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Metformin increases tyrosine kinase activity of the IGF-1/insulin receptor in Xenopus laevis oocytes

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Title:
Metformin increases tyrosine kinase activity of the IGF-1/insulin receptor in Xenopus laevis oocytes
Creator:
Mossel, Cori Ann
Publication Date:
Language:
English
Physical Description:
viii, 42 leaves : illustrations ; 29 cm

Subjects

Subjects / Keywords:
Insulin resistance ( lcsh )
Protein-tyrosine kinase ( lcsh )
Xenopus laevis ( lcsh )
Insulin resistance ( fast )
Protein-tyrosine kinase ( fast )
Xenopus laevis ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 39-42).
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Arts, Biology.
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Cori Ann Mossel.

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Source Institution:
University of Colorado Denver
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Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
37759237 ( OCLC )
ocm37759237
Classification:
LD1190.L45 1996m .M67 ( lcc )

Full Text
Metformin Increases Tyrosine Kinase Activity
of the IGF-1/insulin Receptor in
Xenopus laevis Oocytes
by
Con Aim Mossel
B.A., University of Colorado at Boulder, 1992
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
1996


This thesis for the Master of Arts
degree by
Cori Ann Mossel
has been approved
by

Date


Mossel, Cori Ann (M.A., Biology)
Metformin Increases Tyrosine Kinase Activity of the IGF-l/insulin Receptor in
Xenopus laevis Oocytes
Thesis directed by Associate Professor Bradley J. Stith
ABSTRACT
To study the mechanism of action of the anti-diabetic drug metformin, we
examined whether metformin could stimulate the action of insulin on the Xenopus
laevis oocyte. Metformin (between 0.1 pg/ml and 10 (ig/ml) was found to stimulate
insulin-induced meiotic cell division. These concentrations are similar to therapeutic
concentrations.
Metformin or insulin also stimulated tyrosine phosphorylation of a protein
likely to be the P-subunit of the IGF-l/insulin receptor. In whole oocytes, metformin
stimulation of tyrosine kinase activity was maximal after a 1.5 h incubation. This
response is relatively slow when compared to that of insulin wherein maximal
stimulation of tyrosine kinase activity was obtained by 30 min after insulin addition.
Results of in vivo experiments were compared to in vitro experiments using an
isolated plasma membrane-cortex preparation (PMC), Metformin stimulated tyrosine
kinase activity in the PMC and again showed a maximum response at 10 (ig/ml. Low
concentrations of insulin (e.g. 0.1 nM) induced a strong increase in tyrosine kinase
in


activity but the activity dropped off sharply at 200 nM insulin. The time course for
metformin or insulin stimulation was similar (maximal response by 15 min).
Since the response to metformin was maximal within 15 min of addition to the
PMC, but reached its maximum activity at 1.5 h after drug addition to whole cells, we
hypothesized that metformin slowly crosses the plasma membrane and acts at an
intracellular site. In addition, because metformin stimulates the tyrosine kinase
activity, we suggest that metformin stimulates the receptor tyrosine kinase to fight
diabetes.
This abstract accurately represents the content of the candidate's thesis. I recommend
its publication.
IV


DEDICATION
I dedicate this to my husband for his hard work, patience, and compassion.
I dedicate this to my dad, mom and sister for their continued support and
encouragement.
You all contributed in your own ways to the completion of this manuscript.


CONTENTS
ACKNOWLEDGEMENTS................................................viii
CHAPTER
1. INTRODUCTION.................................................1
Diabetes and Metformin ..................................... 1
TheXenopus Cell Model.................................. 3
Cell Cycle.................................................. 4
Insulin ...........i.........................................4
Insulin Action...............................................6
2. MATERIALS AND METHODS.......................................11
Maintenance.............................................. 11
Priming.................................................... 11
Xenopus oocytes............................................ 11
Homogenization of oocytes...................................12
Bicinchoninic Acid Protein Assay (BC A).....................13
PAGE Protein Separation and Western Blotting................13
Quantification of Tyrosine Phosphorylation..................15
Plasma Membrane Cortices (PMC)..............................16
vi


3. RESULTS .....................................................18
In vivo Experiments..........................................18
Dose Response ..........................................18
Time Course.............................................21
In Vitro Experiments.........................................21
Dose Response...........................................21
Time Course.............................................26
4. DISCUSSION ..................................................29
Dose Response Comparison ....................................29
Time Course Comparison ......................................31
Conclusion.................................................. 32
APPENDIX
A. Raw Data from in vivo Experiments: Density of 95 kDa Band on Western
Blots....................................................33
B. Chemicals, Solutions, and Equipment..........................34
REFERENCES ...................................................... 38
vu


