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Development of ECM for the preservation of adult mouse pancreatic islet function in vitro
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Development of ECM for the preservation of adult mouse pancreatic islet function in vitro
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i
Development of ECM for the preservation of Adult Mouse
Pancreatic Islet Function in vitro
by
Yating Guo
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
December 2015
Copyright
2015
Yating
Guo
ii
Dedication
To
my
grandfather
iii
ACKNOWLEDGMENTS
My deepest thanks to my advisor, Dr. Bangyan L. Stiles, who offered me the precious
opportunity to do the research in her lab and introduce this great project to me. Being a
major transferred student, without her guidance and intellectual knowledge, I could not
have easily completed the transition and this study. Her critical attitude and enthusiasm
for scientific research especially enlighten me on my bioscientific study, and she gave me
the instructive advice, which helped me perform the laboratory tasks that were required
for this work.
I am grateful to my labmates, Zhechu Peng, for her histopathology and cytology
assistance guiding me in studying the H&E and Immumohistochemistry slides and many
other professional techniques and encouraging me to do research. I am grateful to Dr.
Indra Mahajan, who shared numerous valuable ideas and experience with me and whose
professional attitude inspire me all the time. I want to thank all the members in
Dr.Stiles’s lab, Dr. Ni Zeng , Dr. Lina,He , Dr. Chengyou Jia, Dr. Yang Li , Anketse
Kassa, Richa Aggarwal, Joshua Chen, Jingyu Chen, for being supportive and sharing all
the joy and tears with each single day in lab. Others that deserve acknowledgement are
the members of TR Lab of School of Pharmacy Core Facility for allowing me to use the
XF24 extracellular flux analyzer and quantitative RT-PCR for research. I really want to
express my thanks to Dr. Zoltan Tokes, who is the chair of my committee. He accepts me
as a member of Department of BIOC and always gives us encouragement helping us to
pursue academic achievement.
iv
Last but not least, I want to thank my parents and my friends for all their support and
love, without them I would not be able to do anything. My grandfather, help me to keep
an unshakeable faith in pursuing my academic achievements. Without his unconditional
love and blessing, I would not have made it this far.
v
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGMENTS
LIST OF FIGURES
ABBREVIATIONS
ABSTRACT ix
CHAPTER 1: INTRODUCTION 1
1.1 Diabetes and glucose homeostasis
1.2 Islet transplantation and preservation islet function in vitro
1.3 Monolayer culture of adult mouse pancreatic islets on ECM 5
1.4 Role of mitochondria in β-‐cells function and dysfunction 7
1.5 Rationale of study 9
CHAPTER 2: MATERIALS AND METHODS 11
2.1 Animals 11
2.2 Preparation of ECM- coated plates 11
2.3 Islet isolation 12
2.4 Islet culture 13
2.5 Adhersion assay 13
2.6 Glucose-stimulated insulin secretion(GSIS) 14
iii
vii
viii
1
3
vi
2.7 Western blot 14
2.8 RNA isolation and quantified RT-PCR 15
2.9 Immunohistochemistry 15
2.10 Seahorse XF24 metabolic flux analysis 16
CHAPTER 3: RESULTS 18
3.1 Morphological studies of isolated islets 18
3.2 Islet adhesion assay 20
3.3 Glucose stimulated insulin secretion 21
3.4 Gene expression in modifed islet culture 24
3.5 Using BCE coated dishes to study the mitochondrial function of β-cells 26
3.6 Monolayer formation of pancreatic islet on BCE matrix 32
CHAPTER 4 36
Discussion
36
REFERENCE 39
vii
LIST OF FIGURES
Figure 1. Recruitment of β- cells via adjacent cellular proximity 4
Figure 2. The glucose-stimulated insulin secretion 8
Figure 3.XF cell mitochondrial stress test profile and modulation of the ETC 17
Figure 4.
The
bovine corneal endothelia cells coated dishes 18
Figure 5. Contrast
of
purified
islet
within
all
five
groups
Figure 6. Percentage adhesion of islets to each coated surfaces
Figure 7. Insulin content of Pancreatic islets at different time point
Figure 8. Glucose stimulated insulin secretion test of Pancreatic islets
Figure 9 Gene expressions of islets with ECM supporting
Figure 10. PTEN antagonize PI3K activity
Figure 11. PTEN deletion in β-cell maintain the islet function
Figure 12. Increased mitochondrial respiration rate in β-cell-specific 31
PTEN deletion islets
Figure 13. Primary culture of pancreatic islets on BCE-coated plates 33
Figure 14. The monolayer formation of islets on BCE-coated
plates (phase contrast)
19
20
22
23
25
27
29
34
viii
ABBREVIATIONS
ECM : extracellular matrix
BCE : bovine corneal endothelia
FN : fibronectin
BSA: bovine serum albumin
PTEN : phosphatase and tensin homologue deleted on chromosome 10
IE : Islet Equivalent
GSIS : glucose stimulated insulin secretion
H&E : Hematoxylin and eosin
ROS : reactive oxygen species
ETC : mitochondrial electron transport chain
OCR : oxygen consumption rate
ECAR: extracellular acidification
FCCP :p-trifluoromethoxy carbonyl cyanide phenyl hydrazine
BrdU: 5-bromo-2-deoxyuridine
GCK : glucokinase
SUR1: sulfonylurea receptor
GLUT2 : glucose reansproter2
PDX1 : pancreatic and duodenal homeobox
ix
ABSTRACT
Pancreatic islet isolation exposes the islet to a variety of cellular stresses and
disrupts the cell-matrix interaction, which eventually leads to islet apoptosis in vitro. In
this study, various extracellular matrix (ECM) cultures of fragmented pancreatic islets
containing β-cells were studied. Efforts have been made to reestablish the cell growth
environment in order to establish conditions that maintain the islet function and prolong
the survival of in vitro culture. The substrates used in this study to facilitate islet
attachment were collagen I, bovine corneal endothelia(BCE) cells, fibronectin(FN),
poly-L-lysine and bovine serum albumin (BSA) respectively. During the investigation,
the monolayer initiation of adult mouse islets was achieved using BCE substrate.
