# Transthyretin and Amyloid in the Islets of Langerhans in Type-2 Diabetes

Authors: Gunilla T. Westermark; Per Westermark
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Transthyretin (TTR) is a major amyloid fibril protein in certain systemic forms of amyloidosis. It is a plasma protein, mainly synthesized by the liver but expression occurs also at certain minor locations, including the endocrine cells in the islets of Langerhans. With the use of immunohistochemistry and in situ hybridization, we have studied the distribution of transthyretin-containing cells in islets of Langerhans in type-2 diabetic and nondiabetic individuals. TTR expression was particularly seen in alpha (glucagon) cells. Islets from type-2 diabetic patients had proportionally more transthyretin-reactive islet cells, including beta cells. A weak transthyretin immunoreaction in IAPP-derived amyloid occurred in some specimens. In seeding experiments in vitro, we found that TTR fibrils did not seed IAPP while IAPP fibrils seeded TTR. It is suggested that islet expression of transthyretin may be altered in type-2 diabetes.


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## Body

## 1. Introduction

Deposition of amyloid is the single, most common, and characteristic
morphological lesion in islets of Langerhans in individuals with type-2
diabetes. Some degree of islet amyloidosis is found in at least 95% of such
patients [1]. In about two thirds of the cases,
more than 50% of the islets are affected. As the amyloid amount increases, the
percentage of beta cells decreases [2, 3]. The amount of amyloid can be
considerable in a single islet, more or less converting it into amyloid.The islet amyloid fibril consists of
islet amyloid polypeptide (IAPP; amylin) which is a 37-amino acid residues beta cell hormone, stored together with insulin in secretory vesicles
and released with this hormone. The normal molar ratio between IAPP and insulin
in human is less than 5%. Although IAPP is a very fibrillogenic peptide in vitro, it does not fibrillize in
normal islets, possibly due to interaction with insulin which is a potent inhibitor
of IAPP fibril formation [4–6].It is not understood why IAPP forms
amyloid deposits in conjunction with type-2 diabetes. An overexpression of IAPP
does exist in obese type-2 diabetic patients [7], but this alone is most probably
not sufficient for amyloid formation. Thus, transgenic mice, highly
overexpressing human IAPP, do not develop islet amyloidosis unless manipulated
in other ways, for example, feeding a diet high in fat [8–10]. Therefore, additional factors may
operate in the amyloidogenesis.The amyloid diseases constitute a
biochemically and clinically diverse group of disorders. Each amyloid disease
is characterized by one specific amyloid fibril protein and until now, more
than 25 proteins have been found in human amyloidosis [11]. Aggregation of a peptide to
amyloid-like fibrils in vitro is a nucleation-dependent process in which a
nucleus is formed before fibrils start to grow [12–14]. The nature of this nucleus is not
fully understood and the time it takes for its formation, known as the lag
phase, varies depending on protein, concentration, temperature, and other
factors. As soon as a nucleus is present, the growth of amyloid-like fibrils can occur
rapidly. Seeding a solution with preformed fibrils greatly enhances the
fibrillogenesis and reduces the lag phase sometimes close to zero. It is
assumed that protein monomers add to the free end of the fibrils. This
mechanism is believed not only to work in vitro but also in vivo and may be an
important reason why amyloid infiltration in systemic amyloidoses spreads
rapidly as soon as it has started. Seeding capability is generally very
specific and already minor variations in the protein efficiently block the
addition of new monomers [15]. However, in vitro experiments have
shown that preformed fibrils made from heterologous amyloid fibril
proteins sometimes can act as seed [16]. Heterologous amyloid fibrils
may also act as efficient seed in two different murine models of systemic
amyloidosis [17, 18]. That means, at least in
theory, that fibrils of one biochemical nature may be a risk factor for the
formation of amyloid deposits of another kind of amyloidosis.In addition to
IAPP, another major amyloid fibril protein, transthyretin (TTR), is expressed in
considerable amount in the pancreas [19, 20]. TTR is the major amyloid
fibril protein in several systemic familial forms of amyloidosis and in the
prevalent senile systemic amyloidosis [21]. Similar to other types of
systemic amyloidosis, most of the fibril protein in these amyloidoses is
derived from the plasma pool and the major expression site of TTR is the liver [22, 23]. There are, however, a few
minor sites of TTR gene expression and one of them is the endocrine pancreas [24, 25]. The function of TTR in these
localized cells is not known. Furthermore, the possible effect on
amyloidogenesis by these additional expression sites has not yet been studied.Against this
background, we questioned whether IAPP-amyloid could induce TTR-fibril
formation, and also whether deposition of TTR-amyloid in the pancreas leads to
islet amyloidosis, either by deposition of fibrils from TTR or from IAPP. Since
we were not aware of any study of islet TTR in type-2 diabetes, another aim was
to study the immunoreactivity of islet alpha (glucagon) and beta (insulin)
cells in diabetic and nondiabetic individuals.


