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Pten deletion in adult pancreatic beta-cells induces cell proliferation and G1/S cell cycle progression

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Pten deletion in adult pancreatic beta-cells induces cell proliferation and G1/S cell cycle progression

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Content !
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Pten Deletion in Adult Pancreatic Beta-Cells Induces
Cell Proliferation and G1/S Cell Cycle Progression
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By

Kai-Ting Yang

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
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August 2013
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Acknowledgements
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First of all, I want to thank my advisor, Dr. Bangyan Stiles, for her guidance
during my USC life. She not only provided opportunities for me to conduct exciting
research, but also encouraged me to do my best and think outside the box. The
confidence, the tireless work and the passion for science I learn from her will
benefit my whole life.
Next, I would like to thank my committee members, Dr Zoltan Tokes and Dr.
Vijay Kalra. Their support and guidance allowed me to finish my master thesis.
I would also like to thank all the members in Dr. Stiles’ lab, including Drs. Ni
Zeng, Jennifer-Ann Bayan, Vivian Medina, and Lina He, Mr. Yang Li, Ms. Anketse
Kassa, Zhechu Peng and Richa Aggarwal for their technical support and
suggestions. Especially, I want to thank Dr. Ni Zeng. She taught me lots of
professional techniques and encouraged me to do research.
Finally, I would like to thank my family and friends for their consistent support
and company.
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Table of Contents

Acknowledgements………………………………………………………………….ii
List of Figures………………………………………………………………………..vi
Abstract……………………………………………………………………………...vii

Chapter I:
Introduction

I-1 Mass of pancreatic islets………………………………………………………..1
I-2 PI3K/AKT pathway………………………………………………………………3
I-3 PTEN regulation and regeneration…………………………………………….7
I-4 Aging and insulin like signaling…………………………………………………8
1-5 Rationale of the study…………………………………………………………..9

Chapter II:
PTEN loss in adult pancreas results in increased islet size and proliferation
activity

II-1 Introduction and Rationale……………………………………………………11
II-2 Results………………………………………………………………………….12
II-2-1 Generation of adult Pten deletion mice…………………………………12
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II-2-2 increased islet size were observed in both young and aged pten deletion
pancreas ........................................................................................................ 15
II-2-3 Pten deletion in adult pancreas lead to increased proliferation, including
aged onset. .................................................................................................... 19

II-2-4 Rescue proliferated response in aged (>12 months) β-cells when
Pten is deleted. ............................................................................................ 21

II-2-5. Pten deletion in adult pancreas rescues age-induced regeneration
limitation. ...................................................................................................... 21!
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Chapter III:
Endocrine function in adult mice lacking PTEN in β-cells
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III-1 Introduction and Rationale ........................................................................ 23!
III-2 Results ...................................................................................................... 23!
III-2-1 Endocrine function in different genders with Pten deletion in
young mice groups (3 months). .................................................................... 23

III-2-2 Peripheral tissue influenced by endocrine function change. .............. 27
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III-2-3. Aged pancreas (9 months) with Pten deletion maintains higher
insulin levels. ................................................................................................ 30
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Chapter IV:
Molecular mechanism alterations in adult PTEN loss pancreas
IV-1 Introduction and Rationale ....................................................................... 34!
IV-2 Results ..................................................................................................... 35!
IV-2-1. Cell cycle regulation is changed in male and female mice with
Pten deletion at young β-cells (three-month-old). ........................................ 35

IV-2-2. Immunostaining confirms our hypothesis ......................................... 40
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IV-2-3. Pten deletion in old mice result in cell cycle progression. ................ 44
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Chapter V:
Discussion ........................................................................................................ 48
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Chapter VI:
Materials and methods .................................................................................... 54
Bibliography ..................................................................................................... 59
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List of Figures
Figure 1. PI3K/PTEN/AKT pathway 6
Figure 2a. Pten deletion in Pten
loxP/loxP
, Rip-CreER
+
mice treat with
tamoxifen.
13
Figure 2b. Islet size increases after Pten is deleted in both 3-month-old,
and 12-month-old mice.
17
Figure 2c. Pten deletion leads to cell proliferation in adult β-cells. 20
Figure 3a. Endocrine function in PTEN loss adult pancreas. 25
Figure 3b. The influence of Pten deletion pancreas in peripheral tissue. 28
Figure 3c. Endocrine function when lacking Pten in aged pancreas. 32
Figure 4a. Pten deletion in young pancreas leads to change cell cycle
regulators.
38
Figure 4b. Pten deletion in young pancreas leads to change cell cycle
regulators, which is confirmed by immunostaining.
41
Figure 4c. Cell cycle progress when delete Pten in old pancreas. 46

