| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Division of Endocrinology, Departments of
Medicine*
and Biochemistry & Molecular
Biology,
Mayo Clinic, Rochester, Minnesota
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Exogenously added hIAPP is cytotoxic and induces apoptosis when added to a variety of cultured cells, including primary rat pancreatic cells, hippocampal neurons, aortic endothelial cells, COS cells, and PC12 pheochromocytoma cells but neither fibroblasts nor H4 hepatoma cells.11 We previously showed that intracellular amyloid generated from hIAPP was associated with cell death resembling apoptosis in COS-1 cells that have been transfected with an hIAPP expression plasmid.12 The mechanism by which amyloid deposition leads to the loss of pancreatic ß cells is not known. One hypothesis suggests that the IAPP monomers are secreted from the islet ß cells and, at high local concentrations, will aggregate into insoluble fibrils in the extracellular spaces and islet capillaries. These amyloid deposits subsequently come into direct contact with the islet ß cells, resulting in their death by an unknown mechanism.11,13 However, we showed that the level of intracellular IAPP required to kill COS-1 cells was several orders of magnitude less than that required in the cell culture studies,12 indicating that intracellular amyloid formation may be more detrimental.
To eventually understand the mechanism by which intracellular hIAPP causes cell death, we first sought to determine whether transfected COS-1 cells were dying by apoptosis or necrosis. In the current study we demonstrate that the intracellular accumulation of amyloido- genic hIAPP, but not nonamyloidogenic mutant forms of hIAPP or rat (r) IAPP, induces apoptosis in transfected COS-1 cells in a time-dependent manner. Our data suggest that hIAPP amyloid activates specific intracellular signaling pathways that result in apoptosis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The cDNAs for hIAPP, its antisense construct
(hIAPPanti), a mutant form of hIAPP (hIAPPmut),
and rIAPP were cloned into the pMT2 expression
plasmid14
as described previously.12
The
pMT2 vector contains the adenovirus major late promoter with
an SV40 enhancer. The IAPP cDNAs are cloned into an EcoRI
site that attaches the adenovirus tripartite leader sequence allowing
for efficient translation and positions the cDNA upstream of the SV40
early polyadenylation signal. The hIAPPmut was constructed
via inverted polymerase chain reaction mutagenesis15
using
the oligonucleotides 5'-CCCGTTCTCCCACCTACCAACGTGGGATCC-3' and
5'-ACCAAAGTTGTTGCTGG-3' as primers with the pUC18-hIAPP
plasmid12
as template. This results in a cDNA, verified by
direct didexoy sequencing, which converts the sequence
GlyAlaIleLeuSerSer between amino acids 24–29 within the amyloidogenic
domain to the sequence GlyProValLeuProPro, which corresponds to the rat
sequence. The two additional differences between the human and rat
IAPPs (His18 versus Arg18 and
Phe23 versus Leu23) were preserved
in hIAPPmut (see Figure 8
). The pMT2 vector
without insert served as an additional control plasmid.
|
COS-1 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco-BRL, Gaithersburg, MD) supplemented with 10% FetalClone II (Hyclone, Logan, UT), 100 U/ml penicillin (Gibco-BRL), 100 U/ml streptomycin (Gibco-BRL), and 2 mmol/L L-glutamine (Gibco-BRL). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and passaged weekly. Subconfluent cells were harvested by trypsinization and 4 to 5 x 106 cells resuspended in 200 µl cold phosphate-buffered saline (PBS), 20 mmol/L HEPES, with 10 to 15 µg of the plasmid DNA. The cells were incubated on ice for 15 minutes, then electroporated at 900 µfarad and 250 volts in a BIO-RAD Gene Pulser (Richmond, CA) in standard cuvettes with a 4-mm electrode gap. The electroporated cells were cultured in 10 ml of medium in 10-cm tissue culture dishes. At 20 hours after transfection, the medium was replaced to remove nonadherent cells that had been killed or injured during electroporation. Positive controls for apoptosis were obtained by treating with 0.5 µg/ml tunicamycin, an inhibitor of protein glycosylation in the endoplasmic reticulum (ER), for 3 days.
