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
Cell Therapy of Diabetes PowerPoint slide set - Updated 7/06
Proprotein Processing and Pancreatic Islet Function PowerPoint slide
set - Updated 11/06
Stimulus-Secretion Coupling in the Pancreatic Beta-Cell PowerPoint slide set Updated 7/08
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 presentation 1. 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 longterm 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- 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 923 weeks were processed in parallel for
immunohistochemistry and gene expression profiling by Affymetrix
microarray47. At 911 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 1519 weeks, along with clusters of
endocrine cells producing either glucagon or insulin. By 2023 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 910 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
e12.5e15.5. 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 12% 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 915 weeks, tapering off by 1823 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 1123
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 timepoints, 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 neuronlike 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, IA2beta (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 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, cosecreted 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 -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 -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 -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 -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 -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 clathrinindependent retrieval, and complete-collapse with a slower clathrindependent 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 clathrinindependent 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.
Most mammalian insulin binds Zn co-ordinately forming hexamers and can
form large crystalline arrays which promote condensation and aggregation
of the hormone in the acidic core of the secretory granule. Proinsulin
although forming Zn hexamers is prevented from further aggregation by the
highly charged C-peptide, a physical property that may be important in
retaining its solubility in the proximal elements of the secretory pathway. In
the mature secretory granule C peptide exists in equimolar amounts with
insulin in the granule and is co-secreted thus making it a useful marker for
beta cell function in diabetics receiving insulin therapy. Transcription of the
insulin gene is modulated by nutrient secretagogues, a variety of cytokines
and by insulin itself. Insulin signal transduction enhances transcription of
the insulin gene through phosphorylation of insulin receptor substrate
(IRS)-1 and -2, and activation of phosphatidylinositol-3-kinase (PI3K) and it
has been postulated that insulin plays a feed forward role in insulin
biosynthesis ensuring that the stores of insulin are always replenished106.
Exogenous insulin is also shown to hyperpolarize beta cell plasma
membrane in mice by insulin-induced activation of the KATP channels
possible through the PI3K thereby turning off the islet insulin secretion107.
Glucose is also shown to stimulate the production of proinsulin through
rapid activation of translation of a pre-formed pool of mRNA108. Given the
high rates of proinsulin synthesis and the need for proper folding of
proinsulin within the endoplasmic reticulum it has been hypothesized that
pancreatic beta cells are particularly susceptible to apoptosis induced by
endoplasmic reticulum (ER) stress109. Wolfram syndrome (DIDMOAD:
Diabetes Insipidus/Diabetes Mellitus/Optic atrophy/Nerve Deafness) results
from mutations of the WFS1 gene which is a transmembrane protein of the
ER regulated by the unfolded protein response and with a role in ER
calcium transport109.

The proinflammatory cytokines IL-1b, TNFa and IFNg modulate

transcription of the insulin gene. Culturing human beta cells 48 or 72 h with
single cytokines does not affect the cellular content in insulin or proinsulin
whereas the combination of several cytokines (IL-1 + IFN)
disproportionally elevates medium proinsulin levels110. This is caused by a
conserved proinsulin synthesis and a slower hormone conversion possibly
contributed to lower expression of PC1 and PC2 convertases. These
cytokines mediates signal transduction involving binding to specific
receptors, through MAPK and SAPK and mobilization of diverse
transcription factors: NFkB, AP-1 and STAT-1 involved in beta cell
apoptosis 111, 112

Physiological regulation of islet hormone secretion

The blood glucose level is maintained within a narrow range around 5 to 7
mM in the fasting state mainly by the combined and reciprocal action of
insulin and glucagon113, 114. Essentially, when the blood glucose
concentration is elevated after a meal, the beta cells are stimulated to
secrete insulin. Conversely, when blood glucose levels are low the a-cells
are stimulated to secrete glucagon, which leads to glycogenolysis and
gluconeogenesis in the liver and therefore release of glucose to the blood.
Between meals, when the plasma glucose decreases, the release of the
neurotransmitter norepinephrine and the neuropeptide galanin from the
sympathetic nerves will directly activate glucagon release and inhibit insulin
release115. During a meal or the postprandial state, the parasympathetic
nerves potentiate glucose-induced insulin release from islet beta cells. This
is achieved by the release of the neurotransmitter acetylcholine and the
neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP),
vasoactive intestinal polypeptide (VIP), GLP-1 116-119 and GIP113. The
innervation of the islet and its microanatomy is probably important in the
orchestration of these responses. Somatostatin inhibits both insulin and
glucagon secretion and potentially serves as a modulator of islet hormone
release120. However, it is unclear whether it has a physiological role given
the vascular anatomy of the islet in which the secreted products from the aand d-cell enter the blood circulation without reaching the beta cells. The
physiological role of islet PP is unknown, but appears not to affect the
secretion of the other islet hormones except a possible inhibition of islet
insulin release121-123. Rare cells within islets secrete ghrelin, with ghrelin
primarily produced in the gastrointestinal tract and ghrelin under
experimental conditions can influence insulin secretion and is reported to
increase IA-2 beta but not IA-2 message in islets and inhibition of IA-2 beta
with RNAi decreased ghrelin inhibition of insulin secretion124, 125. Much of
the physiological regulation of insulin secretion can be reproduced in vitro
and a wealth of information has been generated from the study of isolated
islets, which appear to behave as autonomous organs. Such studies have
led to the classification of insulin secretagogues into three groups,
initiators, potentiators and inhibitors. Glucose is the most important initiator
in monogastric animals, but other carbohydrates, amino acids and fatty
acids alone can also initiate insulin secretion. Pharmacological agents like
the clinically useful sulphonylureas tolbutamide and glibenclamide also
initiate insulin secretion in a glucose-independent manner. Arginine initiates
insulin secretion by depolarizing the plasma membrane because of its
transport in a positive charge form. It triggers insulin release by increasing
[Ca2+]i through a KATP channel-independent pathway126, 127. Potentiators of
insulin secretion do not initiate insulin secretion by themselves but enhance

stimulation of secretion in the presence of an initiator. Examples of

potentiators are the intestinal glucoincretin hormones glucagon-like peptide
1 (GLP-1)128 and gastric insulinotropic polypeptide (GIP), which provide fine
regulators of insulin secretion in the post-prandial state along with
neurotransmitters. Other neurotransmitters and hormones such as
somatostatin, galanin, adrenalin and even endocannabinoids (or potentially
increase through CB2
receptors 129 reduce insulin secretion
(Fig. 2).
Figure 2. Examples of potentiators,
initiators and inhibitors of insulin
secretion from the pancreatic beta cell.
Abbreviations used: GIP, gastric
insulinotropic polypeptide; VIP,
vasoactive intestinal polypeptide; GLP-1,
glucagon-like peptide 1.

Islet Pathophysiology in type 1 diabetes

Type 1 diabetes is characterized by the appearance of circulating
antibodies targeted at beta cell proteins, which appear many years before
the onset of clinical disease. Proteins such as insulin, GAD, IA2, IA-2 beta
(phogrin) and CPH (carboxy-peptidase H) are specifically targeted but as
yet it is not settled if any one (or unknown targets) of these molecules is
primary or dominant in the autoimmune response. In the NOD mouse
model there is accumulating evidence that insulin may be a primary
autoantigen with knockouts or alterations of specific insulin sequences
preventing diabetes, while knockouts of GAD65, IA-2, IA-2 beta (and even
both IA-2s) do not change the course of development of NOD diabetes130132
. A recent study of lymphocytes from pancreatic lymph node of patients
with type 1 diabetes also implicated insulin133. Insulin is the only molecule in
this group that is specific to the beta cell though the others have the
common feature of being associated with regulated pathway of secretion.
Insulin and CPH are secreted molecules and to some extent remain on the
cell surface following exocytosis. By contrast the dominant epitopes seen
by antibodies reactive to GAD, IA2 and phogrin are intracellular. It is
presently unknown whether the beta cell is targeted directly or indirectly,
whether the antigens are presented by the living cell or through death or
antigen shedding and why the a-cell survives in spite of sharing many
autoantigens with the beta cells and being bathed in the same milieu of
soluble immune effector molecules. One hypothesis is that the beta cell
participates in its own destruction by increased presentation of antigens in

response to glucose, a situation that worsens as beta cell mass decreases

and the remaining cells compensate by increased transcription and
translation of secretory pathway proteins134. Beta and alpha-cells have a
differential sensitivity to cytokines in that inhibition of glucose-stimulated
insulin release by IL-1 is reversible, whereas the effect on glucosemodulated glucagon release is not135. The beta cells are more sensitive to
cytokines than the other three endocrine cell types in the islets. Cytokineinduced free radicals in beta cells such as NO catalyzed by the inducible
nitric oxide synthase136 may be involved in beta cell-specific destruction in
type 1 diabetes137. NO is an important mediator but not the sole mediator of
cytokine-induced cytotoxicity in beta cells as shown by the fact that
inhibition of NO production in human islets did not prevent cytokine-induced
apoptosis, a-cells do not express NO in response to cytokines. Beta cells
but not a-cells induce the expression of heat shock protein 70 (hsp70),
heme oxygenase and MnSOD (manganese superoxide dismutase) upon
exposure to IL-1138. Native beta cells have been shown to possess low
scavenging potential for oxygen-derived free radicals and over expression
in beta cells of scavenger or antioxidant enzymes such as MnSOD,
catalase and glutathione peroxidase has been shown to increase their
survival after exposure to NO and reactive oxygen species137. Production of
oxygen free radicals mediated by macrophages can damage beta cells
directly resulting in type 1 diabetes in NOD mice139. Superoxide dismutase
and catalase protected isolated beta cells against alloxan-induced diabetes
in vivo indicating a role for superoxide radicals and hydrogen peroxide in
the toxicity of alloxan140.
Studies with isolated islets have provided models of how mediators of the
immune response may damage the islet in the autoimmune response in
type 1 diabetes. The macrophage cytokine IL-1b induces an initial phase of
functional stimulation in rodent pancreatic islets, which is followed after 4-7
hours by a progressive inhibition of (glucose-induced) insulin release and
eventually overt damage to the beta cell caused by production of NO136, 141.
The two cytokines, TNFa and INFg potentiate the IL-1b-induced production
of toxic NO and oxygen free radicals which inhibits insulin secretion142, 143.
This has led to the concept that IL-1b in combination with INFg and TNFa
plays an important role for beta cell dysfunction and death. The general
process of cytokine-induced beta cell 'de-differentiation' with impairment of
some of the most differentiated functions of beta cells, such as the
preferential mitochondrial metabolism of glucose and the biosynthesis and
release of insulin is paralleled by the activation of proteins related to cell
survival, such as heat shock proteins and antioxidant enzymes144. There
are important species differences in beta cell response to IL-1. IL-1 has
stimulatory effects on human islets with no formation of NO compared to
rodent islets, which produces NO when exposed to IL-1. Human islets
cultured in the presence of IL-1b alone exhibit a more prolonged

stimulatory phase, which may last up to 48 hours136. But again, with

prolonged exposure of human islets to IL-1b + TNFa + INFg an inhibition of
human islet function is also observed. This difference between species is
probably due to better capacity of human islets to scavenge oxygen free
radicals and to the higher content of hsp70, catalase and superoxide
dismutase in these cells112. Hsp70 has a direct anti-apoptotic role by
inhibiting protein aggregation, decreasing formation of oxygen free radicals
and blocking effector caspases and it could also decrease necrosis by
preventing cellular ATP depletion. The cytokine-induced beta cell dedifferentiation is probably associated to cytokine-induced down-regulation
of pancreas duodenum homeobox gene-1 (PDX-1) and Isl-1. PDX-1 is a
crucial gene for beta cell development and for the maintenance of its
differentiated phenotype (see chapter with Jensen). The decreased
expression of PDX-1 together with the inhibition of Isl-1 expression could
contribute to the observed decreased expression of insulin and glucokinase
mRNA which depend on PDX-1 for their regulated transcription. While IL-1b
inhibits PDX-1 and Isl-1 expression and up-regulate c-myc it does not
trigger beta cell apoptosis112.Plasma type II secreted phospholipase A2
(PLA2) may be closely involved in the pathophysiology of acute
pancreatitis. The serum levels of type II PLA2 has been shown to correlate
with the severity of the disease145. Also, an inhibitor of type II PLA2 protects
the pancreas against tissue damage when pancreatitis is induced in vitro146.
Interestingly PLA2 enzyme isoforms are also produced by inflammatory
cells and by the islets themselves (see below). As in the case of the soluble
immune effector molecules we have little information on the actual
concentrations to which the beta cells are exposed and the actual in vivo
response in terms of regulation of receptors and signaling pathways.
The Stimulus-Secretion Coupling of the Pancreatic beta cell
The pancreatic beta cell is unusual from the biochemical standpoint in that
it has a high rate of glycolytic and oxidative metabolism and that its glucose
metabolism is largely insulin-independent. Most-strikingly it responds to
extracellular glucose concentrations by increased metabolic flux even at
concentrations of the sugar as high as 25 mM. The latter provides a unique
mechanism that couples the availability of metabolizable nutrients to the
control of secretory, translational, and transcriptional processes without the
mediation of ligand-specific cell surface receptors. It may also be the
Achilles heel of the cell in that intracellular proteins are exposed to higher
concentrations of glucose and reactive metabolites than would otherwise
occur in a typical cell that restricts glucose entry and its metabolism to meet
its energetic demands. Within the secretory granule insulin is stored
complexes with zinc and the beta cell has mechanisms to take up
zinc147 and transport it into the secretory granule (ZnT-8[expressed only in

beta cells], a cation diffusion facilitator)148. Two other transport systems that
are abundant in islet beta cells and important for islet physiology are
GPR40 (G protein-coupled receptor 40 a free fatty acid receptor) and 4F2
coupled amino acid transporters. GPR40 mediates the insulin secretory
stimulation of fatty acids such as linoleic acid reportedly through cAMP and
protein kinase A activity, and inhibition of delayed rectifying voltage gated
K+ channels149. The 4F2 heavy chain (CD98 heavy chain) is a component
of heterodimeric amino acid transporters that is highly represented on the
surface of islet beta cells150. Of note this molecule is also an "activation"
antigen and present at high levels on malignant cells151, 152.