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Abstract
β-cells, which produce and release insulin play a major role in maintaining
glucose homeostasis. PTEN is a lipid phosphatase that antagonizes PI3K/AKT
signaling pathway and control cell proliferation, survival, and metabolism.
Previous results from our lab indicate that PTEN controls the age onset
senescence of β-cells through cyclin D1 dependent p16 signaling pathway. The
current study will investigate whether PTEN regulates adult onset β-cell mass and
function especially in aging condition, and similar molecular mechanism are
involved.
Our lab developed a RIP-CreER
+
, Pten
loxP/loxP
mouse model in which we are
able to induce Pten deletion by tamoxifen treatment on mature mice. I observed
evaluated islet mass and functions in mice treated with tamoxifen (Pten null) vs.
those treated with vehicle (Pten control), suggesting that maturity onset deletion
of Pten leads to significant increase of islet mass and proliferation. In addition, no
major difference on fasting and fed glucose or plasma insulin level between Pten
null and control mice are observed with minor hyperglycemia conditions observed
! viii!
in juvenile mice, likely due to muscle insulin resistance. I found that proteins
regulating G1/S cell cycle transition like those previously observed in Pten
L/L
;
Rip-Cre
+
mice are similarly altered in mice when PTEN loss is induced in adult
stage. The pro-proliferation and anti-aging effects of PTEN in pancreatic β-cells
are likely mediated through cyclinD1/Ezh2/p16
ink4a
signaling pathway.
! 1!
Chapter I
INTRODUCTION
I-1. Mass of pancreatic islets
Pancreatic islet mass and function is a key factor for adequate insulin
secretion, and thus maintenance of glucose homeostasis. It is also critical for the
development of autoimmunity-induced type I and insulin resistance-induced type
II diabetes. During early postnatal stage, neogenesis is the major driving process
to expand islet mass rapidly, whereas slow self-replication and death of existing
cells contribute to the stability of islet mass in the adult pancreas. In adult, the islet
size expands with age or increasing insulin demand (Bouwens et al., 2005). In
addition, islet mass and proliferation rate also increase during pancreatic β-cell
injury or metabolic stress. However, this ability diminishes with age of the β-cells.
The increasing β-cells mitotic activity during these stresses is due to the induction
of proliferation signals. However, the signals that regulate this proliferation and
mechanism are not well characterized.
! 2!
There are several growth factors that can influence β-cell mass and functions,
include insulin, insulin-like growth factor (IGF), hepatic growth factor (HGF) and
platelet-derived growth factor (PDGF). It has been shown that IGF-1 can stimulate
islet cell growth, inhibit apoptosis, and regulate insulin biosynthesis and secretion
(Dor et al., 2004). Supplementation of HGF prevent β-cell apoptosis, preserve
β-cell mass and proliferation in diabetic mice induced by STZ (Dai et al., 2003).
Mice with β-cell specific deletion of the HGF receptor displayed smaller islet size,
mild hyperglycemia, and decreased serum insulin levels (Dai et al., 2005).
Similarly, PDGFα knockout islets fail to proliferate and restore β-cell mass after
STZ challenge. Age-dependent attenuation of β-cell Pdgfr-α altered Erk and
Rb/E2F signaling, resulting in β-cell expansion (Chen et al., 2011). Previous
studies from our group indicate that IGF-1/PI3K/PTEN signal control β-cell mass,
proliferation, as well as regeneration after STZ treatment through
Rb/E2F/EZH2/p16 signaling axis (Zeng et al., 2013). These studies suggest that
PI3K/AKT pathway plays a potential candidate to regulate islet mass.
! 3!
I-2 PI3K/AKT pathway
PI3K and AKT is a major downstream target of insulin/IGF-1, HGF and PDGF.
It also represents a key regulator on controlling cell proliferation, survival and
stress response (Katso et al., 2001). Phosphatidylinositide 3-kinases (PI
3-kinases or PI3Ks) are a family of intracellular signal transducer enzymes,
composed of a heterodimer with a catalytic subunit and a regulatory subunit
(PI3Kr) (Carpenter et al., 1990). When growth factors, such as insulin and IGF-1,
are presented, the growth factor receptors are phosphorylated at Tyr residues.
Subsequently, the PI3K complex is recruited and interacts with the phospho-Tyr
on the receptor by SH2 domain of PI3Kr. The activated form of PI3K
phosphorylates the 3-position hydroxyl group of the inositol ring of
phosphatidylinositol (PtdIns). When PIP
2
(phosphatidylinositol (3,4)-bisphosphate,
PtdIns(3,4)P
2
) is phosphorylated to PIP
3
(phosphatidylinositol
(3,4,5)-trisphosphate, PtdIns(3,4,5)P
3
) by PI3K, the plasma membrane docking
sites is presented for proteins containing pleckstrin-homology (PH) domains
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(Figure 1A) (Sellers et al., 2004). One such protein, AKT, transmits
receptor-binding signals to downstream molecules. AKT is a
serine/threonine-specific protein kinase that plays a pivot role on multiple cellular
processes such as glucose metabolism, apoptosis and cell proliferation. It is
active when it binds to PIP3 and interacts with enzymes located at the membrane.
AKT is phosphorylated at Thr308 and Ser473 residues, and then the signals are
passed downstream (Figure 1B) (Vivanco et al., 2002).
AKT suppresses apoptosis activities by phosphorylating and inhibiting
pro-apoptotic protein BAD. It controls cell cycle and cell proliferation through its
direct action on the CDK inhibitors p21 and p27. It also indirectly affects the levels
of cyclin D1, G1/S transition regulator, through inhibiting GSK3β activity.
Moreover, AKT induce cell cycle arrest by p53 through MDM2 phosphorylation
and gene regulation (Figure 1B) (Vivanco et al., 2002). Thus, AKT is a crucial
pro-survival and pro-growth kinase.
The AKT family of kinases includes three isoforms: Akt1 (PKBα), Akt2 (PKBβ)
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and Akt3 (PKBγ). Phenotypic analyses of mice lacking different AKTisoforms
demonstrated their functions in the regulation of cellular growth, glucose
homeostasis and neuronal development. Akt1 knockout mice display growth
retardation and higher rates of cell apoptosis, indicating a crucial role in cell
survival (Chen et al., 2001). Akt2 knockout mice developed type II diabetes-like
phenotype with increased pancreatic islet mass due to increased demand of beta
cells to overcome peripheral insulin resistance (Yao et al., 2011). Finally, Akt3
knockout mice have smaller brain but the mechanism is still not clear.
Large islet size and reduced apoptosis rate is observed in STZ-treated
transgenic mice that express a constitutively active form of AKT (myr-Akt) in
β-cells (Ernesto et al., 2001), consistence with the pro-proliferation regeneration
and anti-apoptosis roles of AKT. Pancreatic β-cells of RIP-N mice, which express
myr-Akt, also displayed increased resistance to apoptosis induced by stress and
were protected from STZ-induced diabetes (Yang et al., 2009).

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A. B.

Figure 1. PI3K/PTEN/AKT pathway
(A). PI3K/PTEN control signal by phosphorylation and dephosphorylation. (B).
PI3K/AKT signal regulates cell cycle, proliferation, apoptosis and survival.