Analysis for Plasma Membrane Alterations in Apoptosis
Cells were harvested by trypsinization at 24, 48, 72, or 96 hours and pooled with their culture medium so that cells that had lost their adherent properties during apoptosis ("floaters") were included in the analysis. Cells were pelleted, washed, and resuspended in 400 µl of binding buffer (BB: 100 mmol/L HEPES (pH 7.4), 1.5 mol/L NaCl, 50 mmol/L KCl, 10 mmol/L MgCl2, 18 mmol/L CaCl2) and 100 µl of cells (1 x 106) aliquoted to 4-ml Falcon tubes for labeling and fluorescence-activated cell sorting (FACS) analysis. Cells were incubated with 1 µg of sample annexin-V-biotin conjugate (Trevigen, Inc, Gaithersburg, MD) at 4°C in the dark for 20 to 30 minutes, washed, and fluorescently labeled with streptavidin-phycoerythrin (PE) (Molecular Probes, Eugene, OR) at 1 µg of sample under the same conditions. Labeled cells were washed and resuspended in 400 µl of BB containing 6 µg/ml 7-amino actinomycin-D (7-AAD, Molecular Probes) and 2% formalin (Sigma, St. Louis, MO). Cells were analyzed on a fluorescence-activated cell sorter (FACSTAR, Becton Dickinson, San Jose, CA) within 2 hours of labeling. Data were analyzed using the PC Lysis program (Becton Dickinson). FACS gating based on forward scatter and side scatter was used to exclude cellular debris and doublets so that typically 14,000 ± 2000 out of 20,000 cells were selected for analysis. Every experiment included control samples that had been transfected with either the pMT2 vector or the pMT2-hIAPPanti gene.
Analysis for DNA Fragmentation by TUNEL
DNA fragmentation in transfected cells were analyzed using the FlowTACS® fluorescein isothiocyanate (FITC) kit from Trevigen and were performed in parallel with annexin-V labeling as described above. Briefly, 1 x 106 cells were fixed in 2% formalin for 10 minutes, washed with PBS, and incubated in Cytopore® (Trevigen) for 10 minutes. DNA fragments were end-labeled with biotinylated nucleotides and terminal deoxynucleotidyl transferase (TdT) for 60 minutes at 37°C, then the biotinylated ends fluorescently tagged with streptavidin-FITC for 20 minutes at room temperature. Cells were briefly fixed in 2% formalin and assayed by FACS within 2 hours. Data were analyzed by the PC Lysis program for cells staining positive for FITC (apoptotic) and those that are FITC negative (nonapoptotic).
Confocal Microscopy
Cells were maintained, harvested, and transfected as described above. Transfected cells (6 to 12 x 103 cells in 400 µl) were added to each well of 8-well culture chamber slides (LabTek, Naperville, IL) that had been precoated with poly-L-lysine. At 48 hours after transfection, the cells were labeled for: 1) intracellular or extracellular IAPP, 2) translocated phosphatidylserine (PS) residues with annexin-V, 3) fragmented DNA with FlowTACS®, 4) specific markers of subcellular organelles by immunohistochemistry, or 5) combinations of the above. Fluorescently labeled cells were analyzed on a dual-beam laser scanning confocal microscope (LSCM, model LSM310, Zeiss, Thornwood, NY).
Labeling of intracellular IAPP on adherent cells was accomplished by fixing the cells for 10 minutes in 3.7% formalin, washing, then blocking for 10 minutes in PBS containing 2 mg/ml goat globulin (NGG, Sigma) and 0.05% saponin (Sigma), designated NGG-Sap-PBS. Cells were incubated on ice for 30 to 60 minutes in 150 µl of a 1:2000 dilution of rabbit anti-amylin antiserum (Peninsula Laboratories, Belmont, CA), washed, and fluorescently labeled in a 1:2000 dilution of a goat anti-rabbit antibody conjugated with the Oregon Green fluorochrome (GAR-FITC, Molecular Probes). Cells were washed, fixed with 3.7% formalin, and mounted using the SlowFade® Light kit (Molecular Probes). Mounted slides were stored in the dark at 4°C and analyzed within 5 days. Labeling to detect extracellular IAPP was performed using the same protocols, except saponin was omitted from the buffer.