Pancreatic β-cells maintain glucose homeostasis via sensing glucose and other nutrients to
regulate insulin exocytosis. BCE substrate is found to better preserve the insulin secretion
function of islets in response to glucose. In addition, expression analysis shows that
expression of β-cell genes are maintained. Finally, the process of insulin secretion is
coupled with mitochondria respiration. We showed that BCE substrate can be used to
support islet culture used for analysis of oxygen consumption though with significantly
reduced respiratory function.
1
CHAPTER 1
INTRODUCTION
1.1 Diabetes and glucose homeostasis
Diabetes mellitus (DM) is a metabolic disorder in which the patients have high
blood glucose level. Diabetes is caused by either the body being unable to produce
sufficient insulin or when they are unable to respond to the normal activities of
insulin(Chiu, et al. 2007). Insulin, the peptide hormone, secreted by the islets of
Langerhans containing β-cells, plays an irreplaceable role in diabetes mellitus. A
persistently high glucose level can cause both acute and long-term complications,
including hyperosmolar coma, cardiovascular disease, kidney failure, stroke and eye
diseases(Kitabchi, et al. 2009).
There are several classes of diabetes, type 1, type 2 and gestational(Mayfield 1998).
Gestational diabetes, as the name suggests, happens during pregnancy, and the mother
can recover from it after the baby is delivered. Thus, type 1 diabetes is considered an
autoimmune disease (where autoantibodies target β-cells) resulting in β-cell loss and
consequently, leading to insulin deficiency. The cause of type 1 diabetes is still
unidentified, however, it can be diagnosed via detection of those autoantibodies
present in the patient’s blood. Type 2 diabetes, in contrast to type 1, is characterized
by two consecutive phases where insulin resistance and insulin deficiency culminate in
severe debilitation of the patients(Vijan 2010). During the insulin resistance phase,
2
although β-cells are still capable of producing insulin, the cells fail to respond
physiologically to the hormone, resulting in lack of glucose uptake from the blood into
the cells, and thus causing the typical glucose accumulation in the blood.
Diabetes is the seventh leading cause of death in the United States. Both lifestyle
and genetic factors contribute to the development of diabetes. Many genes related to
diabetes are associated with the functions of pancreatic β-cells. Besides these 3 major
types of diabetes, there are other subtypes, which are not fully understood and still
under intense investigation. A major concern to public health is diabetes mellitus type
2 due to its association with obesity, which constitutes around 90% of cases of
diabetes.
In summary, all types of DM are marked by reduction in insulin content and/or
by a failure of β-cells to respond properly to glucose. Insulin is critical for glucose
uptake and maintenance of normal blood glucose levels, and prevention of the toxic
effects of hyperglycemia. β-cells are the irreplaceable source of insulin. Thus, a better
understanding of β-cell biology is a major focus of intense investigation in order to
achieve more diabetes treatment alternatives or options. One of the ways to understand
β-cell biology is to study them in in vitro culture. This type of culture not only shed
light on better understanding of β-cell survival but also may be a way to enrich islets
for transplant purpose to treat patients.
3
1.2 Islet transplantation and preservation islet function in vitro
Type 1 diabetes, is an immune-mediated or idiopathic disease leading to the loss of
the insulin-producing β-cells. Islet transplantation, isolated islets from a donor
pancreas into other person, has been regarded as a primary means of restoring
physiological β-cells function in patients with type 1 diabetes (D'Addio, et al. 2014;
Meloche 2007).
Transplanted islets respond to physiological glucose changes to maintain the
glucose homeostasis so that patients will not need to rely on insulin injection.
However, to meet the amount of islets needed for transplant for an adult human (70kg),
one million islets must be isolated from two donor pancreases. Thus, one of the current
limitations is the supply of islets for transplantation. To solve this urgent organ
insufficiency problem, developing ways to “grow” islets and maintain the glucose
regulated insulin release in vitro is necessary. Maintaining the quality of the isolated
islets via in vitro culture is critical to prolonging functionality for transplant
(Narushima, et al. 2005). My goal is to establish protocols for long-term adult mouse
pancreatic islet culture.
The Islet of Langerhans is the region of the pancreas containing the endocrine
cells (Elayat, et al. 1995; Weir, et al. 1984), which secretes insulin, glucagon,
somatostatin and pancreatic polypeptide (Fig.1). Single islet cells can modulate their
actions via autocrine effects, or paracrine effects. Some findings indicate that the
4
interactions of single cells and changes in their relative proportion can influence the
insulin secretion.
Figure 1. Recruitment of β-cells via adjacent cellular proximity. The cellular
communication partly contributes to the regulation of insulin release by pancreatic β-
cells(Dai, et al. 2012). A single β-cell activated by glucose can lead to the recruitment
of adjacent β-cells to an active state where they can promote the glucose utilization.
Loss of vasculature and eventual hypoxia can limit the quantities of oxygen and
nutrients intake from the environment to the islets (Marchetti, et al. 2006; Mikos, et al.
1994) leading to apoptosis, especially occurring with β-cells (Paraskevas, et al. 2000).