## 2. Material and Methods

Paraffin-embedded pancreatic material (corpus or cauda) from 6 individuals
with type-2 diabetes and from 10 nondiabetic individuals was available in the
laboratory files. The inclusion criterion was that specimens had been taken
within 12 hours after death. They were fixed in buffered neutral 4%
formaldehyde solution and embedded in paraffin. Formalin-fixed and
paraffin-embedded tissue from the pancreatic body was also obtained from two
patients with long history of familial TTR-amyloidosis associated with a
V30M-mutation in the TTR gene. Adjacent 5μm sections were taken for alkaline Congo red staining [26] and for immunohistochemistry. The
study was approved by the Ethical Committee at Uppsala University Hospital.Antibodies against insulin (guinea
pig) and glucagon (rabbit) were purchased from DAKO (Glostrup, Denmark).
Rabbit antisera (A110) against rat/mouse
IAPP, which shows complete cross-reactivity with human IAPP [27] and against a recombinant
C-terminal fragment (aa 50–127; antiserum # A1898) of human TTR, have been
characterized previously [28]. A rabbit antiserum, A1899, was
raised against a high-molecular
fraction from a gel separation of ATTR fibrils from an individual with senile
systemic amyloidosis. This antiserum recognizes TTR amyloid, but is unreactive
with AL and AA amyloid in Western blot analysis. Immunohistochemistry was
performed with the antisera diluted 1 : 2000 using the
biotin-streptavidin
system. Sections were developed with
3,3′-diaminobenzidine-tetrahydrochloride (DAB). Double immunolabeling was
performed with antibodies against glucagon and TTR or insulin and TTR,
visualized with rabbit, mouse, or Guinea pig secondary antibodies conjugated to Alexa 488 (analyzed
in blue light) or Alexa 546 (analyzed in green light) (Molecular Probes,
Eugene, Ore, USA).For controls, sections were treated
with antiserum preabsorbed with TTR. Cross-reactivity between TTR or IAPP and
glucagon was ruled out by dot blot analysis for which TTR, IAPP, and glucagon
were dissolved at 3μg/well in 0.1 M sodium carbonate buffer, pH 9.8, spotted on a
nitrocellulose membrane and incubated with the antisera and developed with
biotin-streptavidin followed by DAB. This showed that no cross-reactivity
existed between IAPP and TTR or glucagon and TTR .In order to test
the specificity of the two TTR antisera, sections from an “amyloid
array” were used. In this, 1 mm thick cylinders from amyloid-containing tissues had
been embedded in one block. The array contained the following types of amyloid:
AA-amyloid (7 cases, 9 tissues), AL-amyloid (7 cases, 9 tissues), ATTR-amyloid
(6 cases, 3 tissues), Aβ-amyloid (1
case, brain), and IAPP-amyloid (3 cases, pancreas).


### 2.1. Degree of Islet Amyloidosis

The number of islets with and
without amyloid deposits was determined in Congo red stained sections, analyzed
in polarized light. Usually, at least 50 islets were scrutinized in each
section.