The beta cell Glucose Sensor

The beta cell glucose sensor consists of the combination of two molecules,
which have a restricted tissue distribution, namely Glut2 and glucokinase
(GK). Glucose enters the beta cell through the glucose transporter Glut2
(Km ~17 mM) and is quickly phosphorylated by GK to glucose 6 phosphate
(G6P) (Fig. 3)114. Glut2 expression changes in diabetes and hyperglycemic
states, which seems to underline its importance in the response of the beta
cell, however there is great species variation in the expression of this
molecule and little evidence per se that glucose entry is rate limiting to
glucose metabolism in the beta cell. GK on the other hand, although
present in other tissues such as the liver and kidney appears to be driven
by a pancreatic specific promoter106, 153, 154. Its high expression and
accompanying downregulation of low Km forms of hexokinases155-157 create
a situation where kinetics of the enzyme dictates the kinetics of the
generation of G6P in a cellular context. There has been considerable
debate as to whether the beta cell has a specific glucose 6 phosphatase
(G6Pase) and whether it participates with GK in a regulatory substrate
cycling activity158, 159. The beta cell expresses islet G6Pase related protein
(IGRP) a beta cell specific homolog of the liver G6Pase catalytic subunit
(55% identity) but the molecule is apparently not catalytically active160. Of
note IGRP is a major target of CD8 T lymphocytes of the NOD mouse133.
Other biochemical properties of the beta cell which have been implicated in
its ability to sense metabolic fuels include the absence of lactate
dehydrogenase and thus an inability to re-oxidize cytoplasmically
generated NADH161 and the enhanced expression of the mitochondrial
glycerophosphate shuttle162, 163 which may be involved in the transport of
reducing equivalents in to and out of the mitochondria. Anaplerosis (the
refilling of Krebs cycle intermediates) is shown to be implicated in the
KATP-independent pathways of insulin secretion perhaps via malonyl-CoA
formation and lipid esterification processes or through a pyruvate/malate

shuttle generating NADPH164. Anaplerosis requires conversion of pyruvate

into oxaloacetate by pyruvate carboxylase an enzyme that is abundant in
islet tissue115. Consistent with the view that anaplerosis is an essential
component of beta cell signaling is the fact that the dose dependence of
insulin release highly correlates with the accumulation of citrate, malate,
and citrate-derived malonyl-CoA in the cell164. The fact that the association
of rise in citrate and a-ketoglutarate and initiation of insulin secretion does
not require Ca2+ further indicates that anaplerosis is an early event of beta
cell activation131. A net result of this biochemical machinery is that rates of
glucose metabolism are strongly correlated to changes in the redox state of
pyridine nucleotides (particularly NADPH165, 166 and the ratio of ATP/ADP in
the cell both which may act as intracellular signals for ionic and other
events in the cell (Fig. 3)167. The beta cell is electrically excitable and when
stimulated with glucose it shows oscillations in membrane potential
characterized by bursts of 5-15 sec duration. Increasing glucose lengthen
the burst and shortens the interval leading to continuous spiking over a
base of DY <-40 mV168, 169. The beta cell resting membrane potential of
about -70 mV is mainly determined by the activity of ATP-sensitive K+
channels (KATP)66. When the ATP/ADP ratio increases as a result of
glucose metabolism, the KATP channels in the plasma membrane close170,
170-176
. This causes the cell membrane to depolarize due to the presence of
aninwardly directed cation current that is dominant in the absence of a
reduced KATP-conductance. The nature of this current is currently
unknown but may be a member of the trp family that carries Na+ and Ca2+
ions177. The resulting membrane depolarization leads to opening of the
voltage-dependent L-type Ca2+ channels in the plasma membrane. The
resultant Ca2+ influx elevates the intracellular free Ca2+ concentration
[Ca2+]i and initiates exocytosis of insulin-containing secretory granules
(Fig. 3).
Figure 3. Stimulus-secretion
coupling in the pancreatic
beta cell. Glucose is
transported across the
plasma membrane through
the GLUT2 transporter.
Glucose is metabolized
through the glycolysis and
Krebs (TCA) cycle. The
increased ATP/ADP ratio
leads to closure of the ATPsensitive K+ channel in the
plasma membrane, membrane depolarization and opening of the voltage-dependent
Ca2+ channels. The resulting Ca2+ influx stimulates release of the insulin-containing
granules by exocytosis. [Note: Trp is the transient receptor potential gene family that
first was cloned from Drosophila where it function in photoreception, but which

seems to include Ca2+ store-operated channels and inositol 1,4,5-trisphosphateactivated channels.]

Besides the effect of glucose on the KATP channels glucose is also shown
to affect the stimulus-secretion coupling independently of the KATP
channels thereby amplifying the pathway of glucose-induced insulin
secretion178. This has been shown by the fact that glucose dosedependently increases insulin secretion when the cell is depolarized and
KATP channels cannot be closed (diazoxide plus high K+)179. Under
conditions when the KATP channels are closed by sulfonylureas and
therefore the beta cell plasma membrane is depolarized and insulin
secretion is increased, glucose is shown to be able to further amplify insulin
secretion dose-dependently. This KATP channel-independent (amplifying)
effect of glucose on insulin secretion is Ca2+-dependent but not mediated
by any further rise in [Ca2+]i179. The amplifying pathway serves to augment
the secretory response induced by the triggering signal and creates dosedependent secretory response in the 5-25 mM concentration range.
Glucose may also regulate insulin secretion by a Ca2+-independent
mechanism180-182. Under stringent Ca2+-deprived conditions when protein
kinases A and C are activated simultaneously, glucose stimulates insulin
release in the absence of an elevation of [Ca2+]i. This Ca2+-independent
amplification of glucose-induced insulin release may require GTP late in
stimulus-secretion coupling183, 184. This has been interpreted in terms of two
different mechanism of exocytosis, one driven by Ca2+ and the other by
GTP. Others argue that glucose-induced insulin release is mainly regulated
by the two Ca2+-dependent pathways (rise in beta cell [Ca2+]i and
increase in Ca2+ efficacy) without significant contribution of a Ca2+independent mechanism. In the following, some of the key molecular
components in the beta cell stimulus-secretion coupling will be described.

The ATP-Sensitive K+ Channel Complex

KATP channels are expressed in different cell types including islets, heart,
muscles and ventromedial hypothalamus (VMH) and serve to couple cell
metabolism to membrane excitability. They are composed of a pore-forming
complex consisting of subunits, a specific K+ channel (Kir6.2) surrounded
by regulatory sulphonylurea (SUR) binding subunits. See reviews185-187.
SUR (Mw 140 kDa) is a member of the ABC-transporter superfamily and
Kir6.2 (390 amino acids, 35 kDa) a member of the inward rectifier K+
channel superfamily. Four subunits of Kir6.2 and SUR1 each constitute the
functional channel complex in islets188, 189. KATP channel activity is
modulated by a range of compounds and intracellular metabolites. Channel

activity is reduced by Mg-ATP and sulphonylureas, the most commonly

used class of compounds in the treatment of type 2 diabetes. ADP and
diazoxide conversely activate KATP channel activity. Changes in the beta
cell ATP/ADP ratio are an important physiological regulator of channel
activity with an increase in the blood glucose concentration leading to an
enhanced ATP/ADP ratio and consequently channel closure. Recently,
KATP channels have also been detected in a- and d-cells189-191, whereas
the functional consequence of channel closure in the d-cell is similar to that
in the beta cell (increased hormone release), inhibition of channel activity in
the a-cell leads to inhibition of glucagon release192. The physiologic
importance of this channel in man has become abundantly clear in the past
several years with the discovery that approximately of neonatal diabetes
results from activating mutations of the Kir6.2 molecule. There is a
hierarchy of disease dependent on the severity of the functional effect of
the mutations leading to transient neonatal diabetes, permanent neonatal
diabetes, and finally a syndrome with mental retardation and neonatal
diabetes (DEND syndrome: developmental delay, epilepsy, and neonatal
diabetes193-195. Paternal inactivating mutations of the genes for Kir6.2 and
SUR1 combined with loss of the corresponding maternal chromosomal
(11p15) region in focal areas of the pancreas leads to focal islet lesions,
while autosomal inheritance of mutations results in diffuse pancreatic
lesions, and both cause hyperinsulinemic hypoglycemia185, 196-201. Currently
the genetic bassis of congential hyperinsulinemia have been recognized
due to associations and mutations in eight genes: ABCC8, KCNJ11,
HADH1, GCK, GLUD1, SLC16A1, UPC2 and HNF4a202-205.
HYPERINSULINEMIA GENES
GENE

PROTEIN

LOCUS

INHERITANCE

ABCC8

SUR1

11p15.1

Recessive /Dominant

KCNJ11

Kir6.2

11p15.1

Recessive /Dominant

HADH1

SCHAD

4q22-q26

Recessive

GK

GK

7p15-p13

Dominant

GLUD1

GDH

10q23.3

Dominant

SLC16A1

MCT1

1p13.2-p12

Dominant

UCP2

UCP2

11q13

Dominant

HNF4a

HNF4a

20q12-q13.1

Dominant

Mutations in HADH1 which encodes fatty acid oxidation enzyme SCHAD

causes recessive hyperinsulinemia206. Dominant mutations of GCK cause
hyperinsulinemia that is resistant to therapy207, 208. Dominant mutations of
GLUD1 which encodes mitochondrial glutamate dehydrogenase is
characterized by hyperammonemia209. It is thought that glutamate
dehydrogenase mutations lead to increased ATP in islet beta cells, closing
the K sensitive ATP channel and thus inappropriate insulin secretion and
hypoglycemia. Abnormalities of mitochondria are increasingly recognized
as central to beta cell abnormalities and in particular there are a series of
mitochondrial mutations that lead to diabetes and more recently reports
that mitochondrial "stress" and abnormalities related to mitochondrial
"uncoupling" are important for insulin secretion defects in type 2 diabetes210,
211
.
The Beta Cell Calcium Channels
Membrane depolarization, resulting from KATP channel closure leads to
elevation of cytosolic Ca2+ via voltage-gated Ca2+ channels in the plasma.
The influx of Ca2+ in turn regulates several steps in exocytosis, such as
the size of the readily releasable vesicle pool, the fusion event, and
expansion of the fusion pore212. The Ca2+ concentration required for
initiating insulin secretion in different experimental systems has been
estimated in the range of 10-30 mM213, 214. The resting level is in the
submicromolar range.
Pancreatic beta cells are found to express N-, P/Q- and L-type Ca2+
channels, with the major contribution to Ca2+ influx mediated by the L-type
channel (large and long-lasting)66. The L-type channels are characterized
by the requirement of a strong depolarization to activate. They inactivate
slowly, which is seen in a patch-clamp experiment during a voltage-clamp
depolarization to 0 mV where channel inactivation is maximal (Fig. 4)215.
The L-type channels are by themselves inactivated by Ca2+ ions at the
inner side of the membrane, providing a negative feedback mechanism that
limits Ca2+ entry into the cell216. There is also a voltage-dependent
component in the inactivation The beta cell L-type Ca2+ channel activity is

blocked by dihydropyridines e.g. nifedipine and stimulated by the cyclic

AMP/protein kinase A pathway. Elevated cAMP levels lead to a reduced
rate of Ca2+ channel inactivation whereas the peak current is only
moderately increased217.

Figure 4. Ca2+ current in a mouse beta cell is mediated by L-type Ca2+ channels. The
peak amplitude and the integrated Ca2+ current are depicted resulting from a
membrane depolarization to 0 mV during a voltage-clamp experiment.

Molecular Motors and SNARES

When the Ca2+ channels in the plasma membrane open and Ca2+ flows in
the cell, the onset of exocytosis follows less than 50 ms later. This latency
is shorter than the time required for Ca2+ to equilibrate in the cytosol,
which suggests that the granules are located in the vicinity of the Ca2+
channels and thus sensitive to local [Ca2+]i changes. Indeed, areas with a
high density of Ca2+ channels are found to co-localize with the granules
and represent 'hot spots' of secretion158. A mouse beta cell contains about
13,000 secretory granules218 but only a small fraction (50-75) granules219 in
the readily releasable pool (RRP) are available for immediately release220,
221
. The remainder referred to as the reserve pool needs to be mobilized
into the RRP before they can undergo exocytosis. The number of granules
released seems to be dependent on the phosphorylation state of key
proteins. Activation of protein kinase A and C (PKA and PKC) or inhibition
of protein phosphatases is required for a large exocytotic response217. The
effects of these kinases are additive, which suggests that phosphorylation
of different regulatory proteins are involved. Glucose-stimulated insulin
secretion in vivo is typically biphasic and characterized by a steep transient
first phase is followed by a gradually developing second phase (Fig. 5). In
one model it is explained as the first phase represents release of granules
in RRP, triggered by Ca2+ influx, and the second phase most likely reflects
the ATP-dependent recruitment and release of granules from the reserve
pool221. The electrophysiological response is also biphasic so this
conclusion is open to debate.

Figure 5.
Glucoseinduced insulin
secretion is
characterized
by a rapid first
phase and a
slower second
phase insulin
released to the
bloodstream.
Abbreviation used: iv, intra venous.

Mobilization of granules may involve physical translocation within the beta

cell and/or chemical modification222 and docking of granules near the
plasma membrane. Docked granules need to be primed in order to enter
the RRP. The insulin granules are transported from the reserve pool to the
plasma membrane initially along microtubules and then along the
microfilament networks of the cytoskeleton associated with the plasma
membrane223. An ATP-dependent motor protein such as kinesin is thought
to provide the necessary motive force for transportation. However, the
exact mechanism and its regulation involved in the transport of granules
towards the plasma membrane remains largely unknown224.
The processes by which granules in close proximity to the plasma
membrane bind and fuse to it can be divided into three phases docking,
priming and fusion based on electrophysiological and secretion studies
performed on various neurosecretory tissues and in combination with
biochemical and molecular genetic analysis. Docking describes the initial
reversible interaction of granules with the plasma membrane and
constitutes a pool of unprimed granules in close proximity to the exocytotic
site. There is little morphological evidence of the existence of granule
docking, but the presence of this pool in the beta cell is suggested from the
speed by which RRP is replenished following addition of ATP in the cytosol.
The next priming step may involve ATP-hydrolysis mediated in part by the
ATPase N-ethylmaleimide-sensitive factor (NSF), which is activated by the
soluble NSF-attachment factor (a-SNAP)225. NSF functions as a molecular
chaperone to activate SNAp REceptor (SNARE) proteins destined for the
fusion complex (Fig. 6)224. Another possible ATP-dependent event in
priming is the synthesis of phosphatidylinositol (4,5)-bisphosphate (PIP2),
which is thought to be important in the recruitment and modulation of
proteins implicated in the fusion apparatus - namely the Ca2+ binding
synaptotagmin and calcium-dependent activator protein for secretion
(CAPS)212, 226. Multiple different molecules influence insulin granule
exocytosis. The transcription factor HNF-1 when mutated is a cause of a
severe form of MODY (Maturity Onset Diabetes of Youth) and in addition to
its many effects upon islets and insulin secretrion, the molecule collectrin is

regulated downstream of HNF-1, and collectrin under or over expression

leads to decreased or enhanced insulin secretion227. Insulin secretion is
deficient in a mouse with a mutant Akt/PKB kinase and this protein
regulates exocytosis
in a model
system228.
Figure 6. Docking,
priming and fusion of
insulin-containing
granules. Granules
from the reserve pool
needs to be mobilized
in order to enter RRP
prior to release. The
insert illustrates the
three primary stages of
insulin exocytosis at
the plasma membrane,
docking, priming and
fusion. The granule is tethered at the plasma membrane during docking. Thereafter
the a-SNAP activates NSF, which hydrolyses ATP thereby activating the SNARE
proteins. A second ATP-dependent step in the priming process could be PI4kinase phosphorylation in the generation of PIP2 (after PI-5kinase phosphorylation).
Synaptotagmin is proposed to act as a Ca2+ sensor and a fusion clamp preventing
fusion until a Ca2+ signal arises. When the granule is primed, Ca2+ is the only
requirement for granule fusion with the plasma membrane.

