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University of Southern California Dissertations and Theses
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Protein kinase Bα (AKT1) but not protein kinase Bβ (AKT2) controls pancreatic β cell growth and survival
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Protein kinase Bα (AKT1) but not protein kinase Bβ (AKT2) controls pancreatic β cell growth and survival
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Content
Protein kinase B (AKT1) but not protein kinase B (AKT2) controls
pancreatic cell growth and survival
By
Zhechu Peng
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)
UNIVERSITY OF SOUTHERN CALIFORNIA
Dec 2017
Dedication
To Sun Lin and Shaoyong Peng
Whose tremendous support made it possible for me to complete this work
Acknowledgement
First and foremost, I want to thank my mentor Dr. Bangyan L. Stiles for her
consistent support of my PhD study. Her passion to science, curiosity towards the
unknown, wisdom of conquering difficulties, and patience to her students encourage
everyone in our research group to keep challenging ourselves and achieving higher goals.
Particularly, as a female scientist, her extraordinary skills in balancing work and family
greatly influence and shape my working attitude. I could not have imagined having a better
advisor for my Ph.D study.
Besides my mentor, I would also like to express my gratitude to my thesis
committee: Dr. Curtis Okamoto and Dr. Hooman Allayee, for their insightful comments
and the hard questions which incented me to think deeper. Dr.Okamoto served in my
proposal committee and thesis committee. Every conversation with Dr. Okamoto has
been fruitful and delightful since the first time I met him six years ago. Dr. Allayee served
in my proposal committee and thesis committee as well. He is a brilliant scientist and a
great mentor. Along the way, Dr. Allayee gave me many good advices especially in
performing RNA sequencing experiments.
Also, my sincere thanks go to Dr. Wei Li and Dr. Enrique Cadenas who served in
my proposal committee. Their insightful thoughts and advice widen the perspectives of
my research project. I would also like to express my thanks towards all the past and
current co-workers: Dr. Jennifer Bayan, Dr. Vivian Medina, Dr. Ni Zeng, Dr. Yang Li,
Dr. Anketse Debebe, Dr. Richa Aggarwal, Dr. Lina He, Dr. Indra Mahajan, Joshua Chen,
Jingyu Chen, Fan Fei, and Yating Guo. Without them, this work would not be completed.
I further want to express my appreciation to Californian Institute for Regenerative
Medicine for their generous fellowship support and the Department of Pharmacology and
Pharmaceutical Sciences at USC for their financial support.
I want to especially thank my parents, Shaoyong Peng and Lin Sun, and
grandparents, Ziqing Peng and Zhaodi Yang, for always being supportive. And, I want to
thank my aunts and uncle-in-law, Zhirong Zhang, Zhiying Zhang and Xinfu Wang, for
taking care of my baby Chaonan while I was doing research. I also would like to thank my
parents-in-law, Zhirong Zhang and Dr. Guolin Zhang, for their support to my family.
Last, I would like to deeply thank my husband, Dr.Xianwei Zhang, for being my joy
of life. It is impossible to complete this work without the support from everyone in my
family.
Zhechu Peng
Dec, 2017
University of Southern California
TABLE OF CONTENTS
CHAPTER I. ABSTRACT ............................................................................................... 1
CHAPTER II. INTRODUCTION ...................................................................................... 3
II-1. Overview of Diabetes and Adult Cell Mass............................................................. 3
II-2. Cell Neogenesis And Precursors Differentiation In Maintaining Adult Cell
Mass ............................................................................................................................. 4
II-3. Cell Dedifferentiation And Reprogramming (Novel Mechanisms) ........................ 6
II-4. Overview Of Cell Size ............................................................................................... 8
II-5. Cell Replication ........................................................................................................ 8
II-5.1. Glucose Induced Cell Replication and Involved Signaling .................................. 9
II-5.2. Growth factor induced replication and involved signaling ................................... 10
II-5.2.1. Insulin/Insulin-Like Growth Factor ....................................................................... 10
II-5.2.2. Hepatocyte Growth Factor (HGF) and Platelet-derived growth factor (PDGF) 12
II-5.3. Cell Cycle Regulators Affecting Cell Replication ............................................... 12
II-6. Cell Death ................................................................................................................14
II-6.1. Endoplasmic Reticulum (ER) Stress and Cell Death .......................................... 15
II-7. Rationale, Hypothesis and Aims ...............................................................................17
II-7.1 PI3K/AKT Pathway And β Cell Proliferation ............................................................ 17
II-7.2. AKT In β Cell Proliferation ........................................................................................ 18
II-7.3 Hypothesis and Aims ................................................................................................. 19
CHAPTER III: EFFECTS OF AKTS ON MICE METABOLISM, PANCREATIC Β CELL
MASS AND CELL REPLICATION UNDER PHYSIOLOGICAL CONDITIONS ............ 20
III-1. Introduction and Rationale ........................................................................................20
III-2. Effects of Whole-Body and β-Cell Specific AKT1 or AKT2 Loss on Mice
Metabolism under Physiological Condition .............................................................23
III-3. Effects of Whole-Body and β -Cell Specific AKT1 or AKT2 Loss on Pancreatic β
Cells Under Physiological Conditions ......................................................................29
CHAPTER IV: EFFECTS OF AKTS ON PANCREATIC Β CELL PROLIFERATION
UPON STREPTOZOTOCIN (STZ) INJURY .................................................................. 37
IV-1. Introduction and Rationale ........................................................................................37
IV-2. Effects Of β-Cell Specific AKT2 Loss On β Cell Proliferation .................................38
IV-3. Effects of Whole-Body AKT1 Loss on β Cell Proliferation after STZ injury ...........40
CHAPTER V. EFFECTS OF AKT1 PROTEIN ON PANCREATIC Β CELL
PROLIFERATION UPON HIGH FAT DIET FEEDING .................................................. 44
V-1. Introduction and Rationale ........................................................................................44
V-2. AKT1 Deficiency Blocks The β Cell Proliferation upon Diet Stimulation ...............45
CHAPTER VI. AKT1 LOSS CAUSES ER STRESS IN RESPONSE TO LONGER HFD
EXPOSURE .................................................................................................................. 52
VI-1. Introduction and Rationale ........................................................................................52
VI-2. AKT1 Deficiency Induces ER Stress On β Cells .......................................................54
CHAPTER VII. AKT1 LOSS SENSITIZES Β CELL TO STZ AND HFD INDUCED CELL
APOPTOSIS ................................................................................................................. 60
VII-1. Introduction and Rationale ........................................................................................60
VII-2. Akt1 Deficiency Sensitizes β Cell to STZ Induced Apoptosis in Mice ....................60
VII-3. Akt1 Deficiency Sensitize β Cells to HFD Induced Apoptosis in Animals and in
INS-1 Cells ..................................................................................................................61
CHAPTER VIII. DISCUSSION ...................................................................................... 67
CHAPTER IX. MATERIAL AND METHODS ................................................................ 74
BIBLIOGRAPHY ......................................................................................................... 81
LIST OF FIGURES
Figure 1. 1 whole-body AKT1 loss slightly affect mice metabolism. ..................................26
Figure 1. 2 β-cell specific AKT1 deficient mice has unchanged metabolic profile. ...........27
Figure 1. 3 whole body AKT2 deficiency and β-cell specific AKT2 deficient mice changes
mice metabolic profile. .........................................................................................28
Figure 2. 1 β cell specific AKT1 loss does not affect pancreas phonotype under
physiological condition. .......................................................................................31
Figure 2. 2 whole-body AKT1 loss does not affect pancreas phonotype ...........................32
Figure 3. 1 β cell specific AKT2 loss does not affect pancreas phonotype under
physiological condition ........................................................................................34
Figure 3. 2 whole body AKT2 deficiency induces β cell mass and β cell proliferation. .....36
Figure 4. 1 β cell specific AKT2 loss does not affect pancreas phonotype after STZ injury
...............................................................................................................................39
Figure 5. 1 whole-body AKT1 deficiency causes reduced cell proliferation one week after
STZ induction. ......................................................................................................42
Figure 5. 2 whole-body AKT1 deficiency causes reduced cell proliferation eight weeks
after STZ induction. ..............................................................................................43
Figure 6. 1 β cell specific AKT1 deficient mice have similar metabolic profile compared to
the controls when exposed to high-fat-diet ........................................................47
Figure 6. 2 whole-body AKT1 deficient mice have better glucose tolerance compared to
the controls when exposed to high-fat-diet. .......................................................48
Figure 6. 3 β cell specific AKT1 deficiency block the 2M high-fat diet induced β cell
proliferation. .........................................................................................................49
Figure 6. 4 Whole-body AKT1 deficiency block high-fat diet induced β cell proliferation. 50
Figure 6. 5 AKT1 modulates cyclinD2, cyclinD1,and p27 .....................................................51
Figure 7. 1 increased ER stress in islets from A1KO mice. .................................................57
Figure 7. 2 induced ER stress in islets from βA1KO. ...........................................................58
Figure 8. 1 AKT1 deficiency causes β cell apoptosis ...........................................................64
Figure 8. 2 AKT1 deficiency causes β cell apoptosis in vitro ..............................................65
Figure 8. 3 Inhibition of PI3K/AKT causes β cell apoptosis in vitro ....................................66
Figure 9. 1. AKT2 does not compensate for AKT1 loss in A1KO mice. ...............................72
Figure 10. 1 95% of cells are deficient of AKT1 in Mip-Cre-ERT+, Akt1
lox/lox
, YFP
lox/stop
,
mice .....................................................................................................................73
1
CHAPTER I. ABSTRACT
Insulin producing pancreatic β cells are critical for balancing mammalian
metabolism. β cells are lost or insufficient in Type I diabetes due to autoimmune attack.
Similarly, functional impairment and loss of β cells in Type II diabetic patients promote
disease stage to the point when patients must rely on exogenous insulin. β cell mass is
controlled by β cell replication, differentiation from the progenitors, β cell dedifferentiation,
transformation from other cells, and cell death.
Replication of β cell is vigorous in neonatal period and in the early stage of Type II
diabetic patients when β cells increase population to compensate for metabolic needs.
Researchers have been dedicating to find a good way to regenerate β cells in order to
fight against Diabetes. However, the mechanisms of β cell regeneration remains unclear
which impedes the progressing of using β cells as therapy for Diabetes.
β cells are a group of cells sensitive to nutrients such as glucose and fatty acids.
β cells also respond to hormones and growth factors. How glucose and fatty acids act on
β cells and whether they utilize signaling pathways activated by growth hormones have
not been fully understood. Particularly, how PI3K/AKT pathway which is responsible for
insulin, insulin-like growth factor (IGF), and platelet-derived growth factor(PDGF) involved
in nutrient-stimulated β cells growth has not been well studied.
2
Our lab has previously found Rip-Cre+, Pten
loxP/loxP
mice which are deficient of
PTEN in β cells have increased islet mass and β cell replication through modulating
cyclinD and CDK inhibitors. Akt proteins, a serine/threonine kinase, is encoded by three
different genes, Akt1, Akt2, and Akt3. Thus, it has three isoforms which may function with
their own specificity. To further investigate how AKT and activated PI3K/AKT pathway
upon PTEN loss regulate β cell regeneration, I studied the metabolic phenotype and islet
phenotype of four different mice strains: Akt1-/-(A1KO); Akt2-/-(A2KO); AKT2
loxP/loxP
, Rip-
Cre+ (βA2KO); AKT1
loxP/loxP
, Mip-CreERT+ (βA1KO). The data shows AKT1 or AKT2 is
dispensable for β cells to maintain their replication under physiological condition. Further
animal study revealed that Akt2 protein is not required for injury-induced β cell
proliferation as well. However, streptozotocin(STZ)-induced hyperglycemia and high-fat-
die feeding challenges on A1KO and βA1KO mice identified essential roles of AKT1 in
regulating β cells growth and survival under these stimulated conditions. Particularly, RNA
sequencing data and other evidences showed up-regulated Unfolded Protein Response
(UPR) among the β cells lacking AKT1 protein.
Taken together, my data has suggested that nutrients, like glucose and high-fat
diet, utilize PI3K/AKT signaling to fulfill their effects on β cells particularly through Akt1
protein but not Akt2 protein. Also, my data suggests Akt1 deficiency potentiates β cells to
ER stress and stress-related cell apoptosis.
3
CHAPTER II. INTRODUCTION
II-1. Overview of Diabetes and Adult Cell Mass
Disrupted glucose homeostasis is a feature of diabetes mellitus and is
tightly modulated by insulin. The regulation of insulin release between meals by
pancreatic β cells is a precisely controlled process and the adaptation of insulin
production in respond to metabolic burden is also tightly regulated. It has been well
recognized that insufficient insulin and correspondent insufficient cell mass are the
reasons for both Type I and Type II Diabetes. In Type I Diabetes, autoimmune
attack against pancreatic cells leads to the dramatic loss of cell mass at the onset
of the disease. Although insulin production is sufficient to support metabolic burden
during the “honeymoon” phase in Type I Diabetes, the insulin level and cell mass
drops eventually to the point that patients must rely on insulin injections. In Type
II Diabetes, the pathogenesis is less clear but recent studies have identified
hyperinsulinemia as an initial cause. High demand for expansion of islet mass is
followed by insufficient insulin and reduced islet mass during the progression of
Type II Diabetes. The unbalance of β cell mass together with increased insulin
demand could serve as the mechanisms for Diabetes progression. Furthermore,
previous studies showed the β cell mass would be affected by three aspects: a.
neogenesis and differentiation from precursors; b. change in cell size; c. cell
proliferation and cell death. Recent studies also identified β cell dedifferentiation
to other cell type and α cell transformation to β cell as potential new mechanism
contributing to β cell mass. Although β cell mass is dynamically affected by the
above processes, most of these process has intrinsic limitations and the degree of
4
impacts for each process varies. For example, hypertrophy has been reported for
β cells as an adaptive strategy for more insulin upon glucose stimulation. The
increase of cell size is limited. Among all the biological processes controlling β cell
mass, cell proliferation and cell death are the primary mechanism in the context of
Diabetes. Although the existence of β cell dedifferentiation and transformation
have been reported, they have relatively less impact on β cell mass and thus the
development of Diabetes.