Pervious study shows that the isolation caused loss of per-insular basement membrane
giving rise to pyknic nuclei, increased pro-apoptotic p38 and JNK (C-Jun N-terminal
kinase) activity, reduced pro-survival ERK1/2 (extracellular signal-regulated kinase)
activity and eventually lead to the decreased expression level of integrin accompanied
5
by reducing phenotypic characteristics contributing to apoptosis in vitro(Rosenberg, et
al. 1999). Using Extracellular matrix (ECM) for the fragmented islet culture has
attracted attention as a mean of prolonging the survival of islets. A variety of studies
have been performed to assess their effect of cell-cell, cell-matrix interaction on
β-cells adhesion, motility, and insulin secretion. In addition, the study of the
relationship between the cell and ECM also obtained certain positive result including
the correlation between the expression of integrins and insulin secretion(Cirulli, et al.
2000; Ris, et al. 2002). Among these investigation, collagen and fibrin gels show the
capability to increases the survival in cultured islets (Beattie, et al. 2002). IGF-2, an
effective component in the duct-conditioned medium also prolong the survival of
isolated pancreatic islet suspended in collagen I gels (Ilieva, et al. 1999). Some other
studies to preserve β-cell function include growth factor enriched matrix to induce
proliferation of human β-cells after transplantation (Beattie, et al. 1996).
1.3 Monolayer culture of adult mouse pancreatic islets on ECM
Monolayer cultures, suggested to be superior, can overcome the weakness of
culturing islets where the environment for each single cell varies depending upon its
position in the islet mass. Thus, monolayer culture may allow better assessment of
β-cell functionality and morphology(Kostianovsky, et al. 1974). To acquire the
6
monolayer cells, many efforts have been made to culture β-cells from fetal or neonatal
pancreas(Hopcroft, et al. 1985; Meda, et al. 1980). The initiation of monolayers of
adult islet is the most challenging task, since it demands complicated manipulations,
including providing the supporting surfaces, dispersing the islet mass into the single
cell, and the addition of 3-isobutyl-l-methylxan-thine(Ohgawara, et al. 1978). In
addition, mature β-cells behave differently from either fetal or newborn pancreatic
β-cells and little is known about how adult islet monolayer responds to modulators of
hormone secretion(Freinkel, et al. 1984; Grill, et al. 1981). The over-growth of the
fibroblasts is also regarded as a serious contamination problem(Braaten, et al. 1974).
The different substrate materials used to support monolayer cultures are BCE cells,
collagen I, FN, and poly-L-lysine. Poly-L-lysine is a homoploypeptide that is
commonly used to coat tissue culture. The mechanism of enhancing the cell
attachment is based on the interaction of the positively charged polymer and
negatively charged cells(Mazia, et al. 1975). Originating from the cell basement
membrane is fibronectin (FN) or laminin. FN, as a substrate adhesion molecule, plays
a critical role in cell adhesion, growth and differentiation(Pankov and Yamada 2002).
Also, FN has been shown to be related to cellular morphology, cytoskeletal
assembling and hemostasis. Collagens are ECM proteins with high tensile strength.
Collagen gels are the common substrate that has shown the ability to establish the
physiological environment for cell culture(Montesano, et al. 1983), It may be effective
7
to investigate the effects of ECM originated from rat-tail type I collagen. It has been
reported that ECM from bovine corneal endothelial cells demonstrates notable
enhancement in cell attachment, proliferation and differentiation(Gospodarowicz and
Ill 1980). Therefore, these ECM, regarded as a potential tools, are used to develop the
protocols to help the long-term preservation of adult mouse pancreatic islet function in
vitro.
1.4 Role of mitochondria in β-cells function
Mitochondria, which generate energy for the cells, are involved in cellular
differentiation, signaling and cell death. The process of pancreatic β-cell regulated
insulin secretion demands the mitochondria to fulfill the metabolism-secretion
coupling. Mitochondrial metabolism is critical to β-cells functionality by coupling
with glycolysis to produce ATP (Fig.2).
8
Figure 2. The glucose stimulated insulin secretion. The increased levels of glucose
in blood lead to the oxidative metabolism of glucose. The pancreatic β-cells promote
the glucose up taking via glucose transporter, GLUT2. Consequently, it leads to
increased ATP production and ATP /ADP ratio. The latter results in the closing of the
potassium channel and depolarization of the plasma membrane. The process results in
calcium channels opening and the raising of calcium concentration, which leads to
insulin outside of the β-cell.
Dysfunctional mitochondrial can cause a series of disorders and has been
associated with numerous human diseases, cancer, degenerative disease (Parkinson’s
disease, Alzheimer’s disease), and diabetes. These dysfunctions involve alteration of
mitochondrial respiratory complexes expression level, mutations of mitochondrial
DNA, abnormal reactive oxygen species (ROS) production and abnormal
mitochondrial mass. ROS is suggested to stimulate the oncogenes and facilitate the
9
tumor progression and metastasis (Zager, et al. 2006). It has been investigated that
these reactive oxygen species may be the metabolic coupling factor for glucose
stimulated insulin secretion. In this way, changing of ROS may lead to physiological
control of these insulin-secreting cells. Ungoverned induction of oxidants and decline
of their detoxification ability can result in free radical-mediated chain reactions and
activating pathogenic events (Li, et al. 2008; Pi, et al. 2007). Mitochondrial defects
maybe related to β-cells failure that the final cause of Diabetes mellitus.
1.5 Rationale of the study
The primary aim for this research is to acquire long-term monolayer cultures of adult
mouse islets for prolonging the post-isolation islet survival and preservation of β-cells
functionality in vitro. To do this, I will compare the ability of various ECM : BCE cells,
collagen I, FN, poly-L-lysine and BSA (as control) to support adhesion, viability, and
functionality of the cultured islets. As a readout of β-cell function, I performed gene
analysis and glucose stimulated insulin secretion (GSIS) testing. In addition, the insulin
secretion process is tightly coupled with glucose metabolism and ATP production. The
increased ATP/ADP ratio due to glucose uptake is critical for the closing of the
potassium channel and depolarization of the cell membrane, processes required for
insulin secretion. I will therefore evaluate the glycolysis and mitochondrial function of
10
the cultured cells using the BCE cells coated dishes. My results indicate that BCE coated
matrix has exhibited the ability to boost the attachment and differentiation of sorts of
cells and survival(Gospodarowicz and Tauber 1980).