Fusion of granules
Conserved protein families involved in fusion include the SNAREs, the Rab
GTPases, and the Sec1/munc18-related proteins (also referred to as SM
proteins)229. Granuphilin is reported to be in dense core vesicles of beta
cells and binds to GTP-bound Rab27a230. Rab27a, on the granule
membrane is involved in the regulation of exocytosis of secretory granules.
Granuphilin also directly interacts with plasma membrane bound SNARE
protens230. Insulin granules docked to the membrane are reduced in
granuphilin deficient beta cells231 despite granuphilin null mice having
augmented insulin secretion231. The SNARE proteins were originally
identified as synaptic proteins, but are now generally accepted as
universally involved in the core machinery in all intracellular fusion events.
The primed granules are ready to fuse with the plasma membrane in an
ATP-independent process when the intracellular Ca2+ concentration
[Ca2+]i am sufficiently elevated.
Synaptobrevin (VAMP-2, synaptic vesicle-associated membrane protein) is

located on the beta cell granule as a v-SNARE, and syntaxin 1 and SNAP25 (synaptosome-associated protein of 25 kDa) are located on the plasma
membrane as t-SNARE (Fig. 6)232, 233. Synaptobrevin, SNAP-25 and
syntaxin 1 assemble in a 1:1:1 stoichiometry to form a complex that drives
membrane fusion. Helical portions in the SNARE complex bundle together
(two helical domains from SNAP-25 and one from both syntaxin 1 and
synaptobrevin) and form an extended coiled-coil rod-like structure (see
note), which brings the granule into close proximity with the plasma
membrane, thereby overcoming the free energy barrier for fusion (Fig. 6)234.
[Note: Coiled-coil structure is composed of several a-helices that interact
through hydrophobic residues and stabilizing electrostatic interactions
between the side chains. This results in a very stable structure resembling
a strong rope.]It has been postulated that munc18 functions in a late stage
in the fusion process in chromaffin cells, where its dissociation from
syntaxin 1 determines the kinetics of postfusion events235. However,
a munc18 null mutation leads to a selective defect in the docking of LDCVs
at the target membrane and overexpression ofmunc18 leads to an increase
in number of fusion-competent vesicles without affecting the kinetics of
vesicle fusion. This suggests that munc18 is also important in the docking
step before trans-SNARE complexes have assembled. The essential role
of these proteins in neurosecretion is illustrated by the potent inhibition of
insulin secretion by various botulinum and tetanus toxins that specifically
cleave the different SNARE proteins. Studies with botulinum neurotoxin A
which cleaves VAMP-2 and SNAP-25 demonstrate that VAMP-2 is
absolutely necessary for insulin exocytosis whereas the action of SNAP-25
could be partially reversed by higher levels of Ca2+ or cAMP
potentiation236. Botulinum neurotoxin C1 mainly cleaves syntaxin and
reduces K+-induced insulin release by 95% but glucose stimulated insulin
release only by 25% pointing to further complexity in the exocytotic
mechanism237. The fusion event is highly Ca2+-dependent (>10 mM),
probably due to regulatory proteins that function as Ca2+ sensors.
Synaptotagmin is the best-characterized exocytotic Ca2+ sensor with two
Ca2+-binding regions, the C2 domains (see note) C2A and C2B, each
sharing homology with the C2 regulatory domain of protein kinase C (PKC).
The C2A domain is responsible for the Ca2+-dependent insertion of
synaptotagmin into the beta cell granule membrane at low micromolar (5
mM) free Ca2+ and the binding of synaptotagmin to the core complex
protein syntaxin 1224, 238. CAPS is another Ca2+-binding protein thought to
be important for Ca2+-triggered fusion from neuroendocrine cells, including
the beta cell224. It contains a pleckstrin homology domain (see note) that
exhibits binding specificity towards PIP2, which makes it a specific target
for phospholipid mediators formed during priming. The lipid specificity of
CAPS is switched at elevated Ca2+ concentrations and is thought to help
disrupt the plasma membrane bilayer to permit fusion239. Synaptotagmin is

also found to have an inhibitory function in exocytosis. It has been

suggested to lock the fusion core complex by preventing a-SNAP from
binding until a stimulatory Ca2+ signal arises225, 233. Other components of
the fusion process include syntaphilin that acts as a syntaxin clamp in
regulating assembly of the SNARE complex during membrane fusion
events234 and NSF that may be necessary to dissociate SNARE complexes
formed within a single membrane in favor of complexes between
membranes (trans-SNARE complex)240, 241. [Note: A C2 domain contains
about 120-140 amino acids and has been found in single or multiple copies
in more than 60 proteins. It was first discovered in PKC as a Ca2+dependent, phospholipid-binding domain, but now several other C2
domains have been shown to mediate protein-protein interactions and
Ca2+-independent binding to proteins and phospholipids242, 243. Pleckstrin
homology domain mediates membrane association of kinases with their
target molecules, in some cases by interaction with the headgroup of a
phosphoinositide lipid243.
Rab GTPases represent a large family of homologous Ras-like GTPbinding proteins that direct the vectorial movement of secretory vesicles.
Four isoforms of Rab3 (Rab3A, -B, -C, and -D) have been identified so far.
Rab3A is associated with dense-core insulin-containing secretory granules
and thought to play a negative role in the fusion process by preventing
Ca2+-dependent exocytosis244. Rab3A activation is shown to inhibit Ca2+evoked exocytosis in beta cells by possibly binding to calmodulin at low
stimulatory [Ca2+]i245. Calmodulin is a key Ca2+ sensing protein and this
may be a way for Ca2+ to act in concert with GTP in the insulin exocytotic
mechanism (provides a downstream connection between an intracellular
secondary messenger (Ca2+) and the exocytotic machinery of the
pancreatic beta cell). It is postulated 246 that it is a way to direct calmodulin
to the cytoplasmic face of a b-granule membrane. When [Ca2+]i increases,
calmodulin binds CaMKII more readily than Rab3A and this activates
CaMKII to phosphorylate effector exocytotic molecules and enhance either
the transport of a b-granule to the plasma membrane and/or the
mechanism of insulin exocytosis187.
Ca2+ plays another essential role in triggering endocytosis following
secretion. This may be controlled by the Ca2+/calmodulin-dependent
serine/threonine protein phosphatase calcineurin, which is postulated to be
a calcium sensor for endocytosis in synaptosomes. Calcineurin is activated
by calcium and thereby leads to dephosphorylation of proteins involved in
endocytosis like dynamin, amphiphysin 1, amphiphysin 2, and
synaptojanin247, 248.
Signaling inside the beta cell
Metabolizable secretagogues and extracellular molecules such as
hormones, neurotransmitter that act by binding to receptors on the beta cell

plasma membrane appear to mediate their effects through a common set of

second messengers. These effects include 1) modulation of adenylate
cyclase-cAMP system and activation of protein kinase A (PKA) and recent
evidence that cAMP may influence the ATP-sensitive Potassium channel by
signaling through Epacs (Exchange Proteins Activated by Cyclic AMP)249, 2)
generation of inositol 1,4,5-triphosphate (IP3) and sn-1,2-diacylglycerol
(DAG) resulting in activation of the PKC and finally 3) changes in
intracellular free Ca2+ ions that function through Ca2+-dependent
regulatory proteins such as calmodulin and PKC. Phosphoinositide
signaling appears to also be important for insulin exocytosis with recent
evidence using RNAi technology of effect through phospholipase D1, Ca2+
dependent activator protein for secretion 1, and Munc-18-interacting protein
1250.
Hormones binding to G-protein coupled receptors on the cell surface signal
to a heterotrimeric seven transmembrane G-protein (inhibitory G-protein, Gi
or stimulatory G-protein, Gs), which convey the signal to effector systems
such as adenylate cyclase. When a stimulatory G-protein is activated (e.g.
glucagon stimulation of liver cells) the adenylate cyclase, which resides on
the inner plasma membrane, converts ATP to the second messenger cyclic
AMP (cAMP) that activates the cAMP-dependent serine/threonine kinase
PKA. PKA phosphorylates several proteins at their serine or threonine
residues causing a change in either enzyme activity or protein structure. An
example is the activation of glycogen mobilization by activating glycogen
phosphorylase.
cAMP has a short half-life in the cytoplasm and it is quickly hydrolyzed to
AMP by the action of cAMP phosphodiesterases which is important for
shutting down the stimulation by the second messenger. cAMP also
potentiates Ca2+-dependent insulin secretion251-253. Besides acting on the
Ca2+ channels, the major action of cAMP on insulin release is by a direct
effect on the secretory machinery itself251, 254. This is thought to be a result
of an increased Ca2+ sensitivity of exocytosis by cAMP via an extension of
the distance over which the Ca2+ channels can recruit the secretory
granules for release251. It has been suggested that cAMP acts by increasing
the release probability of granules already in RRP (PKA-independent) and
by accelerating granule mobilization thereby refilling the RRP (PKAdependent)253. A recent study has reported that Epac-selective cAMP
analogs, but not PKA-selective analogs influence K ATP channels. Epac 1
and 2 bind to SUR1 of the ATP K+ channel, and thus cAMP may stimulate
insulin secretion through a novel Epac mediated mechanism249. Other Gprotein receptors (inhibitory and stimulatory G, Go; stimulatory G, Gq) are
coupled to phospholipase C (PLC). Acetylcholine stimulates exocytosis by
activating the cholinergic muscarinic receptor that acts via a stimulating Gprotein (Gq), which is bound to the Ca2+-dependent membrane-bound
PLC. PLC hydrolyses PIP2 in the membrane to yield the two second

messengers IP3 and DAG. IP3 diffuses to the endoplasmic reticulum (ER)
where it binds to and opens a Ca2+ channel in the ER membrane thereby
allowing Ca2+ to exit from the ER stores into the cytosol resulting in
increased [Ca2+]i. The membrane bound DAG stimulates protein kinase C
(PKC) by lowering its requirement for Ca2+. DAG is also produced when
PLC is activated following increases in [Ca2+]i, thus PKC will be activated
under all conditions in which intracellular Ca2+ is elevated254. PKC is shown
to modulate Ca2+-dependent insulin secretion217, 251. However, glucosestimulated insulin secretion is unaffected in PKC-depleted islets, indicating
that this enzyme is not essential for glucose stimulated insulin secretion.
Rather, PKC appears to be involved in the stimulation of insulin secretion
by potentiators of release e.g. acetylcholine and carbamylcholine by
sensitizing the secretory machinery to Ca2+217. Glucose-induced insulin
secretion is associated with inhibition of free fatty acid (FFA) oxidation,
increased esterification and complex lipid formation by pancreatic beta
cells. Increases in the mass of DAG, triglyceride and phosphatidic acid are
observed. Exogenous FFA also potentiate glucose-stimulated secretion of
insulin, possibly by providing additional acyl groups for long chain-CoA
formation or complex lipid synthesis. FFA does not stimulate insulin
secretion in the absence of glucose probably due to their rapid entry into
the mitochondria when malonyl-CoA levels are low. 116 LC-CoA opens the
ATP-sensitive K+ channel whereas the corresponding FFA closes the
channel. Long-chain fatty acids stimulate PKC, but the saturated ones are
ineffective 254 or much less effective than the unsaturated ones255.
Phospholipase A2 Superfamily and Pancreatic beta cell Function
The phospholipase A2 family plays an important role in the inflammatory
response seen in IDDM by generating inflammatory molecules from lipids
substrate. Phospholipase A2 (PLA2) constitutes a growing superfamily of
enzymes that hydrolyze membrane phospholipids at the sn-2 position
generating lipid second messengers as well as structural modulations of
cellular membranes (Fig. 7). The fatty acid released from sn-2 is in most
cases
arachidonic
acid.
Figure 7.
Illustration of
cleavage sites for
phospholipase A2
in the glycerol
backbone of a
phospholipid.
Phospholipase A2
cuts the

phospholipids in the sn-2 position resulting in formation of a fatty acid and a

lysophospholipid, which can be further metabolized to eicosanoids and PAF,
respectively. Abbreviations used: PAF, platelet-activating factor (modified from (171).

Several different types of PLA2 have been identified. They include: 1)

cytosolic PLA2 (cPLA2), 2) secreted PLA2 (sPLA2), 3) Ca2+-independent
PLA2 (iPLA2) and 4) platelet-activating factor acetylhydrolase (PAF-AH).
These groups are further subdivided according to structure, molecular
weight, substrate specificity, cellular localization, and Ca2+ dependency of
the enzyme256. The cytosolic forms of PLA2 (group IV PLA2) are high
molecular weight interfacial PLA2 enzymes (see note). They show a
preference for phosphatidylcholine (PC) with arachidonic acid (AA) in the
sn-2 position and are thought to play an important role in providing free AA
for eicosaniod biosynthesis242, 243, 257. Type IV knock-out mice (IV cPLA2a-/mice) show deficient inflammatory response 258 and peritoneal
macrophages from IV cPLA2a-/- mice fail to produce prostaglandins and
leucotrienes after stimulation259. [Note: Interfacial PLA2 enzymes need to
bind to an aggregated surface in order to access the phospholipid
substrates.]
Group I, IIs (IIA, IIC, IID, IIE, IIF), III, V, IX, X and XI PLA2 comprise a group
of low molecular weight PLA2 (13-18 kDa) interfacial enzymes which are
either secreted or extracellular PLA2 (sPLA2). These enzymes have been
implicated in various physiological and pathological functions, including
lipid digestion, lipid mediator generation, cell proliferation, exocytosis,
antibacterial defense, inflammatory diseases and cancer260.
The Ca2+-independent PLA2 (iPLA2) is a group of cytosolic PLA2 of 85-88
kDa whose major cellular function is the mediation of phospholipid
remodeling and homeostasis246, 261, 262. This enzyme seems to regulate the
incorporation of AA and other fatty acids into membrane phospholipids and
as such may be a limiting factor for eicosanoid biosynthesis though it has
been questioned in the case of the rat pancreatic islet group VIA
enzyme263.
Platelet-activating factor acetylhydrolase (PAF-AH) is another group of
Ca2+-independent PLA2's264. All of them have restricted substrate
specificity and are selective for PAF (see note) and for phospholipids with a
short (or intermediate) acyl chain at the sn-2 position that protects normal
membrane lipids from hydrolysis by PAF-AH. A second group of substrates
is oxidatively fragmented phospholipids. The main function of PAF-AH is to
act as scavenger of bioactive phospholipids especially to regulate the level
of PAF265. (Note: PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a
bioactive phospholipid involved in asthma. It is a mediator of a wide range
of immune and allergic reactions. It is synthesized in a regulated pathway
and activates inflammatory cells. Lysophospholipid can be converted to
PAF.)
Different types of PLA2 in islets and pancreatic beta cells

Pancreatic islets contain at least five different types of PLA2 (Table 2).
Among the secreted form, both type IB and IIA are found with type IB more
abundant. Type IB has been localized to the secretory granules of beta
cells and stimulation with insulin secretagogues results in the co-secretion
of insulin and the enzyme263. Type IB also appears to be regulated by
fasting and ambient glucose263, 266, 267. Furthermore, mRNA for a sPLA2
membrane receptor is found in rat pancreatic islets.

Table 1. PLA2 groups in the pancreatic islet.

