‘gel-free’-based separation methods, 7
‘organelle-specific’ proteomic analyses, 25
aggregation, 16, 23, 26
amylin, 5, 8, 9, 12, 13, 14, 16, 17, 18, 26,
27, 28, 38, 39, 42, 43, 44, 45
amylin aggregation, 17
amylin misfolding, 27
amylin-evoked apoptosis, 16
amylin-generated aggregates, 28
amylin-mediated, 17, 18
amyloid, 16, 17, 18, 28, 29, 38, 39, 41, 42,
43
amyloid fibrils, 17
anti-diabetic compounds, 28
apoptosis, 16
ATF2/p38 MAPK, 17
autoantigens, 10, 12
autoimmune destruction of islet β-cells, 10
autoreactive T-cells, 10
B-cell secretory granules, 10, 14, 25
bioinformatic, 21, 22
Ca2+, 11, 12, 14, 15, 25, 40
Ca2+ efflux, 11
calcium storage, 12
calreticulin, 20, 24, 25, 40
caspase-3, 17
caspase-8, 17
chaperone, 23, 24, 26, 27, 41
chaperones, 23, 27, 28, 41
composition of the β-cell granule, 9
co-purification, 26
C-peptide, 14
cytotoxic oligomers, 9
cytotoxic processes, 28
cytotoxic protein aggregates, 9
decreased β-cell mass, 17
defective insulin secretion, 11, 12
degeneration of the islets of Langerhans, 8
diabetes, 4, 5, 8, 9, 12, 13, 16, 17, 27, 29,
37, 38, 39, 42, 43, 44
disease mechanisms, 5, 7, 8, 13, 27
disease processes, 12
disorders of hormone action, 8
dysregulated amylin folding, 28
dysregulation of pancreatic hormones, 8
electron microscopy, 14
ER, 3, 11, 17, 18, 20, 21, 23, 24, 25, 27
exocytosis, 11, 14, 15, 26, 41
experimental therapeutics, 7
Fas/FasL/FADD, 16, 17, 18
fibrillogenic, 17
fibrils, 16
fuel metabolism, 18
glucagon, 8, 45
glutamate decarboxylase (GAD), 10
glycoisoforms, 7
granule proteins, 5, 10
granule proteome, 5
halo, 13
heat shock protein, 22
heat-shock protein 65 (HSP65), 10
high-resolution methodologies, 5
HSP, 27
human amylin transgenic mice, 17
hyperglycaemia, 17, 28
immunoaffinity purification, 24
immunopurified, 21
in-gel tryptic digestion, 20
INS-1E, 20, 21, 46
insoluble fibrils, 28
insulin, 5, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20,
21, 24, 25, 26, 28, 38, 39, 40, 41, 43, 45,
46
insulin resistance, 18
insulin resistant, 8
insulin therapy, 9
intracellular Ca2+, 11
islet amyloid, 8, 16, 28, 37, 38, 39, 43
islet amyloidosis, 28, 38
islet hormone production and release, 8
islet hormones, 18
islet structure and function, 29
islet β-cell degeneration, 16, 17
islet β-cell dysfunction, 26
islet-cell antigen 69 (ICA69), 10
islet-specific proteins, 18
isobaric tags for relative and absolute
quantitation (iTRAQ), 8
JNK1/cJun, 17
LC-MS2, 21, 22
liquid chromatography (LC), 7
lysosome, 20, 23
lysosomes, 21, 46
misfolding, 9, 12, 16, 18, 23, 26, 27, 28
mitochondria, 20, 21, 22, 25, 26, 41, 46
mitochondrial ATP synthase, 26
molecular mechanisms, 12
molecular pathways, 9
multi-dimensional mass spectrometry, 7
multi-dimensional protein identification
technology (MuDPIT), 8
NAADP, 11, 12
nicotinic acid adenine dinucleotide
phosphate (NAADP), 11
novel islet T-cell antigens, 10
oligomer formation, 16
oligomeric, 7
oligomers, 16, 26, 28
p53/p21WAF1/CIP1, 17
pancreas, 8, 9, 15, 39, 43, 45
pancreatic islet β-cell, 3, 5, 18, 26
pancreatic islets, 8, 15, 27
pancreatic β-cells, 27
pathogenesis of T2DM, 11, 16
Pdx1, 19
pharmacotherapies, 5
physiological processes, 5
post-translational modifications, 6
proinsulin, 14, 15, 40, 41
protein folding, 9
protein identification analysis, 20, 21
proteome, 6, 8, 18, 20, 21, 29, 39, 40
proteomic, 5
proteomic analysis, 3, 5, 6, 9, 12, 15, 19, 27,
28, 29, 43
proteomic methods, 7
proteomics, 6, 8, 12, 15, 16, 18, 19, 23, 41
protofibrils, 16, 28
PTM, 3, 7
PTMs, 6, 7
purification steps, 20
Rab GTPases, 11
recombinant human insulin, 15
regulated insulin secretion, 12
regulated secretion, 5, 8, 12, 28
regulation of fuel metabolism, 8
regulation of insulin and amylin secretion,
12
regulation of islet hormone secretion, 28
regulation of metabolism, 5, 8
role of insulin, 19
ryanodine receptor (RyR) I, 11
RyR, 3, 11
SDS-PAGE, 20
secretory granule, 3, 5, 6, 8, 9, 10, 11, 12,
13, 15, 18, 19, 20, 23, 24, 26, 28
secretory vesicle, 12
semi-quantitative comparisons, 8
subcellular, 11, 13, 23, 27
subproteomes, 6
T1DM, 3, 9, 10, 28
T2DM, 4, 9, 10, 12, 16, 17, 18, 26, 28, 29
T-cell, 10
therapeutic interventions for diabetes, 13
T-lymphocytes, 10, 25
transgenic models of amylin-mediated
diabetes, 17
two-dimensional gel electrophoresis
(2DGE), 7
type-1 diabetes (T1DM), 9
type-2 diabetes (T2DM), 9
ultracentrifugation, 20, 21
VAMP, 13
VAMP2, 11
western blotting, 20
zinc-containing crystals, 13
Zn2+, 24
β-cell, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29,
38, 39
β-cell death, 28
β-cell degeneration, 9, 17, 23, 29
β-cell dysfunction, 26, 29
Β-cell granule proteins, 5
β-cell granule-specific T-cell lines, 10
β-cell secretory granules, 10, 11, 13, 14, 19,
20, 21, 25, 27, 28
β-conformers, 16
β-sheet, 16, 26
Title: Proteomic analysis of the pancreatic islet β-cell secretory granule:
current understanding and future opportunities
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
2
Au: Garth J S Cooper
School of Biological Sciences
Faculty of Science
University of Auckland
NEW ZEALAND
Email:g.cooper@auckland.ac.nz
and
Department of Pharmacology,
Division of Medical Sciences
University of Oxford
Mansfield Road
Oxford OX1 3QT
UNITED KINGDOM
Email: garth.cooper@pharm.ox.ac.uk
Nonstandard abbreviations: 2DGE, two-dimensional gel electrophoresis; AFM, atomic force
microscopy; ER, endoplasmic reticulum; FasL, Fas ligand; FADD, Fas-associated death domain
protein; GAD, glutamate decarboxylase; HSP, heat-shock protein; ICA, islet-cell antigen;
iTRAQ, isobaric tags for relative and absolute quantitation; MALDI-TOF, matrix-assisted laserdesorption-ionization time-of-flight; MS, mass spectrometry; MuDPIT, multi-dimensional
protein identification technology; NAADP, nicotinic acid adenine dinucleotide phosphate; PC1,
proprotein convertase 1; PTM, post-translational modification; RyR, ryanodine receptor; T1DM,
type-1 diabetes mellitus; T2DM, type-2 diabetes mellitus; VAMP, vesicle-associated membrane
protein
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
3
Chapter 17
Proteomic analysis of the pancreatic islet β-cell secretory granule: current
understanding and future possibilities
Garth J S Cooper
“To every complex question there’s a simple answer, and it’s always wrong”