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I-3 PTEN regulation and regeneration
Phosphatase and tensin homolog (PTEN), a dual lipid and protein
phosphatase, is a well-known negative regulator of PI3K/AKT pathway. It acts as
an anti-proliferation signal by dephosphorylating PIP3 back to PIP2. As a result,
AKT is unable to be activated and transmit growth receptor signals.
Two exciting studies found that PTEN controls pancreatic β-cells proliferation
and function. First, Irs2
-/-
; Pten
+/-
mice were utilized to determine whether
decreased Pten expression could restore β-cell function and prevent diabetes in
Irs2
-/-
mice. They found that the insufficiency of PTEN is able to improve insulin
sensitivity in Irs2
-/-
mice. Irs2
-/-
; Pten
+/-
mice displayed small islets but insulin
production from these islets is sufficient to maintain normal glucose tolerance and
allow the mice to survive without diabetes (Kushner et al., 2005). Another study
suggests that Pten deletion specifically in pancreatic β-cells increase islet size
and mass and induce β-cells regeneration in STZ-treated mice; the β-cells mitotic
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activity increased significantly in these β-cell specific Pten null mice during
embryogenesis (Stiles, 2005). This was collaborated with a later study, which
demonstrated that Pten deletion in pancreatic β-cell led to increased islet mass,
improved insulin sensitivity and glucose tolerance without compromise of β-cell
function. (Nguyen et al., 2006)
1-4 Aging and insulin like signaling
Aging refers to a multidimensional process of physical change. Roughly
100,000 people worldwide die each day of age-related causes that has attracted
dramatically attention in scientific research. The first lifespan regulator gene, daf-2,
was found in nematode, Caenorhabditis elegans (Larsen, 1993). Mutations of
daf-2, which encodes the nematode ortholog of insulin receptor/IGF-1 receptor
(Kimura et al., 1997), and age-1, encoding a PI3K ortholog (Morris et al., 1996),
result in profound extension of life span. Recent studies also demonstrated that
manipulating IGF-1 receptor in mice affects their lifespan and aging-induced
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stresses response. (Bokov et al., 2011) These studies indicate that PI3K/AKT
pathway is crucial for the aging regulation.
How adult β-cell mass can be maintained by this age-related
PI3K/AKT/PTEN signal axis, however, is not clear.
1-5 Rationale of the study
My thesis focus on how PTEN is involved in regulating adult pancreatic β-cell
mass, proliferation, regeneration, endocrine function and molecular mechanism.
Previous studies from our group demonstrate that β-cells in Pten

null mice display
increased ability of cell proliferation during development and regeneration during
injury (Zeng et al., 2013). Increases of both islet size and number were
observed. However, we don’t know if the same phenotype can be observed in
mice post development and particularly in aged mice where regeneration capacity
is restricted. Therefore, we bred the Pten
loxp/loxp
mice with insulin promoter driving
CreER lines to delete Pten in pancreas at selective age. Using these mice model,
my thesis defined that PTEN maintain β-cell mass through proliferation in mice of
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different age, especially in old mice.
In chapter II, I investigated the phenotypical changes in vivo using the
Pten
loxP/loxP
, Rip-CreER mice model. Increasing islet mass by manipulating
PI3K/AKT pathway through Pten deletion was examined in juvenile
(three-month-old) and old (more than 12-months-old) mice. Two possible reasons
to cause expansion of β-cells are increased proliferation and decreased apoptosis.
Both are also investigated. In chapter III, I investigated if the expansion islet can
maintain glucose homeostasis in juvenile as well as aged mice. Finally, analyzing
molecular mechanism involve in rescues aging β-cells through our model allowed
me to demonstrate how Pten deletion control β-cell mass and cellular senescence
during aging effect.
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Chapter II
PTEN LOSS IN ADULT PANCREAS RESULTS IN INCREASED ISLET SIZE
AND PROLIFERATION ACTIVITY
II-1 Introduction and Rationale
Pancreatic β-cells are the major target of diabetes treatment. Under normal
condition, it proliferates in a very low rate and undergoes aging process with
reduced replication and proliferation even with growth factor stimulation in adult
individual, which is consistent with the higher incidence of diabetes in aged
population. Our group found that more and large islets are observed in mice
lacking PTEN specifically in the pancreatic β-cells. Moreover, the proliferation and
regeneration rate also increased dramatically. However, it is not characterized if
the same phenotype can be observed in adult β-cells. To define whether and how
PTEN/PI3K pathway control β-cell mass particularly in adult tissues, we use a
mouse model (Pten
loxP/loxP
, Rip-CreER), which can selectively delete PTEN in
pancreatic β-cells in adult individuals.
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II-2 Results
II-2-1 Generation of adult Pten deletion mice
In Pten
loxP/loxP
, Rip-CreER mouse model, tamoxifen treatment leads to the
activation of the cells expressing insulin. Cre is then able to enter nucleus and
induce recombination specifically at the loxP site. The exon 5 region of Pten, the
catalytic core motif, is located between the loxP sites. As a result, Pten is deleted
only in the pancreatic β-cells at the age tamoxifen is introduced.
We show here that 5 dose of 6mg tamoxifen injection in 2 weeks is sufficient
to induce Pten deletion in mice at different age (3 months old and 12 months old).
These are confirmed by immunostaining, and PTEN was not detected in the islet
of Pten null mice (Pten
loxP/loxP
; Cre
+
; Tamoxifen injection) (figure 2a).

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A

B

C

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Figure 2a. Pten deletion in Pten
loxP/loxP
, Rip-CreER
+
mice treat with
tamoxifen
(A). Representative image of pancreas from control (top panel) and Pten null
(bottom panel) mice. Insulin (green), Pten (red) and DAPI (blue) staining of
pancreas sections from three months and three months post-tamoxifen injection
(six months old) is presented. Pten is unable to detect on the Pten null
(Pten
loxP/loxP
; Cre
+
; Tamoxifen injection) section. (B). Immunofluorescence image
from three months mice and six months post-tamoxifen injection (nine months old).
(C). Section was analyzed by Immunofluorescence from 12 months mice and 12
months post-tamoxifen injection (two years old). Representative image of more
than 3 mice are confirmed in each age.