II-2. Cell Neogenesis And Precursors Differentiation In Maintaining Adult Cell
Mass
Earlier researches proposed the idea that neogenesis plays a major role in
maintaining adult cell mass in the situation when insulin demands exceeds the
productive ability of islets. The cell neogenesis is thought to be extensively
involved in cell replenishment in several mouse models such as INF, TGF, and
gastrin over-expressing models, partial pancreatectomy model, main duct ligation
models and glucose infusion models (Bonner-Weir et al., 1989; Bonner-Weir et al.,
1983; Campbell et al., 1994; Gu and Sarvetnick, 1993; Higuchi et al., 1992;
Rosenberg and Vinik, 1992). Particularly, Paris M. showed 48 hours of glucose
infusion successfully induce cell neogenesis as indicated by new islets budding
from ducts (Paris et al., 2003). Besides the existence of progenitor cells in
pancreas, another focus of this area is to identify the location and identity of true
pancreatic stem cells. Most researchers thought the progenitor cells are ductal
cells differentiating to cells or cells that reside inside ducts (Bisgaard and
5
Thorgeirsson, 1991; Teitelman et al., 1987). One loophole across all these studies
is the lack of reliable methods to measure islets initiated from activated progenitor
cells. Most researchers considered small islets (islets have 1-10 cells) or islets
close to ductal area derived from progenitor activation. With the advancement of
techniques, researchers have successfully isolated ductal progenitor cells
expressing CD133 and c-Met from mouse pancreas and characterized their
proliferative capability and multi-potency (Oshima et al., 2007). Further study by
Harry Heimberg’s group identified Ngn3+ cells located in ductal lining as progenitor
cells that could give rise to newly formed islets in the partial duct ligation model
(Xu et al., 2008).
On the other side, Dor et al disputed the importance of progenitor cells in
compensatory cell growth by studying a lineage tracking of genetically labeled
cells in mice and concluded the self-replication served as the primary event for
adult cell hyperplasia under stimulated conditions. This study cast doubts on the
view that adult stem cell plays a significant role in cell replenishment (Dor et al.,
2004). This idea was also supported by another study from Jake A Kushner’s
group in which they used CIdU/IdU double labeling system to demonstrate self-
replication was the primary mechanism for cell replenishment in the case of 50%
pancreatectomy, pregnancy, and exendin-4 treatment (Dor et al., 2004). Although
these studies questioned the significance of progenitor cells in cell regeneration
upon stimulated conditions, the results still indicated and confirmed the existence
of progenitor cell activation at a minor level.
6
Based on currently available evidence, it is very likely that both replication
and progenitor cell differentiation exist under stimulated conditions, but replication
plays the major role rather than progenitor cell differentiation.
II-3. Cell Dedifferentiation And Reprogramming (Novel Mechanisms)
Very recent studies have demonstrated novel mechanisms contributing to
cell mass. The discovery of the conversion between cells and other
endocrine/exocrine cells reshape the notion of pancreas plasticity.
The phenomenon of cell dedifferentiation was first observed in an in vitro
study in which cultured islets with YFP labelled cells were observed for nearly a
month(Weinberg et al., 2007). Later on, using lineage tracing technique, Talchai,
C. et al observed a substantial percentage of cells underwent a series of identity
change instead of apoptosis which contributes to the decrease of cell mass in
respond to FOXO1 loss and long-term hyperglycemic condition. More specifically,
they pointed out, in the condition of FOXO1 deficiency and hyperglycemia, cells
first become insulin granule negative cells and then these cells further reprogram
to Neurog3 expressing endocrine progenitors and Sox9+ pre-endocrine
progenitors (Talchai et al., 2012). Similar technique and observation were seen in
KATP-gain-of-function mutant mouse which simulates neonatal KATP-dependent
diabetes (Wang et al., 2014). Moreover, two studies using clinical samples
confirmed the existence of cell dedifferentiation among Type 2 Diabetic patients,
7
but the significance of cell dedifferentiation on cell mass was considered to be
minor (Butler et al., 2016; Cinti et al., 2016).
Another newly discovered source of cells is potential conversion of cells
to cells. and cells were used to considered as two mature lineages with little
possibility to convert from one to the other. However, a study published in 2009
discovered overexpressing a transcription factor, Pax4, could force cells convert
to cells (Collombat et al., 2009). Another study used transgenic mice expressing
diphtheria toxin receptor also identified to cell conversion when almost the
entire cell population was removed by treating mice with diphtheria toxin (Thorel
et al., 2010). Similar observation was found in PDL model and PDL+alloxn models
(Chung et al., 2010). Additionally, Dr. Melton’s group developed an in vivo
reprogramming strategy to convert exocrine cells to cells partially resemble by
expressing Mafa, Pdx1 and Ngn3 (Zhou et al., 2008). Up to now, this conversion
has only observed in experimental settings. The significance and relevance to
diabetes remain to be illusive.
Overall, the mechanism of cell expansion seems to be dependent on the
type and the severity of injury. Many novel findings proposed new possible ways
to regenerate cells. However, more and more researches need to be done to
confirm the applicability of these mechanisms in the development of human
diabetes.
8
II-4. Overview Of Cell Size
cell hyperplasia and hypertrophy both contribute to the overall regulation
of cell mass. A study on followed the cell morphology of rats across their entire
lifespan and concluded cell hypertrophy only contributes to the increased islet
mass found in old rats when cell replication becomes minimum (Montanya et al.,
2000). Comparing to the studies understanding on cell hyperplasia, less studies
are focusing on understanding the mechanisms controlling cell size. Up to date,
researchers have shown that insulin/IGF axis, particularly PI3K, AKT, and S6K, is
the main regulators for cell size. One study showed S6-kinase-deficient mice
presented phenotype of hypoinsulinaemia, glucose intolerance and diminished
cell size (Pende et al., 2000).
II-5. Cell Replication
A well-balanced maintenance of β cell mass is crucial for the regulation of
plasma glucose under normal physiological condition. In adults, cells sense and
respond to various nutrients such as glucose, amino acids and fatty acids. The
adaptive response of β cells to nutrients could be viewed as compensatory
strategies to maintain normality of metabolism in condition like insulin resistance
and pregnancy. Nonetheless, this compensation is usually limited and eventually,
fail to adapt to excessive-nutrients caused metabolic burden which would result in
the onset of disease like Diabetes.
9
II-5.1. Glucose Induced Cell Replication and Involved Signaling
The glucose-stimulated increase of cell mass has been reported since
1972. Although cell presented low cell turnover rate in physiological condition,
several studies have showed increase replicative capability of cells correlated
with direct glucose exposure (Bonner-Weir et al., 1989; Hoorens et al., 1996;
Kaung, 1983). This observation was mirrored by discoveries of increased cell
proliferation events in several hyperglycemic mouse models, such as NOD mice,
Zucker Diabetic rats, streptozotocin-treated rodents, allxon treated rodents,
animals with partial pancreatectomy and high fat diet treated rodents(Golson et al.,
2010; Sreenan et al., 1999; Wang et al., 1994)(Tyrberg et al., 2001). In the in vivo
studies, the effects of glucose are usually combined with other factors, such as
gene mutations or surgical injury. Thus, the observed increase in cell replication
has to be viewed as a combination results of hyperglycemia and other factors.
The studies explaining mechanisms on how glucose acts on cells
replication are usually complicated by the combined effects of glucose and other
factors in animal models. A few studies tried to demonstrate the direct effects of
glucose on cells by performing short-term glucose infusion study on animals or
treating cell lines/isolated islets with glucose of different concentrations. Two
studies by Dr. Yuval Dor’s group identified cyclinD2 as a potential regulator for
glucose-stimulated cell replication. In the first study, Alonso, L. et al observed
increase in cell proliferation and cyclin D1/D2 protein level but not at mRNA level
when comparing saline control group to glucose infusion group, indicating a
10
correlation between glucose exposure and increased cyclinD2 (Alonso et al., 2007).
The second study confirmed the upregulation of nucleic cyclinD2 in relation to
glucose exposure but also proposed that cyclinD2 is downregulated in dividing S-
G2-M phase cells while gets upregulated in glucose-exposed quiescent cells
(Salpeter et al., 2011). The latter study also pointed out the high cyclinD2 level was
maintained by high levels of glucose metabolism which is thought to be a critical
process in glucose-induced cell replication (Salpeter et al., 2011).
II-5.2. Growth factor induced replication and involved signaling
Several growth factors, including Platelet-derived growth factor (PDGF),
insulin/insulin-like growth factor (IGF), and Glucagon-like peptide-1(GLP-1), have
been widely studied and identified as regulators for cell replication.
II-5.2.1. Insulin/Insulin-Like Growth Factor
Insulin and insulin growth factor (IGF) are well-known regulators for
metabolism and cell growth. Insulin promotes cell growth by binding with insulin
receptor (IR) while IGF binds to IGF receptors. Both IR and IGF1R are receptor
tyrosine kinases with tyrosine kinase in β-chain and ligand binding in α-chain of
the transmembrane receptor. Composed by two subunits which have extracellular
α-chain and intracellular β-chain, these two types of receptors share similar
structures and sometimes form hybrid receptors (Nakae et al., 2001). In the in vitro
studies using isolated islets and cell lines, it has been demonstrated that both IGF-
11
I and IGF-II enhance cell replication (Lingohr et al., 2002; Ohsugi et al., 2005).
Also, it is thought that IGF acts synergistically with glucose to induce cell
replication(Hugl et al., 1998). In mouse models, studies show that the function of
Insulin, IGF and their receptors are involved in metabolism and cell growth. Mice
lacking IR are born with growth retardation, followed by impaired metabolic control,
β cell failure and further death of animals in diabetic ketoacidosis (Kulkarni et al.,
1999). Specific deletion of IR in β cells leads to insulin secretory defect and a
progressive impairment of glucose tolerance accompanied with decreased islet
size (Duvillie et al., 2002; Kulkarni et al., 1999). Global deletion of Igf1 and Igf1r
causes growth retardation. Also, Igf1r null mice have also been reported to develop
metabolic abnormalities and decreased islet size (Lu et al., 2004; Petrik et al.,
1999).
The activated insulin receptor recruits insulin receptor substrate1/2 (IRS1
and IRS2) and phosphorylates them which further triggers PI3/AKT pathway. This
signaling node in liver, muscle and fat cells allows them to sense plasma insulin,
mediate translocation of glucose transporters to the cell membrane and initiate
downstream signaling related to glucose metabolism. In β cells, this signaling
pathway acts as a dominant effector for Insulin and IGF’s pro-proliferative function.
Manipulation of molecules in this signaling pathway in rodents has significant
effects on β cells. For example, global knockout of Irs1 results in defective insulin
secretion in response to glucose, while Irs2 deletion leads to impaired β cell
proliferation (Leibiger et al., 2001; Withers et al., 1998).
12
II-5.2.2. Hepatocyte Growth Factor (HGF) and Platelet-derived growth factor
(PDGF)
Early work by Hayek’s group identified HGF as a potential mitogen for in
vitro monolayer-cultured human islets (Beattie et al., 1996). An in vitro study in
INS-1 cells indicated HGF could accelerate β cell proliferation under low glucose
condition possibly through activating PI3K/AKT and atypical PKC signaling(Gahr
et al., 2002). In vivo studies using genetic manipulated mouse models further
showed HGF stimulates β cell proliferation. Mice overexpressing HGF in β cells
using rat insulin II promoter (RIP) display an increase in β cell mass and
proliferation, better glucose tolerance and resistance to streptozotocin(STZ)
(Garcia-Ocana et al., 2000; Garcia-Ocana et al., 2001). More recent studies
showed the signaling involved in HGF-induced β cell proliferation depends on c-
Met and p65/NF-kB(Mellado-Gil et al., 2011).
PDGF signaling has been shown to regulate β cell proliferation in an age
dependent manner. Chen,H et al reported deficiency of PDGF receptor specifically
in β cells reduced β cell mass by controlling the expression of E2F, EZh2, and
retinoblastoma protein (Rb) (Chen et al., 2011).
II-5.3. Cell Cycle Regulators Affecting Cell Replication
Replication by altering cell cycle of β cells has been investigated in various
mouse models. Together, these studies show that cell cycle regulators controlling
13
G1/S transition are the major players in regulating β cell replication. A prevalent
consensus has been established that cyclinD1 and cyclinD2 are expressed in
pancreatic islets across different species. Genetically modified animal models
have revealed the role of cyclinDs in regulating β cell cycle entry and β cell mass.