H. L. Mencken (attrib.)
Abstract ― The pancreatic islet β-cell granule has been the subject of intense study for decades,
in part because it serves as the vehicle for the regulated secretion of insulin and amylin, through
which it exerts regulation of metabolism. Β-cell granule proteins have been closely linked to
disease mechanisms in both major types of diabetes, and recent findings from genome-wide
association studies have reinforced the importance of these linkages for understanding disease
mechanisms. Granule proteins have also proven to be of major interest in pharmaceutics, since
two of them, insulin and amylin have each served as the basis for the development of antidiabetic pharmacotherapies. In spite of all the attention this enigmatic granule has received to
date, many fundamental questions about its molecular structure and function remain unanswered.
In the past few years, high-resolution methodologies have begun to unravel the granule proteome
in ever-increasing detail. Emerging data complements the results from the other approaches that
have been applied to understand the granule. This chapter will explore the current state of
knowledge in the field, and the implications of emerging proteomic data for the study of
physiological processes and disease mechanisms in diabetes.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
4
1. Introduction: proteomics and the β-cell secretory granule
1.1 Proteomes and proteomics: definitions The proteome may be defined as the complete set of
proteins expressed by a genome, cell, tissue or organism. Proteomes typically vary according to
developmental stage and in response to environmental or genetic influences.
Subproteomes may also be defined. They may, for example, comprise all the metalbinding proteins, the phospho-proteins, the glycosylated proteins, or the membrane proteins
expressed by an organism, organ, tissue, cell or organelle, to mention a few of the myriad
possibilities. There are clearly many different ways in which such subproteomes can be
delineated.
Proteomics is the study of proteomes. Proteomic analysis frequently begins with the
study of whole organs or tissues, and then, according to the findings and emerging focus,
proceeds to the examination of sub-cellular fractions or organelles, for example the
‘mitochondrial proteome’, with increasing degrees of resolution [1]. Proteomics is frequently
employed in the first instance in its so-called ‘hypothesis-generating’ or ‘hypothesis-free’ mode,
where it can be extremely effective in generating hypotheses, for example by comparisons
between related states [2]. Thereafter, it can be switched into its ‘hypothesis-driven’ mode,
where hypotheses generated from analysis of the first phase of investigation can be explored in
ever-increasing detail in a series of follow-up experimental designs.
Amongst its many beneficial properties, proteomics is particular adept at identifying and
characterising post-translational modifications (PTMs) in proteins [3, 4]. This ability to detect
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
5
and quantify the contributions made to the modification of protein structure by the many possible
post-translational modifications, is now pointing the way towards levels of regulation in
biological systems that are far more complex than has previously been envisaged [5, 6]. The
multiple, regulated glycoisoforms of the protein adiponectin and the variable oligomeric
structures that they generate, provide a useful example of the complexity that is increasingly
being unveiled by the systematic application of proteomic PTM analysis [2, 7, 8]. In future,
proteomic PTM investigation is expected to contribute substantively to our understanding of
disease aetiopathogenesis, and to deliver many new targets for research into disease mechanisms
and the generation of experimental therapeutics [3].
1.2 Proteomic methods: a very brief overview Previously, proteomic methods frequently
employed two-dimensional gel electrophoresis (2DGE) to perform the required separation of
complex mixtures of proteins, followed by multi-dimensional mass spectrometry and informatics
for protein identification. Although 2DGE-based methods can yield important information [9,
10], for example through the detection and characterisation of groups of closely-related proteins
that differ in the quantity or quality of their similar PTMs [4, 11], they are also subject to the
many shortcomings imposed by the physical properties of the many classes of proteins that
cannot be resolved adequately using gels (for example, those with very high or low molecular
weights, membrane proteins, those with high or low pI values, fibrous, very large or crosslinked
proteins such as those occurring in the ECM, and those of low abundance). These limitations
mean that most proteins present in most proteomes are inaccessible to analysis by 2DGE-based
methods. Therefore there has been an increasing shift towards liquid chromatography (LC)- or
‘gel-free’-based separation methods applied to proteolytic digests of whole proteomes with
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
6
subsequent fractionation and labelling, for example those based on multi-dimensional protein
identification technology (MuDPIT). These approaches have the additional advantage that, with
the introduction of approaches such as isobaric tags for relative and absolute quantitation
(iTRAQ), semi-quantitative comparisons between multiple related proteomes have become
feasible (for examples, see [12, 13]).
1.3 The islet β-cell and its secretory granule: targets for proteomics What is the possible
relevance of proteomics to the insulin secretory granule? The pancreatic islets play a key role in
the regulation of metabolism through their regulated secretion of the peptide hormones insulin,
amylin, and glucagon. They are of fundamental interest in the study of a broad range of disease
mechanisms, including those characterised by dysregulation of pancreatic hormones as well as
by disorders of hormone action, such as occur in insulin resistant states [14].
