# Amyloid Deposition in Transplanted Human Pancreatic Islets: A Conceivable Cause of Their Long-Term Failure

Authors: Arne Andersson; Sara Bohman; L. A. Håkan Borg; Johan F. Paulsson; Sebastian W. Schultz; Gunilla T. Westermark; Per Westermark
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Following the encouraging report of the Edmonton group, there was a rejuvenation of the islet transplantation field. After that, more pessimistic views spread when long-term results of the clinical outcome were published. A progressive loss of theβ-cell function meant that almost all patients were back on insulin therapy after 5 years. More than 10 years ago, we demonstrated that amyloid deposits rapidly formed in human islets and in mouse islets transgenic for human IAPP when grafted into nude mice. It is, therefore, conceivable to consider amyloid formation as one potential candidate for the long-term failure. The present paper reviews attempts in our laboratories to elucidate the dynamics of and mechanisms behind the formation of amyloid in transplanted islets with special emphasis on the impact of long-term hyperglycemia.


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## Body

## 1. Introduction

The  discovery  of insulin in the early 1920s greatly improved the
prognosis for type 1 diabetes patients and by such means patients with diabetes
could survive a previously fatal disease. Because of the substantial
improvements in insulin therapy, most patients nowadays can handle their
treatment themselves and risks for the crippling long-term complications have
become extensively reduced. This, however, requires strict blood glucose
control and life style restrictions. These latter insufficiencies of the
present treatment together with the fact that a subgroup of patients is still
disturbed by frequent hypoglycaemic attacks have meant that there is
considerable interest in pancreatic islet transplantation. For long replacement of the
destroyedβ-cells in type 1 diabetes with new β-cells, this has attracted much attention. Paul Lacy’s pioneering work  with his collagenase-based method for rat
islet isolation paved the way for islet transplantation experiments. Clinical
trials were carried out in the 80s and 90s but only about 10% of islet
recipients achieved normoglycemia without insulin therapy. However, in their
report in the year 2000 James Shapiro et al. reported a handful of diabetes patients all
of whom became normoglycemic after two or three intraportal implantations of noncultured human islets [1].  Given a steroid-free
immunosuppression, these patients remained off insulin for at least one year.
In an international trial of this so-called Edmonton protocol, 36 subjects with
type 1 diabetes underwent this type of treatment at nine international sites [2]. While 16 of them (44%) were
insulin free after one year only 5 (14%) remained so after one more year. It
was concluded that there was a progressive loss of islet function in most
subjects, who had all become insulin independent initially.For long, it has been postulated
that long-term hyperglycemia might influenceβ-cell function in a negative way.
Numerous in vitro and in vivo studies have indicated that so is the case but
the molecular mechanisms are still unclear. We, therefore, found it conceivable
to consider amyloid formation as one potential candidate. This paper reviews
attempts in our laboratory to elucidate the fate of transplanted human islets
with a special view on their morphology and function and especially so under
influence of prolonged hyperglycemic stress.


## 2. Islet Amyloid Polypeptide and Islet Amyloid

Although islet amyloid was discovered already in 1901 [3, 4], its impact in the pathogenesis of type 2
diabetes has been questioned for a long period of time. However, there are
several lines of evidence for the importance of the amyloid formation for the β-cell
lesion in type 2 diabetes (for reviews, see [5, 6]). The exact mechanisms are still not very well
understood but aggregated IAPP is toxic to β-cells
[7, 8].IAPP was discovered by purification and
analysis of amyloid, first from a human insulinoma [9, 10] and later from islets of Langerhans [11, 12]. The same peptide was found to form amyloid in
apes [13, 14] and cats [11, 15]. Human IAPP is a 37-amino acid residue peptide, expressed as a
prepromolecule. After removal of the signal peptide, the 67-amino acid
propeptide is further processed at two double basic residues by the prohormone
convertases PC2 and PC1/3 which remove two short peptides N- and C-terminally
(Figure 1). The remaining peptide is C-terminally amidated and there is a
disulfide bridge between residues 2 and 7.Figure 1
Processing at double basic amino acid residues
of proinsulin and proIAPP by the prohormone convertases PC 1/3 and PC2.IAPP is expressed byβ-cells and is stored and
released together with insulin. IAPP is very aggregation-prone in vitro and
rapidly forms amyloid-like fibrils. This does not normally happen in vivo,
where there must be mechanisms which hinder this. Binding to insulin may be
such a mechanism [16, 17]. However, it is not understood why IAPP
aggregates into amyloid in conjunction with type 2 diabetes. Experiments with
transgenic mice, overexpressing human IAPP, clearly indicate that an increased
production of IAPP is not the single explanation but that other factors must
contribute.


