The two most common forms of diabetes in man (Type 1A and Type 2) have very different etiologies and different clinical presentation1. Nevertheless, the underlying loss of islet beta cell function has similar consequences in terms of glycemic control and the emergence of long-term complications. type 1 Diabetes (T1D) is a polygenic T-cell dependent autoimmune disease, characterized by the selective destruction of the ß-cells of the islets of Langerhans1-6 and that susceptible individuals have inherent defects in critical immunomodulatory mechanisms7 that increase the risk of a pathogenic rather than protective immune response to self6, 8, 9. Type 2 Diabetes is typically linked to dysmetabolism or metabolic syndrome and the presence of insulin resistance, however a large subset of T1D patients routinely exhibits insulin resistance10-13 contributing to the metabolic distress in islets. With the rising incidence of T1D and T2D, it is now being argued that both T1D and T2D are essentially disorders of altered insulin resistance set against the backdrop of genetic susceptibility14 and the inflammatory process; in T1D brought about by the autoimmune component of the disease process15.
In type 1A (autoimmune diabetes) the loss of beta cells is often close to absolute with less than 1% of beta cells remaining in patients with long-term diabetes16-18 with prolonged C peptide production19. In most patients with almost no remaining beta cells, essentially all of the islets are devoid of beta cells while islets contain cells expressing glucagon and somatostatin. Such islets are termed pseudoatrophic islets20. Nevertheless, some beta cells remain often as scattered single cells in the parenchyma and ducts. In a small subset of patients, even with long-term type 1A diabetes, significant C-peptide is present and lobules of pancreas remain where all the islets contain beta cells and appear essentially normal in terms of expression of insulin while the rest of the pancreas is devoid of beta cells in islets20-22. The figure below illustrates a section of one such pancreas from the nPOD collection (jdrfnpod.org) where the pancreatic lobule on the right is stained dark and at higher power one can observe that all of the islets in this lobule lack insulin23. In contrast to the lobule on the left, all of the islets contain insulin. The dark staining of the right lobule likely results from pancreatic acinar atrophy that occurs with severe loss of pancreatic insulin. Shrinkage in overall pancreatic mass in patients with type 1 diabetes has long been noted24-26. Analysis of decreased pancreatic volume was recently combined with imaging of iron particle pancreatic accumulation to help distinguish patients with type 1 diabetes from normal controls27-29.
In fact, C-peptide secretion in long-standing diabetic patients has now been explained by two different patterns of beta cell survival, which possibly reflect different subsets of type 1 diabetes. In a recent study20 associated Pattern A with type 1A diabetes that histologically had lobular retention of islet areas with “abnormal’ beta cells producing the apoptosis inhibitor BIRC5 (survivin) and HLA class I. In pattern B, 100% of all islets contained normal-appearing but quantitatively reduced beta cells without survivin or HLA class I. Baculoviral IAP repeat 5 BiRC530 is an apoptosis inhibitor that is produced in the beta cells of fetal human pancreas31, but not in adult islets. It is also found in the beta cells in areas of pancreatitis32. The presence of survivin, in all surviving islet beta cells Pattern A patients, may result from 1) inflammatory changes that did not result in beta cell destruction of a subset of islets or 2) be protected from destruction and further lymphocytic infiltration. An alternative hypothesis extended by the authors is that lobular regions with beta cells of Pattern A pancreas represent areas of beta cell regeneration. Although the sudden onset of type 1A belies the fact that the underlying loss of beta cell mass is the culmination of many years of gradual and progressive loss of beta cells in the face of autoimmune attack which is first evident with the appearance of autoantibodies to islet proteins in the preceding years (see other chapters)33-38. In the NOD mouse the infiltration of the islets with immune and inflammatory cells that initiates the disease first appears in the islets of the pancreatic periphery, affects a subpopulation of islets and is possibly benign or at least kept in check by the presence of regulatory T cells39-42. The invasive insulitis seen in NOD mice closer to disease onset may reflect a change in the balance of destructive and protective responses in favor of the former. The histological changes in man are comparatively mild and may reflect the slower progression of the disease or possibly a different immune process. The islet tends to be viewed as the source of autoantigen that supports or initiates the immune attack and ultimately the victim of the crime.
