# Islet Specific Wnt Activation in Human Type  II Diabetes

Authors: Seung-Hee Lee; Carla Demeterco; Ifat Geron; Annelie Abrahamsson; Fred Levine; Pamela Itkin-Ansari
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

The Wnt pathway effector gene TCF7L2 has been linked to type II diabetes, making it important to study the role of Wnt signaling in diabetes pathogenesis. We examined the expression of multiple Wnt pathway components in pancreases from normal  individuals and type II diabetic individuals. Multiple members of the Wnt signaling pathway, including TCF7L2, Wnt2b,β-catenin, pGSK3β, TCF3, cyclinD1, and c-myc, were undetectable or expressed at low levels in islets from nondiabetic individuals, but were  also upregulated specifically in islets of type II diabetic patients. Culture of pancreatic tissue and islet isolation led to Wnt activation that was reversed by the Wnt antagonist sFRP, demonstrating that Wnt activation in that setting was due to soluble Wnt factors. These data support a model in which the Wnt pathway plays a dynamic role in the pathogenesis of type II diabetes and  suggest  manipulation of Wnt signaling as a new approach to β-cell-directed diabetes therapy.


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## Body

## 1. Introduction

Theβ-cell
is a major target organ in the pathogenesis of type II diabetes  as evidenced by
the fact that overt diabetes does not occur until β-cell
dysfunction/loss has proceeded to the point where hypersecretion of insulin can
no longer compensate for peripheral insulin resistance [1].
The predominant model of the molecular events leading to β-cell
failure is that the diabetic environment, in particular chronically high levels
of glucose and free fatty acids, has toxic effects on the β-cell
[2].  A number of signaling pathways have been implicated in β-cell
failure, including insulin signaling [3]
and oxidative stress [4].Wnt signal transduction is one of
the central pathways that control organismal growth and differentiation [5]. In the best-studied branch of
the Wnt pathway, termed the canonical pathway, Wnts bind to frizzled receptors
in conjunction with LRP family coreceptors. Resultant pathway activation prevents
GSK3-mediated phosphorylation of β-catenin and its subsequent ubiquitin-mediated degradation. Stabilized β-catenin translocates into the nucleus where it interacts
with transcription factors of the TCF family to activate target genes,
including c-myc and cyclin D family members.In addition to the canonical
pathway, an alternative, noncanonical pathway with multiple branches exists. A calcium-dependent pathway acts through
calcium/calmodulin-dependent kinase II (CamKII) primarily to control cell
movement. In some cell types, activation of the noncanonical pathway leads to
activation of the NFAT transcription factor [6], which plays an important role
in the maintenance of functional β-cells [7].Wnt signaling is
critical in early foregut and pancreatic development [8–10].
Moreover, homozygous mutation of LRP5 in mice leads to defective glucose-stimulated
insulin secretion from isolated islets in
vitro [11]. Components of the Wnt pathway are
present in the adult pancreas, and in particular multiple members of the
frizzled family of Wnt receptors have been identified in the islet [8].
There are discrepant data on the role of Wnt signaling in mature islets. While
most studies have found little evidence that Wnt signaling is involved in
endocrine differentiation or in the adult islet [9, 10, 12],
there is some evidence for an effect of Wnt signaling on β-cell
replication [13].To study the role of Wnt
signaling in human diabetes, we systematically examined components of this
pathway in the pancreas of normal and type II diabetic individuals. Strikingly,
Wnt2b, which activates the canonical Wnt pathway, was highly upregulated in
type II diabetes. Other mediators of the Wnt pathway, including pGSK3β,
TCF/Lef factors, c-myc, and cyclinD1, were also induced in type II diabetes,
specifically in the β-cells. β-catenin,
in contrast to the mouse, in which it is highly expressed in normal islets [14],
was low or absent in normal human islets in
situ. However, it was highly upregulated in an islet-specific manner in
human type II diabetes. Most α-cells
did not exhibit Wnt pathway activation and instead expressed high levels of the
noncanonical Wnt, Wnt4, which can act to repress canonical Wnt signaling [15].
The upregulation of TCF factors, including TCF7L2, is particularly interesting
as linkage studies have identified a polymorphism in that gene as being tightly
linked to type II diabetes in humans and there is some evidence that this
polymorphism affects β-cell
function [16–20].Ectopic
overexpression of the Wnt target gene c-myc in mice has been shown to causeβ-cell
apoptosis and diabetes [21].
To determine whether c-myc was a candidate for being an early effector in
diabetes pathogenesis, we examined the effect of high-fat diet on c-myc expression
in the murine pancreas. Interestingly, mice fed a high-fat diet for three
months exhibited upregulated c-myc at a time when the mice were only mildly
obese and not yet diabetic.The finding that the Wnt
pathway is activated in human type II diabetes and that the Wnt target gene c-myc
is also upregulated early in progression to diabetes in rodents has important
implications for understanding the mechanism ofβ-cell
compensation and failure in diabetes and provides impetus for the development
of new therapies targeted to that pathway.


## 2. Methods

### 2.1. Tissue

Paraffin-embedded
specimens from human pancreases (5 nondiabetic and 9 type II diabetic) were
previously described [22].
Freshly isolated and cultured pancreases from three nondiabetic individuals
were obtained from the Islet Resource center in (Seattle, Wash, USA) immediately prior to islet
isolation. These pancreases were obtained postmortem by UNOS
and shipped to Seattle in University of Wisconsin solution (UW
solution). Specimens were collected in 4% paraformaldehyde (PFA) and shipped to
us overnight followed by 30% sucrose/PBS, embedded in OCT freezing media
(Sakura, Calif, USA) and snap-frozen at −80°C. Isolated human islets (cultured
islets) were obtained through the Islet Procurement Resource Program (JDRF).
Islets were shipped in CMRL 1066 medium with serum-free supplements. Upon
arrival, islets were fixed in PFA or washed and cultured in suspension in RPMI
supplemented with 5.5 mM glucose, penicillin (100 U/mL), streptomycin (100 ug/mL),
and 10% bovine serum. Twenty four hours later, they were treated
with 0, 100, 500 ng/mL rhsFRP-1(R&D systems, Minn, USA) for 48 hours,
harvested by fixing in 4% neutral phosphate-buffered formalin, followed by 30%
sucrose/PBS, embedded in OCT freezing media (Sakura, Calif, USA), and snap-frozen at
−80°C.Human
fetal pancreases at 18–24 gestational
weeks were obtained from Advanced Bioscience Resource, in accordance with University of California San Diego
Institutional Review Board regulations (cold ischemia 12–24 hours).Murine pancreases were obtained from
anesthetized Balb/c or C57/bl mice and collected in 4% PFA followed by paraffin
embedding.Preparation of nonendocrine
pancreatic cells (NEPCs) clusters was as described [23].
The unpurified islet fractions and Cobe bag fractions were obtained from the Universities
of Edmonton, Minnesota, Miami, Washington, the Whittier
Institute for Diabetes, and the City of Hope Medical Center.


### 2.2. Immuno-Histochemistry

Both paraffin and frozen samples were sectioned to a mean thickness of 5μm, washed four
times with PBS, treated with 0.3% Triton in PBS for 10 minutes, and incubated
in blocking solution (PBS with 0.1% Triton X-100/5% normal goat serum) for 30
minutes at room temperature. They were then treated by alcohol series and
antigen retrieval with CitriSolvTM (Fisher Scientific, Pa, USA). Sections were incubated in an
antibody solution overnight at 4°C as described Table 1. For fluorescent
imaging, slides were incubated with Alexa 488 (Molecular Probs, Ore, USA)/Rhodamine or Alexa 596 (Jackson Immuno Research, 1:300) fluor-labeled
anti-Goat/mouse/rabbit for 45 minutes at room temperature. They were mounted
in Vectashield (Vector Labs, Burlingame, Calif, USA).
Digital images of stained sections were captured using a fluorescence
microscope with a digital camera (Nikon, Tokyo, Japan) and deconvoluted (nearest neighbor) using
SlideBook software (Intelligent Imaging Innovations, Denver, Colo, USA).
Confocal microscopy was performed with
an MRC 1024 MP laser scanning microscope (Bio-Rad Laboratories, Inc., Richmond,
Calif, USA) equipped with krypton/argon laser and a Millenia-Tsunami two-photon
Ti-Sapphire femtosecond laser system (Spectra-Physics, Mountain View, Calif,
USA). Fluorescent intensity signals in confocal images from slides from
nondiabetic and type II diabetic patients were scored blindly and quantitated
using Image J histogram analysis. Statistical analysis was performed using
Student's t-test to determine mean +/−SE.Table 1
Antibodies.
AntibodyCat. No.SupplierConc.AmylaseSC-12821Santa Cruz1:300β-cateninSC-7963Santa Cruz1:200β-cateninSC-7199Santa Cruz1:200β-catenin610154Pharmingen1:200c-myc554205Pharmingen1:200c-mycNCL-CMYCNovocastra1:100Cyclin D1SC-718Santa Cruz1:200E-cadherinSC-7870Santa Cruz1:200γ-cateninSC-8415Santa Cruz1:200γ-cateninSC-7900Santa Cruz1:200GlucagonG-2654Sigma1:1000GlucagonAB932Chemicon1:500InsulinSC-8033Santa Cruz1:200InsulinSC-9168Santa Cruz1:200InsulinI7660US Biological1:200Pdx-1Dr. Chris Wright1:1000pGSK3βSC-11757Santa Cruz1:200sFRP-1AF1384R&D1:200SomatostatinSC-13099Santa Cruz1:200TCF-3SC-8635Santa Cruz1:200TCF7L2SC-8631Santa Cruz1:200Wnt2bAF3900R&D1:100Wnt4AF475R&D1:100