### 2.1. Transgenic Animals Overexpressing Human IAPP

Mice and rats do not develop islet amyloid, depending on differences in
the IAPP sequence. Proline residues in the amyloid-forming core of IAPP abolish
the fibril formation in both species [18]. Several groups have, therefore, created
transgenic mouse lines expressing human IAPP under regulation of an insulin
promoter. In spite of overexpression of human IAPP, islet amyloid generally
does not develop. However, amyloid does appear when such animals are fed a diet
high in fat [19, 20] or are crossed with ob/ob [21] or agouti [22] mice. We are working with a mouse line,
overexpressing human IAPP behind rat insulin 1 promoter but devoid of mouse
IAPP. Animals of this strain do not spontaneously develop islet amyloid at any
age but in male mice, when fed a diet with high content of fat, amyloid
deposits occur at an age of 11 months [20]. The amyloid is mainly found extracellularly
but intracellular deposits do occur [23].


### 2.2. Amyloid Development in Cultured Human and Transgenic Mouse Islets

Interestingly, islets isolated from our transgenic mouse strain develop
amyloid deposits rapidly when cultured in vitro [24]. A similar experience was obtained with
another human IAPP transgenic mouse strain [25]. Furthermore, in contrast to what is found in
islets in type 2 diabetes, where the amyloid is extracellular [26], intracellular aggregation of IAPP initially
takes place in cultured human islets [27]. The exact compartmental position has been
difficult to determine but is probably the endoplasmic reticulum or Golgi
apparatus [28].


### 2.3. Aberrant Processing and Amyloid Formation

There is evidence that the intracellular amyloid contains
proIAPP and a defect processing of this
precursor to mature IAPP may play a role in the pathogenesis of amyloid
formation [23]. β-Cell stress that occurs in the initial phase of type 2 diabetes results
in a disproportional secretion of unprocessed or partially processed proinsulin
(des 32-33 C-peptide-A-chain fragment) [29]. This shift can mirror an increase
in granule turnover, or, perhaps more interestingly is a sign of incomplete processing due to
convertase deficiency. Also the prohormone convertases PC 1/3 and PC2
themselves must undergo cleavage to become active, and therefore, aberrant
activation of convertases can lead to incomplete processing. Proinsulin is
processed by PC1/3 at the B-chain/C-peptide junction followed by PC2 cleavage
at the C-peptide/A-chain junction while PC1/3 and PC2 processing of proIAPP
results in the removal of the C-terminal and N-terminal flanking peptides, respectively [30]. In vitro, IAPP is one of the more
aggregation-prone amyloid peptides known and insulin has been shown to exert a
concentration-dependent inhibitory effect on IAPP fibril formation at neutral
pH. We have produced human IAPP and partially processed proIAPP, lacking the
C-terminal flanking peptide (NIAPP) with recombinant technology [31]. In the following, previously
unpublished study, IAPP or NIAPP (20 μM) and insulin (40 μM) were dissolved in 25 mM phosphate buffer with 50 mM glycine at pH 7 and
pH 5.2. Aliquots were analyzed
for the presence of amyloid fibrils after Congo red staining. We conferred our
earlier findings that addition of insulin to IAPP delays fibril formation at pH
7.0 and this was also true for NIAPP. However, at pH 5.2 the fibril formation
was triggered for both IAPP and NIAPP. Semiquantitative analysis of amyloid
amount, based on Congo red staining and electron microscopical analyses, showed
that NIAPP was more prone to form amyloid-like fibrils than mature IAPP. Since
both NIAPP and the des 32-33
C-peptide-A-chain proinsulin derivative are expected to appear in the secretory
granules as a consequence of reduced PC2 processing, we also expressed des 32-33
C-peptide-A-chain proinsulin. NIAPP and 32-33
C-peptide-A-chain proinsulin were solubilized as described above and mixed 1:1
and 1:4. It was then shown that addition of 32-33
C-peptide-A-chain proinsulin to NIAPP promotes fibril formation. These
previously unpublished results show that the intragranular composition of
prohormones and processing metabolites is of importance and changes of the
equilibrium can be a factor that causes IAPP to aggregate. Transfection of human proIAPP to cell lines
missing one or both of the processing enzymes has supported this conclusion
since the aberrant processing resulted in increased amyloid formation [32, 33].