Histopathological examination of pancreata from diabetic organ donors procured from nPOD was examined with the goal to provide a foundation for the informed selection of potential therapeutic targets within the chemokine/receptor family43. CCL5, CCL8, CCL22, CXCL9, CXCL10 and CX3CL1 were the major chemokines transcribed and translated by human islet cells in response to in vitro inflammatory stimuli. CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and T1D patients, while CCL5, CCL8, CXCL9 and CX3CL1 proteins were present at lower levels in the islets of both species. Importantly, additional expression of the same chemokines in human acinar tissues emphasized an underappreciated involvement of the exocrine pancreas in the natural course of T1D that will require consideration for further T1D pathogenesis and immune intervention studies.
Undoubtedly, much more needs to be learned about the reaction of the islet to cytokine mediators of the immune response and about how the beta cell manages to survive so long or replenish its population from progenitor cells in the pancreas. Since the mechanism of autoimmune destruction by effector cells may be mediated by CD4+ cells, and thus indirect, there is also the question of whether the beta cell is uniquely susceptible to oxygen and nitrogen free radicals or cytokine mediators of cell death which may account for the fact that other islet cells exposed to same molecules survive while the beta cell dies.
The focus of the following review is to discuss the wealth of information regarding the physiological and pathophysiological responses of the islet to nutrient secretagogues and pharmacological agents and to emphasize how the beta cell differs from its neighbors and from other endocrine tissues and how it may participate in its own demise in type 1 diabetes. The review also illustrates the challenges faced by investigators wishing to genetically engineer non-b cells for cellular therapy of type 1 diabetes or wanting to introduce specific genes into the beta cell population to afford it greater protection from autoimmune attack.
Development of the Human Pancreas
Similar to the mouse pancreas, the human pancreas develops from two endodermal diverticula, the dorsal and ventral44, which fuses around 56 days post coitum of development45 . The pancreas comprises of 3 important cell lineages: Endocrine, acinar and ductal (which together make up the exocrine pancreas). The morphogenesis of the endocrine tissue, however, is unlikely to be equivalent given the differences in gestation (260 vs 20 days) and the larger relative volume of the human pancreas46. Human fetal pancreases obtained at gestational ages 9–23 weeks were processed in parallel for immunohistochemistry and gene expression profiling by Affymetrix microarray47. At 9–11 weeks, the pancreas was made up principally of mesenchymal tissue interspersed with PDX1 positive branched epithelial structures containing scattered hormone-negative neurogenin3-positive endocrine cells. Protoacinar structures marked by carboxy esterase lipase (CEL) expression were noted by 15–19 weeks, along with clusters of endocrine cells producing either glucagon or insulin. By 20–23 weeks, vascularized islet-like structures appeared. Analysis of Ki67 immunoreactivity showed that the replicative rate of endocrine cells was low and suggested that the endocrine expansion was derived from hormone-negative precursors. Insulin, glucagon, somatostatin, ghrelin and pancreatic polypeptide transcripts were present at 9–10 weeks as confirmed by quantitative PCR and increased progressively, commensurate with the expansion of endocrine cell volume. The human equivalent of a mouse endocrine secondary transition was not evident, neither in terms of morphology nor in dramatic changes in endocrine-specific transcriptional regulators. By contrast, exocrine genes showed a marked transition at around 11 weeks, associated with a greater than six-fold increase in exocrine gene transcripts. The terminal differentiation of human endocrine tissue into late gestation and the presence of NEUROG3 are in contrast with findings in the mouse, where neurog3 is transiently expressed from e12.5–e15.5. This indicates that the human fetal pancreas could provide an abundant islet precursor cell population that could be expanded ex vivo for therapeutic transplantation for the treatment of brittle and unstable type 1 diabetes. The ductal cells also develop from the PDX1 expressing primordial pancreatic epithelium and expresses Cytokeratin 19 (CK19), cystic fibrosis transmembrane receptor, DBA lectin, Carbonic anhydrase 248. (For images of the human fetal pancreas development, please refer to power-point slides).
the islet of Langerhans
The endocrine pancreas is arranged in clusters of secretory cells the Islets of Langerhans scattered throughout the exocrine glandular tissue (Fig. 1)49, 50. In man, the pancreas contains around one million islets that comprise 1-2% of the total mass of the gland. The islets are separated from the exocrine tissue by a capsule made up of connective tissue fibers and by glial like cells and human islets vary in size from less than 50 up to several thousand cells. Four different endocrine cell types are contained in the islets; beta cells, which produce insulin and constitute 60-80% of the endocrine cell mass, glucagon secreting a-cells (10-20%), somatostatin producing d-cells (~5%) and pancreatic polypeptide secreting PP-cells (<1%)44. The cells within islets are critically dependent upon a series of transcription factors during embryonic development and of note, in knockouts of the homeodomain protein nkx2.2, almost all islet cells are replaced with ghrelin producing cells51. In parallel, in the human pancreas, the endocrine genes insulin, glucagon and somatostatin steadily increase from 9–15 weeks, tapering off by 18–23 weeks47. This was in tandem with other dense core secretory granule markers such as PCSK1, PCSK1N, PTPRN, SLC30A8, IAPP, CHGA and CHGB that peak in the later phase of development. Components of the stimulus secretion coupling machinery such as GCK and GLUT2 (SLC2A4) are however more variable. The high signal intensity observed for insulin, glucagon and somatostatin correspond to the abundance of immunopositive cells detected by immunohistochemistry. Low signals for ghrelin (GHRL) and pancreatic polypeptide correspond to the lower frequency of these cells in the histological analysis.