Dual immunohistochemical labeling to specifically determine the subcellular localization of amylin was accomplished by co-incubating blocked cells with a 1:2000 dilution of rabbit anti-amylin antiserum and diluted mouse monoclonal antibodies against the subcellular markers protein disulfide isomerase (PDI), an ER resident protein, p58, a Golgi resident protein, and Mann II, an ER/Golgi marker protein. Anti-PDI and anti-Mann II antibodies were diluted 1/500, and anti-p58 antibodies were diluted 1/200. Immunohistochemical labeling was in NGG-Sap-PBS on ice for 60 minutes. Sandwich labeling was done concurrently with GAR-FITC (for anti-amylin labeling) and goat-anti-mouse-Rhodamine-Red (GAM-RHOD, Molecular Probes) under the same conditions. Final fix, mount, and analysis were as described above.
To directly correlate the accumulation of intracellular amyloid with the appearance of apoptosis markers, transfected cells were double-labeled with anti-IAPP antiserum and either annexin-V or FlowTACS®. Extracellular PS residues were labeled first by incubating adherent cells with 0.2 µg/sample annexin-V in BB, fixing with ice-cold methanol for 20 minutes and blocking with NGG-Sap-PBS. Secondary labeling of extracellular annexin-V was accomplished by co-incubating with streptavidin-Rhodamine-X conjugate (0.2 µg/sample) and primary labeling of intracellular IAPP with a 1:2000 dilution of rabbit anti-IAPP antiserum in NGG-Sap-PBS. Secondary labeling of intracellular IAPP was done by incubating with a 1:2000 dilution of GAR-FITC. Labeled cells were fixed in 2% formalin, mounted (SlowFade®), and analyzed by LSCM, as described above.
To correlate intracellular IAPP with fragmented nuclear DNA characteristic of apoptosis, cells were first fixed in 3.7% formalin at room temperature, and the membranes were permeabilized by incubating in NGG-PBS (2 mg/ml) containing either a 50:50 dilution of Neuropore (Trevigen) or 0.05% saponin. Nuclear DNA was labeled first using TdT and biotinylated nucleotides (FlowTacs®). Biotin-labeled DNA fragments were fluorescently tagged concurrent with labeling for intracellular IAPP by a co-incubation with 1.5 µg/ml streptavidin-Rhodamine-X and a 1:2000 dilution of rabbit anti-amylin antibodies for 20 minutes at room temperature. Anti-amylin antibodies were then fluorescently labeled by incubation with a 1:2000 dilution of GAR-FITC. Cells were fixed in 3.7% formalin and mounted as described above. Mounted slides were stored at 4°C in the dark and analyzed within 5 days.
Statistical Analyses
In all cases the data from at least three independent experiments were subjected to multivariate analysis of variance using a post hoc Bonferroni t-test to evaluate the significance of individual variables in experiments containing multiple variables.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Because amyloid has been previously shown to accumulate in transfected
COS-1 cells after 72 to 96 hours and the cells only exhibited pycnotic
nuclei within this time period,12
we first examined
annexin-V and 7-AAD labeling by FACS of COS-1 cells that had been
transfected with the pMT2, pMT2-hIAPP, and
pMT2-hIAPPanti genes and cultured for 72 to 96
hours. Annexin-V binds to PS residues that have undergone a
translocation from the cytoplasmic side of the plasma membrane to the
extracellular surface early in the apoptotic pathway.16
The
PS-bound annexin-V-biotin is then fluorescently labeled with
streptavidin-PE (
ex = 488 nm, em = 578
nm). The DNA-specific fluorescent stain 7-AAD (ex = 488
nm, em = 647 nm) is excluded from live cells
the membranes of which are intact but will leak through the compromised
membranes of cells that have died from necrosis and those that are in
the final stage of apoptosis.18
This labeling technique
allows separation of cells that are in the early and mid stages of
apoptosis (annexin positive) from those in late apoptosis (positive for
both annexin and 7-AAD), from necrotic (7-AAD positive only), and live
cells (negative for both 7-AAD and annexin).