Proteomic investigation of the pancreas has been motivated by several objectives. One of
these is to improve our understanding of the mechanisms of islet hormone production and
release, and their linkages to the regulation of fuel metabolism [14]. Another is to identify
proteins and, through them, processes that might provide better understanding of diseases that
directly impact on the pancreatic tissues, chief amongst which are diabetes mellitus, pancreatitis
and pancreatic cancer [15-22].
At one level, investigation of the pancreatic proteome has arguably been underway for
most of the last hundred years, driven in large part by the need to understand and reverse the
processes that lead to or cause diabetes. Islet amyloid or ‘hyaline’ (Figure 1) was the original
observation that linked degeneration of the islets of Langerhans to the causation of the form of
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
7
the disease now known as type-2 diabetes (T2DM) [23, 24]. Fundamentally important results
from early protein chemical studies of the pancreas led to the isolation and characterisation of
insulin, and the development of insulin therapy, initially for type-1 diabetes (T1DM), and later
for T2DM [25].
Decades later, granule proteins were identified as likely targets of immune mechanisms
related to the aetiology and pathogenesis of T1DM [26].
(Figure 1 near here)
1.4 Granule-associated pathogenic processes and the origins of diabetes Increasing evidence has
implicated misfolding of the β-cell hormone amylin [27-29] to generate cytotoxic oligomers [30,
31], as potentially responsible for β-cell degeneration in T2DM [32]. These phenomena provide
a clear rationale for elucidation of the composition of the β-cell granule at high resolution, with
the aim of indentifying intrinsic molecular pathways that might mediate as-yet unknown granule
functions [20, 22] – for example those relating to the control of protein folding within the
granule, and possible defects that might contribute to the formation of cytotoxic protein
aggregates [20, 33].
Evidence underpinning facets of this emerging pathogenetic mechanism is developed in
the following section, to provide an example of but one of the important unsolved mysteries of
the β-cell secretory granule, which may prove amenable to proteomic study.
There are at least two other well-recognized pathobiological questions of fundamental
importance relating to the granule, where proteomic analysis could also have a part to play.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
8
1.5 Granule proteins as putative autoantigens in T1DM The first of these questions relates to the
nature of the component proteins that act as autoantigens in the autoimmune destruction of islet
β-cells, for example in T1DM. Numbers of proteins have been identified as candidate
autoantigens with potential relevance to the mechanisms of autoimmune β-cell destruction, either
through studies in animal models such as the non-obese diabetic (NOD) mouse, or in human
patients [34-37]. Most are components of β-cell secretory granules, although they may also exist
in other organelles, and frequently in other cell types as well. Several of these candidate
autoantigens are recognized by T-lymphocytes, including insulin, glutamate decarboxylase
(GAD) 65 and GAD 67, heat-shock protein 65 (HSP65), and islet-cell antigen 69 (ICA69) [38].
Nevertheless, there remains uncertainty concerning the nature of another group of autoantigens
associated with the secretory granule [39]. Indeed, there is evidence for recognition of novel islet
T-cell antigens by β-cell granule-specific T-cell lines from new-onset T1DM patients, where a
fraction of islet β-cells appear to be targeted predominantly by autoreactive T-cells [39].
These considerations point to a need for improved understanding of the protein
components of the secretory granule for the following reasons: (i), to identify new potential
autoantigens, and (ii), to better elucidate the mechanisms that evoke T-cell activation and T-cellmediated autoimmune β-cell destruction in T1DM.
1.6 Granule proteins and hormone secretion The second question relates to defective insulin
secretion in T2DM. B-cell secretory granules have been studied for decades, with the major aim
of elucidating the mechanisms of insulin processing and secretion [40-42]. Recent results from
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
9
genome-wide association studies (GWAs) [43] have reinforced linkages between defective
insulin secretion, β-cell granule proteins, and the pathogenesis of T2DM [44, 45].
Many studies of the role of β-cell granules in insulin secretion have focussed on the
mechanisms by which insulin is processed and stored [41, 42], and the roles of ion channels [4648] and other proteins in the regulation of their exocytosis [49-58]. Some of the proteins that
mediate exocytosis, for example the small Rab GTPases and VAMP2, are intrinsic to
substructures within the β-cell secretory granule [59, 60], whereas others reside in other parts of
the cell and may therefore not co-purify with granules.
In recent years, increasing evidence has pointed to the secretory granule itself as playing
a leading role in the triggering of its own secretion. In particular, ryanodine receptor (RyR) Imediated Ca2+-induced Ca2+ release from the β-cell secretory granule, possibly potentiated by
nicotinic acid adenine dinucleotide phosphate (NAADP), is increasingly seen to play an essential
role in the activation of insulin secretion [61, 62]. Receptors for NAADP, a novel intracellular
Ca2+-mobilizing agent [63, 64], may represent an alternative pathway for Ca2+ efflux from β-cell
secretory granules [65]. Islets and MIN6 β-cells express two RyR isoforms, RyR I and RyR II,
which display distinct subcellular localizations. Whereas type-I RyRs were present in
approximately equal density in a mixed vesicle/mitochondrial fraction and in microsomes, RyR
II was considerably more abundant on ER membranes [62]. Functional NAADP-sensitive Ca2+stores are also present in human β-cells [66].
Dantrolene, a selective inhibitor of RyR I, increased steady-state free [Ca2+] in β-cell
secretory granules but not in the ER, consistent with the presence on granules of a further
activator or channel capable of amplifying the effects of RyRs on Ca2+ release. Receptors for
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
10
NAADP may thus serve this role, and insulin secretory vesicles, but not the ER, may comprise
an NAADP-responsive Ca2+ store [62]. Regulated calcium storage in the insulin secretory
granule has thus been implicated in the mechanisms of regulated insulin secretion.
This emerging picture implicates Ca2+ regulation within the insulin secretory vesicle
itself as pivotal to the regulated secretion of insulin and amylin, and points to areas where
proteomic analysis might be able to contribute to the elucidation of molecular mechanisms.
Questions that arise include those of the nature of the proteins that might mediate aspects of this
emerging process, the nature and roles of putative Ca2+-binding proteins in the insulin secretory
granule, and ultimately the nature of the molecular defects in T2DM that generate defective
insulin secretion.
In order to understand such processes at the molecular level, it would help to know which
proteins and pathways are actually present in the granule, so as to understand which may
possibly be implicated in disease processes that might occur therein.