ACKNOWLEDGEMENTS
I wish to thank Dr. Brad Stith for providing me with the opportunity to extend
my knowledge and acheive a very personal goal. For offering guidance and resources
when needed but also allowing me autonomy to improve my critical thinking skills and
pursue what I hope to be my career of teaching. I enjoyed being a part of Dr. Stith's
laboratory and all that I take with me will prove to be invaluable.
I wish to thank Dr. Dixon and Dr. Brockway for accepting to be a part of my
committee and graduate career. Their advice and support played a critical part in the
completion of this thesis.
I also would like to thank Keith Woronoff, Mitch Espinoza, Laura Whitworth and
Tony Ferdensi for their time and efforts.
This work was made possible by a National Science Foundation grant awarded
to Dr. Stith.
Vlll


CHAPTER 1
INTRODUCTION
Diabetes and Metformin
The impact of diabetes on the general population is substantial. It is the
fourth leading cause of death with one out of sixteen individuals affected by the
disease (6). Late onset non-insulin dependent diabetes mellitus (NIDDM), also
known as Type II diabetes, accounts for 90-95% of diabetes cases. It is believed to
be caused in part by impaired insulin action or insulin resistance in peripheral tissues
(9). In comparison, the juvenile onset Type I diabetes is caused by impaired insulin
release (9).
Metformin, the N,N' dimethylbiguanide (Figure 1.1) has been extensively
used in the treatment of NIDDM in Europe and Canada since 1979 (17), but has only
recently been introduced to the U.S.. Metformin does not lower blood sugar in
nondiabetic subjects, but is generally associated with a fall in fasting glucose levels of
20-30% in NIDDM individuals (6).
Early research concluded that in the presence of insulin, metformin increases
insulin-stimulated glucose uptake and translocation of glucose transporters (7,21), the
metabolic clearance of glucose, or decreases insulin resistance (6). Metformin may
1


CH, H
Figure 1.1: Structure of metformin (MW = 129.17). Adapted from Merck Index.
2


act through an alteration of membrane fluidity (8). Metformin does not increase
insulin binding (25). The initial steps by which the drug stimulates insulin action is
not known.
The Xenopus Cell Model
There are many advantages to using the Xenopus oocyte as a model system
for the study of both insulin and metformin action. First, oocytes are large cells that
can divide very rapidly and synchronously when insulin, insulin-like growth factor
(IGF-1), or progesterone are added. Second, the Xenopus oocyte contains both the
IGF-1 and insulin receptor. High (> 100 nM) levels of insulin can bind and activate
the IGF-1 receptor and high levels of IGF-1 can bind the insulin receptor. Lastly,
since it is thought that Type II diabetics have "bad" insulin receptors and Xenopus
oocytes contain many normal IGF-l/insulin receptors, it has been an excellent model
in which to study the insulin receptor.
Since high levels of insulin or low levels of IGF-1 are required for induction
of meiotic cell division and elevation of intracellular pH (29), it is believed that an
active IGF-1 receptor is present. Due to binding studies (20), it is thought that the
insulin receptor is present but inactive. The IGF-1 and insulin receptors show high
levels of sequence homology and both act through similar mechanisms (11). At
times when it is not known if the receptor is the IGF-1 or the insulin receptor, we will
use the term IGF-l/insulin receptor.
3


Cell Cvcle
In complex multicellular organisms, different cells divide at very different
rates (4). The variability in the length of the cell cycle occurs mainly in G1 and G2
(4).
Xenopus oocytes (Stage IV; diameter 1.2 mm) are arrested in early M-phase
until they are stimulated with insulin, IGF-1, or progesterone to progress through
Meiosis I to arrest at metaphase of Meiosis II (Figure 1.2). This process is called
maturation of the oocyte to the egg. Oocytes have a dark animal pole where the
nucleus and many enzymes reside and a light vegetal pole that contains mainly yolk
proteins. This egg can be visualized by the appearance of a white spot in the animal
pole due to movement of the nucleus to just below the cell surface as it pushes away
pigment granules. The white spot is also associated with germinal vesicle breakdown
(or GVBD) (28). Upon addition to the oocyte, insulin triggers matauration after 8-12
hours (11). Metformin stimulates the ability of insulin to induce meiotic cell division
without changing insulin binding (10). In addition, the attachment of metformin to
beads prevents the entry of the drug into the oocyte and prevents metformin
stimulation of insulin-induced GVBD (17).
Insulin
Insulin is a polypeptide hormone produced in the p cells of the islets of
Langerhans of the vertebrate pancreas (22). It is synthesized as a single chain
4