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II-2-2 Increased islet size were observed in both young and aged Pten
deletion pancreas.
We manipulated Pten deletion on three-month-old mice β-cells by tamoxifen
delivery. Mice were randomly assigned to -Tam or +Tam groups. -Tam group
receives vehicle treatment (corn oil) whereas +Tam group receive 30mg
tamoxifen treatment (5 doses of 6mg in two weeks). After that, mice were
euthanized at different time point to analyze the islet mass and function due to the
effect of Pten deletion. Group 1 (Grp 1) mice were euthanized after one month
post-tamoxifen treatment; Group 2 (Grp 2), 3 months post-treatment; and Group 3
(Grp3), 6 months post-treatment (Figure 2b-A). Phenotype represented by H&E
staining showed that islet size increases significantly in Pten null group (EXP)
(Figure 2b-B). The islet to pancreas ratio calculated by our group suggests that
Pten deletion led to more than 2-fold increasing of islet size at all three time points.
The ratio also increases with post-treatment time.
Previous studies show that adaptive β-cell proliferation and regeneration are
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severely restricted in aged mice (> 8 months) even with growth factors stimulation
(Rankin et al., 2009). The PTEN/PI3K signal was found to stimulate the expansion
of islet mass (Stiles, 2005). However, it is not characterized whether activation of
PI3K/AKT pathway by Pten deletion can overcome this age obstacle. To address
this question, we delete Pten using the same protocol of tamoxifen delivery on
12-month-old mice. Mice were euthanized at one month (Group 1,Grp 1), 9
months (Group 2, Grp 2), and 12 months (Group 3, Grp 3) after 5 doses of
tamoxifen injection (total 30mg) (Figure 2b-C). Similar to 3-month-old mice EXP
groups, islet size increased dramatically in the Pten null mice (Figure 2b-D,
bottom panel) when compare to control group (top panel). Quantitative analysis of
islet/pancreas ratio calculated by our group shows that islet mass increase 1.5 to
2 fold in EXP groups.

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A.

B.

C.

D.

Group!1 Group!3 Group!2
Control Pten!null
50µm
Group!1 Group!2 Group!3
Control Pten!null
50µm
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Figure 2b. Islet size increases after Pten is deleted in both 3-month-old, and
12-month-old mice.
(A). Experiment protocol figure. Mice were treat with 5 doses tamoxifen (6mg per
dose, total 30mg) in two weeks at three months old and then euthanized at
different lag times—1 month (Group 1, Grp 1), 3 months (Group 2, Grp2), and 6
months (Group 3, Grp 3). Control (CON) group include CreER- mice with corn oil
or tamoxifen treatment and CreER+ mice with corn oil treatment, which show
intake Pten expression. (B). Represented H&E pancreas staining image of control
(CON, top panel) and Pten null (EXP, bottom panel) mice for all three groups. (C).
Experiment protocol of aged onset mice. Same tamoxifen delivery protocol was
administered on 12-month-old mice. After 1 month (Group 1, Grp 1), 9 months
(Group 2, Grp 2), and 12 months (Group 3, Grp3) lag time, mice were euthanized.
(D). H&E staining images of control (CON, top) and Pten null (EXP, bottom) mice
from group 1, 2, and 3, representatively.

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II-2-3 Pten deletion in adult pancreas lead to increased proliferation,
including aged onset.
We explored two possible reasons that can lead to the expansion of islet
β-cell mass—cell proliferation increasing, cell death decreasing.
5-bromo-2-deoxyuridine (BrdU) incorporation assay was used to examine
proliferated activity. Three-month-mice with different post-tamoxifen injection lag
time (Group1,2,3) were fed with BrdU containing water for five days before
euthanize. Subsequently, analysis by immunostaining and quantification of BrdU
positive β-cells confirm that Pten deletion increase β-cell proliferation indeed
(Figure 2c). The proliferation rate increases with lag time. A moderate increase
(1.5 times) of BrdU positive β-cells in group 1, two and three fold increase in
groups 2 and 3, were observed by our group in EXP group than CON group.

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A.

Figure 2c. Pten deletion leads to cell proliferation in adult β-cells.
Representative immunofluorescence images of control (CON, left panel) and Pten
null (EXP, right panel) mice pancreas. Tissues from group 1, 2 and 3 were
analyzed by insulin (red), BrdU (green) and DAPI (blue). Group 1 (top panel) were
analyzed one month after 5 dose tamoxifen injection in three-month-old mice.
Group 2 and 3 images indicate 3 months and 6 months post-treatment in
three-month-old mice (n=5-6 per genotype, per group).
Group 1 Group 2 Group 3
CON EXP
Insulin!+!BrdU!+!DAPI
!
!
!
50µm
! 21!
II-2-4 Rescue proliferated response in aged (>12 months) β-cells when Pten
is deleted.
We next studied whether Pten deletion in adult mice can induce mitotic
activity and rescue the loss of proliferation response due to aging. The
experiments were carried out on 12-months-old mice which were euthanized after
1 month (Group, Grp 1), 9 months (Group 2, Grp 2), and 12 months (Group 3, Grp
3) lag time following the last dose of tamoxifen injection. Our group executed the
same BrdU incorporation assay and analyzed cell proliferation through
immunofluorescence. The results demonstrate that the cell proliferation increases
in Pten null β-cells. The percentage of insulin-BrdU double positive cells expand
approximately 4 times in all three EXP groups than CON groups.
II-2-5. Pten deletion in adult pancreas rescues age-induced regeneration
limitation.
Previous reports from our lab show that apoptotic cells are rare and hard to
detect in mice with or without PTEN. To examine cell death rate when Pten is
! 22!
deleted in adult pancreas, our group induced β-cells death by Streptozotocin
(STZ), a chemical that is particularly toxic to the pancreatic β-cells. Subsequently,
cell death analyzed by TUNEL assay indicates that three times more death of
β-cells were observed in the control groups than experiment groups, suggesting
that PTEN loss can prevent apoptosis induced by injury. The plasma glucose
levels in STZ-treated CON groups are exceedingly higher than non-STZ-treated
CON groups (approximately 300mg/dl vs. 120mg/dl respectively in all three
groups). In STZ treated Pten null model, plasma glucose levels remain low in the
Pten null (EXP) groups. Therefore, β-cell function can be maintained during injury
to regulate plasma glucose levels when Pten is deleted.

! 23!
Chapter III
ENDOCRINE FUNCTION IN ADULT MICE LACKING PTEN IN β-CELLS
III-1 Introduction and Rationale
Incidence of insulin resistance increases when animal becomes old.
Peripheral tissues fail to response to the normal insulin action, thus enhancement
of insulin secretion is reqired. To achieve increased requirement of insulin level,
more insulin production and enhanced β-cell number are needed. However, aged
β-cells lose their mitotic activity and fail to increase cell mass in response to such
demand, leading to hyperglycemia. In our model, Pten deletion in adult β-cells
results in increased proliferative activity. Therefore, we examined the potential
endocrine function change due to the expansion of β-cells.
III-2 Results
III-2-1 Endocrine function in different genders with Pten deletion in young
mice groups (3 months).
To define the function of increased islet mass, we examine the fasting and
! 24!
random glucose levels in both male and female mice (n=5-6 per genotype, per
group). In juvenile (3 months) mice where PTEN loss is induced, we observed
slightly lower, but no significand differences of random glucose levels in either
gender. Moderate, but statistically signficant higher fasting glucose levels are
observed in EXP male mice but not in female mice (Figure 3a-A). No difference in
plasma insulin levels between control and experiment mice is observed in either
gender in these juvenile mice (Figure 3a-B).
Moreover, we performed the glucose tolerance test (GTT) and insulin
tolerance test (ITT) to understand how Pten deletion in β-cells affect glucose
metabolism. In GTT, higher glucose level is detected in experiment group of male
mice, which indicate lower sensitivity at cleaning glucose than control group.
There is no significant different in female mice between control and experiment
group (Figure 3a-C). On the other hand, both male and female experiment mice
display slightly lower insulin sensitivity than control mice in ITT (Figure 3a-D).