Figure 1. The pancreas is located close to the duodenum. The enlarged part of the pancreas shows the exocrine acinar cells next to the islet endocrine cells. The islets consist of four different cell types, which secrete different peptide hormones. The majority of the islets are beta cells that secrete insulin. The a-cells secrete glucagon, d-cells secrete somatostatin and PP cells secrete pancreatic polypeptide.
Most mammalian islets have a beta cell rich core surrounded by a mantle of alpha-, delta- and PP-cells with some controversy as to applicability to human islets. Afferent arterioles enter the beta cell rich medulla where the arterioles split into a branching system of capillaries that traverse the beta cell mass before reaching the mantle of a- and d-cells. Thus, the beta cells are the first endocrine islet cell type to sense blood-borne metabolic changes. Insulin is known to inhibit glucagon secretion and glucagon to stimulate insulin secretion. Somatostatin will inhibit the secretion of both insulin and glucagons. Somatostatin's inhibition of insulin secretion is reported to result from its inhibition of glucose metabolism of the beta cell (14) and glucagon; hence the anatomical relationship of the islet cells is critical to the function of the organ. The islets are innervated by autonomic nerve fibers, containing both parasympathetic and sympathetic nerves that enter the islets and terminate in proximity to the endocrine cells.
The architecture of the human islets is however more heterogeneous. A progressive increase in the endocrine cell population is evident from 11–23 weeks, reflecting both the progressive expansion of the pancreatic epithelium and an increase in the density of endocrine cells within the epithelium47. With increasing fetal age evidence of formation of endocrine cell clusters especially from 15 weeks onwards is noted, although solitary endocrine cells were still observed at all time-points. The endocrine cell clusters at early time-points displayed a significant homotypic association of either insulin- or glucagon-positive cells. At later developmental time-points, heterotypic endocrine cell clustering and the appearance of typical islet-like structures is a feature.
The Beta Cell as a Neuron-like Cell
Though islet beta cells are derived from endoderm and not ectoderm they share many properties with neurons that probably relate to their function in terms of secreting a peptide hormone stored in dense core secretory granules and having microvesicles with "neurotransmitters". The neuron-like properties include presence of microvesicles containing molecules such as GABA gamma-Aminobutyric acid (GABA)52, an inhibitory neurotransmitter, expression of enzymes such as Glutamic acid decarboxylase (GAD, both GAD65 and GAD67), IA-2 (ICA512)53, 54, IA-2beta (phogrin)55. Surface expression of complex neuronal/Oligodendrocyte gangliosides such as GQ gangliosides (e.g. target of monoclonal A2B5) and the targets of monoclonals 3G556 and R2D657, and expression of type 2 monoamine vesicular transporters(VMAT2)58, 59 is also seen. These shared neuronal/neuroendocrine properties are of importance in considering the pathophysiology of autoimmune beta cell destruction (e.g. though GAD65 autoantibodies are characteristic of type 1 diabetes of man, only beta cells and not the many other cell types that express GAD65 are destroyed in type 1A diabetes and in contrast in Stiff Man Syndrome neurologic disease predominates in association with very high levels of GAD65 autoantibodies) and recently for the in vivo detection of islets, potentially for determining beta cell mass. PAM peptidylglycine alpha-amidating monooxygenase gene encodes a multifunctional protein with two enzymatically active domains with catalytic activities; peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL)60-62. These catalytic domains work sequentially to catalyze neuroendocrine peptides to active alpha-amidated products is also found in hypothalamic magnocellular neurons, the hippocampal formation, and olfactory cortex63 and human endocrine pancreas64. PAM is localized primarily in the secretory granules of alpha cells not in the beta, PP and Delta cells. Alpha cells secrete GLP1, which possess a carboxy terminal amide group65 while beta and delta cells that secrete insulin and somatostatin respectively which are not terminally amidated. Investigators have utilized [(11)C]Dihydrotetrabenazine ([(11)C]DTBZ) that binds specifically to VMAT2, with PET scanning to estimate beta cell mass in the BB type 1 diabetes rat59. It is possible that many of the technologies developed to interrogate the brain (e.g. MRI spectroscopy) will be applicable to studies of islet beta cells, if the goal is determination of approximate mass rather than direct visualization of the relatively small islets, given the shared biochemistry of neurons and islets.