Figure 1.2: The cell cycle. Interphase includes Gap-1 (Gl), synthesis of DNA (S),
and Gap-2 (G2), but does not include meiosis or mitosis (M). Oocytes
arrest in oogenesis at the G2/M block. Hormone releases oocytes from
G2/M block and allows oocyte to enter prophase I.
5


polypeptide precursor, proinsulin, which is then converted by proteolysis to insulin
(22). Insulin (Figure 1.3) is a very large protein with two distinct chains. It is
secreted directly into the bloodstream where it regulates carbohydrate metabolism,
influences the synthesis of protein and of RNA, and the formation and storage of
neutral lipids (22,24). It is also unique in that it is a hormone that affects almost all
tissues of the body and is responsible for inducing a wide range of both metabolic and
trophic responses in cells (24).
Both the IGF-1 and insulin receptors are very closely related (ie. show high
sequence homolgy), and are composed ot two a- and two P-subunits (15). The a-
subunits are extracellular and have a molecular weight of approximately 135 kDa.
They contain the actual hormone binding site. The P-subunit spans the membrane
and has a molecular weight of approximately 95 kDa. Its intracellular portion has the
tyrosine kinase domain. Insulin binding to the a-subunits causes auto-
phosphorylation on tyrosine residues of the P-subunits (15,24), and this
autophosphorylation is required for full activation of the receptor.
Insulin Action
The study of the transduction of the mitogenic signal from transmembrane
growth hormone receptors to those enzymes directly controlling cell cycle
progression is a rapidly expanding field. Many details of the signal transduction
pathway downstream of the receptor have yet to be elucidated. Therefore, the role
6


Gly
He
I
Val
I
Glu
Chain A
Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
\ |
His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys
L
G
Asn
Chain B
Gly
I
Glu
Val
Pile
Arg
Thr-Lys-Pro-Thr-Tyr-Phe-Phe-Gly
Figure 1.3: Structure of insulin (MW = 6000). Adapted from Merck Index.
7


played by tyrosine phosphorylation in other processes of normal cell growth and
differentiation remains a subject of intense investigation. Tyrosine phosphorylation
can involve autophosphorylation (kinase phosphorylates itself) or kinase action on a
second protein. Phosphorylation of specific tyrosine residues of other proteins can
result in the activation of the other protein. Tyrosine phosphorylated proteins are
involved in signal transduction and in the regulation of cell proliferation. Antibodies
to phosphotyrosine residues can detect these phosphorylated proteins through a
Western blot. Phospholipase C (PhLC) may be a substrate for the IGF-l/insulin
receptor and may be responsible for some of the events downstream from the
receptor. PhLC, once activated, causes phosphatidylinositol 4,5-bisphosphate (PIP2)
to be cleaved into inositol trisphosphate (IP3) and sn 1,2-diacylglycerol (DAG).
Previous studies (Stith & Proctor, 1989) have shown that IP3 releases intracellular
Ca2+ but that elevated [Ca 2+]in (cytosolic free calcium ion concentration) is not
sufficient by itself to induce GVBD. DAG activates protein kinase C (PKC) which in
turn activates additional downstream proteins that are believed to result in the
induction of GVBD (28).
The current proposed model of IGF-1 or insulin action in theXenopus oocyte
begins with the hormone binding to its membrane receptor tyrosine kinase. This
stimulates the receptor to autophosphorylate at tyrosine residues located on the inside
of the cell membrane. The receptor then becomes able to phosphorylate substrates
such as PhLC (Figure 1.4). Insulin or IGF-1 stimulation of the receptor in oocytes
8


Extracellular
Insulin
Metformin V
IGF-1 Receptor Tyrosine Kinase.
PIP2 DAG
A K. U
4 | ^ IP3 ^ PKC
A Inc. Ca2" V Protein Activation 7