! 25!
A.

B.

C.

D.

! 26!
Figure 3a. Endocrine function in PTEN loss adult pancreas.
(A). Plasma glucose levels were determined from non-fasting (left-panel) and 16
hours overnight fasting (right-panel) mice (n=5-6 per genotype, per gender group)
at indicated age. (B). Plasma insulin levels were obtained from fed (left-panel) and
16 hours overnight fasting (right-panel) mice (n=5-6 per genotype, per gender
group). (C). Glucose tolerance test (GTT) from young mice. Left panel is male
mice glucose levels during GTT from control and experiment Pten null groups;
right panel is female mice GTT results from control and experiment groups. (D).
Insulin tolerance test (ITT) from young mice. Glucose levels changes during ITT
were performed from male (left panel) and female (right panel) in control and
experiment groups.

! 27!
III-2-2 Peripheral tissue influenced by endocrine function change
Since the GTT and ITT data suggest that the EXP mice display lower insulin
sensitivity, we investigate peripheral tissues that might be influenced by endocrine
function change, possibly due to feedback adaptation. To address this possibility,
we examine GLUT4, insulin-regulated glucose transporter in adipose tissue and
striated muscles; and p-AKT, in the liver and muscle using immunobloting (Figure
3b). In the liver, phospho-AKT increased in male experiment group, suggesting
that higher insulin produced by Pten deletion in islet might contribute to the
activated PI3K/AKT pathway in the liver. No difference in female experiment liver
is observed when compared with control age-matched mice (Figure 3b-A). In the
muscle, there is no difference on phospho-AKT levels. However, lower GLUT4
expression level in both male and female pancreatic Pten deletion groups in
muscle were observed (Figure 3b-B). This downregulation of GLUT4 level in Pten
null muscle may be a reason for the insulin resistance in our model.

! 28!
A.

B.

Male
Control Experiment
GLUT 4
P-AKT (Ser473)
β-Actin
Control Experiment
P-AKT (Ser473)
Female
GLUT 4
β-Actin
Male
Control Experiment
GLUT 4
P-AKT (Ser473)
GAPDH
Control Experiment
P-AKT (Ser473)
Female
GLUT 4
GAPDH
! 29!
Figure 3b. The influence of Pten deletion pancreas in peripheral tissue.
(A). Immunobloting analysis of glucose transporter (GLUT4) and p-AKT involved
in insulin signaling in liver. (B). Immunobloting analysis in skeleton muscle of both
genders in control vs. experiment groups. Quantification by image J.
Standardized basal protein in control groups as 1 and compared relative protein
levels in experiment groups.

! 30!
III-2-3. Aged pancreas (9 months) with Pten deletion maintains higher
insulin levels.
In aged pancreas, increased islet mass is also observed when Pten is
deleted. To determine whether the rescued islet mass are capable of maintaining
normal endocrine functions in the aged mice, glucose and insulin levels were also
determined in nine-month-old mice with one-month post Pten deletion mice.
There were non-significant different on random and fasting glucose levels
between control and experiment mice in either male or female (Figure 3c-A).
Significantly higher fasting plasma insulin was observed in Pten null mice,
indicating likely more insulin secretion by expansion of β-cells. However, only
moderately higher levels were observed when we analyze male and female
separately, possibly due to low sample size (Figure 3c-B). We analyzed the
glucose metabolism in aged pancreas with one-month post Pten deletion using
glucose tolerance test (GTT) and insulin tolerance test (ITT). In female
experiment group, mice were more sensitive in GTT to increased glucose than
! 31!
control group, which is consistent with higher insulin level, while there was no
difference in male groups (Figure 3c-C). In addition, insulin tolerance test data
show that Pten null female mice display lower insulin sensitivity compared with
control group (Figure 3c-D).

! 32!
A.

B.

C.

D.

! 33!
Figure 3c. Endocrine function when lacking Pten in aged pancreas.
(A). Random glucose level (left panel) and 16 overnight fasting glucose level (right
panel) were obtained from aged mice in control and experiment groups. Male and
female mice plasma glucose concentrations were performed separately. (n=2 per
genotype, per group. Same test performed twice in a mouse in another week) (B).
Plasma insulin levels were determined from fed (left panel) and 16 overnight
fasting (right panel) male and female mice in control and experiment groups. (C).
Glucose tolerance test (GTT) from aged mice. Glucose levels from control and
experiment mice altered during GTT were recorded. (D). Insulin tolerance test
(ITT) from aged male and female mice.