By use of different inhibitors of PLA2, it has been demonstrated that Ca2+dependent PLA2 may participate in acetylcholine-induced activation of the
granule movement in pancreatic beta cells268. It is proposed that the type IV
cPLA2 couples the secretagogue induced increase in intracellular Ca2+ to
the liberation of AA and the subsequent release of insulin269. Both
exogenous sPLA2 and products of its action have also been shown to
suppress KATP channel activity in excised and cell-attached patches of
plasma membranes from HIT insulinoma cells270.
In glucose-stimulated islets, hydrolysis of AA from membrane phospholipids
appears in part to be mediated by a PLA2 that is catalytically active in the
absence of Ca2+ and inactivated by the iPLA2 inhibitor HELSS
(haloenollactone suicide substrate)224. However, iPLA2 is not required for
AA incorporation or phospholipid remodeling in beta cells, suggesting that
iPLA2 plays another role in these cells. iPLA2 in rat and human beta cells
is implicated as a putative glucose sensor, which can couple alterations in
glycolytic metabolism to the generation of biologically active eicosanoids
and thereby facilitate glucose-induced insulin secretion271. iPLA2 is
activated by ATP and other nucleotides and it has been postulated that it
may respond to the increase in the ATP/ADP ratio after glucose stimulation,
however this function has been questioned.
Besides playing an important role in the stimulus-secretion coupling in the
beta cell different types of PLA2 may be involved in inflammatory
responses. Type II sPLA2 and type IV cPLA2 mRNA is upregulated in
areas with histologic damage in pancreatitis suggesting that these isoforms
might contribute to the morphologic changes that occur272. In addition,
several proinflammatory cytokines such as IL-1, IL-6 and TNFa have been

shown to induce transcription of the type IIA sPLA2 gene in a variety of

cells. TNF priming of human neutrophils (PMN) caused marked increase in
fMLP stimulated AA release in parallel to enhanced activity and secretion of
sPLA2 and activity of cPLA2273. Type I sPLA2, on the contrary, is non-toxic
to acinar cells in both severe and mild pancreatitis. This may indicate that
type II but not type I sPLA2 have pathophysiologic importance in severe
acute pancreatitis associated with systemic inflammatory response
syndrome.

Arachidonic acid in cell signaling

Arachidonic acid is a major constituent of islet phospholipids comprising
about 34-39% of the sn-2 fatty acyl mass in islet glycerophospholipids 271,
274, 275
compared to 13% in rat brain and heart and 1% in rat liver276. The
intracellular level of free AA in islets is normally in the low micromolar
range, but rises to a level of 50-200 mM during glucose stimulation277, 278.
Such changes could result by the action of PLA2, but also by the action of
diacylglycerol lipase and monoacylglycerol lipase on diacylglycerol (DAG),
generated from the action of phospholipase C (Fig. 10)279, 280. Phosphatidic
acid (PA), generated by phospholipase D hydrolysis of PL, is also a
potential source of AA279.

Figure 8. Arachidonic acid can

be made in different ways, but
also degraded in several ways.
AA can be reesterified into
membranes or metabolized by
the lipoxygenase pathway to
leucotrienes, by the
cyclooxygenase pathway to
prostaglandins and
thromboxanes or by the
cytochrome P450
monooxygenase enzyme to
epoxyeicosatrienoic acid.
Abbreviations used: PAPH,
phosphatidic acid
phosphohydrolase.

Inhibition of AA release prevents insulin secretion281. Increases in the

availability of AA (or its metabolites) by itself, however is probably
insufficient to initiate insulin release, instead it seems that AA potentiates

the action of other secretagogue282. In different cell types, AA has been

associated with several specific cellular processes such as regulation of
PKC and PLC and modulation of intracellular Ca2+ transients283-286. In beta
cells, it has been suggested that both Ca2+-dependent and Ca2+independent components are involved in PLA2-induced insulin secretion.
The Ca2+-independent component could be a result of AA or a
lipoxygenase product affecting late events in stimulus-secretion coupling287,
288
. The AA-induced Ca2+-dependent event is biphasic with an early rise
probably reflecting release of Ca2+ from intracellular stores and a
sustained phase, which could reflect extracellular Ca2+ entry possible
mediated by voltage-sensitive Ca2+ channels 289
Role of arachidonic acid metabolites in cell signaling
When AA is generated, it can be reesterified into phospholipids by
acyltransferases or metabolized by the cyclooxygenase (COX),
lipoxygenase (LPX) or cytochrome P450 epoxygenase pathways
generating prostaglandins and thromboxanes, leukotrienes and lipoxins, or
epoxyeicosatrienoic acids290. Prostaglandins made by the cyclooxygenasepathway acting on AA have been demonstrated, in cell-attached patches on
a beta cell line, to be involved in insulin secretion by increasing KATP
channel activity270. E Prostaglandins inhibit insulin secretion and a pertussis
toxin insensitive G alpha i family member G alpha z apparently mediates
this effect291. The lipoxygenase metabolites of AA are potential mediators
of insulin secretion. During nutrient-induced insulin secretion 12hydroxyeicosatetraenoic acid (12-HETE; a product of 12-lipoxygenase
metabolized AA) is augmented282 and a lipoxygenase inhibitor
(nordihydroguaiaretic acid, NDGA) inhibits glucose-induced insulin
secretion292. Products of the lipoxygenase pathway have also been
demonstrated to play a role in apoptosis induced by Ca2+ store depletion
in beta cells by using the inhibitor NDGA293.
The cytokine IL-1 also augments islet production of 12-HETE and PGE2276.
This is not caused by an increased production of AA but by suppression of
reesterification of AA into islet phospholipids. This might occur as a result
the IL-1-induced production of NO and inhibition of ATP-dependent
conversion of the fatty acid to a coenzyme A thioester intermediate276. 12HPETE, a precursor of 12-HETE, has been shown to induce apoptosis of
cells with reduced levels of glutathione peroxidase. Thus the effects of IL-1
to increase substrate flux through the lipoxygenase pathway and to
augment NO production may interact cooperatively to inflict injury on beta
cells, and at the same time stimulate insulin release in the short term.
Pathophysiogical role of lipid metabolites and lysophospholipids on
beta cell signaling

Inflammatory response in the pancreas not only plays an important role in

the destruction of islet tissue in autoimmune diabetes (see chapter by
Mandrup Poulsen), but also in the ability of the pancreatic beta cell to
recover from such insult. Pancreatitis induced by cellophane wrapping
(CW), surgery, coxsackievirus B4, cerulein treatment or with other
chemicals294-296 for example can lead to islet regeneration. Locally produced
INFg causes lymphocyte infiltration and islet cell destruction and while IFNg gene linked to an insulin promoter produces diabetes new cells are
formed from duct epithelial cells and they differentiate into endocrine
cells297. The proinflammatory cytokines, IL-1, TNF and perhaps IL-6 is
shown to induce PLA2 expression and release298. The proinflammatory
action of TNF depends in part on activation of PLA2299. In humans with
acute pancreatitis, there is a correlation between elevated serum PLA2
levels and inflammation and lipid metabolites are also potential mediators
in these events. In this regard it will be of interest to see whether selective
inhibition of PLA2 can be used as a therapeutic approach against type 1
diabetes. Recently, components of the sphingolipid pathways have been
thought to be important contributors to the pathophysiology of diabetes300302
. PLA2 hydrolysis of membrane phospholipids also generates a
lysophospholipid (lysoPL). Several types of lysoPL's exist in the islet
membrane: lysophosphatidylcholine (lysoPC), -ethanolamine (lysoPE),
-serine (lysoPS) and -inositol (lysoPI). LysoPL's and phosphatidic acid (PA)
are shown to promote insulin release when the cellular content of either
lipid increases234. LysoPL's affect stimulus-secretion coupling at a number
of points247, 248, 303, 304. They have been observed to inhibit directly the KATP
channel activity in the pancreas305. LysoPC and lysophosphatidylglycerol
can also effectively mobilize Ca2+ from intracellular stores304. LysoPAF has
been demonstrated to circumvent the inhibition of glucose-induced insulin
release caused by phospholipase inhibitors306.

Micro RNA and Diabetes

There may be as many as 1,000 different micro RNAs in the human
genome, Micro RNAs, following production in the nucleus by the enzyme
Drosha are exported to the cytoplasm, and processed by the enzye DICER.
They then are incorporated into the RNA-induced silencing complex150 and
function to either silence by binding to messenger RNA or by directing the
cleavage of mRNA. It is estimated that there are 68 miRNAs in beta cells.
MiRNAs play essential roles in many biological processes in mammals,

including insulin secretion307, beta-cell development308-311, and adipocyte

differentiation312. The function of one islet specific miRNA has recently been
elucidated. MiRNA-375 is reported to inhibit insulin secretion by inhibiting
the production of the protein myotrophin and thereby decreasing granule
exocytosis312. In fact more MiRNAs have been recently reported to be
enriched in the islets (miR-127-3p, miR-184, miR-195 and miR-493) of
which many of them are differentially expressed in healthy and glucose
intolerant subjects313. Further support also comes from MiRna that have
been identified to play a role in insulin biosynthesis and signaling314-319.
Concluding remarks
Despite considerable progress in defining the molecular physiology of
insulin secretion there remain many unanswered questions. It is also
important to recognize that some current techniques may introduce
complexity, such as the recent report that the RIP-Cre transgene itself
produces mice with impaired glucose tolerance320. Nevertheless given our
current knowledge it is clear that the pancreatic beta cell is an excitable cell
that is critically dependent upon oxidative metabolism 321 and dependent on
the influx of Ca2+ through gated channels and its subsequent removal from
the cell by intracellular sequestration or plasma membrane pumping leaves
it in a precarious position when faced with agents that disrupt Ca2+
homeostasis in the cell. In a similar way, its susceptibility to drugs such as
the DNA alkylating agent streptozotocin or the free radical generator
alloxan may be the consequence of the mechanism of sugar transport,
which can carry these agents into the cell. Such phenomenon may explain
in part why so many of the agents that one associates with pancreatic beta
cell damage and death turn out to be specific secretagogues at least in the
short-term. The challenge is to discover which interactions are part of the
normal responsiveness of the cell to metabolizable substrates, peptides
and neurotransmitters and which are truly deleterious and thus a legitimate
target for pharmacological intervention. It is likely that the concepts that
there are "good" or a "bad" cytokines in the context of autoimmune
diabetes is no more valid than that of glucose being "good" or "bad" in the
context of glucotoxicity and type 2 diabetes. The short-term effects of
secretagogues on islet function have been extensively documented but
there is also the need to consider the broader perspective of how the cell
responds in the longer term to inflammatory stimuli or physiological
changes in energy homeostasis such as accompany pregnancy and
starvation (Fig. 9).

Figure 9. Yin and Yan

representation of the pancreatic
beta cell in terms of induction of
either cell death or secretory
response to secretagogues
such as inflammatory cytokines and PLA2.

Reference List
1. Eisenbarth GS. Type 1 diabetes: molecular, cellular and clinical
immunology. Adv Exp Med Biol 2004;552.
2. Wucherpfennig KW. Insights into autoimmunity gained from
structural analysis of MHC-peptide complexes. Curr Opin Immunol
2001;13(6).

3. Yoon JW, Jun HS. Cellular and molecular pathogenic mechanisms

of insulin-dependent diabetes mellitus. Ann N Y Acad Sci 2001;928.
4. Rosmalen JG, Leenen PJ, Pelegri C, Drexhage HA, HomoDelarche F. Islet abnormalities in the pathogenesis of autoimmune
diabetes. Trends Endocrinol Metab 2002;13(5).
5. Dib SA, Gomes MB. Etiopathogenesis of type 1 diabetes mellitus:
prognostic factors for the evolution of residual beta cell function.
Diabetol Metab Syndr 2009;1(1).
6. Zhang L, Gianani R, Nakayama M et al. Type 1 diabetes: chronic
progressive autoimmune disease. Novartis Found Symp 2008;292.
7. Rossini AA. Autoimmune diabetes and the circle of tolerance.
Diabetes 2004;53(2).
8. Arif S, Tree TI, Astill TP et al. Autoreactive T cell responses show
proinflammatory polarization in diabetes but a regulatory phenotype
in health. J ClinINVEST 2004;113(3).
9. Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and
clinical interventions in type 1 diabetes. Nature 2010;464(7293).
10. Llaurado G, Gallart L, Tirado R et al. Insulin resistance, low-grade
inflammation and type 1 diabetes mellitus. Acta Diabetol 2012.
11. Nadeau KJ, Regensteiner JG, Bauer TA et al. Insulin resistance in
adolescents with type 1 diabetes and its relationship to
cardiovascular function. J Clin Endocrinol Metab 2010;95(2).
12. Rodrigues TC, Biavatti K, Almeida FK, Gross JL. Coronary artery
calcification is associated with insulin resistance index in patients
with type 1 diabetes. Braz J Med Biol Res 2010;43(11).
13. Wilkin TJ. Insulin resistance and progression to type 1 diabetes in
the European Nicotinamide Diabetes Intervention Trial (ENDIT):
response to Bingley et al. Diabetes Care 2008;31(4).
14. Wilkin TJ. The accelerator hypothesis: a review of the evidence for
insulin resistance as the basis for type I as well as type II diabetes.
Int J Obes (Lond) 2009;33(7).
15. Atkinson MA, Eisenbarth GS. Type 1 diabetes: new perspectives on
disease pathogenesis and treatment. Lancet 2001;358(9277).

16. Butler AE, Galasso R, Meier JJ, Basu R, Rizza RA, Butler PC.
Modestly increased beta cell apoptosis but no increased beta cell
replication in recent-onset type 1 diabetic patients who died of
diabetic ketoacidosis. Diabetologia 2007;50(11):2323-2331.
17. Meier JJ, Lin JC, Butler AE, Galasso R, Martinez DS, Butler PC.
Direct evidence of attempted beta cell regeneration in an 89-year-old
patient with recent-onset type 1 diabetes. Diabetologia
2006;49(8):1838-1844.
18. Atkinson MA, Gianani R. The pancreas in human type 1 diabetes:
providing new answers to age-old questions. Curr Opin Endocrinol
Diabetes Obes 2009;16(4):279-285.
19. Wang L, Lovejoy NF, Faustman DL. Persistence of Prolonged Cpeptide Production in Type 1 Diabetes as Measured With an
Ultrasensitive C-peptide Assay. Diabetes Care 2012;35(3).
20. Gianani R, Campbell-Thompson M, Sarkar SA et al. Dimorphic
histopathology of long-standing childhood-onset diabetes.
Diabetologia 2010;53(4).
21. Keenan HA, Sun JK, Levine J et al. Residual insulin production and
pancreatic ss-cell turnover after 50 years of diabetes: Joslin Medalist
Study. Diabetes 2010;59(11):2846-2853.
22. Foulis AK, Stewart JA. The pancreas in recent-onset type 1 (insulindependent) diabetes mellitus: insulin content of islets, insulitis and
associated changes in the exocrine acinar tissue. Diabetologia
1984;26(6):456-461.
23. Eisenbarth GS. Banting Lecture 2009: An Unfinished Journey:
Molecular Pathogenesis to Prevention of Type 1A Diabetes. Diabetes
2010;59(4):759-774.
24. Henderson JR. Why are the islet of Langerhans? Lancet
1969;2(7618):469-470.
25. Williams JA, Goldfine ID. The insulin-pancreatic acinar axis.
Diabetes 1985;34(10):980-986.
26. Williams AJ, Chau W, Callaway MP, Dayan CM. Magnetic
resonance imaging: a reliable method for measuring pancreatic
volume in Type 1 diabetes. Diabet Med 2007;24(1):35-40.