CyclinD1 overexpressing in pancreatic β cells increases β cell mass by almost
three fold with more proliferating cells indicated by PCNA staining and reduced
apoptosis indicated by TUNEL staining(Zhang et al., 2005). Overexpressing CDK4
or cyclinD1 in human and rat islets with adenovirus induces phosphorylation of
retinoblastoma protein and thus increases the proliferation rate (Cozar-Castellano
et al., 2004). Conversely, globally cyclinD2 knockout mice presents intolerance for
glucose with 4 fold reduction of β cell mass and less proliferation detected by BrdU
staining(Georgia and Bhushan, 2004). Further reduction of β cell mass and
proliferation rate are observed in mice with combined deletion of cyclinD1 and
cyclinD2 (Kushner et al., 2005).
Studies based on human islets and mice further shows cyclin-dependent
kinase, D-type cyclins, CDK inhibitors (CDKIs) are the major regulators for β cell
proliferation. CDK4 knockout mice display phenotype of reduced pancreatic islet
mass, insulin deficient diabetes and growth retardation in multiple tissues including
the pancreas (Rane et al., 1999). Transgenic mice expressing a mutated form of
CDK4 without affinity to CDK inhibitor p16 shows islet hyperplasia and elevated
proliferation(Martin et al., 2003). Also, this is the earliest study indicating CDK
inhibitor p16 also regulates β cell replication. P16 belongs to the INK4/ARF protein
14
family encoded by CDNK2A gene. It expresses more in adult mice than in young
mice which provide evidence that p16 is a biomarker for β cell aging. P16-/- mice
can better endure β cell-specific toxin streptozotocin (STZ) induced damage. Other
types of CDK inhibitors such as p21, p27 have also been studied in β cells. Mice
studies showed p27 has similar function with p16 in adult mice but its level doesn’t
change during aging (Georgia and Bhushan, 2004). P27 has been considered to
be a key regulator for β cells proliferation by keeping differentiated β cells in their
quiescent state. p27-/- mice have increased islet mass 21 days after birth
compared with WT and they are also less susceptible to STZ-induced
diabetes(Wolf et al., 2005). To sum up, regulation of cell cycle regulator controls
the replication and cell survival signaling of β cells. D-type cyclins and several CDK
inhibitors are the major molecules in determining the growth or death of β cells.
II-6. Cell Death
β cell apoptosis is a biological event discovered in both experimental
models of diabetes and diabetic patients. It has been suggested that apoptotic β
cells are cleared from pancreas really fast which made it very difficult to detect the
cells undergoing apoptosis in in vivo diabetic animal models or preserved pancreas
tissue of diabetic patients (Eizirik and Mandrup-Poulsen, 2001). In spite of this
difficulty, rare occurrence of apoptotic cells has been observed by detecting (TdT)-
mediated dUTP nick-end labelling (TUNEL) method in NOD mice and BB rats
(Augstein et al., 1998; Lally et al., 2001; O'Brien et al., 1997). As expected,
increased β cell apoptosis was observed in type I diabetes since the primary
15
mechanism for type I diabetes is autoimmune toxicity (Meier et al., 2005). CD8+ T
cells and activated CD4+ and CD8+ T cells posted autoimmune attack on β cells
(Mandrup-Poulsen, 2001). In type II diabetic patients, β cell apoptosis was also
found in β cells and contributes to the disease development (Butler et al., 2003).
II-6.1. Endoplasmic Reticulum (ER) Stress and Cell Death
There are three ER stress sensors discovered so far, inositol-requiring
protein 1α, protein kinase RNA-like endoplasmic reticulum(ER) kinase(PERK), and
activating transcription factor 6(ATF6), transduce the status of protein biosynthesis
in ER to the cytosol and nucleus. During this process, glucose-regulated protein
78 kDa (Grp78, Bip) dynamically interacts with these three sensors and
inappropriate folded protein, and then activates IRE1α, PERK, or ATF6 by
phosphorylation process. Activated IRE1α further signals NF-kB, JNK signaling
and enzymatically cut XBP1u mRNA to its active form XBP1s mRNA, which further
targets DNA and initiates unfolded protein response (UPR) target genes. Activation
of PERK causes the autophosphorylation of itself and affects the translocation of
ATF4 to nucleus, which also initiates UPR target genes(Hetz, 2012). Mutations in
PERK has a result of neonatal diabetes and β cell specific PERK knockout study
showed it is required for the maintenance of β cell mass, normal proinsulin
trafficking, and insulin secretion(Zhang et al., 2006) (Gao et al., 2012). These
stress triggered signaling aims at helping cells to restore cell homeostasis. The
inability to adapt to ER stress would trigger cell apoptosis mechanisms involving
C/EBP-homologous protein(CHOP), BCL2, and GADD34(Hetz, 2012).
16
It has been indicated that long-term hyperglycemia and free fatty acids
could cause ER stress in β cells. Under high glucose condition for long time,
overwhelmed need for insulin biosynthesis stresses on endoplasmic reticulum and
causes dysregulation of the protein synthesis. Activation of typical ER stress
signaling pathways, such as IRE1α and PERK signaling, was observed in isolated
rat islets exposed to glucose (Elouil et al., 2007; Lipson et al., 2006). These
signaling pathways eventually activate the expression of CCAAT-enhancer-
binding protein (C/EBP) homologous protein (CHOP), which leads to β cell
apoptosis(Hou et al., 2008; Jonas et al., 2009). Chronically, activated ER stress,
also potentially affects the production of reactive oxygen species (ROS) and
contributes to cell death (Malhotra and Kaufman, 2007). The link between glucose
exposures to ER stress is not clear yet. One study on INS-1 cells indicated sterol
regulatory element-binding protein 1(SREBP-1) may involve in the glucose-
induced ER stress (Wang et al., 2005).
The role of free fatty acids on the ER stress of β cells seems to depend on
the type of fatty acids. Palmitate acids, not oleate acids, has also been shown to
be involved in the induction of ER stress and cell death in β cell lines (Karaskov et
al., 2006; Lupi et al., 2002) Overexpression of Grp78(bip) in β cells could protect
against high fat diet induced apoptosis (Teodoro-Morrison et al., 2013). FFA was
proposed to affect ER-to-Golgi trafficking, up-regulating CHOP, activate JNK
pathway, and caspase-12 (Kharroubi et al., 2004; Preston et al., 2009).
17
II-7. Rationale, Hypothesis and Aims
II-7.1. PI3K/AKT Pathway And β Cell Proliferation
Several growth factors and hormones, such as IGF-I/II, insulin, and exendin-4,
have been proposed utilizing PI3K/AKT pathway to signal β cell growth. PI3K/AKT
pathway is activated by Insulin/IR/IRS and IGF1/IGF1R activation. The
phosphatidylinositol 3 kinases (PI3K) are conserved lipid kinases which
phosphorylate 3’-hydroxyl group of phosphatidylinositol and phosphoinositides.
Phosphorylated lipids provide docking domain to downstream targeting including
kinases Phosphoinositide-dependent kinase-1(PDK1) and AKT and allow them to
access the cell membrane. Membrane AKT is phosphorylated by PDK1 and further
by mTOR2 to become fully activated. Activated AKT functions as serine/threonine
kinase and initiates a wide variety of downstream signaling that are responsible for
the characterized pro-survival and pro-growth functions of AKT. PTEN (the
phosphatase and Tensin homolog deleted on chromosome 10) acts as a negative
regulation of PI3K/AKT pathway. PTEN is often mutated and lost in various
cancers. Loss of PTEN in β cells does not cause tumor phenotype but leads to a
dramatic increase in islet mass (Zeng et al., 2013). Further studies show β cells
express more PTEN in the high-fat diet induced diabetic model and in db/db mice
model. Furthermore, PTEN deletion in mice β cells protects β cell dysfunctions
under high-fat diet condition (Wang et al., 2010).
18
II-7.2. AKT In β Cell Proliferation
Particularly, a series of transgenic and knockout studies have partially
shown the role of AKT in β cells and metabolism. For example, β cell specific
overexpression of a constitutively active AKT1 form, which lacks the PH domain
(AktΔ4-129) but contains an N-terminal Src myristolation signal (myrAktΔ4-129),
leads to increased islet mass and beta cell size by nearly 3 fold (Tuttle et al., 2001).
Akt2-/- mice develop diabetes mellitus-like syndrome. Their islet mass are almost
3 times larger than WT mice at 5 months of age (Cho et al., 2001). Conversely,
ectopic expression of dominant negative AKT in β cells leads to impairment of
insulin secretion and susceptibility to diabetes (Bernal-Mizrachi et al., 2004; Tuttle
et al., 2001). These studies strongly suggest a pro-growth/survival role and an anti-
growth/survival role of AKT1 and AKT2 respectively. In addition, loss of PTEN, a
phosphotase that antagonizes the PI3K/AKT signaling pathway(Carracedo and
Pandolfi, 2008), results in increased β cell mass, proliferation, improved glucose
tolerance with mild hypoglycemia. Our lab further discovered that PTEN reverses
the aging process of β cells by up-regulating cyclinD1 while down-regulating
p16(Stiles et al., 2006; Zeng et al., 2013). Since PTEN is a negative regulator of
PI3K/AKT pathway, the increased islet mass and β cell proliferation observed in
PTEN deletion mice model is likely mediated by PI3K/AKT pathway. Taken
together, these studies suggest that PI3K/AKT signaling is involved in regulating
islet mass. Nevertheless, how AKTs contribute to this regulation and the divergent
roles of the AKT isoforms remains.
19
II-7.3. Hypothesis and Aims
Based on previous publications, I hypothesize that AKT1 and AKT2
isoforms have different function on regulating β cell growth and survival.
Aim1. Determine how AKTs regulate β cell growth under physiological condition.
Aim2. Determine how AKTs regulate β cell growth and survival after
Streptozotocin (STZ) treatment.
Aim3. Study how AKTs involve in High-fat-diet induced β cell growth and survival.
20
Chapter III: EFFECTS OF AKTS ON MICE METABOLISM, PANCREATIC
Β CELL MASS AND CELL REPLICATION UNDER PHYSIOLOGICAL
CONDITIONS
III-1. Introduction and Rationale
Since first discovered as oncogene in mouse leukemia virus, protein kinase
B, or AKT, has been widely studied and defined as a downstream signaling node
for growth factors, cytokines, and a lot of other cellular stimuli. As a
serine/threonine kinase, Akts response to PI3K and their substrates govern many
cell functions. For example, AKT’s substrate, mTORC complex 1 (mTORC1) has
been thought to be a conservative regulator for cell size (Wullschleger et al., 2006).
Akts also block the pro-apoptotic functions of several Bcl-2 homology domain
(BH3) proteins, such as BAD (Franke et al., 2003). Gain of function mutations in
Akt are found in breast cancers and its upstream PI3K is frequently mutated in
human cancers as well (Samuels et al., 2004; Stemke-Hale et al., 2008). Thus, Akt
is a critical enzyme governing cell growth and survival.
Meanwhile, as the downstream effector of insulin signaling, Akt’s role in
metabolism has been demonstrated in important insulin-target tissues like muscle,
fat, liver, and pancreas (Whiteman et al., 2002). Overall, in respond to insulin, Akts
promote the storage of nutrients following food intake to liver, muscle, and fat cells,
while inhibiting the catabolism of fuel reserves. In muscle and adipose tissue, Akts
involve in redirecting glucose transporter type 4 (GLUT4) to the cell membrane and
21
stimulate the glucose uptake. An in vitro study in 3T3-L1 adipocytes showed
constitutively active Akt kinase stimulated GLUT4 translocation (Kohn et al., 1996).
Another high-fat-diet rat study showed deficiency of GLUT4 translocation
correlated with decreased Akt signaling in skeletal muscle cells (Tremblay et al.,
2001). In liver tissue, it is generally proposed that insulin signaling suppresses the
feeding-related glucose output through the activation of PI3K/AKT pathway. Akt2
whole-body deletion mice presented hyperglycemic condition with increased
hepatic glucose output (Cho et al., 2001). Mice with liver specific Akt1 and Akt2
combined deletion presented metabolic abnormalities featured with glucose
intolerance and the deletion of FOXO1 protein could partially reverse the
phenotype resulting from Akt deficiencies (Lu et al., 2012).
In pancreas, Akts have been shown to regulate β cell mass and insulin
secretion which directly links to the metabolism control. Akts have three isoforms
Akt1, Akt2, Akt3, which share at least 80% of sequence similarity. According to the
phenotypes of mice lacking different Akt isoforms, it is generally believed that the
three isoforms have their own specificity. However, how this specificity is achieved
remains elusive in β cells, the functions of three Akt isoforms are not clear.
AKT1 has been studied in the regulation of β cell physiology using gain-of-
function approach. Overexpression of a constitutively active form of AKT1 (Myr-
Akt1) in β cells increases cell size, islet mass and induces β cell proliferation. To
address a physiological role of AKT1, AKT1 global knockout mice were evaluated.
22
These mice have smaller body size with increased apoptotic process in testes and
thymus at 3 month of age. The β cells of these mice presented similar proliferative
ability when compared to the controls at 3-month old. Nonetheless, the β cell
proliferation is lower among those mice when they are old (>12-month old) (Figure
2.2C). These studies contradicts with each other leading to inconclusive role of
AKT1 in the regulation of β -cell mass. Moreover, we have found Akt1 germline
knockout mice presented moderate improved glucose tolerance due to unclear
peripheral effects which could affects β cells (Figure 1.1C). In order to exclude
these peripheral effects, we generated and studied the morphology and cell
replication of β cells among conditional AKT1 knockout mice (Akt
loxP/loxP
, MIP-
ERT+, βA1KO).