1.7 Potential future contributions by proteomics Phenomena, such as those which potentially
link aspects of insulin secretory granule function to the misfolding of amylin, the regulation of
insulin and amylin secretion, the generation of β-cell autoantigens, and through these processes
to the pathogenesis of the major types of diabetes, provide a clear motivation and focus for the
systematic, ongoing investigation of this organelle.
Proteomic analysis is but one of a number of hypothesis-generating methodologies that
are now being brought to bear on questions concerning the origins and mechanisms of diabetes,
and the roles of granule-associated pathways in these processes. One of its advantages in this
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
11
case is that it can be selectively targetted at the granule itself, as explained below. Other
hypothesis-generating methods include GWAs, whole-genome transcriptomics, and
metabolomics, which together contribute different aspects of the information available in the
broader field of systems biology, are broader but less focussed in their scope.
One of the challenges that will need to be met in the next phase of the application of
systems biology to the insulin secretory granule, is the integration and interpretation of the large
data sets currently being generated by these complementary but distinct methodologies, with the
objective of generating testable hypotheses for the targetted dissection of disease mechanisms,
and the use of these in the generation of new, integrated hypotheses whose final goal must be the
generation of new and improved therapeutic interventions for diabetes.
2. β-cell secretory granules : structural regions and functional specialization
The β-cell granule performs a specialized subcellular function in the storage and secretion of
insulin and amylin. It is a complex intracellular organelle containing many proteins with
different catalytic activities and messenger functions [24, 67, 68] along with other components
including adenine nucleotides, inorganic phosphate and bivalent metal ions [69].
The granule itself comprises several distinct structural regions, including the dense core
with its component insulin- and zinc-containing crystals, the halo, and the enveloping, VAMPcontaining outer membrane [60, 68, 70-72]. There is evidence for differential distribution of
component proteins between these different regions, which subserve distinct functions. These
different structures could possibly be targetted individually in future proteomic studies, with
consequent increases in resolution and improved understanding of protein distribution and
function within the granule.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
12
B-cell secretory granules can be visualized by electron microscopy as spheroidal
structures of about 200-300 nm in diameter (Figure 2), and comprise a crystalline core of
zinc/insulin-containing crystals surrounded by a mantle of less dense material, enwrapped by a
phospholipid bilayer membrane [68, 69]. The granule is, however, far more than just a cellular
repository for processed insulin. For example, its membrane contains a series of proteins
involved in the integration of its trafficking and docking to the cell membrane and (as discussed
above), it contributes actively to its own secretion through regulation of cell-Ca2+ metabolism
[64].
In the early 1980s, Hutton reported that β-cell granules may contain more than 150
distinguishable proteins in addition to their major constituents, which were considered to be
insulin and its connecting-peptide (C-peptide) [73], and that a number of these are secreted in
addition to insulin [74]. Additional granule components were noted to include proteinases
implicated in proinsulin-to-insulin conversion, intermediates in that conversion process, minor
co-secreted peptides, membrane proteins that mediate granule movement and exocytosis, and
ion-translocating proteins involved in the regulation of the within-granule environment.
More recently, amylin (designated also as IAPP), was found to be a second major
hormone packaged in the β-cell secretory granules , which is also mainly β-cell-specific [27, 75].
Interestingly, amylin may be predominantly present in the granule haloes, whereas processed
insulin resides mainly in the dense cores.
(Figure 2 near here)
Typical β-cells contain about 104 insulin secretory granules, but less than 1% of these are
thought to be available for immediate release [71]. All the rest are considered to be immature,
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
13
and must be primed and then recruited to the cell membrane before they can undergo exocytosis.
These processes require several ATP-, Ca2+-, and phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2)-dependent steps, which culminate in pore formation and granule release to the cell
exterior [70, 76].
2.1 Before proteomics: major protein components of the β-cell secretory granule
The protein composition of the endocrine pancreas has arguably been under investigation for
most of the past 100 years. The initial impetus was provided by the search for the hypoglycaemic
principle, which turned out to be insulin [25]. For many years after its discovery, until the advent
of recombinant human insulin manufactured in microbial expression systems, bovine and
porcine pancreases were the only feasible sources of insulin for clinical use [77]. The extraction
of pharmacologically-active insulin from the pancreas, wherein the predominant exocrine cells
are replete with proteolytic hormones, was said to be one of the most challenging of all
extractions of natural products for pharmaceutical purposes [77].
The considerable heterogeneity of highly-purified insulin preparations was demonstrated
by application of various chromatographic and electrophoretic methods well before the discovery
of proinsulin. Biochemical analysis of the pancreatic islets subsequently yielded the insulinprecursor, proinsulin [42, 78, 79], and with it in time the understanding of insulin release by
enzyme-catalysed conversion from proinsulin [42].
These pre-proteomic era studies provided a platform on which current proteomic analysis
of the β-cell secretory granule may be anchored. Modern proteomic studies are thus seen as an
extension of this earlier work.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
14
(Figure 3 near here)
3. Evolution of a question that might be addressed by proteomics: “How might amylin
misfolding cause T2DM?”
Islet amyloid is formed mainly by misfolded human amylin [27], a physiological resident of the
islet β-cell granule (Figures 1, 3). Aggregation of the human hormone into small soluble β-sheetcontaining oligomers is linked to islet β-cell degeneration and the pathogenesis of T2DM [3133]. Islet amyloid is associated with substantial reductions in relative β-cell mass in type-2
diabetes (on average ~60 %), probably due to increased apoptosis compared with obese and lean
non-diabetic humans [80].
Several lines of evidence now provide compelling support for the idea that processes
associated with amylin aggregation contribute to β-cell degeneration and the onset of T2DM.
First, in vitro studies with synthetic amylin show that fibrillar structures assemble
spontaneously through self-association of monomers into protofibrils and higher-order fibrillar
structures [81, 82]. Studies with time-dependent atomic force microscopy have enabled direct in
vitro visualization of this process. These studies show that oligomer formation can take place
within minutes [31], a time-course that matches the activation of the β-cell membrane
Fas/FasL/FADD-activated pathway in β-cells destined to undergo amylin-evoked apoptosis [33].