Representative dot plot profiles generated by FACS analysis of
transfected COS-1 cells 96 hours after transfection are shown in Figure 1
in which annexin-V fluorescence is read
on the x axis and 7-AAD on the y axis. Figure 1, A and B
are typical for cells transfected with the control plasmids,
pMT2 and pMT2-hIAPPanti, respectively
and show that approximately 90% of the cells are viable (lower left
panel) with 9.2 and 11.2% of the cells exhibiting positive staining
for annexin (both upper and lower right panels), indicating they are
apoptotic. These results are consistent with the expected amount of
cell death in cultured cells and are virtually identical with those
from untransfected cell cultures (data not shown). In contrast, cells
that have been transfected with the amyloidogenic pMT2-hIAPP
gene have approximately threefold more (29.9%) annexin positive
(apoptotic) cells (Figure 1C)
. In all experiments, the percentage of
necrotic cells (upper left panel) in pMT2-,
pMT2-hIAPPanti-, and
pMT2-hIAPP-transfectants were virtually identical
(approximately 3 to 5%), indicating that expression of neither
amyloidogenic nor nonamyloidogenic forms of IAPP cause cell death by
necrosis. It should be emphasized that because only approximately 50%
of the cells are transfected, the number of cells undergoing
hIAPP-induced apoptosis approaches 60% of the transfected cells that
express hIAPP.
|
compares cell number on the
y axis with the relative fluorescence value, ie, annexin-V
binding, on the x axis with apoptotic cells defined by bar
M1. In Figure 2
there is a time-dependent increase in the number of
apoptotic cells in pMT2-hIAPP transfectants (Figure 2A)
but
not pMT2-hIAPPanti (Figure 2B)
. Apoptosis in
both hIAPP (Figure 2A)
and control (Figure 2B)
transfectants is
negligible at 24 and 48 hours transfection, but in hIAPP-expressing
cells, apoptosis increased to two- to threefold greater than the
control at 72 and 96 hours. These data correlate with the previously
described time course for amyloid generation after transfection of
COS-1 cells with the pMT2-hIAPP gene in which amyloid was
only detectable by electron microscopy at 72 to 96 hours12
and follows the intracellular expression and accumulation of hIAPP
(discussed below).
|
. Both the negative
(pMT2-hIAPPanti) and positive
(pMT2-hIAPPsense) controls in these
experiments behaved as previously described in Figure 1
in which hIAPP
expression results in high levels of apoptosis (25 to 30%) and control
transfectants have only background levels (10 to 12%).
Immunohistochemical detection of hIAPP showed only low level hIAPP
expression in a much smaller population of cells in COS-1 cells
co-transfected with both pMT2-hIAPPanti and
pMT2-hIAPPsense vectors than with the
pMT2-hIAPPsense vector alone (Figure 4D
, discussed below). Thus, hIAPP
expression is blocked by its antisense mRNA, thereby protecting the
cells from hIAPP-induced apoptosis. These data indicate that the
apoptotic events induced in the hIAPP transfected cells is dependent
upon the specific expression of the hIAPP protein.