On the other hand, opposite from the predicted pro-growth/survival role for
AKT kinases, germline Akt2 deletion mice (Akt2-/-, A2KO) presented a phenotype
of increased β cell mass, implying it may suppress the growth of β cells.
Additionally, the A2KO mice has insulin resistance and hyperglycemic condition
which further complicated the interpretation of the results. Thus, to exclude the
peripheral’s effects due to Akt2 deletion, we developed Akt2
loxP/loxP
, Rip-Cre
+
( A2KO) mice with specific AKT2 deletion on β cells.
Together, these studies demands more detailed analysis of AKT1 and 2 in
the regulation of β -cell biology.
23
III-2. Effects of Whole-Body and β-Cell Specific AKT1 or AKT2 Loss on Mice
Metabolism under Physiological Condition
According to previous animal studies, Akt isoforms are generally believed
to have their own specificity. Specifically, Akt2’s substrates or its tissue distribution
pattern are thought to be more related to metabolism because Akt2-/- (A2KO) mice
develop severe hyperglycemia.
It was reported previously that Akt1 is dispensable for physiological
regulation of β-cell mass based on loss-of-function studies even though topical
expression of AKT1 can induce β-cell growth. However, the Akt1 germline
knockout mice were reported to have increased plasma leptin level and is more
resistant to high fat diet induced insulin resistance. Such effects of AKT1 loss on
peripheral tissues can affect glucose level and in turn β cell mass due to the
regulated response of b-cell mas by glucose (Wan et al., 2012). Our data generally
supported this idea but we do find that A1KO mice have slightly better glucose
tolerance when challenged by glucose. The AKT1 germline knockout mice (Akt1-
/-, A1KO) are slightly smaller than control mice (Fig.1.1A). They displayed a
metabolic profile that is indifferent from that observed with the control mice when
they are at 3 month of age. Both fasting and random glucose are comparable
between control and A1KO mice, so are their response to insulin challenge
(Figure.1.1B-D). By the time they are at one year old, A1KO mice become
significantly smaller than the control mice (Fig.1.1E). The fasting glucose, insulin
sensitivity, and glucose tolerance are comparable between A1KO and control mice
24
at 12 month old (Fig.1.1F-H). However, the A1KO mice are moderately more
sensitive in an intraperitoneal glucose tolerance test at 3 month old, indicating a
potential unclear function of Akt1 on peripheral tissue (Fig.1.1C,G). This
observation is consistent with the result from Dr. Birnbaum’s group who found
increased energy expenditure in A1KO mice might contribute to this phenotype
(Wan et al., 2012).
Previous study (Cho et al., 2001) has reported hyperglycemic conditions
developed in Akt2 whole body deficient mice as early as 3-month old. We
confirmed this observation by studying both 3-month old and 6-month old mice. At
3 and 6 month old, A2KO mice present a significantly higher fasting plasma
glucose level and they display glucose intolerance in intraperitoneal glucose
tolerance test (Fig.1.3A,B).
As a key player in regulating body metabolism, β cells are very sensitive to
any metabolic change, especially the change in plasma glucose. When plasma
glucose level increase at the early phase of diabetes, β cells usually are able to
adapt to the new metabolic burden by inducing cell proliferation and increasing β
cell mass. Thus, any metabolic changes caused by peripheral tissue, especially
changes in plasma glucose, have to be excluded in the studies aiming at β cells.
Here, both A1KO and A2KO mice have moderate to severe changes in glucose
metabolism. Thus, mouse models where restricted gene deletion studies are
needed. In order to achieve this goal, we generated β-cell specific AKT1 deficient
25
mice line (βA1KO: Akt1
loxP/loxP
, MIP1-CreERT +) and β cell restricted AKT2
deficient mice line ( A2KO: Akt2
loxP/loxP
, Rip-Cre
+) mice. According to previous
report, in βA1KOs, about 90% of β cells stops expressing AKT1 upon tamoxifen
treatment (Oropeza et al., 2015). And in βA2KOs, the mice stop Akt2 expressing
once insulin starts to be produced in β cells which should be around E12.5 for
mice. Both βA1KO and βA2KO mice present similar fasting glucose, glucose
tolerance, and insulin tolerance when challenged by intraperitoneal glucose or
insulin solution (Fig. 1.2A-E, Fig.1.3C-E).
26
A. B.
C.
D.
Figure 1. 1 whole-body AKT1 loss slightly affect mice metabolism.
A,E. Body weight of WT and A1KO mice (A: WT:n=17, A1KO:n=21;E: WT:n=13,
A1KO:n=18); B.F.Fasting glucose level was measured through tail vein blood
sampling after 16 hours of fasting. (B: WT:n=17, A1KO:n=21;F: WT:n=13 for,
A1KO:n=18); C,G. intraperitoneal glucose tolerant test of Control and A1KO mice.
Mice was fasted 16 hours before tests (C: WT:n=16, A1KO:n=24;G: WT:n=7 for,
A1KO:n=14) D,H. insulin tolerance test of Control and A1KO mice. Mice was fasted
5 hours before tests (D: WT:n=16, A1KO:n=24;H: WT:n=7 for, A1KO:n=14) .
F.
H.
E.
G.
27
βA1KO: Akt1
loxP/loxP
, MIP1-CreERT +
Con: Akt1
loxP/loxP
, MIP1-CreERT -
5 injections
of Tamoxifen
(250mg/kg)
1 Month
Euthanize
Figure 1. 2 β-cell specific AKT1 deficient mice has unchanged metabolic profile.
A. illustrative image showing the induction strategy of inducing β-cell specific AKT1
deletion. B. Body weight of WT and βA1KO mice; C. Fasting glucose level was
measured through tail vein blood sampling after 16 hours of fasting. D. intraperitoneal
glucose tolerant test of Control and βA1KO mice. Mice was fasted 16 hours before
tests E. insulin tolerance test of Control and βA1KO mice. Mice was fasted 5 hours
before tests. (A-E: n=5/group)
A.
B.
C.
D.
E.
3-4 Month
28
A.
B.
C.
D.
E.
Figure 1. 3 whole body AKT2 deficiency and β-cell specific AKT2 deficient mice
changes mice metabolic profile.
A. Fasting glucose level was measured through tail vein blood sampling after 16 hours
of fasting.(n=5 for Con, n=8 for A2KO) B. intraperitoneal glucose tolerant test of
Control and βA2KO mice. Mice was fasted 16 hours before tests (n=7 for Con, n=8
for A2JO) C. Fasting glucose level was measured through tail vein blood sampling
after 16 hours of fasting (n=21 for Con, n=10 for βA2KO); D. intraperitoneal glucose
tolerant test of Control and βA1KO mice. Mice was fasted 16 hours before tests (n=23
for Con, n=11 for βA2KO) E. insulin tolerance test of Control and βA2KO mice. Mice
was fasted 5 hours before tests (n=19 for Con, n=10 for βA2KO).
29
III-3. Effects of Whole-Body and β -Cell Specific AKT1 or AKT2 Loss on
Pancreatic β Cells Under Physiological Conditions
β cell mass is a critical measurement for diabetes. It is determined by β cell
replication, neogenesis, hypertrophy versus dedifferentiation and apoptosis. Over
the years, many researchers tried to understand the mechanisms controlling β cell
mass and develop therapies that can boost β cell mass in fighting against diabetes.
As mentioned above, it is worthy to investigate the functions of PI3K/AKT pathway
on β cells as this signaling stands at the crossroad of several important growth
factors and nutrients. Here, we try to demonstrate the role of Akt isoforms on β
cells by investigating the pancreatic phenotypes of genetic modified mouse models,
particularly focusing on β cell mass and proliferation.
As mentioned earlier, both Akt1 and Akt2 whole-body knockout mice have
some unclear peripheral changes which affect β cells. On the other hand, although
β cell specific Akt1/2 knockout mice we generated overcomes these impactful
peripheral changes, some minor imperfections do exist when using these mouse
models to study β cell biology. For example, the RIP-CRE line used to breed
βA2KO (Akt2
loxP/loxP
, RIP-CRE) mice has been proposed to display glucose
intolerance (Lee et al., 2006). And in βA1KO mice (Akt1
loxP/loxP
, MIP-CRE-ERT),
Tamoxifen injection achieves Akt1 deletion in 90 or higher percentage of β cells
which means β cells without Akt1 deletion could affect the overall results. Thus, I
decided to investigate the pancreas and β cells in both whole-body knockout mice
and β cell specific ones. In this way, one observed β cell phenotype could be
30
confirmed using the other mouse model. More reliable conclusion could be
achieved.
We first investigated the role of AKT1 in β cell regulation using βA1KO mice
where AKT1 protein is lacking in β cells. At 3-4 month old, no significant difference
is observed for either islet area or β-cell proliferation between wild type and βA1KO
mice (Fig.2.1A-C). However, in A1KO mice whose AKT1 protein is deficient in all
cells, β cell mass is moderately lower in A1KOs than in the controls. And more
importantly, this difference is enlarged in 12 month old mice (Figure2.2A,B). This
difference seems to be caused by lower cell proliferation in A1KO mice identified
in 12 month old group (Figure2.2C). One explanation for this result is that because
the cell turnover rate is extremely low for β cells under physiological condition, the
anti-proliferative function of AKT1 deficiency could only be revealed by a long-time
accumulation of this effect. Another explanation for the phenotype lies on the
improved glucose tolerance of A1KO mice. It is also possible that reduced
metabolic burden among A1KO mice lowered the need for insulin and thus results
in the reduced β cell mass and proliferation (Figure 1.1 A-H). Thus, it is
inconclusive for the role of AKT1 protein on β cells based on these evidence.
31
B.
Insulin / Ki67/ DAPI
Figure 2. 1 β cell specific AKT1 loss does not affect pancreas phonotype
under physiological condition.
A. Representative Haemotoxylin and Eosin (H&E) staining pictures of pancreas from Control
and βA1KO mice (scale bar:100μm). Ki67/insulin/DAPI triple staining picture of Control mice
and A1KO mice (Red:insulin; Green:Ki67; Blue: DAPI, scale bar: 100μm ) B. Islet area was
calculated by dividing islet area over total pancreas area. Three sections (240μm apart) were
analyzed for each mice. (n=3/group); C. Ki67 ratio was calculated by dividing the number of
Ki67/insulin positive cells over total insulin positive cells. Three sections (240μm apart) were
analyzed for each mice. n=4-5/group.
βA1KO
Con
A.
C.
32
Control AKT1 KO
Insulin / BrdU / DAPI
100um 100um
A.
B.
C.
Figure 2. 2 whole-body AKT1 loss does not affect pancreas phonotype
A. Representative Haemotoxylin and Eosin(H&E) staining pictures of pancreas from Control
and A1KO mice (scale bar:100μm). BrdU/insulin/DAPI triple staining picture of Control mice
and A1KO mice (Red:insulin; Green:BrdU; Blue: DAPI, scale bar: 100μm ) B. Islet area was
calculated by dividing islet area over total pancreas area. Three sections (240μm apart) were
analyzed for each mice. (n=3/group); C. BrdU ratio was calculated by dividing the number of
Ki67/insulin positive cells over total insulin positive cells. Three sections (240μm apart) were
analyzed for each mice. n=3/group.
33
Next, we investigated at βA2KO mice whose β cells lack AKT2 protein. At
3-month old, islet mass is slightly but significantly higher in A2KO mice with a
comparable β cell proliferative rate (Fig. 3.1A-C). It is unclear why A2KO mice
have higher islet mass. Here, we could draw a primary conclusion that AKT2
protein is not required for β cell proliferation. Additionally, when we investigated
A2KO mice whose AKT2 protein is deficient in all cells, we found increased islet
mass and cell proliferative events in A2KO mice at both 3 and 6 month old
(Fig.3.2A-D) (Cho et al., 2001). This increase in β cell mass and proliferation could
be due to two factors: a. the deletion of AKT2 protein; b. the hyperglycemic
condition of A2KO mice since it is known that hyperglycemia increases β cell mass.
If this increase in β cell mass results from AKT2 deletion, then AKT2 protein must
not be required for β cell proliferation. If this increase in β cell mass results from
the hyperglycemic condition, then AKT2 protein must not be required for glucose-
induced β cell proliferation which also means it is not involved in controlling the β
cell mass in the development of Type II diabetes. Thus, not matter what reason
causes the increased β cell mass and proliferation in A2KO mice, it is likely that
AKT2 does not involve in controlling β cell proliferation. Whether AKT2 protein
posts an anti-growth effect on β cells is not clear.
34
A.
100μm 100μm
βA2KO
Con
Insulin / Ki67 / DAPI
B. C.
Figure 3. 1 β cell specific AKT2 loss does not affect pancreas phonotype under
physiological condition
A. Representative Haemotoxylin and Eosin(H&E) staining pictures of pancreas from Control and
βA2KO mice (scale bar:100μm). Ki67/insulin/DAPI triple staining picture of Control mice and A2KO
mice (Red:insulin; Green:Ki67; Blue: DAPI, scale bar: 100μm ) B. Islet area was calculated by
dividing islet area over total pancreas area. Three sections (240μm apart) were analyzed for each
mice. (n=3/group); C. Ki67 ratio was calculated by dividing the number of Ki67/insulin positive cells
over total insulin positive cells. Three sections (240μm apart) were analyzed for each mice.
n=3/group.
35
AKT2 KO WT
3M
6M
A.
36
3M
6M
B.
Insulin / BrdU / DAPI
C.
D.
Figure 3. 2 whole body AKT2 deficiency induces β cell mass and β cell
proliferation.