Cytotoxic amylin preparations contain few preformed fibrils, but undergo time-dependent
aggregation into soluble β-conformers [83]. Islet β-cell toxicity evoked by aggregating
extracellular amylin, occurs through an apoptotic mechanism [84, 85] mediated via a pathway
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
15
comprising initial activation of a membrane-bound Fas/FasL/FADD/caspase-8 complex [33, 86]
followed by a three-pronged downstream cascade comprising JNK1/cJun [86], ATF2/p38
MAPK [87], and p53/p21WAF1/CIP1 [85], that results ultimately in activation of caspase-3 [86]
and consequent apoptosis. In addition, parallel amylin-mediated activation of ER-stress related
pathways might also contribute to islet β-cell degeneration [88].
Second, associations between human amylin aggregation and decreased β-cell mass have
been reported from in vivo studies in several murine transgenic models of amylin-mediated
diabetes [29, 32, 89-93] (Figure 5). Similar associations are present in primates, whose wild-type
amylin molecules contain an amyloidogenic sequence [24, 94, 95], are fibrillogenic, and form
islet amyloid [96-99]. By contrast, murine amylin molecules are not aggregation-prone [100], so
diabetic phenotypes in human amylin transgenic mice develop in a background devoid of
amyloid formed by the wild-type murine hormone. Obese human amylin-transgenic mice have
been reported to replicate pathological findings in T2DM, showing non-ketotic hyperglycaemia,
amyloid deposition, and decreased β-cell mass, possibly via increased apoptosis [92].
(Figure 5 near here)
Amylin aggregation could thus mediate β-cell degeneration in T2DM. However, the
significance of tissue aggregates comprising mature amyloid fibrils in T2DM pathogenesis
remains uncertain, since some studies have implied that amyloid fibrils themselves may be toxic,
or that human amylin-transgenic mice may not develop spontaneous diabetes [91, 101-104]. The
latter discrepancies may well be explained however, by a requirement for permissive genetic
background-human amylin transgene interactions to manifest full-blown β-cell degeneration and
a diabetic phenotype [32, 93, 105].
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
16
Finally, human amylin purified from islet amyloid deposits is a potent inducer of insulin
resistance in ex vivo rat skeletal muscle [106, 107] (Figure 5). This finding provides a potential
link between islet β-cell dysfunction and the induction of peripheral insulin resistance.
(Figure 6 near here)
These observations, and the ongoing uncertainty concerning precise mechanistic linkages
between amylin aggregation, β-cell degeneration, the regulation of systemic fuel metabolism,
and T2DM onset [108], provide fertile ground for future proteomic investigation. For example,
the exact location and mechanism of amylin misfolding is unknown, as is the site of origin and
nature of the amylin-mediated death-initiating signal – is it cell membrane-bound
Fas/FasL/FADD activation [33], ER stress [88], or some other process that might occur
elsewhere [109, 110]?
One key question that is yet to be answered, is whether amylin-mediated misfolding
occurs prior to, within, or after amylin secretion from the pancreatic islet β-cell granule. Granulefocussed studies are expected to prove crucial in answering this key question.
4. The β-cell secretory granule proteome
Available proteomic studies of whole mouse [111] and human [112] islets have identified groups
of islet-specific proteins. The bulk of proteins detected overlap with those in other tissue types,
but islet hormones were also identified. The resulting peptide reference libraries are seen as
providing a resource for future higher-throughput and quantitative studies of islet biology, which
may be useful in the study of T2DM mechanisms, for example.
Proteomics has been applied to analyse the effects of insulin signalling in isolated murine
and human islets, in a study that provides a useful example of how this methodology can be
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
17
integrated with other methods to address a complex research question – in this case, the role of
insulin in β-cell apoptosis [113]. In this study, combined data, to which proteomic analysis made
a significant contribution, indicated that insulin can act as a master regulator of islet survival by
regulating Pdx1.
4.1 Proteomic approach to the β-cell secretory granule Two systematic proteomic analyses of βcell secretory granules have been reported in the past few years [20, 22]. Results from one of
these are detailed here (Table 1) as a basis for the following comparisons and contrasts [20].
(Table 1 near here)
These studies reported the molecular identities of 51 and 130 ‘granule-related’ proteins
[20, 22], respectively. In each case, most of the proteins reported were newly identified as
potential granule components.
Without doubt, these findings usher in a new era of studies of the β-cell secretory
granule. These studies point to numerous proteins and pathways that have not previously been
identified in β-cell secretory granules, and thus have the potential to greatly increase our
knowledge of the intricate functionality of these organelles.
However, comparisons and contrasts between the two are illustrative of some of the
challenges confronted by application of proteomics to the β-cell secretory granule at present, and
caution is warranted in their interpretation and application. Intensive follow-up work is required
before the status of each newly-identified putative granule protein is confirmed and clarified.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
18
Both groups analysed granules purified from insulin-secreting rat INS-1E cells, which
came initially from the same source. Comparisons and contrasts between the two studies are
therefore informative for several reasons, so they are now described in greater detail.
4.2 Method comparisons Both studies identified many proteins that had previously been
associated with β-cell secretory granules by pre-proteomic methods, and there were considerable
overlaps between the two data sets [20, 22].
However, Brunner et al reported many proteins usually considered to be lysosomeassociated, as components of their purified β-cell secretory granule proteome. By contrast,
Hickey et al described numbers of proteins more usually associated with ER and mitochondria,
some of which did not appear in the data set of Brunner et al. These differences deserve further
consideration, since they highlight potential challenges faced by current proteomic techniques.
In order to focus exclusively on β-cell secretory granule-associated proteins, both groups
employed several purification steps prior to protein identification analysis. These studies
illustrate the principle that increased sensitivity and specificity can potentially be achieved by
prior organellar purification.
Brunner et al employed two sequential ultracentrifugation steps: a first-dimensional
layered-discontinuous Nycodenz gradient followed by a Percoll cushion [22]. They monitored
protein recovery by using an insulin immunoassay (granule marker) and western blotting for
calreticulin and betagranin, respectively as markers for ER and β-cell secretory granules . They
then separated proteins by using one-dimensional SDS-PAGE and sectioning gels into 28
consecutive pieces, followed by in-gel tryptic digestion and protein identification analysis using
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
19
LC-MS2 followed by bioinformatic methods. They also monitored relative purity of granule
preparations by western blotting for a series of markers associated with various extra-granular
organelles. These findings pointed to the presence of lysosomes within their granule
preparations, so the significance of the putative assignments of lysosomal proteins as
components of the granule proteome, remains to be confirmed.