27. Gaglia JL, Guimaraes AR, Harisinghani M et al. Noninvasive

imaging of pancreatic islet inflammation in type 1A diabetes patients.
J Clin Invest 2011;121(1):442-445.
28. Jiao Y, Peng ZH, Xing TH, Qin J, Zhong CP. Assessment of islet
graft survival using a 3.0-Tesla magnetic resonance scanner. Anat
Rec (Hoboken) 2008;291(12).
29. Wang P, Yigit MV, Medarova Z et al. Combined small interfering
RNA therapy and in vivo magnetic resonance imaging in islet
transplantation. Diabetes 2011;60(2).
30. Altieri DC. Molecular cloning of effector cell protease receptor-1, a
novel cell surface receptor for the protease factor Xa. J Biol Chem
1994;269(5).
31. Liggins C, Orlicky DJ, Bloomquist LA, Gianani R. Developmentally
regulated expression of Survivin in human pancreatic islets. Pediatr
Dev Pathol 2003;6(5).
32. Hasel C, Bhanot UK, Heydrich R, Strater J, Moller P. Parenchymal
regression in chronic pancreatitis spares islets reprogrammed for the
expression of NFkappaB and IAPs. LabINVEST 2005;85(10).
33. Achenbach P, Bonifacio E, Koczwara K, Ziegler AG. Natural history
of type 1 diabetes. Diabetes 2005;54 Suppl 2.
34. Achenbach P, Bonifacio E, Ziegler AG. Predicting type 1 diabetes.
Curr Diab Rep 2005;5(2).
35. Bingley PJ, Gale EA. Progression to type 1 diabetes in islet cell
antibody-positive relatives in the European Nicotinamide Diabetes
Intervention Trial: the role of additional immune, genetic and
metabolic markers of risk. diabetol 2006.
36. Bottazzo GF, Bosi E, Bonifacio E, Mirakian R, Todd I, Pujol-Borrell
R. Pathogenesis of type I (insulin-dependent) diabetes: possible
mechanisms of autoimmune damage. Br Med Bull 1989;45(1).
37. Eisenbarth GS. Italian Society of Diabetology Mentor Award. The
stages of Type 1A diabetes: retrospective and prospective. Diabetes
Nutr Metab 2004;17(6).
38. Palmer JP, Fleming GA, Greenbaum CJ et al. C-Peptide Is the
Appropriate Outcome Measure for Type 1 Diabetes Clinical Trials to
Preserve beta-Cell Function: Report of an ADA Workshop, 21-22
October 2001. diab 2004;53(1).

39. Andre I, Gonzalez A, Wang B, Katz J, Benoist C, Mathis D.

Checkpoints in the progression of autoimmune disease: lessons from
diabetes models. Proc Natl Acad Sci U S A 1996;93(6).
40. Bresson D, Togher L, Rodrigo E et al. Anti-CD3 and nasal proinsulin
combination therapy enhances remission from recent-onset
autoimmune diabetes by inducing Tregs. Journal of Clinical
Investigation 2006;116(5).
41. Dai YD, Jensen KP, Lehuen A et al. A peptide of glutamic acid
decarboxylase 65 can recruit and expand a diabetogenic T cell clone,
BDC2.5, in the pancreas. J Immunol 2005;175(6).
42. Liu W, Putnam AL, Xu-Yu Z et al. CD127 expression inversely
correlates with FoxP3 and suppressive function of human CD4+ T
reg cells. J Exp Med 2006;203(7).
43. Sarkar SA, Lee CE, Victorino F et al. Expression and regulation of
chemokines in murine and human type 1 diabetes. Diabetes
2012;61(2).
44. Slack JM. Developmental biology of the pancreas. Development
1995;121(6).
45. Piper K, Brickwood S, Turnpenny LW et al. Beta cell differentiation
during early human pancreas development. J Endocrinol
2004;181(1).
46. Richardson MK, Hanken J, Gooneratne ML et al. There is no highly
conserved embryonic stage in the vertebrates: implications for
current theories of evolution and development. Anat Embryol (Berl)
1997;196(2).
47. Sarkar SA, Kobberup S, Wong R et al. Global gene expression
profiling and histochemical analysis of the developing human fetal
pancreas. Diabetologia 2008;51(2).
48. Reichert M, Rustgi AK. Pancreatic ductal cells in development,
regeneration, and neoplasia. J ClinINVEST 2011;121(12).
49. Iburi T, Izumiyama H, Hirata Y. [Endocrine glands of pancreas].
Nihon Rinsho69 Suppl 2.
50. Youos JG. The role of alpha-, delta- and F cells in insulin secretion
and action. Diabetes Res Clin Pract93 Suppl 1.

51. Prado CL, Pugh-Bernard AE, Elghazi L, Sosa-Pineda B, Sussel L.

Ghrelin cells replace insulin-producing beta cells in two mouse
models of pancreas development. Proc Natl Acad Sci U S A
2004;101(9).
52. Sorenson RL, Garry DG, Brelje TC. Structural and functional
considerations of GABA in islets of Langerhans. Beta-cells and
nerves. Diabetes 1991;40(11).
53. Wiest-Ladenburger U, Hartmann R, Hartmann U, Berling K, Bohm
BO, Richter W. Combined analysis and single-step detection of
GAD65 and IA2 autoantibodies in IDDM can replace the
histochemical islet cell antibody test. Diabetes 1997;46(4).
54. Aanstoot HJ, Kang SM, Kim J et al. Identification and
characterization of glima 38, a glycosylated islet cell membrane
antigen, which together with GAD65 and IA2 marks the early phases
of autoimmune response in type 1 diabetes. J ClinINVEST
1996;97(12).
55. Hawkes CJ, Wasmeier C, Christie MR, Hutton JC. Identification of
the 37-kDa antigen in IDDM as a tyrosine phosphatase-like protein
(phogrin) related to IA-2. diab 1996;45(9).
56. Bartholomeusz RK, Campbell IL, Harrison LC. Pancreatic islet
A2B5- and 3G5-reactive gangliosides are markers of differentiation in
rat insulinoma cells. Endocrinology 1989;124(6).
57. Alejandro R, Shienvold FL, Hajek SAV, Pierce M, Paul R, Mintz DH.
A ganglioside antigen on the rat pancreatic B cell surface identified
by monoclonal antibody R2D6. J ClinINVEST 1984;74.
58. Anlauf M, Eissele R, Schafer MK et al. Expression of the two
isoforms of the vesicular monoamine transporter (VMAT1 and
VMAT2) in the endocrine pancreas and pancreatic endocrine tumors.
J Histochem Cytochem 2003;51(8).
59. Harris PE, Ferrara C, Barba P, Polito T, Freeby M, Maffei A. VMAT2
gene expression and function as it applies to imaging beta-cell mass.
J Mol Med (Berl) 2008;86(1).
60. Eipper BA, Green CB, Mains RE. Expression of prohormone
processing enzymes in neuroendocrine and non-neuroendocrine
cells. J Natl Cancer Inst Monogr 1992;(13).
61. Eipper BA, Milgram SL, Husten EJ, Yun HY, Mains RE.
Peptidylglycine alpha-amidating monooxygenase: a multifunctional

protein with catalytic, processing, and routing domains. Protein Sci

1993;2(4).
62. Eipper BA, Perkins SN, Husten EJ, Johnson RC, Keutmann HT,
Mains RE. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase.
Purification, characterization, and expression. J Biol Chem
1991;266(12).
63. Schafer MK, Stoffers DA, Eipper BA, Watson SJ. Expression of
peptidylglycine alpha-amidating monooxygenase (EC 1.14.17.3) in
the rat central nervous system. J Neurosci 1992;12(1).
64. Martinez A, Montuenga LM, Springall DR, Treston A, Cuttitta F,
Polak JM. Immunocytochemical localization of peptidylglycine alphaamidating monooxygenase enzymes (PAM) in human endocrine
pancreas. J Histochem Cytochem 1993;41(3).
65. Orskov C, Bersani M, Johnsen AH, Hojrup P, Holst JJ. Complete
sequences of glucagon-like peptide-1 from human and pig small
intestine. J Biol Chem 1989;264(22).
66. Ashcroft FM, Rorsman P. Electrophysiology of the pancreatic betacell. Prog Biophys Mol Biol 1989;54(2).
67. Colombo C, Porzio O, Liu M et al. Seven mutations in the human
insulin gene linked to permanent neonatal/infancy-onset diabetes
mellitus. J ClinINVEST 2008;118(6).
68. Hodish I, Liu M, Rajpal G et al. Misfolded proinsulin affects
bystander proinsulin in neonatal diabetes. J Biol Chem 2011;285(1).
69. Hodish I, Absood A, Liu L et al. In vivo misfolding of proinsulin below
the threshold of frank diabetes. Diabetes 2011;60(8).
70. Atkinson MA, Bluestone JA, Eisenbarth GS et al. How does type 1
diabetes develop?: the notion of homicide or beta-cell suicide
revisited. Diabetes 2011;60(5).
71. Grimaldi KA, Siddle K, Hutton JC. Biosynthesis of insulin secretory
granule membrane proteins. Control by glucose. Biochem J
1987;245(2).
72. Orci L, Ravazzola M, Storch MJ, Anderson RG, Vassalli JD,
Perrelet A. Proteolytic maturation of insulin is a post-Golgi event
which occurs in acidifying clathrin-coated secretory vesicles. Cell
1987;49(6).

73. Hutton JC, Davidson HW, Peshavaria M. Proteolytic processing of

chromogranin A in purified insulin granules. Formation of a 20 kDa Nterminal fragment (betagranin) by the concerted action of a Ca2+dependent endopeptidase and carboxypeptidase H (EC 3.4.17.10).
Biochem J 1987;244(2).
74. Orci L, Ravazzola M, Amherdt M et al. Conversion of proinsulin to
insulin occurs coordinately with acidification of maturing secretory
vesicles. J Cell Biol 1986;103(6 Pt 1).
75. Orci L, Ravazzola M, Perrelet A. (Pro)insulin associates with Golgi
membranes of pancreatic B cells. Proc Natl Acad Sci U S A
1984;81(21).
76. Lazure C, Seidah NG, Pelaprat D, Chretien M. Proteases and
posttranslational processing of prohormones: a review. Can J
Biochem Cell Biol 1983;61(7).
77. Lernmark A, Chan SJ, Choy R et al. Biosynthesis of insulin and
glucagon: a view of the current state of the art. Ciba Found Symp
1976;41.
78. Dodson G, Steiner D. The role of assembly in insulin's biosynthesis.
Curr Opin Struct Biol 1998;8(2).
79. Emdin SO, Dodson GG, Cutfield JM, Cutfield SM. Role of zinc in
insulin biosynthesis. Some possible zinc-insulin interactions in the
pancreatic B-cell. Diabetologia 1980;19(3).
80. Clifford KS, MacDonald MJ. Survey of mRNAs encoding zinc
transporters and other metal complexing proteins in pancreatic islets
of rats from birth to adulthood: similar patterns in the SpragueDawley and Wistar BB strains. Diabetes Res Clin Pract 2000;49(2-3).
81. Vallee BL, Falchuk KH. The biochemical basis of zinc physiology.
Physiol Rev 1993;73(1).
82. Chimienti F, Aouffen M, Favier A, Seve M. Zinc homeostasisregulating proteins: new drug targets for triggering cell fate. Curr
Drug Targets 2003;4(4).
83. Bloc A, Cens T, Cruz H, Dunant Y. Zinc-induced changes in ionic
currents of clonal rat pancreatic -cells: activation of ATP-sensitive K+
channels. J Physiol 2000;529 Pt 3.

84. Franklin I, Gromada J, Gjinovci A, Theander S, Wollheim CB. Betacell secretory products activate alpha-cell ATP-dependent potassium
channels to inhibit glucagon release. Diabetes 2005;54(6).
85. Ishihara H, Maechler P, Gjinovci A, Herrera PL, Wollheim CB. Islet
beta-cell secretion determines glucagon release from neighbouring
alpha-cells. Nat Cell Biol 2003;5(4).
86. Kim BJ, Kim YH, Kim S et al. Zinc as a paracrine effector in
pancreatic islet cell death. Diabetes 2000;49(3).
87. Kambe T, Narita H, Yamaguchi-Iwai Y et al. Cloning and
characterization of a novel mammalian zinc transporter, zinc
transporter 5, abundantly expressed in pancreatic beta cells. J Biol
Chem 2002;277(21).
88. Eide DJ. Metal ion transport in eukaryotic microorganisms: insights
from Saccharomyces cerevisiae. Adv Microb Physiol 2000;43.
89. Seve M, Chimienti F, Devergnas S, Favier A. In silico identification
and expression of SLC30 family genes: an expressed sequence tag
data mining strategy for the characterization of zinc transporters'
tissue expression. BMC Genomics 2004;5(1).
90. Sim BK, Fogler WE, Zhou XH et al. Zinc ligand-disrupted
recombinant human Endostatin: potent inhibition of tumor growth,
safety and pharmacokinetic profile. Angiogenesis 1999;3(1).
91. Sim DL, Yeo WM, Chow VT. The novel human HUEL (C4orf1)
protein shares homology with the DNA-binding domain of the XPA
DNA repair protein and displays nuclear translocation in a cell cycledependent manner. Int J Biochem Cell Biol 2002;34(5).
92. Palmiter RD, Findley SD. Cloning and functional characterization of
a mammalian zinc transporter that confers resistance to zinc. EMBO
J 1995;14(4).
93. Wenzel HJ, Cole TB, Born DE, Schwartzkroin PA, Palmiter RD.
Ultrastructural localization of zinc transporter-3 (ZnT-3) to synaptic
vesicle membranes within mossy fiber boutons in the hippocampus of
mouse and monkey. Proc Natl Acad Sci U S A 1997;94(23).
94. Wenzlau JM, Juhl K, Yu L et al. The cation efflux transporter ZnT8
(Slc30A8) is a major autoantigen in human type 1 diabetes. Proc Natl
Acad Sci U S A 2007;104(43).