### 2.3. Western Blot

Whole-cell extracts were prepared
by incubation in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, 100 mg/mL PMSF, 1 mg/mL aprotinin, 1% Triton X-100) for 30 minutes on ice, followed
by 15 minutes of centrifugation at 12 000 g, 4°C as previously described [24].
Extracts were mixed with loading buffer and electrophoresed on 4–15% acrylamide
gel (ReadyGel Bio-Rad Laboratories, Inc. Hercules, Calif, USA)
in Tris/glycine/SDS buffer. Proteins were transferred onto double
nitrocellulose membranes (Immobilon-Psq, Millipore, Bedford, Mass, USA)
in Tris/glycine/methanol buffer for 1 hour at 100 V. After overnight blocking
in PBS-Tween (PBST) with 3% milk, membrane was incubated for 2 hours at room
temperature with antibodies: anti-β-catenin
(Pharmingen), anti-CK19 (cytokeratin-19) (DAKO), anti-Actin (Sigma, St  Louis, Mo, USA). After extensive washing in PBST, the membrane  was incubated with secondary antibody conjugated to horseradish peroxidase
diluted 1:2000  (Amersham/GE, Buckinghamshire, UK), washed in PBST, and revealed by ECL (Amersham/GE, Buckinghamshire, UK).


### 2.4. Mouse Studies

Male C57BL/6J mice were purchased from Harlan Sprague Dawley, Inc.
(Indianapolis, Ind, USA). The animals were obtained at 4 weeks of age weighing 17.9–21.2 g at the
start of the study and kept four per cage on a 12:12-hour light dark schedule. The study was approved by the UCSD IACUC. One week after arrival, mice were
divided into two groups and were fed either a high-fat diet (Research Diets,
New Brunswick, NJ, USA) or received continuous feeding of a normal diet  for up to 14 weeks. The
high-fat diet consisted of 45% fat, 35% carbohydrate, and 20% protein (4.73 Kcal/g), whereas the normal diet contained 5% fat, 57% carbohydrate, and 18%
protein (3.4 Kcal/g). A caveat to high-fat feeding experiments is the
possibility that a high-fat diet differs from normal chow in multiple
constituents, potentially confounding results [25].For intraperitoneal glucose tolerance test
(IPGTT), 8-hour fasted mice received an intraperitoneal injection with
D-glucose (1.5 g/kg). Blood samples were obtained from the tail vein
immediately before and at 15, 30, 60, 90, and 120 minutes after the glucose
load. Blood glucose levels were measured with a Precision XtraTM glucometer
(Abbott Laboratories, Alameda, Calif, USA).


## 2.1. Tissue

Paraffin-embedded
specimens from human pancreases (5 nondiabetic and 9 type II diabetic) were
previously described [22].
Freshly isolated and cultured pancreases from three nondiabetic individuals
were obtained from the Islet Resource center in (Seattle, Wash, USA) immediately prior to islet
isolation. These pancreases were obtained postmortem by UNOS
and shipped to Seattle in University of Wisconsin solution (UW
solution). Specimens were collected in 4% paraformaldehyde (PFA) and shipped to
us overnight followed by 30% sucrose/PBS, embedded in OCT freezing media
(Sakura, Calif, USA) and snap-frozen at −80°C. Isolated human islets (cultured
islets) were obtained through the Islet Procurement Resource Program (JDRF).
Islets were shipped in CMRL 1066 medium with serum-free supplements. Upon
arrival, islets were fixed in PFA or washed and cultured in suspension in RPMI
supplemented with 5.5 mM glucose, penicillin (100 U/mL), streptomycin (100 ug/mL),
and 10% bovine serum. Twenty four hours later, they were treated
with 0, 100, 500 ng/mL rhsFRP-1(R&D systems, Minn, USA) for 48 hours,
harvested by fixing in 4% neutral phosphate-buffered formalin, followed by 30%
sucrose/PBS, embedded in OCT freezing media (Sakura, Calif, USA), and snap-frozen at
−80°C.Human
fetal pancreases at 18–24 gestational
weeks were obtained from Advanced Bioscience Resource, in accordance with University of California San Diego
Institutional Review Board regulations (cold ischemia 12–24 hours).Murine pancreases were obtained from
anesthetized Balb/c or C57/bl mice and collected in 4% PFA followed by paraffin
embedding.Preparation of nonendocrine
pancreatic cells (NEPCs) clusters was as described [23].
The unpurified islet fractions and Cobe bag fractions were obtained from the Universities
of Edmonton, Minnesota, Miami, Washington, the Whittier
Institute for Diabetes, and the City of Hope Medical Center.


## 2.2. Immuno-Histochemistry

Both paraffin and frozen samples were sectioned to a mean thickness of 5μm, washed four
times with PBS, treated with 0.3% Triton in PBS for 10 minutes, and incubated
in blocking solution (PBS with 0.1% Triton X-100/5% normal goat serum) for 30
minutes at room temperature. They were then treated by alcohol series and
antigen retrieval with CitriSolvTM (Fisher Scientific, Pa, USA). Sections were incubated in an
antibody solution overnight at 4°C as described Table 1. For fluorescent
imaging, slides were incubated with Alexa 488 (Molecular Probs, Ore, USA)/Rhodamine or Alexa 596 (Jackson Immuno Research, 1:300) fluor-labeled
anti-Goat/mouse/rabbit for 45 minutes at room temperature. They were mounted
in Vectashield (Vector Labs, Burlingame, Calif, USA).
Digital images of stained sections were captured using a fluorescence
microscope with a digital camera (Nikon, Tokyo, Japan) and deconvoluted (nearest neighbor) using
SlideBook software (Intelligent Imaging Innovations, Denver, Colo, USA).
Confocal microscopy was performed with
an MRC 1024 MP laser scanning microscope (Bio-Rad Laboratories, Inc., Richmond,
Calif, USA) equipped with krypton/argon laser and a Millenia-Tsunami two-photon
Ti-Sapphire femtosecond laser system (Spectra-Physics, Mountain View, Calif,
USA). Fluorescent intensity signals in confocal images from slides from
nondiabetic and type II diabetic patients were scored blindly and quantitated
using Image J histogram analysis. Statistical analysis was performed using
Student's t-test to determine mean +/−SE.Table 1
Antibodies.
AntibodyCat. No.SupplierConc.AmylaseSC-12821Santa Cruz1:300β-cateninSC-7963Santa Cruz1:200β-cateninSC-7199Santa Cruz1:200β-catenin610154Pharmingen1:200c-myc554205Pharmingen1:200c-mycNCL-CMYCNovocastra1:100Cyclin D1SC-718Santa Cruz1:200E-cadherinSC-7870Santa Cruz1:200γ-cateninSC-8415Santa Cruz1:200γ-cateninSC-7900Santa Cruz1:200GlucagonG-2654Sigma1:1000GlucagonAB932Chemicon1:500InsulinSC-8033Santa Cruz1:200InsulinSC-9168Santa Cruz1:200InsulinI7660US Biological1:200Pdx-1Dr. Chris Wright1:1000pGSK3βSC-11757Santa Cruz1:200sFRP-1AF1384R&D1:200SomatostatinSC-13099Santa Cruz1:200TCF-3SC-8635Santa Cruz1:200TCF7L2SC-8631Santa Cruz1:200Wnt2bAF3900R&D1:100Wnt4AF475R&D1:100