! 34!
Chapter IV
MOLECULAR MECHANISM ALERATIONS IN ADULT PTEN LOSS
PANCREAS
IV-1 Introduction and Rationale
There are several molecule regulators that influence β-cell mass and
proliferation including cell cycle regulators, cyclin D1, cyclin D2, p16 and p27.
Previous studies suggest that constitutively active AKT induces β-cells
proliferation by increasing cyclin D1 and D2 expression (Fatrai et al., 2006). In
adult murine islets, basal cyclin D1 mRNA expression is very low; beta-cell
proliferation, and glucose tolerance were decreased in adult cyclin D2
-/-
and cyclin
D1
-/+
D2
-/-
mice (Kushner et al., 2005). Another cell cycle regulator, P27, a cell
cycle inhibitor, can regulate the transition of β-cells from quiescence to
proliferation. β-cells from p27
-/-
mice could retain the ability to reenter cell cycle
after STZ-induced diabetes (Georgia et al., 2006). The PI3K/AKT pathway have
been shown to control these cell cycle regulators directly in several cell types
! 35!
(Charles et al., 2002), indicating that PI3K/AKT pathway may control these G1/S
transition molecules to regulate β-cell mass, proliferation and regeneration.
Previous reports from our lab indicate that PTEN controls the age onset
senescence of β-cells through cyclin D1 dependent p16 signaling pathway (Figure
7A) (Zeng et al., 2013). Cyclin D1, cell cycle G1/S transition regulator, induce the
expression of E2F, transcription factor that bind Ezh2 promotor region, and thus
stimulate Ezh2 to inhibit p16 level by histone methylation. PTEN is able to block
this pathway and inhibit cell proliferation by inducing p16 expression. In this
chapter, we investigated whether the same mechanism of cell proliferation is
conrolled by PTEN in adult pancreas by evaluating cell cycle regulators and
cyclinD1/Ezh2/p16 pathway.
IV-2 Results
IV-2-1. Cell cycle regulation is changed in male and female mice with Pten
deletion at young β-cells (three-month-old).
A novel pathway demonstrated by our lab indicates that PTEN mediate
! 36!
proliferation and anti-aging effect through cyclinD1/Ezh2/ p16
Ink4a
(Figure 4a-A)
(Zeng et al., 2013). To explore how PTEN regulates β-cells proliferation and
anti-aging effect in molecular mechanism level in our model, we investigated G1/S
transition proteins – cyclin D1, D2, cell cycle promoter, and p16, p27, cell cycle
inhibitor as well as the cyclinD1/Ezh2/p16
Ink4a
pathway. First of all, we examined
protein expression profile in young mice (three-month-old) with one-month post
treatment with tamoxifen or vehicle. The results revealed that both of cyclin D1
and D2 levels were induced when Pten was deleted in male and female mice. In
male EXP group, cyclin D1 and cyclin D2 increased approximately 30% and
100% when compare to age-matched control group. Concomitantly, cell cycle
inhibitors, p16, was downregulated when PTEN loss in both genders (Figure
4a-B).
Previous studies from our group showed that cyclin D1 and p16, but not
cyclin D2 and p27, are altered in islets lacking PTEN, particularly when mice get
older. In order to investigate whether PTEN regulates p16 expression in adult
! 37!
pancreas through the same pathway demonstrated by our group in Pten null mice,
we examine Ezh2 level which is a polycomb group protein and able to inhibit gene
expression by methylation. As shown in figure 4a-B, Ezh2 expression increases
when Pten was absent in both male and female mice. This change is more
dramatic in male mice compared with female Pten deletion mice. This data
verifies that inducing deletion of Pten in adult mice can alter the same mechanism
that governs β-cell proliferation as we have previously defined using mice lacking
PTEN at birth.

! 38!
A.

B.

! 39!
Figure 4a. Pten deletion in young pancreas leads to change cell cycle
regulators.
(A). CyclinD1/E2F/Ezh2/p16 signaling pathway. (B). Protein profiles in isolated
islets from one month Pten null lag time after three-month-old male and female
mice (upper panel). Quantification of protein levels change (protein level/actin
level). Standardized protein level/actin level in control group as 1, and compared
relative protein levels change in experiment group. Image J was used to quantify
immunobloting band to numeral.

! 40!
IV-2-2. Immunostaining confirms our hypothesis
Consistently, immunostaining results show that lower levels of p16 in adult
male pancreas (three-month-old) with three months (Figure 4b-A, upper panel)
and six months (Figure 4b-A, bottom panel) Pten deletion lag time compared to
the age-match control mice. Cyclin D1 and D2 levels also increased in Pten null
male islet, shown in the immunostaining images, supporting our previous finding
in Pten
L/L
; Rip-Cre mice (Figure 4b-B). Surprising, the expression of p27
increased 80% in male and 36% in female mice when induce Pten deletion in a
shorter time lag of one month in adult pancreas (Figure 4a-B); however, it did
decrease in three-month-old mice with six months PTEN loss lag time (Figure
4b-B). These results verifies that the same signaling through cyclin D1, D2 and
p16 may regulate β-cell mass in mice where PTEN loss is induced in adult onset.

! 41!
A.

! 42!
B.

! 43!
Figure 4b. Pten deletion in young pancreas leads to change cell cycle
regulators, which is confirmed by immunostaining
(A). Islet Immunohistostaining of p16, insulin and DAPI in a lag time of three
months (upper panel), and six months (bottom panel) Pten deletion after three
month old. (B). Islet Cyclin D1 (upper panel), Cyclin D2 (middle panel), and p27
(bottom panel) expression index shown by represented immunofluorescence
images from mice deleted Pten for six months after three-month-old.

! 44!
IV-2-3. Pten deletion in old mice result in cell cycle progression.
To further investigate whether PTEN loss in aged pancreas overcome
proliferation restriction through the same mechanism, we knockout Pten in
nine-months-old mice and analysis molecule levels change after one month.
Increasing cyclin D1, D2, Ezh2 and decreasing p16 protein level were observed in
these mice (Figure 4c-A upper panel). Consistent with our hypothesis,
age-induced cell cycle arrest can be rescued through Pten deletion. It induces
cyclin D1 and D2 expression and stimulates Ezh2 expression likely through E2F
transcription factor binding on its promoter. Subsequently, Ezh2 inhibit p16
expression level by methylation mechanism, and thus, β-cells mitotic activity is
restored (Zeng et al., 2013).
Furthermore, the relative protein level in experiment group changes with age
within male group (Figure 4a-B bottom left panel vs. Figure 4c-A bottom left panel).
The order experiment male group has much dramatically relative protein level
change in cell cycle regulators and cyclinD1/Ezh2/p16 pathway; while there is no
! 45!
difference in female young v.s. old group. Together with data demonstrate in
Chapter II, these analyzed of cell cycle regulators suggests that Pten deletion in
male aged pancreas have capacity to overcome age-induced lower mitotic activity.
Besides, lower p16 expression in experiment group can be maintained in male
mice with one-year post Pten deletion in one-year-old mice (Figure 4c-B).

! 46!
A.

B.