95. Chimienti F, Devergnas S, Favier A, Seve M. Identification and

cloning of a beta-cell-specific zinc transporter, ZnT-8, localized into
insulin secretory granules. Diabetes 2004;53(9).
96. Chimienti F, Devergnas S, Pattou F et al. In vivo expression and
functional characterization of the zinc transporter ZnT8 in glucoseinduced insulin secretion. J Cell Sci 2006;119(Pt 20).
97. An S, Zenisek D. Regulation of exocytosis in neurons and
neuroendocrine cells. Curr Opin Neurobiol 2004;14(5).
98. Tsuboi T, McMahon HT, Rutter GA. Mechanisms of dense core
vesicle recapture following "kiss and run" ("cavicapture") exocytosis
in insulin-secreting cells. J Biol Chem 2004;279(45).
99. Artalejo CR, Elhamdani A, Palfrey HC. Secretion: dense-core
vesicles can kiss-and-run too. Curr Biol 1998;8(2).
100. Wu LG. Kinetic regulation of vesicle endocytosis at synapses. Trends
Neurosci 2004;27(9).
101. Artalejo CR, Elhamdani A, Palfrey HC. Sustained stimulation shifts
the mechanism of endocytosis from dynamin-1-dependent rapid
endocytosis to clathrin- and dynamin-2-mediated slow endocytosis in
chromaffin cells. Proc Natl Acad Sci U S A 2002;99(9).
102. Artalejo CR, Henley JR, McNiven MA, Palfrey HC. Rapid endocytosis
coupled to exocytosis in adrenal chromaffin cells involves Ca2+,
GTP, and dynamin but not clathrin. Proc Natl Acad Sci U S A
1995;92(18).
103. Straub SG, Shanmugam G, Sharp GW. Stimulation of insulin release
by glucose is associated with an increase in the number of docked
granules in the beta-cells of rat pancreatic islets. Diabetes
2004;53(12).
104. Straub SG, Sharp GW. Hypothesis: one rate-limiting step controls the
magnitude of both phases of glucose-stimulated insulin secretion. Am
J Physiol Cell Physiol 2004;287(3).
105. Daniel S, Noda M, Straub SG, Sharp GW. Identification of the
docked granule pool responsible for the first phase of glucosestimulated insulin secretion. Diabetes 1999;48(9).
106. Leibiger B, Leibiger IB, Moede T et al. Selective insulin signaling
through A and B insulin receptors regulates transcription of insulin
and glucokinase genes in pancreatic beta cells. Mol Cell 2001;7(3).

107. Leibiger IB, Leibiger B, Moede T, Berggren PO. Exocytosis of insulin

promotes insulin gene transcription via the insulin receptor/PI-3
kinase/p70 s6 kinase and CaM kinase pathways. Mol Cell 1998;1(6).
108. Schuit FC, In't Veld PA, Pipeleers DG. Glucose stimulates proinsulin
biosynthesis by a dose-dependent recruitment of pancreatic beta
cells. Proc Natl Acad Sci U S A 1988;85(11).
109. Fonseca SG, Fukuma M, Lipson KL et al. WFS1 is a novel
component of the unfolded protein response and maintains
homeostasis of the endoplasmic reticulum in pancreatic beta-cells. J
Biol Chem 2005;280(47).
110. Hostens K, Pavlovic D, Zambre Y et al. Exposure of human islets to
cytokines can result in disproportionately elevated proinsulin release.
J ClinINVEST 1999;104(1).
111. Cnop M, Welsh N, Jonas JC, Jorns A, Lenzen S, Eizirik DL.
Mechanisms of Pancreatic {beta}-Cell Death in Type 1 and Type 2
Diabetes: Many Differences, Few Similarities. Diabetes 2005;54
Suppl 2:S97-S107.
112. Eizirik DL, Mandrup-Poulsen T. A choice of death - the signaltransduction of immune-mediated beta-cell apoptosis. diabetol
2001;44(12).
113. Schuit FC, Huypens P, Heimberg H, Pipeleers DG. Glucose sensing
in pancreatic beta-cells: a model for the study of other glucoseregulated cells in gut, pancreas, and hypothalamus. Diabetes
2001;50(1).
114. Matschinsky FM. Banting Lecture 1995. A lesson in metabolic
regulation inspired by the glucokinase glucose sensor paradigm.
Diabetes 1996;45(2).
115. Schuit F, De Vos A, Farfari S et al. Metabolic fate of glucose in
purified islet cells. Glucose-regulated anaplerosis in beta cells. J Biol
Chem 1997;272(30).
116. Drucker DJ. The biology of incretin hormones. Cell Metab 2006;3(3).
117. Hay CW, Sinclair EM, Bermano G, Durward E, Tadayyon M, Docherty
K. Glucagon-like peptide-1 stimulates human insulin promoter activity
in part through cAMP-responsive elements that lie upstream and
downstream of the transcription start site. J Endocrinol 2005;186(2).

118. Park S, Dong X, Fisher TL et al. Exendin-4 uses Irs2 signaling to

mediate pancreatic beta cell growth and function. J Biol Chem
2006;281(2).
119. Triplitt C, Wright A, Chiquette E. Incretin mimetics and dipeptidyl
peptidase-IV inhibitors: potential new therapies for type 2 diabetes
mellitus. Pharmacotherapy 2006;26(3).
120. de Sa SV, Correa-Giannella ML, Machado MC et al. Somatostatin
receptor subtype 5 (SSTR5) mRNA expression is related to
histopathological features of cell proliferation in insulinomas. Endocr
Relat Cancer 2006;13(1).
121. Degano P, Peiro E, Miralles P, Silvestre RA, Marco J. Effects of rat
pancreatic polypeptide on islet-cell secretion in the perfused rat
pancreas. Metabolism 1992;41(3).
122. Lundquist I, Sundler F, Ahren B, Alumets J, Hakanson R.
Somatostatin, pancreatic polypeptide, substance P, and neurotensin:
cellular distribution and effects on stimulated insulin secretion in the
mouse. Endocrinology 1979;104(3).
123. Szecowka J, Tatemoto K, Rajamaki G, Efendic S. Effects of PYY and
PP on endocrine pancreas. Acta Physiol Scand 1983;119(2).
124. Doi A, Shono T, Nishi M, Furuta H, Sasaki H, Nanjo K. IA-2beta, but
not IA-2, is induced by ghrelin and inhibits glucose-stimulated insulin
secretion. Proc Natl Acad Sci U S A 2006;103(4).
125. Takahashi H, Kurose Y, Kobayashi S et al. Ghrelin enhances
glucose-induced insulin secretion in scheduled meal-fed sheep. J
Endocrinol 2006;189(1).
126. Henquin JC. Triggering and amplifying pathways of regulation of
insulin secretion by glucose. Diabetes 2000;49(11).
127. Stiernet P, Guiot Y, Gilon P, Henquin JC. Glucose acutely decreases
pH of secretory granules in mouse pancreatic islets: mechanisms
and influence on insulin secretion. J Biol Chem 2006;.
128. Johnson KH, O'Brien TD, Betsholtz C, Westermark P. Islet amyloid
polypeptide: mechanisms of amyloidogenesis in the pancreatic islets
and potential roles in diabetes mellitus. Lab Invest 1992;66.
129. Juan-Pico P, Fuentes E, Javier Bermudez-Silva F et al. Cannabinoid
receptors regulate Ca(2+) signals and insulin secretion in pancreatic
beta-cell. Cell Calcium 2006;39(2).

130. Kubosaki A, Nakamura S, Notkins AL. Dense Core Vesicle Proteins

IA-2 and IA-2{beta}: Metabolic Alterations in Double Knockout Mice.
diab 2005;54 Suppl 2.
131. Moriyama H, Abiru N, Paronen J et al. Evidence for a primary islet
autoantigen (preproinsulin 1) for insulitis and diabetes in the
nonobese diabetic mouse. Proc Natl Acad Sci U S A 2003;100(18).
132. Nakayama M, Abiru N, Moriyama H et al. Prime role for an insulin
epitope in the development of type 1 diabetes in NOD mice. Nature
2005;435(7039).
133. Han B, Serra P, Amrani A et al. Prevention of diabetes by
manipulation of anti-IGRP autoimmunity: high efficiency of a lowaffinity peptide. Nat Med 2005;11(6).
134. Rotig A, Cormier V, Chatelain P, et al. Deletion of mitochondrial DNA
in a case of early-onset diabetes mellitus, optic atrophy, and
deafness (Wolfram syndrome, MIM 222300). J Clin Invest 1993;91.
135. Rabinovitch A, Pukel C, Baquerizo H. Interleukin-1 inhibits glucosemodulated insulin and glucagon secretion in rat islet monolayer
cultures. Endocrinology 1988;122(6).
136. Chen MC, Paez-Espinosa V, Welsh N, Eizirik DL. Interleukin-1beta
regulates phospholipase D-1 expression in rat pancreatic beta-cells.
Endocrinology 2000;141(8).
137. Nielsen K, Karlsen AE, Deckert M et al. Beta-cell maturation leads to
in vitro sensitivity to cytotoxins. Diabetes 1999;48(12).
138. Strandell E, Buschard K, Saldeen J, Welsh N. Interleukin-1 beta
induces the expression of hsp70, heme oxygenase and Mn-SOD in
FACS-purified rat islet beta-cells, but not in alpha-cells. Immunol Lett
1995;48(2).
139. Horio F, Fukuda M, Katoh H et al. Reactive oxygen intermediates in
autoimmune islet cell destruction of the NOD mouse induced by
peritoneal exudate cells (rich in macrophages) but not T cells.
Diabetologia 1994;37(1).
140. Jorns A, Tiedge M, Lenzen S, Munday R. Effect of superoxide
dismutase, catalase, chelating agents, and free radical scavengers
on the toxicity of alloxan to isolated pancreatic islets in vitro. Free
Radic Biol Med 1999;26(9-10).

141. Eizirik DL, Sandler S, Welsh N, Juntti-Berggren L, Berggren PO.

Interleukin-1 beta-induced stimulation of insulin release in mouse
pancreatic islets is related to diacylglycerol production and protein
kinase C activation. Mol Cell Endocrinol 1995;111(2).
142. Karlsen AE, Sparre T, Nielsen K, Nerup J, Pociot F. Proteome
analysis--a novel approach to understand the pathogenesis of Type 1
diabetes mellitus. Dis Markers 2001;17(4).
143. Karlsen AE, Storling ZM, Sparre T et al. Immune-mediated beta-cell
destruction in vitro and in vivo-A pivotal role for galectin-3. Biochem
Biophys Res Commun 2006;344(1).
144. Eizirik DL. Interleukin-1 induced impairment in pancreatic islet
oxidative metabolism of glucose is potentiated by tumor necrosis
factor. Acta Endocrinol (Copenh) 1988;119(3).
145. Miura M, Endo S, Kaku LL et al. Plasma type II phospholipase A2
levels in patients with acute pancreatitis. Res Commun Mol Pathol
Pharmacol 2001;109(3-4).
146. Uhl W, Schrag HJ, Schmitter N, Aufenanger J, Nevalainen TJ,
Buchler MW. Experimental study of a novel phospholipase A2
inhibitor in acute pancreatitis. Br J Surg 1998;85(5).
147. Gyulkhandanyan AV, Lee SC, Bikopoulos G, Dai F, Wheeler MB. The
Zn2+-transporting Pathways in Pancreatic beta-Cells: A ROLE FOR
THE L-TYPE VOLTAGE-GATED Ca2+ CHANNEL. J Biol Chem
2006;281(14).
148. Chimienti F, Favier A, Seve M. ZnT-8, a pancreatic beta-cell-specific
zinc transporter. Biometals 2005;18(4).
149. Itoh Y, Hinuma S. GPR40, a free fatty acid receptor on pancreatic
beta cells, regulates insulin secretion. Hepatol Res 2005.
150. Srikanta S, Telen M, Posillico JT et al. Monoclonal antibodies to a
human islet cell surface glycoprotein: 4F2 and LC7-2. Endocrinology
1987;120(6).
151. Kanai Y, Endou H. Heterodimeric amino acid transporters: molecular
biology and pathological and pharmacological relevance. Curr Drug
Metab 2001;2(4).
152. Kim dK, Ahn SG, Park JC, Kanai Y, Endou H, Yoon JH. Expression of
L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc)

in oral squamous cell carcinoma and its precusor lesions. Anticancer

Res 2004;24(3a).
153. Magnuson MA. Tissue-specific regulation of glucokinase gene
expression. J Cell Biochem 1992;48(2).
154. Watada H, Kajimoto Y, Miyagawa J et al. PDX-1 induces insulin and
glucokinase gene expressions in alphaTC1 clone 6 cells in the
presence of betacellulin. Diabetes 1996;45(12).
155. Ferber S, BeltrandelRio H, Johnson JH et al. GLUT-2 gene transfer
into insulinoma cells confers both low and high affinity glucosestimulated insulin release. Relationship to glucokinase activity. J Biol
Chem 1994;269(15).
156. German MS. Glucose sensing in pancreatic islet beta cells: the key
role of glucokinase and the glycolytic intermediates. Proc Natl Acad
Sci U S A 1993;90(5).
157. Halban PA, Praz GA, Wollheim CB. Abnormal glucose metabolism
accompanies failure of glucose to stimulate insulin release from a rat
pancreatic cell line (RINm5F). Biochem J 1983;212(2).
158. Khan A, Chandramouli V, Ostenson CG et al. Glucose cycling is
markedly enhanced in pancreatic islets of obese hyperglycemic mice.
Endocrinology 1990;126(5).
159. Ling ZC, Khan A, Delauny F et al. Increased glucocorticoid sensitivity
in islet beta-cells: effects on glucose 6-phosphatase, glucose cycling
and insulin release. Diabetologia 1998;41(6).
160. Ebert DH, Bischof LJ, Streeper RS et al. Structure and promoter
activity of an islet-specific glucose-6-phosphatase catalytic subunitrelated gene. Diabetes 1999;48(3).
161. Sekine N, Cirulli V, Regazzi R et al. Low lactate dehydrogenase and
high mitochondrial glycerol phosphate dehydrogenase in pancreatic
beta-cells. Potential role in nutrient sensing. J Biol Chem
1994;269(7).
162. Giroix MH, Rasschaert J, Sener A et al. Study of hexose transport,
glycerol phosphate shuttle and Krebs cycle in islets of adult rats
injected with streptozotocin during the neonatal period. Mol Cell
Endocrinol 1992;83(2-3).

163. Sener A, Malaisse WJ. Hexose metabolism in pancreatic islets.

Ca(2+)-dependent activation of the glycerol phosphate shuttle by
nutrient secretagogues. J Biol Chem 1992;267(19).
164. Farfari S, Schulz V, Corkey B, Prentki M. Glucose-regulated
anaplerosis and cataplerosis in pancreatic beta-cells: possible
implication of a pyruvate/citrate shuttle in insulin secretion. Diabetes
2000;49(5).
165. Malaisse WJ, Hutton JC, Kawazu S, Herchuelz A, Valverde I, Sener
A. The stimulus-secretion coupling of glucose-induced insulin
release. XXXV. The links between metabolic and cationic events.
Diabetologia 1979;16(5).
166. Newgard CB, McGarry JD. Metabolic coupling factors in pancreatic
beta-cell signal transduction. Annu Rev Biochem 1995;64:689-719.
167. DeFronzo RA, Prato SD. Insulin resistance and diabetes mellitus. J
Diabetes Complications 1996;10(5).
168. Rorsman P, Bokvist K, Ammala C, Eliasson L, Renstrom E, Gabel J.
Ion channels, electrical activity and insulin secretion. Diabete Metab
1994;20(2).
169. Williams JA. Electrical correlates of secretion in endocrine and
exocrine cells. Fed Proc 1981;40(2).
170. Ashcroft FM. K(ATP) channels and insulin secretion: a key role in
health and disease. Biochem Soc Trans 2006;34(Pt 2).
171. Ashcroft FM, Rorsman P. ATP-sensitive K+ channels: a link between
B-cell metabolism and insulin secretion. Biochem Soc Trans
1990;18(1).
172. Ashcroft SJ, Ashcroft FM. Properties and functions of ATP-sensitive
K-channels. Cell Signal 1990;2(3).
173. Bryan J, Aguilar-Bryan L. The ABCs of ATP-sensitive potassium
channels: more pieces of the puzzle. Curr Opin Cell Biol 1997;9(4).
174. Cook DL, Satin LS, Ashford ML, Hales CN. ATP-sensitive K+
channels in pancreatic beta-cells. Spare-channel hypothesis.
Diabetes 1988;37(5).
175. Loussouarn G, Pike LJ, Ashcroft FM, Makhina EN, Nichols CG.
Dynamic sensitivity of ATP-sensitive K(+) channels to ATP. J Biol
Chem 2001;276(31).