## 2.1. Transgenic Animals Overexpressing Human IAPP

Mice and rats do not develop islet amyloid, depending on differences in
the IAPP sequence. Proline residues in the amyloid-forming core of IAPP abolish
the fibril formation in both species [18]. Several groups have, therefore, created
transgenic mouse lines expressing human IAPP under regulation of an insulin
promoter. In spite of overexpression of human IAPP, islet amyloid generally
does not develop. However, amyloid does appear when such animals are fed a diet
high in fat [19, 20] or are crossed with ob/ob [21] or agouti [22] mice. We are working with a mouse line,
overexpressing human IAPP behind rat insulin 1 promoter but devoid of mouse
IAPP. Animals of this strain do not spontaneously develop islet amyloid at any
age but in male mice, when fed a diet with high content of fat, amyloid
deposits occur at an age of 11 months [20]. The amyloid is mainly found extracellularly
but intracellular deposits do occur [23].


## 2.2. Amyloid Development in Cultured Human and Transgenic Mouse Islets

Interestingly, islets isolated from our transgenic mouse strain develop
amyloid deposits rapidly when cultured in vitro [24]. A similar experience was obtained with
another human IAPP transgenic mouse strain [25]. Furthermore, in contrast to what is found in
islets in type 2 diabetes, where the amyloid is extracellular [26], intracellular aggregation of IAPP initially
takes place in cultured human islets [27]. The exact compartmental position has been
difficult to determine but is probably the endoplasmic reticulum or Golgi
apparatus [28].


## 2.3. Aberrant Processing and Amyloid Formation

There is evidence that the intracellular amyloid contains
proIAPP and a defect processing of this
precursor to mature IAPP may play a role in the pathogenesis of amyloid
formation [23]. β-Cell stress that occurs in the initial phase of type 2 diabetes results
in a disproportional secretion of unprocessed or partially processed proinsulin
(des 32-33 C-peptide-A-chain fragment) [29]. This shift can mirror an increase
in granule turnover, or, perhaps more interestingly is a sign of incomplete processing due to
convertase deficiency. Also the prohormone convertases PC 1/3 and PC2
themselves must undergo cleavage to become active, and therefore, aberrant
activation of convertases can lead to incomplete processing. Proinsulin is
processed by PC1/3 at the B-chain/C-peptide junction followed by PC2 cleavage
at the C-peptide/A-chain junction while PC1/3 and PC2 processing of proIAPP
results in the removal of the C-terminal and N-terminal flanking peptides, respectively [30]. In vitro, IAPP is one of the more
aggregation-prone amyloid peptides known and insulin has been shown to exert a
concentration-dependent inhibitory effect on IAPP fibril formation at neutral
pH. We have produced human IAPP and partially processed proIAPP, lacking the
C-terminal flanking peptide (NIAPP) with recombinant technology [31]. In the following, previously
unpublished study, IAPP or NIAPP (20 μM) and insulin (40 μM) were dissolved in 25 mM phosphate buffer with 50 mM glycine at pH 7 and
pH 5.2. Aliquots were analyzed
for the presence of amyloid fibrils after Congo red staining. We conferred our
earlier findings that addition of insulin to IAPP delays fibril formation at pH
7.0 and this was also true for NIAPP. However, at pH 5.2 the fibril formation
was triggered for both IAPP and NIAPP. Semiquantitative analysis of amyloid
amount, based on Congo red staining and electron microscopical analyses, showed
that NIAPP was more prone to form amyloid-like fibrils than mature IAPP. Since
both NIAPP and the des 32-33
C-peptide-A-chain proinsulin derivative are expected to appear in the secretory
granules as a consequence of reduced PC2 processing, we also expressed des 32-33
C-peptide-A-chain proinsulin. NIAPP and 32-33
C-peptide-A-chain proinsulin were solubilized as described above and mixed 1:1
and 1:4. It was then shown that addition of 32-33
C-peptide-A-chain proinsulin to NIAPP promotes fibril formation. These
previously unpublished results show that the intragranular composition of
prohormones and processing metabolites is of importance and changes of the
equilibrium can be a factor that causes IAPP to aggregate. Transfection of human proIAPP to cell lines
missing one or both of the processing enzymes has supported this conclusion
since the aberrant processing resulted in increased amyloid formation [32, 33].