## 2.3. Western Blot

Whole-cell extracts were prepared
by incubation in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, 100 mg/mL PMSF, 1 mg/mL aprotinin, 1% Triton X-100) for 30 minutes on ice, followed
by 15 minutes of centrifugation at 12 000 g, 4°C as previously described [24].
Extracts were mixed with loading buffer and electrophoresed on 4–15% acrylamide
gel (ReadyGel Bio-Rad Laboratories, Inc. Hercules, Calif, USA)
in Tris/glycine/SDS buffer. Proteins were transferred onto double
nitrocellulose membranes (Immobilon-Psq, Millipore, Bedford, Mass, USA)
in Tris/glycine/methanol buffer for 1 hour at 100 V. After overnight blocking
in PBS-Tween (PBST) with 3% milk, membrane was incubated for 2 hours at room
temperature with antibodies: anti-β-catenin
(Pharmingen), anti-CK19 (cytokeratin-19) (DAKO), anti-Actin (Sigma, St  Louis, Mo, USA). After extensive washing in PBST, the membrane  was incubated with secondary antibody conjugated to horseradish peroxidase
diluted 1:2000  (Amersham/GE, Buckinghamshire, UK), washed in PBST, and revealed by ECL (Amersham/GE, Buckinghamshire, UK).


## 2.4. Mouse Studies

Male C57BL/6J mice were purchased from Harlan Sprague Dawley, Inc.
(Indianapolis, Ind, USA). The animals were obtained at 4 weeks of age weighing 17.9–21.2 g at the
start of the study and kept four per cage on a 12:12-hour light dark schedule. The study was approved by the UCSD IACUC. One week after arrival, mice were
divided into two groups and were fed either a high-fat diet (Research Diets,
New Brunswick, NJ, USA) or received continuous feeding of a normal diet  for up to 14 weeks. The
high-fat diet consisted of 45% fat, 35% carbohydrate, and 20% protein (4.73 Kcal/g), whereas the normal diet contained 5% fat, 57% carbohydrate, and 18%
protein (3.4 Kcal/g). A caveat to high-fat feeding experiments is the
possibility that a high-fat diet differs from normal chow in multiple
constituents, potentially confounding results [25].For intraperitoneal glucose tolerance test
(IPGTT), 8-hour fasted mice received an intraperitoneal injection with
D-glucose (1.5 g/kg). Blood samples were obtained from the tail vein
immediately before and at 15, 30, 60, 90, and 120 minutes after the glucose
load. Blood glucose levels were measured with a Precision XtraTM glucometer
(Abbott Laboratories, Alameda, Calif, USA).


## 3. Results

### 3.1. TCF7L2 Is Upregulated in Islets of Type II Diabetic Patients

Recently, TCF7L2 has been genetically linked to type II
diabetes in multiple populations [16–19]. However, the
mechanism by which it contributes to diabetes pathogenesis is unknown. The mRNA
level of TCF7L2 is higher in cultured isolated islets from type II patients
versus normal individuals [20], but expression in situ has not been examined. We
found that TCF7L2 protein was barely detectable in the pancreas of normal individuals,
but it was highly upregulated in an islet-specific manner in patients with type
II diabetes (Figures1(a)–1(e)). In addition, the expression of TCF3, which has
not been linked to type II diabetes, was also upregulated (Figures 1(f)–1(j)).Figure 1
Expression of TCF factors in type II diabetes. TCF7L2 (red in (a)–(d)) and TCF3 (red
in (f)–(i)) are absent from islets of nondiabetic individuals (a), (b), (f), and (g), but are present in islets from
type II diabetics (c), (d), (h), and (i). Islets are identified by insulin (green in (b), (d), (g), and (i)). Quantitation of
TCF7L2 (e) and TCF3 (j) expressions in nondiabetic (n=4) and type II (n=7) islets: error
bars = mean +/−s.e.m. *P<.05. Scale bars = 50 μm.


### 3.2. Wnt2b Is Upregulated in Type II Diabetes

Because TCF7L2 is both an
effector as well as a downstream target of Wnt signaling [26], the induction of TCF factors suggested that a more global
activation of Wnt signaling may be occurring. To test that hypothesis, we examined
the expression of soluble Wnt factors and the initiators of Wnt signaling. Based
upon the reported pattern of expression of soluble Wnt factor mRNA in the
pancreas, we examined Wnt2b, which was a good candidate as its RNA has been
reported to be present in isolated islets [8, 27].
In nondiabetic individuals, little or no Wnt2b expression was detectable
(Figures 2(a), 2(b), 2(c)). However, there was dramatic upregulation in the islets
of type II diabetics. Robust expression of Wnt2b occurred in both β-cells
(Figures 2(c), 2(d), 2(e)) and α-cells
(not shown) of diabetic patients.Figure 2
Expression of Wnt2b,β-catenin, cyclinD1, and
c-myc in type II diabetes. Wnt2b (red in (a), (b), (d), and (e)), β-catenin
(red in (f), (g), (i), and (j)), cyclinD1 (red in (k), (l), (n), and (o)), and c-myc
(red in (q) and (s)) are absent from islets of nondiabetic individuals (a), (b), (f), (g), (k), (l), (p) and (q), but are present in islets from type II diabetics (d), (e), (i), (j), (n), (o), (r), and (s). Islets are identified by insulin (green in (a), (d), (f), (i), (p), and (r)) or glucagon (l) and (o) and are outlined by a dashed line in (g) to better demonstrate the absence of
β-catenin. Quantitation of Wnt2b (c), β-catenin (h), and cyclinD1 (m) expression in non-diabetic (n=5) and type
II (n=7) islets: error bars = mean +/−s.e.m. *P<.05. Scale
bars = 100 μm except for f.g.i.j which are 50 μm.


### 3.3. Humanβ-Cells Lack Detectable β-Catenin Expression But It Is Strongly Upregulated  in Type II Diabetes

Activation of frizzled receptors by
soluble Wnts results in the stabilization ofβ-catenin. β-catenin
in human islets of all five nondiabetic individuals examined was markedly lower
than in the surrounding exocrine tissue, where it was strongly expressed (Figures
2(f)–2(h), Supplementary Figures 1(a), 1(b) available online at doi:10.1155/2008/728763). The same pattern was observed in
the human fetal pancreas, with high levels of β-catenin
expression in nonendocrine epithelial cells and very low levels in endocrine
cells (Supplementary Figures 1(c), 1(d)). Colocalization studies with PDX-1 revealed that β-catenin
was substantially restricted to nonendocrine PDX-1
positive, cells in the human fetal pancreas (Supplementary Figure 1(e)).
Because PDX-1 serves as a
precursor for both exocrine and endocrine compartments, β-catenin
expression must be repressed during human, but not murine, endocrine
differentiation.To determine whetherβ-catenin plays a role in β-cell
dysfunction and/or loss in human diabetes, its expression was examined in the
pancreases of 9 patients with type II diabetes. In contrast with nondiabetic
pancreases (Figures 2(f)–2(h)), it was strongly expressed in the islets of all
9 patients, to a level approximately half that of the surrounding exocrine
tissue (Figures 2(h)–2(j)). In contrast, there was no noticeable effect of type
II diabetes on the expression of E-cadherin, which is the major islet cadherin
and may also influence the level of β-catenin
protein in the cell by sequestering it from degradation (not shown).Becauseβ-catenin is regulated by GSK3β, we examined the state of GSK3β
activation in normal and type II diabetic pancreases. The inactive,
phosphorylated form of GSK3β, pGSK3β,  that leads to stabilization of β-catenin,
was limited to islets in both nondiabetic and diabetic
individuals (Supplementary Figure 2). In type II diabetics, 95% of islets expressed pGSK3β,
while 80% of islets from nondiabetic individuals expressed pGSK3β.
Thus, the majority of normal islets expressed pGSK3β
despite having low or undetectable levels of β-catenin,
suggesting that the upregulation of β-catenin
in islets is not controlled in a simple way by the level of pGSK3β.


### 3.4.γ-Catenin Is Expressed in Human β-, But Not α-, Cells

The low level ofβ-catenin in normal human islets in vivo
prompted us to investigate whether another molecule might be substituting for
it in mediating its signaling and/or structural roles in the β-cell.
Lower organisms such as Drosophila have a single cadherin-binding catenin,
Armadillo. However, in mammals, there are two such catenins, β-
and γ-catenin, the latter also called plakoglobin [28].
In murine β-cells, γ-catenin was upregulated when β-catenin
was deleted, but this was not noted to have any functional significance [10].
In the adult human pancreas, we found that γ-catenin
was expressed in a highly selective manner in β-cells
(Supplementary Figures 1(f)–1(h)), in contrast to α-cells
and the surrounding exocrine tissue, where it was low or undetectable (Supplementary Figures
1(i)–1(k)). Thus, α-cells
do not appear to exhibit high level expression of either catenin.The pattern of catenin expression in the adult was mirrored in the human
fetal pancreas, withγ-catenin being expressed in β-, but not α-cells (Supplementary
Figures 1(l)–1(q)). Interestingly, in the human fetal pancreas, γ-catenin
was also expressed in nonendocrine PDX-1 positive cells, being absent in PDX-1
negative cells (Supplementary Figures 1(r)–1(u)). Thus, in the human fetal pancreas, β-
and γ-catenin are both expressed in PDX-1 expressing progenitors, but are inversely regulated as those progenitors
diverge to form mature exocrine and β-cells.


### 3.5. Terminal Effectors of Wnt Signaling Are Upregulated in Human Type II Diabetes

TCF/LEF factors activate a number of terminal effectors of Wnt
signaling. A well-studied member of that group is c-myc [29].
Strikingly, c-myc was expressed in the islets from pancreases from patients
with type II diabetes, but not in islets from nondiabetic individuals (Figures 2(p)–2(s)).
Despite the role of c-myc in promoting cell proliferation, no increase in islet
cell proliferation was observed, as measured by Ki67 staining (data not shown).
In addition to c-myc, cyclinD1 is an important regulator of proliferation that
is induced by Wnt signaling [30].
It was also highly upregulated in the islets of type II diabetic patients (Figures
2(k)–2(o)).


### 3.6. Wnt Target Expression Is Limited toβ-Cells, with α-Cells Being Unaffected

While upregulation of Wnt2b occurred throughout the islet,
activation of Wnt downstream signaling was limited toβ-cells,
with α-cells
in type II diabetic islets lacking expression of activation markers, including
cyclinD1 (Figures 2(m)–2(o)). The lack of Wnt signaling in α-cells
is interesting, as significant α-cell
hyperplasia was observed in the type II diabetic pancreases (Figures 3(a), 3(b))
and is consistent with previous reports of the effect of type II diabetes on
the endocrine pancreas [31].α-cell hyperplasia in type II diabetes and expression of Wnt4 in α-cells.
Immunostaining of pancreas from nondiabetic (a), (c), and (e) and type II diabetic (b), (d), and (f) individuals
demonstrates α-cell hyperplasia in type II diabetes (a) and (b) and expression of Wnt4 (red in (c)–(f))
predominantly in α-cells (c)–(f). Scale bar in (a) and (b) = 200 μm and in (c)–(f) = 50 μm.
(a)(b)(c)(d)(e)(f)Mechanistically, the fact that Wnt2b was upregulated inα-cells,
but downstream signaling was not activated, suggested that the pathway is
defective or repressed in those cells. Occasional α-cells
expressing a low level of cyclinD1 and c-myc was more consistent with a
repressor being present than with α-cells
having a completely defective pathway. Members of the Wnt family that mediate
noncanonical Wnt signaling repress signaling through the canonical pathway
under some circumstances [15].
Thus, we studied the expression of the noncanonical Wnt, Wnt4, which microarray
studies had suggested was expressed at a high level in human islets
(unpublished). Consistent with the model, Wnt4 was expressed in α-,
but not β-cells (Figures 3(c)–3(f)).


### 3.7. Islet Isolation Mimics the Effect of Type II Diabetes onβ-Catenin Expression

To pursue the role of Wnt activation in the islet, it would
be desirable to have an in vitro
model. Thus, we examined Wnt activation in isolated islets. Surprisingly, when
cultured human islets were examined by Western blotting,β-catenin,
which is low or absent in the islet compared with surrounding tissue in situ, was expressed at a higher
level than in the nonendocrine pancreatic cells (NEPCs) [23]
(Figure 4(a)). To determine whether this was due to the process of islet
isolation or subsequent in vitro
culture in medium containing fetal bovine serum, we examined fragments from
whole pancreas that were fixed following transport using the bilayer method to
the islet isolation center [32],
but prior to islet isolation itself. In those fragments, β-catenin
induction occurred to a similar extent as in isolated islets (cf. Figures 4(b),
4(c)) marking it as an early event in the tissue procurement process. To
determine whether upregulation of β-catenin
in isolated islets indicated active canonical Wnt signaling, we examined them
for c-myc expression, finding that it was highly expressed (Figures 4(k), 4(m)).Figure 4
Wnt signaling is induced in islets in
vitro. Western blot analysis (a) indicates that β-catenin
is expressed in both cultured purified islets
and cultured purified nonendocrine pancreatic
clusters (NEPCs). Islets were dithizone picked to 99% purity, verified by qPCR as previously reported (23). As
expected, the duct marker CK 19 is expressed at
higher levels in NEPCs than in purified islets. Actin
expression confirmed equivalent sample loading.
(b) and (c) β-catenin (green in (b) and (c)) and somatostatin (red
to visualize islets) staining demonstrate a low or
absent β-catenin expression in islets of pancreatic
tissue removed and fixed immediately postmortem
(b) compared with high β-catenin
expression in islets of pancreas tissue removed
and fixed after the entire pancreas was shipped
using the bilayer method to an islet isolation center
(c). β-catenin is downregulated in cultured human
islets following transplantation under the kidney
capsule of Scid mice (d) and (e). Scale bars for (b)–(e):
50 μm. Islets isolated from nondiabetic individuals
(n=3) were cultured for 48 hours in the absence
(f), (h), and (k) or presence (g), (i), and (l) of 500 ng/mL of the Wnt
inhibitor sFRP. Immunohistochemical analysis of
insulin (green) and anti-sFRP (red in (f) and (g)) detects
sFRP on islet cells only when sFRP has been
added to the culture media. sFRP exposure led to
inhibition of β-catenin ((h) versus (i), quantitated in (j)) and
c-myc ((k) versus (l), quantitated in (m)). Error bars = mean
+/−s.e.m. *P<.05. Scale bars in (f)–(m): 25 μm.


### 3.8. The Wnt Inhibitor sFRP Reducesβ-Catenin and c-myc Expression in Human Islets

The model suggested by the data in type II diabetes and in
isolated islets is that upregulation of soluble Wnts by some aspect of the
environment in type II diabetic individuals or in isolated islets then activates
the Wnt signaling pathway. However, alternative mechanisms for Wnt pathway
activation are possible as well. For example, insulin, which is hypersecreted
at early stages in type II diabetes in response to end organ unresponsiveness,
is a potent inhibitor of GSK3 and thus a potential activator of the Wnt pathway
[33].To distinguish between Wnt-dependent and Wnt-independent
activation of downstream signaling, human islets were cultured with or without soluble
frizzled receptor protein (sFRP), which represses Wnt signaling by
sequestration of Wnt proteins [34].
sFRP bound at high levels to islets (Figure 4(g)). Consistent with canonical
activation of the pathway by secreted Wnts interacting with frizzled receptors,
sFRP caused a reduction in both cytoplasmic and membrane-associated β-catenin
(Figures 4(h)–4(j)) and c-myc (Figures 4(k)–4(m)). Thus, a Wnt inhibitor
restored human beta cells to the β-catenin
and c-myc negative state associated with normal tissue in situ, demonstrating clearly that Wnt pathway activation is
occurring and suggesting that the process of Wnt activation in isolated islets
mimics what occurs in type II diabetes.Activation of Wnt signaling in harvested islets has
implications for islet transplantation. To determine whether Wnt activation was
reversible following transplantation, three cultured islet preparations
(containing some nonendocrine material) were transplanted under the kidney
capsule of immunodeficient mice and analyzed three months later.β-catenin
expression in the islets had reverted back to levels much lower than in the
surrounding duct structures within the graft (Figures 4(d), 4(e)), similar to
the pattern found in the normal human pancreas.


### 3.9. Wnt Effectors Are Inversely Correlated with Insulin Expression in Type II Diabetes

In two pancreases from patients with type II diabetes but
none of the nondiabetic controls, there were two distinct types of islets,
exhibiting a 3.5-fold difference in the level of insulin expression in theβ-cells.
Interestingly, β-catenin
upregulation was present to a much greater degree in β-cells
with diminished insulin expression (Figures 5(a)–5(e)). This indicates either
that local factors are generated as a result of the diabetic environment or
that islets are heterogeneous, with some being more susceptible to effects of
factors that are homogeneously distributed.Figure 5
β-catenin is upregulated in islets from type II diabetic patients.
Insulin (a) and (c) and β-catenin (b) and (d) are inverely expressed in the pancreas of type II diabetic patients (a)–(d). Islets
with weak insulin expression (solid lines) had the highest levels of β-catenin and islets with strong insulin expression
(dashed lines) had levels of β-catenin intermediate between the weak insulin expressing islets and islets from
nondiabetic individuals. Quantitation of β-catenin expression in β-cells from nondiabetic and type II diabetic
individuals with strong (S) and weak (W) insulin staining (e). Insulin staining of weak islets was 3.5-fold less
intense than in islets with strong insulin staining. More than 200 islets were examined (P<.05). Amylase (red in (f))
and insulin (green in (g)) colocalized in weak insulin-expressing islets in type II diabetes (solid line) but not in high
insulin expressing islets (dashed line) (merged in (h)). Weak insulin expressing islets (marked with solid lines) retained
PDX-1 expression (red in (i)) but lost γ-catenin ((j) and (k), islet with strong insulin marked with arrowheads). Weak insulin expressing
islets (solid line) contained normal glucagon expressing cells that did not express amylase (l).
Scale bars: 50 μm. All images are confocal.Sinceγ- and β-catenin were inversely regulated in the normal pancreas, the upregulation in some type II
diabetic pancreases of β-catenin
in islets with lower insulin expression suggested the possibility that γ-catenin
expression might be downregulated in those islets. This proved to be true (Figures
5(j), 5(k)). Thus, the weak insulin-expressing islets present in the diabetic
state mimicked the pattern of catenin expression in the exocrine pancreas,
where β-catenin protein is abundant and γ-catenin is not expressed.To pursue the finding thatβ-cells
with low-insulin expression had a pattern of catenin expression resembling that
in the exocrine pancreas, pancreas sections were immunostained for the acinar
marker amylase as well as insulin, revealing that low-insulin β-cells
coexpressed amylase (Figures 5(f), 5(g), 5(h)). The insulin/amylase
double-positive cells expressed PDX-1
(Figure 5(i)), which in the adult pancreas is restricted to β-cells
and is never expressed in mature acinar cells, indicating that the weak insulin
expression was not artifactual. The finding of cells expressing both insulin
and amylase is consistent with a proposal by some investigators that endocrine
and exocrine cells can transdifferentiate [35].
Insulin-amylase double-positive cells have been found in models of β-cell
regeneration [36–38].To further explore whether the areas containing the
insulin/amylase double-positive cells arose by alteration of preexistingβ-cells
or by induction of insulin expression in preexisting exocrine cells, as has
been described in some β-cell
regeneration models [37–39],
we examined those areas for glucagon expression. Consistently, high levels of
glucagon and a lack of amylase were observed in all α-cells,
whether the islets exhibited high or low insulin expression (Figure 5(l)). Thus,
the α-cells
appeared normal, even in low-insulin expressing islets. Overall, these data
suggest that one effect of type II diabetes on the β-cell
is to promote an aberrant differentiation state in which β-cells
lose insulin expression and begin to express exocrine markers.


### 3.10. Expression of the Wnt Target Gene c-myc Is an Early Response to High-Fat Diet

To begin to address the question of whether Wnt activation was
an early or late event in diabetes pathogenesis, we moved to a mouse model. Mice
were placed on either a normal chow (low-fat) or high-fat diet and harvested
pancreases were examined for c-myc expression after 12 weeks of high-fat diet, at
which point the mice were obese (Figure6(g)), but had a normal fasting blood
glucose. Intraperitoneal glucose tolerance tests revealed mild glucose
intolerance, being marginally statistically different from the control group
only at the 90-minute time point (P=.04) (Figure 6(h)).Figure 6
Wnt signaling in high-fat fed mice. In normal mouse pancreas, β-catenin (green in (a) and (b))
is expressed in islets as identified by somatostatin (red in (a)) and colocalizes with insulin (red in
(c)). c-myc (red in (d) and (e)) was not expressed in islets of normal mice (marked by dotted lines and
glucagon in green in (d)) but was induced in islets and some ducts of high-fat fed mice (islets
marked by glucagon in green). C-myc expression is quantitated in (f). At 12 weeks, the time of
analysis, high-fat mice were obese (g), and nondiabetic but mildly glucose-intolerant as
measured by IPGTT (h). Number of normal mice = 3 and number of high-fat mice = 4 for (f), (g), and (h).
Error bars = mean +/−s.e.m. *P<.05. Scale bars: 100 μm.Consistent with previous studies [14],
we found that the murine pancreas exhibited a pattern of β-catenin
expression opposite from that in the human, with mouse islets expressing high
levels of β-catenin and the exocrine pancreas having less β-catenin
(Figures 6(a)–6(c)). However, the high level of β-catenin
expression even in the normal chow group did not reflect Wnt activation, as
c-myc was not detectable in either the endocrine or exocrine pancreas (Figures
6(d), 6(f)). In contrast, the high-fat fed animals exhibited a high level of
c-myc in islets (Figures 6(e), 6(f)).  Additionally, some but not all ducts in the
high-fat fed animals exhibited c-myc expression (Figure 6(e)). Unlike in the
human, where we were able to identify Wnt2b and Wnt4 as being expressed in the
islet, we have thus far been unable to identify the soluble Wnts responsible
for activating Wnt targets in the adult mouse pancreas. However, combined with
the human data, the results in the mouse suggest that obesity alone may be sufficient
to induce Wnt activation, which would mark it as an early event in the
pathogenesis of type II diabetes.


## 3.1. TCF7L2 Is Upregulated in Islets of Type II Diabetic Patients

Recently, TCF7L2 has been genetically linked to type II
diabetes in multiple populations [16–19]. However, the
mechanism by which it contributes to diabetes pathogenesis is unknown. The mRNA
level of TCF7L2 is higher in cultured isolated islets from type II patients
versus normal individuals [20], but expression in situ has not been examined. We
found that TCF7L2 protein was barely detectable in the pancreas of normal individuals,
but it was highly upregulated in an islet-specific manner in patients with type
II diabetes (Figures1(a)–1(e)). In addition, the expression of TCF3, which has
not been linked to type II diabetes, was also upregulated (Figures 1(f)–1(j)).Figure 1
Expression of TCF factors in type II diabetes. TCF7L2 (red in (a)–(d)) and TCF3 (red
in (f)–(i)) are absent from islets of nondiabetic individuals (a), (b), (f), and (g), but are present in islets from
type II diabetics (c), (d), (h), and (i). Islets are identified by insulin (green in (b), (d), (g), and (i)). Quantitation of
TCF7L2 (e) and TCF3 (j) expressions in nondiabetic (n=4) and type II (n=7) islets: error
bars = mean +/−s.e.m. *P<.05. Scale bars = 50 μm.


## 3.2. Wnt2b Is Upregulated in Type II Diabetes

Because TCF7L2 is both an
effector as well as a downstream target of Wnt signaling [26], the induction of TCF factors suggested that a more global
activation of Wnt signaling may be occurring. To test that hypothesis, we examined
the expression of soluble Wnt factors and the initiators of Wnt signaling. Based
upon the reported pattern of expression of soluble Wnt factor mRNA in the
pancreas, we examined Wnt2b, which was a good candidate as its RNA has been
reported to be present in isolated islets [8, 27].
In nondiabetic individuals, little or no Wnt2b expression was detectable
(Figures 2(a), 2(b), 2(c)). However, there was dramatic upregulation in the islets
of type II diabetics. Robust expression of Wnt2b occurred in both β-cells
(Figures 2(c), 2(d), 2(e)) and α-cells
(not shown) of diabetic patients.Figure 2
Expression of Wnt2b,β-catenin, cyclinD1, and
c-myc in type II diabetes. Wnt2b (red in (a), (b), (d), and (e)), β-catenin
(red in (f), (g), (i), and (j)), cyclinD1 (red in (k), (l), (n), and (o)), and c-myc
(red in (q) and (s)) are absent from islets of nondiabetic individuals (a), (b), (f), (g), (k), (l), (p) and (q), but are present in islets from type II diabetics (d), (e), (i), (j), (n), (o), (r), and (s). Islets are identified by insulin (green in (a), (d), (f), (i), (p), and (r)) or glucagon (l) and (o) and are outlined by a dashed line in (g) to better demonstrate the absence of
β-catenin. Quantitation of Wnt2b (c), β-catenin (h), and cyclinD1 (m) expression in non-diabetic (n=5) and type
II (n=7) islets: error bars = mean +/−s.e.m. *P<.05. Scale
bars = 100 μm except for f.g.i.j which are 50 μm.


## 3.3. Humanβ-Cells Lack Detectable β-Catenin Expression But It Is Strongly Upregulated  in Type II Diabetes

Activation of frizzled receptors by
soluble Wnts results in the stabilization ofβ-catenin. β-catenin
in human islets of all five nondiabetic individuals examined was markedly lower
than in the surrounding exocrine tissue, where it was strongly expressed (Figures
2(f)–2(h), Supplementary Figures 1(a), 1(b) available online at doi:10.1155/2008/728763). The same pattern was observed in
the human fetal pancreas, with high levels of β-catenin
expression in nonendocrine epithelial cells and very low levels in endocrine
cells (Supplementary Figures 1(c), 1(d)). Colocalization studies with PDX-1 revealed that β-catenin
was substantially restricted to nonendocrine PDX-1
positive, cells in the human fetal pancreas (Supplementary Figure 1(e)).
Because PDX-1 serves as a
precursor for both exocrine and endocrine compartments, β-catenin
expression must be repressed during human, but not murine, endocrine
differentiation.To determine whetherβ-catenin plays a role in β-cell
dysfunction and/or loss in human diabetes, its expression was examined in the
pancreases of 9 patients with type II diabetes. In contrast with nondiabetic
pancreases (Figures 2(f)–2(h)), it was strongly expressed in the islets of all
9 patients, to a level approximately half that of the surrounding exocrine
tissue (Figures 2(h)–2(j)). In contrast, there was no noticeable effect of type
II diabetes on the expression of E-cadherin, which is the major islet cadherin
and may also influence the level of β-catenin
protein in the cell by sequestering it from degradation (not shown).Becauseβ-catenin is regulated by GSK3β, we examined the state of GSK3β
activation in normal and type II diabetic pancreases. The inactive,
phosphorylated form of GSK3β, pGSK3β,  that leads to stabilization of β-catenin,
was limited to islets in both nondiabetic and diabetic
individuals (Supplementary Figure 2). In type II diabetics, 95% of islets expressed pGSK3β,
while 80% of islets from nondiabetic individuals expressed pGSK3β.
Thus, the majority of normal islets expressed pGSK3β
despite having low or undetectable levels of β-catenin,
suggesting that the upregulation of β-catenin
in islets is not controlled in a simple way by the level of pGSK3β.


## 3.4.γ-Catenin Is Expressed in Human β-, But Not α-, Cells

The low level ofβ-catenin in normal human islets in vivo
prompted us to investigate whether another molecule might be substituting for
it in mediating its signaling and/or structural roles in the β-cell.
Lower organisms such as Drosophila have a single cadherin-binding catenin,
Armadillo. However, in mammals, there are two such catenins, β-
and γ-catenin, the latter also called plakoglobin [28].
In murine β-cells, γ-catenin was upregulated when β-catenin
was deleted, but this was not noted to have any functional significance [10].
In the adult human pancreas, we found that γ-catenin
was expressed in a highly selective manner in β-cells
(Supplementary Figures 1(f)–1(h)), in contrast to α-cells
and the surrounding exocrine tissue, where it was low or undetectable (Supplementary Figures
1(i)–1(k)). Thus, α-cells
do not appear to exhibit high level expression of either catenin.The pattern of catenin expression in the adult was mirrored in the human
fetal pancreas, withγ-catenin being expressed in β-, but not α-cells (Supplementary
Figures 1(l)–1(q)). Interestingly, in the human fetal pancreas, γ-catenin
was also expressed in nonendocrine PDX-1 positive cells, being absent in PDX-1
negative cells (Supplementary Figures 1(r)–1(u)). Thus, in the human fetal pancreas, β-
and γ-catenin are both expressed in PDX-1 expressing progenitors, but are inversely regulated as those progenitors
diverge to form mature exocrine and β-cells.


## 3.5. Terminal Effectors of Wnt Signaling Are Upregulated in Human Type II Diabetes

TCF/LEF factors activate a number of terminal effectors of Wnt
signaling. A well-studied member of that group is c-myc [29].
Strikingly, c-myc was expressed in the islets from pancreases from patients
with type II diabetes, but not in islets from nondiabetic individuals (Figures 2(p)–2(s)).
Despite the role of c-myc in promoting cell proliferation, no increase in islet
cell proliferation was observed, as measured by Ki67 staining (data not shown).
In addition to c-myc, cyclinD1 is an important regulator of proliferation that
is induced by Wnt signaling [30].
It was also highly upregulated in the islets of type II diabetic patients (Figures
2(k)–2(o)).


## 3.6. Wnt Target Expression Is Limited toβ-Cells, with α-Cells Being Unaffected

While upregulation of Wnt2b occurred throughout the islet,
activation of Wnt downstream signaling was limited toβ-cells,
with α-cells
in type II diabetic islets lacking expression of activation markers, including
cyclinD1 (Figures 2(m)–2(o)). The lack of Wnt signaling in α-cells
is interesting, as significant α-cell
hyperplasia was observed in the type II diabetic pancreases (Figures 3(a), 3(b))
and is consistent with previous reports of the effect of type II diabetes on
the endocrine pancreas [31].α-cell hyperplasia in type II diabetes and expression of Wnt4 in α-cells.
Immunostaining of pancreas from nondiabetic (a), (c), and (e) and type II diabetic (b), (d), and (f) individuals
demonstrates α-cell hyperplasia in type II diabetes (a) and (b) and expression of Wnt4 (red in (c)–(f))
predominantly in α-cells (c)–(f). Scale bar in (a) and (b) = 200 μm and in (c)–(f) = 50 μm.
(a)(b)(c)(d)(e)(f)Mechanistically, the fact that Wnt2b was upregulated inα-cells,
but downstream signaling was not activated, suggested that the pathway is
defective or repressed in those cells. Occasional α-cells
expressing a low level of cyclinD1 and c-myc was more consistent with a
repressor being present than with α-cells
having a completely defective pathway. Members of the Wnt family that mediate
noncanonical Wnt signaling repress signaling through the canonical pathway
under some circumstances [15].
Thus, we studied the expression of the noncanonical Wnt, Wnt4, which microarray
studies had suggested was expressed at a high level in human islets
(unpublished). Consistent with the model, Wnt4 was expressed in α-,
but not β-cells (Figures 3(c)–3(f)).


## 3.7. Islet Isolation Mimics the Effect of Type II Diabetes onβ-Catenin Expression

To pursue the role of Wnt activation in the islet, it would
be desirable to have an in vitro
model. Thus, we examined Wnt activation in isolated islets. Surprisingly, when
cultured human islets were examined by Western blotting,β-catenin,
which is low or absent in the islet compared with surrounding tissue in situ, was expressed at a higher
level than in the nonendocrine pancreatic cells (NEPCs) [23]
(Figure 4(a)). To determine whether this was due to the process of islet
isolation or subsequent in vitro
culture in medium containing fetal bovine serum, we examined fragments from
whole pancreas that were fixed following transport using the bilayer method to
the islet isolation center [32],
but prior to islet isolation itself. In those fragments, β-catenin
induction occurred to a similar extent as in isolated islets (cf. Figures 4(b),
4(c)) marking it as an early event in the tissue procurement process. To
determine whether upregulation of β-catenin
in isolated islets indicated active canonical Wnt signaling, we examined them
for c-myc expression, finding that it was highly expressed (Figures 4(k), 4(m)).Figure 4
Wnt signaling is induced in islets in
vitro. Western blot analysis (a) indicates that β-catenin
is expressed in both cultured purified islets
and cultured purified nonendocrine pancreatic
clusters (NEPCs). Islets were dithizone picked to 99% purity, verified by qPCR as previously reported (23). As
expected, the duct marker CK 19 is expressed at
higher levels in NEPCs than in purified islets. Actin
expression confirmed equivalent sample loading.
(b) and (c) β-catenin (green in (b) and (c)) and somatostatin (red
to visualize islets) staining demonstrate a low or
absent β-catenin expression in islets of pancreatic
tissue removed and fixed immediately postmortem
(b) compared with high β-catenin
expression in islets of pancreas tissue removed
and fixed after the entire pancreas was shipped
using the bilayer method to an islet isolation center
(c). β-catenin is downregulated in cultured human
islets following transplantation under the kidney
capsule of Scid mice (d) and (e). Scale bars for (b)–(e):
50 μm. Islets isolated from nondiabetic individuals
(n=3) were cultured for 48 hours in the absence
(f), (h), and (k) or presence (g), (i), and (l) of 500 ng/mL of the Wnt
inhibitor sFRP. Immunohistochemical analysis of
insulin (green) and anti-sFRP (red in (f) and (g)) detects
sFRP on islet cells only when sFRP has been
added to the culture media. sFRP exposure led to
inhibition of β-catenin ((h) versus (i), quantitated in (j)) and
c-myc ((k) versus (l), quantitated in (m)). Error bars = mean
+/−s.e.m. *P<.05. Scale bars in (f)–(m): 25 μm.


## 3.8. The Wnt Inhibitor sFRP Reducesβ-Catenin and c-myc Expression in Human Islets

The model suggested by the data in type II diabetes and in
isolated islets is that upregulation of soluble Wnts by some aspect of the
environment in type II diabetic individuals or in isolated islets then activates
the Wnt signaling pathway. However, alternative mechanisms for Wnt pathway
activation are possible as well. For example, insulin, which is hypersecreted
at early stages in type II diabetes in response to end organ unresponsiveness,
is a potent inhibitor of GSK3 and thus a potential activator of the Wnt pathway
[33].To distinguish between Wnt-dependent and Wnt-independent
activation of downstream signaling, human islets were cultured with or without soluble
frizzled receptor protein (sFRP), which represses Wnt signaling by
sequestration of Wnt proteins [34].
sFRP bound at high levels to islets (Figure 4(g)). Consistent with canonical
activation of the pathway by secreted Wnts interacting with frizzled receptors,
sFRP caused a reduction in both cytoplasmic and membrane-associated β-catenin
(Figures 4(h)–4(j)) and c-myc (Figures 4(k)–4(m)). Thus, a Wnt inhibitor
restored human beta cells to the β-catenin
and c-myc negative state associated with normal tissue in situ, demonstrating clearly that Wnt pathway activation is
occurring and suggesting that the process of Wnt activation in isolated islets
mimics what occurs in type II diabetes.Activation of Wnt signaling in harvested islets has
implications for islet transplantation. To determine whether Wnt activation was
reversible following transplantation, three cultured islet preparations
(containing some nonendocrine material) were transplanted under the kidney
capsule of immunodeficient mice and analyzed three months later.β-catenin
expression in the islets had reverted back to levels much lower than in the
surrounding duct structures within the graft (Figures 4(d), 4(e)), similar to
the pattern found in the normal human pancreas.


## 3.9. Wnt Effectors Are Inversely Correlated with Insulin Expression in Type II Diabetes

In two pancreases from patients with type II diabetes but
none of the nondiabetic controls, there were two distinct types of islets,
exhibiting a 3.5-fold difference in the level of insulin expression in theβ-cells.
Interestingly, β-catenin
upregulation was present to a much greater degree in β-cells
with diminished insulin expression (Figures 5(a)–5(e)). This indicates either
that local factors are generated as a result of the diabetic environment or
that islets are heterogeneous, with some being more susceptible to effects of
factors that are homogeneously distributed.Figure 5
β-catenin is upregulated in islets from type II diabetic patients.
Insulin (a) and (c) and β-catenin (b) and (d) are inverely expressed in the pancreas of type II diabetic patients (a)–(d). Islets
with weak insulin expression (solid lines) had the highest levels of β-catenin and islets with strong insulin expression
(dashed lines) had levels of β-catenin intermediate between the weak insulin expressing islets and islets from
nondiabetic individuals. Quantitation of β-catenin expression in β-cells from nondiabetic and type II diabetic
individuals with strong (S) and weak (W) insulin staining (e). Insulin staining of weak islets was 3.5-fold less
intense than in islets with strong insulin staining. More than 200 islets were examined (P<.05). Amylase (red in (f))
and insulin (green in (g)) colocalized in weak insulin-expressing islets in type II diabetes (solid line) but not in high
insulin expressing islets (dashed line) (merged in (h)). Weak insulin expressing islets (marked with solid lines) retained
PDX-1 expression (red in (i)) but lost γ-catenin ((j) and (k), islet with strong insulin marked with arrowheads). Weak insulin expressing
islets (solid line) contained normal glucagon expressing cells that did not express amylase (l).
Scale bars: 50 μm. All images are confocal.Sinceγ- and β-catenin were inversely regulated in the normal pancreas, the upregulation in some type II
diabetic pancreases of β-catenin
in islets with lower insulin expression suggested the possibility that γ-catenin
expression might be downregulated in those islets. This proved to be true (Figures
5(j), 5(k)). Thus, the weak insulin-expressing islets present in the diabetic
state mimicked the pattern of catenin expression in the exocrine pancreas,
where β-catenin protein is abundant and γ-catenin is not expressed.To pursue the finding thatβ-cells
with low-insulin expression had a pattern of catenin expression resembling that
in the exocrine pancreas, pancreas sections were immunostained for the acinar
marker amylase as well as insulin, revealing that low-insulin β-cells
coexpressed amylase (Figures 5(f), 5(g), 5(h)). The insulin/amylase
double-positive cells expressed PDX-1
(Figure 5(i)), which in the adult pancreas is restricted to β-cells
and is never expressed in mature acinar cells, indicating that the weak insulin
expression was not artifactual. The finding of cells expressing both insulin
and amylase is consistent with a proposal by some investigators that endocrine
and exocrine cells can transdifferentiate [35].
Insulin-amylase double-positive cells have been found in models of β-cell
regeneration [36–38].To further explore whether the areas containing the
insulin/amylase double-positive cells arose by alteration of preexistingβ-cells
or by induction of insulin expression in preexisting exocrine cells, as has
been described in some β-cell
regeneration models [37–39],
we examined those areas for glucagon expression. Consistently, high levels of
glucagon and a lack of amylase were observed in all α-cells,
whether the islets exhibited high or low insulin expression (Figure 5(l)). Thus,
the α-cells
appeared normal, even in low-insulin expressing islets. Overall, these data
suggest that one effect of type II diabetes on the β-cell
is to promote an aberrant differentiation state in which β-cells
lose insulin expression and begin to express exocrine markers.


## 3.10. Expression of the Wnt Target Gene c-myc Is an Early Response to High-Fat Diet

To begin to address the question of whether Wnt activation was
an early or late event in diabetes pathogenesis, we moved to a mouse model. Mice
were placed on either a normal chow (low-fat) or high-fat diet and harvested
pancreases were examined for c-myc expression after 12 weeks of high-fat diet, at
which point the mice were obese (Figure6(g)), but had a normal fasting blood
glucose. Intraperitoneal glucose tolerance tests revealed mild glucose
intolerance, being marginally statistically different from the control group
only at the 90-minute time point (P=.04) (Figure 6(h)).Figure 6
Wnt signaling in high-fat fed mice. In normal mouse pancreas, β-catenin (green in (a) and (b))
is expressed in islets as identified by somatostatin (red in (a)) and colocalizes with insulin (red in
(c)). c-myc (red in (d) and (e)) was not expressed in islets of normal mice (marked by dotted lines and
glucagon in green in (d)) but was induced in islets and some ducts of high-fat fed mice (islets
marked by glucagon in green). C-myc expression is quantitated in (f). At 12 weeks, the time of
analysis, high-fat mice were obese (g), and nondiabetic but mildly glucose-intolerant as
measured by IPGTT (h). Number of normal mice = 3 and number of high-fat mice = 4 for (f), (g), and (h).
Error bars = mean +/−s.e.m. *P<.05. Scale bars: 100 μm.Consistent with previous studies [14],
we found that the murine pancreas exhibited a pattern of β-catenin
expression opposite from that in the human, with mouse islets expressing high
levels of β-catenin and the exocrine pancreas having less β-catenin
(Figures 6(a)–6(c)). However, the high level of β-catenin
expression even in the normal chow group did not reflect Wnt activation, as
c-myc was not detectable in either the endocrine or exocrine pancreas (Figures
6(d), 6(f)). In contrast, the high-fat fed animals exhibited a high level of
c-myc in islets (Figures 6(e), 6(f)).  Additionally, some but not all ducts in the
high-fat fed animals exhibited c-myc expression (Figure 6(e)). Unlike in the
human, where we were able to identify Wnt2b and Wnt4 as being expressed in the
islet, we have thus far been unable to identify the soluble Wnts responsible
for activating Wnt targets in the adult mouse pancreas. However, combined with
the human data, the results in the mouse suggest that obesity alone may be sufficient
to induce Wnt activation, which would mark it as an early event in the
pathogenesis of type II diabetes.


## 4. Discussion

The studies presented here are based primarily upon the
examination of pancreas samples from normal and type II diabetic humans, from
which we conclude that Wnt signaling effectors are upregulated in the islets in
type II diabetes.A number of components in the Wnt pathway have been found to
be associated with type II diabetes or obesity in linkage studies. Our finding
that TCF3 and TCF7L2 are induced in islets from type II diabetics is
particularly interesting in light of recent linkage association studies in
which TCF7L2 has been identified as the gene most strongly linked to type II
diabetes [17–19].
TCF7L2 is also a direct downstream target of β-catenin
[26],
which we found to be induced in type II diabetes. Similarly, lrp5, a component
of the Wnt receptor signaling complex is associated with obesity phenotypes [40]
and Wnt5b is associated with susceptibility to type II diabetes [41].
Wnt pathway genes have also been linked to type I diabetes [42].
Whether the involvement of Wnt signaling on predisposition to diabetes occurs
through effects in the islet or in peripheral tissue has not been determined.
However, we and others have established that c-myc has substantial effects on β-cell
function, exemplified by its ability to repress hormone expression and to
induce both proliferation and apoptosis [43, 44].Of relevance to the specificity of the changes in Wnt signaling
that were observed, alterations in the level of Wnt pathway components in the
human pancreas were restricted to the islet, with little change in the exocrine
pancreas. Consistent changes in Wnt pathway components were evident despite the
fact that the human pancreases were obtained from widely divergent patients.
The “normal” controls were mostly not from young healthy individuals, but
rather from older individuals with significant illness [22].
This is likely to have minimized the extent of any differences, so it is
striking that we still observed large changes in the expression of Wnt pathway
components in the samples from diabetic patients.While no single Wnt downstream effector or pathway
intermediate is absolutely specific for the Wnt pathway given the extensive cross-talk
between Wnt and other signaling pathways, the evidence for activation of Wnt signaling
inβ-cells
is strong, with pathway components at all levels being affected, including
upregulation of β-catenin,
TCF3, TCF7L2, cyclinD1, c-myc, and Wnt2b. Upregulation of Wnt2b is important,
as there cannot be true pathway activation in the absence of an initiating
signal. The downregulation of the downstream target c-myc by sFRP provides
direct evidence that Wnt activation through a soluble Wnt occurs in islets,
though the stimulus for upregulation of the soluble Wnt may or may not be the
same in isolated islets and type II diabetes.While Wnt2b was induced throughout the islet, canonical Wnt activation
was found predominantly inβ-cells.
In α-cells, the pathway remained inactive, a state that correlated with α-cell-specific
expression of Wnt4, a member of the Wnt family that mediates noncanonical
signaling and that can repress canonical signaling [15, 45].
While we observed changes in expression of Wnt2b and Wnt4, the Wnt family is
large. Attempts to examine other members are ongoing but have been limited by
the lack of high-quality antibodies. It is likely that additional complexity
will be revealed as other members of the Wnt family and other components of the
pathway are examined.Wnt binding to frizzled receptors leads to stabilization ofβ-catenin
by inactivation of GSK3β, which
is itself under complex control, including distinct regulation by growth
factors as well as Wnt signaling [46].
Of relevance to diabetes, GSK3β is
inhibited by insulin signaling through the PI3 kinase pathway [47].
Thus, hyperinsulinemia, a classic feature of type II diabetes that occurs early
in the disease, could lead quite directly to upregulation of β-catenin.
Interestingly, it has been proposed, on the basis of its involvement in insulin
signaling, that inhibitors of GSK3 may be effective in improving insulin
sensitivity in rodent models of type II diabetes [48].
However, based on data presented here, such inhibitors might lead to Wnt activation
in the islet. The inverse correlation between Wnt pathway activation and
insulin gene expression that we found suggests that, at least in the long term,
Wnt activation could be deleterious to islet function. In contrast to what is
believed to occur in the peripheral tissue, it is possible that Wnt inhibition
and consequent activation of GSK3β
could have positive effects on islet function. Thus, in one tissue, GSK3
inhibition might prove beneficial, while in another, harmful.β-cell specific Wnt effector upregulation occurred in the context of striking
differential expression of Wnt pathway components in the endocrine versus
exocrine pancreas in nondiabetic humans and mice, as well as significant
interspecies differences. The best example of this is β-catenin,
which was expressed in an inverse pattern in the normal human and murine pancreases. In humans, β-catenin
was virtually absent in islets and expressed at high levels in the exocrine
pancreas, while in the murine pancreas the reverse pattern was found. In the
human exocrine pancreas, β-catenin
appears to be localized to the plasma membrane, which is a pattern that is
consistent with its role in mediating connections between cadherins and the
actin cytoskeleton. Despite decreased β-catenin
expression in islets, only the human endocrine pancreas expressed high levels
of phosphorylated GSK3β, which in the canonical model of Wnt signaling should
have led to stabilization and upregulation of β-catenin. Unfortunately, we were unable to assess the expression of the unphosphorylated,
active form of GSK3β due to limitations of the available antibodies.γ-catenin was expressed in a converse fashion to β-catenin
in islets from nondiabetic individuals. Further, the dynamic regulation of β-
and γ-catenin, with β-catenin being induced and γ-catenin
repressed in islets with activated Wnt signaling, suggests that these molecules
play an important role in diabetes pathogenesis. In some tissues, β-
and γ-catenin act antagonistically, with β-catenin tending to promote cell
growth, while γ-catenin acts as a tumor suppressor [49, 50]. While both β- and γ-catenin
can bind to TCF/LEF transcription factors, γ-catenin
does so much less efficiently [51].
In addition, we have found that TCF genes are specifically activated by β-,
but not γ-catenin (unpublished results).Wnt pathway activation in isolated islets has implications
for islet transplantation. Islet dysfunction and loss is a major problem in
islet transplantation, both in the immediate posttransplant period and
chronically [52].
The finding that the process of isolation induces some changes that are also
seen in type II diabetes raises the possibility that the isolation process
could have deleterious effects. If, as indicated by the known undesirable
effects of c-myc on islet biology [43, 44, 53],
Wnt activation has a negative effect on β-cell
function and/or survival, inhibiting that upregulation in transplanted islets could
be beneficial. The demonstration here that β-catenin
upregulation is reversible following renal subcapsular transplantation or by inhibiting
Wnt signaling with sFRP provides a direct means of testing that hypothesis and
could also provide a model system to study the effects of Wnt pathway
activation in type II diabetes.A question of major importance is the functional role of Wnt
signaling in diabetes pathogenesis, that is, is it part of an adaptive response
or a pathologic response? These are not mutually exclusive. One possibility is
that Wnt activation plays an adaptive role early in type II diabetes, perhaps
in promotingβ-cell proliferation [13],
while chronic pathway activation leads to cell death, a well-recognized
function of c-myc [44, 54].
The mouse studies suggest but do not yet prove definitively that Wnt activation
could be an early event in diabetes pathogenesis that is associated with high-fat
diet.The striking activation of Wnt signaling in human type II
diabetes, albeit in a small number of patients,  provides insight into the molecular effects of
diabetes on theβ-cell and offers a novel potential route to prevent β-cell
failure. Direct manipulation
of the activation state of the pathway is required to dissect out the complex
roles of the Wnt pathway in diabetes. In addition, diabetes has been shown to be a
strong risk factor for cancer, including pancreatic cancer [55, 56]. Moreover, overexpression of  β-catenin in the mouse pancreas promoted pancreatic cancer [57]. Thus, activated Wnt signaling may be a
critical mechanism of pathogenesis common to both diabetes and pancreatic
cancer.



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*Source: 728763-2009-01-20.xml*