### 2.2. Localization of TTR in Islets of Langerhans

A cDNA library was constructed from
human liver with the aid of a cDNA synthesis kit (Amersham Bioscience, Uppsala, Sweden).
A 237-nucleotide
long fragment corresponding to amino acid residues 2–106 of TTR was amplified
by polymerase chain reaction. The achieved fragment was ligated into the
multiple cloning site of pGEM4Z (Promega, SDS-Biosciences, Falkenberg, Sweden).
The vector was linearized in front of the SP6 or T7 promoters and
digoxigenin-labelled RNA probes were produced according to the manufacturer
(Roche, Bromma, Sweden). In situ hybridization was
performed as described [29].For
ultrastructural studies, pancreatic tissue was available from one patient with type-2
diabetes and one normal control, fixed in 2% glutaraldehyde in phosphate buffer
and embedded in epon. For double immunolabeling, ultrathin sections on
formvar-coated nickel grids, were incubated with the primary antibodies (rabbit
anti TTR50-127) and mouse antiglucagon (DAKO). TTR was visualized with 5 nm
gold particles and glucagon with 10 nm gold particles (British Biocell, Cardiff, UK).


### 2.3. Seeding Experiments

Full length human TTR was a kind
gift from Dr. Tom Pettersson (Danderyd, Sweden) and human IAPP was synthesized by Keck
Laboratory, New Haven, Connecticut. TTR (0.5 mg) and IAPP (0.125 mg) were dissolved separately in 25μl methyl sulfoxide (DMSO). To the
solutions, 100μl distilled water was added. After
incubation for 2 days at room temperature, 0.5μl samples were taken from each and
studied electron microscopically (negative contrast) for presence of
amyloid-like fibrils. Since typical fibrils were seen in both solutions, they
were diluted to 2 ml with 0.05 M sodium phosphate buffer, pH 7.2. To wells of a 96-well plate with either 25μl of TTR or IAPP fibrils, 50μm newly dissolved TTR or IAPP in 0.05 M phosphate buffer
containing 2% DMSO was added, followed by thioflavin T to 10μM. The final volume was 100μl. Fluorescence was measured every 20
minutes in a fluoroscan as described [28].


### 2.4. Statistics

Values are given as mean  ±  SD.
Comparison between groups was performed with Mann Whitney test.


## 2.1. Degree of Islet Amyloidosis

The number of islets with and
without amyloid deposits was determined in Congo red stained sections, analyzed
in polarized light. Usually, at least 50 islets were scrutinized in each
section.


## 2.2. Localization of TTR in Islets of Langerhans

A cDNA library was constructed from
human liver with the aid of a cDNA synthesis kit (Amersham Bioscience, Uppsala, Sweden).
A 237-nucleotide
long fragment corresponding to amino acid residues 2–106 of TTR was amplified
by polymerase chain reaction. The achieved fragment was ligated into the
multiple cloning site of pGEM4Z (Promega, SDS-Biosciences, Falkenberg, Sweden).
The vector was linearized in front of the SP6 or T7 promoters and
digoxigenin-labelled RNA probes were produced according to the manufacturer
(Roche, Bromma, Sweden). In situ hybridization was
performed as described [29].For
ultrastructural studies, pancreatic tissue was available from one patient with type-2
diabetes and one normal control, fixed in 2% glutaraldehyde in phosphate buffer
and embedded in epon. For double immunolabeling, ultrathin sections on
formvar-coated nickel grids, were incubated with the primary antibodies (rabbit
anti TTR50-127) and mouse antiglucagon (DAKO). TTR was visualized with 5 nm
gold particles and glucagon with 10 nm gold particles (British Biocell, Cardiff, UK).


## 2.3. Seeding Experiments

Full length human TTR was a kind
gift from Dr. Tom Pettersson (Danderyd, Sweden) and human IAPP was synthesized by Keck
Laboratory, New Haven, Connecticut. TTR (0.5 mg) and IAPP (0.125 mg) were dissolved separately in 25μl methyl sulfoxide (DMSO). To the
solutions, 100μl distilled water was added. After
incubation for 2 days at room temperature, 0.5μl samples were taken from each and
studied electron microscopically (negative contrast) for presence of
amyloid-like fibrils. Since typical fibrils were seen in both solutions, they
were diluted to 2 ml with 0.05 M sodium phosphate buffer, pH 7.2. To wells of a 96-well plate with either 25μl of TTR or IAPP fibrils, 50μm newly dissolved TTR or IAPP in 0.05 M phosphate buffer
containing 2% DMSO was added, followed by thioflavin T to 10μM. The final volume was 100μl. Fluorescence was measured every 20
minutes in a fluoroscan as described [28].