A. Representative Haemotoxylin and Eosin(H&E) staining pictures of pancreas from
Control and A2KO mice at either 3-month(3M) or 6-month(6M)(scale bar:100μm). B.
BrdU/insulin/DAPI triple staining picture of Control mice and A2KO mice (Red:insulin;
Green:BrdU; Blue: DAPI, scale bar: 100μm ) C. Islet area was calculated by dividing
islet area over total pancreas area. Three sections (240μm apart) were analyzed for
each mice. (n=3/group); D. BrdU ratio was calculated by dividing the number of
Ki67/insulin positive cells over total insulin positive cells. three sections (240μm apart)
were analyzed for each mice. n=3/group.
37
CHAPTER IV: EFFECTS OF AKTS ON PANCREATIC Β CELL
PROLIFERATION UPON STREPTOZOTOCIN (STZ) INJURY
IV-1. Introduction and Rationale
The results of studying AKT deficient mice in the previous section show a
potential role of AKT1 protein in controlling β cell proliferation while the AKT2
protein might cast no effect on β cell growth. These observed effects could be
biased by the slow cell turnover of β cells if most β cells are not in the cell cycle
when the study was performed. Thus, this extreme slow cell turnover of β cells
makes the study hard to cover enough time frame to reflect the difference. Thus,
applying a stimuli to spurt β cell growth could help us shorten and narrow down
the study window. Here, I applied streptozotocin (STZ) on mice as a stimuli.
Steptozotocin(STZ) injury model is a classic diabetic animal model. STZ
specifically damages β cells and the regeneration process after STZ injury involves
a cocktail of events, such as β cell proliferation, β cell neogenesis, and cell death.
This model has been widely used as an experimental tool to introduce β cell
regeneration(Like and Rossini, 1976). On one hand, using this model on AKT1 and
AKT2 deficient models could help us evaluate whether these proteins participate
in and which biological events they involve during the regeneration of β cells. On
the other hand, STZ model mimics the scenario happened in diabetes. By applying
STZ on AKT1 and AKT2 deficient models, we can better understand how AKT1
and AKT2 proteins contribute to the development of diabetes.
38
IV-2. Effects Of β-Cell Specific AKT2 Loss On β Cell Proliferation
As βA2KO mice have larger islet mass and the hyperglycemic condition of
A2KO mice seems not affecting β cell mass and proliferation, I hypothesize AKT2
is dispensable for β cell regeneration upon injury. To confirm this hypothesis, I
challenged the βA2KO mice with streptozotozin (STZ). One week after STZ
treatment, both Control and βA2KO mice presented elevated plasma glucose level
because of the β cell death (Fig.4.1C). However, βA2KO mice displayed similar
replicative capability as the control mice after STZ injury (Fig.4.1B,D). This
experiment further confirms that AKT2 is dispensable for β-cell proliferation in both
physiological maintenance condition and injury induced regenerative condition.
39
βA2KO: Akt2
loxP/loxP
, Rip-Cre+
Con: Akt2
loxP/loxP
, Rip-Cre-
5 daily injections of
STZ (50mg/kg)
3 Month
Euthanize
WT
βA2KO
Figure 4. 1 β cell specific AKT2 loss does not affect pancreas phonotype after
STZ injury
A. Illustration demonstrating the STZ treatment of conditional AKT1/AKT2 depletion in pancreatic
β cells. B. Fasting glucose level of Control and A2KO mice before and one week after STZ
treatment (n=5/group). C. Representative pictures showing BrdU/insulin double staining. BrdU
percentage is calculated by dividing BrdU+Ins+ β cells over total β cells. Three sections (240μm
apart) were analyzed for each mouse. n=5/group.
A.
B. C.
D.
40
IV-3. Effects of Whole-Body AKT1 Loss on β Cell Proliferation after STZ injury
Based on previous results of the reduced islet mass in old A1KO mice and
the insignificant reduction of islet mass in 3-month old A1KO, I hypothesized that
AKT1 involves in β cell growth. As mentioned before, injury model could allow us
better target study window and here, STZ was applied to A1KO mice. Because
βA1KO mice do not have 100% Akt1 deficiency, βA1KO mice were not chosen to
perform this experiment since the cells with normal AKT1 protein could over-grow
the ones without AKT1 and prevented us to observe the results.
In response to STZ treatment, a dramatic increase in plasma glucose was
observed starting from 1 week after STZ treatment in both the A1KO and Control
mice (Fig.5.1B). The percentage increase of the plasma glucose was significantly
higher in A1KO mice as fasting plasma glucose are lower in these A1KO mice
before STZ treatment (Fig.5.1C). I analyzed the pancreas samples at two time
points: 1 and 8 weeks after the STZ injections. The destruction to the islets was
not obvious after one week of STZ treatment, but the destruction was very obvious
after eight weeks of STZ treatment (Fig. 5.2A). In order to understand why the
percentage of changed glucose was higher in A1KO group and why the islets were
more damaged in A1KO mice, I evaluated the cell proliferation rate of β cells by
counting the percentage of BrdU+Insulin+ cells because these mice were fed with
BrdU containing water before euthanized. In the control group, the percentage of
BrdU labeled β cells was around 1.5% at 1 week after STZ treatment. This number
is higher than the 1% BrdU labeled β cells observed in the non-injured islets
41
(Fig.2.1C, Fig.5.1E), indicating an enhanced proliferative capability occurred in
respond to the injury. In A1KO group, this value dropped to 0.5% 1 week after STZ
treatment (Fig 5.1E) and remained until 8 weeks after STZ treatment (Fig.5.2B).
As expected, when comparing the percentage of BrdU labelled β cells between
A1KO group and Control group at 1 week and 8 weeks after STZ treatment, the
control mice exhibited three times higher percentage of BrdU labeled β cells than
A1KO mice did, suggesting that the A1KO islets are unable to compensate for the
loss of islet mass by inducing cell proliferation (Fig.5.1E, Fig.5.2B). This
observation further proves that AKT1 protein deficiency in β cells would reduce the
proliferative capability of β cells.
A1KO: Akt1
-/-
WT: wild type
5 daily
injections of
STZ (50mg/kg)
3 Month
Euthanize
8 weeks
after STZ
1 week
after STZ
Euthanize
A.
42
Control
Insulin/ BrdU/ DAPI
Control A1KO
B. C.
D.
E.
Figure 5. 1 whole-body AKT1 deficiency causes reduced cell proliferation one
week after STZ induction.
A. illustration demonstrating the implementation of STZ treatment. B. Plasma glucose level at
indicated time point (n=7 for Con, n=6 for A1KO). C. Changed percentage of glucose is calculated
by dividing the glucose level at the time of measurement to the initial value before the experiment
started.(n=5/group) D, Representative H&E staining pictures and representative BrdU/Insulin
staining pictures of mice one week after STZ treatment. (Red: insulin, Green:BrdU, Blue:DAPI). E
The percentage of BrdU Insulin double positive cells over total β cells. Three sections (240um
apart) per mouse is included in the analysis. At least 1000 cells were counted for each mouse.
*
*
*
*
*
*
*
43
Control
A1KO
Insulin / BrdU / DAPI
A.
Figure 5. 2 whole-body AKT1 deficiency causes reduced cell proliferation eight
weeks after STZ induction.
A. Representative H&E staining pictures and representative BrdU/Insulin staining pictures of mice
one week after STZ treatment. (Red: insulin, Green:BrdU, Blue:DAPI). (n=7 for Con, n=6 for A1KO)
B The percentage of BrdU Insulin double positive cells over total β cells. Three sections (240um
apart) per mouse is included in the analysis. At least 1000 cells were counted for each mouse.
B.
44
CHAPTER V. EFFECTS OF AKT1 PROTEIN ON PANCREATIC Β
CELL PROLIFERATION UPON HIGH FAT DIET FEEDING
V-1. Introduction and Rationale
High fat diet feeding on rodents is a model for studying mechanisms and
treatment of impaired glucose tolerance and Type 2 Diabetes (Winzell and Ahren,
2004). The high-fat diet model partially mimics the development of Type 2 Diabetes
in humans and serves as another stimuli for β cell replication (Golson et al., 2010).
High fat diet feeding for certain time would induce obesity and insulin resistance
on animals which further induces elevated blood glucose. In response to insulin
resistance, β cells produce more insulin by secreting more insulin per β cell and
by increasing β cell mass. Intensified insulin signaling could lead to increased level
of PIP3 and phosphorylate AKTs which further activates cell cycle regulators and
initiates cell replication (Golson et al., 2010).
Based on the results from STZ experiments described before, AKT1 protein,
instead of AKT2, should be the one that is responsible for controlling β cell
proliferation induced by stimuli. Although STZ model could be a quick way to test
whether and how AKT proteins participated in the β cell regeneration upon injury,
this model is not physiological relevant to diabetes. A better way to investigate
whether AKT proteins also involve in the development of diabetes could be
applying high-fat-diet model on AKT deficient mice. Since I already ruled out the
involvement of AKT2 protein in β cell proliferation based on earlier evidence, I
decided to apply high-fat-diet on βA1KO mice.
45
Another concern on the experimental design was the incomplete AKT1
deletion in tamoxifen induced AKT1 deletion model. Because a small portion of β
cells might still express AKT1, it is possible that these cells fast proliferate in
respond to HFD and override the reduced cell proliferation of AKT1 deficient β cells.
Thus, I also applied HFD on A1KO mice. This design could allow me cross-validate
the results in A1KO and βA1KO mice.
V-2. AKT1 Deficiency Blocks The β Cell Proliferation upon Diet Stimulation
To address the role of AKT1 in adaptive growth of β-cells, we exposed the
βA1KO mice to high fat diet (HFD) feeding. βA1KO mice respond to HFD very well.
βA1KO mice gained weight at the same rate as the controls when exposed to HFD
(Fig.6.1B). HFD feeding is also capable of inducing hyperglycemia as it did in the
control mice (Fig.6.1C). The intraperitoneal GTT and ITT tests showed βA1KO
mice were insulin resistant and glucose intolerant in the same way as the controls
after HFD (Fig.6.1D,E).
When I applied HFD on A1KO mice, similar changes in metabolism were
observed as compared to βA1KO mice. HFD induced hyperglycemic condition on
A1KOs (Fig.6.2C). However, A1KO mice gained less weight in respond to HFD.
Intraperitoneal GTT and ITT tests also indicated that A1KO mice on either 2
months or 4 months of HFD are more tolerant to glucose likely due to enhanced
peripheral sensitivity to insulin (Fig.6.2D-G). This peripheral sensitivity has been
46
implicated previously by report demonstrating higher energy expenditure in the
muscle of A1KO mice(Wan et al., 2012).
In pancreas, HFD-induced islet mass increase was observed among control
mice but not βA1KO mice (Fig.6.3C). This observation was further supported by
the significant increase in islet mass in Control but not A1KO mice exposed to both
2-month and 4-month HFD (Fig.6.4A,C). This result indicated AKT1 deficiency
could block the expansion of β cells upon HFD feeding. Staining with
Bromodeoxyuridine (BrdU) further revealed 2-5 fold increase in -cell proliferation
by HFD feeding among control mice (Fig.6.3D, Fig.6.4D).
In neither βA1KO nor A1KO mice, HFD could induce β cell proliferation. The
percentages of BrdU or Ki67 positive β cells are similar to the number observed in
mice fed on chow diet, indicating the HFD induced cell proliferation is blocked due
to AKT1 deficiency (Fig.6.3D, Fig.6.4D). As introduced previously, cell cycle
regulators such as cyclinD1/D2, p16, p21, CDK4, and p27 have been found to
participate in the regulation of β cell proliferation. Thus, I tested whether these
molecules were responsible for the HFD induced β cell proliferation and whether
AKT1 deficiency affects their expression. Indeed, I found AKT1 deficiency reduced
cyclinD1and cyclinD2 levels of β cells upon HFD stimulation but increased p27
levels, which further explains the mechanism of how AKT1 block the cell
proliferation event of β cells (Fig.6.5A-C).
47
βA1KO: Akt1
loxP/loxP
, MIP1-CreERT +
Con: Akt1
loxP/loxP
, MIP1-CreERT -
5 injections
of Tamoxifen
(250mg/kg)
1 Month
High fat diet
2 weeks 12 weeks
Euthanize
A.
B.
C.
D. E.
Figure 6. 1 β cell specific AKT1 deficient mice have similar metabolic profile
compared to the controls when exposed to high-fat-diet
A. Illustration showing the strategy for treatments. B. Bodyweight of mice (n=5/group) C.
Fasting glucose level of Control and βA1KO mice before HFD feeding and after HFD
feeding. (n=5/group) D. 2mg/kg glucose solution (prepared with sterile PBS) was injected
to mice intraperitoneally after 16 hrs of fasting. Blood samples were collected tho ugh tail
vein at certain time point (n=4/group) E. insulin tolerance test: 0.5U/g insulin prepared in
PBS was injected to mice intraperitoneally after 5 hours of fasting. Blood glucose level
was tested at correspondent time points. (n=4/group, *<0.05)
*
48
*
*
*
*
*
*
*
*
*
*
*
*
A.
B.
D. E.
F.
G.
Figure 6. 2 whole-body AKT1 deficient mice have better glucose tolerance
compared to the controls when exposed to high-fat-diet.