## 3. Influence of Hyperglycemia on Grafted Human Islets

### 3.1. Electron Microscopical Appearance

In general, the ultrastructure of human islets grafted into
normoglycemic mice remains normal 4 weeks after implantation [34]. The β-cells are in great majority.
A 4-week hyperglycaemic period induces well-known signs of β-cell hyperactivity
such as marked degranulation and also signs of the development of an abundant
rough endoplasmic reticulum (Figure 2). We also observed signs of glycogen
particles accumulating in the β-cells. These glycogen depositions disappear when
transferring the islets to a normoglycemic milieu by curing the recipient by
means of implantation of a second islet graft. Interestingly, the mitochondria
residing in the hyperglycaemic, noncured recipients are often swollen (Figure 2).Figure 2
Electron micrograph of human islets transplanted under the kidney
capsule of an alloxan-diabetic athymic nude mouse four weeks after
implantation. Note the extensive degranulation, the abundant endoplasmic
reticulum (star), glycogen particles (black arrows), and swollen mitochondria
(white arrow).Taken together, these previous
ultrastructural investigations show that the transmission electron
microscopical tool is of utmost importance when elucidating the impact of
different functional loads put on human islets. Obviously, the knowledge on the
classical “hydropic degeneration," later referred to as “ballooning degeneration" described by Weichelbaum
and Stangl, Allen, Toreson, and Lazarus and Volk [35], in reality has become extended by
the findings of the glycogen accumulations described above. Likewise, the very
early reports on hyalinization of the islets of patients with diabetes by Opie,
in 1901 [3], have formed the platform for extensive studies, both morphological
and biochemical, on the formation of amyloid deposits (described below).


### 3.2. Functional Properties

The ultrastructural findings were corroborated by measurements of the
islet graft insulin content (Figure3). Thus, the high glucose-exposed islet
grafts contained about one tenth of the insulin found in the normoglycemic
control grafts indicating a parallelism between low insulin content and
extensive β-cell degranulation. In graft perfusion experiments, where test
substances were infused via the renal artery and effluents collected from the
ureter and renal vein [36],
we found that a high glucose challenge in the test medium increased the
insulin concentration of the effluent medium in a biphasic mode when the graft
had resided in a normoglycemic recipient not treated with alloxan. Quite in
contrast, islet grafts exposed to a high (more than 20 mM) glucose concentration
in vivo for 4 weeks displayed a blunted insulin secretion. In fact, the
integrated area under the curve, that is, the amount of insulin secreted during
the 30-minute stimulation period, was less than 5% of that observed for the
control, normoglycemic grafts (Figure 3). Interestingly, this extensively
impaired glucose-stimulated insulin secretion was only marginally returned to
normal after a 2-week period of normoglycemia effected by a second intrasplenic
mouse islet graft (Figure 3). This was despite a nearly total reconstitution of
the insulin content of the graft.Figure 3
Total insulin secretion in effluent medium
collected from human islet graft-bearing kidneys during 30-minute perfusion
with 16.7 mM glucose (black bars), and insulin content of renal subcapsular
islet grafts (grey bars). Values are means ± SEM.In further studies of this defective
glucose-induced insulin release of the human islet grafts, we found that also
arginine-stimulated secretion was heavily impaired [37]. Neither impaired glucose
metabolism nor decreased (pro)insulin biosynthesis could explain the
deleterious effects of the diabetic state on human islet graft insulin
secretion. It is tempting to speculate that formation of intracellular amyloid
deposits might be one hitherto neglected reason for this functional impairment.
With our present knowledge, attention should be paid to functional
abnormalities also in IAPP biosynthesis and secretion. One process of
particular interest in this context might be the enzymatic cleavage of pro-IAPP
by the converting enzymes PC 2 and PC 1/3 [38].