By contrast, Hickey et al chose to isolate β-cell secretory granules using a conservative
methodology that initially employed two sequential orthogonal steps with concomitant
monitoring for several, potentially-confounding organelles including lysosomes, ER,
mitochondria and cytosol [20]. The first of these used ultracentrifugation after overlayering INS1E culture supernatants onto preformed, continuous OptiPrep gradients with subsequent
fractionation and separation of granule-containing fractions, as determined by their hormone
content [114], whose buoyant density was 1.10-1.11. They monitored this step using an insulin
immunoassay as a granule marker coupled with multiple enzyme assays to track potential
contamination with other organelles, including aryl sulphatase (lysosomal marker), NADH
cytochrome C reductase (ER), citrate synthase (mitochondria), and lactate dehydrogenase
(cytosol) (Figure 7).
(Figure 7 near here)
In the following, orthogonal step, they immunopurified granules by using anti-VAMP2
antibody-conjugated magnetic Dynal beads [20], and then undertook protein identification
analysis that comprised the following sequential steps: reduction and S-carboxymethylation;
snap-freezing and concentration by vacuum centrifugation; trypsinization; peptide recovery by
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
20
batch reversed-phase chromatography; and LC-MS2 with stringent bioinformatic criteria for
inclusion in the final list.
4.3 How to account for contrasting conclusions? Although there are areas of substantive overlap
in the results and conclusions, there were also broad areas of divergence between these two
studies [20, 22].
Why might this be so? Both groups went to considerable lengths to ensure the purity of
the granular preparations, whose contents they subsequently analysed. Hickey et al assigned
proteins to specific sub-cellular locations or functions by application of currently-accepted rules.
By contrast, Brunner et al simply listed numerous mitochondrial proteins under the category of
‘Other Proteins’ (for example: 10-kDa heat shock protein; mitochondrial cystatin-C precursor;
malate dehydrogenase, mitochondrial precursor; superoxide dismutase (whether this was SOD1
or SOD 2 they did not specify – there is evidence from others that both forms localize in
mitochondria); voltage-dependent anion-selective channel protein 1 (VDAC); and ATP synthase
alpha chain, mitochondrial precursor), without allocating proteins to specific locations or tasks.
Their findings with respect to this sub-group of proteins were generally consistent with those of
Hickey et al, who also reported significant numbers of mitochondria-associated proteins in their
granule preparations. Brunner et al did not specify NCBI accession numbers for their individual
proteins, so the exact molecular identity of some, for example, ‘superoxide dismutase’, remains
uncertain.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
21
Brunner et al. also reported an abundance of lysosomal proteins, which were annotated as
hydrolases or lysosomal membrane proteins [22]. However, by contrast, none of these was
detected by Hickey et al [20].
4.4 Possible explanations for between-study divergence Why were there such major differences
between the two reports? Clearly, the two studies employed different isolation methods, so it was
possible that there would be some differences between the sets of proteins identified. However,
the divergences between large groups of proteins with shared prior subcellular localizations,
indicates that more systematic factors were probably also at work. The lack of lysosomeassociated proteins in the second study, might reflect the use of enzyme markers during the
fractionation which, when coupled with the following orthogonal immunopurification, permitted
exclusion of lysosome-rich fractions from the final preparation.
In the reverse direction, 20% of the proteins identified by Hickey et al were chaperones,
whose main agreed functions reside in the ER, and a further 20% comprised other proteins with
known ER- or Golgi-related functions (Figure 8). Clearly, the existence of a large number of
chaperone-related proteins in the β-cell secretory granule, if confirmed, could have important
implications for disorders triggered by protein misfolding, such as aggregation-associated β-cell
degeneration [32].
(Figure 8 near here)
However, the conclusions from these two preliminary forays into the field of β-cell
secretory granule proteomics may best be viewed as providing the basis for further, intensive
research.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
22
The immunoaffinity purification procedures employed by Hickey et al may have purified not
only granules, but also aspects of the cytoskeletal apparatus responsible for granule transport,
which therefore may adhere to them (for example, actin, tubulin, and Rab GTP-binding
proteins). Similarly, the presence of ER and cytosolic components in those results may follow
from intracellular associations between secretory granules and aspects of the associated
structures. As one example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was
also detected in that study, has several cellular roles and locations, and is known to bind to
microtubules [115].
However, there is evidence that attachments between secretory granules and components
of the ER and cytoskeleton are physiological, so the findings of ER- and cytoskeleton-related
proteins in purified granule preparations may well reflect these associations.
4.5 Calreticulin: putative assignment as an insulin-granule protein or indicator of ER-protein
contamination? Hickey et al identified calreticulin in their β-cell secretory granule isolates [20],
whereas Brunner et al specifically excluded it from their ‘immature secretory granule (ISG)’
preparations [22]. The latter findings can be interpreted to indicate that the former preparations
were contaminated with ER-derived proteins. Alternatively, calreticulin might be associated with
a subset of granules (ISGs) not purified in Brunner et al’s approach.
What does the balance of currently-available evidence suggest concerning this interesting
and potentially significant difference? There are reports that calreticulin fulfils distinct roles in
different organelles. It has three functional domains (termed N-, P-, and C-). The first binds
KXFFKR motifs, thereby acting as a chaperone [116], and also binds Zn2+ [117], which is noted
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
23
to be present at high concentrations in the granule core. The latter two domains contain Ca2+binding sites and may thus function as local Ca2+ stores [118]. Although a COOH-terminal
KDEL sequence could be seen as consistent with specific targetting to the ER, there is available
evidence that calreticulin also localizes to cytoplasmic granules in T-lymphocytes [117] and
sperm acrosomes, and to the Golgi complex [119], and that it is systemically secreted to circulate
in the plasma [120]. B-cell secretory granules contain a high-Ca2+ environment necessary for
proteolytic processing [64, 73, 121, 122], so Hickey et al’s findings could also be consistent with
a role for calreticulin as a newly-recognised Ca2+-storage protein in β-cell secretory granules.
The preceding analysis points to a series of specific and testable hypotheses. Evidently,
an early consideration is that assignment of calreticulin as a putative insulin-granule protein
requires independent confirmation.
4.6 How might the appearance of ‘mitochondrial proteins’ in ‘insulin-granule-specific’
preparations be interpreted? The presence in insulin-granule preparations of proteins usually
associated with mitochondria requires comment.
It is now becoming evident that apparently discrete organelles do not necessarily contain
discrete protein sets. Interestingly, ‘organelle-specific’ proteomic analyses have frequently
demonstrated multiple locations for up to perhaps 40% of all component proteins [123].