! 47!
Figure 4c. Cell cycle progress when delete Pten in old pancreas.
(A). Isolate islet to investigate protein level changes in aged male and female
mice (nine-month-old) after tamoxifen-induced PTEN loss for one month.
Quantification by image J to analysis relative protein levels change compare to
basal (actin). Protein level/actin level in control groups were standardized to 1,
and thus compare with the relative protein levels in experiment group. (B). Islet
immunostaining of p16 from one year plus one year Pten deletion male mice.

! 48!
Chapter V
DISCUSSION
Maintaining pancreatic β-cell mass and function are key factors to prevent
diabetes and preserve glucose homeostasis. Diabetes is characterized by either
an absolute (type I) or relative (type II) insufficiency of insulin production by β-cells.
Previously, islet transplantation has been administrated to treat type I diabetes
patients. Currently, differentiate β-cells from precursor populations and expansion
of β-cells in vitro for transplantation or in vivo were two major targets to treat
diabetes. To achieve this goal, understanding the molecular regulation of β-cell
mass development, maintenance, and expansion are important.
In adult murine, β-cells proliferate at a low rate and this rate gradually decline
with age. Previous data show that 30% to less than 1% of β-cells replicate per day
in mice from the first week after birth to 1 year of age (Teta et al. 2005). During
adulthood, β-cell apoptosis rate also stay in a very low value, while the average
size and mass of the islets increases with age. The expanded islet mass is due to
! 49!
age-related body weigh increasing and insulin resistance (Montanya et al., 2000).
Young mice responded to high-fat diet by increasing islet mass through β-cell
proliferation to maintain normoglycemia. However, old mice do not display any
increases in β-cell mass or proliferation in response to high fat diet and became
diabetic (Tschen et al., 2009). Overcoming the block for expanding β-cell mass
through increasing cell proliferation might be a strategy to overcome age-induced
insulin resistance.
Previous studies showed that PTEN loss specifically in mice pancreatic
β-cells led to increased β-cell mass and proliferative activity, even under
STZ-induced injury condition (Stiles et al., 2006). It can also improve insulin
sensitivity, glucose tolerance and preserve β-cell function without tumorigenesis
(Nguyen et al., 2006). These studies indicate that PTEN controled PI3K/AKT
pathway play a critical role in maintaining β-cells proper function during
hyperglycemia and injury. However, it is not clear that Pten deletion can rescues
age-induced cell senescence and relative insufficiency of insulin production of
! 50!
β-cells. To address the question, using Pten
loxP/loxP
; RIP-CreER
+
mice model
where Pten deletion is induced after tamoxifen injection can evaluate the effects
of PTEN loss specifically in adult β-cells. We showed that Pten deletion and
PI3K/AKT signal activation in adult pancreas can increase β-cell proliferation and
islets mass. The experiments were performed in three-month-old and
one-year-old mice, and we observed increased mitotic activity and islet mass
phenomena not only observed in younger groups (3 months), but also in aged
mice that fail to induce proliferation during physiological stimulation. The results
suggest that PTEN/PI3K pathway control and maintain β-cell mass and response
to peripheral stimulation even in aging phenotype.
To examine the endocrine function in Pten deletion in adult β-cell, we
evaluated glucose metabolism in three-month-old and nine-month-old mice and
peripheral insulin signal downstream molecules after one-month Pten deletion.
The plasma glucose and insulin results indicated non-significant difference
between control and experiment group at either age and genders, except younger
! 51!
male onset on fasting glucose test. GTT and ITT suggest that experiment groups
are less sensitivity to insulin level change. Our analysis in muscle and liver
indicated insulin signal alteration in response to Pten deletion in the β-cell. Insulin
resistance observed in experiment group may be due to decrease GLUT4
membrane expression in the muscle. However, longer Pten deletion duration time
might need to see the significant different and how this GLUT4 downregulation
occurs is not clear.
In proliferating cells, cyclin D-Cdk4/6 complex accumulate to drive the cell
cycle from G1 to S phase. Our results indicate that several G1/S transition
molecules are altered when PTEN is loss. Especially, cyclin D1 and cyclin D2 are
induced dramatically in Pten null islets, which is consistent with previous studies
showing that islet mass and glucose tolerance were decreased in adult cyclin
D2(-/-) and cyclin D1(-/+) D2(-/-) mice (Kushner et al., 2005).
Furthermore, our results show that p16 and p27, CDK/cyclin D inhibitors, are
downregulated in PTEN loss mice. Even though p27 slightly increase in
! 52!
one-month Pten deletion groups, it decreases in PTEN loss of six months lag time
onsets. These observations are supported by studies that demonstrated
decreased islet proliferation in p16
INK4a
overexpression mice and increased islet
regeneration and proliferation in p16
INK4a
-deficient old mice (Krishnamurthy et al.,
2006). The same paper also suggest that p16
INK4a
play a crucial role on β-cell
aging. We know that p16 expression increases with age. Our data indicates that
Pten deletion in one-year-old mice can still reduce p16 level and increase
proliferate rate. Besides, previous reports from our lab demonstrated that PTEN
regulate the age onset β-cell regeneration and proliferation through
cyclinD1/E2F/Ezh2/p16 signal pathway. In our model, Pten deletion in adult
pancreas is able to induce proliferate of β-cells. The molecules change observed
through western blot confirm that the same pathway is induced when PTEN
deletion is induced. Thus, Pten deletion in aged β-cells is able to overcome the
cell cycle block and allow β-cells to retain proliferation potential.
! 53!
In summary, our study demonstrates that PTEN, PI3K/AKT pathway
antagonizer, is able to regulate β-cell proliferation in both young and old age
through G1/S transition molecules and cyclinD1/E2F/Ezh2/p16 signal pathway.
Due to the fact that most studies shown that many growth factors and signaling
pathway that induce beta cell proliferation and mass expansion in juvenile mice
have little effect in old mice, our study may be a new direction to understanding
aging-induced insulin resistance-related diseases.