Hormone Response ^
Intracellular
Figure 1.4: IGF-1/insulin receptor tyrosine kinase pathway. Addition of insulin and
metformin stimulate kinase activity by acting at different sites; insulin
binds at an extracellular site while metformin binds intracellularly.
DAG diacylglycerol, IPS inositol trisphosphate, PIP2 -
phosphatidylinositol bisphosphate, PhLC phospholipase C, PKC -
protein kinase C, Inc. Ca2+ increase in calcium, IGF-1 insulin-like
growth factor 1.
9


results in an increase in IP3 (Stith, et al. 85), DAG (Stith, et al. 85). internal pH
(Stith, et al. 85), protein synthesis (Stith, et al. 85), activation of ribosomal S6 (Stith,
et al. 85), and increase in glucose.uptake (16). As will be discussed, we also suggest
that metformin stimulates the P-subunit of the receptor.
10


CHAPTER 2
MATERIALS AND METHODS
Maintenance
Female Xenopus laevis frogs (Xenopus One, Ann Arbor, MI) were fed with a
diet of 2 g of ground beef heart every other day. The water in which the frogs lived
was replaced every day with 15 gallons fresh water in which 10 ml of Nov Aqua
(Novatek, Haywood, CA) was added.
Priming
Selected female frogs were primed four days in advance of each experiment
with 35 I.U. of pregnant mare serum gonadotropin (PMSG) (Sigma, St. Louis, MO).
Priming stimulates the growth of the ovarian follicles and the formation of the corpus
luteum which increases the response and shortens the time required for treated
oocytes to enter into prophase I of meiosis (28).
Xenopus Oocvtes
Oocytes were manually dissected from ovarian fragments in OR2 solution (83
mM NaCl, 0.5 mM CaCl2,1 mM MgCl2, 10 mM HEPES) at room temperature. Stage
11


VI oocytes (1.2 mm or greater) were selected for each experiment, since they are
competent to enter meiosis. Dissected oocytes were placed in 12-well plates
containing 3 ml OR2 solution. Each group contained 35 oocytes. Fatty acid-free and
insulin-free bovine serum albumin (BSA) was added to each well of 0R2 at a
concentration of 0.5% to prevent the insulin and metformin from sticking to the walls
of the 12-well plate. Experimental cells were incubated with varying concentrations
made with a 131 pM insulin stock, dilutions included 0.001, 0.01, 0.1,1.0, and 10
nM, or 0.988 pg/ml metformin stock, dilutions included 0.01, 0.1,1.0, and 10 pg/ml.
For the in vivo insulin and metformin time course, 1 pM or 10 pg/ml were used
respectively.
Homogenization of Oocvtes
All homogenizations were done at 5C in order to slow protein degradation.
Cells were homogenized by placing each group of 35 oocytes into a glass mortar with
homogenizing buffer [a final concentration of 6.5 mM Tris, pH 7.0 at room
temperature, 1 mM phenyl methylsulfonyl fluoride (PMSF) (an irreversible inhibitor
of serine proteases), 1 mM EGTA (a chelating agent selective for Ca2*)]. Thorough
homogenization was done with a glass pestle. The solution was transferred to 1.5 ml
Eppendorf tubes and (6.5 mM Tris, 1 mM PMSF) and 600 pi 1,1,2-trichloro-1,2,2-
trifluoroethane (Freon) were added. The Freon was a delipidizer (removes the yolk).
All samples were vortexed for 30 sec and centrifuged for 10 min at 4C. The top
12


layer that contained the proteins of interest was removed and placed in a clean 1.5 ml
Eppendorf tube. At this time, 30 pi was removed from each sample and placed in
glass culture tubes and set aside for protein concentration measurements. To prepare
the samples for electrophoresis, sample buffer (0.5 M Tris-HCI, pH 6.8, 10%
glycerol, 0.36 mM SDS, 0.02 mM bromophenol blue, 25% P-mercaptoethanol) was
added to the remainder of each sample. Samples were boiled for 10 min then placed
at -20 C for storage.
Bicinchoninic Acid Protein Assay ('BCA'i
Protein concentrations were measured using the BCA method. Proteins
reduce alkaline Cu (II) to Cu (I) in a concentration dependent manner. Bicinchoninic
acid binds Cu (I) to form a purple complex with an absorbance maximum at 562 nm
(Tikhonov, et al. and Maonski, et ah). Standards were prepared using 0 pg, 20 pg,
40 pg, 60 pg, 80 pg, 100 pg of highly purified bovine serum albumin and plotted on a
graph. The Y-axis was plotted as absorbance at 562 nm and the X-axis was the
protein standard in micrograms. This plot was used to determine the amount of
protein in the unknown samples.
PAGE Protein Separation and Western Blotting
Proteins in the homogenate (stored at -20 C) were separated by
polyacrylamide gel electrophoresis (PAGE). Equal amounts of protein were added
13