## 2.4. Statistics

Values are given as mean  ±  SD.
Comparison between groups was performed with Mann Whitney test.


## 3. Results

In the pancreata of all individuals
with type-2 diabetes and in 7 out of 10 individuals without diabetes, amyloid
was found in islets of Langerhans. All of the diabetic individuals had a very
pronounced islet amyloidosis with some islets with little remaining endocrine
cells (Figure1). In the nondiabetic patients, usually only small amyloid deposits
were detected but in three individuals amyloid affected more than 20% and in
one as much as 38% of the islets. Comparison of the percentage of affected
islets in the two groups when only individuals with some degree of islet
amyloidosis were included, showed that
significantly more islets contained deposits in the diabetic individuals (98  ±  3%
and 13  ±  14%, P=0.001). The islet amyloid had typical staining properties with
Congo red and was strongly labeled with antiserum against IAPP (not shown).Figure 1
Islet in a type-2 diabetic patient. Most of the islet has been
converted into amyloid; Congo red, bar 20μm.


### 3.1. Hormone and Transthyretin Reactivity in Islet Cells of Non-Diabetic Individuals

In sections of pancreata without
amyloid from nondiabetic individuals, both antisera A1898 and A1899 labeled
islet cells in a similar way. A strong reaction was seen in all islets with
cells with a preferentially peripheral distribution (Figure2(a)) When compared
with sections immunostained for glucagon, an identical distribution was seen
(Figure 2(b)). In addition, the majority of remaining islet cells, mainly beta
cells, were also labeled but only weakly (Figure 2(a)). In situ hybridization
exhibited clear expression of TTR in cells which had a distribution of alpha
cells only and no certain reactivity was found with beta cells
(Figure 2(c)).
Electron microscopically
glucagon cells are characterized by secretory vesicles with a rounded electron
dense core often situated somewhat eccentrically on the rest of the granule. Glucagon
immunoreactivity occurred in both these areas of the granules while TTR labeling
was mainly seen in the less electron dense parts of the alpha cell granules
(Figure 2(d)). Small gold particles were also seen in the translucent areas of beta
cells but only to a small extent (not shown.). Antisera against insulin and IAPP
labeled normal beta cells as described [30].Normal human islets immunolabeled with antisera against: (a)
transthyretin and (b) glucagon. In (c) is shown a normal human
islet, subjected to in situ hybridization with a TTR probe,
visualized with immunohistochemistry. Note that positive cells have
a distribution indicative of glucagon cells. Bar 20μm. (d)
shows a part of a glucagon cell with typical granules. The section
was double immunolabeled for glucagon (10 nm gold particles) and TTR
(5 nm gold particles). Immunolabeling for both these substances is
seen on the granules, bar 500 nm.
(a)(b)(c)(d)Double labeling
for glucagon and TTR showed that all glucagon cells also exhibited TTR
immunoreactivity (Figures3(a) and 3(b)). In addition, a large number of islet
cells, negative for glucagon, showed an evident TTR-reactivity
(Figure 3(c)). Colocalization of insulin and TTR was seen in many, but not all, beta cells (Figure 3(d)).In (a), (b), and (c), an islet double labeled for glucagon (red)
and TTR (green). Yellow color indicates colocalization. A large
number of islet cells show TTR but not glucagon content. The islet
in (d) is double labeled for insulin (green) and TTR (red). Many beta
cells exhibit both TTR and insulin immunoreactivity (yellow). The
amyloid (A) is weakly labeled for TTR. Bar (a)–(c) 25μm, (d) 200μm.
(a)(b)(c)(d)Amyloid-rich islet from a type-2 diabetic individual, in (a)
immunolabeled for TTR, and in (b) for insulin. Note virtually
identical distribution of immune reactive cells. No nuclear
staining, bar 20μm.
(a)(b)


### 3.2. Endocrine Cells in Islets with Amyloid

Both insulin and glucagon
immunoreactive cells were identified in islets will all degrees of amyloid
deposits. As described earlier [31, 32], beta cells in amyloid-laden
islets generally were devoid of IAPP-immunoreactivity. Many cells showed a
strong labeling with antibodies against TTR. From adjacent sections, stained
for TTR, insulin, and glucagon, it was obvious that not only alpha cells but
also beta cells were strongly TTR-positive (Figure 3).


