obese diabetic mouse model of type I diabetes, we also characterized the infiltration of CD45 (also known as PTPRC)-labeled leukocytes in islets. At 14 weeks, 40% of the small islets, but more than 80% of large islets, showed leukocyte infiltration. These results demonstrate how quantitative light-sheet imaging can capture changes in individual islets to help pharmacological research in diabetes.

Species differences in pancreatic binding of DO3A-VS-Cys40-Exendin4

AimsRadiolabeled Exendin-4 has been proposed as suitable imaging marker for pancreatic beta cell mass quantification mediated by Glucagon-like peptide-1 receptor (GLP-1R). However, noticeable species variations in basal pancreatic uptake as well as uptake reduction degree due to selective beta cell ablation were observed.MethodsIn vitro and ex vivo autoradiography studies of pancreas were performed using [177Lu]Lu-DO3A-VS-Cys40-Exendin4, in order to investigate the mechanism of uptake as well as the islet uptake contrast in mouse, rat, pig, and non-human primate. The autoradiography results were compared to the in vivo pancreatic uptake as assessed by [68Ga]Ga-DO3A-VS-Cys40-Exendin4 Positron Emission Tomography (PET) in the same species. In vitro, ex vivo, and in vivo data formed the basis for calculating the theoretical in vivo contribution of each pancreatic compartment.Results[177Lu]Lu-DO3A-VS-Cys40-Exendin4 displayed the highest islet-to-exocrine pancreas ratio (IPR) in rat (IPR 45) followed by non-human primate and mouse at similar levels (IPR approximately 5) while pigs exhibited negligible IPR (1.1). In vivo pancreas uptake was mainly GLP-1R mediated in all species, but the magnitude of uptake under basal physiology varied significantly in decreasing order: non-human primate, mouse, pig, and rat. The theoretical calculation of islet contribution to the total pancreatic PET signal predicted the in vivo observation of differences in pancreatic uptake of [68Ga]Ga-DO3A-VS-Cys40-Exendin4.ConclusionsIPR as well as the exocrine GLP-1R density is the main determinants of the species variability in pancreatic uptake. Thus, the IPR in human is an important factor for assessing the potential of GLP-1R as an imaging biomarker for pancreatic beta cells.

Inside the pancreas: progress and challenges of human beta cell mass quantification

The accurate quantification of beta cell mass in humans is one of the key challenges in understanding the role of beta cell loss and dysfunction in the pathogenesis of diabetes mellitus. Autopsy studies indicate that beta cell loss is not only a hallmark of autoimmune diabetes but also plays a pivotal role in type 2 diabetes, owing to the toxic effects of lipids, glucose and cytokines. Thus, there is an urgent need for non-invasive clinical techniques for beta cell mass quantification, which should be optimally integrated into standard diagnostic equipment in hospitals. In this issue of Diabetologia (Brom et al DOI 10.1007/s00125-014-3166-3) it is reported that single photon emission computed tomography (SPECT) data with (111)indium-labelled glucagon-like peptide-1 (GLP-1) receptor agonist exendin-3 correlate with the morphometric analysis of beta cell mass in a rat model of alloxan-induced diabetes. With this validation, the authors were able to demonstrate a significant loss of beta cell mass in C-peptide-negative type 1 diabetic patients. Thus, (111)indium-labelled exendin-3 could serve as a model tracer for future studies of larger cohorts of diabetic patients to monitor the dynamics of beta cell loss and regeneration. Despite the recent progress from SPECT imaging data there remain open questions that await clarification in the near future such as variations in GLP-1 receptor density and physiological variation of beta cell mass in relation to beta cell function. The use of GLP-1-based tracer analysis may open new clinical avenues for non-invasive quantification of beta cell mass in patients with newly diagnosed type 1 diabetes and prediabetic individuals with high titres of autoantibodies.