A. Illustration showing the strategy for treatments. B. Bodyweight of mice (n=5/group) C. Fasting
glucose level of Control and A1KO mice before HFD feeding and after HFD feeding. (n=5/group)
D,F. 2mg/kg glucose solution (prepared with sterile PBS) was injected to mice intraperitoneally
after 16 hrs of fasting. Blood samples were collected though tail vein at certain time point
(n=4/group) E,G. insulin tolerance test: 0.5U/g insulin prepared in PBS was injected to mice
intraperitoneally after 5 hours of fasting. Blood glucose level was tested at correspondent time
points. (n=4/group, *<0.05)
A1KO: Akt1
-/-
WT: wild type
3 Month
High-fat diet
16 weeks 8 weeks
Euthanize
Euthanize
C.
49
Con
HFD
NC
βA1KO
NC
HFD
B.
A.
C.
D.
Figure 6. 3 β cell specific AKT1 deficiency block the 2M high-fat diet induced β
cell proliferation.
A. Representative H&E staining pictures of pancreas from Control mice and A1KO mice on either
normal chow or high fat diet for 2 months. (scale bar:100μm). B. representative pictures of
BrdU/Insulin/DAPI staining of pancreas from indicated mice group. (Red: Insulin, Green: BrdU,
Blue:DAPI) C. islet area of control and A1KO mice on either normal chow or high fat diet for 2
months. Three sections (240um) apart were analyzed for each mouse. ( n=2 for A1KO NC group,
n=3 for other groups). D: Quantification of the percentage of BrdU-positive cells in indicated
group. Three sections (240um) apart were analyzed for each mouse. At least 1000 cells was
counted for each mice ( n=2 for A1KO NC group, n=3 for other groups).
50
A1KO
Con
4M HFD 4M NC
100 μm 100 μm
100 μm 100 μm
2M HFD
Con
A1KO
2M NC
Insulin/ Ki67/DAPI
A.
B.
C.
D.
Figure 6. 4 Whole-body AKT1 deficiency block high-fat diet induced β cell
proliferation.
A. Representative H&E staining pictures of pancreas from Control mice and A1KO mice on either
normal chow or high fat diet for 4 months. (scale bar:100μm). B. representative pictures of
BrdU/Insulin/DAPI staining of pancreas from indicated mice group. (Red: Insulin, Green: BrdU,
Blue:DAPI) C. islet area of control and A1KO mice on either normal chow or high fat diet . Three
sections (240um) apart were analyzed for each mouse. (n=3/groups). D: Quantification of the
percentage of BrdU-positive cells in indicated group. Three sections (240um) apart were
analyzed for each mouse. At least 2000 cells was counted for each mice ( n=3/groups).
51
βA1KO
βA1KO HFD Con Con HFD
CyclinD2
CyclinD1
p27
Insulin / p27 / DAPI
Insulin / CyclinD1 / DAPI
Insulin / CyclinD2 / DAPI
Figure 6. 5 AKT1 modulates cyclinD2, cyclinD1,and p27
cyclin D1,cylin D2 and P27 immunohistochemical staining on islets of indicated groups.
52
CHAPTER VI. AKT1 LOSS CAUSES ER STRESS IN RESPONSE TO
LONGER HFD EXPOSURE
VI-1. Introduction and Rationale
One mechanism proposed for the effect of HFD feeding on β-cell survival is
through regulation of endoplasmic reticulum stress (ER stress). ER stress is
triggered by the existence of misfolded proteins or the condition of overloaded
proteins in ER. The stress condition of endoplasmic reticulum results in the
activation of unfolded protein response (UPR) which aims at resolving the ER by
slowing down the translation process. ER stress and UPR are easily triggered for
β cells whose primary function is to produce and secrete protein in response to
environmental conditions(Biden et al., 2014). UPR signaling initiates from proteins
reside in ER lumen. There are three classic UPR signaling arms: protein kinase
RNA(PKR)-like ER-associated kinase (PERK), the serine/threonine-protein
kinase/endoribonuclease(IRE1) and the transcription factor activating transcription
factor (ATF6), and. It was thought all these three proteins are inactivated by the
binding with protein chaperon Bip/Grp78 at unstressed state. At the stressed state,
Bip/78 protein binds to misfolded proteins and is released from PERK, ATF6, and
IRE1 which activates the downstream signaling. Once IRE1 gets activated, it
triggers the alternative splicing of X-box binding protein 1 mRNA to its active form
which encodes and relocates XBP-1 protein to the nucleus(Ueki and Kadowaki,
2011). As transcription factor, XBP-1 initiates transcription for chaperons, ER-
associated degradation (ERAD), and other proteins involve in UPR response. This
arm of regulation is relatively longer because it involves several steps and the
53
transcriptional initiation for a panel of related proteins. On the other side, when
PERK gets autophosphorylation by disassociating with Bip/Grp78. It initiates a
transient but fast-acting blockade of protein translation by phosphorylating the
translational initiation factor eukaryotic initiation factor 2 (eIF2)-α (Biden et al.,
2014).
The role of ER stress on the development of diabetes has been well
proposed and tested. At the beginning, in vitro studies and animal models link
lipotoxicity of free fatty acids to β cell ER stress and apoptosis. Palmitate acids
exposure was shown to induce ER stress in MIN6 cells and further cause cell
apoptosis(Laybutt et al., 2007). Induced ER stress was also identified in the β cells
of the Zucker diabetic mice which further linked ER stress to β-cell
dysfunction(Omikorede et al., 2013). Later, more studies confirmed that ER stress
was induced in the islets of type 2 diabetic patients which further supports the idea
that ER stress contributes to β cell failure and diabetes(Laybutt et al., 2007;
Marchetti et al., 2007).
In order to figure out the signaling pathways get affected by HFD and AKT1
deficiency, I performed high performance RNA-seq analysis for islets extracted
from the βA1KO and control mice fed either chow or HFD. Ingenuity pathway
analysis of the RNA-seq data identified EIF2 (eukaryotic initiation factor) signaling
pathway as the most significantly altered signaling induced by HFD feeding. EIF2
is required in the initiation of protein translation. It mediates the binding of
54
tRNAMet to the ribosome in a GTP-dependent manner and is respond to various
signaling activated by environmental changes. As I mentioned above, one of the
changes that would affect EIF2 signaling is intensified ER stress. Additionally, it
has been proposed that, in the condition such as HFD where nutrients is
consistently excessive, alterations in metabolism bring toxic effects on β cells,
gradually impairing insulin signaling, inducing ER stress and eventually causing β
cells apoptosis. This process also is thought to relate with the disease progression
of Type II diabetes. Thus, we decided to investigate more into the ER stress status
of the islets and further performed gene set enrichment analysis (GSEA) on the
RNA-sequencing data (Fig. 7.2A). Several ER markers genes, such as HSPA5
which encodes Grp78 and EIF2AK3 which encodes PERK, were shown to be up-
regulated when AKT1 protein is deletion in β cells. HYOU1, which was also up-
regulated in βA1KO mice encodes a protein involves in protein folding and
secretion in ER. Suppression of this gene is also thought to accelerate apoptosis.
CALR encodes calreticulin, a protein locate in ER and affect the Ca
2+
signaling.
Thus, the RNA-sequencing data imply the dysregulation of ER function in β cells
when lacking AKT1 protein.
VI-2. AKT1 Deficiency Induces ER Stress On β Cells
Under hyperglycemic condition, glucose rapidly stimulates Ins gene
transcription, which produces preproinsulin. In the ER, preproinsulin is synthesized,
folded, and cleaved to form proinsulin, which later moves to Golgi and packages
into secretion granules. In these granules, proinsulin is further cleaved to form
55
mature insulin. High need of insulin could result in protein overload in ER and
generates ER stress condition. ER stress triggered UPR response could cause
transient functional pause on ER which further causes reduced efficacy of insulin
processing and inappropriate proinsulin secretion to plasma(Ashcroft and
Rorsman, 2012). Thus, blood proinsulin level and proinsulin over insulin ratio have
been reported to be indicators for β cells ER function (Pfutzner and Forst, 2011;
Yoshioka et al., 1988).
To specifically determine if AKT1 loss indeed affects the ER status and
triggers UPR response, we first tested the proinsulin level in β cells and mice
plasma. In our model, the plasma proinsulin level of control mice slightly increased
in response to 4 months of HFD, indicating normal ER function in islets. Conversely,
in A1KO mice, the plasma proinsulin level increased dramatically in response to
HFD and the level was significantly higher than the plasma proinsulin level in the
control mice fed with HFD (Figure.7.1A). This result indicates potential ER
dysfunction in β cells at A1KO HFD group. At the same time, this high plasma
proinsulin level was mirrored by stronger proinsulin staining in β cells at A1KO HFD
group (Fig.7.1D). The plasma insulin level could reflect β cell mass since larger
islet mass could predict higher insulin level in general. When control mice were on
HFD, their plasma insulin level increased which was consistent to the increased
islet mass (Fig6.4C, Fig.7.1B). Conversely, when A1KO mice were on HFD, their
plasma insulin level increased at a less significant level because the islet mass of
A1KO did not increase in response to HFD (Fig.6.4C, Fig.7.1B). As another
56
indicator, the plasma proinsulin over insulin ratio could reflect the efficiency for
proinsulin processing and the severity of ER stress in β cells. A1KO mice fed with
16 weeks of high fat diet presented highest level of proinsulin to insulin ratio, further
confirmed the existence of ER dysfunction in their β cells. (Fig.7.1B,C).
In order to preclude the peripheral effects in A1KO mice, we further
evaluated proinsulin, proinsulin/insulin ratio among βA1KO mice exposed to 12
weeks of high fat diet. Slightly different than the results in A1KO and their controls,
the βA1KO and their control mice both had increased proinsulin level in respond
to HFD (Figure 7.2C). The plasma proinsulin level in βA1KO HFD mice were higher
than Control HFD mice which indicated more severe β cell dysfunction in A1KO
mice. The plasma insulin level of the control and βA1KO mice could reflect their
islet mass in respond to HFD. HFD induced islet mass and plasma insulin level
among the control mice while HFD failed to induce islet mass and plasma insulin
level among the βA1KO mice (Figure 7.2D). Lastly, the plasma proinsulin/insulin
ratio were significantly higher in βA1KO HFD group than other groups which further
proved the existence of ER dysfunction in β cells when AKT1 protein is deficient
(Figure 7.2E).
57
Proinsulin/ DAPI
Con
4M HFD
A1KO
4M HFD
GRP 78
GAPDH
p-PERK
P-eIF2a ser 51
t-eIF2a
t-PERK
AKT1
WT HFD A1KO HFD WT NC A1KO NC
1.6 1.0
1.77 1.59 GRP78/GAPDH
1.2
1.0 1.5 1.5 P-eIF2a/eIF2a
P-PERK/PERK
1.2 1.0 1.3 0.6
A. B.
C.
D.
E.
Figure 7. 1 increased ER stress in islets from A1KO mice.
A. plasma proinsulin level of mice at indicated group. (n=5/group) B. plasma insulin level of mice
at indicated group. (n=5/group) C. ratio of plasma proinsulin over plasma insulin level. D.
representative staining picture of Proinsulin/DAPI staining (Green:proinsulin, Blue:DAPI) E.
western blots of isolated islets from mice in indicated group. Protein quantification was performed
for p-PERK, PERK, GRP78, p-EIF2a and total EIF2a
*
*
*
*
58
FDR q-value: 0.35
NES:1.17
Figure 7. 2 induced ER stress in islets from βA1KO.
A. top gene list identified by Gene set enrichment analysis, each lane indicates for one mice B.
Enrichement plots of A1KO NC versus WT NC and A1KO HFD versus WT HFD. C-E. Plasma
proinsulin level, insulin level and proinsulin/insulin ratio of Control and A1KO mice after 14 weeks
of HFD feeding (NC: Normal Chow, HFD: High fat diet) (n=4/group)
A.
B.
C.
D. E.
*
*
*
59
To further elucidate the molecular mechanisms of intensified ER
dysfunction in AKT1 protein deficient β cells, I isolated islets from control and
A1KO mice either at normal chow or HFD and determined the levels of several
proteins related with ER stress among the protein lysates from the islets.
Animal studies on PERK have revealed its importance in regulating β cell
ER stress and function. Mice lack PERK protein develop severe hyperglycemic
condition with reduced β cell mass (Harding et al., 2001). PERK activation directly
inactivates eIF2 and inhibiting mRNA translation. As a chaperon protein binding
to misfolded proteins, GRP78 (Bip) is often served as an indicator for ER stress.
Comparing with Control group, phosphorylated PERK, BIP and
phosphorylated EIF2a were upregulated in A1KO NC islets, indicating an
increased ER stress at basal level when AKT1 is deficient (Fig.7.1E). According to
the quantification result of these proteins, HFD could induce the activation of UPR
response by increase p-PERK, p-EIF2a, and Grp78. However, since these protein
levels were already higher in A1KO NC group, HFD did not increase the level of
them in A1KO group which means that AKT1 deficiency potentially disables the
initiation of UPR response. With the increased metabolic need resulting from HFD,
this inability to launch UPR response eventually resulted in ER dysfunction and the
secretion of proinsulin to plasma.
60
Collectively, based on the plasma levels of proinsulin and proinsulin/insulin
ratio as well as the western blot analysis on the isolated islets, it is likely to
conclude that AKT1 protein deficiency in β cells results in a dysregulation of ER
function.
CHAPTER VII. AKT1 LOSS SENSITIZES Β CELL TO STZ AND HFD
INDUCED CELL APOPTOSIS
VII-1. Introduction and Rationale
As β cell mass contributes to the development of diabetes, the negative
regulator for β cell death- β cell apoptosis- becomes a critical event for diabetes.
