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Pten regulates beta-cell regeneration intrinsically and independently of development

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Pten regulates beta-cell regeneration intrinsically and independently of development

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i

PTEN REGULATES BETA-CELL REGENERATION INTRINSICALLY AND
INDEPENDENTLY OF DEVELOPMENT

by

Jennifer-Ann Bayan

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

August 2012

Copyright 2012 Jennifer-Ann Bayan

ii

Table of Contents

List of Figures iii
Abstract v
Chapter I: Overview of β-cell function and regeneration, PI3K/PTEN/
AKT signaling pathway, and stellate cell contribution

1
I-1 β-cell function, regeneration and maintenance
I-2 PI3K/PTEN/AKT signaling and β-cell growth
I-3 β-Cells and the extracellular matrix

1
5
9
Chapter II: Adult onset deletion of PTEN increases proliferation of
islet β-cells

11
II-1 Introduction
II-2 Results
II-2-1 Generation of Pten
loxP/loxP
; Rosa
lacZ
; Rip-CreER mice
II-2-2 Deletion of Pten in adult pancreas results in increased
islet area and enhanced endocrine function
II-2-3 Pten deletion in adult mice leads to increase proliferation
and blocks cell death induced by STZ
II-2-4 Pten deletion rescues the loss of proliferation response
in aged β-cells
II-2-5 Cell-cycle regulation is altered when Pten in deleted in
aged β-cells
II-3 Discussion

11
13
13
18

23

28

31

36
Chapter III: Activation of extracellular matrix (ECM) in response to
deletion of Pten in β-cells

43
III-1 Introduction
III-2 Results
III-2-1 A paracrine effect of β-cell PTEN loss on STZ-treated
mice
III-2-2 Stellate Cells support the growth of β-cells
III-3 Discussion

43
45
45

54
59

Chapter IV: Overall Discussion

63

Chapter V: Materials and Methods

Bibliography
68

76

iii

List of Figures

Figure 1: Schematic representation of β-cell mass correlated with age

Figure 2: Schematic representation of PI3K/PTEN/AKT signaling

Figure 3: Efficiency of Pten deletion in Pten
loxP/loxP
; Rosa26
lacZ
; RIPCreER
+
mice
treated with tamoxifen

Figure 4: Body and pancreas weight of control and Pten null animals

Figure 5: Deletion of Pten in adult β-cells leads to increased relative islet area
and improved glucose homeostasis

Figure 6: Expression of β-gal in β-cells of tamoxifen-treated Pten null group

Figure 7: Glucose and insulin tolerance of Control and Pten null animals

Figure 8: Deletion of Pten in adult β-cells leads to increased cell proliferation

Figure 9: Analysis of apoptosis in control and Pten null mice

Figure 10: Deletion of Pten in adult β-cells prevents STZ-induced apoptosis

Figure 11: Deletion of Pten in aged (>1 year) β-cells leads to expansion of
relative islet area and improved glucose homeostasis

Figure 12: Deletion of Pten in aged (>1 year) β-cells leads to increased cell
proliferation

Figure 13: Deletion of Pten in adult β-cells leads to changes is cell-cycle
regulators.

Figure 14: Long-term deletion of Pten (15 months) does not lead to
development of insulinomas

Figure 15: Mitotic activity is observed in non-insulin positive cells adjacent to the
islets

Figure 16: Peri-islet proliferating cells are confined to extra-islet tissues

Figure 17: Proliferating activity is mostly confined to β-cells in non-treated mice

iv

Figure 18: Peri-islet proliferating cells induced by STZ treatment do not express
insulin

Figure 19: Generation of Pten
loxP/loxP
; Rosa26
lacZ
; RIP-Cre mice

Figure 20: Dedifferentiation of β-cells is unlikely to contribute to the peri-islet
proliferating activity induced by STZ

Figure 21: Exocrine or other stem/progenitor cell types do now contribute to the
extensive mitotic activity surrounding the islets in the STZ-treated Pten null
pancreas

Figure 22: Inflammatory cell infiltration in the STZ-treated mice

Figure 23: PTEN loss leads to expansion of surrounding mesenchymes and
pancreatic stellate cells

Figure 24: Stellate cells support the growth of β-cells in vitro

Figure 25: β-cell regeneration is maintained in Pten-null β-cells without
involvement of developmental factors and injury

v

Abstract

The pancreatic β-cells are responsible for producing insulin and maintaining
glucose homeostasis. Loss of β-cells or their inability to compensate for insulin
resistance is the major cause for type 1 and type 2 diabetes, respectively.
Increasing β-‐cell mass or generating optimally functional islets in vitro for
transplantation could potentially improve or cure type 1 diabetic conditions.
Thus, efforts have been focused on improving β-cell mass by understanding and
manipulating the mechanisms involved in their differentiation, proliferation and
regeneration. Phosphatase and tensin homolog on chromosome 10 (PTEN) is a
lipid phosphatase that antagonizes the function of the phosphatidylinositol-3-
kinase (PI3K) signaling pathway. Targeted deletion of Pten in insulin producing
cells led to a significant increase in total islet mass. This study suggests that β-
cell regeneration may be under mitogenic regulation and that PTEN may be
regulating β-cell regeneration in a paracrine fashion. Using a conditional knock-
out mouse model inducing the deletion of Pten, the data show that this deletion in
adult and aged β-cells induced their proliferation and increased their islet mass.
These mice showed enhanced metabolic function and resistance to
streptozotocin (STZ)-induced diabetes. There was also evidence of G1/S protein
involvement and no insulinoma development. The role of stellate cells in the
growth and regeneration of pancreatic β-cells was then examined. Deletion of
Pten was accompanied by an increase in activity surrounding islets treated with
STZ. The cells surrounding the islets were activated mesenchymal cells

vi

displaying markers of pancreatic stellate cells. Further studies demonstrated that
these activated stellate cells support the proliferation of β-cells. Therefore, the
loss of PTEN specifically in the β-cells leads to a paracrine effect inducing the
proliferative activity of surrounding mesencymal cells. Together, our present
study suggests that the signaling regulated by PTEN plays a key role in β-cell
proliferation and maintenance and β-cell regeneration may be under mitogenic
regulation with PTEN regulating β-cell regeneration in a paracrine fashion.

1

Chapter I
Overview of β-cell function, regeneration, PI3K/PTEN/AKT signaling
pathway

I-1 β-cell function, regeneration and maintenance
Diabetes is a growing epidemic worldwide. It is a syndrome that is
characterized by metabolic dysregulation and hyperglycemia resulting from either
low levels of insulin or insulin resistance coupled with insufficient insulin secretion
to compensate (Saltiel and Kahn, 2001). Diminished insulin production by β-cells
to meet metabolic demands is a hallmark for the development of diabetes
mellitus. Loss of β-cell mass or inadequate function of β-cells is the major reason
for the diminished insulin production. Type 1 diabetes is characterized by an
autoimmune attack of pancreatic β-cells. Type 2 diabetes develops from
peripheral insulin resistance and β-cell failure (Donath et al., 2005; Gianani and
Eisenbarth, 2005). Currently, treatment for type 1 diabetes is limited to insulin
replacement therapy and islet transplantation, both with significant drawbacks
and side effects. Type 2 diabetes can be controlled with diet and exercise or with
oral pharmaceutical agents (i.e. secretagogues or biguanides), however if not
properly regulated, insulin replacement therapy must be initiated (Mastrandrea
and Quattrin, 2006).
Replenishing β-cell mass is a key factor of any therapeutic intervention for
insulin-dependent diabetes. The induction of stem cells to a β-cell-like
phenotype is the primary strategy currently under investigation (D'Amour et al.,
2006; Kelly et al., 2011; Prabakar et al., 2011). However, there are number of

2

other approaches with great therapeutic potential. Three of these approaches
focus on the utilization and development of cells that reside in the pancreas,
including, but not limited to, progenitor cells that can be used for endogenous
regeneration, acinar or ductal cells that can be used for reprogramming, and
residual β-cells for self-renewal or expansion.
The pathways and mechanisms that underlie islet cell growth and
replication are largely unknown. However, various factors have been associated
with β-cell growth. These include nutrients such as glucose, hormones (i.e.
glucagon-like peptide-1), and growth factors (i.e. insulin-like growth factors (IGF),
fibrobast growth factors (FGF), and hepatocyte growth factors (HGF)) (Kulkarni,
2004). Although during embryogenesis replication and neogenesis contribute to
the growth and development of the pancreas, in the adult pancreas, the
proliferation rate is relatively low, with approximately 0.2% mitotic activity per day
(Teta et al., 2005). It is only during times of metabolic stress and/or pancreatic/β-
cell injury that the mitotic activity of β-cells increases and β-cell regeneration
occurs. Unfortunately, the precise model of β-cell origin and the mechanism(s) of
adult β- cell regeneration still need elucidation. To date, several theories of β-cell
origin and pathways of regeneration have been postulated. One theory suggests
that β-cells regenerate from a pancreatic stem or progenitor cell. Early
immunohistochemical analyses have provided evidence for the presence of
pancreatic stem/progenitor cells, or markers for these cells, in both islets and
ducts (Bonner-Weir and Sharma, 2002). A second theory suggests that

3

transdifferentiation from other cell types residing in the pancreas leads to
regeneration of β-cells (De Haro-Hernandez et al., 2004; Simeone et al., 2006).
De Haro-Hernandez, et al. have shown evidence of β-cell regeneration and
neogenesis from small clusters and acinar cells (De Haro-Hernandez et al.,
2004). Through the use of direct lineage analysis, a third theory was proposed.
This study demonstrated that the adult β-cells are formed by self-duplication
rather than stem cell differentiation (Brennand et al., 2007; Dor et al., 2004; Teta
et al., 2007). Genetic lineage tracing studies using the tamoxifen inducible
Cre/lox system pioneered by Dor, et al, suggests that terminally differentiated β-
cells retain a significant proliferative capacity in vivo and are responsible for β-
cell regeneration (Dor et al., 2004). These latter studies challenge the theory that
adult stem cells are responsible for β-cell regeneration under normal
physiological conditions.
Due to the slow turn-over of β-cells, the endocrine pancreas is considered
to be primarily post-mitotic (Teta et al., 2005). The number of insulin-producing
cells was thought to remain unchanged throughout adult life, unless reduced by
aging or disease. However, this theory no longer holds true based on research
and evidence that β-cells have the ability to adapt their mass and proliferative
activity in a dynamic fashion in response to a number of stimuli. In humans,
evidence has demonstrated the capability of dynamic perinatal β-cell expansion
and regeneration (Meier et al., 2008). This is primarily based on the observation
that there are residual β-cells in patients with type 1 diabetes even decades after

4

disease onset (Meier et al., 2006). Pathological factors that dictate the adaptive
potential of β-cell mass include hyperglycemia (Bonner-Weir et al., 1989), obesity
(Butler et al., 2003a), mutations (Kassem et al., 2010), or pregnancy (Kjos and
Buchanan, 1999). Other factors are the result of experimental interventions such
as streptozotocin treatment (Fernandes et al., 1997), partial pancreatectomy
(Bonner-Weir et al., 1993), and ductal ligation (Xu et al., 2008).
Increasing β-cell mass or generating optimally functional islets in vitro for
transplantation are strategies that can improve or cure diabetic conditions. Thus,
efforts have been focused on improving β-cell mass by understanding and
manipulating the mechanisms involved in their differentiation, proliferation and
regeneration (Figure 1).

Figure 1. Schematic representation of β-cell mass correlated with age. During
embryogenesis, replication and neogenesis contribute to the growth and development of the
pancreas. In the adult pancreas, the proliferation rate is relatively low. β-cells have the ability to
adapt their mass and proliferative activity in a dynamic fashion in response to a number of stimuli,
including type 1 and 2 diabetes, obesity, and pregnancy.

5

I-2 PI3K/PTEN/AKT signaling and β-cell growth

Several growth factors in the insulin signaling pathway, including insulin,
insulin-like growth factor (IGF) and hepatic growth factor (HGF), have been
shown to play important roles in regulating insulin secretion from β-cells and
determining cell and organ size. The actions of insulin and insulin-like growth
factor-1 (IGF-1) are mediated, in part, by the PI3K/PTEN/AKT pathway and play
a critical role in regulating β-cell mass and function (Nguyen et al., 2006; Tuttle et
al., 2001). Studies done where the insulin or IGF-1 receptor was deleted,
revealed an impairment of differentiated β-cell function, and identified the insulin
receptor substrate 2 (IRS-2) as a key factor in determining β-cell mass (Nguyen
et al., 2006). Beta-cell specific deletion of IGF-1 under the insulin 1 promoter
didn’t increase islet mass, but there was a three-fold increase in β-cell
proliferation in the islets (George et al., 2002). Studies in which IGF-2 is over
expressed showed a three- to five-fold increase in islet mass with enhanced
proliferation of α- and β-cells (Devedjian et al., 2000; Petrik et al., 1999). Specific
deletion of the β-cell insulin receptor (Ir) exhibits impaired glucose tolerance,
reduced β-cell mass and decreased islet number (Kulkarni et al., 1999; Otani et
al., 2004). These studies showed that when the functional receptors for insulin
are lost specifically in β-cells, this leads to defects in postnatal β-cell growth. In
other genetic studies, mice lacking HGF receptor, specifically in the β-cells, suffer
from enhanced apoptosis and smaller islet mass (Dai et al., 2005; Mellado-Gil et
al.; Roccisana et al., 2005). Conversely, overexpressing HGF leads to increased

6

proliferation as well as enhanced islet function. Furthermore, other studies have
shown that hyperactivation of the PI3K/AKT pathway enhance β-cell survival,
proliferation, size, and increase islet mass and number (Bernal-Mizrachi et al.,
2001; Tuttle et al., 2001). Although many studies have illustrated the importance
of the IGF-1/PI3K/AKT signaling pathway in β-cell growth and survival, little is
known about the negative regulation of this pathway.
Phosphatase and tensin homolog on chromosome 10 (PTEN) is a key cell
growth regulator that functions as a lipid phosphatase that antagonizes the
functions of phosphatidylinositol-3-kinase (PI3K) by dephosphorylating
phosphatidylinositol triphosphate (PIP3) and overall negatively regulates the
phosphatidylinositol-3- kinase (PI3K)/Protein kinase B (AKT) signaling pathway
(Stiles et al., 2006). AKT pathway activation has been shown to lead to a
number of cellular responses including cell growth, survival, and metabolism
(Manning and Cantley, 2007). AKT is an important mediator in cell growth and
proliferation. Increased β-cell size has been shown to contribute to the
augmented β-cell mass observed in the constitutively active Akt transgenic mice
(Bernal-Mizrachi et al., 2001; Tuttle et al., 2001). These mice also demonstrated
an increase in the number of islets as well as increased efficiency of insulin
secretion when challenged with glucose. Conversely, loss of Akt lead to increase
apoptosis and decreased proliferation (Elghazi et al., 2006; Stiles et al., 2002).
Beta-cells expressing a kinase-dead dominant-negative form of AKT resulted in
decreased insulin secretion, albeit without changes in β-cell mass (Bernal-

7

Mizrachi et al., 2004). Genetic deletion of AKT2 displayed mild hyperglycemia
along with an increase in β-cell mass, which indicates that the β-cells are able to
compensate for the increased glucose levels (Cho et al., 2001). Other factors
regulated by AKT involved in β-cell proliferation, cells size, and insulin signaling
are those regulated by TSC1/2, including mTOR and S6K. Studies performed
using mice with β-cell-specific Tsc2 deletion have demonstrated that mTORC1 is
an important factor in regulating β-cell size. These mice are hypoglycemic,
display increased β-cell proliferation and improved glucose metabolism (Rachdi
et al., 2008; Shigeyama et al., 2008). S6K1-deficient mice studies suggest that
S6K is major candidate involved in regulating β-cell function. S6K1-deficient mice
exhibit hypoinsulinemia, impaired insulin secretion, glucose intolerance and
reduced β-cell size (Pende et al., 2000). The β-cell phenotype is also controlled
by factors that regulate AKT upstream of the PI3K pathway. Furthermore, AKT
mediates survival via inactivating phosphorylations of the pro-apoptotic proteins
BAD, as well as caspases 3 and 9 and regulating cell cycle modulators p21, p27
and cyclin D in order to stimulate β-cell proliferation (Cozar-Castellano et al.,
2006).
In a double transgenic mouse study of Pten and Irs2 (Irs2-/-; Pten+/-), the
authors found improved peripheral insulin sensitivity, rescued islet growth, and
sustained islet function in Irs2-/- mice with Pten haploinsufficiency (Kushner et
al., 2005b). These results were substantiated by studies done by Stiles, et al and
Nguyen, et al, who found that tissue-specific knockout of Pten in β-cells is

8

essential in glucose metabolism and is able to increase islet mass and maintain
resistance to streptozotocin (STZ)-induced diabetes without compromising β-cell
function (Nguyen et al., 2006; Stiles et al., 2006). Therefore, the manipulation of
the IGF-1/PI3K/AKT signaling pathway may be a promising approach to regulate
β-cell maintenance (Figure 2).