Non-invasive quantification of the beta cell mass by SPECT with 111In-labelled exendin

A reliable method for in vivo quantification of pancreatic beta cell mass (BCM) could lead to further insight into the pathophysiology of diabetes. The glucagon-like peptide 1 receptor, abundantly expressed on beta cells, may be a suitable target for imaging. We investigated the potential of radiotracer imaging with the GLP-1 analogue exendin labelled with indium-111 for determination of BCM in vivo in a rodent model of beta cell loss and in patients with type 1 diabetes and healthy individuals. The targeting of (111)In-labelled exendin was examined in a rat model of alloxan-induced beta cell loss. Rats were injected with 15 MBq (111)In-labelled exendin and single photon emission computed tomography (SPECT) acquisition was performed 1 h post injection, followed by dissection, biodistribution and ex vivo autoradiography studies of pancreatic sections. BCM was determined by morphometric analysis after staining with an anti-insulin antibody. For clinical evaluation SPECT was acquired 4, 24 and 48 h after injection of 150 MBq (111)In-labelled exendin in five patients with type 1 diabetes and five healthy individuals. The tracer uptake was determined by quantitative analysis of the SPECT images. In rats, (111)In-labelled exendin specifically targets the beta cells and pancreatic uptake is highly correlated with BCM. In humans, the pancreas was visible in SPECT images and the pancreatic uptake showed high interindividual variation with a substantially lower uptake in patients with type 1 diabetes. These studies indicate that (111)In-labelled exendin may be suitable for non-invasive quantification of BCM. ClinicalTrials.gov NCT01825148, EudraCT: 2012-000619-10.

Characterisation of Age-Dependent Beta Cell Dynamics in the Male db/db Mice

AimTo characterise changes in pancreatic beta cell mass during the development of diabetes in untreated male C57BLKS/J db/db mice. MethodsBlood samples were collected from a total of 72 untreated male db/db mice aged 5, 6, 8, 10, 12, 14, 18, 24 and 34 weeks, for measurement of terminal blood glucose, HbA1c, plasma insulin, and C-peptide. Pancreata were removed for quantification of beta cell mass, islet numbers as well as proliferation and apoptosis by immunohistochemistry and stereology. ResultsTotal pancreatic beta cell mass increased significantly from 2.1 ± 0.3 mg in mice aged 5 weeks to a peak value of 4.84 ± 0.26 mg (P < 0.05) in 12-week-old mice, then gradually decreased to 3.27 ± 0.44 mg in mice aged 34 weeks. Analysis of islets in the 5-, 10-, and 24-week age groups showed increased beta cell proliferation in the 10-week-old animals whereas a low proliferation is seen in older animals. The expansion in beta cell mass was driven by an increase in mean islet mass as the total number of islets was unchanged in the three groups. Conclusions/InterpretationThe age-dependent beta cell dynamics in male db/db mice has been described from 5-34 weeks of age and at the same time alterations in insulin/glucose homeostasis were assessed. High beta cell proliferation and increased beta cell mass occur in young animals followed by a gradual decline characterised by a low beta cell proliferation in older animals. The expansion of beta cell mass was caused by an increase in mean islet mass and not islet number.

PS18 - 2. Quantification of the beta cell mass based on 111In-Exendin imaging in patients with type 1 diabetes and healthy controls

Various tests exist to quantify beta cell (BC) function, but do not necessarily provide information on BC mass (BCM), while BCM seems an important predictor of future BC failure. A method for reliable BCM quantification would be a tremendous asset to better elucidate the pathophysiology of type 1 and type 2 diabetes (T1D and T2D), and to determine effects of therapeutic interventions.