|
|
). Conversely, 32% of
pMT2-hIAPP transfectants are apoptotic. These data indicate
that only the amyloidogenic form of hIAPP will induce apoptosis,
suggesting that a unique structure within this molecule is responsible
for triggering the apoptosis signaling pathway. To verify that the analysis for apoptosis using annexin-V labeling of translocated PS residues in hIAPP-transfected COS cells is consistent with other markers characteristic of apoptosis, we also used a technique in which the free ends of fragmented DNA are labeled with fluorescently tagged nucleotides (TUNEL) and analyzed by FACS. Although TUNEL does not strictly differentiate apoptotic cells from necrotic cells, this assay verifies that cells are undergoing DNA fragmentation, which is characteristic of apoptosis. Annexin-V labeling and TUNEL were performed in parallel on cells from the same transfection cultures and gave remarkably similar results (data not shown). The percentage of cells that were apoptotic in control cultures varied by less than 1% between TUNEL and annexin-V staining (9 to 10%), and in the hIAPP transfectants, the percentage of apoptotic cells averaged 31% by annexin-V staining and 29% by TUNEL at 96 hours. Because we analyzed necrosis by 7-AAD staining in parallel samples and showed that hIAPP expression does not increase background necrosis, we are confident that TUNEL is a reliable marker of apoptosis in these experiments. These data confirm that the expression of amyloidogenic hIAPP in transfected COS cells induces these cells to undergo programmed cell death by classic apoptotic mechanisms that include both translocation of PS residues and fragmentation of nuclear DNA.16,17
We wanted to identify the cellular location of the expressed hIAPP in
transfected COS cells to correlate cellular location with the onset of
apoptotic events. Intracellular expression of hIAPP was confirmed using
immunohistochemical labeling with antiamylin antiserum in the presence
of the mild detergent saponin. As additional control, cells from the
same transfections were also analyzed for apoptosis and necrosis by
annexin-V and 7-AAD labeling and FACS to correlate IAPP expression with
cell death. Figure 4, A and B
shows pMT2-hIAPP-transfected
cells in both phase contrast and immunofluorescent microscopy. Figure 4A
shows the intracellular accumulation of amylin immunoreactivity in
distinct granular structures in the immediate perinuclear region of
pMT2-hIAPP transfected cells (green fluorescence) and that
approximately one-half of the cells are labeled (compare with Figure 4B
). Conversely, cells that have been transfected with the
pMT2-hIAPPanti plasmid (Figure 4C)
or
mock-transfected controls (data not shown) exhibit no amylin
immunoreactivity. When cells were co-transfected with
pMT2-hIAPP and pMT2-hIAPPanti
plasmids, the number of cells exhibiting immunoreactive amylin was
greatly reduced, and the level of intracellular hIAPP expression was
decreased (Figure 4D)
, demonstrating that co-transfection of the hIAPP
antisense expression vector reduced hIAPP expression significantly.
Intracellular hIAPP immunoreactivity could be detected as early as 24
hours transfection (data not shown) and is optimal at 48 hours. By 72
hours the very high immunoreactive signal and cellular destruction by
apoptosis limits the use of immunohistochemical labeling and LSCM in
these studies.
We also transfected COS-1 cells with the two nonamyloidogenic variants
of IAPP mentioned previously, rat IAPP (pMT2-rIAPP)
and a mutant form of human IAPP
(pMT2-hIAPPmut), and analyzed them for
expression of intracellular amylin. Both rat IAPP (Figure 4E)
and
mutant hIAPP (Figure 4F)
show a pattern of immunoreactivity that is
comparable with that of hIAPP (Figure 4A)
with localization and
clustering in the perinuclear region, whereas the antisense control
exhibits no amylin immunoreactivity (Figure 4C)
. It is very interesting
that although both the rat and mutant forms of IAPP are being
synthesized and accumulate in subcellular organelles at levels
comparable with the amyloidogenic hIAPP, they do not induce apoptosis
as indicated by annexin-V labeling (Figure 3B
, discussed above). This
data indicate that the amyloidogenic region (amino acids 20–29) within
the hIAPP protein is unique, making it capable of triggering
the apoptotic signaling pathways, whereas the nonamyloidogenic forms do
not despite the high levels of accumulation within subcellular