11
CHAPTER 2
MATERIALS AND METHODS
2.1 Animals –We used wild type mouse with the C57 background. Targeted deletion of
Pten in β-cells was obtained by crossing Pten
loxP/loxP
mice (C57/129/J background)
(Lesche et, al, 2002) with mice expressing Cre recombinase under the rat insulin
promoter (RIP-Cre, C57 background) (Postic, 2001). F1 generation was composed of
heterozygous animals that were backcrossed with Pten
loxP/loxP
mice to produce F2
generation Pten
loxP/loxP,
Cre+ animals which were used in the experiments. Animals were
genotyped via tail DNA extraction followed by standard genomic PCR techniques.
Animals were housed in a temperature-, humidity-, and light-controlled room (12-h
light/dark cycle), allowing free access to food and water. All experiments were conducted
strictly under the Institutional Animal Care and Use Committee of the University of
Southern California research guidelines.
2.2 Preparation of ECM- coated plates – Bovine corneal endothelia cells were cultured
on 60-mm Falcon plates (Falcon) with an initial density of 10
6
Cells, and were plated at a
1:6 split ratio in Dulbecco’s modified Eagle’s medium containing 10% (vol/vol) fetal
bovine serum (FBS), and 1% (vol/vol) Penicillin-Streptomycin. After the cultures became
confluent (around 7-12 days), the BCE cells were removed by 0.5% Triton X-100 in
sterilized Water and then washed with phosphate-buffered saline (PBS) 5 times with
12
1.5ml each per well. The BCE cell coated plates were stored filled with PBS at 4°C,
ready for use. Collagen I gels (Gibco) were acquired from rat tail. Collagen I was diluted
to 50 µg/ml in 20mM acetic acid. The collagen solution was dispensed into the dishes at
5 µg/cm
2
. Coated dishes were incubated at 37 °C in humidified incubator for 40 minutes
and then were raised with sterile PBS and RPMI-1640 medium several times. Fibronectin
(Sigma) was diluted to the coating concentration of 5 µg/cm
2
using the media without
Ca
2+
and Mg
2+
(avoiding the formation of hydroxide precipitate). The coated dishes were
incubated at room temperature for 1 hour and then the remaining solution were removed.
The dishes were rinsed with PBS and media several times. The dishes were dispensed
with the poly-L-lysine (Sigma) concentration of 1.0 ml/25 cm
2
. The solution was
removed via aspiration after 10 minutes. The dishes were rinsed with PBS and media and
allowed to dry before seeding cells.
2.3 Islet Isolation – Pancreases were perfused by HEPES-buffered Hanks’ Balanced
Salt Solution (HBSS), pH 7.4, 0.8mg/ml collagenase P (Roche) injection via the
pancreatic duct. The tissue was digested by interval shaking for 15-20 min in a 37 °C
water bath (Carter, et al. 2009). Then tissue fragments were washed with HBSS and
purified by Ficoll gradient centrifugation. Ficoll layers had densities of 1.108, 1.096,
1.069 and 1.037 g/ml (Cellgro). The islets were individually pipetted out under the
microscope and further purified with RPMI-1640 medium.
13
2.4 Islet culture and Islet Equivalent (IE) - For the design of this experiment, the total
number of islets was divided into four groups. All cells we incubated at 37 °C,
humidified environment of 5% CO2, 95% air and RPMI-1640 medium was changed on
alternative days.
Table
1. Islet equivalent
(IE)
conversion*
*Determination
of
islet
volume
for
each
50µ diameter range and relative conversion
into equivalent number of islets with a diameter of 150 µ.
The islet equivalent (IE) is the measurement applied to estimate the yield of islet
isolated, since IE can normalize the amount of islets between preparations for functional
assays like GSIS(Ricordi 1991). In these studies, the islets were picked with the diameter
range from 50 micron to 350 micron. Converting all the isolated islet number into IE
(Table 1).
2.5 Adhesion assay – This assay was performed to illustrate the cell-matrix interactions
(Weinel, et al. 1992). The protocol was modified to test the effect of islets bound to each
14
ECM coated dish. The groups were divided into BCE, Collagen I, FN and BSA control,
Each group was harvested after 48h. At the end of the incubation period, unattached islets
were washed off by PBS. The attached islets were stained with dithizon and each group
of islets was counted under a light microscope to count the amount in the four groups.
2.6 Glucose-stimulated insulin secretion (GSIS) – To obtain the baseline of insulin
secretion, the RPMI-1640 medium was replaced by Krebs-Ringer bicarbonate buffer
(KRB buffer), pH 7.4, containing 10 mM HEPES, 0.25% BSA, a basal glucose
concentration (3.3 mM) and pre-incubated for 1hour at 37
0
C. Then cells had the
Kerbs-Ringer Buffer (KRB) containing 16.7 mM glucose and incubated for an hour. The
media was collected for insulin detection by ELISA. The amount of insulin released in
the test was measured with the enzyme –linked immunosorbent ELISA kit (ALPCO ).
The islets were lysed and their protein concentration was measured in order to normalize
the values for each sample.
2.7 Western blot -The pancreatic islets were lysed in cell lysis buffer (Rountree, et al.
2009) over ice. Supernatants of the lysates were normalized by protein concentration and
subjected to SDS-PAGE and then transferred to PVDF membranes for immunoblotting.
15
The primary antibodies used were as follows: PTEN (Cell Signaling Tech., #9552),
GLUT2 (Abcam), GCK ,GAPDH (Santa Cruz); ECL mouse IgG, HRP-linked whole
antibody (GE healthcare Life Sciences, NA931).