### 3.3. Transthyretin Reactivity in Islet Amyloid

Islet amyloid from diabetic and
nondiabetic individuals exhibited a strong immunolabeling with antiserum
against IAPP (not shown). Since we have shown previously that commercially
available antibodies against TTR often do not recognize TTR in fibrillar (i.e.,
amyloid) form, we developed two different rabbit antisera which both strongly
labeled cellular TTR and TTR in amyloid. The two TTR antisera labeled islet
amyloid weakly in some cases (Figures3(d) and 5(a)). This staining was even
and no areas with strong reaction were seen. In the electron microscopic study,
IAPP antiserum labeled amyloid in the diabetic case, but no certain binding of
TTR antibodies to fibrils was seen. In order to study the specificity of the
reaction in amyloid, we used an amyloid array with tissues from several
patients in one block. The two TTR antisera showed reaction only with amyloid
of known TTR origin (i.e., Swedish familial amyloidosis and SSA) but not with
amyloid of AA, AL, AMed, or Aβ nature, showing that
TTR-immunoreactivity is not a general feature of amyloid deposits.In (a) is shown an islet from a type-2 diabetic individual. There
is pronounced IAPP amyloid infiltration, weakly labeled with
antibodies against TTR. (b) shows a small islet in a nondiabetic
individual with familial TTR-amyloidosis. There are heavy deposits
of TTR-amyloid outside the islet but no islet amyloid. A  =  amyloid.
Immunolabeled with antiserum against TTR.
(a)(b)The pancreatic
tissue from the individuals with familial TTR-amyloidosis contained varying
amounts of amyloid, irregularly distributed in the exocrine parenchyma and surrounding
connective tissue. Although amyloid was seen very close to some pancreatic
islets, TTR-amyloid did not occur within these islets (Figure5(b)). Neither was
IAPP-amyloid seen here.


### 3.4. IAPP Fibrils Seed Fibril Formation from TTR

Fibril formation of many amyloid
proteins, including TTR and IAPP, occurs
spontaneously in vitro after a lag phase which varies in length depending on
protein. Seeding with preformed fibrils can shorten the lag phase considerably,
which was seen most evidently when a solution of IAPP was seeded with IAPP fibrils
(Figure6, inserted). TTR fibrils did not induce any increase in fluorescence
signal when incubated with newly dissolved TTR or solubilized IAPP (Figure 6).
However, IAPP fibrils had a clear effect on TTR after a lag phase of about 3
hours (Figure 6).Figure 6
In vitro seeding and cross-seeding experiments with TTR and IAPP
measured with thioflavin T. Preformed fibrils of IAPP seeded TTR
(blue circles) and IAPP (red triangles, insert). No other increase
in signals occurred. Thus, TTR fibrils did not seed IAPP (red
circles).