organelles. Further evidence that the mechanism of cell death proceeds
by apoptosis was indicated by the finding of cells exhibiting
immunoreactive apoptotic bodies (Figure 4G)
and membrane blebbing
(Figure 4H)
. These structures are hallmark morphological features of
apoptosis that represent different stages of the organized breakdown of
cells, whereby the cellular contents are packaged in membrane-bound
apoptotic bodies that are phagocytosed and destroyed by neighboring
cells without eliciting an immune reaction.19,20
To determine that the expressed IAPP is intracellular and not a
secreted extracellular form, we performed immunohistochemical labeling
of control pMT2-hIAPPanti- versus
pMT2-hIAPP-transfected COS cells in the presence and absence
of the detergent saponin. Cells were fixed so that insoluble,
extracellular IAPP, if present, would remain in place. In Figure 5
there is no evidence of extracellular
immunoreactivity in nonpermeabilized pMT2-hIAPP- or
pMT2-hIAPPanti-transfected cells (Figure 5, A and C
, respectively) or intracellular immunoreactivity in permeabilized
control cells (Figure 5D)
. However, in permeabilized
pMT2-hIAPP transfectants there is a high concentration of
localized intracellular immunoreactivity in the immediate perinuclear
region (Figure 5B)
clearly demonstrating the intracellular accumulation
of hIAPP. Combined with the previous studies demonstrating that
apoptosis correlates with hIAPP expression, these data demonstrate that
apoptosis is initiated by the intracellular accumulation of hIAPP as
opposed to its secretion and extracellular aggregation. Consistent with
this interpretation, we had earlier shown that the secreted levels of
hIAPP were insufficient to kill COS-1 cells.12
To test this
experimentally, we incubated COS-1 cells that had been transfected with
the control pMT2 and pMT2-hIAPP vector for 96
hours in either normal medium or conditioned medium from COS-1 cells
that had been transfected with the pMT2-hIAPP expression
vector for 96 hours. As shown in Figure 5E
, incubating COS-1 cells
transfected with pMT2 vector in conditioned medium did not
induce apoptosis. Similarly, the conditioned medium did not increase
the amount of apoptosis in cells that had been transfected with the
pMT2-hIAPP plasmid (Figure 5E)
.
|
, which demonstrates strong amylin
immunoreactivity (Figure 6, A and C
, green) in combination with either
annexin-V labeling (Figure 6A
, red) or intense TUNEL staining of
fragmented nuclear DNA (Figure 6C
, red) in
pMT2-hIAPP-transfected COS-1 cells. Conversely, cells that
have been transfected with the pMT2-hIAPPanti
plasmid have only very low levels of background staining with either
annexin-V or TUNEL (Figure 6, B and D
, respectively). These data link
the accumulation of intracellular amylin with distinct markers
characteristic of apoptosis in the same cell.
|
demonstrates colocalization of amylin (Figure 7C
, green) with the Golgi
marker p58 (Figure 7A
, red) in pMT2-hIAPP-transfected cells.
Intracellular amylin is not detected in either control or
nontransfected cells, although p58 immunoreactivity is present (not
shown). Likewise in Figure 7E
there is clear co-localization (yellow)
of amylin immunoreactivity (Figure 7F
, green) with the ER-Golgi marker
Mann II (Figure 7D
, red). Amylin immunoreactivity is not present in
nontransfected cells, an example of which can be seen just above dual
stained cells in Figure 7, D and E
. Amylin also co-localizes (Figure 7H
, yellow) with the endoplasmic reticulum marker, PDI (Figure 7G
, red)
and amylin (Figure 7I
, green). These data confirm that the expression
of hIAPP in pMT2-hIAPP-transfected cells results in the
accumulation of amylin immunoreactivity within the ER and Golgi
apparatus. Because the immunolabeling was performed on cells 48 hours
after being transfected with the hIAPP expression vector, the
subcellular accumulation of amylin within the ER and Golgi apparatus
precedes the onset of apoptosis (72 to 96 hours).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Significant amounts of hIAPP immunoreactivity were detected in both the
ER and Golgi apparatus by 48 hours after transfection of COS-1 cells
with the pMT2-hIAPP gene (Figure 7)
, although electron
microscopy revealed that the cells exhibited intact plasma membranes
and intracellular organelle structures with normal chromatin patterns
and lacked evidence for amyloid formation.12
Conversely, at