2.8 RNA isolation and quantitative RT-PCR – RNA isolation was performed using
TRIzol (Invitrogen, CA) under the protocol guideline. cDNA was reverse transcribed
using the Omniscript RT kit (Qiagen, Mississauga, ON). To evaluate the expression
levels of the specific genes, primer for insulin, glucagon,somatostain, pancreatic and
duodenal homeobox 1 (PDX1 ), glucokinase (GCK), sulfonlurea receptor 1 (SUR1),
glucose transporter 2 (GLUT2), and glyceraldehye-3-phosphate dehydrogenase (GAPDH)
were used for QPCR using SYBR green chemistry. All expression levels of these genes
were normalized to GAPDH.
2.9 Immunohistochemistry - Zinc-formalin fixed overnight and paraffin embedded
sections were stained as previously reported (Stiles, et al. 2006). Hematoxylin and Eosin
(H&E ) staining was used for islet morphological study. The antibodies used were: PTEN
16
(Cell Signaling Tech. #9559), insulin (Invitrogen), and Brdu (BDPharmingen).
Antibodies for other pancreatic hormones were provided by Zymed.
2.10 Seahorse XF-24 metabolic flux analysis XF stress test includes series of assays
using XF Analyzer (Seahorse Bioscience) to measure mitochondrial respiration,
glycolysis and fatty acid oxidation in cells. In my studies, both XF cell mitochondrial
stress test and XF glycolysis stress test were used to detect the mitochondrial biogenesis
of Pten-null and wild type islets.
Mitochondrial (ATP production) and glycolysis function are measured as oxygen
consumption rate (OCR) and extracellular acidification rate (ECAR), respectively using a
Seahorse XF-24 metabolic flux analyzer. As depicted in Figure 3, the basal respiration is
the parameter to depict the oxygen consumption required for cellular ATP production.
The injection of the oligomycin inhibits ATP synthase and leads to the decline of OCR.
Thus the difference in OCR before and after addition of oligomycin indicates the part of
basal respiration that was used to produce the ATP. H
+
(Proton) leak, which does not
produce ATP accounts for the basal respiration being uncouple from ATP production..
The maximal respiration is stimulated by addition of FCCP to the cell. FCCP uncouples
the proton gradient and ATP production. Therefore, the respiration does not rely on ATP
production. In this case, the OCR measured after FCCP addition indicates the maximum
17
capacity the cells have to respire. The non-mitochondrial respiration results from the
cellular enzymes that still consume oxygen after injection of oligomycin, which destroys
the mitochondria.
Figure 3. XF cell mitochondrial stress test profile and modulation of the ETC. A) the
injections of Oligomycin, FCCP, Rotenone & antimycin A aim to measure the basal
respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity
and non-mitochondrial respiration illustrated via labeling. B) These compounds targets
the respective complexes of the electron transport chain and eventually modulate the ATP
formation.
A
B
18
CHAPTER 3
RESULTS
3.1 Morphological studies of isolated islets
To develop a culture condition for islet β-cells, I tested several different conditions.
Group-I islets were seeded in BCE-coated dishes; Group–II islets were grown on the
dishes coated with collagen I from rat tail extract; Group- III islets were seeded in
Fibronectin (FN) coated dishes; Group-IV islets were placed in bovine serum albumin
(BSA, as control), and Group-V islets were seeded in poly-L-lysine coated dishes. The
islet diameter ranged from 50µ to 500µ. Yield was approximately 300 IE per mouse islet,
the purified isolated islets plated on the five groups of ECM-coated dishes gradually
started adherent during the first 12-24 h. Dithiazone staining was applied before
counting. BCE cells (Fig.4) were seed at the six-well plates. Cells were trypsinize and
removed after they reach 100% confluence, leaving behind the cell matrix.
Figure
4.
The
bovine corneal endothelia cell coated dishes(BCE). The BCE cells
were seeded on the six well plates. A: day 2, the BCE cells well spread.(x20) B: day 8,
the BCE progresses quickly and obtains a 100% confluence.
19
Figure 5. Contrast of purified islet within all five groups. A)the freshly
isolated islet B-F, islets 3 days after culturing on B)BCE coated dish, C) on
Collagen I gel, D) on FN, E) on Poly-L-lysine. F) on BSA dishes floated in the
medium and appeared to undergo cell death. 20x magnification.
The freshly isolated islet had a loose appearance with an irregular border
(Fig.5A). After 3 days of the primary culture, medium was changed in all groups,
the attached islet appeared to be smoother, flattened and spherical shape with trim
20
periphery in groups I-III (Fig.5 B-D). However, the poly-L-lysine coated culture groups
and the control group show less adherent ability, floated in the medium quickly
appeared to undergo apoptosis (Fig.5E-F). Among these groups, BCE and collagen I
groups showed less contamination and part of the contaminated of fibroblasts and part of
the contaminated cells eventually underwent spontaneous degradation.
3.2 Islet adhesion analysis
To address how well the ECM layers supported the attachment of islets, I performed
adhesion analysis to complement optical microscopy investigation, This was done for
groups I-IV ECM and not poly-L-lysine as poly-L-lysine was not able to keep the islets
intact. As shown in Figure 6, the most effective ECM protein to support adhesion was
group I, BCE cell. It led to maximize adhesion that up to 79 % of the islets got well
attached after 2 days of the initial day. Collagen I and fibronectin were also shown to
increase the capability of adhesion when compared with BSA control coated dishes with
approximately 50-60% adhesion. While the BSA group as control exhibited only 20%
islet adhesion.