Therefore, many proteins, which until now have not been associated with granules, may in fact
be localized in or adherent to them, and thus serve roles in granule function additional to those
already assigned to them in other organelles.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
24
For example, both studies [20, 22] detected component chains of the mitochondrial ATP
synthase in purified granules, whereas until recently these subunits have mainly been considered
to play roles in oxidative phosphorylation. However, the α-chain of ATP-synthase was recently
shown to act as a receptor for apolipoprotein A-1-mediated hepatic LDL endocytosis [124].
Thus, the current findings might signal that ATP-synthase chains play further, hitherto
unsuspected roles in β-cell secretory granule homeostasis.
A further explanation for the presence of a number of ‘mitochondrial proteins’ in β-cell
insulin-granule preparations is that of the physical attachment between the two organelles that
causes their obligatory co-purification. There is evidence that mitochondria are attached to
cytoskeletal structures in hepatocytes, ciliary desmosomes [125], PC12 cells [126], and
neurosecretory vesicles [127], and to pancreatic islet β-cell granules [62]. The proton-pumping
requirements for granule maturation [128], within-granule chaperone activity, and insulin
exocytosis are dependent on mitochondria-derived ATP [126]. Furthermore, mutations in
mitochondrial DNA can cause β-cell dysfunction and rare forms of T2DM [129]. The close
proximity of mitochondria to granules could enhance efficient transfer of ATP to drive granuleassociated processes, for example their intracellular translocation, and within-granule protein
processing, maturation and exocytosis.
4.7 Chaperones in the β-cell secretory granule: possible implications Aggregation of human
amylin into β-sheet-containing oligomers has been linked to islet β-cell dysfunction and the
causation of the common form of T2DM [32, 33, 93]. The existence of amylin misfolding in
T2DM [108], points to a defect in regulation of the β-cell protein-folding pathways as a key
target in the search for the underlying cause of islet β-cell dysfunction.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
25
Within this context, the identification of specific chaperones in the β-cell granule
provides a clear mechanistic linkage between protein misfolding in diabetes and a newlyemergent, putative granule function. For example, alterations in the function of specific
chaperones and their interactions with amylin can now be investigated as potential contributors
to amylin misfolding. These findings represent a major potential contribution of proteomic
analysis to the search for the molecular basis of disease mechanisms in diabetes.
In the study of Hickey et al, ER- and Golgi-associated proteins accounted for almost 40%
of all those identified (Table 1), consistent with close association between β-cell secretory
granules , and elements of the ER and Golgi apparatus. Particularly abundant in these
preparations were recognised chaperone proteins, which comprised ~20% of all identified
proteins in their preparations [20]. These data are consistent with the idea that β-cells may act as
a significant source of secreted/circulating chaperone proteins. The localization of chaperones in
β-cell granules might be considered unremarkable, given that they are abundant in most tissues
and organelles. Consistent with these data, others have reported that HSPs and other chaperone
proteins are plentiful in pancreatic islets [17, 130], where some are co-localized in secretory
granules [56, 57, 131] or synaptophysin-containing microvesicles [132]. HSPs are also localized
in the extracellular compartment [133] and their serum concentrations can vary according to
physiological status [134].
Different chaperone molecules act cooperatively [134], consistent with the observed
diversity in the β-cell secretory granules . HSP90 localizes in neuronal tissues [135], and a recent
subcellular proteomic dissection of neuromelanin granules also reported high levels of ERderived chaperones [136], consistent with its presence in pancreatic β-cells.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
26
The apparent abundance of chaperones in β-cell secretory granules highlights their likely
roles in preserving intact and well-regulated secretion of the hormones, amylin and insulin, and
protecting the cell from the biological effects of protein misfolding [20, 32, 33]. The amylingenerated aggregates present in T2DM [27, 108] are similar to those that occur in Alzheimer’s,
Parkinson’s and Huntington’s diseases [137]. At high concentrations, dysregulated amylin
folding occurs independently of protein convertases (PC2, or PC1/3), to potentially seed the
amyloid fibrils that in turn form the tissue aggregates designated as ‘islet amyloid’ [81, 100,
138]. There is no direct evidence, however, for the presence of aberrantly-processed human
amylin in pancreatic islet amyloid [108]. Further proteomic analysis will be required to
determine whether aberrant amylin processing is a contributor to islet amyloidosis and the
origins of T2DM.
During its transition from soluble oligomers through protofibrils to insoluble fibrils [30,
31, 81, 100, 139], misfolding human amylin can elicit cytotoxic processes that can cause or
result in β-cell death [83, 139]. This process in turn can cause decreased pancreatic β-cell mass
and insulin secretory capacity, resulting in hyperglycaemia and, ultimately, T2DM [32, 33, 93,
140]. It may also serve as a target in the development of new classes of anti-diabetic compounds
[32, 141].
5. Next steps
This chapter has presented reasons why it is desirable to undertake further detailed proteomic
studies of the islet β-cell secretory granule. In particular, it is expected that important insights
concerning the physiological regulation of islet hormone secretion and β-cell death, and the
origins and mechanisms of T1DM and T2DM, might be obtained through such analysis.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
27
However, the significant discrepancies between the two data sets discussed above [20,
22], show that currently-available approaches have clear limitations. In particular, it is apparent
that each new protein assignment to the islet β-cell granule proteome must be regarded as no
more than putative until substantive confirmatory evidence is obtained, by application of more
selective proteomic analysis as well as one or more orthogonal methods, such as immunological
co-localization of proteins to the granule. Results from even the latter must be viewed with
caution, however, since limits of sensitivity and specificity of individual antibodies may provide
limitations that must be taken into account.
At present, there are no available proteomic studies of β-cell granules from diabetic
humans, so inferences concerning diabetes-related changes in granule composition, and how
such effects might contribute to the phenomena of diabetic β-cell degeneration must be drawn
from other lines of investigation. There is a clear need for more precise information to
characterise the changes in islet structure and function that occur in relation to the origins of βcell dysfunction, islet amyloid formation and β-cell disappearance in T2DM.
It is to be hoped that the ongoing rapid development of new and improved proteomic
approaches will enable such studies in the near future. Indeed, it is expected that one of the
exciting outcomes of the current assembly of expert views in this volume will be the creation of
linkages and methodologies by which these important goals can be facilitated.