Figure 2. Schematic representation of PI3K/PTEN/AKT signaling. PTEN is a lipid
phosphatase that negatively regulates the PI3K signaling pathway. Deletion of Pten activates
AKT, which controls a number of downstream factors that influence proliferation, cell survival,
translation, cell size, and cell metabolism.

Caspase
BAD

PIP2
PIP3

p27
p21

S6K
Cyclin D
GSK3α/β
TSC

9

I-3 β-Cells and the extracellular matrix

The extracellular matrix (ECM) is composed of a framework of collagen
proteins that make up the basement membrane (BM), which is a sheet-like ECM
associated with epithelia and various specialized cell types, including the cells of
the endocrine pancreas. In addition to the ECM’s role in structure, cellular
differentiation, behavior and function, it is known to bind to and act as a reservoir
for growth factors that affect cell behaviors and regulate growth factor activity
(Ramirez and Rifkin, 2009; Wang et al., 2008; Zacchigna et al., 2006; Zhu et al.,
1999). The pancreatic islets are densely vascularized structures that are unable
to form their own BM. Instead they work in conjunction with the BM laid down by
islet endothelial cells. In mice, signals that affect β-cell function and its
proliferative potential involve interactions between β-cell surface β1-integrins and
laminins of the endothelial BM (Nikolova et al., 2006). Various studies have
shown that BM components (i.e. laminin and collagen IV) have important effects
on β-cells, including increase in insulin gene transcription, insulin secretion, and
β-cell survival rates and proliferation (Kragl and Lammert, 2010).
In the pancreas, pancreatic stellate cells are a type of myofibroblast that
can be activated during injury. The stellate cells, when activated, can proliferate
and migrate to the site of tissue injury (Omary et al., 2007). They lay down
extracellular matrix proteins to promote repair of damaged tissues. Stellate cells
have been shown to display different gene expression profiles, including glial
fibrillary acidic protein (GFAP) and nestin when dormant and smooth muscle

10

actin α (SMAα) and desmin when activated (Omary et al., 2007).
Following STZ-induced β-cell injury, a vast increase in cell proliferation
surrounding the islets is observed. Through lineage analysis, we have shown that
these proliferating cells are not β-cells undergoing self-duplication. This cell
population that we have identified expresses SMAα and desmin but not GFAP or
nestin, indicating that the stellate cells are in an activated state. The morphology
of this cell population characterized by elongated fiber-like bodies also resembles
stellate cells found at site of injury (Masamune et al., 2009). Our data indicates
that PTEN loss may signal these activated stellate cells to proliferate in order to
promote the regeneration of the β-cells. Research from the pancreatic cancer
field suggests that there is a symbiotic relationship between cancer growth and
activation of SMAα expressing activated stellate cells (Omary et al., 2007). Such
a relationship may also exist for the growth of islets in the β-cell-specific Pten
deletion model and the surrounding stellate cells. How loss of PTEN in the β-cell
compartment led to the response in the stellate cell compartment is unclear. In
chicken pancreas, intercalated islet stellate cells were found to contribute to the
mass of β-cells (Datar and Bhonde, 2009). To what extent this interaction occurs
in mammalian tissues remains unclear.

11

Chapter II

Adult onset deletion of PTEN increases proliferation of islets β-cells
II-1 Introduction
The mass of the pancreatic islets is an important factor in the maintenance
of glucose homeostasis. In the adult pancreas, maintenance of islet mass
depends on the balance between newly formed islet cells and death of existing
cells. Both proliferation and apoptosis rates are low in the adult endocrine
pancreas during physiological maintenance (Lee and Nielsen, 2009). During
times of metabolic stress and/or pancreatic/β-cell injury, the mitotic activity of β-
cells and β-cell mass both increase. Presumably, the increase of β-cell mitotic
activity during these stress conditions results from signals that may potentially
induce their proliferation. However, the signals that maintain β-cell mass and
those that stimulate their growth are not well characterized (Bouwens and
Rooman, 2005; Elghazi et al., 2006). In the last few years, studies using
transgenic animals implicated a major role for the G1/S cell cycle transition
machinery in the growth of β-cells (Elghazi et al., 2007). However, how these
factors may be regulated by growth signals that dictate maintenance of adult β-
cells is not understood.
Several growth factors have been found to play roles in β-cell function,
including insulin, insulin-like growth factor (IGF) and hepatic growth factor (HGF)
(Dai et al., 2005; Dai et al., 2003; Garcia-Ocana et al., 2000; Garcia-Ocana et al.,
2001; Mellado-Gil et al.; Roccisana et al., 2005; Vila et al., 1995). Islets from

12

mice lacking HGF receptor suffer from smaller islet mass and enhanced
apoptosis (Dai et al., 2005; Mellado-Gil et al.; Roccisana et al., 2005).
Overexpression of HGF, on the other hand, leads to enhanced islet function with
increased proliferation. Similarly, IGF-1 treatment prevents apoptosis and is
thought to be a mitogen for β-cell growth (Agudo et al., 2008; George et al., 2002;
Robertson et al., 2008; Yu et al., 2003). These studies suggest that the
PI3K/AKT pathway plays a mitogenic role in β-cell growth as both IGF-1, insulin,
and HGF signal through the PI3K/AKT signaling pathway (Bernal-Mizrachi et al.,
2004; Holst et al., 1998; Liu et al., 2002; Tuttle et al., 2001). Consistently, mice
overexpressing a constitutive active form of AKT displayed larger islets and
reduced apoptosis rate in response to streptozotocin (STZ) treatment (Bernal-
Mizrachi et al., 2001). Therefore, the PI3K/AKT signaling pathway appears to
play prominent roles in regulating β-cell growth.
Phosphatase and tensin homolog on chromosome 10 (PTEN) is a lipid
phosphatase that antagonizes the functions of PI3K by dephosphorylating
phosphatidylinositol triphosphate (Stiles et al., 2004). Heterozygous deletion of
Pten was previously demonstrated to rescue the dysfunctional islets in IRS2
-/-

mice (Kushner et al., 2005b). Our group and others reported that mice lacking
PTEN specifically in the β-cells have more and larger islets when compared to
mice with intact PTEN (Nguyen et al., 2006; Stiles et al., 2006). Our group
showed that β-cell mitotic activity is significantly increased in the Pten null mice
during embryogenesis, as well as in response to STZ-induced regeneration of β-

13

cells (Stiles et al., 2006). However, in adult islets, how PTEN and PI3K signal
may control the maintenance of adult β-cells is not clear. In 2-month-old Pten
null islets, β-cell proliferation rate is only moderately higher that that of controls
(Stiles et al., 2006). An effect of PTEN loss on β-cell proliferation in older mice
was not investigated. To define whether and how PTEN/PI3K signal maintains
adult β-cell mass, we engineered a mouse model where Pten is deleted
specifically in adult β-cells. This model allows us to evaluate the effect of PTEN
loss and PI3K activation specifically in adult β-cells without the complications of
developmental effects. We show here that manipulating the PI3K signaling
through deletion of Pten is capable of inducing the growth of β-cells in adult
animals at both 3 and 12 months of age. Analysis of the downstream signaling
showed upregulation of D cyclins and downregulation of p27 cell cycle inhibitor,
suggesting a role of this G1/S transition machinery in the maintenance of adult β-
cell mass by PTEN/PI3K signaling.
II-2 Results
II-2-1 Generation of Pten
loxP/loxP
; Rosa
lacZ
; Rip-CreER mice
The growth of pancreatic β-cells decreases significantly in adult individuals
(Teta et al., 2005). Others and we have shown that deletion of the negative
mitotic regulator Pten results in increased islet mass in murine models (Nguyen
et al., 2006; Stiles et al., 2006). To determine if PTEN loss can also inhibit the
adult-onset slowing of proliferation, we generated a mouse model in which Pten
can be deleted specifically in the β-cells of adult pancreas (Pten
loxP/loxP
; Rosa
lacZ
;

14

Rip-CreER
+
). In this model, treatment of tamoxifen (+Tam) induces the activation
of Cre recombinase and thus deletion of Pten due to this activity. The addition of
Rosa
lacZ
reporter alleles allowed us to optimize the dose of tamoxifen needed to
induce robust expression of Cre and deletion of Pten in islet β-cells. We show
here that a total of 30mg tamoxifen delivery (5 doses of 6mg) is sufficient to allow
a majority of the cells that express insulin (β-cells) to be labeled with β-gal,
indicating that Cre recombinase is sufficiently expressed in these cells (Figure
3A). Compared to CreER
-
mice treated with or without tamoxifen or CreER
+
mice
treated with vehicle as controls, tamoxifen treatment in CreER
+
mice is the only
one showing expression of Rosa lacZ in the islets, as indicated by the blue
staining. This result suggests that Cre recombinase is active in the islets of the
CreER
+
mice treated with tamoxifen and inactive in the three control groups.
Minimum staining is observed in the exocrine pancreas, indicating specificity of
the Cre expression and thus Pten deletion specifically and solely in the islets.
Genomic PCR analysis of DNA isolated from islets of Tam vs. vehicle
treated (-Tam) mice demonstrated that tamoxifen treatment indeed induced
deletion of Pten as indicated by the appearance of the Δ5 band, while no Pten
deletion is observed in vehicle treated mice (Figure 3B, left panel). Analysis of
protein isolated from mouse islets confirmed deletion of Pten in the +Tam group
compared to the -Tam group, as shown in the significant reduction of the PTEN
protein expression in the +Tam group (Figure 3B, right panel). As a result, the
phosphorylated form of AKT is increased in the same islets. We further

15

confirmed that PTEN is lost from islet β-cells using indirect immunofluorescence
(Figure 3C). In control pancreas, PTEN staining is observed in the nucleus of the
β-cells (red, co-stained with insulin (green)). This nuclear staining is lacking in
most of the islets from the Tam treated mice.
Unlike the Pten
loxP/loxP
; Rip-Cre
+
mice, adult onset deletion of Pten did not
affect the body weight or pancreas weight of the mutant mice (Figure 4). Partial
deletion of PTEN in the hypothalamus of the Pten
loxP/loxP
; Rip-Cre
+
mice has been
attributed to be the cause of the smaller body size (Nguyen et al., 2006).
Consistently, analysis of DNA isolated from brain tissue of the +Tam mice show
no deletion of Pten (Exon Δ5 band) (Fig 3B, left panel). We also found no
difference in PTEN protein expression in brain tissue lysates from the +Tam and
-Tam groups (Figure 3B, right panel). Thus, the Pten
loxP/loxP
; Rosa
lacZ
; Rip-
CreER
+
model can be used for evaluating the effect of PTEN loss on β-cell
proliferation in adult pancreas.

16

B
A
!-Actin
-Tam +Tam
PTEN
p-AKT
RIP-CreER+
Tamoxifen Treated
RIP-CreER-
Tamoxifen Treated
RIP-CreER+
Vehicle Treated
RIP-CreER-
Vehicle Treated
H&E
In Situ
!-gal staining
Pten
loxP/loxP
; Rosa
lacZ/lacZ

C
+ Tamoxifen - Tamoxifen
PTEN Insulin DAPI Merged
50 µm
PTEN
!-Actin
- Tam +Tam
loxP
"5
- Tam +Tam
Islets Brain
Islets
Brain

Figure 3: Efficiency of Pten deletion in Pten
loxP/loxP
; Rosa26
lacZ
; RIPCreER
+
mice treated with
tamoxifen. (A) Top panels, H&E images of pancreas sections from control (Pten
loxP/loxP
;
Rosa26
lacZ
; RIPCreER
-
treated with or without Tamoxifen; first three columns from the left) and
Pten null (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
treated with tamoxifen; last column) mice. Bottom
panels, in situ β-gal staining (blue). Blue stain indicates Pten-deleted β-cells. (B) Genomic PCR
(left panel) and western blot (right panel) analysis of control (-Tamoxifen, -Tam) and Pten null
(+Tamoxifen, +Tam) DNA and protein, respectively, isolated from mouse islets and brain tissue.
Immunofluorescent staining (right figure) of pancreata from control and mutant mice using
markers for PTEN (red), insulin (green) and DAPI (blue).

17

Pancreas Weight(g) Group 1 Group 2 Group 3
Body Weight(g)
Pancreas : Body
Weight Ratio
0
10
20
30
40
0
10
20
30
40
50
0
10
20
30
40
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
0
0.005
0.015
0.025
0
0.005
0.015
0.025
0
0.005
0.015
0.025
-Tam +Tam
-Tam +Tam -Tam +Tam
-Tam +Tam -Tam +Tam
-Tam +Tam
-Tam +Tam -Tam +Tam -Tam +Tam

Figure 4. Body and pancreas weight of control and Pten null animals. Body weight (top
panel) and pancreas weight (middle panel) for control (Pten
loxP/loxP
; Rosa
lacZ/lacZ
; CreER
-
) and
mutant (Pten
loxP/loxP
; Rosa
lacZ/lacZ
; CreER
+
) mice treated with (+Tam) or without (-Tam) tamoxifen in
the three different groups (n=5-7 per genotype per group). The ratio of pancreas to body weight
(bottom panel) was calculated. The error bars represent the SEM for all animals analyzed in each
group.

18

II-2-2 Deletion of Pten in adult pancreas results in increased islet area and
enhanced endocrine function

Using the same protocol of tamoxifen delivery, we deleted Pten from
three-month-old mouse β-cells. The Pten
loxP/loxP
; Rosa
lacZ
; Rip-CreER
+
mice were
randomly assigned to -Tam and +Tam groups where the -Tam groups received
vehicle treatment (corn oil) and the +Tam groups received 30mg tamoxifen. Mice
were then euthanized at various time points to evaluate the effects of adult Pten
deletion on islet mass and function (Figure 5A). Group 1 was euthanized one
month after the last dose of tamoxifen treatment (Grp 1); Group 2, 3 months after
treatment (Grp 2); and Group 3, 6 months after treatment (Grp 3) (Figure 5A).
Immunofluorescent analysis of β-gal demonstrates robust β-gal staining in islets
in all three groups of tamoxifen-induced mice. These data suggests that Cre is
efficiently active in islets from all three groups and that Pten is effectively deleted
(Figure 6). We determined the relative islet area by measuring the size of the
islets vs. the size of the pancreas (islet vs. pancreas ratio). In the control mice,
this ratio is low in all three groups of mice (0.005-0.01%). Deletion of Pten led to
more than 2-fold increase of islet/pancreas ratio at all three time points. The
islet/pancreas ratio also continues to increase with time laps. Islet vs. pancreas
ratio in Grp 2 and Grp 3 mice are significantly higher than that of Grp 1 mice
(Figure 5B).

19

A
B
Group 1
Lag time =
1 Mon.
Group 2
Lag time
= 3 Mon.
Group 3
Lag time =
6 Mon.
2 wk TX 3 Months
Islet : Non-Islet Ratio
0
0.01
0.02
*
*
*
Grp 1 Grp 2 Grp 3
-Tamoxifen
+Tamoxifen
- Tam + Tam
Group 1
Group 2 Group 3
50 µm
**
**
NS
!

Figure 5. Deletion of Pten in adult β-cells leads to increased relative islet area and
improved glucose homeostasis. (A) Schematic figure of the treatment timeline. At three
months of age, mice were treated with tamoxifen (30mg total dose) or corn oil (vehicle) and then
euthanized for analysis at 1 month (Group 1, Grp 1), 3 months (Group 2, Grp 2), and 6 months
(Group 3, Grp 3) post treatment. (B) Representative image of control (-Tam) and Pten null
(+Tam) mouse pancreata stained with H&E for all three time points (left panel). Islet to non-islet
area ratio was calculated to determine the relative islet area (right panel) (n=5 per genotype, per
group). Corn oil-treated control (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
-
; open bars) and tamoxifen-
treated Pten null (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
; closed bars). The error bars represent the
SEM for all animals analyzed for that group. p<0.05 is considered to be statistically significant.
The * represents significance within the group, whereas ** represents significance of the mutants
between groups.

20

C
Fasting Glucose (mg/dl) 0
40
80
120
160
*
*
*
Random Glucose (mg/dl)
0
40
80
120
160
*
* *
-Tamoxifen
+Tamoxifen
-Tamoxifen
+Tamoxifen
Grp 1 Grp 2 Grp 3
Grp 1 Grp 2 Grp 3
Fasting Plasma Insulin (ng/ml)
0
0.04
0.08
0.12
0.16
-Tam +Tam
*
0
0.05
0.15
0.25
*
-Tam +Tam
0
0.2
0.4
0.6
0.8
*
-Tam +Tam
Group 1 Group 2 Group 3

Figure 5, continued. Deletion of Pten in adult β-cells leads to increased relative islet area
and improved glucose homeostasis. (C) Plasma glucose levels were determined from fasted
(top left panel) and non-fasted (top right panel) mice (n=5-7 per genotype, per group). Plasma
from the control and Pten null mice was obtained after overnight fasting and insulin levels were
determined (bottom panels) (n=3-5 per genotype, per group). Corn oil-treated control
(Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
-
; open bars) and tamoxifen-treated Pten null (Pten
loxP/loxP
;
Rosa26
lacZ
; RIPCre
+
; closed bars). The error bars represent the SEM for all animals analyzed for
that group. p<0.05 is considered to be statistically significant. The * represents significance
within the group, whereas ** represents significance of the mutants between groups.

21

Group 1 Group 2 Group 3
Pten
loxP/loxP
; Rosa
lacZ/lacZ
; RIP-CreER+
Pten
loxP/loxP
; Rosa
lacZ/lacZ
;
RIP-CreER-
Insulin + !-gal + DAPI
!

Figure 6. Expression of β-gal in β-cells of tamoxifen-treated Pten null group. Control
(Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
-
; top panel) and Pten null (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
;
bottom three panels) pancreas were sectioned and costained with various markers 1 month
(group 1), 3 months (group 2), and 6 months (group 3) after tamoxifen treatment. β-gal (red),
insulin (green) and DAPI (blue).

22

To address the functional significance of the increased relative islet area,
we determined fasting and non-fasting blood glucose levels. We found that both
fasting and random fed glucose decreased significantly in the tamoxifen-treated
mice across all the groups (Figure 5C, top two panels). A 40% decrease in
fasting glucose levels was observed in Grp 2 and 3 when Pten is deleted (Figure
5C, top left panel). A constant 15% decrease of random glucose is also
observed with all groups of tamoxifen-induced mice where Pten is deleted
(Figure 5C, top right panel). Consistent with the lowered glucose levels, we also
observed lower fasting plasma insulin levels in the tamoxifen-treated Pten null
mice (Figure 3C, bottom panels), indicating β-cells lacking PTEN are responsive
to peripheral glucose changes.
To assess the effect of β-cell Pten deletion on glucose metabolism, we
subjected the –Tam and +Tam mice to glucose (GTT) and insulin tolerance tests
(ITT). In all three experiments (Group 1, 2, & 3), mice from the +Tam groups are
more sensitive at clearing glucose than those from the –Tam group (Figure 7A).
Analysis of plasma insulin during GTT indicate that insulin levels tracked that of
glucose during GTT except for the two time points at peak glucose levels (15 and
30 mins) for the Grp 1 and 2 animals (Figure 7B). In group 3, insulin levels
tracked glucose levels throughout GTT from 15 min to 2 hour post-glucose
injection. In mice from group 1, 30 min post glucose injection, plasma insulin
levels are the same in the +Tam as compared to the –Tam mice even though
glucose levels are lower in the +Tam group at this time point. Similarly, in mice

23

from group 2, plasma insulin levels are the same in +Tam and –Tam mice 15 min
after glucose injection. At this time point, glucose levels in the +Tam mice is 30%
lower than that of the –Tam mice. In both groups 1 and 2 experiments, the
insulin/glucose ratios are higher in the +Tam group (Goup1: 0.0009% (+Tam) vs.
0.0007% (-Tam); Group 2: 0.002% (+Tam) vs. 0.001% (-Tam)) at peak glucose
measurement. These analyses suggest that pancreatic islets from the +Tam
group may be producing more insulin for the same glucose maintenance upon
stimulation, consistent with previous observations (Nguyen et al., 2006). We also
performed ITT tests on these mice. Consistent through all experimental groups,
+Tam mice displayed higher insulin sensitivity than the –Tam mice (Figure 7C),
indicating that the mice are also more sensitive at clearing glucose from the
peripheral tissues.
II-2-3 Pten deletion in adult mice leads to increased proliferation and blocks
cell death induced by STZ

In order to determine whether deletion of Pten in adult pancreas is
capable of inducing their mitotic activity, we evaluated the mitotic rate of islet β-
cells from all three groups using BrdU incorporation (Figure 8). Our data
demonstrated increased proliferation rate in islets where Pten is deleted vs. islets
where Pten is present. A moderate increase (1.5X) of BrdU incorporation is
observed for β-cells 1 month after tamoxifen injection (Figure 8B, top panel). In
groups 2 and 3 where lag time is longer, proliferation rate measured by BrdU
incorporation is two and three times higher in islets lacking PTEN (+Tam) than
control islets (-Tam), respectively (Figure 8B, bottom two panels).

24

A
C
Time after glucose
injection (min) Time after glucose
injection (min) *
*
*
*
*
*
*
*
!
"!
#!
$!
%!
&!!
&"!
! '! $! (! &"!
!
"!
#!
$!
%!
&!!
&"!
! '! $! (! &"!
Time after glucose
injection (min) Glucose (mg/dl) *
*
*
*
*
!
"!
#!
$!
%!
&!!
&"!
! '! $! (! &"!
B
Plasma Insulin (ng/ml) Time after glucose
injection (min) Time after glucose
injection (min) Time after glucose
injection (min) *
*
* *
*
* *
*
*
*
*
*
!
!)!*
!)&
!)&*
!)"
!)"*
!)'
!)'*
!)#
! '! $! (! &"!
!
!)!*
!)&
!)&*
!)"
!)"*
!)'
!)'*
!)#
!)#*
! '! $! (! &"!
!
!)"
!)#
!)$
!)%
&
! '! $! (! &"!
Group 1 Group 2 Group 3
Time after glucose
injection (min) Time after glucose
injection (min) *
*
*
*
*
*
*
*
*
Glucose (mg/dl) Time after glucose
injection (min) *
*
*
!
*!
&!!
&*!
"!!
"*!
'!!
'*!
! '! $! (! &"!
!
*!
&!!
&*!
"!!
"*!
'!!
'*!
! '! $! (! &"!
!
*!
&!!
&*!
"!!
"*!
'!!
'*!
! '! $! (! &"!
-Tam
+Tam
-Tam
+Tam
-Tam
+Tam

Figure 7. Glucose and insulin tolerance of Control and Pten null animals. (A) Glucose
tolerance test of control or Pten null mice treated with (+Tam) or without (-Tam) tamoxifen. (B)
Insulin levels of control or Pten null mice treated with (+Tam) or without (-Tam) tamoxifen. (C)
Insulin tolerance test of control or Pten null mice treated with (+Tam) or without (-Tam) tamoxifen.
Control (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
-
; open circles) and Pten null (Pten
loxP/loxP
; Rosa26
lacZ
;
RIPCre
+
; closed squares) (n=3-4 per genotype, per group). The error bars represent the SEM for
all animals analyzed in each group. *p<0.05 is considered to be statistically significant. The *
represents significance between the two groups at the indicated time point.

25

- Tamoxifen + Tamoxifen
Group 1 Group 2 Group 3
!-gal + BrdU + DAPI
0
0.1
0.2
0.3
0.4
0.5
-Tam +Tam
% !-Cells Positive for BrdU
*
0
0.1
0.2
0.3
0.4
0.5
-Tam +Tam % !-Cells Positive for BrdU
*
0
0.1
0.2
0.3
0.4
0.5
-Tam +Tam
% !-Cells Positive for BrdU
*
A B
!

Figure 8. Deletion of Pten in adult β-cells leads to increased cell proliferation. (A)
Representative image of pancreas from control (-Tamoxifen, left column) and Pten null
(+Tamoxifen, right column) mice. Sections were analyzed for β-gal (red), BrdU (green) and DAPI
(blue) using immunofluorescence. Dashed circles indicate islet area. (B) Quantification of BrdU
and insulin double positive cells. Data reported as percentage of double positive vs. total insulin
positive cells. The bars represent the average percentage of BrdU-positive β-cells in control and
Pten null mouse pancreata. Group 1 (top panel) is analyzed 1 month after last dose of tamoxifen
treatment in 3-month-old mice. Groups 2 & 3 (middle and bottom panels) are 3 months and 6
months post treatment, respectively (n=5-6 per genotype, per group). The error bars represent
the SEM for all animals analyzed for that group. *p<0.05 is considered to be statistically
significant from control (-Tam) group.

26

We also determined cell death rate in response to Pten deletion in adult
islets. Like previously reported (Stiles et al., 2006), apoptotic cells (measured by
TUNEL) are rare and difficult to detect in mice with or without PTEN (Figure 9).
Thus, we evaluated the extent of cell death in response to treatment by a β-cell
toxin STZ. In the STZ model, β-cell death is observed in the -Tam groups where
PTEN is intact (Figure 10A, top panel). When PTEN is lost (+Tam groups),
substantially fewer cells are positive for TUNEL, indicating less apoptosis when
Pten is deleted (Figure 10A, bottom panel). Concomitantly, plasma glucose
levels in the Pten null mice (+Tam) remained low in the STZ-treated mice (Figure
10B), indicating functional β-cells and protection from STZ-induced β-cell death.
Plasma glucose levels are significantly higher in the STZ-treated control mice
than those observed previously with non-STZ-treated controls in Figure 3
(approximately 300mg/dl vs. 120mg/dl respectively in all three groups of mice).
Plasma glucose levels in the STZ-treated Pten null mice remained at
approximately 100mg/dl level. Together, these data suggest that inhibiting PTEN
function in adult islets is capable of inducing proliferation, blocking death of β-
cells induced by injury, and preserving the functions of islets.

27

Group 1 Group 2 Group 3
- Tamoxifen + Tamoxifen
TUNEL + DAPI
50 µm

Figure 9. Analysis of apoptosis in control and Pten null mice. Control (-Tamoxifen) and
Mutant (+Tamoxifen) pancreas were sectioned and analyzed for apoptosis using an
immunoflorescence TUNEL assay 1 month (group 1), 3 months (group 2), and 6 months (group 3)
after tamoxifen treatment. TUNEL (green) and DAPI (blue).

- Tamoxifen + Tamoxifen
TUNEL (STZ) + DAPI
Group 1 Group 2
A
50
150
250
350
0
Random Glucose (mg/dl)
*
-Tam +Tam
Group 1
*
50
150
250
350
0
Random Glucose (mg/dl)
-Tam +Tam
Group 2
*
50
150
250
350
0
Random Glucose (mg/dl)
-Tam +Tam
Group 3 B
50 µm
Group 3

Figure 10. Deletion of Pten in adult β-cells prevents STZ-induced apoptosis. (A)
Representative image of TUNEL analysis. STZ-treated control (-Tamoxifen) and Pten null
(+Tamoxifen) pancreas were sectioned and analyzed for apoptosis using an immunofluorescence
TUNEL assay; TUNEL (green) and DAPI (blue). Dashed circles indicate islet areas. Higher
magnification images of areas within the squares are show on the right panels. (B) Random
plasma glucose levels in STZ-treated control (-Tam) and Pten null (+Tam) mice 10 days after the
initial dose of STZ. Group 1 is analyzed 1 month after last dose of tamoxifen treatment in 3-
month-old mice. Groups 2 & 3 are 3 months and 6 months post treatment, respectively (n=4-5
per genotype, per group). *p<0.05 is considered to be statistically significant from control (-Tam)
group.

28

II-2-4 Pten deletion rescues the loss of proliferation response in aged β-
cells

Previous studies have shown that β-cell regeneration and islet mass are
severely restricted in older mice even in the presence of physiological stimulation
signals (Rankin and Kushner, 2009; Tschen et al., 2009). In response to
physiological stimulation, islets adapt by expanding the β-cell population. This
adaptive β-cell proliferation diminishes with age and becomes limited in old
animals (>8 months). The PI3K/AKT signal regulated by PTEN is one signal that
can stimulate the expansion of islet mass (Elghazi et al., 2006; Elghazi and
Bernal-Mizrachi, 2009). However, it is not clear whether it is sufficient to
overcome this age onset inhibition. In order to determine whether deletion of
Pten can override this blockage, we deleted Pten in 12-month-old mice (Figures
11 & 12), where adaptive proliferation is severely restricted in β-cells (Rankin and
Kushner, 2009). The same protocol (30mg of tamoxifen) is used to delete Pten
in the 12-month-old mice as that in 3-month-old mice. Mice were euthanized
after a 1 month (Group 1, Grp 1), 9 month (Group 2, Grp 2) and a 12 month lag
time (Group 3, Grp 3) following the last tamoxifen injection (Figure 11A). We
determined whether the relative islet area is similarly increased as in our first set
of experiments where Pten was deleted from 3-month-old mice (Figure 3).
Quantitative analysis of H&E stained sections showed that all tamoxifen-treated
mice (bottom panels) had higher islet/pancreas ratio compared to the vehicle-
treated controls (top panels) (Figure 11B). Similar to what was observed for the

29

3-month-old mice, islet/pancreas ratio is about 1.5 to 2 fold higher in the
tamoxifen treated groups vs. the vehicle treated groups.
2 wk TX 12 MONTHS
Group 1
Lag time
= 1 Mon.
Group 2
Lag time
= 9 Mon.
Group 3
Lag time =
12 Mon.
Group 1 Group 2 Group 3
Islet : Non-Islet Ratio
- Tamoxifen + Tamoxifen
A
B
C
0
0.01
0.02
0.03
*
Grp 1 Grp 2 Grp 3
-Tamoxifen
+Tamoxifen
Fasting Glucose (mg/dl)
0
40
80
120
*
*
*
-Tamoxifen
+Tamoxifen
Grp 1 Grp 2 Grp 3
Random Glucose (mg/dl)
0
40
80
120
160
*
*
*
Grp 1 Grp 2 Grp 3
-Tamoxifen
+Tamoxifen
*
*
**
**
NS
!

Figure 11. Deletion of Pten in aged (>1 year) β-cells leads to expansion of relative islet
area and improved glucose homeostasis. (A) Schematic figure of the treatment timeline. At
twelve months of age mice were treated with tamoxifen (30mg) or corn oil (vehicle) and then
euthanized for analysis at 1 month (Group 1, Grp 1), 9 months (Group 2, Grp 2), and 12 months
(Group 3, Grp 3) after the last injection. (B) Representative image of control (-Tamoxifen) and
Pten null (+Tamoxifen) mouse pancreata stained with H&E (left panel). Islet to non-islet area ratio
was analyzed and calculated (right panel) (n=3-4 per genotype per group). (C) Plasma glucose
levels were determined from fasted (left panel) and non-fasted (right panel) mice (n=3-4 per
genotype per group).

30

Time after glucose
injection (min) Time after glucose
injection (min) 1M 6M *
*
*
*
*
*
!
"!!
#!!
$!!
%!!
! $! &! '! "#!
!
"!!
#!!
$!!
%!!
! $! &! '! "#!
*
D
Glucose (mg/dl)
Glucose (mg/dl) Plasma Insulin (ng/ml)
!
!(#
!(%
!(&
!()
"
! $! &! '! "#!
* *
0
*
!
!(#
!(%
!(&
!()
"
"(#
! $! &! '! "#!
*
*
!
*!
"!!
"*!
! $! &! '! "#!
*
*
*
!
*!
"!!
"*!
! $! &! '! "#!
*
*
Time after glucose
injection (min) Time after glucose
injection (min) Time after glucose
injection (min) Time after glucose
injection (min) -Tam
+Tam
-Tam
+Tam
-Tam
+Tam

Figure 11, continued. Deletion of Pten in aged (>1 year) β-cells leads to expansion of
relative islet area and improved glucose homeostasis. (D) Glucose tolerance test of control
or Pten null mice treated with (+Tam) or without (-Tam) tamoxifen (top panel); Insulin levels of
control or Pten null mice treated with (+Tam) or without (-Tam) tamoxifen (middle panel); Insulin
tolerance test of control or Pten null mice treated with (+Tam) or without (-Tam) tamoxifen
(bottom panel) (n=3-4 per genotype, per group). Corn oil-treated control (Pten
loxP/loxP
; Rosa26
lacZ
;
RIPCre
-
; open bars/circles) and tamoxifen-treated Pten null (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
;
closed bars/squares). The error bars represent the SEM for all animals analyzed for that group.
p<0.05 is considered to be statistically significant. The * represents significance within the group
whereas ** represents significance of the mutants between groups.

31

Analysis of the fasting and non-fasting glucose of control and Pten null
mice showed that both fasting and random fed glucose levels are significantly
lower in Pten null mice (+Tam) compared to the controls (-Tam) (Figure 11C).
Moderately improved glucose tolerance is also observed in these older mice
when Pten is deleted, though not as obvious as those observed with the young
mice. Consistent with the observation in young mice, +Tam mice displayed
slightly improved insulin sensitivity than the -Tam mice (Figure 11D), indicating
that these mice also have improved ability to clear glucose from the peripheral
tissues.
Analysis of mitotic rate (percentage of BrdU-positive β-cells) shows that
deletion of Pten in this age consistently induces the proliferation of β-cells (Figure
12). BrdU incorporation is increased by approximately 4-fold in all three
tamoxifen treated groups vs. vehicle groups (Figure 12B).
II-2-5 Cell-cycle regulation is altered when Pten in deleted in aged β-cells
To explore the molecular mechanism for the PTEN regulated β-cell
proliferation, we investigated the cell cycle regulatory mechanisms at G1/S
transition. Molecules that regulate G1/S transition including D type cyclins, cell
cycle inhibitor p27, and Rb family of pocket proteins have been shown to play a
role in the proliferation capacity of β-cells (Harb et al., 2009; Kushner et al.,
2005a; Rachdi et al., 2006). The PTEN/AKT pathway directly regulates one or
more of these cell-cycle regulators in various cell types, particularly tumor cells
(Elghazi et al., 2006; Elghazi and Bernal-Mizrachi, 2009). We determined the

32

levels of cyclin D1, D2 and p27 in one-year-old mice 3 months after initial
treatment with tamoxifen or vehicle. We found that both cyclin D1 and D2 levels
are induced in islets of the tamoxifen-treated groups where PTEN is lost vs. the
controls with intact PTEN. Concomitantly, induced PTEN loss in these mice led
to downregulation of cell cycle inhibitor p27 (Fig 13 A & C). Together, these
G1/S transition proteins may compose the downstream regulatory network that
mediates PTEN/PI3K regulated β-cell proliferation.
+ Tamoxifen - Tamoxifen
Insulin + BrdU + DAPI
Group 1 Group 2 Group 3
A
50 µm

Figure 12. Deletion of Pten in aged (>1 year) β-cells leads to increased cell proliferation.
(A) Pancreas from control (-Tamoxifen, -Tam) and Pten null (+Tamoxifen, +Tam) mice were
analyzed for insulin (red), BrdU (green) and DAPI (blue) using immunofluorescence. (B)
Quantification of BrdU and insulin double positive cells. Data reported as percentage of double
positive vs. total insulin positive cells. Group 1 is analyzed 1 month after last dose of tamoxifen
treatment in 12-month-old mice. Groups 2 & 3 are 9 months and 12 months post treatment,
respectively. The error bars represent the SEM for all animals analyzed for that group (n=3-4 per
genotype per group). *p<0.05 is considered to be statistically significant from –Tam group.
B
% !-Cells Positive for BrdU
0
0.1
0.2
0.3
0.4
0.5
-Tam +Tam
*
0
0.1
0.2
0.3
0.4
0.5
-Tam +Tam
*
0
0.1
0.2
0.3
0.4
0.5
-Tam +Tam
*
Group 1 Group 2 Group 3

33

- Tamoxifen + Tamoxifen
Cyclin D1+Insulin+DAPI Cyclin D2+Insulin+DAPI p27+Insulin+DAPI
A
PDX-1+Insulin
+DAPI
C-Myc+Insulin
+DAPI
C
Cyclin D1
Cyclin D2
p27
PDX-1
!-Actin
T (-) T (+)
C-Myc
B
- Tamoxifen + Tamoxifen
!
!
Figure 13. Deletion of Pten in adult β-cells leads to changes is cell-cycle regulators. (A)
Representative image of pancreas from control (-Tamoxifen, top panel) and Pten null
(+Tamoxifen, bottom panel) mice. Sections were analyzed for Insulin (red), Cyclin D1, Cyclin D2,
p27 (green), and DAPI (blue) using immunofluorescence. (B) Representative image of pancreas
from control (-Tamoxifen, left column) and Pten null (+Tamoxifen, right column) mice. Sections
were analyzed for Insulin (red), PDX-1, C-Myc (green), and DAPI (blue) using
immunofluorescence. (C) Western blot analysis of control (-Tamoxifen, T(-)) and Pten null
(+Tamoxifen, T(+)) protein isolated from mouse islets.

34

Furthermore, we determined the levels of Pdx-1, a progenitor marker
known to regulate glucose induced insulin response in adult β-cells (Kaneto et al.,
2008), and C-myc, an early responsive gene known to regulate proliferation in
multiple cell types (Cheung et al., 2010; Radziszewska et al., 2009). We
observed no significant difference in either Pdx-1 or C-Myc expression when
PTEN is lost (Figure 13 B & C). This observation is also consistent with the
previously reported lack of tumor development in the Pten
loxp/loxP
; Rip-Cre
+
model
(Nguyen et al., 2006; Stiles et al., 2006) as PTEN loss in Pdx-1 positive cells led
to pancreatic ductal carcinoma (Stanger et al., 2005) whereas C-myc is found to
play a role in insulinoma development (Pelengaris and Khan, 2001). Our result
from the current study supports this lack of tumor observation. In mice treated
with tamoxifen at 3 months of age (Figure 7A), we observed normal islet
morphology with well-defined boundaries 15 months after deletion of Pten
(Figure 14B). Hormone expression in these islets is also distributed in the same
manner as those of the control islets (Figure 14C).

35

2 wk TX 3 Months
15 MONTHS
18 MONTHS
A
B
- Tamoxifen + Tamoxifen
H&E Insulin + !-gal + DAPI
50 µm
C
Merged
- Tamoxifen + Tamoxifen
Insulin Hormone Mix
!
!
Figure 14. Long-term deletion of Pten (15 months) does not lead to development of
insulinomas. (A) Schematic figure of the treatment timeline. At three months of age mice were
treated with tamoxifen or corn oil (vehicle) and then euthanized 15 months later for analysis at 18
months of age. n=3. (B) Control (-Tamoxifen, top panel) and Pten null (+Tamoxifen, bottom panel)
mice pancreata were analyzed with H&E staining (left column) and immunofluorescence using
markers for insulin (red), β-gal (green), and DAPI (blue) (right column). (C) Islets were stained
with markers for insulin (red) and a hormone mixture (glucagon, somatostatin, pancreatic
polypeptide)) (green) (right column).

36

II-3 Discussion
Preserving the function and mass of β-cells in adult pancreas is important
and necessary for maintaining euglycemia and preventing the development of
diabetes. During adult maintenance of β-cells, replication of β-cells are found to
be the major mechanism to sustain the islet mass while neogenesis may play a
role in other circumstances (Dor et al., 2004; Georgia and Bhushan, 2004; Teta
et al., 2007). In murine models, β-cell mitotic rate decreases rapidly 2-3 weeks
after birth from approximately 30% in the first week after birth to less than 1% at
2 months of age (Finegood et al., 1995; Teta et al., 2005). A similar observation
was made in human pancreas where a drop of 1.3% to 0.08% mitotic activity was
observed between perinatal and 6 months of age (Kassem et al., 2000). The
mitotic activity is then maintained at this low rate unless stimulation such as
obesity, pregnancy, or injury conditions are present (Bernard-Kargar and Ktorza,
2001; Flier et al., 2001; Green et al., 1981; Parsons et al., 1992; Saisho et al.,
2007). These studies suggest that the proliferation of islet β-cells may be
governed by two mechanisms: those that control its physiological maintenance
and those responsible for their response to stimulation, such as pregnancy,
obesity and injury, and during early postnatal development. Under STZ-induced
injury conditions, we showed previously that loss of PTEN and activation of PI3K
signaling led to increased proliferation (Stiles et al., 2006). In high fat diet
induced obese mice and db/db mice, β-cells lacking PTEN displayed enhanced
ability to respond to hyperglycemia (Nguyen et al., 2006; Wang et al., 2010).

37

These studies suggest that PTEN regulated PI3K signals are important for the
physiological response of β-cells to hyperglycemia and injury. We demonstrate
here that Pten deletion also controls the maintenance of β-cells in physiological
conditions without stimulation. Using the Pten
loxP/loxP
; Rosa
lacZ
; Rip-CreER
+

model where Pten deletion is induced by injection of tamoxifen, we evaluated the
effects of Pten deletion and PI3K activation in adult β-cells only. This model
allowed us to specifically investigate the effect of activating PI3K signaling in β-
cells after maturity onset. We showed that deleting Pten and upregulation of
PI3K/AKT signal in adult (3 months and older) β-cells induces their mitotic activity
and increases the islet mass. Furthermore, in mice older than 1 year when
physiological stimulations failed to induce proliferation of β-cells in wild type mice
(Rankin and Kushner, 2009), deletion of Pten is still capable of enhancing
proliferation and islet mass. These results suggest that the PTEN and PI3K
pathway not only regulates signals that control the response of β-cells to
stimulation, but also their maintenance when stimulation signals are no longer
present.
The PI3K/AKT signaling is a major mitogenic signaling pathway that
controls growth and survival regulated by growth factors/hormones including IGF,
insulin and HGF. Manipulation of these growth factors has led to alterations in β-
cell function (Butler AA, 1998; Dai et al., 2003; Garcia-Ocana et al., 2000; Holst
et al., 1998; Kulkarni et al., 2002; Lu et al., 2004; Okada et al., 2007; Robertson
et al., 2008). Although mice lacking IGF-1 receptor (or insulin receptor) alone did

38

not display significant abnormality in islet mass, combined deletion of insulin
receptor (IR) and IGF-1 receptor in β-cells significantly decreased postnatal islets
mass (Ueki et al., 2006). In addition, inactivation of insulin receptor substrate
(Irs2), the target of these receptors in β-cells also leads to β-cell failure due to
decreased proliferation and increased apoptosis (Kubota et al., 2000). Together,
these studies suggest that the IGF-1 signal and its downstream targets PI3K/AKT
likely play important roles in maintaining β-cell function. Consistently,
overexpressing a constitutively active form of AKT in pancreatic β-cells resulted
in an increase in β-cell mass, proliferation, neogenesis and cell size (Bernal-
Mizrachi et al., 2001; Tuttle et al., 2001). Conversely, reducing AKT1 activity by
overexpressing a kinase-dead mutant in β-cells resulted in glucose intolerance,
reduced basal insulin levels and insulin secretory defect (Bernal-Mizrachi et al.,
2004). Mice null in Akt2 also develop diabetes due to a reduction in insulin-
stimulated glucose uptake and β-cell failure (Cho et al., 2001). Furthermore,
genetic studies of downstream molecules including mTOR, TSC1/2, S6K and
FOXO (Balcazar et al., 2009; Buteau and Accili, 2007; Buteau et al., 2007;
Pende et al., 2000; Rachdi et al., 2008; Shigeyama et al., 2008) support a major
role of the PI3K signaling pathway in the regulation of β-cell function. We and
others showed that deleting Pten, the negative regulator of PI3K signaling, leads
to activation of AKT and increased islet mass(Nguyen et al., 2006; Stiles et al.,
2006). While a change in β-cell proliferation was not observed in young adult
mice with Pten deletion, the mitotic rate of β-cells was significantly higher in

39

embryos and in STZ-treated mice, suggesting a role of PTEN/PI3K signal in
postnatal islet growth. In this study, using a model where we can manipulate this
signaling pathway specifically in adult tissues, we showed that the PI3K/PTEN
signal indeed controls the growth and proliferation of adult β-cells. Our data
indicate that deletion of Pten in adult tissues significantly alters the mass and
proliferation rate of β-cells. More over, this effect is not only observed in young
adult (3 months) but also aged mice (12 months).
PTEN, being the negative regulator of the PI3K signaling, is critical for cell
growth and apoptosis. Loss of PTEN or activation of PI3K and its downstream
targets (e.g. AKT) unequivocally leads to advanced cell cycle progression
(Elghazi et al., 2006; Elghazi et al., 2007). Conversely, loss of AKT signals and
inhibition of PI3K leads to apoptosis and reduced abilities for cells to proliferate
(Elghazi et al., 2006; Stiles et al., 2002). These effects are partially due to the
role of AKT in regulating cell cycle proteins such as p27 and cyclin D (Kushner et
al., 2005a; Rachdi et al., 2006). In pancreatic β-cells, overexpressing a
constitutive forms of AKT leads to enlarged islet mass (Bernal-Mizrachi et al.,
2001). The accelerated β-cell proliferation rate observed in this model appears
to dependent on the presence of CDK4 (Fatrai et al., 2006). Being the major
kinase acting at G1 phase of the cell cycle, CDK4 functions to drive the cell cycle
from G1 to S phase. Observations from our study show that several molecules
at the G1/S transition are altered when PTEN is lost. We show that the major
partner for CDK4, the D-type cyclins (particularly cyclin D1), is robustly induced

40

when Pten is deleted. Furthermore, p27, the inhibitor for CDK4/cyclin D complex
is downregulated as a result of PTEN loss. This observation is supported by
previous studies showing that deletion of Cyclins D1 and D2 resulted in a
decrease in islet mass, β-cell proliferation and glucose tolerance (Kushner et al.,
2005a). Conversely, deletion of the cell cycle inhibitor p27 showed an increase
in islet mass, β-cell proliferation, and improved glucose tolerance (Rachdi et al.,
2006). Concomitant deletion of the pocket proteins controlling G1/S-phase cell
cycle transition pRb and p130 also resulted in accelerated β-cell replication and
apoptotic cell death. The pancreas and β-cell mass in these double mutant mice
were significantly reduced (Harb et al., 2009). These studies, though not
performed in a β-cell specific manner, are consistent with our observation that
the machineries controlling the G1/S transition play a major role in the regulation
of β-cell mass. Our signaling analysis done in 12-month-old mice where
physiological changes (pregnancy, obesity, or injury to β-cells) are unable to
stimulate the β-cell regeneration response, highlighted the importance of the
G1/S signal network in adult maintenance of β-cell mass. Whether one or the
other cell cycle regulators is more important in this effect requires further studies
that directly manipulate these signals specifically in adult β-cells.
The ability to sustain β-cell function is crucial for maintaining glucose
homeostasis. In individuals with peripheral insulin resistance, β-cells increase
insulin production to compensate for increased insulin demands. This is
achieved by the increase in both the mass of β-cells and their ability to secrete

41

insulin (Butler et al., 2003b; Chen et al., 2004; Goran et al., 2004). Thus, the
overall function of β-cells is critical for the physiology of glucose homeostasis,
particularly their ability to compensate peripheral insulin resistance. Recently,
expression analysis demonstrated increased PTEN protein and mRNA in islets
from high fat diet (HFD) and db/db mice compared to controls (Wang et al., 2010).
This observation suggests that PTEN and signals controlled by PTEN play a
physiological role in maintaining β-cell function, particularly in
hyperglycemia/insulin resistant conditions. In our study, we showed that PTEN
loss in islets led to hypoglycemia accompanied by hypoinsulinemia. This
phenotype observed together with increased islet/pancreas ratio confirmed that
islets in Pten null mice are subjected to physiological regulation, consistent with
previous observations from our and other labs (Nguyen et al., 2006; Stiles et al.,
2006). In addition, we also observed increased insulin to glucose ratio at peak
glucose levels during GTT and ITT tests, suggesting that the glucose-induced
insulin secretion is enhanced when PTEN is lost. This observation is in
agreement with the previous observation that β-cell specific PTEN loss protects
mice from development of hyperglycemia in HFD and db/db mice but has no
significant effects on control mice fed with chow diet. This previous study also
indicated that PTEN loss sensitizes the HFD and db/db islets to glucose induced
insulin secretion but has no effects on islets isolated from chow fed wild type
mice (Wang et al., 2010). Together, this evidence suggest that PTEN regulated
signals may control the responses of islets to glucose changes and play a role in

42

crosstalk between endocrine pancreas and peripheral responses. In agreement,
GTT and ITT test indicated improved peripheral insulin sensitivity when Pten is
deleted in adult β-cells. This effect is not due to Pten deletion in the
hypothalamus as has been implicated from previous studies using Pten
loxP/loxP
;
Rip-Cre
+
mice. Here, we showed that tamoxifen treatment in adult Pten
loxP/loxP
;
Rip-CreER
+
mice led to islet specific Pten deletion without affecting the
hypothalamus. This approach still resulted in improved insulin secretion in
peripheral tissues. Thus, the effects of β-cell specific Pten deletion on glucose
homeostasis may partially result from the crosstalk between the altered β-cells
and peripheral tissues. How this crosstalk occurs between PTEN manipulated
islets and peripheral tissues remains to be understood.
In summary, our study showed that manipulating PI3K signaling by
deleting its negative regulator PTEN significantly induces the proliferation
capacity of adult β-cells in young adult as well as mice of advanced age. This
data suggests that activation of PI3K signaling (or inhibition of PTEN function)
may overcome the inability of aged β-cells to proliferate in response to
physiological stimulation. This signaling pathway likely controls the molecular
signals that regulate the age-onset proliferation decay of β-cells in addition to its
role in mitogenic stimulation.

43

Chapter III
Activation of extracellular matrix (ECM) in response to deletion of Pten in β-
cells

III-1 Introduction
Regeneration of injured tissues is a complicated process that involves
participation from multiple cell types. In particular, the myofibroblasts that
compose the extracellular matrix (ECM) produce critical factors needed for the
replication and replenishment of lost cells (Hinz et al., 2007). In the pancreas
and liver, injury to the parenchymal results in activation of myofibroblasts such as
the stellate cells. These myofibroblasts secrete and deposit matrix proteins to
build and restructure the ECMs. Similarly, chronic inflammatory disease such as
pancreatitis are also accompanied by the activation of these myofibroblasts,
particularly the resident stellate cells (PSCs) (Talukdar and Tandon, 2008).
While the mechanisms for the activation of the myofibroblasts are not clear, the
activated PSCs are found to secrete and express extracellular matrix (ECM)
proteins including smooth muscle actin α (SMAα) and collagens, respectively.
The activated myofibroblasts also produce enzymes such as metalloproteases
and their inhibitors, of which the functions are to construct the ECMs. In addition,
PSCs produce inflammatory cytokines and growth factors that stimulate the
proliferation of various cell types to repair the injured pancreas. In line with this
observation, the growth and proliferation of tumor cells in pancreatic ductal
adenocarcinoma appears to be promoted by the activated PSCs (Vonlaufen et al.,

44

2008). While the function of the myofibroblasts has not been specifically defined,
the fact that they reconstitute ECM and restructure the tissue by producing
growth factors suggests a role for these cells in supporting growth and
proliferation of local parenchymals.
Pancreatic β-cells are the major endocrine cell types in the islets.
Diminished production of insulin by β-cells (due to loss of mass or function) to
meet the metabolic demand leads to diabetes mellitus, a growing epidemic
worldwide. Recent studies suggest that PSCs may serve as tissue stem cells in
the pancreas and can be induced to become insulin producing endocrine cells
that respond to glucose stimulation. Isolated stellate cells from lactating rat
pancreas were found to have properties of progenitor cells expressing the
multidrug resistant gene ABCG2 (Docherty, 2009). In culture, these cells
produce insulin when the culture condition is appropriate. Similar observations
were found in chicks where islet-derived stellate cells form islet structures
expressing c-peptides in culture. Here, we investigated whether stellate cells
contribute to the regeneration of β-cells observed in a mouse model that we
previously developed (Stiles et al., 2006). PTEN (phosphatase and tensin
homologue deleted on chromosome 10) is a negative regulator of the
insulin/IGF/PI3K signaling pathway. We showed previously that PTEN loss in
insulin producing β-cells (Pten
loxP/loxP
; Rip-Cre
+
) results in increased islet mass
and resistance to β-cell toxin, STZ-induced islet injury (Stiles et al., 2006). While
the deletion of Pten is confined to cells producing insulin because of the use of

45

rat insulin promoter to drive transcription of Cre recombinase, we observed
enhanced mitotic activity both within and in the extracellular matrix surrounding
the regenerating islets (Stiles et al., 2006). We found here that the peri-islet
proliferating cells are mesenchymal cell types displaying stellate cell markers and
they support the growth of β-cells through a paracrine action.
III-2 Results
III-2-1 A paracrine effect of β-cell PTEN loss on STZ-treated mice
We, and others, have reported previously that loss of the phosphatase
PTEN leads to resistance to STZ-induced hyperglycemia and diabetes (Stiles et
al., 2006). We showed that deletion of Pten protected β-cells from undergoing
apoptosis in multiple low dose STZ-treated mice. In addition, we showed using
BrdU short term labeling (6hrs) that proliferation rate in the pancreas is increased.
We also observed enhanced mitotic activity peripheral to the islets (Figure 15).
Our preliminary analysis showed that some of these peri-islets proliferating cells
are glucagon positive β-cells while most of them are negative for either insulin or
glucagon. Under defined experimental conditions, cells expressing glucagon
have been reported to be precursors for regenerating β-cells (Thorel et al., 2010).
Other cell types in the pancreas, e.g. exocrine cells and PSCs are also found to
have the ability to differentiate into β-cells (Datar and Bhonde, 2009; Docherty,
2009).
To understand how PTEN regulated signals control β-cell maintenance
and the relationship of β-cells with adjacent tissues during their regeneration, we

46

performed long term BrdU labeling (5 days in drinking water) to increase the
number of cells labeled for BrdU at the time of observation since mitotic activity is
low in the islets (Teta et al., 2007). We stained the control and Pten null pancreas
with glucagon (α-cells) as well as other endocrine cell markers: PP (pancreatic
polypeptide cells), and somatostatin (δ-cells) together with anti-BrdU (Figure 16
& 17). Compared to β-cells, these other cell populations are present in much
lower numbers in the islets and are physically located surrounding the β-cell
clusters. In the untreated mice, very few or none of the peri-islet proliferating
cells are positive for any of the endocrine hormones (Figure 17). STZ treatment
significantly induced proliferation in the islet area. Under these conditions, we
still did not observe significant overlap of BrdU positive cells with the non-insulin
endocrine positive cells (Figure 16). This result suggests that the peri-islet
proliferating cells are not pancreatic endocrine cell types and glucagon positive
cells do not contribute to the mass of β -cells in the Pten null model.
Supplemental Figure 1 Mitotic activity is observed in non-insulin positive
cells adjacent to the islets. Top, insulin (red) and BrdU (green) staining
indicating proliferating cells are not !-cells. Bottom, glucagon (red) and BrdU
(green) staining indicate some proliferating cells are glucagon positive. Blue,
DAPI.
Insulin+BrdU+DAPI
Glucagon+BrdU+DAPI
Bayan et al Supplemental Figure 1

Supplemental Figure 1 Mitotic activity is observed in non-insulin positive
cells adjacent to the islets. Top, insulin (red) and BrdU (green) staining
indicating proliferating cells are not !-cells. Bottom, glucagon (red) and BrdU
(green) staining indicate some proliferating cells are glucagon positive. Blue,
DAPI.
Insulin+BrdU+DAPI
Glucagon+BrdU+DAPI
Bayan et al Supplemental Figure 1

Figure 15. Mitotic activity is observed in non-insulin positive cells adjacent to the islets.
Left, insulin (red) and BrdU (green) staining indicating proliferating cells are not β-cells. Right,
glucagon (red) and BrdU (green) staining indicate some proliferating cells are glucagon positive.
Nuclear DAPI staining (blue).

47

BrdU; Glucagon; DAPI BrdU; Somatostatin; DAPI BrdU; PP; DAPI
Pten
+/+
; RIP-Cre
+
Pten
loxP/loxP
; RIP-Cre
+
Bayan et al Figure 1
50 !m

Figure 16. Peri-islet proliferating cells are confined to extra-islet tissues. Control (Pten
+/+
;
Rosa26
lacZ
; RIPCre
+
) and Pten null (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
) were treated with STZ for 5
consecutive days. Pancreas were dissected and costained with endocrine cell markers:
glucagon, pancreatic polypeptide, somatostatin (red), BrdU (green), and DAPI (blue). Islets are
designated in dashed circles. Original magnification, 20X.

48

Glucagon;
BrdU; DAPI PP; BrdU; DAPI
Somatostatin;
BrdU; DAPI
Bayan et al Supplemental Figure 2
a.
Pten
+/+
; RIP-Cre
+
Pten
loxP/loxP
; RIP-Cre
+
50 !m
Supplemental Figure 2: Proliferating activity are mostly confined to "-cells in non-
treated mice. Pancreatic tissues were stained for glucagon, pancreatic polypeptide or
somatostatin (Red) and costained with BrdU (Green) and DAPI (Blue) for nuclei. Very
little BrdU positive cells are outside the islet confine (dotted circles). Right panels are a
magnification of the area within the dashed squares.

Figure 17. Proliferating activity is mostly confined to β-cells in non-treated mice.
Pancreatic tissues were stained for glucagon, pancreatic polypeptide or somatostatin (red) and
costained with BrdU (green) and DAPI (blue) for nuclei. Very little BrdU positive cells are outside
the islet confine (dotted circles). Right panels are a magnification of the area within the dashed
squares.

With this long term BrdU labeling, however, we found that PTEN loss in β-
cells led to extensive mitotic activity surrounding the regenerating islets in the
STZ-treated mice. Indeed, majority of the proliferating cells in the pancreas are
negative for insulin or endocrine hormones and are clustered around the islets
(Figure 16 & 18). Quantitation of the number of islets with this peri-islet
proliferation phenotype showed that 54.3%±4.7 of the islets within the Pten null
pancreas have this phenotype compared to 6.8%±3.6 in the controls (Figure 18).

49

Pten
loxP/loxP
;Rosa26
lacZ/lacZ
;
RIP-Cre
-
ten
loxP/loxP
;Rosa26
lacZ/lacZ
;
RIP-Cre
+
Bayan et al Figure 2
50 !m
B.
Percentage of Islets with
Peri-Islet Proliferation (%)
0
10
20
30
40
50
60
Control Pten null
*
A.
50 !m
Insulin + BrdU + DAPI
Insulin + BrdU + DAPI
Pten
loxP/loxP
;Rosa26
lacZ/lacZ
;
RIP-Cre
-
Pten
loxP/loxP
;Rosa26
lacZ/lacZ
;
RIP-Cre
+
Insulin + BrdU + DAPI

Figure 18: Peri-islet proliferating cells induced by STZ treatment do not express insulin.
(A) Control (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
-
) and Pten null (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
)
mice were treated with either vehicle sodium citrate (top row) or STZ to induce β-cell injury
(bottom row) and the pancreas was analyzed with the endocrine β-cell marker insulin (red),
proliferating cell marker BrdU (green) and nuclear cell marker DAPI (blue). Top row: The left
panel is the control (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
-
) islet. The right panel is the Pten null
(Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
) islet. Original magnification, 20X. Bottom row: The left panel is
the control (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
-
) islet. The middle panel is the Pten null (Pten
loxP/loxP
;
Rosa26
lacZ
; RIPCre
+
) islets. Original magnification, 20X. The right panel is a higher magnification
of the area within the dashed square. (B) Graph summarizing the ratio of the number of islets
with increased per-islet proliferation compared to the number of islets with little to no peri-islet
proliferation (n=4). The bars represent the average percentage of peri-islet proliferation for the
controls and mutants. The error bars represent the SEM for all animals analyzed for that group.
*p≤0.05 is considered to be statistical significant.

50

This observation prompted us to test further whether these peri-islet cells
may contribute to the regeneration potential of the Pten null β-cells. We explored
the possibility that the peri-islet cells are de-differentiated β-cells due to PTEN
loss and activation of PI3K signaling pathway. We hypothesized that the rapidly
proliferating β-cells in the Pten null pancreas need to reduce the burden of
secretory granule (Cozar-Castellano et al., 2008). To test this hypothesis and
determine whether these proliferating cells were β-cells that lost their insulin
expression, we crossed the Pten
loxP/loxP
; Rip-Cre
+
mice with a reporter strain
Rosa26
lacZ
. In this model, all cells that expressed insulin at any given point
during their existence are labeled with β-gal. Immunochemical analysis of
pancreatic sections confirmed the expression of lacZ reporter gene in the islets of
Langerhans (Figure 19). In the Pten
loxP/loxP
; Rosa
lacZ
; Rip-Cre
+
mice, insulin and
β-gal are colocalized to the same cell (Figure 19). This result suggests that loss
of Pten, as indicated by expression of lacZ (positive for β-gal) is confined to the
insulin positive β-cells in the pancreas as predicted. Similar to the results
previously reported with the β-cell specific Pten-deletion model (Stiles et al.,
2006), islets in the newly developed model are larger. The number of islets is
also considerably higher in the Pten null mice than the controls (data not shown),
confirming that addition of lacZ reporter gene did not affect the phenotype of Pten
deletion.

51

Bayan et al Supplemental Figure 3
d.
Pten
loxP/loxP
;
Rosa
lacZ/lacZ
; RIP-Cre+
Pten
loxP/loxP
;
Rosa
lacZ/lacZ
; RIP-Cre-
H&E
In Situ
!-gal staining
Supplemental Figure 3: Generation of Pten
loxP/loxP
;
Rosa26
lacZ
;RIPCre mice. Control (Pten
loxP/loxP
;
Rosa26
lacZ
;RIPCre
-
)and Pten null (Pten
loxP/loxP
;
Rosa26
lacZ
;RIPCre
+
) mice. pancreata were analyzed with
H&E staining (top panel) and in situ !-gal staining (bottom
panel). Blue stain indicates Pten-deleted !-cells. Original
magnification, 20X.

Figure 19. Generation of Pten
loxP/loxP
; Rosa26
lacZ
; RIP-Cre mice. Control (Pten
loxP/loxP
;
Rosa26
lacZ
; RIP-Cre
-
) and Pten null (Pten
loxP/loxP
; Rosa26
lacZ
; RIP-Cre
+
) mice. Pancreata were
analyzed with H&E staining (top panel) and in situ (bottom panel). Blue stain indicates Pten-
deleted β-cells. Original magnification, 20X

Using β-gal staining, we determined if the BrdU positive and insulin
negative cells express β-gal. A positive result would suggest that these cells
were β-cells that lost their insulin staining. Our results indicate that none of the
surrounding cells are positive for β-gal (Figure 20 a-d). This is true for both the
control (Fig 20 a,c) and the Pten null (Figure 20 b,d) pancreas. In addition, the
majority of the BrdU positive cells within the islets are also negative for β-gal
(Figure 20 e). This result suggests that loss of PTEN in the β-cells produced a
paracrine effect to induce the proliferation of surrounding non-β-cells under the
STZ-induced injury condition.

52

Bayan et al Figure 3
!-Gal + BrdU+ DAPI
Pten
loxP/loxP
;Rosa26
lacZ/lacZ
;
RIP-Cre
+
Pten
+/+
;Rosa26
lacZ/lacZ
;
RIP-Cre
+
a
b
cd
e
50 "m

Figure 20. Dedifferentiation of β-cells is unlikely to contribute to the peri-islet proliferating
activity induced by STZ. Mice were treated with STZ to induce β-cell injury and the pancreas
was analyzed with β-gal (red), BrdU (green) and DAPI (blue). (a) Control islet (Pten
+/+
;
Rosa26
lacZ
; RIPCre
+
). (b) Pten null islet (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
). Original magnification,
20X. (c) Higher magnification of area within the dashed square of control islet. (d) Higher
magnification of area within the dashed square of mutant islet. (e) Higher magnification of area
within the solid square of mutant islet. Dashed circles indicate islet area.

53

Further, we stained the pancreatic sections with amylase and CK to mark
the acinar cells and ductal cells, respectfully. Our data shows that the
proliferating BrdU positive, insulin negative cells are not these two common
exocrine cell types. Very little colocalization was found for BrdU with amylase or
CK (Figure 21). We also stained the sections with several potential progenitor
cell markers including Pdx-1, a pancreatic progenitor cell marker and Ngn3, a
pancreatic endocrine progenitor cell marker. Our staining shows that none of the
proliferating cells are these cell types (Figure 21).
Pten
+/+
; Rosa26
lacZ/lacZ
; RIP-Cre
+
Pten
loxP/loxP
; Rosa26
lacZ/lacZ
;RIP-Cre
+
Bayan et al supplemental Figure 4
BrdU; Amylase; DAPI
BrdU; Amylase; DAPI BrdU; Pan-CK; DAPI
BrdU; Pan-CK; DAPI
A.
BrdU; Pdx-1
BrdU; GFAP; DAPI
BrdU; Ngn3; DAPI BrdU; Nestin; DAPI
Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-Cre
+
B.
50 !m
50 !m
Supplemental Figure 4: Exocrine or other stem/progenitor cell types do not
contribute to the extensive mitotic activity surrounding the islets in the STZ
treated Pten null pancreas. (A) STZ-treated control (Pten
+/+
; Rosa26
lacZ
;RIPCre
+
)
and mutant (Pten
loxP/loxP
;Rosa26
lacZ
;RIPCre
+
)pancreaswerecostainedwitheitherthe
acinar cell marker amylase or the ductal cell marker pan-cytokeratin (red) and BrdU
(green) and DAPI (blue). The left two panels are the controls and the right two panels
are the mutants. Original magnification, 20X. (B) Mutant pancreas (Pten
loxP/loxP
;
Rosa26
lacZ
;RIPCre
+
)werecostainedwithvariouspotentialprogenitor cellmarkers:
pancreatic progenitor cell markers Ngn3 and PdX-1, neuronal stem cell marker nestin
and astrocyte cell marker GFAP (red), and BrdU (green) and DAPI (blue). Top panel is
the original magnification 20X. Bottom panel is higher magnification of the
corresponding IHC. Dashedcircles indicateislet area.

Figure 21. Exocrine or other stem/progenitor cell types do now contribute to the extensive
mitotic activity surrounding the islets in the STZ-treated Pten null pancreas. (A) STZ-
treated control (Pten
loxP/loxP
; Rosa26
lacZ
; RIP-Cre
-
) and mutant (Pten
loxP/loxP
; Rosa26
lacZ
; RIP-Cre
+
)
pancreas were costained with either the acinar cell marker amylase or the ductal cell marker pan-
cytokeratin (red) and BrdU (green) and DAPI (blue). The left two panels are the controls and the
right two panels are the mutants. Original magnification, 20X. Dashed circles indicate islet area.

54

Pten
+/+
; Rosa26
lacZ/lacZ
; RIP-Cre
+
Pten
loxP/loxP
; Rosa26
lacZ/lacZ
;RIP-Cre
+
Bayan et al supplemental Figure 4
BrdU; Amylase; DAPI
BrdU; Amylase; DAPI BrdU; Pan-CK; DAPI
BrdU; Pan-CK; DAPI
A.
BrdU; Pdx-1
BrdU; GFAP; DAPI
BrdU; Ngn3; DAPI BrdU; Nestin; DAPI
Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-Cre
+
B.
50 !m
50 !m
Supplemental Figure 4: Exocrine or other stem/progenitor cell types do not
contribute to the extensive mitotic activity surrounding the islets in the STZ
treated Pten null pancreas. (A) STZ-treated control (Pten
+/+
; Rosa26
lacZ
;RIPCre
+
)
and mutant (Pten
loxP/loxP
;Rosa26
lacZ
;RIPCre
+
)pancreaswerecostainedwitheitherthe
acinar cell marker amylase or the ductal cell marker pan-cytokeratin (red) and BrdU
(green) and DAPI (blue). The left two panels are the controls and the right two panels
are the mutants. Original magnification, 20X. (B) Mutant pancreas (Pten
loxP/loxP
;
Rosa26
lacZ
;RIPCre
+
)werecostainedwithvariouspotentialprogenitor cellmarkers:
pancreatic progenitor cell markers Ngn3 and PdX-1, neuronal stem cell marker nestin
and astrocyte cell marker GFAP (red), and BrdU (green) and DAPI (blue). Top panel is
the original magnification 20X. Bottom panel is higher magnification of the
corresponding IHC. Dashedcircles indicateislet area.

Figure 21, continued. Exocrine or other stem/progenitor cell types do now contribute to
the extensive mitotic activity surrounding the islets in the STZ-treated Pten null pancreas.
(B) Mutant pancreas were costained with various potential progenitor cell markers: pancreatic
progenitor cell markers Ngn3 and Pdx-1, neuronal stem cell marker nestin, and astrocyte cell
marker GFAP (red), and BrdU (green) and DAPI (blue). Top panel is the original magnification,
20X. Bottom panel is higher magnification of the corresponding IHC. Dashed circles indicate
islet area.

III-2-2 Stellate cells support the growth of β-cells.
A well-established effect of low dose STZ treatment is the induction of
inflammatory response and lymphocyte infiltration. We show here evidence of
inflammatory cell infiltration in the STZ-treated mice (Figure 22). CD45 positive
immune cells are evident in some but not all islets.

55

Bayan et al supplemental Figure 5
CD45+BrdU+DAPI
Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-Cre
+
Supplemental Figure 5: Inflammatory cell infiltration in
the STZ treated mice. STZ-treated mutant (Pten
loxP/loxP
;
Rosa26
lacZ
;RIPCre
+
)pancreas were costained with CD45
(leukocyte cell marker) (red), BrdU (green) and DAPI (blue).
Top panel is the original magnification, 20X. Bottom panel is
higher magnification of the corresponding IHC within the
indicated square.
!
Bayan et al supplemental Figure 5
CD45+BrdU+DAPI
Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-Cre
+
Supplemental Figure 5: Inflammatory cell infiltration in
the STZ treated mice. STZ-treated mutant (Pten
loxP/loxP
;
Rosa26
lacZ
;RIPCre
+
)pancreas were costained with CD45
(leukocyte cell marker) (red), BrdU (green) and DAPI (blue).
Top panel is the original magnification, 20X. Bottom panel is
higher magnification of the corresponding IHC within the
indicated square.

Figure 22. Inflammatory cell infiltration in the STZ-treated mice. STZ-treated mutant
(Pten
loxP/loxP
; Rosa26
lacZ
; RIP-Cre
+
) pancreas were costained with CD45 (leukocyte cell marker)
(red), BrdU (green) and DAPI (blue). Top panel is the original magnification, 20X. Bottom panel
is the higher magnification of the corresponding IHC within the indicated square.

Infiltration of inflammatory cells suggests that the Pten null pancreas
sustained extensive injury. In the pancreas, injury induced inflammatory
conditions such as pancreatitis promote growth of epithelial cells by stimulating
the activation of ECM cells. To determine if ECM in the STZ-treated Pten null
pancreas is activated, we evaluated the pancreatic sections with mesenchymal
marker smooth muscle actin α (SMAα) and desmin. We found that both SMAα
and desmin stainings are significantly induced in the Pten null pancreas (Figure

56

23). Particularly, these SMAα and desmin positive cells are physically located at
the peri-islet area where proliferation activities are high. Desmin is also a marker
for pancreatic stellate cells (PSCs).
Stellate cells from ECM are found to support the growth of epithelial cells
in malignant pancreas (Vonlaufen et al., 2008). We investigated the hypothesis
that activation of stellate cells may support the growth of β-cells using a co-
culture system of stellate cells and β-cells. Hepatic stellate cells (HSCs) are
more abundant and 99.9% similar to PSCs (Kordes et al., 2009). Using a β-cell
line (β-TC-6) cells and rat HSC, we observed that β-TC-6 cells co-cultured with
HSCs grow much faster than those grown on mono layer single population
cultures. Cell count indicated a 2-fold increase (from 1.2X10
5
to 2.1X10
5
) in cell
number 3 days after initial co-culturing (Figure 24A). We followed this
experiment by determining if this phenomenon requires cell-cell contact. We
collected conditioning medium from the HSC cultures and applied them to the β-
TC-6 culture. The conditioned medium similarly induced the proliferation of β-
TC-6 cells. Cell count increased from 0.8X10
5
to 1.8X10
5
when conditioned
medium were added. To address whether this increase in cell number is due to
increased cell proliferation, we performed
3
H-thymidine incorporation study.
When β-TC6 cells are cultured in conditioned medium collected from HSC culture,
more than 2-fold higher
3
H-thymidine were incorporated into the cells than when
they were cultured with standard medium (Figure 24B), confirming the increased
proliferation.

57

Bayan et al Figure 4
Need the TIFF for this
SMA!+BrdU+DAPI
B.
A.
Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-Cre
+
Pten
loxP/loxP
;
Rosa26
lacZ/lacZ
; RIP-Cre
-
Pten
loxP/loxP
;
Rosa26
lacZ/lacZ
; RIP-Cre
-
Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-Cre
+
Desmin+BrdU+DAPI

Figure 23: PTEN loss leads to expansion of surrounding mesenchymes and pancreatic
stellate cells. (A) STZ-treated control (Pten
+/+
; Rosa26
lacZ
; RIPCre
+
; left panel) and Pten null
(Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
; middle and right panels) pancreas sections were costained with
smooth muscle actin α (SMAα) and BrdU. Original magnification, 20X. (B) STZ-treated control
(Pten
+/+
; Rosa26
lacZ
; RIPCre
+
; top panel/row) and Pten null (Pten
loxP/loxP
; Rosa26
lacZ
; RIPCre
+
;
middle and bottom rows) pancreas sections were costained with Desmin and BrdU. Middle row
indicates original magnification, 20X. Bottom row indicates higher magnification of mutants.
Islets are designated in dashed circles.

58

!-T6 Cells + HSC !-T6 Cells + Control
0
5
10
15
20
25
Control Conditioned Media
0
5
10
15
20
25
Control Co-Culture
*
*
Number of Cells (x10
4
)
Number of Cells (x10
4
)
Bayan et al Figure 5
A.
0
5
10
15
20
25
30
Contol Conditioned Media
Averge CMP
*
B.

Figure 24. Stellate cells support the growth of β-cells in vitro. (A) β-TC-6 cells were cultured
either with a control insert containing no cells (left panel) or with isolated HSCs (right panel) and
incubated for three days. Original magnification, 20X. (B) β-TC-6 cells treated with conditioned
media (left graph) or co-cultured with HSCs (right graph) and incubated for three days. The bars
represent the average number of cells (x10
4
/mL) counted for the controls and experimental
conditions. The error bars represent the SEM for all animals analyzed for that group. *p≤0.05 is
considered to be statistical significant. (C) Graph is representative of
3
H thymidine incorporation
in proliferating β-TC-6 cells. 3H incorporation is performed at least three times with different
incubation time. The bars represent the average counts per minute (CPM) for the controls and
conditional medium cultures. The error bars represent the SEM for all animals analyzed for that
group. *p≤0.05 is considered to be statistical significant.

59

III-3 Discussion
Tissue repair involves a dynamic sequence of events that leads to
regeneration of the injured tissues. One of the early events after the insult is
stopped is the building and deposition of extracellular matrix (ECM) to support
the regeneration of lost cells. This process, while not well understood, involves
the crosstalk between injured cells and other resident cells, as well as the
infiltrating immune cells. In this study, we showed that activation of surrounding
mesenchymal cells occurs simultaneously with regeneration of the injured islet β-
cells. The fast regenerating β-cells in the Pten null model are accompanied by
significantly more accumulation of activated stellate cells surrounding the islets
compared to the slower regenerating β-cells in mice with intact PTEN. These
mesenchymal cells, likely pancreatic stellate cells (PSCs) support the growth of
the injured β-cells by producing soluble factors.
Pancreatic stellate cells (PSCs) are a type of myofibroblast that can be
activated during injury. When activated, PSCs can proliferate and migrate to the
site of tissue injury (Omary et al., 2007). They lay down extracellular matrix to
promote repair of damaged tissues. These stellate cells express an array of
markers including GFAP and nestin when dormant and SMAα and desmin when
activated (Omary et al., 2007). The peri-islet cell population with high mitotic
activity that we have identified expresses SMAα and desmin, but not GFAP or
nestin, indicating that they might be activated stellate cells. The morphology of
this cell population with elongated fiber-like body also resembles activated

60

stellate cells found at site of injury (Masamune et al., 2008). Our data suggests
that PTEN loss in β-cells may signal these activated stellate cells to proliferate in
order to promote the regeneration of the β-cells. Research from the pancreatic
cancer field suggests that there is a symbiotic relationship between cancer cell
growth and activation of SMAα expressing stellate cells (Omary et al., 2007). We
showed here that such a relationship may also exist for the growth of injured
islets and the surrounding stellate cells. In the Pten null mice, the fast
regenerating β-cells may induce the activation of myofibroblasts in an effort to
support their rapid growth. How loss of PTEN in the epithelial β-cell
compartment led to such response in the stellate cell compartment however, is
unclear. Speculations from the pancreatic cancer and fibrosis field suggest the
involvement of TGFβ in this process (Omary et al., 2007). While TGFβ and
PTEN signals do crosstalk (Assinder et al., 2009), whether the results seen in
this model are due to an altered response in TGFβ signaling needs to be further
explored.
Recently, it was shown that β-cells may reduce their insulin secretion
when proliferating (Cozar-Castellano et al., 2008). Although this study uses
insulinoma cell lines, the implication may extend to β-cells in vivo. The primary
function of β-cells is to secrete insulin. The cytoplasm of β-cells is laden with
insulin granules. In order to make space in the cytoplasm for β-cells to replicate,
it is not unlikely that β-cells need to shed their insulin granules. Human β-cells
cultured in vitro can go through epithelial-mesechymal transition (EMT) to

61

proliferate (Gershengorn et al., 2004). Cells produced in vitro through such EMT
process were shown to produce high levels of C-peptide in SCID mice capable of
rescuing hyperglycemia. Here, we demonstrate in vivo that the proliferating cells
surrounding the regenerating islets in the STZ-treated Pten null mice are not β-
cells that lost their insulin granules in an effort to proliferate. Our lineage analysis
clearly show that in either maintenance or STZ-stimulated stage, fast proliferating
cells surrounding the islets are negative for β-gal staining, an indication of non-
β-cell origin. Therefore, a potential “dedifferentiation” process does not occur in
vivo in β-cell maintenance or in this STZ-treated stage in our mouse model. This
conclusion is in agreement with the in vitro study using mouse islets showing that
migrating cells from cultured islets are indeed fibroblasts rather than β-cells going
through EMT (Atouf et al., 2007). Proliferating β-cells may reduce their insulin
burden as shown in vitro with insulinoma cell lines (Cozar-Castellano et al.,
2008). However, they do not change their cell fate in the effort to proliferate even
in the Pten deletion mice. These studies, however, suggest that communication
between β-cells and surrounding mesenchymal cells are critical for maintaining
the growth of both cell types.
Regenerating pancreatic β-cells is a long-term goal for regenerative
medicine and cell-based therapy given the ever-growing incidence of diabetes.
Recent studies highlighted the plasticity of β-cells in their ability to grow and
proliferate and also adapt and alter their identity (Dor et al., 2004; Elghazi et al.,
2008; Teta et al., 2007). Recent advances in the stem cells field also highlighted

62

the plasticity of somatic cells. By manipulating key transcriptional factors,
exocrine cells are capable of producing insulin (Zhou et al., 2008). However,
they do not migrate and form clusters like the β-cells do. In chicken pancreas,
intercalated islet stellate cells were found to contribute to the mass of β-cells
(Datar and Bhonde, 2009). During embryogenesis, development of the islets is
coordinated with surrounding tissues. Communication between mesoderm and
endoderm help set the pattern for pancreas development (Kumar et al., 2003).
Similarly, regeneration of islets in adult tissues may also rely on such
communications. Here, we showed that myofibroblasts such as stellate cells
promotes the growth and proliferation of β-cells in culture. In vivo, islet β-cells in
the Pten null mice are surrounded by more myofibroblasts and display higher
mitotic indexes in response to injury. This evidence suggests that myofibroblasts
support the growth and regeneration of pancreatic β-cells. Our study indicated
that communication between the stellate cell-like cell population and β-cells may
be important for the maintenance of islets. Future studies are needed to further
dissect the specific signals that promote this effect.

63

Chapter IV

Overall Discussion

Due to their essential role in maintaining euglycemia and metabolic
functionality, β-cells are an important target for diabetes therapies. There are
many studies trying to understand how β-cell activity is regulated, especially how
β-cell regeneration can be achieved because it could provide a possible source
for β-cells that can be transplanted into diabetic patients. Although the
pathological defects that result in diabetic conditions are abundant, the extent of
the loss of pancreatic β-cell mass is a critical determinant of disease
development (Kahn, 2001). Studies have shown that β-cell mass is dictated by
the dynamic balance between neogenesis, proliferation, cell size and apoptosis
(Bonner-Weir, 2000). Growth factors and nutrients, such as insulin, insulin
growth factors, and glucose, play crucial roles in β-cell mass regulation.
Furthermore, the capacity of β-cells to expand in response to insulin resistance
and pancreatic injury is critical in maintaining glucose homeostasis.
The PTEN regulated PI3K signaling pathway is a primary mitogenic
pathway for many cell types. Evidence from the IGF/Insulin-IRS studies has
suggested a role for this pathway in the regulation of β-cell homeostasis (Nguyen
et al., 2006; Tuttle et al., 2001). Studies targeted at AKT, the major downstream
effector of PI3K also indicated a dominant role of AKT in β-cell homeostasis
(Bernal-Mizrachi et al., 2001; Tuttle et al., 2001). Regeneration of β-cells is the
major target for diabetes treatment. Understanding the molecular mechanism for

64

maintaining β-cell plasticity and homeostasis is crucial for the ultimate goal of
stimulating β-cell regeneration.
Restoring insulin production can be achieved by increasing islet mass or
restoring their function. Recent studies on the cell-cycle biology of β-cells have
demonstrated that the proliferative capacity of β-cells can be modified (Cozar-
Castellano et al., 2006). While these studies clearly demonstrated that restoring
insulin production from remnant β-cells is possible, they underscored the
contribution of other cell types to the enhancement of β-cell mass. In addition, it
is entirely unclear if the increased islet mass phenotype is intrinsic to β-cells with
no contribution from other cell types in these genetically manipulated models. To
address this question, we developed a Pten
lox/lox
; Rosa26
lacZ
; Rip-CreER
+
mouse
model, which permits Pten deletion by treatment of tamoxifen. We evaluated
islet mass and function as well as β-cell proliferation in adult mice treated with
tamoxifen (Pten deleted) vs. mice treated with vehicle (Pten control). We showed
that deletion of Pten in adult β-cells significantly induced their proliferation and
increased islet mass. The expansion of islet mass occurred concomitantly with
lowered fasting and fed glucose levels as well as lowered circulating insulin
levels, suggesting enhanced ability of the Pten deleted mice to maintain
euglycemia. In addition, islets with adult onset Pten deletion are also more
resistant to streptozotocin (STZ)-induced apoptosis. In older mice, deletion of
Pten similarly induced increases of islet mass and β-cell proliferation. This later
experiment suggests that PTEN regulated mechanisms may override the age

65

onset diminished ability of β-cells to respond to mitogenic stimulation. We also
showed that regulatory proteins involved in G1/S transition may play a role in the
network that mediates PTEN/PI3K regulated β-cell proliferation and sustained
long-term deletion of Pten in β-cells does not lead to insulinoma development.
The increase in total islet mass phenotype seem in Pten null mice was
accompanied by an increase in peri-islet mitotic activity, particularly in islets
injured by STZ. Therefore, we then investigated the role of myofibroblasts,
particularly stellate cells, in the growth and regeneration of pancreatic β-cells. We
showed that mesenchymals surrounding the pancreatic islets are activated
(induced to proliferate) in the Pten null mice compared to the control mice.
These mesenchymals display markers of pancreatic stellate cells such as
smooth muscle actin α and desmin positive pancreatic stellate cells. Further
experiments showed that activated stellate cells support the proliferation of β-
cells. Our data suggest that loss of PTEN in the β-cells resulted in a paracrine
effect to induce the proliferative activity in the surrounding mesenchymes. These
mesenchymal cells may support the regeneration of the islets. Identifying how
this communication occurs may provide clinically relevant mechanism for
inducing β-cell regeneration. Together, our present study suggests that the
signalings regulated by PTEN play a key role in β-cell proliferation and
maintenance and β-cell regeneration may be under mitogenic regulation with
PTEN regulating β-cell regeneration in a paracrine fashion.
Overall, targeting and manipulating the PI3K/PTEN/AKT signaling

66

pathway by deleting its negative regulator Pten has the capacity to induce the
proliferation of adult and aged β-cells. The mitogenic signaling that regulates
PTEN may regulate β-cell proliferation and regeneration in a cell intrinsic manner
and play an important role in the maintenance of islet mass. During times of
pancreatic stress or injury, mesenchymals become activated in order to stimulate
the regeneration capacity of the injured β-cells. Communication between the
stellate cell population and β-cells may play an important role in the maintenance
of the islets. Therefore, understanding how β-cells regenerate can lead to a
potential therapeutic approach to treating diabetic conditions. The PTEN
regulated signaling pathway could be a possible target for therapeutic
interventions targeted at type 1 diabetes.
Furthermore, although there are several theories as to how β-cells
regenerate (self-duplication, differentiation of ductal stem/progenitor cells, acinar
transdifferentiation, stem/progenitor cell differentiation) there still appears to be
no definitive answer as to which theory is preferred. In the mouse model, self-
duplication seems to be the mechanism of choice for normal beta-cell turnover.
However in response to specific insults, the pancreas exhibits a degree of
plasticity making it hard to determine the mechanism(s) involved. Although, it
appears that there may be resident stem cells (i.e. pancreatic stellate cells) that
have the ability to protect beta-cell damage when exposed to a toxin (Figure 25).

67

Figure 25: β-cell regeneration is maintained in Pten-null β-cells without involvement of
developmental factors and injury. Targeted deletion of Pten specifically in adult and aged β-
cells leads to a regeneration phenotype without the influence of developmental factors. In
increase in islet mass, proliferation, and glucose metabolism are observed. In a non-injury
model, the β-cells mass expands via self-duplication. However, when the β-cells are subjected to
the β-cell toxin STZ, quiescent resident pancreatic stellate cells (PSCs) become activated and
support the growth and regeneration of the β-cells. These mice are resistant to STZ-induced
diabetes, display increase islet mass and β-cell maintenance is observed.

68

Chapter V

Materials and Methods
Animals. Targeted deletion of Pten in β-cells was achieved by crossing
Pten
loxP/loxP
mice with rat insulin promoter-CreER
+
(RIP-CreER
+
) mice (Dor et al.,
2004). F
1
generation compound heterozygous animals were backcrossed with
Pten
loxP/loxP
mice to produce F
2
generation experimental animals. These mice
were then crossed with Rosa
lacZ
reporter mice to generate Pten
loxP/loxP
;
Rosa26
lacZ
; RIP-CreER mice. Of the offspring generated, we used male
Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-CreER
+
mice (with Tamoxifen) as our mutants
(Pten null) and male Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-CreER
-
(with Tamoxifen) or
Pten
loxP/loxP
and Rosa26
lacZ/lacZ
; RIP-CreER
+
mice (with Corn Oil) as our controls.
Targeted deletion of Pten in β-cells was achieved by crossing Pten
loxP/loxP

mice with rat insulin promoter-Cre (RIP-Cre) mice (Stiles et al., 2006). F
1
generation compound heterozygous animals were backcrossed with Pten
loxP/loxP

mice to produce F
2
generation experimental animals. These mice were then
crosses with Rosa
lacZ
reporter mice to generate Pten
loxP/loxP
; Rosa26
lacZ
; RIP-Cre
mice. Of the offspring generated, we used male Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-
Cre
+
mice as our mutants and male Pten
loxP/loxP
; Rosa26
lacZ/lacZ
; RIP-Cre
-
or
Pten
+/+
and Rosa26
lacZ/lacZ
; RIP-Cre
+
mice as our controls.
Animals were genotyped from tail DNA using standard genomic PCR
techniques (Lesche et al., 2002). Islets were isolated using the islet isolation
protocol described below and brain tissue was removed and lysed overnight.

69

Genotypes of these tissues were obtained using standard genomic PCR
techniques. Animals were housed in a temperature-, humidity-, light-controlled
room (12-h light/dark cycle), allowing free access to food and water. All
experiments were conducted according to the Institutional Animal Care and Use
Committee of the University of Southern California research guidelines.

Immunohistochemistry. Pancreatic tissue was fixed overnight in Zn-Formalin
solution and embedded in paraffin, sectioned in 4µm slices. Hemotoxylin and
Eosin (H&E) staining was performed for morphological analysis.
Immunohistochemistry and indirect immunofluorescence was performed using
the following antibodies: anti-pten (Cell Signaling, Danvers, MA), p-AKT (Cell
Signaling, Danvers, MA), anti-insulin (Invitrogen, Carlsbad, CA); anti-β-
galactosidase (Abcam, San Francisco, CA); anti-BrdU (BD Pharmingen, San
Jose, CA), anti-glucagon (Abcam, San Francisco, CA), anti-somatostatin
(Invitrogen, Carlsbad, CA), and anti-pancreatic polypeptide (Invitrogen, Carlsbad,
CA), anti-Pdx-1 (Abcam, San Francisco, CA), anti-C-myc (Abcam, San
Francisco, CA), anti-cyclin D1 (Santa Cruz, Santa Cruz, CA), anti-cyclin D2
(Santa Cruz, Santa Cruz, CA), anti-p27 (Santa Cruz, Santa Cruz, CA), anti-
amylase (Zymed); anti-pan-cytokeratin (Dako); anti-ngn3 (generous gift from
Michael German); anti-nestin (Chemicon); anti-GFAP and anti-SMAα (Dako);
anti-desmin (Thermo Scientific); anti-CD45 (BD Pharminigen).

70

In Situ X-gal Staining. Fresh pancreatic tissue was rinsed with a mild detergent
used to aid in permeability of tissue to fixative and stain. Tissue was then fixed
with Zn-Formalin for one hour and stained with 1mg/ml X-gal (Sigma-Aldrich, St.
Louis, MO) for 24 hours at 30
o
C on rocker. The following day the tissue was
rinsed with PBS + 3% DMSO and paraffin embedded for sectioning. Sections
were counterstained with hematoxylin and eosin.

Determination of Cell Proliferation. Cell proliferation was evaluated on different
groups of adult mice via bromodeoxyuridine (BrdU) (Sigma-Aldrich, St. Louis,
MO) pulse-labeling. BrdU (1mg/ml) was administered in the drinking water. Mice
were allowed free access to BrdU containing water for five continuous days. On
the sixth day, the mice were sacrificed and the pancreatic tissue was processed
for immunohistochemical analysis using anti-BrdU antibody. Sections were co-
stained with insulin or β-gal to visualize islets and β-cells. For quantitative
analysis, BrdU-positive cells are counted from 5 mice. Two sections per mouse
60µm apart are used for quantitative analysis. All insulin or β-gal positive cells
from the two slides are counted. Cells double positive for both BrdU and insulin/
β-gal are also counted. Mitotic index is determined using percentage of
BrdU+insulin/ β-gal positive cells vs. total insulin/ β-gal positive cells.

Determination of Cell Apoptosis. Beta-cell apoptosis was evaluated using
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).

71

Paraffin-embedded slides we stained and examined using the TUNEL assay kit
(Roche, Indianapolis, IN).

Streptozotocin Treatment. Three-month-old male mice were injected i.p. with 5
subdibetogenic doses of streptozotocin (STZ) (Sigma-Aldrich, St. Louis, MO) at a
dose of 50mg/kg body weight daily for five consecutive days to produce a β-cell
injury model followed by BrdU labeling. Following BrdU pulse-labeling (5 days in
drinking water), mice are sacrificed for tissue collection and assessed for β-cell
proliferation and apoptosis.

Tamoxifen Injection. Tamoxifen (Sigma-Aldrich, St. Louis, MO) was prepared in
corn oil at a concentration of 20mg/ml. Mice were injected intraperitoneally (i.p.)
with either corn oil as control or tamoxifen (6mg per dose every 3 days for 5
doses, 30mg total) and then euthanized and dissected after 1 month to evaluate
the effectiveness of the injection on inducing Pten deletion or at indicated time
points for analysis of β-cell proliferation and phenotypes.

Islet Isolation and Western Immunoblotting. Collagenase P enzyme solution
for rodent islet isolation (Roche, Indianapolis, IN) was infused at 0.8 mg/ml into
the pancreas via the pancreatic duct. Inflated pancreata were then removed and
incubated in Collagenase P solution for 18 min at 37°C. Islets were dissociated
from the exocrine tissues by shaking vigorously several times, followed by Ficoll

72

(Sigma-Aldrich, St. Louis, MO) gradient. Islets were handpicked and lysed by
tissue extraction buffer (Invitrogen, Carlsbad, CA). Lysates with equal amounts of
protein were resolved by SDS-PAGE, followed by transferring to polyvinylidene
fluoride membrane for immunoblotting. The membranes were probed with
specific antibodies against PTEN (Cell Signaling, Danvers, MA), p-AKT (Cell
Signaling, Danvers, MA), anti-pdx-1 (Abcam, San Francisco, CA), anti-c-myc
(Abcam, San Francisco, CA), anti-cyclin D1 (Santa Cruz, Santa Cruz, CA), anti-
cyclin D2 (Santa Cruz, Santa Cruz, CA), anti-p27 (Santa Cruz, Santa Cruz, CA),
and β-actin (Sigma-Aldrich, St. Louis, MO).

Plasma Assays. Glucose levels are determined using a commercially available
Therasense glucometer from tail veil puncture blood sampling. Fasting glucose
is determined from overnight fasted mice. Blood samples are obtained through
cardiac puncture at the time of sacrifice. Plasma is separated from the blood
samples and used for insulin determination using an insulin Elisa kit (Alpco,
Salem, NH). Blood was collected during GTT at 0, 15, 30, 60, 120 minutes after
injection of glucose via orbital eye bleed. Plasma is separated from the blood
samples and used for insulin determination.

Glucose Tolerance Tests. The glucose tolerance test (GTT) was performed on
mice fasted for 16 hours at the groups indicated in the results.

Mice were given a
single dose (2g/kg body weight) of d-dextrose (Sigma-Aldrich, St. Louis, MO) by

73

i.p. injection after a baseline glucose check. Circulating glucose levels were
measured at indicated time after injection using the Therasense glucometer.

Insulin Tolerance Tests. The insulin tolerance test (ITT) was performed on
mice fasted for 16 hours at the groups indicated in the results.

Mice were given a
single dose (0.5U/kg body weight) of human regular insulin (Novo Nordisk,
Princeton, NJ) by i.p. injection after a baseline glucose check. Circulating glucose
levels were measured at 0, 15, 30, 60, 120 minutes after injection using the
Therasense glucometer.

Relative Islet Area Determination. Islet mass was measured using the
Axiovision 4.5 software. Islet and non-islet tissue were measured separately
from two sections per mouse 60µm apart and used for quantitative analysis. The
islet to non-islet ratio was calculated and graphed.

Cell Culture. β-TC-6 cells were obtained from ATCC. Cells were maintained
DMEM medium with 20% FBS, penicillin and streptomycin. Rat HSCs were
isolated from the liver tissues of 500g-550g Wistar rats (Charles River,
Wilmington, MA) under the approval of the Institutional Animal Care and Use
Committee of the University of Southern California. Primary cells were
maintained in DMEM medium with 10% FBS, penicillin and streptomycin.

74

HSC Conditioned Media with β-TC-6 cells. Isolated rat HSCs were cultured in
DMEM medium supplemented with 10% FBS, penicillin and streptomycin. After
three days of incubation, the HSC conditioned media was removed. β-TC-6 cells
(3 X 10
5
cells/well) were seeded in 6-well culture plates (BD Bioscience) in
DMEM medium supplemented with 20% FBS, penicillin and streptomycin. The
following day, the β-TC-6 cell media was removed, and the HSC conditioned
media, supplemented with glucose and FBS, was added to the β-TC-6 cells, and
incubation was continued up to 3 days. At the end of the incubation period, the
cells were trypsinized and counted.

Indirect Co-Culture of β-TC-6 cells and Hepatic Stellate Cells. β-TC-6 cells
(3 X 10
5
cells/well) were seeded in 6-well culture plates (BD Bioscience) in
DMEM medium supplemented with 20% FBS, penicillin and streptomycin. Rat
HSCs (2 X 10
5
cells/culture insert) were seeded into culture inserts of 1.0 µm
pore size in DMEM medium with 10% FBS, penicillin and streptomycin. The
following day, the culture inserts seeded with HSCs were placed into the 6-well
plates containing the β-TC-6 cells, and incubation was continued up to 3 days.
At the end of the incubation period, the cells were trypsinized and counted.

Tritiated Thymidine Incorporation Assay. β-TC-6 cells (3 X 10
5
cells/well)
were seeded in 6-well culture plates (BD Bioscience) in DMEM medium
supplemented with 20% FBS, penicillin and streptomycin. The following day the

75

β-TC-6 cell media was removed, and the HSC conditioned media, supplemented
with glucose and FBS, was added to the β-TC-6 cells. Each well received 0.5
µCi of Thymidine [Methyl-
3
H] (Perkin Elmer, San Jose, CA) and was incubated
for 8, 24 and 30 hours at 37
o
C. After incubation the media was removed and
cells were washed with phosphate-buffered saline, 0.5 mL of 10% trichloroacetic
acid was then added to each well for 1 hour to precipitate the DNA. Unbound
thymidine was removed by a gentle wash with 4
o
C phosphate-buffered saline.
The DNA was then dissolved in 500 µL of 0.1% Triton X
100
in 10% sodium
hydroxide. The final lysates were transferred to a PET flexible microplate (Perkin
Elmer, San Jose, CA) containing 600 µL of scintillation fluid. Measurements
were obtained using the 1450 LSC Microbeta TriLux Counter from Perkin Elmer.

Statistics. Student’s t tests were performed to compare the differences between
control and Pten null mice on quantified data. Data are presented as
Mean+SEM. Statistical analysis was calculated by a one-way ANOVA with
Bonferroni correction as a post-test n order to compare the differences between
groups on quantified data.

76

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Abstract (if available)

Abstract The pancreatic β-cells are responsible for producing insulin and maintaining glucose homeostasis. Loss of β-cells or their inability to compensate for insulin resistance is the major cause for type 1 and type 2 diabetes, respectively. Increasing β-cell mass or generating optimally functional islets in vitro for transplantation could potentially improve or cure type 1 diabetic conditions. Thus, efforts have been focused on improving β-cell mass by understanding and manipulating the mechanisms involved in their differentiation, proliferation and regeneration. Phosphatase and tensin homolog on chromosome 10 (PTEN) is a lipid phosphatase that antagonizes the function of the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Targeted deletion of Pten in insulin producing cells led to a significant increase in total islet mass. This study suggests that β-cell regeneration may be under mitogenic regulation and that PTEN may be regulating β-cell regeneration in a paracrine fashion. Using a conditional knockout mouse model inducing the deletion of Pten, the data show that this deletion in adult and aged β-cells induced their proliferation and increased their islet mass. These mice showed enhanced metabolic function and resistance to streptozotocin (STZ)-induced diabetes. There was also evidence of G1/S protein involvement and no insulinoma development. The role of stellate cells in the growth and regeneration of pancreatic β-cells was then examined. Deletion of Pten was accompanied by an increase in activity surrounding islets treated with STZ. The cells surrounding the islets were activated mesenchymal cells displaying markers of pancreatic stellate cells. Further studies demonstrated that these activated stellate cells support the proliferation of β-cells. Therefore, the loss of PTEN specifically in the β-cells leads to a paracrine effect inducing the proliferative activity of surrounding mesencymal cells. Together, our present study suggests that the signaling regulated by PTEN plays a key role in β-cell proliferation and maintenance and β-cell regeneration may be under mitogenic regulation with PTEN regulating β-cell regeneration in a paracrine fashion.

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Asset Metadata

Creator Bayan, Jennifer-Ann (author)

Core Title Pten regulates beta-cell regeneration intrinsically and independently of development

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Publication Date 08/02/2012

Defense Date 06/06/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag beta-cells,Diabetes,extracellular matrix,OAI-PMH Harvest,pancreatic stellate cells,PTEN,Regeneration

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Stiles, Bangyan L. (committee chair), Maxson, Robert E., Jr. (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email bayan@usc.edu,jennifer.bayan@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-84203

Unique identifier UC11289055

Identifier usctheses-c3-84203 (legacy record id)

Legacy Identifier etd-BayanJenni-1114.pdf

Dmrecord 84203

Document Type Dissertation

Rights Bayan, Jennifer-Ann

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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