21
Figure 6. Percentage adhesion of islets to each coated surfaces after 48h
(n=6, mean ± STD, *P<0.01 versus BSA control) in RPMI-1640 medium. BCE
cells modified surface leads to maximal adhesion. Collagen I and fibronectin
induced relatively equal level of adhesion.
3.3 Glucose stimulated insulin secretion
To compare the ability of BCE to support the function of the islets and b-cells, insulin
content of islets in each group’s islets was detected at the isolation day and the 1, 2, 4, 6
days after the isolation respectively. The total amount of insulin of BSA group dropped
more than 55% by day 6 when cultured on BSA coated dishes. In contrast, islet cultured
22
on BCE coated dishes showed increased insulin content reaching 121% (p< 0.05) of
original after 2 days of culturing. This number started to drop by day 6 but still maintain
approximately 90% of original, suggesting the BCE protein modified surface is better at
maintaining the function of the cultured islets. Collagen I and FN exhibited varied
effects on islet insulin content with declines of around 19% and 40% by day 6 of
culturing , respectively(Fig .7)
Figure 7. Insulin content of Pancreatic islets at different time points With each
time point from day 0 to day 6 , the four group’s islets exhibit different extended
drops. The BCE group shows an optimistic result that has a 121% increase by day 2
and only drop less than 10% compared to day 0. The BSA control group has drop of
more than 55% by day 6.
23
Figure 8. Glucose stimulated insulin secretion test of Pancreatic islets.
A) Insulin secretion from three groups of islet culture was stimulate with
basal (3mM) and stimulatory glucose(16.7mM) level. B) the change of
different concentration of glucose stimulated insulin release were
calculated (n-fold) of A) ( *p< 0.05)
As a previous study found in human islets, the monolayer formation of the islet
eventually loses their phenotype. It was critical to investigate the glucose stimulated
insulin secretion (Fig.8A). The result exhibited that islets cultured on BCE protein
24
modified surfaces responded better to glucose stimulation. Addition of 16.7mM glucose
to the BCE cultured islets led to approximately 2 fold increased insulin secretion from
an average basal value of 2.38 ± 0.55(ng insulin/ ml protein) to 4.44± 0.8 (ng insulin/ ml
protein). The Fibronectin group didn’t show a significant difference from the BSA
control group, which had no distinguished response to the lower and higher glucose
stimulation.
3.4 Gene expression in modified islet culture
To further characterize the effects of the ECMs on islet function, the expression of
specific genes were analyzed. The expression of insulin, glucagon, somatostatin and
pancreatic polypeptide genes is reported in Fig.13A. Similar to what was observed for
GSIS, islets cultured on BCE cells and collagen I express significantly higher levels of
insulin than those cultured on either BSA controls or fibronectin coated dishes. Insulin
expression in islets cultured on fibronectin is similar to that of BSA controls, suggesting
that the islet phenotype is lost quickly in this culture. Expression of other islet hormones
did not change significantly though culturing with BCE may moderately reduce gamma
and pp cells as indicated by the reduced expression of somatostatin and pp in islets
cultured on BCE.
25
A
Figure 9. Gene expressions of islets with ECM supporting. The islets were isolated
on each ECM substrates on the day 3. All the gene expression was relatively measured
BSA control group (*p < 0.05 ). GAPDH is used for loading control. The selective genes
were A) insulin, glucagon, somatostatin and pancreatic polypeptide, and B) GLUT2,
GCK, Pdx 1 and SUR1. BCE cells modified culture islet has the highest insulin,
glucagon, GLUT2 and glucagon gene expression. The collagen I came in second place
B
26
and also show a significant high level on these genes. SUR1 was down regulated in the
three protein modulated islet cultures.
We additionally evaluated the expression of glucokinase (GCK ), glucose
transporter 2 (GLUT2),pancreatic and duodenal homeobox (PDX1) and sulfonylurea
receptor 1 (SUR1) (Fig.9B). These genes are involved in the development of the
pancreas and the maturation of β- cells. Correlating with increased GSIS, expression of
GLUT2 is higher in islets cultured on BCE and Collagen. No significant changes were
observed for other genes. Pdx 1and SUR1 showed low levels genes expression in each
group.
Glycolysis and oxidative phosphorylation are main ways for cells to produce energy.
Islets can adapt either way to produce energy dynamic depending on their environment.
During glycolysis, glucose in the islet is converted to pyruvate, which is used by the
mitochondria during oxidative phosphorylation to produce ATP. The pyruvate can also
be transformed to lactate. The buildup of lactate leads to a flux of protons into the
extracellular medium, which increases the acidification of the environment.
3.5 Using BCE coated dishes to study the mitochondrial function of β-cells
To validate the usefulness of BCE, the best substrate in studies of islet functions, we
used two different groups of islets, Pten control and deleted islets. Phosphatase and
27
Tensin Homolog Deleted on Chromosome 10 (PTEN) is a lipid phosphatase involved in
insulin signaling (Stokoe 2001; Worby and Dixon 2014).
The activity of PTEN closely associates with its lipid phosphatase activity, which
antagonize PI3K/AKT activation (Figure 10). PTEN regulates proliferation, survival,
energy metabolism, cellular architecture, and motility. Pervious study in our lab has
demonstrated that in the liver-specific deletion of PTEN model, the consequently
activated PI3K/AKT signaling pathway is involved with elevated oxidative stress ,
increased mitochondrial mass, and enhanced respiration accompanied by promoted
glycolysis(Li, et al. 2013).
Figure 10. PTEN antagonizes PI3K activity. In response to extracellular signals,
receptor tyrosine (Y) kinases are activated. Phosphorylated Y residues recruit the
catalytic subunit of PI3K( p110) to the membrane through the Src homology 2
28
domains interacting with its regulatory subunit (p85). PI3K catalyzes
phosphatidylinositol 4,5-bisphospate [PI(4,5)P2] to [PI(3,4,5)P3] ,which appeals
AKT and PDK to the membrane. Activated AKT then can promote cell
proliferation, enhancement of cellular glycolytic flux, and inhibition of
apoptosis(Worby and Dixon 2014).