96 hours the hIAPP-transfected cells exhibited intense intracellular
hIAPP immunogold labeling associated with intracellular amyloid, highly
condensed chromatin and disrupted plasma membranes, all characteristic
of apoptotic cells.12
In the current study, significant
apoptosis was induced at 72 to 96 hours but not at earlier time points
(Figures 1 to 3)
, indicating that amyloid formation is correlated with
and precedes the induction of apoptosis. Although the expression and
accumulation of hIAPPmut and rIAPP within the ER is
comparable with that of the amyloidogenic hIAPP (Figure 4)
,12
neither of these proteins is cytotoxic as indicated
by annexin-V and 7-AAD labeling (Figure 3)
. In addition,
hIAPPmut reverts the amyloidogenic domain of hIAPP between
amino acids 20 and 294
(Figure 8
, GAIL), to the nonamyloidogenic domain
of the rIAPP peptide while preserving the two other amino acid
differences between these two species. Thus the amyloidogenic domain of
hIAPP must be responsible for activation of the apoptotic signaling
events. Our data provide evidence that the accumulation of amyloid
within the ER and/or Golgi apparatus provides a specific stimulus for
the induction of apoptosis. Whereas further work will be required to
ascertain the nature of this signal, a likely starting point might well
be the various ER quality control mechanisms that insure the orderly
trafficking of correctly folded polypeptides. Such mechanisms include
the unfolded protein response21,22
and ER overload response
pathways.23,24,25
Both of these pathways provide for the
induction of ER chaperones, including Bip/Grp78, Grp94, calnexin,
calreticulin, peptidyl prolyl isomerase, and protein disulfide
isomerase that are involved in protein folding.26
In
addition, the degradation of malfolded proteins by the
ubiquitin-proteosome pathway is an essential component of the ER
quality control systems, and the regulation of this pathway also
involves molecular chaperones.27,28
Some preliminary
evidence for the involvement of these quality control pathways in the
regulation of amyloid already exists. For example, in the studies of
human insulinomas, which provided the first evidence for intracellular
IAPP amyloid formation, ubiquitin immunoreactivity was observed as
punctate intracellular labeling in association with intracellular as
well as extracellular amyloid deposits.29
In addition, we
have demonstrated previously that small heat shock proteins inhibit the
in vitro formation of amyloid by the Alzheimer
Aß1–42 polypeptide, suggesting that chaperones might be
involved in inhibiting the formation of certain peptide conformations
that lead to amyloid formation.30
While the exact mechanism of ß-cell death in NIDDM is unknown and the
role of amyloid in ß-cell death is controversial,3,9
the
present studies suggest that intracellular amyloid-induced apoptosis
may contribute to the loss of ß-cell mass in NIDDM. Recent evidence
that strongly supports a direct role of IAPP in the genesis of NIDDM is
the finding that a missense mutation of the IAPP gene
(S20G) is associated with early onset (
35 years)
NIDDM in a high proportion of individuals carrying the
mutation.10
Thus NIDDM bears similarities to Alzheimer
disease in which rare mutations of the APP gene are
associated with early onset disease and increased amyloid deposition in
the brains of afflicted individuals.31-33
A number of investigators have examined hIAPP expression in transgenic mice and have observed conflicting results. Janson et al34 reported that transgenic mice homozygous for the hIAPP gene (RHF-hIAPP+/+) express high levels of hIAPP and spontaneously develop diabetes mellitus. In the RHF-hIAPP+/+ mice amyloid deposits are not observed, but intra- and extra-cellular amorphous aggregates of hIAPP are present, suggesting that structural forms of hIAPP in addition to or other than amyloid may be cytotoxic.34 Treatment of the hemizygous RHF-hIAPP+/- mice with growth hormone and dexamethasone to induce insulin resistance for 4 weeks did cause islet amyloidosis, including intra- and extra-cellular deposits that preceded ß-cell dysfunction;6 however, the untreated RHF-hIAPP+/- mice did not spontaneously develop any signs of pancreatic dysfunction or glucose intolerance.6,34 D'Alessio et al35 reported initially that their transgenic mice lacked islet amyloid deposits but had higher levels of islet insulin and proinsulin.36 Interestingly, when these animals were moved to another institution and subjected