176. Song DK, Ashcroft FM. ATP modulation of ATP-sensitive potassium

channel ATP sensitivity varies with the type of SUR subunit. J Biol
Chem 2001;276(10).
177. Sakura H, Ashcroft FM. Identification of four trp1 gene variants
murine pancreatic beta-cells. Diabetologia 1997;40(5).
178. Gembal M, Detimary P, Gilon P, Gao ZY, Henquin JC. Mechanisms
by which glucose can control insulin release independently from its
action on adenosine triphosphate-sensitive K+ channels in mouse B
cells. J Clin Invest 1993;91(3).
179. Gembal M, Gilon P, Henquin JC. Evidence that glucose can control
insulin release independently from its action on ATP-sensitive K+
channels in mouse B cells. J Clin Invest 1992;89(4).
180. Komatsu M, Noda M, Sharp GW. Nutrient augmentation of Ca2+dependent and Ca2+-independent pathways in stimulus-coupling to
insulin secretion can be distinguished by their guanosine
triphosphate requirements: studies on rat pancreatic islets.
Endocrinology 1998;139(3).
181. Komatsu M, Schermerhorn T, Noda M, Straub SG, Aizawa T, Sharp
GW. Augmentation of insulin release by glucose in the absence of
extracellular Ca2+: new insights into stimulus-secretion coupling.
Diabetes 1997;46(12).
182. Komatsu M, Sharp GW, Aizawa T, Hashizume K. Glucose stimulation
of insulin release without an increase in cytosolic free Ca2+
concentration: a possible involvement of GTP. Jpn J Physiol 1997;47
Suppl 1:S22-4.
183. Li GD, Luo RH, Metz SA. Effects of inhibitors of guanine nucleotide
synthesis on membrane potential and cytosolic free Ca2+ levels in
insulin-secreting cells. Biochem Pharmacol 2000;59(5).
184. Regazzi R, Li G, Ullrich S, Jaggi C, Wollheim CB. Different
requirements for protein kinase C activation and Ca2+-independent
insulin secretion in response to guanine nucleotides. Endogenously
generated diacylglycerol requires elevated Ca2+ for kinase C
insertion into membranes. J Biol Chem 1989;264(17).
185. Ashcroft FM, Gribble FM. ATP-sensitive K+ channels and insulin
secretion: their role in health and disease. Diabetologia 1999;42(8).
186. Babenko AP, Aguilar-Bryan L, Bryan J. A view of sur/KIR6.X, KATP
channels. Annu Rev Physiol 1998;60:667-87.

187. Polak M, Shield J. Neonatal Diabetes Mellitus -- genetic aspects

2004. Pediatr Endocrinol Rev 2004;2(2).
188. Miki T, Nagashima K, Seino S. The structure and function of the ATPsensitive K+ channel in insulin-secreting pancreatic beta-cells. J Mol
Endocrinol 1999;22(2).
189. Suzuki M, Fujikura K, Inagaki N, Seino S, Takata K. Localization of
the ATP-sensitive K+ channel subunit Kir6.2 in mouse pancreas.
Diabetes 1997;46(9).
190. Bokvist K, Olsen HL, Hoy M et al. Characterisation of sulphonylurea
and ATP-regulated K+ channels in rat pancreatic A-cells. Pflugers
Arch 1999;438(4).
191. Gopel SO, Kanno T, Barg S, Rorsman P. Patch-clamp
characterisation of somatostatin-secreting -cells in intact mouse
pancreatic islets. J Physiol 2000;528(Pt 3).
192. Gopel SO, Kanno T, Barg S, Weng XG, Gromada J, Rorsman P.
Regulation of glucagon release in mouse -cells by KATP channels
and inactivation of TTX-sensitive Na+ channels. J Physiol
2000;528(Pt 3).
193. Hattersley AT, Ashcroft FM. Activating mutations in Kir6.2 and
neonatal diabetes: new clinical syndromes, new scientific insights,
and new therapy. diab 2005;54(9).
194. Koster JC, Remedi MS, Dao C, Nichols CG. ATP and sulfonylurea
sensitivity of mutant ATP-sensitive K+ channels in neonatal diabetes:
implications for pharmacogenomic therapy. diab 2005;54(9).
195. Proks P, Girard C, Haider S et al. A gating mutation at the internal
mouth of the Kir6.2 pore is associated with DEND syndrome. EMBO
Rep 2005;6(5).
196. Nestorowicz A, Glaser B, Wilson BA et al. Genetic heterogeneity in
familial hyperinsulinism [published erratum appears in Hum Mol
Genet 1998 Sep;7(9):1527]. Hum Mol Genet 1998;7(7).
197. Flanagan S, Damhuis A, Banerjee I et al. Partial ABCC8 gene
deletion mutations causing diazoxide-unresponsive
hyperinsulinaemic hypoglycaemia. Pediatr Diabetes 2011.
198. Flanagan SE, Kapoor RR, Banerjee I et al. Dominantly acting ABCC8
mutations in patients with medically unresponsive hyperinsulinaemic
hypoglycaemia. Clin Genet 2011;79(6).

199. Ismail D, Smith VV, de Lonlay P et al. Familial focal congenital

hyperinsulinism. J Clin Endocrinol Metab 2011;96(1).
200. Kapoor RR, Flanagan SE, James CT et al. Hyperinsulinaemic
hypoglycaemia and diabetes mellitus due to dominant
ABCC8/KCNJ11 mutations. Diabetologia 2011;54(10).
201. Kumaran A, Kapoor RR, Flanagan SE, Ellard S, Hussain K.
Congenital hyperinsulinism due to a compound heterozygous ABCC8
mutation with spontaneous resolution at eight weeks. Horm Res
Paediatr 2010;73(4).
202. James C, Kapoor RR, Ismail D, Hussain K. The genetic basis of
congenital hyperinsulinism. J Med Genet 2009;46(5).
203. Kapoor RR, Flanagan SE, James C, Shield J, Ellard S, Hussain K.
Hyperinsulinaemic hypoglycaemia. Arch Dis Child 2009;94(6).
204. Kapoor RR, James C, Hussain K. Hyperinsulinism in developmental
syndromes. Endocr Dev 2009;14.
205. Poon M, Hussain K. Postprandial hyperinsulinaemic hypoglycaemia
and type 1 diabetes mellitus. BMJ Case Rep 2009;2009.
206. Kapoor RR, James C, Flanagan SE, Ellard S, Eaton S, Hussain K. 3Hydroxyacyl-coenzyme A dehydrogenase deficiency and
hyperinsulinemic hypoglycemia: characterization of a novel mutation
and severe dietary protein sensitivity. J Clin Endocrinol Metab
2009;94(7).
207. Cuesta-Munoz AL, Huopio H, Otonkoski T et al. Severe persistent
hyperinsulinemic hypoglycemia due to a de novo glucokinase
mutation. Diabetes 2004;53(8).
208. Osbak KK, Colclough K, Saint-Martin C et al. Update on mutations in
glucokinase (GCK), which cause maturity-onset diabetes of the
young, permanent neonatal diabetes, and hyperinsulinemic
hypoglycemia. Hum Mutat 2009;30(11).
209. Kawajiri M, Okano Y, Kuno M et al. Unregulated insulin secretion by
pancreatic beta cells in hyperinsulinism/hyperammonemia syndrome:
role of glutamate dehydrogenase, ATP-sensitive potassium channel,
and nonselective cation channel. Pediatr Res 2006;59(3).
210. Chan CB, Saleh MC, Koshkin V, Wheeler MB. Uncoupling protein 2
and islet function. diab 2004;53 Suppl 1.

211. Joseph JW, Koshkin V, Saleh MC et al. Free fatty acid-induced betacell defects are dependent on uncoupling protein 2 expression. J Biol
Chem 2004;279(49).
212. Ann K, Kowalchyk JA, Loyet KM, Martin TF. Novel Ca2+-binding
protein (CAPS) related to UNC-31 required for Ca2+-activated
exocytosis. J Biol Chem 1997;272(32).
213. Bokvist K, Eliasson L, Ammala C, Renstrom E, Rorsman P. Colocalization of L-type Ca2+ channels and insulin-containing secretory
granules and its significance for the initiation of exocytosis in mouse
pancreatic B-cells. EMBO J 1995;14(1).
214. Wiser O, Trus M, Hernandez A et al. The voltage sensitive Lc-type
Ca2+ channel is functionally coupled to the exocytotic machinery.
Proc Natl Acad Sci U S A 1999;96(1).
215. Nilius B, Hess P, Lansman JB, Tsien RW. A novel type of cardiac
calcium channel in ventricular cells. Nature 1985;316(6027).
216. Gutnick MJ, Lux HD, Swandulla D, Zucker H. Voltage-dependent and
calcium-dependent inactivation of calcium channel current in
identified snail neurones. J Physiol 1989;412:197-220.
217. Ammala C, Eliasson L, Bokvist K et al. Activation of protein kinases
and inhibition of protein phosphatases play a central role in the
regulation of exocytosis in mouse pancreatic beta cells. Proc Natl
Acad Sci U S A 1994;91(10).
218. Dean PM. Ultrastructural morphometry of the pancreatic -cell.
Diabetologia 1973;9(2).
219. Eliasson L, Renstrom E, Ding WG, Proks P, Rorsman P. Rapid ATPdependent priming of secretory granules precedes Ca(2+)-induced
exocytosis in mouse pancreatic B-cells. J Physiol 1997;503(Pt 2).
220. Gromada J, Hoy M, Renstrom E et al. CaM kinase II-dependent
mobilization of secretory granules underlies acetylcholine-induced
stimulation of exocytosis in mouse pancreatic B-cells. J Physiol
1999;518(Pt 3).
221. Rorsman P, Eliasson L, Renstrom E, Gromada J, Barg S, Gopel S.
The Cell Physiology of Biphasic Insulin Secretion. News Physiol Sci
2000;15:72-77.
222. Rorsman P. The pancreatic beta-cell as a fuel sensor: an
electrophysiologist's viewpoint. Diabetologia 1997;40(5).

223. Huang JD, Brady ST, Richards BW et al. Direct interaction of

microtubule- and actin-based transport motors. Nature
1999;397(6716).
224. Easom RA. Beta-granule transport and exocytosis. Semin Cell Dev
Biol 2000;11(4).
225. Sollner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE. A
protein assembly-disassembly pathway in vitro that may correspond
to sequential steps of synaptic vesicle docking, activation, and fusion.
Cell 1993;75(3).
226. Rupnik M, Kreft M, Sikdar SK et al. Rapid regulated dense-core
vesicle exocytosis requires the CAPS protein. Proc Natl Acad Sci U S
A 2000;97(10).
227. Fukui K, Yang Q, Cao Y et al. The HNF-1 target collectrin controls
insulin exocytosis by SNARE complex formation. Cell Metab
2005;2(6).
228. Evans GJ, Barclay JW, Prescott GR et al. Protein kinase B/Akt is a
novel cysteine string protein kinase that regulates exocytosis release
kinetics and quantal size. J Biol Chem 2006;281(3).
229. Jahn R, Sudhof TC. Membrane fusion and exocytosis. Annu Rev
Biochem 1999;68:863-911.
230. Izumi T, Gomi H, Torii S. Functional analysis of Rab27a effector
granuphilin in insulin exocytosis. Methods Enzymol 2005;403.
231. Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T. Granuphilin
molecularly docks insulin granules to the fusion machinery. J Cell Biol
2005;171(1).
232. Jacobsson G, Bean AJ, Scheller RH et al. Identification of synaptic
proteins and their isoform mRNAs in compartments of pancreatic
endocrine cells. Proc Natl Acad Sci U S A 1994;91(26).
233. Lang J. Molecular mechanisms and regulation of insulin exocytosis
as a paradigm of endocrine secretion. Eur J Biochem 1999;259(1-2).
234. Lao G, Scheuss V, Gerwin CM et al. Syntaphilin: a syntaxin-1 clamp
that controls SNARE assembly. Neuron 2000;25(1).
235. Fisher RJ, Pevsner J, Burgoyne RD. Control of fusion pore dynamics
during exocytosis by Munc18. Science 2001;291(5505).

236. Huang X, Kang YH, Pasyk EA et al. Ca(2+) influx and cAMP
elevation overcame botulinum toxin A but not tetanus toxin inhibition
of insulin exocytosis. Am J Physiol Cell Physiol 2001;281(3).
237. Land J, Zhang H, Vaidyanathan VV, Sadoul K, Niemann H, Wollheim
CB. Transient expression of botulinum neurotoxin C1 light chain
differentially inhibits calcium and glucose induced insulin secretion in
clonal beta-cells. FEBS Lett 1997;419(1).
238. Li C, Ullrich B, Zhang JZ, Anderson RG, Brose N, Sudhof TC.
Ca(2+)-dependent and -independent activities of neural and nonneural synaptotagmins. Nature 1995;375(6532).
239. Loyet KM, Kowalchyk JA, Chaudhary A, Chen J, Prestwich GD,
Martin TF. Specific binding of phosphatidylinositol 4,5-bisphosphate
to calcium-dependent activator protein for secretion (CAPS), a
potential phosphoinositide effector protein for regulated exocytosis. J
Biol Chem 1998;273(14).
240. Broer A, Friedrich B, Wagner CA et al. Association of 4F2hc with light
chains LAT1, LAT2 or y+LAT2 requires different domains. Biochem J
2001;355(Pt 3).
241. Xu T, Ashery U, Burgoyne RD, Neher E. Early requirement for alphaSNAP and NSF in the secretory cascade in chromaffin cells. EMBO J
1999;18(12).
242. Clark JD, Lin LL, Kriz RW et al. A novel arachidonic acid-selective
cytosolic PLA2 contains a Ca(2+)-dependent translocation domain
with homology to PKC and GAP. Cell 1991;65(6).
243. Sharp JD, White DL, Chiou XG et al. Molecular cloning and
expression of human Ca(2+)-sensitive cytosolic phospholipase A2. J
Biol Chem 1991;266(23).
244. Iezzi M, Escher G, Meda P et al. Subcellular distribution and function
of Rab3A, B, C, and D isoforms in insulin-secreting cells. Mol
Endocrinol 1999;13(2).
245. Kajio H, Olszewski S, Rosner PJ, Donelan MJ, Geoghegan KF,
Rhodes CJ. A low-affinity Ca2+-dependent association of calmodulin
with the Rab3A effector domain inversely correlates with insulin
exocytosis. Diabetes 2001;50(9).
246. Balsinde J, Balboa MA, Insel PA, Dennis EA. Regulation and
inhibition of phospholipase A2. Annu Rev Pharmacol Toxicol
1999;39:175-89.