Pten-null islets exhibit comparable ability to secrete insulin after the stimulation of
low (3.3 mM) and high (16.7 mM) concentration glucose(Fig .11A), compared with the
wild type mouse islet (Con). The change was calculated and no statistically significant
changes in insulin level between the Pn and Con group., The Pten-null islet shows the
same distribution of insulin and the non-insulin-producing cells (Fig.11D).
Insulin-producing cells centralize in islets, while non-insulin-producing cells were
predominantly scattering the periphery of the islets. In some staining slides, We
observed that non-insulin-producing cells were surrounding the b-cells in both control
and Pten-null islets. In adult mouse islets, the proliferation rate of β-cells is relatively
low and rarely undergoes the cell death. In this study, 5-bromo-2-deoxyuridine (BrdU)
labeling was used to investigate the proliferation of β-cells in mutant islets. The 3-month
old mice were fed with water containing the BrdU in five days. The mice were sacrificed
for staining with insulin and BrdU. The BrdU positive cells were counted. The β-cells
proliferation in Pn group was as twice as much as the Co group islet in Fig.11F.
29
30
Figure 11. PTEN deletion in β-cell maintains the islet function. The selective
Deletion of Pten in β-cells does not significantly affect glucose and insulin homeostasis.
A) .GSIS test of Wild type vs. Pten-null 3-month adult mouse pancreatic islets. B). the
change of insulin secretion in responses to different concentrations of glucose
stimulation were calculated (n-fold) of A) & C). The hematoxylin eosin (H&E) staining
indicates the Pten-null (Pn)l mice preserve the normal cellular morphology and D)
maintain a insulin secretion Con and pancreas stained with insulin (red) and sorts of
endocrine hormones (green, cocktail of glucagon , somatostatin and pancreatic
polypeptide). E) Pancreas sections immunostained with antibodies against BrdU (green),
insulin (red) and counterstained with DAPI (blue). (50 µm) F). BrdU positive beta
cells were quantified in E). * p < 0.05. N=6.
Freshly isolated islets from Pten-null (PN) and wild type mice (CON) are seeded
directly on BSA or BCE cell coated XF24 microplates. The Pten-null (PNM) and wild
type mouse islet (CONM) were seeded for 1 week in culture ( Fig.15A). As predicted,
islets from Pten deletion mice showed significantly enhanced OCR rates than those from
the control mice. Both basal (25%) and maximal OCRs (30%) were higher when Pten is
deleted. Similar results were observed in islets cultured on BCE though with lower OCR
regardless of genotype. The PNM islets displayed 15% and 25% enhanced basal and
maximal oxygen consumption, respectively, when compared to the CONM islets.
However, the BCE cultured islets exhibited a 50% lower basal and maximal OCR
compared to the freshly isolated islets, regardless of genotypes.
31
32
Figure
12.
Increased
mitochondrial
respiration
rate
in
β-cell-specific PTEN
deletion islets. A) The basal and maximal oxygen consumptions (OCR) of freshly
isolated Pten null (Pn) and control (con) mouse islets as well as islets cultured for 7
days on BCE (Pnm, Pten null; Conm, Control). B) Quantitate baseline OCR in Pn and
Con groups of islets . C) Quantitate baseline OCR in Conm and Pnm group of islets
culture with BCE substrate. D) the extracellular consumptions rate (ECAR) was
tested simultaneously with OCR. Pten null islets either freshly isolated or cultured have
higher levels of ECAR.
In the baseline phase, both the Pten-null cell and those supported with BCE cell
coating showed the high ECAR rate. After the injection of FCCP, they also boost OCR
in a significant manner, compared with the control group. These results suggest that
PTEN and PTEN-regulated signals may contribute to Mitochondrial respiration and
glycolysis rate in pancreatic islet, and this modulation is independent of their metabolic
status.
3.6 Monolayer formation of pancreatic islet on BCE matrix
I further evaluated the BCE cells surface-modified dishes for long-term culturing as
BCE supported more initial islet adhesion and the morphology of the adhered islets are
more intact. In Fig.13A, the majority of the cultured islets were within 50µ< diameter<
250µ. They kept the original appearance after the isolation. One common phenomenon
after 3 days culture for most of the large islets (with diameter >250 ) more in both the
uncoated and coated group exhibited various degrees of cell necrosis. (Fig. 13B) The
problem of loss of necrotic cells in central area of the large islet could be avoided by
trypsin digestion. Fibroblasts grew surrounding the islets in some dishes of each group at
33
five days. Without any treatment, later the fibroblast haunted everywhere and inhibited
the spreading and flattening of the endocrine cells (Fig.13C). Some islets appeared an
irregular and dispersed morphology for an unknown reason.(Fig. 13D)
Figure 13. Primary culture of pancreatic islets on BCE-coated plates. One batch of
isolated islet after 3 days culture: A) the relative small islet shows the normal
morphology; B) the large islet core displayed the cell necrosis. In some cultures, C) the
fibroblast growth around the islet severely. D) Islet appeared with an irregular and
dispersed morphology with the vacant space in the central of the islet.
20x
magnification.
34
Figure 14. Monolayer formation of pancreatic islet on BCE-coated plates (phase
contrast) the same one A) the periphery of islet forms the monolayer, Day 7.B) entire
monolayer formation completed, day 18. C) zoom in of image D)The monolayer islet
accompanied with fibroblast , day 23. 20x magnification.