to a change in diet, extensive islet amyloid deposits were observed in male transgenic mice that were older than 13 months but were only observed in 11% of the female mice.13 In these animals, half of those that had islet amyloid deposits were hyperglycemic with plasma glucose levels >11 mmol/L. In younger animals (6 to 9 months), islet amyloid deposits were rarely observed; however, their occurrence was associated with severe hyperglycemia with plasma glucose levels >22 mmol/L.13 Two other studies of transgenic animals expressing hIAPP were largely negative with failure to demonstrate islet amyloid or effect changes in circulating glucose levels.37,38 In one of these studies, some amyloid in secretory granules was observed.38 In the other study, when the islets were cultured in vitro in relatively high concentrations of glucose (11 to 22 mmol/L), amyloid was observed to develop within 7 days.37 More recently, Soeller et al,39 crossed RHF-hIAPP+/- with the insulin-resistant agouti viable yellow (Avy/a) mice, and the resultant hybrids developed a slow onset form of diabetes that was associated with the accumulation of islet amyloid and a reduction of ß-cell mass. Taken together, these studies support the hypothesis that islet amyloid plays a role in ß-cell dysfunction that leads to hyperglycemia; however, other factors, including the absolute levels of IAPP expression, the specific conformation of the IAPP aggregates, diet, and insulin resistance appear to play important conditional roles in the development of these abnormalities.
Because the cell destruction that occurs in NIDDM appears to be confined solely to the ß cells, the concept that the cells are killed through an intracellular apoptotic mechanism is attractive, as apoptosis would be expected to minimize inflammatory effects that could damage the surrounding tissue. Whereas much work remains to unravel the mysteries of hIAPP-induced cell death, this COS cell model will be useful in establishing the intracellular signaling pathway that leads to apoptosis and provide insights about the mechanisms by which intracellular amyloid exerts its cytotoxic action.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by grants from the National Institutes of Health (AG08031 and AG14522, NLE), a gift from the Quade Amyloidosis Research Fund (NLE), and Mayo Foundation Research Funds.
Accepted for publication November 30, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  M. Apostolidou, S. A. Jayasinghe, and R. Langen Structure of {alpha}-Helical Membrane-bound Human Islet Amyloid Polypeptide and Its Implications for Membrane-mediated Misfolding J. Biol. Chem., June 20, 2008; 283(25): 17205 - 17210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. M. Engel, L. Khemtemourian, C. C. Kleijer, H. J. D. Meeldijk, J. Jacobs, A. J. Verkleij, B. de Kruijff, J. A. Killian, and J. W. M. Hoppener Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane PNAS, April 22, 2008; 105(16): 6033 - 6038. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Marzban, C. J. Rhodes, D. F. Steiner, L. Haataja, P. A. Halban, and C. B. Verchere Impaired NH2-Terminal Processing of Human Proislet Amyloid Polypeptide by the Prohormone Convertase PC2 Leads to Amyloid Formation and Cell Death. Diabetes, August 1, 2006; 55(8): 2192 - 2201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. Paulsson and G. T. Westermark Aberrant Processing of Human Proislet Amyloid Polypeptide Results in Increased Amyloid Formation Diabetes, July 1, 2005; 54(7): 2117 - 2125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Butler, J. Janson, W. C. Soeller, and P. C. Butler Increased {beta}-Cell Apoptosis Prevents Adaptive Increase in {beta}-Cell Mass in Mouse Model of Type 2 Diabetes: Evidence for Role of Islet Amyloid Formation Rather Than Direct Action of Amyloid Diabetes, September 1, 2003; 52(9): 2304 - 2314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sakagashira, H. J. Hiddinga, K. Tateishi, T. Sanke, T. Hanabusa, K. Nanjo, and N. L. Eberhardt S20G Mutant Amylin Exhibits Increased in Vitro Amyloidogenicity and Increased Intracellular Cytotoxicity Compared to Wild-Type Amylin Am. J. Pathol., December 1, 2000; 157(6): 2101 - 2109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W.M. Hoppener, B. Ahren, and C. J.M. Lips Islet Amyloid and Type 2 Diabetes Mellitus N. Engl. J. Med., August 10, 2000; 343(6): 411 - 419. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