The role of Raf-1 kinase inhibitor protein in the regulation of pancreatic beta cell proliferation in mice

Manoeuvres aimed at increasing beta cell mass have been proposed as regenerative medicine strategies for diabetes treatment. Raf-1 kinase inhibitor protein 1 (RKIP1) is a common regulatory node of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and therefore may be involved in regulation of beta cell homeostasis. The aim of this study was to investigate the involvement of RKIP1 in the control of beta cell mass and function. Rkip1 (also known as Pebp1) knockout (Rkip1 (-/-)) mice were characterised in terms of pancreatic and glucose homeostasis, including morphological and functional analysis. Glucose tolerance and insulin sensitivity were examined, followed by assessment of glucose-induced insulin secretion in isolated islets and beta cell mass quantification through morphometry. Further characterisation included determination of endocrine and exocrine proliferation, apoptosis, MAPK activation and whole genome gene expression assays. Capacity to reverse a diabetic phenotype was assessed in adult Rkip1 (-/-) mice after streptozotocin treatment. Rkip1 (-/-) mice exhibit a moderately larger pancreas and increased beta cell mass and pancreatic insulin content, which correlate with an overall improvement in whole body glucose tolerance. This phenotype is established in young postnatal stages and involves enhanced cellular proliferation without significant alterations in cell death. Importantly, adult Rkip1 (-/-) mice exhibit rapid reversal of streptozotocin-induced diabetes compared with control mice. These data implicate RKIP1 in the regulation of pancreatic growth and beta cell expansion, thus revealing RKIP1 as a potential pharmacological target to promote beta cell regeneration.

Molecular imaging for diagnosis and management of diabetes - a review

Diabetes incidence is increasing due to population growth, aging, urbanisation, and increasing prevalence of obesity and physical inactivity. This has spurred efforts to apply imaging techniques to gain better understanding of pathogenesis of diabetes. In particular, efforts have been focused on imaging the pancreatic islets for insulin-producing beta-cells. As insulin is the primary hormone involved in glucose regulation, variations in pancreatic beta-cell mass (BCM) are central to the etiology of diabetes. Location of pancreas and sparse distribution of beta-cells within the pancreas have thus far posed challenges in accurate BCM quantification for early diabetes detection. However, recent advances in the application of molecular imaging techniques exhibit potential in early detection and prevention of diabetes progression. They have been used with promising success for visualisation and quantification of BCM in experimental and clinical settings. We review the strengths and weaknesses of molecular imaging modalities as applied towards diagnosis and management of diabetes.

Decreased defluorination using the novel beta-cell imaging agent [18F]FE-DTBZ-d4 in pigs examined by PET

BackgroundFluorine-18 dihydrotetrabenazine [DTBZ] analogues, which selectively target the vesicular monoamine transporter 2 [VMAT2], have been extensively studied for in vivo quantification of beta cell mass by positron-emission tomography [PET]. This study describes a novel deuterated radioligand [18F]fluoroethyl [FE]-DTBZ-d4, aimed to increase the stability against in vivo defluorination previously observed for [18F]FE-DTBZ.Methods[18F]FE-DTBZ-d4 was synthesized by alkylation of 9-O-desmethyl-(+)-DTBZ precursor with deuterated [18F]FE bromide ([18F]FCD2CD2Br). Radioligand binding potential [BP] was assessed by an in vitro saturation homogenate binding assay using human endocrine and exocrine pancreatic tissues. In vivo pharmacokinetics and pharmacodynamics [PK/PD] was studied in a porcine model by PET/computed tomography, and the rate of defluorination was quantified by compartmental modeling.Results[18F]FE-DTBZ-d4 was produced in reproducible good radiochemical yield in 100 ± 20 min. Radiochemical purity of the formulated product was > 98% for up to 5 h with specific radioactivities that ranged from 192 to 529 GBq/μmol at the end of the synthesis. The in vitro BP for VMAT2 in the islet tissue was 27.0 ± 8.8, and for the exocrine tissue, 1.7 ± 1.0. The rate of in vivo defluorination was decreased significantly (kdefluorination = 0.0016 ± 0.0007 min-1) compared to the non-deuterated analogue (kdefluorination = 0.012 ± 0.002 min-1), resulting in a six fold increase in half-life stability.Conclusions[18F]FE-DTBZ-d4 has similar PK and PD properties for VMAT2 imaging as its non-deuterated analogue [18F]FE-DTBZ in addition to gaining significantly increased stability against defluorination. [18F]FE-DTBZ-d4 is a prime candidate for future preclinical and clinical studies on focal clusters of beta cells, such as in intramuscular islet grafts.