After one week in BCE culture, monolayer formation of pancreatic islets was
achieved. In the ECM modified culture group, the periphery of the islets started to grow
out as monolayer, indicated as the red arrow in Fig. 14A. After 20 days of culture, the
entire monolayer formation was completed and mostly restored the morphology about
one month (Fig. 14B-C). The islet cells displayed epithelial cell morphology with tight
junctions. In most of the cultures, the monolayer islets are accompanied with fibroblasts
35
in the end (Fig.14D). In each experiment, the plating efficiency and monolayer
formation had subtle differences.
36
CHAPTER 4
DISCUSSION
The primary goal of this study is to investigate the effect of extracellular matrix in
supporting the long-term in vitro culture of adult mouse pancreatic islets. With the basic
islet culture techniques, the freshly isolated islet can only survive for four to five days
without any ECM modified culture. Poly-L-lysine coated dishes have been used to
prolong the islet survival in vitro culture. However, islets quickly loose their
morphological characteristics under these conditions, similar to the uncoated condition.
In my study, several ECM proteins were used and shown protective effects against
β-cells apoptosis. The Bovine corneal endothelium cells (BCE) modified islet culture
shows the greatest adhesion. This matrix helps the islet settle down and promotes cell
spreading, and finally monolayer formation. Also, with the BCE and collagen I coated
dishes, islets shows a slower decline of insulin content and more persistent glucose
stimulated insulin secretion, comparing with FN and BSA control groups. The genes also
involved in the development of the pancreatic and maturation of β-cells are evaluated.
The gene expression levels show different extend of change in each group of ECM
supported islet culturing ,with BCE being the best at maintaining islet gene expression.
The monolayer initiation of adult mouse pancreatic islets was obtained using BCE
substrate. Regarding the tight and dense structure of islets, the cells in the central area of
islet are confronted with oxygen deficit and shortage of nutrition. Thus, the monolayer
37
cell culture allows cells to be exposed to the same condition and allow perfusion for
growth factor or drugs. In our initiated monolayer culture, the islet cells can quickly
stretch and become smooth and regular shape when cultured on BCE. When BCE
culturing is used, this culture condition, islet survival increased significantly from five
days up to forty-one days in the primary culture. The islet cells cultured on BCE also
have more sensitive response to the acute change of glucose when compared to other
matrices. Thus, BCE coating is a better monolayer culture mechanism for preserving the
islet function. In addition, the gene expression profile further confirms that the BCE
matrix preserves islet function. Using BCE substrates initiated monolayer culturing, our
lab members investigate oxidation of exogenous and endogenous free fatty acids of
pancreatic β-cells. Although collagen I matrix exhibits certain capability at supporting
islet cells survival, the effects are less than BCE matrix supported group in each
assessment and can rarely initiate the monolayer formation. Fibronectin did not show any
potential to protect the islet cells against apoptosis.
Lingering questions remain for improving the islet culture conditions. The condition
of islet varies from each batch of culture. The fibroblasts that grow together with the
islets may present problems for long-term culturing. The fibroblast growth factor
(FGF)-saporin conjugates are effective to remove the fibroblasts, whereas the FGF
complex may be noxious to the islets(Thivolet, et al. 1985). This maybe considered for
future improvement of islet culturing.
38
The ultimate aim of promoting the pancreatic islet culture is to restore the β-cells
mass which is critical for maintaining hormone homeostasis and preventing
diabetes mellitus(Ackermann and Gannon 2007). Pancreatic β-cells serve as sensors
of nutrients and associate with neurohormonal signals, which claim the metabolic
activation. In this study, the PTEN deleted Pancreatic β-cells model were used to monitor
the islet function. As the results indicate, in the mutant model the islet maintains the
normal function in the proliferation and insulin secretion. I investigated whether the BCE
culturing can be used to study the differences between control and Pten null islets. The
Pten-null islets displayed an overall increased OCAR and ECAR in the mitochondrial
stress test compared with the will-type islet. This difference is maintained when islets are
cultured with BCE matrix even though both OCAR and ECAR were significantly lower
in the BCE matrix groups regardless of Pten status.
In summary, I have established BCE coating as a superb culture condition for islets
compared with other coating conditions. Using this culture condition, I showed that
monolayer culturing of adult mouse pancreatic islets can be initiated. This culture
condition is also better at preserving normal islet function and enhancing the long-term
survival of islets in culture. Since monolayer culture is superior to the traditional culture
at promoting oxygen and nutrient uptake, the BCE matrix can be regarded as an excellent
culture condition to support the isolated islet culture for both laboratory and clinical
research.
39
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Abstract (if available)
Abstract
Pancreatic islet isolation exposes the islet to a variety of cellular stresses and disrupts the cell‐matrix interaction, which eventually leads to islet apoptosis in vitro. In this study, various extracellular matrix (ECM) cultures of fragmented pancreatic islets containing β‐cells were studied. Efforts have been made to reestablish the cell growth environment in order to establish conditions that maintain the islet function and prolong the survival of in vitro culture. The substrates used in this study to facilitate islet attachment were collagen I, bovine corneal endothelia (BCE) cells, fibronectin (FN), poly‐L‐lysine and bovine serum albumin (BSA) respectively. During the investigation, the monolayer initiation of adult mouse islets was achieved using BCE substrate. Pancreatic β-cells maintain glucose homeostasis via sensing glucose and other nutrients to regulate insulin exocytosis. BCE substrate is found to better preserve the insulin secretion function of islets in response to glucose. In addition, expression analysis shows that expression of β‐cell genes are maintained. Finally, the process of insulin secretion is coupled with mitochondria respiration. We showed that BCE substrate can be used to support islet culture used for analysis of oxygen consumption though with significantly reduced respiratory function.
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Guo, Yating
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Development of ECM for the preservation of adult mouse pancreatic islet function in vitro
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Keck School of Medicine
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Master of Science
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Biochemistry and Molecular Biology
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11/06/2015
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