The Biosynthesis of Insulin
The islet beta cell is the principal cell in the adult mammal able to transcribe the insulin gene. The insulin receptor by comparison is widely distributed even on cells that are not thought to be insulin responsive and in the case not even exposed to significant concentrations of the hormone. This may reflect the evolution of the molecule from primarily a neurotransmitter to an endocrine function66. Of note a series of rare mutations which result in misfolded insulin and neonatal diabetes have now been identified67. Beta cell death may be the result of ER stress68-70.
Insulin mRNA is translated to a pre-pro-insulin peptide, which is rapidly processed in the rough endoplasmic reticulum to pro-insulin by removal of the N-term signal peptide. Proinsulin contains the A and B chains of insulin linked by the C peptide, a structure that helps to align the disulfide bridges that are generated between the A and B chain. Proinsulin is transported through the Golgi apparatus and thereafter further processed in the maturing granule to insulin by excision of the C peptide by the endopeptidases, PC1 and PC2 (prohormone convertases), and carboxypeptidase H. The bioactive insulin molecule consists of an A and B chain (21 and 30 amino acids, respectively), linked intramolecularly by disulfide bridges71-77.
Role of Zinc in insulin biosynthesis (Leah Sheridan, PhD)
Insulin secretion is tightly regulated by extracellular secretagogues/inhibitors, as well as intracellular signaling pathways. It is packaged in dense core vesicles (DCVs) as a hexamer bound to two Zn2+ ions and remains stable in this configuration at pH5.5 until fusion of the DCV and plasma membranes during exocytosis78, 79 . The complex is then released into the pH neutral circulation, and dissociates, allowing both insulin and Zn2+ to act independently.
The Zn2+ content of pancreatic b-cells is one of the highest in the body80. This is evidently due to its additional role in insulin crystal formation, beyond its various fundamental roles in protein stability and function, DNA replication, metabolic enzyme activity, and cellular protection against apoptosis and oxidative stress81, 82. In addition, co-secreted Zn2+ is believed to play both an autocrine and paracrine role by activating KATP channels, thereby inhibiting the secretory process83, 84, and regulating alpha cell glucagon secretion84, 85. Zn2+ is also implicated in b-cell death via a paracrine mechanism86. Although it is apparent that mammalian cells require Zn2+ for many cellular processes, it can also be toxic. The regulation of intracellular Zn2+ is therefore of great importance to maintain homeostatic cellular physiology. One such regulatory mechanism involves controlling the influx and efflux of Zn2+ across membranes, both cellular and subcellular, by Zn2+ transporters.
Mammalian Zn2+ transporters were first discovered in the mid-1990s, and were shown to transport Zn2+ and other metal ions into the cytoplasm either from the extracellular space, or from the organellar lumen. These were members of the SLC39 (also known as ZIP) family of Zn2+ transporters. Later, the SLC30 (also known as ZnT) family of Zn2+ transporters were identified that worked counter to ZIPs, in that they transport Zn2+ out of the cytoplasm, either out of the cell into the extracellular space, or into the lumen of organelles87, 88. Mammals carry genes for 10 homologous Zn2+ export proteins (ZnT1 through 10). The overall homology of ZnT9, however, to other members of the ZnT family is very low89, and may not function in Zn2+ transport, but potentially in DNA excision-repair90, 91. ZnT8 and ZnT10 were the latest members to be identified, and akin to ZnT1-7, share the same predicted structure of six transmembrane (TM)-spanning domains with cytoplasmic amino (N) and carboxy (C) terminals (see above cartoon), and contain a histidine-rich intracellular loop between TM IV and V which may act as the Zn2+ binding region. ZnT6 is an exception, in that it retains the prokaryotic serine-rich loop. Despite the similar topological structure of the ZnT family, the overall sequence similarities are relatively low, with the highest being 53.5% between ZnT2 and ZnT889. This sequence divergence may reflect the different localizations of each transporter (i.e. ZnT1-plasma membrane, ZnT2-endosome/lysosome, ZnT3-synaptic vesicles, etc)92, 93 and correspondingly the different tasks and means of regulation in which each transporter partakes; a combined bioinformatics/molecular engineering strategy to identify potential molecular targets of circulating type 1 diabetic autoantibodies94. In such, they identified 51 candidate islet autoantigens from microarray mRNA expression profiling experiments on isolated mouse and human pancreatic islets, FACS sorted b-cells and mouse pancreatic tumor cell lines. Among the candidates, this study identified the Zn2+ transporter ZnT8 as a novel type 1 diabetes (T1D) autoantigen, and furthermore, localized the epitope of immunoreactivity to the C-terminus of the protein. Among the other major known targets of diabetes autoantibodies (insulin, GAD, IA2, and phogrin) identified by the study, ZnT8 was found to be b-cell specific, displayed moderate to abundant islet expression, and is associated with the regulated secretory pathway.