Acknowledgements I wish to acknowledge my wife, Margaret Cameron-Cooper, for her
unswerving loyalty and steadfast support throughout the past 30 years of scientific endeavour;
Professor Sir P John Scott for his guidance and wise counsel; Cynthia Tse for her valuable help
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
28
with preparation of the manuscript; and all my scientific colleagues, who have contributed to our
group’s studies in so many different ways.
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
29
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41
7. Figure Legends
Figure 1 Microscopic view of an original haematoxylin and eosin-stained pancreatic section
wherein islet ‘hyaline’ (now known as amyloid) was first identified by Dr Eugene Opie, in a
post-mortem study of a patient with type-2 diabetes mellitus [23]. The islets, which are usually
replete with endocrine cells, have largely been replaced by the amorphous, faintly pink-staining
islet amyloid. [Reproduced with permission of Robert D. Hoffman, M.D., Ph.D., Department of
Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD].
Figure 2 Electron micrographs illustrating secretory granules from cultured murine insulinsecreting βC6-F7 cells. A, Structures in the perinuclear region, show characteristic membranelimited granules in different stages of maturation (arrowed). B, Mature secretory granules near
the cellular periphery show characteristic electron-dense cores and adjacent electron-lucent
haloes (arrowed). Abbreviation: N, nucleus; Bars = 300 nm. (Reproduced with permission from
C. M. Buchanan et al.: Biochem J 360, 431, 2001 [114]).
Figure 3 Original chromatographic purification of human amylin from pancreatic extracts of
type-2 diabetic patients provides an early example of the application of comparative proteomic
analysis to tissues. A, HPLC gel filtration in 6 M guanidine hydrochloride of an extract from an
amyloid-containing pancreas taken at post mortem from a patient with type-2 diabetes. Amylin
was present in the region indicated by the bar. B, Reversed-phase HPLC of material from the
region indicated by the bar in A: unreduced amylin was present in peak 3. C, Reversed-phase
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
42
HPLC of a control extract from a control pancreas from a nondiabetic patient, as in B. Peaks 1
and 2 corresponded in elution time and amino acid composition to 1 and 2 in B. D, Repurification by reversed-phase chromatography of peak 3B after reduction and alkylation of
cysteine residues and [14C]-radiolabelling of cysteinyl residues. Peaks 4 and 5 had amino acid
compositions distinct from that of 3RA, which was reduced and alkylated amylin. E, Separation
of product peptides after tryptic digestion of reduced and alkylated amylin by reversed-phase
HPLC. Peak 6 was the smaller, more hydrophilic peptide amylin1-11, and peak 7 the larger, more
hydrophobic amylin12-37. All radiolabel was present in peak 6. Identity of peaks was confirmed
by quantitative amino acid analysis and by gas-phase peptide sequencing. The ratio of peak
heights is consistent with the relative lengths of the amylin-derived peptides. (Reproduced with
permission from G. J. S. Cooper et al.: Proc Natl Acad Sci USA 84, 8628, 1987 [27]).
Figure 4 Time-lapse atomic force microscopy (AFM) showing a human amylin oligomer
growing into a fibril. Droplets of a human amylin solution were placed on a mica surface and
studied by AFM using published methods [31]. Oligomers are seen to grow in height prior to
extensive elongation into fibrils, and consist of ~16 monomers when first visualized (left-hand
panel). The height of the oligomer (arrow) is seen to increase with each scan. The time points
and height (h) and length (l) measurements are as indicated in each image. (Reproduced with
permission from J. D. Green et al.: J Biol Chem 279, 12206, 2004 [31]).
Figure 5 Amyloid visualized by light microscopy was dissociable from occurrence of diabetes in
hemizygous human amylin transgenic mice. Photomicrographs show serial pancreatic islet
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
43
sections from non-transgenic and human amylin transgenic animals. Left photomicrographs from
top three panels show insulin- (green) and glucagon- (red) immunoreactivity. Bottom two left
panels show islet sections incubated with antisera to somatostatin and glucagon, revealing brown
cytoplasmic staining. Middle and right panels show corresponding light- and polarizedmicroscopic field views of adjacent islet sections stained with Congo red. Amyloid birefringence
is apple green whereas that corresponding to collagen is silvery. The scale bar (50 μm) shown in
top left photomicrograph applies to all images except for those corresponding to the 600 day
non-diabetic hemizygous mouse (second to bottom row) which represents 100 μm. (Reproduced
with permission from J. F. Aitken et al.: Diabetes, Epub ahead of print September 30th, 2009;
doi:10.2337/db09-0548 [32]).
Figure 6 Human amylin extracted from the pancreas of type-2 diabetic patients elicits dosedependent inhibition of insulin-stimulated glycogen synthesis in ex vivo rat soleus muscle. The
human amylin used in these studies was purified and characterised by the methods of Cooper et
al. [27]. Values shown are means of at least four separate incubations. Statistically-significant (p
< 0.05, Student’s t-test) decreases from control values are indicated by *, (control against 10-9 M
amylin); †, (10-9 M amylin against 10-8 M amylin).
Methods. Muscle preparation and incubation methods were as described [106]. Typically, a
submaximal concentration of insulin (100 μU.ml-1) stimulates the rate of glycolysis and glycogen
synthesis, by ~50% and 125% above basal rates respectively, in this preparation. (Reproduced
with permission from B. Leighton and G. J. S. Cooper: Nature 335, 632, 1988 [106]).
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
44
Figure 7 Fractionation of lysates from cultured INS-1E β-cells was monitored by serial enzyme
or hormone assays, as described [20]. Markers used were NADH cytochrome C reductase
(endoplasmic reticulum), citrate synthase (mitochondria), lactate dehydrogenase (cytosol),
insulin (granules), and aryl sulphatase (lysosomes). Insulin was concentrated within specific
fractions (broken line). Insulin-containing fractions were then subjected to immunoaffinity
purification using VAMP2. (Reproduced with permission from A. R. Hickey et al.: J Proteome
Res 8, 178, 2009 [20]). Abbreviations: VAMP, vesicle-associated membrane protein
Figure 8 Pie chart summarizing the cellular location of proteins identified in anti-VAMP2
antibody-conjugated magnetic Dynal bead-purified granule proteins, as listed in Table 2.
(Reproduced with permission from A. R. Hickey et al.: J Proteome Res 8, 178, 2009 [20]).
Abbreviations: VAMP, vesicle-associated membrane protein
Garth J S Cooper, School of Biological Sciences, University of Auckland, Private Bag 92-019,
Auckland, New Zealand E-mail: g.cooper@auckland.ac.nz
45