Type 1 Diabetes: Cellular, Molecular & Clinical Immunology
Chapter 2 - The Pancreatic Beta-Cell
Suparna A. Sarkar, 3/1/2012 Update of chapter by Kirstine Juhl, John C. Hutton
and George S. Eisenbarth
Human Fetal Pancreas Development slide set Updated 03/12
Introduction
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 §-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 ÒabnormalÕ 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
9Ð23 weeks were processed in parallel for immunohistochemistry and gene
expression profiling by Affymetrix microarray47. At 9Ð11 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 15Ð19 weeks, along with clusters of endocrine
cells producing either glucagon or insulin. By 20Ð23 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 9Ð10 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 e12.5Ðe15.5. 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).
Physiology of
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 9Ð15 weeks,
tapering off by 18Ð23 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 11Ð23 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 (Òkiss-and-runÓ vs.
Òcomplete-collapseÓ)97-99; has its endocytic counterpart that Òkiss-and-runÓ events are
associated with rapid clathrin-independent retrieval, and Òcomplete-collapseÓ
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