## 3.1. Hormone and Transthyretin Reactivity in Islet Cells of Non-Diabetic Individuals

In sections of pancreata without
amyloid from nondiabetic individuals, both antisera A1898 and A1899 labeled
islet cells in a similar way. A strong reaction was seen in all islets with
cells with a preferentially peripheral distribution (Figure2(a)) When compared
with sections immunostained for glucagon, an identical distribution was seen
(Figure 2(b)). In addition, the majority of remaining islet cells, mainly beta
cells, were also labeled but only weakly (Figure 2(a)). In situ hybridization
exhibited clear expression of TTR in cells which had a distribution of alpha
cells only and no certain reactivity was found with beta cells
(Figure 2(c)).
Electron microscopically
glucagon cells are characterized by secretory vesicles with a rounded electron
dense core often situated somewhat eccentrically on the rest of the granule. Glucagon
immunoreactivity occurred in both these areas of the granules while TTR labeling
was mainly seen in the less electron dense parts of the alpha cell granules
(Figure 2(d)). Small gold particles were also seen in the translucent areas of beta
cells but only to a small extent (not shown.). Antisera against insulin and IAPP
labeled normal beta cells as described [30].Normal human islets immunolabeled with antisera against: (a)
transthyretin and (b) glucagon. In (c) is shown a normal human
islet, subjected to in situ hybridization with a TTR probe,
visualized with immunohistochemistry. Note that positive cells have
a distribution indicative of glucagon cells. Bar 20μm. (d)
shows a part of a glucagon cell with typical granules. The section
was double immunolabeled for glucagon (10 nm gold particles) and TTR
(5 nm gold particles). Immunolabeling for both these substances is
seen on the granules, bar 500 nm.
(a)(b)(c)(d)Double labeling
for glucagon and TTR showed that all glucagon cells also exhibited TTR
immunoreactivity (Figures3(a) and 3(b)). In addition, a large number of islet
cells, negative for glucagon, showed an evident TTR-reactivity
(Figure 3(c)). Colocalization of insulin and TTR was seen in many, but not all, beta cells (Figure 3(d)).In (a), (b), and (c), an islet double labeled for glucagon (red)
and TTR (green). Yellow color indicates colocalization. A large
number of islet cells show TTR but not glucagon content. The islet
in (d) is double labeled for insulin (green) and TTR (red). Many beta
cells exhibit both TTR and insulin immunoreactivity (yellow). The
amyloid (A) is weakly labeled for TTR. Bar (a)–(c) 25μm, (d) 200μm.
(a)(b)(c)(d)Amyloid-rich islet from a type-2 diabetic individual, in (a)
immunolabeled for TTR, and in (b) for insulin. Note virtually
identical distribution of immune reactive cells. No nuclear
staining, bar 20μm.
(a)(b)


## 3.2. Endocrine Cells in Islets with Amyloid

Both insulin and glucagon
immunoreactive cells were identified in islets will all degrees of amyloid
deposits. As described earlier [31, 32], beta cells in amyloid-laden
islets generally were devoid of IAPP-immunoreactivity. Many cells showed a
strong labeling with antibodies against TTR. From adjacent sections, stained
for TTR, insulin, and glucagon, it was obvious that not only alpha cells but
also beta cells were strongly TTR-positive (Figure 3).


## 3.3. Transthyretin Reactivity in Islet Amyloid

Islet amyloid from diabetic and
nondiabetic individuals exhibited a strong immunolabeling with antiserum
against IAPP (not shown). Since we have shown previously that commercially
available antibodies against TTR often do not recognize TTR in fibrillar (i.e.,
amyloid) form, we developed two different rabbit antisera which both strongly
labeled cellular TTR and TTR in amyloid. The two TTR antisera labeled islet
amyloid weakly in some cases (Figures3(d) and 5(a)). This staining was even
and no areas with strong reaction were seen. In the electron microscopic study,
IAPP antiserum labeled amyloid in the diabetic case, but no certain binding of
TTR antibodies to fibrils was seen. In order to study the specificity of the
reaction in amyloid, we used an amyloid array with tissues from several
patients in one block. The two TTR antisera showed reaction only with amyloid
of known TTR origin (i.e., Swedish familial amyloidosis and SSA) but not with
amyloid of AA, AL, AMed, or Aβ nature, showing that
TTR-immunoreactivity is not a general feature of amyloid deposits.In (a) is shown an islet from a type-2 diabetic individual. There
is pronounced IAPP amyloid infiltration, weakly labeled with
antibodies against TTR. (b) shows a small islet in a nondiabetic
individual with familial TTR-amyloidosis. There are heavy deposits
of TTR-amyloid outside the islet but no islet amyloid. A  =  amyloid.
Immunolabeled with antiserum against TTR.
(a)(b)The pancreatic
tissue from the individuals with familial TTR-amyloidosis contained varying
amounts of amyloid, irregularly distributed in the exocrine parenchyma and surrounding
connective tissue. Although amyloid was seen very close to some pancreatic
islets, TTR-amyloid did not occur within these islets (Figure5(b)). Neither was
IAPP-amyloid seen here.