The identification of ZnT8 with roots in the pathogenesis of T1D creates opportunities for the development of improved diagnostic capabilities as well as provides an additional strategy for the creation of therapeutic interventions. Like the other four main autoantigens associated with T1D studied to date (insulin, GAD, IA2, and phogrin), ZnT8 has been shown to be associated with the regulated pathway of secretion, having been localized to intracellular vesicles in HeLa cells, and co-localized with insulin granules in INS-1 cells and insulin in human islets95, 96. The importance of such an association with the secretion pathway remains to be elucidated; however, one can speculate that trafficking of these antigens to and from the surface membrane may play a role in the diabetic pathology. Due to the specific function of insulin secretion by pancreatic b-cells, proteins specifically related to that function are potentially more likely to be targeted by the immune system. Several possible mechanisms come to mind: 1) Proteins associated with DCVs (i.e. insulin, phogrin) that get trafficked to and from the surface in response to insulin secretagogues, expose extracellular epitopes that are recognized by T Cell receptors or circulating antibodies secreted by B cells; 2) Epitopes may be exposed to the immune system during normal tissue turnover, especially for intracellular proteins (i.e. GAD and IA2) that would not expose surface epitopes; 3) Recycling of secretory pathway associated proteins enables access to post-golgi compartments where the protein could be cut up by proteosomes and presented on the cell surface by MHC I for recognition by T Cells. These are only a few of many potential cellular mechanisms by which a protein may become antigenic. In light of the recent discovery of ZnT8 and its identification as a T1D autoantigen, it is essential to investigate its role in pancreatic b-cells to gain insight into its mechanism of antigen presentation, and furthermore, to generally learn why many other autoantigens are associated with the DCV (i.e. insulin, CPE and amylin). One study has been conducted on the functionality of ZnT8 to date, which showed that its overexpression does not play a role in Zn2+ toxicity, and protected INS-1 cells, an insulin-secreting cell line, from death during Zn2+ depletion (86). ZnT8 therefore appears to be a functional channel that actively participates in intracellular Zn2+ transport. However, much remains to be discovered about this transporter. It has been shown to co-localize with insulin95, but whether ZnT8 is trafficked to the surface membrane with insulin upon stimulation, and therefore truly associated with DCVs remains to be shown. In addition, if ZnT8 is trafficked to the surface membrane, how long it lingers there before being re-endocytosed, and where it is trafficked to following its retrieval is unknown. The processes of DCV exocytosis and endocytosis are well studied. DCVs undergo regulated exocytosis in response to signals that elevate cytosolic Ca2+, a process that must ultimately be coupled to endocytosis of the granule membrane to avoid significant increases in cell surface area. The concept that exocytosis can occur by two related, but mechanistically distinct mechanisms (“kiss-and-run” vs. “complete-collapse”)97-99; has its endocytic counterpart that “kiss-and-run” events are associated with rapid clathrin-independent retrieval, and “complete-collapse” with a slower clathrin-dependent mechanism97, 98, 100. In neuroendocrine tissues the type of event that occurs is dictated by the strength and duration of the stimulus101, and possibly related to the prevailing cytosolic Ca2+ level97, 102. Thus clathrin-independent endocytosis predominates in the initial phases of the secretory response, while chronic stimulation (> 5 min) leads to a shift to a mainly clathrin-dependent mechanism and full fusion of DCVs101. Insulin secretion in vivo is biphasic, which may reflect the release of docked vs. recruited DCVs, changes in the stimulus intensity as reflected by plasma membrane potential and cytosolic Ca2+, or indeed the mechanism of granule fusion and retrieval as discussed above103-105