Elevated β cell apoptosis was found in both type I and type II diabetic patients. It
was also proposed that increased ER stress in β cells could contribute to β cell
apoptosis and eventually cell death (Laybutt et al., 2007).
Based on the above results, ER stress was induced when AKT1 protein is
deficient in β cells. We proposed that this stress condition potentiate β cells to
premature death state.
VII-2. Akt1 Deficiency Sensitizes β Cell to STZ Induced Apoptosis in Mice
STZ has a similar structure to glucose and could be selectively transported
by GLUT2 transporter which is mainly expressed in β cells. When given to animal
61
at low doses, STZ selectively accumulates in β cells and elicits immune and
inflammatory reaction by presenting autoantigens to the immune system. Thus,
under the low dose condition, the destruction of β cells is associated with both
inflammatory infiltrations of lymphocytes and the hyperglycemic condition of the
animals (Graham et al., 2011). In order to evaluate the cell apoptosis event when
mice were given STZ, terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) staining was performed on the pancreas samples from mice
treated with STZ. A 2-fold increase in TUNEL positive β cells was observed among
A1KO mice treated with STZ but not the controls (Figure 8.1 C,D). Quantification
on the percentage of TUNEL positive β cells and β cell morphology revealed that
AKT1 loss in β cells make cells more prone to STZ caused cell apoptosis. It is
unknown whether this is because of affecting the inflammatory infiltration of
lymphocytes or through mechanisms related with glucotoxicity.
VII-3. Akt1 Deficiency Sensitize β Cells to HFD Induced Apoptosis in
Animals and in INS-1 Cells
Since chronic ER stress links to β cell failure, we further investigated at cell
apoptosis among mice exposed to HFD. Indeed, β cell apoptotic events tested by
TUNEL staining were significantly higher among A1KO mice receiving 4 months
of high fat diet. A 4-fold increase in TUNEL positive β cells was observed in A1KO
mice treated with HFD while the TUNEL positive β cells remained unchanged in
the control group (Fig.8.1A,B).
62
In order to verify that increased cell apoptosis is indeed caused by AKT1
deficiency, we generated an INS-1 cell line that is deficient for AKT1 expression
detected by antibody using CRISPR/Cas9 technique. The guidance RNA was
designed in the exon 1 of AKT1 gene. After single cell selection process and
screening of about 100 cell lines, one cell line was identified to have mutation in
the targeted sequence and was named #56 (Fig.8.2 A,B). The #56 cell displays
similar morphology as INS-1 but it’s slightly more susceptible to high glucose
induced cell apoptosis indicated by the percentage of Annexin/Propidium iodide(PI)
positive cells when exposed to high glucose media for 48 hours (Fig.8.2 B,C).
Furthermore, when we treated INS-1 cells and #56 cells with Tunicamycin (Tuni),
the phosphorylated level of Eif2a is slightly higher than in #56 cells, indicating
potential severer ER stress (Fig.8.2D). In order to validate this data, we further
used LY294002 inhibitor to mimic the downregulation of phosphorylated AKT
levels found in #56 cells and compared the p-eIF2a level of INS-1 cell treated with
Tuni versus INS-1+LY+Tuni. Indeed, inhibition of PI3/AKT pathway using
LY294002 compound could increase the p-eIF2a level of INS-1 cells (Fig.8.3B).
This result was further validated by flowcytometry studies showing dramatically
increased cell apoptosis events in INS-1+LY+Tuni group as Annexin IV positive,
and Propidium iodine double positive cells are extremely high in INS-1+LY+Tuni
group (Fig.8.3A,C).
63
Collectively, the results in STZ treatment experiment, HFD experiment,
and in vitro studies all point out that AKT1 loss potentiate β cell to apoptosis and
this is likely due to AKT1-deficiency induced ER stress.
64
4M HFD
4M NC
Insulin/ TUNEL / DAPI
Con
A1KO
Insulin/ TUNEL/ DAPI
Con+STZ
A1KO+STZ
Figure 8. 1 AKT1 deficiency causes β cell apoptosis
A. Representative picture of TUNEL/insulin double staining on islets exposed to HFD/NC. (Red:
insulin, Green: TUNEL, Blue: DAPI) C. Representative picture of TUNEL/insulin double staining
on islets one week after STZ treatment. (Red: insulin, Green: TUNEL, Blue: DAPI)B,D Percentage
of TUNEL positive cells over total counted cells. Three sections (240um apart) were analyzed for
each mouse, at least 3000 cells were counted for each mouse.
A.
B.
C.
D.
65
INS-1 INS-1 #56 #56
AKT1
Actin
3mM glucose 11mM glucose 33mM glucose
Propidium iodide
Annexin
p-AKT
p-eIF2a
Actin
Figure 8. 2 AKT1 deficiency causes β cell apoptosis in vitro
A. sequence of #56 cells aligned to the original rat INS-1 cells. The indicated sequence was where
the guidance RNA targets. B. Western blot shows the down-regulation of AKT1 in #56 cell line. C.
Flow cytometry results of treating INS-1 cells and #56 cells treated to different glucose
concentration. D. western blot shows the p-AKT T308 level and p-eIF2a level of INS-1 cells and
#56 cell to either tunicamycin or not.
A. B.
C.
D.
66
FSC-H, SSC-H subset
INS-1
Event Count: 19982
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
0.53 5.78
2.91 90.8
FSC-H, SSC-H subset
INS-1+LY
Event Count: 19972
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
2.15 4.07
2.06 91.7
FSC-H, SSC-H subset
INS-1+TUNI
Event Count: 19760
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
5.05 13
5.84 76.1
FSC-H, SSC-H subset
INS-1+TUNI+LY
Event Count: 19891
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
7.75 28
12.7 51.5
FSC-H, SSC-H subset
INS-1
Event Count: 19982
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
0.53 5.78
2.91 90.8
FSC-H, SSC-H subset
INS-1+LY
Event Count: 19972
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
2.15 4.07
2.06 91.7
FSC-H, SSC-H subset
INS-1+TUNI
Event Count: 19760
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
5.05 13
5.84 76.1
FSC-H, SSC-H subset
INS-1+TUNI+LY
Event Count: 19891
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
7.75 28
12.7 51.5
FSC-H, SSC-H subset
INS-1
Event Count: 19982
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
0.53 5.78
2.91 90.8
FSC-H, SSC-H subset
INS-1+LY
Event Count: 19972
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
2.15 4.07
2.06 91.7
FSC-H, SSC-H subset
INS-1+TUNI
Event Count: 19760
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
5.05 13
5.84 76.1
FSC-H, SSC-H subset
INS-1+TUNI+LY
Event Count: 19891
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
7.75 28
12.7 51.5
FSC-H, SSC-H subset
INS-1
Event Count: 19982
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
0.53 5.78
2.91 90.8
FSC-H, SSC-H subset
INS-1+LY
Event Count: 19972
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
2.15 4.07
2.06 91.7
FSC-H, SSC-H subset
INS-1+TUNI
Event Count: 19760
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
5.05 13
5.84 76.1
FSC-H, SSC-H subset
INS-1+TUNI+LY
Event Count: 19891
10
0
10
1
10
2
10
3
10
4
FL1-H: A NNEXIN-FITC
10
0
10
1
10
2
10
3
10
4
FL3-H: propidium iodide
7.75 28
12.7 51.5
p-AKT
p-eIF2a
Actin
Figure 8. 3 Inhibition of PI3K/AKT causes β cell apoptosis in vitro
A. Flow cytometry results of treating INS-1 cells treated with LY294002, Tunicamycin, or LY29002
and tunicamycin together. B.western blot shows the p-AKT T308 level and p-eIF2a level of INS-1
cells exposed to LY294002, tunicamycin, LY294002 and tunicamycin. C. quantification of the
percentage of Annexin+, propidium iodide positive, or Annexin and proidium iodide positive cells;
three repeated experiments.
A.
B.
C.
67
CHAPTER VIII. DISCUSSION
PI3K/AKT pathway has been well-studied for its function in promoting
cancer cell proliferation. Meanwhile, its role in fulfilling insulin’s function and
maintaining healthy human metabolism also attracts attention from many
researchers. Animal metabolism is tightly regulated by insulin concentration in
plasma which correlates with β cell mass. The data in this thesis reemphasizes
the importance of PI3K/AKT pathway in controlling pancreatic β cell growth, animal
metabolism and further reveals distinct functions of AKT isoforms. First, we
confirms that manipulation on Akt1 and Akt2 gene would affect mice metabolism.
Second, our data suggest attenuating AKT level in β cells does not interfere with
the normal growth of β cells at physiological condition but it does involve in the
adaptive response of β cells to different stimuli. Third, we found AKT1 but not AKT2
is required for β cell to regenerate in injury-stimulated and high-fat-diet condition.
And last, AKT1 protein deficiency would affect the ER stress condition of β cells
which relates with β cell apoptosis.
Similar to the phenotype descripted in earlier publication, a trend of better
glucose tolerance in A1KO mice was observed here in 3-month old mice but not
12-month old at C57BL/6 background (Figure1.1 G,H) (Wan et al., 2012). Wan et
al reported mice presented an increase in energy expenditure resulting from the
germline deletion of Akt1. Particularly, Wan et al found the locomotor activity of
Akt1 germline knockout mice was higher during night. In order to identify the
reason for this phenotype, AKT1 conditional deletion mice was developed with
68
promoters driving deletion of AKT1 in either muscle, liver, or brain tissue. However,
none of these mouse strains presented increased energy expenditure. Another
Akt1 germline knockout mice (Pkbα-/-) was developed by a group in Zuirich on a
mixed 129/Ola and C57BL/6 background. Using this mouse strain, Buzzi et al
reported statistically improved glucose tolerance and insulin tolerance due to the
deficiency of AKT1 protein. They also reported increased plasma insulin level
without significance among these mice and potential dysregulated insulin secretion
in Pkbα-/- mice when they stimulated isolated islets with glucose (Buzzi et al.,
2010). Buzzi et al also found that the glucose uptake of perigonadal fat cells
isolated from Pkbα-/- mice were more sensitive to insulin. In our A1KO mice, we
tested the plasma insulin level at different time points after glucose injection in GTT
test. However, no statistical significance was observed. Consistent to our study,
none of the other two studies found changes in β cell mass and β cell proliferation
under physiological condition. Based on three studies mentioned here, it is still
inconclusive on why germline AKT1 deficient mice has better glucose sensitivity.
Several possibilities exist: 1. Better glucose tolerance is caused by increased
energy expenditure which is a result of synergetic deletion of AKT1 on two or
multiple types of tissues. 2. Sensitivity to insulin is modulated by the deletion of
AKT1 in metabolic tissues such as liver, adipocytes, and muscle cells. In order to
understand why AKT1 deletion has such effect on animal metabolism, more
detailed studies on conditionally Akt1 knockout mice need to be performed.
69
Manipulation in the upstream of PI3K/AKT, such as insulin receptor deletion,
insulin receptor substrate 2 deletion, or PTEN deletion, affects β cell mass and
insulin release at physiological condition. Pancreatic specific deletion of IR or IRS2
reduces β cell mass at 3-4 month old mice (Cantley et al., 2007; Kulkarni et al.,
1999). Pancreatic specific deletion of PTEN, which results in the activation of PI3K,
causes increased β cell proliferation as well (Stiles et al., 2006; Zeng et al., 2013).
Yet, deletion of AKT1 or AKT2 in β cells does not cause any dramatic phenotypic
change in β cells at normal condition. This suggests besides AKT, other molecules
in the downstream of insulin receptor, insulin receptor substrate, and PI3K may
coordinate with AKT to maintain the normal β cell population. Indeed, our group
tried to delete AKT1 at an activate PI3K background by cross β cell PTEN deficient
mice with AKT1 deficient mice. The β cell mass of these mice was lower than β
cell specific PTEN deficient mice but still higher than normal wild type mice, which
suggests that other molecules are also involved in β cell proliferation when PI3K
is activated (data not shown).
According to previous study, AKT2 deficiency causes hyperglycemia
condition in mice. The study did not focus on β cells and the hyperglycemia
condition in A2KO mice served as a cofounding factor for β cell growth which
complicated the data interpretation. Consistent with previous findings, we
confirmed that global AKT2 deficiency does cause hyperglycemia (Cho et al.,
2001). Additionally, we investigated β cell proliferation in detail here and eliminated
the effects of hyperglycemia in A2KO mice by investigating β cell specific AKT2
70
knockout mice. These mice have normal fasting and random glucose level. It is
unclear why the βA2KO mice displays slightly larger islet areas. One possible
reason could be the dysfunction of incorrect Phase I insulin release since we did
observe higher glucose level only at 15 and 30 mins of IPGTT challenge and the
plasma insulin level of βA2KO mice at 0 mins and 15 mins are slightly lower than
the control mice without significance. The mild hyperplasia phenotype in those
mice might be a compensatory effects of insulin secretion inability among those
mice with unknown mechanism.
By investigating AKT1 and AKT2 knockout mice, it seems like that, both
AKTs are not required for maintaining the β cell mass at physiological condition.
However, we investigated at β cell phenotype in aged A1KO mice (>1year old) and
discovered a reduction in cell proliferation and cell mass in those mice (data not
shown). It is possible that the reason we failed to observe any difference of islets
in AKT knockouts is due to low β cell turn-over. It is likely that AKT1 deficiency
affects β cell proliferation in a mild way that can only be observed at stimulated
conditions such as injury or fat feeding conditions or at longer time scale.