Human beta cell proliferation by glucose—a complex scenario

0.05 cells per islet [3] or in the range of 0–1.2% of the beta cells in adults [4]. The mechanisms for proliferation of human beta cells are unknown, as sensitive and robust clinical methods for the quantification of beta cell mass are not available for humans. Transplantation of human islets under the kidney capsule of immunodeficient mice may serve as valuable tool to investigate the effects of defined

Seeing is believing: How the MIP-luc mouse can advance the field of islet transplantation and β-cell regeneration

In the last five years, bioluminescence has emerged as a vital tool in the non-invasive quantification of beta-cell mass following islet transplantation in murine models. The successes achieved in bioluminescence imaging in islet transplantation has prompted its use in the discipline of beta-cell regeneration. Employing bioluminescence permits quantifying changes in β-cell mass in an individual mouse over time, which can provide valuable insights into the kinetics of beta-cell regeneration.

Taking a closer look at the pancreas

Our current understanding of how pancreatic beta cell mass fluctuates during the course of diabetes development is largely based on indirect evidence. Various approaches based on in vivo metabolic testing have been described, each reflecting specific aspects of beta cell function [1]. These measurements are correlated with actual beta cell mass only to a certain extent, and were developed to allow estimates of mass following surgery, therapy or transplantation. Direct visualisation of beta cell mass by means of novel imaging technologies is anticipated to provide more detailed insight into the true kinetics of key processes such as physiological beta cell turnover, islet graft loss and the pathophysiology of type 1 and type 2 diabetes. Likewise, a substantial part of what we know about the autoimmune component of type 1 diabetes has historically been derived from ex vivo, cross-sectional studies in rodents. Improved intravital microscopy techniques could also shed new light on the in situ behaviour of immune cells—after all, seeing is believing. In this issue of Diabetologia, Holmberg and Ahlgren review recent progress in the area of optical imaging technologies that is specifically applicable to monitoring the pancreas [2]. Although most investigators may be familiar with conventional non-optical imaging modalities such as magnetic resonance imaging (MRI) and positron emission tomography (PET), and the ongoing endeavour to optimise these technologies for non-invasive monitoring of beta cell mass in patients, there are a number of lesser known optical strategies that are likely to contribute essential information in the near future. In an effort to improve our understanding of emerging technologies such as optical projection tomography (OPT), bioluminescence imaging, confocal microscopy and optical coherence tomography, the authors outline the characteristics, benefits and limitations of each technique. Perhaps one of the most elegant examples of this new generation of imaging modalities is optical projection tomography, an approach that has recently been advanced by the authors to allow for detailed whole organ assessment and accurate quantification of beta cell mass ex vivo. The three-dimensional OPT image provided in their current review showcases the remarkable quantitative resolution that is characteristic of OPT, enabling us to observe individual islets and their position relative to each other. The authors conclude that, although OPTand the other technologies discussed are at present merely suitable for pre-clinical research, they could prove clinically relevant in the long run. So which questions need to be addressed by optical imaging? (see text box: Questions to be addressed by optical imaging). We and others have recently proposed a cyclical model for type 1 diabetes, describing the disease as a relapsing–remitting condition, based on our understanding of its key cellular constituents [3]. According to this hypothesis, the kinetics of beta cell degeneration are directed by the degree of epitope spreading, the rate of beta cell proliferation in response to autoimmune attack and the immune modulatory action of regulatory T cells (Tregs). As a result of the dominance of the autoreactive effector T cell responses, beta cell mass will gradually decrease in atrisk individuals, ultimately leading to disease manifestation once a critical threshold has been passed. Support for the existence of this delicate balance was obtained in the NOD mouse, still the most widely studied animal model of type 1 diabetes. Sensitive longitudinal detection of alterations in beta cell mass during diabetes development in these animals may put this theory to the test and may Diabetologia (2008) 51:2145–2147 DOI 10.1007/s00125-008-1181-y