S U M M A R YThe recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets ␣ and ␦ cells resided at the periphery of the  -cell core. However, human islets were markedly different in that ␣ ,  , and ␦ cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of ␣ ,  , and ␦ cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.
To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and -cell mass, mice with -cell-reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of -cell secretagogues to islets, we showed that reduced insulin output is not a result of -cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation. Diabetes
Complete lack of transcription factor PDX-1 leads to pancreatic agenesis, whereas heterozygosity for PDX-1 mutations has been recently noted in some individuals with maturity-onset diabetes of the young (MODY) and in some individuals with type 2 diabetes. To determine how alterations in PDX-1 affect islet function, we examined insulin secretion and islet physiology in mice with one PDX-1 allele inactivated. PDX-1 ؉/؊ mice had a normal fasting blood glucose and pancreatic insulin content but had impaired glucose tolerance and secreted less insulin during glucose tolerance testing. The expression of PDX-1 and glucose transporter 2 in islets from PDX-1 ؉/؊ mice was reduced to 68 and 55%, respectively, whereas glucokinase expression was not significantly altered. NAD(P)H generation in response to glucose was reduced by 30% in PDX-1 ؉/؊ mice. The in situ perfused pancreas of PDX-1 ؉/؊ mice secreted about 45% less insulin when stimulated with 16.7 mM glucose. The K m for insulin release was similar in wild type and PDX-1 ؉/؊ mice. Insulin secretion in response to 20 mM arginine was unchanged; the response to 10 nM glucagonlike peptide-1 was slightly increased. However, insulin secretory responses to 10 mM 2-ketoisocaproate and 20 mM KCl were significantly reduced (by 61 and 66%, respectively). These results indicate that a modest reduction in PDX-1 impairs several events in glucose-stimulated insulin secretion (such as NAD(P)H generation, mitochondrial function, and/or mobilization of intracellular Ca 2؉ ) and that PDX-1 is important for normal function of adult pancreatic islets.The islet transcription factor PDX-1 (also known as IPF-1, IDX-1, and STF-1) was originally discovered as an activator of the insulin and somatostatin genes and from developmental work in the frog (1-9). Moreover, several laboratories have demonstrated that PDX-1 plays a key role in pancreatic development and that animals and humans lacking PDX-1 have pancreatic agenesis (1-3, 6 -10). PDX-1 also regulates transcription of other islet genes such as GLUT2, glucokinase (GK), 1 and islet amyloid polypeptide (IAPP) (1, 2, 9, 11-14). How PDX-1 functions in concert with other islet transcription factors and leads to normal pancreatic exocrine and islet development is unknown.Mutations in PDX-1 lead to abnormalities in islet function and diabetes in humans and mice (1,9,(15)(16)(17)(18)(19). Individuals who are heterozygous for the Pro63fsdelC mutation develop maturity-onset diabetes of the young (MODY4) and have impaired insulin secretion in response to glucose (19). This mutation is thought to reduce PDX-1 activity by creating an alternate internal translation start site that produces a dominant negative isoform of PDX-1 (20). Some recent reports indicate that mutations in PDX-1 may predispose individuals to late onset type 2 diabetes mellitus (15)(16)(17)21). Similarly in murine models both conventional and postnatal, cell-specific inactivation of one PDX-1 allele leads to impaired glucose tolerance (2,6,8,18). In addition, Edlund and co-workers (...
OBJECTIVEConditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain.RESEARCH DESIGN AND METHODSTransgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry.RESULTSAll transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)1Lphi mice were the only line that lacked Cre activity in the brain.CONCLUSIONSCre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.
A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood–derived T cells1–3, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis4–8, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors9 and isolation and identification of DQ8 and DQ2–DQ8 heterodimer–restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D10. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.