247. Bauerfeind R, Takei K, De Camilli P. Amphiphysin I is associated with

coated endocytic intermediates and undergoes stimulationdependent dephosphorylation in nerve terminals. J Biol Chem
1997;272(49).
248. Lai MM, Hong JJ, Ruggiero AM et al. The calcineurin-dynamin 1
complex as a calcium sensor for synaptic vesicle endocytosis. J Biol
Chem 1999;274(37).
249. Kang G, Chepurny OG, Malester B et al. cAMP Sensor Epac As A
Determinant Of ATP-Sensitive Potassium Channel Activity In Human
Pancreatic Beta Cells And Rat INS-1 Cells. J Physiol 2006.
250. Waselle L, Gerona RR, Vitale N, Martin TF, Bader MF, Regazzi R.
Role of phosphoinositide signaling in the control of insulin exocytosis.
Mol Endocrinol 2005;19(12).
251. Ammala C, Ashcroft FM, Rorsman P. Calcium-independent
potentiation of insulin release by cyclic AMP in single beta-cells.
Nature 1993;363(6427).
252. Hamilton PB, Stevens JR, Gidley J, Holz P, Gibson WC. A new
lineage of trypanosomes from Australian vertebrates and terrestrial
bloodsucking leeches (Haemadipsidae). Int J Parasitol 2005;35(4).
253. Renstrom E, Eliasson L, Rorsman P. Protein kinase A-dependent
and -independent stimulation of exocytosis by cAMP in mouse
pancreatic B-cells. J Physiol 1997;502(Pt 1).
254. Ashcroft FM, Proks P, Smith PA, Ammala C, Bokvist K, Rorsman P.
Stimulus-secretion coupling in pancreatic beta cells. J Cell Biochem
1994;55 Suppl:54-65.
255. Murakami K, Chan SY, Routtenberg A. Protein kinase C activation by
cis-fatty acid in the absence of Ca2+ and phospholipids. J Biol Chem
1986;261(33).
256. Tischfield JA. A reassessment of the low molecular weight
phospholipase A2 gene family in mammals. J Biol Chem
1997;272(28).
257. Leslie CC. Properties and regulation of cytosolic phospholipase A2. J
Biol Chem 1997;272(27).
258. Sapirstein A, Bonventre JV. Specific physiological roles of cytosolic
phospholipase A(2) as defined by gene knockouts. Biochim Biophys
Acta 2000;1488(1-2).

259. Bonventre JV, Huang Z, Taheri MR et al. Reduced fertility and

postischaemic brain injury in mice deficient in cytosolic
phospholipase A2. Nature 1997;390(6660).
260. Murakami M, Nakatani Y, Kuwata H, Kudo I. Cellular components
that functionally interact with signaling phospholipase A(2)s. Biochim
Biophys Acta 2000;1488(1-2).
261. Balsinde J, Dennis EA. Function and inhibition of intracellular
calcium-independent phospholipase A2. J Biol Chem 1997;272(26).
262. Winstead MV, Balsinde J, Dennis EA. Calcium-independent
phospholipase A(2): structure and function. Biochim Biophys Acta
2000;1488(1-2).
263. Ramanadham S, Ma Z, Arita H, Zhang S, Turk J. Type IB secretory
phospholipase A2 is contained in insulin secretory granules of
pancreatic islet beta-cells and is co-secreted with insulin from
glucose-stimulated islets. Biochim Biophys Acta 1998;1390(3).
264. Dennis EA. The growing phospholipase A2 superfamily of signal
transduction enzymes. Trends Biochem Sci 1997;22(1).
265. Stafforini DM, McIntyre TM, Zimmerman GA, Prescott SM. Plateletactivating factor acetylhydrolases. J Biol Chem 1997;272(29).
266. Loweth AC, Scarpello JH, Morgan NG. Phospholipase A2 expression
in human and rodent insulin-secreting cells. Mol Cell Endocrinol
1995;112(2).
267. Metz S, Holmes D, Robertson RP, Leitner W, Draznin B. Gene
expression of type I phospholipase A2 in pancreatic beta cells.
Regulation of mRNA levels by starvation or glucose excess. FEBS
Lett 1991;295(1-3).
268. Niwa T, Matsukawa Y, Senda T, Nimura Y, Hidaka H, Niki I.
Acetylcholine activates intracellular movement of insulin granules in
pancreatic beta-cells via inositol trisphosphate-dependent [correction
of triphosphate-dependent] mobilization of intracellular Ca2+.
Diabetes 1998;47(11).
269. Parker KJ, Jones PM, Hunton CH, Persaud SJ, Taylor CG, Howell
SL. Identification and localisation of a type IV cytosolic phospholipase
A2 in rat pancreatic beta-cells. J Mol Endocrinol 1996;17(1).

270. Eddlestone GT. ATP-sensitive K channel modulation by products of

PLA2 action in the insulin-secreting HIT cell line. Am J Physiol
1995;268(1 Pt 1).
271. Gross RW, Ramanadham S, Kruszka KK, Han X, Turk J. Rat and
human pancreatic islet cells contain a calcium ion independent
phospholipase A2 activity selective for hydrolysis of arachidonate
which is stimulated by adenosine triphosphate and is specifically
localized to islet beta-cells. Biochemistry 1993;32(1).
272. Kashiwagi M, Friess H, Uhl W et al. Phospholipase A2 isoforms are
altered in chronic pancreatitis. Ann Surg 1998;227(2).
273. Seeds MC, Jones DF, Chilton FH, Bass DA. Secretory and cytosolic
phospholipases A2 are activated during TNF priming of human
neutrophils. Biochim Biophys Acta 1998;1389(3).
274. Ma Z, Ramanadham S, Kempe K, Chi XS, Ladenson J, Turk J.
Pancreatic islets express a Ca2+-independent phospholipase A2
enzyme that contains a repeated structural motif homologous to the
integral membrane protein binding domain of ankyrin. J Biol Chem
1997;272(17).
275. Turk J, Wolf BA, Lefkowith JB, Stump WT, McDaniel ML. Glucoseinduced phospholipid hydrolysis in isolated pancreatic islets:
quantitative effects on the phospholipid content of arachidonate and
other fatty acids. Biochim Biophys Acta 1986;879(3).
276. Ma Z, Zhang S, Turk J, Ramanadham S. Stimulation of insulin
secretion and associated nuclear accumulation of iPLA(2)beta in
INS-1 insulinoma cells. Am J Physiol Endocrinol Metab 2002;282(4).
277. Wolf BA, Pasquale SM, Turk J. Free fatty acid accumulation in
secretagogue-stimulated pancreatic islets and effects of arachidonate
on depolarization-induced insulin secretion. Biochemistry
1991;30(26).
278. Wolf BA, Turk J, Sherman WR, McDaniel ML. Intracellular Ca2+
mobilization by arachidonic acid. Comparison with myo-inositol 1,4,5trisphosphate in isolated pancreatic islets. J Biol Chem 1986;261(8).
279. Bell RL, Stanford N, Kennerly DA, Majerus PW. Diglyceride lipase: a
pathway for arachidonate release from human platelets. Adv
Prostaglandin Thromboxane Res 1980;6:219-24.
280. Konrad RJ, Major CD, Wolf BA. Diacylglycerol hydrolysis to
arachidonic acid is necessary for insulin secretion from isolated

pancreatic islets: sequential actions of diacylglycerol and

monoacylglycerol lipases. Biochemistry 1994;33(45).
281. Konrad RJ, Jolly YC, Major C, Wolf BA. Inhibition of phospholipase
A2 and insulin secretion in pancreatic islets. Biochim Biophys Acta
1992;1135(2).
282. Metz SA. Pancreatic islet phospholipase A2: differential, Ca2+dependent effects of lysophospholipids, arachidonic acid, and its
lipoxygenase-derived metabolites on insulin release. Adv
Prostaglandin Thromboxane Leukot Res 1987;17B:668-76.
283. Damron DS, Van Wagoner DR, Moravec CS, Bond M. Arachidonic
acid and endothelin potentiate Ca2+ transients in rat cardiac
myocytes via inhibition of distinct K+ channels. J Biol Chem
1993;268(36).
284. Graber MN, Alfonso A, Gill DL. Ca2+ pools and cell growth:
arachidonic acid induces recovery of cells growth-arrested by Ca2+
pool depletion. J Biol Chem 1996;271(2).
285. McPhail LC, Clayton CC, Snyderman R. A potential second
messenger role for unsaturated fatty acids: activation of Ca2+dependent protein kinase. Science 1984;224(4649).
286. Shuttleworth TJ. Arachidonic acid activates the noncapacitative entry
of Ca2+ during [Ca2+]i oscillations. J Biol Chem 1996;271(36).
287. Metz SA. Exogenous arachidonic acid promotes insulin release from
intact or permeabilized rat islets by dual mechanisms. Putative
activation of Ca2+ mobilization and protein kinase C. Diabetes
1988;37(11).
288. Metz SA. The pancreatic islet as Rubik's Cube. Is phospholipid
hydrolysis a piece of the puzzle? Diabetes 1991;40(12).
289. Ramanadham S, Gross R, Turk J. Arachidonic acid induces an
increase in the cytosolic calcium concentration in single pancreatic
islet beta cells. Biochem Biophys Res Commun 1992;184(2).
290. Hirabayashi T, Shimizu T. Localization and regulation of cytosolic
phospholipase A(2). Biochim Biophys Acta 2000;1488(1-2).
291. Kimple ME, Nixon AB, Kelly P et al. A role for G(z) in pancreatic islet
beta-cell biology. J Biol Chem 2005;280(36).

292. Kato R, Yamamoto S, Nakadate T, Nakaki T. Possible involvement of

phospholipase A2 activation and lipoxygenase product(s) in the
mechanism of insulin secretion. Adv Prostaglandin Thromboxane
Leukot Res 1983;12:265-70.
293. Zhou YP, Teng D, Dralyuk F et al. Apoptosis in insulin-secreting cells.
Evidence for the role of intracellular Ca2+ stores and arachidonic
acid metabolism. J Clin Invest 1998;101(8).
294. Frossard JL, Bhagat L, Lee HS et al. Both thermal and non-thermal
stress protect against caerulein induced pancreatitis and prevent
trypsinogen activation in the pancreas. Gut 2002;50(1).
295. Ramsingh AI, Chapman N, Tracy S. Coxsackieviruses and diabetes.
Bioessays 1997;19(9).
296. Sevilla N, Homann D, von Herrath M et al. Virus-induced diabetes in
a transgenic model: role of cross-reacting viruses and quantitation of
effector T cells needed to cause disease. J Virol 2000;74(7).
297. Gu D, Sarvetnick N. Epithelial cell proliferation and islet neogenesis
in IFN-g transgenic mice. Development 1993;118(1).
298. Pruzanski W, Vadas P. Phospholipase A2--a mediator between
proximal and distal effectors of inflammation. Immunol Today
1991;12(5).
299. van Puijenbroek AA, Wissink S, van der Saag PT, Peppelenbosch
MP. Phospholipase A2 inhibitors and leukotriene synthesis inhibitors
block TNF-induced NF-kappaB activation. Cytokine 1999;11(2).
300. Deevska GM, Nikolova-Karakashian MN. The twists and turns of
sphingolipid pathway in glucose regulation. Biochimie 2011;93(1).
301. Fox TE, Bewley MC, Unrath KA et al. Circulating sphingolipid
biomarkers in models of type 1 diabetes. J Lipid Res 2011;52(3).
302. Ortsater H. Arachidonic acid fights palmitate: new insights into fatty
acid toxicity in beta-cells. Clin Sci (Lond) 2011;120(5).
303. Metz SA. Mobilization of cellular Ca2+ by lysophospholipids in rat
islets of Langerhans. Biochim Biophys Acta 1988;968(2).
304. Rustenbeck I, Lenzen S. Effects of lysophosphatidylcholine and
arachidonic acid on the regulation of intracellular Ca2+ transport.
Naunyn Schmiedebergs Arch Pharmacol 1989;339(1-2).

305. Gasser KW, Holda JR. ATP-sensitive potassium transport by

pancreatic secretory granule membrane. Am J Physiol 1993;264(1 Pt
1).
306. Metz SA. Ether-linked lysophospholipids initiate insulin secretion.
Lysophospholipids may mediate effects of phospholipase A2
activation on hormone release. Diabetes 1986;35(7).
307. Fernandez-Valverde SL, Taft RJ, Mattick JS. MicroRNAs in beta-cell
biology, insulin resistance, diabetes and its complications. Diabetes
2012;60(7).
308. Joglekar MV, Parekh VS, Hardikar AA. New pancreas from old:
microregulators of pancreas regeneration. Trends Endocrinol Metab
2007;18(10).
309. Joglekar MV, Parekh VS, Mehta S, Bhonde RR, Hardikar AA.
MicroRNA profiling of developing and regenerating pancreas reveal
post-transcriptional regulation of neurogenin3. Dev Biol 2007;311(2).
310. Joglekar MV, Joglekar VM, Hardikar AA. Expression of islet-specific
microRNAs during human pancreatic development. Gene Expr
Patterns 2009;9(2).
311. Joglekar MV, Parekh VS, Hardikar AA. Islet-specific microRNAs in
pancreas development, regeneration and diabetes. Indian J Exp Biol
2011;49(6).
312. Cuellar TL, McManus MT. MicroRNAs and endocrine biology. J
Endocrinol 2005;187(3).
313. Bolmeson C, Esguerra JL, Salehi A, Speidel D, Eliasson L, Cilio CM.
Differences in islet-enriched miRNAs in healthy and glucose
intolerant human subjects. Biochem Biophys Res Commun404(1).
314. Davalos A, Goedeke L, Smibert P et al. miR-33a/b contribute to the
regulation of fatty acid metabolism and insulin signaling. Proc Natl
Acad Sci U S A108(22).
315. Fred RG, Bang-Berthelsen CH, Mandrup-Poulsen T, Grunnet LG,
Welsh N. High glucose suppresses human islet insulin biosynthesis
by inducing miR-133a leading to decreased polypyrimidine tract
binding protein-expression. PLoS One5(5).
316. Frost RJ, Olson EN. Control of glucose homeostasis and insulin
sensitivity by the Let-7 family of microRNAs. Proc Natl Acad Sci U S
A108(52).

317. Gallagher IJ, Scheele C, Keller P et al. Integration of microRNA

changes in vivo identifies novel molecular features of muscle insulin
resistance in type 2 diabetes. Genome Med2(2).
318. Hennessy E, Clynes M, Jeppesen PB, O'Driscoll L. Identification of
microRNAs with a role in glucose stimulated insulin secretion by
expression profiling of MIN6 cells. Biochem Biophys Res
Commun396(2).
319. Kalis M, Bolmeson C, Esguerra JL et al. Beta-cell specific deletion of
Dicer1 leads to defective insulin secretion and diabetes mellitus.
PLoS One6(12).
320. Lee JY, Ristow M, Lin X, White MF, Magnuson MA, Hennighausen L.
RIP-Cre revisited, evidence for impairments of pancreatic beta-cell
function. J Biol Chem 2006;281(5).
321. Wiederkehr A, Wollheim CB. Implication of mitochondria in insulin
secretion and action. Endocrinology 2006.