## 3.1. Electron Microscopical Appearance

In general, the ultrastructure of human islets grafted into
normoglycemic mice remains normal 4 weeks after implantation [34]. The β-cells are in great majority.
A 4-week hyperglycaemic period induces well-known signs of β-cell hyperactivity
such as marked degranulation and also signs of the development of an abundant
rough endoplasmic reticulum (Figure 2). We also observed signs of glycogen
particles accumulating in the β-cells. These glycogen depositions disappear when
transferring the islets to a normoglycemic milieu by curing the recipient by
means of implantation of a second islet graft. Interestingly, the mitochondria
residing in the hyperglycaemic, noncured recipients are often swollen (Figure 2).Figure 2
Electron micrograph of human islets transplanted under the kidney
capsule of an alloxan-diabetic athymic nude mouse four weeks after
implantation. Note the extensive degranulation, the abundant endoplasmic
reticulum (star), glycogen particles (black arrows), and swollen mitochondria
(white arrow).Taken together, these previous
ultrastructural investigations show that the transmission electron
microscopical tool is of utmost importance when elucidating the impact of
different functional loads put on human islets. Obviously, the knowledge on the
classical “hydropic degeneration," later referred to as “ballooning degeneration" described by Weichelbaum
and Stangl, Allen, Toreson, and Lazarus and Volk [35], in reality has become extended by
the findings of the glycogen accumulations described above. Likewise, the very
early reports on hyalinization of the islets of patients with diabetes by Opie,
in 1901 [3], have formed the platform for extensive studies, both morphological
and biochemical, on the formation of amyloid deposits (described below).


## 3.2. Functional Properties

The ultrastructural findings were corroborated by measurements of the
islet graft insulin content (Figure3). Thus, the high glucose-exposed islet
grafts contained about one tenth of the insulin found in the normoglycemic
control grafts indicating a parallelism between low insulin content and
extensive β-cell degranulation. In graft perfusion experiments, where test
substances were infused via the renal artery and effluents collected from the
ureter and renal vein [36],
we found that a high glucose challenge in the test medium increased the
insulin concentration of the effluent medium in a biphasic mode when the graft
had resided in a normoglycemic recipient not treated with alloxan. Quite in
contrast, islet grafts exposed to a high (more than 20 mM) glucose concentration
in vivo for 4 weeks displayed a blunted insulin secretion. In fact, the
integrated area under the curve, that is, the amount of insulin secreted during
the 30-minute stimulation period, was less than 5% of that observed for the
control, normoglycemic grafts (Figure 3). Interestingly, this extensively
impaired glucose-stimulated insulin secretion was only marginally returned to
normal after a 2-week period of normoglycemia effected by a second intrasplenic
mouse islet graft (Figure 3). This was despite a nearly total reconstitution of
the insulin content of the graft.Figure 3
Total insulin secretion in effluent medium
collected from human islet graft-bearing kidneys during 30-minute perfusion
with 16.7 mM glucose (black bars), and insulin content of renal subcapsular
islet grafts (grey bars). Values are means ± SEM.In further studies of this defective
glucose-induced insulin release of the human islet grafts, we found that also
arginine-stimulated secretion was heavily impaired [37]. Neither impaired glucose
metabolism nor decreased (pro)insulin biosynthesis could explain the
deleterious effects of the diabetic state on human islet graft insulin
secretion. It is tempting to speculate that formation of intracellular amyloid
deposits might be one hitherto neglected reason for this functional impairment.
With our present knowledge, attention should be paid to functional
abnormalities also in IAPP biosynthesis and secretion. One process of
particular interest in this context might be the enzymatic cleavage of pro-IAPP
by the converting enzymes PC 2 and PC 1/3 [38].


## 4. Amyloid Deposits in Transplanted Pancreatic Islets Influence of
Implantation Site, Functional Activity, and Microencapsulation

In our first report on the rapid deposition of amyloid in human islets
transplanted into nude mice, our primary aim was to study the occurrence of IAPP-positive
cells in the grafts [39]. Not surprisingly, comparisons of
adjacent human islet graft sections stained for insulin and IAPP, respectively,
indicated that the antisera stained the same cells. However, while the insulin staining
was fairly even, both strongly and weakly labelled cells occurred after
staining for IAPP. Interestingly, we found a lower percentage of IAPP-positive
cells in the grafts of hyperglycaemic mice, suggesting that the storage of the
substance was decreased after hyperglycemia.By means of Congo red staining, we
found amyloid deposits in human islet transplants in six out of eight normoglycaemic and two
out of four hyperglycaemic recipients. All these islet grafts had resided under
the kidney capsule of the nude mice for no more than two weeks, demonstrating
the rapidity of the process. Thus, no amyloid was found in sections of the
donor pancreata collected before they were processed for islet isolation. The
amyloid deposits were usually multiple and small and located extracellularly
but some faintly stained deposits were also found in the cytoplasm of the islet
cells.Electron microscopical
investigations showed explicitly that IAPP immunoreactivity normally was
confined to the secretory granules of theβ-cells, while α- and δ-cells were
negative. Moreover, as in the light microscopical study, accumulation of
amyloid material, strongly labelled with antisera to IAPP, was found in eight
of the twelve grafted mice (Figures 4(a) and 4(b)). Large amounts of amyloid
fibrils were easily recognized (Figure 4(c)) but sometimes the material also
had a granular appearance.Intra- and extracellular amyloid in an islet
graft implanted under the renal capsule of a nude mouse. (a) In the overview,
it is seen that the amyloid (arrow) is present in the periphery of degranulatedβ-cells (star). (b) At higher magnification, it is obvious that the
amyloid forms a network, presumably due to presence in the endoplasmic
reticulum. (c) At high magnification, the fibrillar ultrastructure of the
amyloid is evident as well as its specific immunolabelling with antibodies
against IAPP, visualized with 10 nm gold particles.
(a)(b)(c)It is worthy of note that in a
comparative study elucidating the amyloid deposition in islets of transgenic
mice expressing hIAPP and in human islets implanted into nude mice, we found
considerable differences
[27]. Thus, in human islets amyloid was
mainly formed intracellularly (Figure 4), whilst in islets from transgenic mice
the amyloid was exclusively deposited extracellularly. Later studies have
shown, however, that also in these animals the first amyloid occurs within β-cells [38].Descriptions of amyloid formation in
grafted islets in this paper have all referred to studies using the subcapsular
renal space as implantation site. Since essentially all clinical islet
transplantations are
performed by intraportal infusion, we were interested in investigating
intraportally grafted islets as well. Again, nude mice were used as recipients
and indeed amyloid exhibiting affinity for Congo red was found in 8 of 9
islet-containing livers (a total of 10 mice were implanted with human islets) [28]. Both quantitatively and
qualitatively, the formation of amyloid seemed to occur to the same extent and
similarly to that seen in the subcapsularly grafted islets. Separate studies of
intrasplenic islet grafts showed that also such islets contained amyloid with
the same appearance as in the intraportally implanted human islets.While we were unable to demonstrate
an effect of hyperglycemia on the amount of amyloid formed in our first study
when using both normoglycemic and alloxan-diabetic recipients long-term (14 d)
culture of the human islets prior to transplantation seemed to considerably
enhance the amyloid formation [28]. At least this was the case in
specimens observed for a short-time period—the grafts were evaluated already after 2 weeks.
Taken together with results from studies of grafts kept under the kidney
capsule for half a year [28] where rather large extracellular
deposits were found, it appears that the first amyloid is formed
intracellularly and that amyloid at a later stage acts as a nidus for further
extracellular deposition. For some reason, however, the process halts and
therefore the heavy amyloid deposition as seen in the islets of type 2 diabetes
patients never develops.
The reasons for this are still unknown but obviously the present experimental
model offers unique opportunities for such studies.One circumstance that might explain
the rapid deposition of amyloid in the grafted islets is their fairly low
vascular density as compared with the endogenous islets in the pancreas [40]. Such a relative lack of blood
vessels providing for an efficient export of the secretory products thus might
facilitate the accumulation of IAPP and formation of amyloid. The ultimate test
of that hypothesis would be to look for the presence of amyloid deposits in
microencapsulated islets, which exemplify a totally nonvascularized islet
graft. For that purpose, we encapsulated both human islets and hIAPP transgenic
mouse islets in a high-guluronic alginate solution [41]. These capsules were subsequently
transplanted into the renal subcapsular space of normoglycemic nude mice [42]. Indeed, preliminary results
suggest that encapsulated human islet grafts that were retrieved one month
after implantation contained considerable amounts of amyloid (Figure 5). Obviously,
under these specific conditions amyloid deposits develop, thus demonstrating
that a sustained blood supply is not a prerequisite for their formation. It
also seems feasible to use the microencapsulated islets as a tool for more
detailed studies of the amyloid formation process under forced circumstances.Figure 5
Polarized light microscopic image of a Congo
red stained microencapsulated human islet residing in the renal subcapsular
space of an athymic nude mouse for four weeks. The black arrow points out
amyloid in and outside a normal islet cell, whereas the white arrow indicates
amyloid in the central necrotic part of the islet. Surrounding the islet is the
alginate capsule (AC), and in the lower part of the image the renal capsule
(RC).


## 5. Clinical Implications

At present, reports on the pathology of clinically grafted islets are
very scarce and to our knowledge amyloid has not been looked for specifically
except for our recent study [43]. There are methodological difficulties, one of
which might be the fairly long ischemic periods before liver tissue can be
harvested. Nevertheless, studies aiming at the localization and
characterization of the implanted islets are highly warranted. Since the
identification of the amyloid material is often laborious, consultations with
groups experienced in this field of research might be desirable. During the
final preparation of this manuscript, we published data indeed demonstrating
widespread amyloid deposition in clinically transplanted human islets [43]. A patient with type 1 diabetes for
more than 35 years died in a myocardial infarction 5 years after the first of
three intraportal islet infusions. In almost every second of a total of 89
islets found in the liver tissue blocks, amyloid deposits, most of  them being
extracellular, were identified. Immunoelectron microscopy demonstrated amyloid
fibrils that were positive for antibodies against IAPP. Indeed, these findings
highly strengthen the validity of our hypothesis.


## 6. Future Perspectives

Long-term results with clinical islet transplantation are fairly
discouraging. There is evidence to suggest that this is caused by a progressive
loss of the graftedβ-cells. Knowledge on the nature of that process is,
however, meagre. Therefore, the importance of performing necropsies of as many
as possible of deceased patients with islet grafts, functioning or
nonfunctioning, cannot be enough underlined. Pathologists, experienced in
different aspects of islet pathology, including islet amyloidosis, should be
consulted when judging the harvested material. By such means, further insights
on the nature of the destructive process(es) should be gained.As regards, the pathogenic
mechanisms of islet amyloidosis, islet transplantation models might offer
unique possibilities to study them in more detail. We have very much focussed
on the first intracellular IAPP aggregation and the role of proIAPP and proIAPP
intermediates in that process. It remains to be established that under
circumstances when concentrations of such molecules are high, there is an
enhanced amyloid formation in vivo.In the Edmonton protocol,
preparative islet culture was not used perhaps because such manoeuvres might
decrease the viability of the isolated islets. Although that view is
controversial, it cannot be ruled out that amyloid develops during culture of
human islets or mouse islets transgenic for hIAPP. Indeed, there is some
evidence in support of that view [28, 44]. However, it has to be proven that
such pretransplant deposits indeed stimulate a further and more extensive
formation of amyloid in the islets once they have become transplanted.Finally, it is still an open
question as to whether enhanced insulin production, as under hyperglycemic conditions,
promotes amyloid growth in the transplanted islets. A general suppression ofβ-cell function by means of insulin treatment or, at least under experimental
conditions, drugs like somatostatin and its analogs or diazoxide might be of
value to test. In this context, other types of medical intervention against
IAPP aggregation should be of interest as well. One such substance is
eprodisate, which recently was shown to slow the decline of renal function in
patients with AA amyloidosis [45].



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*Source: 562985-2009-03-05.xml*