## 3.4. IAPP Fibrils Seed Fibril Formation from TTR

Fibril formation of many amyloid
proteins, including TTR and IAPP, occurs
spontaneously in vitro after a lag phase which varies in length depending on
protein. Seeding with preformed fibrils can shorten the lag phase considerably,
which was seen most evidently when a solution of IAPP was seeded with IAPP fibrils
(Figure6, inserted). TTR fibrils did not induce any increase in fluorescence
signal when incubated with newly dissolved TTR or solubilized IAPP (Figure 6).
However, IAPP fibrils had a clear effect on TTR after a lag phase of about 3
hours (Figure 6).Figure 6
In vitro seeding and cross-seeding experiments with TTR and IAPP
measured with thioflavin T. Preformed fibrils of IAPP seeded TTR
(blue circles) and IAPP (red triangles, insert). No other increase
in signals occurred. Thus, TTR fibrils did not seed IAPP (red
circles).


## 4. Discussion

This study confirms that TTR is
normally expressed by pancreatic alpha (glucagon) cells. As earlier suggested [33], beta cells may also produce
TTR but at a low degree, not detectable by in situ hybridization. TTR is stored
in the secretory vesicles and is therefore most likely released together with
hormones at exocytosis. TTR in plasma is a transporter of thyroxine and
indirectly of retinol, but the function of the protein in the secretory
granules is unknown. One can only speculate about the possibility that TTR
binds to the glucagon precursor and may affect its processing.IAPP and TTR are
both well-known amyloid fibril proteins. Formation of amyloid fibrils is a
nucleation dependent phenomenon which can be shown in vitro [14]. After a lag phase, which can
last for several days, generation of fibrils usually is rapid. Seeding a
fibrillogenic protein solution with preformed fibrils shortens or abolishes
this lag phase. Cross-seeding, that is, the same effect achieved by addition of
fibrils of a different biochemical nature, can occur with some proteins [16]. In this study, IAPP fibrils
seeded IAPP efficiently. This was not seen with TTR which is in accordance with
a previous report [34]. TTR fibrils did not seed
IAPP but, interestingly, IAPP fibrils evidently induced fibril formation from
TTR. Therefore, the weak immunoreaction of IAPP amyloid with antibodies against
TTR in some cases may indicate existence of mixed fibrils in islet deposits as
a result of a specific interaction between IAPP and TTR, although a passive
diffusion of the latter molecule into the amyloid cannot be ruled out. While
insulin is an inhibitor of IAPP fibril formation, effects of TTR is not known.
It has been suggested that molecules may exist that lead to pathological
protein folding and aggregation and these have been called “pathological
chaperones” [35]. Therefore, the possible
interaction between TTR and IAPP has to be studied further.In the present
study, we also evaluated the TTR immunoreactivity in alpha and beta cells in
conjunction with type-2 diabetes. No such studies seem to have been published.
Generally, islets in type-2 diabetic patients contained proportionally more
strongly TTR-reactive cells in accordance with the known loss of beta cells in
that disease [2, 3]. In addition, an interesting
and unexpected finding was an increased TTR immunoreactivity of beta cells in
pancreatic islets with heavy amyloid deposits. What this means is presently not
understood but we have shown previously that IAPP in beta cells in islets with
amyloid obtains altered immunoreactive properties [32]. Thus, IAPP immunoreactivity
with a polyclonal IAPP antiserum was lost, although labeling with a monoclonal
antibody was retained. This finding was interpreted as a sign of modification
of the beta cell IAPP in type-2 diabetes. The finding here that IAPP fibrils
interact with TTR is interesting in this respect. The observation in the
present study that beta cells in amyloidotic islets are strongly TTR
immunoreactive may indicate that the intragranular milieu is altered which may affect the ability of
IAPP to aggregate.



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*Source: 429274-2008-09-24.xml*