Meantime, unlike AKT1, since the β cells lacking AKT2 could proliferate at a
comparable rate with control mice, we predict the lack of AKT2 for longer time
would not reduce β cell proliferation.
Our results shows AKT1 but not AKT2 is required for injury and fat-feeding
induced adaptive response of β cells. The explanation for this phenomenon could
71
be isoform specificity of AKTs. The isoform specificity of AKT has been discovered
and discussed for more than 10 years. The hypothesis was raised from the
observations from AKT isoform specific knockout studies in which different AKT
knockout mice have dramatic phenotypic differences (Gonzalez and McGraw,
2009). Our current study further confirms the existence of this isoform specificity.
Several hypothesis has been raised to answer the question how AKT isoforms
achieve this specificity and this question remains unsolved till today. One
possibility is the differential expression of AKT isoforms in specific cell type. In our
system, we detected either AKT1 or AKT2 protein in islets from WT mice (Figure
9). However, our RNA sequencing data on mice islets suggest mRNA levels of
AKT1 is about 2-4 fold to AKT2 mRNA (data not shown). Thus, assuming all AKT1
and AKT2 mRNAs get translated, it can be estimated that more AKT protein would
be deficient when we manipulated AKT1 in our system. And this expressional
difference could serve as a potential reason to explain why the deletion of AKT1
cause more dramatic phenotypic change in β cell proliferation.
In this thesis, data shows signaling pathway involving PI3K/AKT,
particularly AKT1, could be important to maintain the adaptive growth of pancreatic
β cells. This work unveils the surface of a complicated molecular signaling network
contributing to the process of β cell growth. More work focusing on studying the
components within this signaling network would benefit the development of
therapies against both Type I and Type II diabetes in the future.
72
Figure 9. 1. AKT2 does not compensate for AKT1 loss in A1KO mice.
Islets were extracted from WT mice or A1KO mice.
73
Tamoxifen (50mg/kgX5) Corn Oil
Mip-Cre-ERT+, Akt1
lox/lox
, YFP
lox/stop
Figure 10. 1 95% of cells are deficient of AKT1 in Mip-Cre-ERT+, Akt1
lox/lox
,
YFP
lox/stop
, mice
Mip-Cre-ERT+, Akt1
lox/lox
, YFP
lox/stop
mice were injected either tamoxifen or corn oil for 5 times
at dosage of 50mg/kg. Mice pancreas was collected and embed in paraffin at the end of
experiment. IHC staining (Red: Insulin, Green:YFP, Blue:DAPI performed on the pancreas slide
later shows above 90% β cells was labeled with YFP, indicating above 90% of β cells was AKT1
protein deficient.
74
CHAPTER IX. MATERIAL AND METHODS
Animals
Several mice lines were used for the experiments in this paper. Akt1 -/-(A1KO) and
Akt2 -/- (A2KO) were used as experimental mice and correspondingly, Akt1 +/+
and Akt2 +/+ mice were used as controls (Con). Akt1
loxP/loxP
, Mip-Cre-ERT + mice
with tamoxifen treatments ( A1KO) (250mg/kg, every the other day for 5 shots)
and Akt2
loxP/loxP
, Rip-Cre-ER +, (A2KO) mice were compared with Akt1
loxP/loxP
,
Mip-Cre-ER – (Con) mice and Akt2
loxP/loxP
, Rip-Cre – mice (Con) respectively. Akt1
germline knockout mice (A1KO), A1KO mice were with C57BL/6 background.
Pten
loxP/loxP
, Rip-Cre + mice were originally with C57BL/6 background, which later
outbred with BALB/c mice because of breeding difficulties. All mice were housed
in a temperature-, humidity- and light-controlled room (12 h light/dark cycle), and
were allowed 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.
BrdU, Tamoxifen injection, STZ injection and High Fat Diet feeding
BrDU (1mg/ml concentration) was given to mice in water for 5 days before ending
the study. Mice were euthanized after the 5 days and organs were collected as per
the experiment. Tamoxifen (Sigma-Aldrich, St Louis, MO, USA) was prepared in
corn oil at a concentration of 20 mg/ml. The stock solution was filtered by 0.22 um
75
filter and kept in -20 ℃ before injection. Akt1
loxP/loxP
, Mip-Cre-ERT + mice were
given subcutaneous injections of tamoxifen (a dose of 250mg/kg was given every
2 days for 5 doses). Same dose and injections were given to Akt1
loxP/loxP
(Akt1
loxp/+
),
Mip-Cre-ER – as control. The effectiveness of the injection was assessed by
injecting YFP
loxP/loxP
, Mip-Cre-ERT + with same dosing strategy which shows
nearly 95% efficiency in deletion. Streptozotocin (STZ, Sigma) injection is
conducted daily for 5 days at the dosage of 50mg/kg body weight. The STZ powder
is diluted in 0.1 N sodium citrate buffer right before the injection. The high fat diet
were purchased from PicoLab 5053. 60.3% of calories in this diet is provided from
fat content while 18.4% and 21.3% of calories is from protein and carbohydrates
respectively.
Kcal% Normal Chow (NC) High Fat Diet (HFD)
CHO 62 21
Fat 13 60
Protein 24.5 19
Plasma assays
Glucose levels were determined by measuring tail vein blood sample with the
Abbott Freestyle glucose monitoring system. Fasting glucose was determined from
overnight (16 hours) fasted mice. Random glucose was determined while animal
have free access to food. For the glucose tolerance test, glucose (2 mg/kg body
weight) was injected intraperitoneally after 16-hour fasting. For the insulin
tolerance test, insulin (0.5 U/ml body weight) was injected intraperitoneally after 5
hour fasting. The plasma glucose level was evaluated at indicated time points after
the injection.
76
Insulin/Proinsulin Concentration
Blood samples were obtained through orbital eye bleeding during glucose
tolerance test and cardiac puncturing when the mice were sacrificed. Plasma was
separated from the blood samples by centrifuging at 2,200rpm for 20 mins. Mouse
insulin ELISA kit and mouse proinsulin ELISA kit (Alpco, Salem, NH, USA) was
used to determine the concentration accordingly.
Relative islet area determination
Pancreatic tissue was fixed overnight in Zn-Formalin (10%) solution containing 1%
Zn sulfate in 4 ℃, embedded in paraffin and sectioned into 4 µ m slices. H&E
staining was performed for morphological analysis. Islet and pancreas area was
measured using the Axiovision 4.5 software (Zeiss, Thornwood, NY, United States)
and ImageJ. Islet and pancreas areas were measured from three sections per
mouse, 240 µ m apart, for quantitative analysis. The islet to-pancreas ratio was
calculated and graphed.
Determination of cell proliferation and cell death.
Cell proliferation was evaluated with either BrdU or Ki67 immunohistochemical
staining. Specifically, BrdU (1 mg/ml; Sigma-Aldrich) was administered in the
drinking water for five consecutive days before the mice were sacrificed. Paraffin
embedded tissue sections were co-stained with insulin to visualize cells. Cell
77
proliferation was then evaluated with immunohistochemical analysis using anti-
BrdU antibody or anti-Ki67 antibody and reported as the percentage of BrdU or
Ki67 positive cells among total cells in each slide. For quantitative analysis, at
least three sections with 240 µ m apart from each mouse was used for quantitative
analysis. A minimum of 3,000 cells were counted for each mouse. All insulin
positive cells from the slides were counted. The mitotic index was determined
using the percentage of BrdU and insulin positive cells (or, Ki67 and Insulin positive)
vs total insulin positive cells. Β cell apoptosis was evaluated by using TUNEL
assay kit on paraffin embedded tissue sample (Roche, Indianapolis, IN, USA). At
least 5,000 insulin positive cells from tissue sections 240 µ m apart were counted
for cell death assay. The cell apoptosis ratio was determined using the percentage
of TUNEL/insulin positive cells vs total insulin positive cells.
Immunohistochemistry
Zn-formalin fixed and paraffin embedded sections were stained as reported (Stiles
et al. 2006)
The following antibodies were used: insulin (Abcam, Carlsbad, CA, USA);
BrdU(1:100) (BD Pharmingen, San Jose, CA, USA), Cyclin D1 (Santa Cruz, sc-
8396), Cyclin D2 (Santa Cruz, sc-593), Ki-67 monoclonal Ab (CST#12202), p27
(Santa Cruz, sc-1641),
78
Mouse islet isolation, protein extraction and RNA extraction
The pancreas was perfused with collagenase P solution (0.8 mg/ml in HBSS buffer
with 1mM HEPES and 1mM MgCl2 ) and digested at 37 ° C for 17 mins. Islets were
then washed with HBSS buffer containing 1mM HEPES, 1mM CaCl2, and 1mM
MgCl2 three times. The achieved tissue samples were further purified using Ficoll
gradients with densities of 1.108, 1.096, 1.069 and 1.037 (Cellgro) (Stiles et al.
2006). At last, islets were handpicked from the dishes either for protein extraction
or RNA extraction. Protein extraction was done by adding protease-inhibitor
containing cell lysis buffer to islets followed by centrifuging. RNA extraction was
done by using RNeast mini kit (QIAGEN).
RNA sequencing
cDNA library preparation and sequencing was done by University of Southern
California molecular Genomics’ core. We prepared 12 mice islet RNA (3 for each
group) for sequencing. DNA libraries are pooled and sequenced on an Illumina
HiSeq2500 with a single 75 bp read, 25 million reads per sample. FASTQ files
were generated by CASAVA (v1.8.2) and further analyzed by Patrek Flow software.
Reads were mapped to the mouse transcriptome (RefGene v1.1.17) using
TOPHAT2 allowing two mismatches and a maximum of 20 multiple hits. The gene
expression values ( Quratile) were used for analysis.
79
Western immunoblotting
Cell or islet tissue lysates with equal amounts of protein were subjected by SDS-
PAGE, followed by transferring to polyvinylidene fluoride membrane for
immunoblotting. The membranes were probed with specific antibodies against: p-
PERK(Thr980)(Cell Signaling, #3179), total PERK(Cell Signaling, #3192), p-
Eif2a(Ser41) (Cell Signaling, #9721), total Eif2a(Cell Signaling, #9722), Grp78
(Santa Cuz,sc-13968), p27 (Santa Cruz, sc-1641), Actin (Sigma),GAPDH(Cell
Signaling) . All antibodies were 1 to 1000 diluted with 1%BSA (in PBST).
PI/ANNEXIN V FACS
INS-1 cells were seeded at density (2.5x105 cells/well) in six-well plates in RPMI
media. After 48 hours of seeding, cells were treated with 1% BSA or 0.4mM
Palmitic Acid for 48 hours. Media containing detached and floating cells was
collected and the rest of the cells were trypsinized, and washed once with PBS.
Cells were then washed and suspended in 1X Annexin Binding Buffer (ABB).
Diluted Annexin (400ng per 1x106 cells) was then added to cells and incubated for
8 minutes at room temperature away from light. Propidium Iodide was then added
to each sample (2.5ug/ml per sample) and incubated for approximately 2 minutes.
FACS analysis was then performed.
80
Statistics
Student’s t tests were performed to compare the differences between control and
experimental 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-hoc test for comparing the differences between groups on quantified
data.
81
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Abstract (if available)
Abstract
Insulin producing pancreatic β cells are critical for balancing mammalian metabolism. β cells are lost or insufficient in Type I diabetes due to autoimmune attack. Similarly, functional impairment and loss of β cells in Type II diabetic patients promote disease stage to the point when patients must rely on exogenous insulin. β cell mass is controlled by β cell replication, differentiation from the progenitors, β cell dedifferentiation, transformation from other cells, and cell death. ❧ Replication of β cell is vigorous in neonatal period and in the early stage of Type II diabetic patients when β cells increase population to compensate for metabolic needs. Researchers have been dedicating to find a good way to regenerate β cells in order to fight against Diabetes. However, the mechanisms of β cell regeneration remains unclear which impedes the progressing of using β cells as therapy for Diabetes. ❧ β cells are a group of cells sensitive to nutrients such as glucose and fatty acids. β cells also respond to hormones and growth factors. How glucose and fatty acids act on β cells and whether they utilize signaling pathways activated by growth hormones have not been fully understood. Particularly, how PI3K/AKT pathway which is responsible for insulin, insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) involved in nutrient-stimulated β cells growth has not been well studied. ❧ Our lab has previously found Rip-Cre+, Ptenˡᵒˣᴾ/ˡᵒˣᴾ mice which are deficient of PTEN in β cells have increased islet mass and β cell replication through modulating cyclinD and CDK inhibitors. Akt proteins, a serine/threonine kinase, is encoded by three different genes, Akt1, Akt2, and Akt3. Thus, it has three isoforms which may function with their own specificity. To further investigate how AKT and activated PI3K/AKT pathway upon PTEN loss regulate β cell regeneration, I studied the metabolic phenotype and islet phenotype of four different mice strains: Akt1-/-(A1KO)
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Peng, Zhechu
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Protein kinase Bα (AKT1) but not protein kinase Bβ (AKT2) controls pancreatic β cell growth and survival
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Molecular Pharmacology and Toxicology
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AKT1 and AKT2,beta cells,cell proliferation,Diabetes,ER stress,high fat diet,OAI-PMH Harvest,Pancreas,PI3K/AKT signaling,unfolded protein response (UPR)
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AKT1 and AKT2
beta cells
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ER stress
high fat diet
PI3K/AKT signaling
unfolded protein response (UPR)