! 54!
Chapter VI
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. We used
Pten
loxP/loxP
; RIP-CreER
+
mice with Tamoxifen injection at indicated times as Pten
null experiment groups and Pten
loxP/loxP
; RIP-CreER
+
mice or Pten
loxP/loxP
;
RIP-CreER
-
mice with corn oil injection as control groups. Genotyped from tail
DNA by standard genomic PCR techniques were performed. Animals of indicated
age in control or experiment group were injected i.p. with 50mg/kg streptozotozin
(STZ) for five concecutive days to induce β-cells injury. Five continuous days of
drinking water with BrdU were also administrated to mice at indicated age to
determine cell proliferation capacity. Animals were housed in a temperature-,
humidity-, light-controlled room (12-h light/dark cycle), allowing free access to
! 55!
food and water. All experiments were conducted according to the Institutional
Animal Care and Use Committee of the University of Southern California research
guidelines.
Tamoxifen Injection – Tamoxifen (Sigam-Aldrich, St. Louis, MO) was dissolved
in corn oil at 20mg/ml concentration before usage, and storage at -20 degree.
Mice were injected with either corn oil as a control groups or tamoxifen as
experiment Pten null groups intraperitoneally (i.p.) every three days for 5 doses
(6mg tamoxifen per dose, 30 mg total). Subsequently, mice were euthanized at
indicate time points to analysis β-cells proliferation and phenotypes as well as
muscle and liver protein levels change.
Immunohistochemistry – Pancreatic tissue was fixed overnight in Zn-Formalin
solution and embedded in paraffin, sectioned in 4µm slices (Stiles et al., 2006).
Hemotoxylin and Eosin (H&E) staining was performed for morphological analysis.
Antibodies used were: PTEN (Cell signaling Tech. #9559), cyclin D1 (Santa Cruz,
sc-8396), cyclin D2 (NeoMarkers, MS-221-PO), p27 (Santa Cruz, sc-1641),
! 56!
p16
lnk4a
(Santa Cruz, sc-1661), BrdU (BD Pharmingen), insulin (Abcam, ab7842).
Cell Proliferation Determination – Bromodeoxyuridine (BrdU) labeling assay
was administrated to evaluate cell proliferation. Mice had free access to drinking
water with BrdU (1mg/ml) (Sigma-Aldrich) for five continuous days. On the sixth
day, mice were euthanized and pancreas tissues were collected to analyze by
immunohistochemistry using BrdU antibody and insulin to visualize islet and
β-cells.
Plasma Insulin Determination – Random insulin and fasting insulin were
collected in the morning around the same time in different date to avoid the
difference caused by circadian rhythm. Fasting insulin was obtained from 16
hours overnight fasted mice. Blood samples were collected via orbital eye bleed.
After that, separate plasma from blood samples and analyze insulin levels by
insulin Elisa kit (Alpco, 80-INSMSU-E01).
Glucose Tolerance Test – Glucose levels were determined using Therasense
Glucose Meter from tail veil puncture blood sampling. All of the random glucose,
! 57!
fasting glucose and glucose tolerance test (GTT) results were performed in the
morning around the same time to avoid circadian rhythm difference. After 16
hours overnight fasting, fasting glucose levels were checked before a single dose
(2g/kg body weight) of d-dextrose (Sigma-Aldrich) i.p. injection on mice.
Subsequently, circulating glucose levels were measured at indicated times.
Insulin Tolerance Test – All of insulin tolerance test (ITT) were administrated in
the afternoon around the same time to rule out the possibility of circadian rhythm
influence. Mice were fasted for 5 hours and given one dose (0.5U/kg body weight)
of human regular insulin (Novo Nordisk) by i.p. injection before baseline glucose
measure. Circulating glucose levels were obtained at indicated times by glucose
meter.
Islet Isolation – Pancreas were perfused by collagenase P solution (0.8mg/ml,
Roche) via the pancreatic duct and then digested at 37°C for 18-25 min. Islets
were purified from endocrine tissues by using Ficoll gradients with densities of
1.108, 1.096, 1.069 and 1.037 (Cellgro). Handpick islets were lysed by cell lysis
! 58!
buffer previous to western blot analysis.
Western blot – Mice islets, muscle and liver were collected and lysed in cell lysis
buffer. Lysates with equal amounts of protein were subjected to SDS-PAGE and
then transferred to PVDF membranes for immunoblotting. Antibodies used: PTEN
(Cell signaling Tech. #9559), cyclin D1 (Santa Cruz, sc-8396), cyclin D2
(NeoMarkers, MS-221-PO), p27 (Santa Cruz, sc-1641), p16
lnk4a
(Santa Cruz,
sc-1661), Ezh2 (Cell signaling Tech, #4905), pAKT ser473 (Cell signaling Tech,
#4060S), GLUT4 (Abcam, ab65267), GAPDH (Santa Cruz), β-actin (Sigma).

! 59!
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Abstract (if available)

Abstract β-cells, which produce and release insulin play a major role in maintaining glucose homeostasis. PTEN is a lipid phosphatase that antagonizes PI3K/AKT signaling pathway and control cell proliferation, survival, and metabolism. Previous results from our lab indicate that PTEN controls the age onset senescence of β-cells through cyclin D1 dependent p16 signaling pathway. The current study will investigate whether PTEN regulates adult onset β-cell mass and function especially in aging condition, and similar molecular mechanism are involved. ❧ Our lab developed a RIP-CreER⁺, PtenloxP/loxP mouse model in which we are able to induce Pten deletion by tamoxifen treatment on mature mice. I observed evaluated islet mass and functions in mice treated with tamoxifen (Pten null) vs. those treated with vehicle (Pten control), suggesting that maturity onset deletion of Pten leads to significant increase of islet mass and proliferation. In addition, no major difference on fasting and fed glucose or plasma insulin level between Pten null and control mice are observed with minor hyperglycemia conditions observed in juvenile mice, likely due to muscle insulin resistance. I found that proteins regulating G1/S cell cycle transition like those previously observed in PtenL/L

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PTEN loss antagonizes aging through promoting regeneration and prevents oxidative stress induced cell death

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Asset Metadata

Creator Yang, Kai-Ting (author)

Core Title Pten deletion in adult pancreatic beta-cells induces cell proliferation and G1/S cell cycle progression

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 08/05/2013

Defense Date 06/07/2013

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

Tag beta-cell,cell proliferation,OAI-PMH Harvest,PTEN

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Stiles, Bangyan L. (committee chair), Kalra, Vijay K. (committee member), Tokes, Zoltan A. (committee member)

Creator Email kelly20.yang@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-316863

Unique identifier UC11293652

Identifier etd-YangKaiTin-1966.pdf (filename),usctheses-c3-316863 (legacy record id)

Legacy Identifier etd-YangKaiTin-1966.pdf

Dmrecord 316863

Document Type Thesis

Format application/pdf (imt)

Rights Yang, Kai-Ting

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...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA