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Diabetes Mellitus Type II
Beta Cell Failure in DM T2
signaling pathways implicated in β-cell failure
Controls organismal
growth and differentiation
Wnt Signaling Pathway
Wnt signalling Pathway and DM T2
1. Homozygous mutation of LRP5 in mice
leads to defective glucose-stimulated
insulin secretion from isolated islets in vitro.
2. 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.
3. TCF7/L2 gene polymorphism affects B-cell
function
While most studies have found little evidence
that Wnt signaling is involved in endocrine
differentiation or in the adult islet,
there is some evidence for an
effect of Wnt signaling on
β-cell replication.
Main Objective
To study the role of Wnt signaling
in human diabetes
Systematically examined
components of the
pathway in the pancreas
of normal & DMT2
Mouse Studies
METHODS
TISSUE PREPARATION
1. Paraffin-embedded from human pancreases: 5 non-diabetic & 9 DM T2
2. Freshly isolated & cultured pancreases: 3 non-diabetic (Isolated human islets)
3. Human Fetal Pancreases in 18-24 gestational weeks
4. Murine Pancreaes (Balb/c or C57/bl mice)
5. NEPCs
IMMUNO-HISTOCHEMISTRY
1. Paraffin samples
2. Frozen samples
WESTERN BLOT
1. Whole-cell extracts
I. Objective:
To determine if
TCF7L2
is upregulated in islets
of type II diabetic patients
•TCF7L2 is a protein acting as a
transcription factor.
•Several SNPs of the gene are
associated with Type 2 DM
TCF/L2
TCF3
TCF7L2 is upregulated in
islets of type II diabetic
patients
II. Objective:
To determine if
Wnt2b
is upregulated in
type II diabetes
To test the hypothesis that the induction
of TCF factors, being a both effector
as well as a downstream target of
Wnt Signaling, is a result of a more
global activation of Wnt signaling
B Catenin
Wnt2b
Wnt 2b is upregulated in
Type 2 Diabetes
In islets
III. Objective:
To determine if
B-catenin
is upregulated in
type II diabetes
B-catenin
B-catenin
insulin
Human B-cellsB-Catenin
lack
In islets
Detectable B-catenin
expression but it
is highly upregulated in
DM T2
islet
Normal
Normal
Beta-catenin in human islets of all
5 nondiabetics was markedly lower
Than in the surrounding exocrine
Tissue where it was strongly
Expressed (figs. 2f-2h)
insulin
insulin
DM T2
B-catenin
B-catenin
DM T2
Beta-catenin is strongly expres
in the islets of all DM T2 to a
Level approximately half that of the
Surrounding exocrine tissue
(figs 2h-2j)
IV. Objective:
To determine if the terminal effectors
(c-myc & cyclin D) are upregulated in DM T2
TCF/LEF factors activate a number of
terminal effectors Of Wnt signaling
(c-myc and cyclinD1)
c
NORMAL
Terminal effectors of
CyclinD1
C-myc
Wnt signaling
In islets
DM T2
In(c-myc
islets & cyclinD1)
are upregulated in
human type 2 Diabetes
MOUSE MODEL
V. Objective:
To determine whether Wnt
activation was an early or late
event in diabetes pathogenesis
normal chow
high-fat diet
12 weeks
OBESE BUT WITH NORMAL FBS
Harvested pancreases examined
for c-myc expression
High-fat
Expression of the
Wnt target gene c-myc
is an early response
to high-fat diet
Normal
B -catenin
Normal
C-myc
Normal
Normal
Islets
High-Fat
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).
Summary
1. B-catenin in human islets of all 5 nondiabetics
was markedly lower than in the surrounding
exocrine tissue, where it was strongly
expressed.
2. Expression of the Wnt2b, B-catenin, TCF7/L2, &
terminal effectors (c-myc & cyclinD1) are all
upregulated in the islets in type II diabetes
3. The mouse model suggested 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.
Conclusion
Type II Diabetes activates the
Wnt signaling pathway
specifically in the Beta cells
of the islets of Langerhans
Thank
You!!!
• 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.
• 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, βcatenin, 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 pursue the finding that β-cells with lowinsulin 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 βcells coexpressed amylase (Figures 5(f), 5(g),
5(h)).
• The insulin/amylase doublepositive 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.
• To further explore whether the areas containing
the insulin/amylase double-positive cells arose by
alteration of preexisting β-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)).
• Figure 1. Schematic representation of WNT/TCF signalling. Secreted
WNTs bind to FZD and LRP receptors, which in turn inactivate the
degradation complex comprising AXIN, DVL and GSK3B.
• This results in non-phosphorylated b-catenin entering into the nucleus
and binding to TCF7L2, thus activating a wide variety of genes.
• TCF7L2 could regulate several genes – tissue specifically influencing
both insulin secretion and insulin sensitivity. For example, TCF7L2
regulates b-cell growth and differentiation. TCF7L2 also activates the
expression of proglucagon gene, which encodes the GLP-1 (glucagons
like peptide-1) and thus promotes insulin secretion. Alterations in this
pathway (in TCF7L2 risk variants) could lead to reduced secretion of
GLP-1 and hence defective insulin secretion.
• In addition, altered WNT signalling (in TCF7L2 risk variants) could be
expected to influence adipose tissue growth and development and thus
BMI. Increased pro-inflammatory signals (IL-6, TNF-a) and altered
adiponectin from the adipocytes (through their endocrine function)
might result in skeletal muscle insulin resistance. With regard to
microvascular complications of diabetes, TCF7L2 may also influence
mesangial cell expansion and retinal neovascularization.