to an 8% resolving gel and 4% stacking gel. 1% Ammonium persulfate, a catalyst,
and 0.1% TEMED (N,N,N!,N'-tetramethylethylenediamine), a cross-linker, were
added to induce polymerization. Electrode buffer (12.4 mM Tris base, 100 mM
glycine, 1.7 mM SDS) was added to the top and bottom reservoirs of the
electrophoresis chamber and electrophoresis was carried out overnight using a
programmable power supply (6 mA for 18 hours; 30 mA for 2.5 hours; 6 mA for 18
hours).
The polyacrylamide gel was thoroughly rinsed twice with standard transfer
buffer (25 mM Tris base, 192 mM glycine, 20% MeOH). Filter papers (Whatman
chromatography paper) were also wet in the standard transfer buffer and placed
around the gel. Electrophoretic transfer of the proteins in the gel onto membrane
utilized a Genie apparatus. For the best resolution of our larger proteins,
polyvinylidene difluoride membrane (PVDF) with a pore size is 0.2 pm was used (as
compared to nitrocellulose with a pore size of 0.05 pm). The membrane was charged
by soaking briefly in methanol. The protein transfer was carried out for 1.5 h at 24
volts.
After completion of the transfer, the PVDF membrane was placed in
blocking buffer (2% BSA, 10 mM Tris, 100 mM NaCl, 0.1% Tween 20) and allowed
to float freely for 30 min at 37 s C. Incubation with a 1:2000 dilution of the anti-
phosphotyrosine antibody was carried out at room temperature for 1 h in a sealed
plastic bag with rolling metal balls on a shaker. The anti-phosphotyrosine antibody
14


with alkaline phosphatase conjugate has high specificity and binds only to
phosphorylated tyrosine residues (not serine or threonine). The membrane was
washed in buffer (10 mM Tris, 100 mM NaCl, 0.1% Tween 20) for 15 min at 37C,
briefly rinsed with dH20 and then placed in 20 ml 5-bromo-4-chloro-3-indolyl
phosphate/nitro blue tetrazolium (BCIP/NBT) color developer. This solution reacts
with the alkaline phosphatase conjugate of the primary antibody and allows
visualization by producing a purple color.
Quantification of Tvrosine Phosphorylation
As described above, tyrosine phosphorylated proteins could be visualized by
BCIP/NBT color development. Since volumes for each sample were corrected so that
equal amounts of protein were loaded onto the polyacrylamide gel, direct
comparisons in the color development could allow quantification of tyrosine kinase
activity in each sample.
Our positive control was provided by Transduction Laboratories which was
composed of A431 cell lysates (a human epidermoid carcinoma cell line which
express 106 epidermal growth factor receptors at the cell surface). The cells were
stimulated with epidermal growth factor (EGF), a hormone that increases tyrosine
kinase activity. Our negative control was the group of oocytes labeled as "control" in
which no insulin or metformin was added. The band intensity produced by the
negative control after color development was then used as a baseline in which the
15


experimental groups could be compared. An increase in the intensity of the 95 kDa
P-subunit band of the IGF-l/insulin receptor band would indicate an increase in
tyrosine phosphorylation.
Quantification of the bands was accomplished by first scanning the PVDF
membrane using an H-P Scanjet IICX followed by computer analysis (Sigma Gel,
Jandel Scientific). The intensity of the 95 kDa band (IGF-l/insulin receptor) was
calculated and integrated and these values were then plotted on a graph. The number
of times each experiment was replicated is represented in the legend of each graph as
Plasma Membrane Cortices ('PMC')
PMC experiments were used to provide in vitro results to compare with the in
vivo Xenopus oocyte preparation. Experiments were completed by Keith Woronoff
with assistance from me. Oocytes (35 per group) were manually dissected from
ovarian tissue in OR2 at room temperature. They were then placed in 4C Regular
Sadler Buffer (10 mM NaCl, 10 mM HEPES, pH 7.2). Oocytes were opened by
piercing with closed forceps and allowing the forceps to open. They were then
flattened, yolk side down, and placed on ice for 20 min to allow the yolk and
intracellular material to dissociate from the plasma membrane. Receptors,
transmembrane proteins and membrane associated proteins should remain intact and
retain normal function. The PMC was washed away from the intracellular material
16


and placed in a 0.5 ml Eppendorf tube. They were pelleted by brief (seconds)
centrifugation in a microfuge and excess liquid was removed. Modified Sadler
Buffer (50 pi Regular Sadler Buffer with 5 mM MgCL) and 0.5 mM RR-SRC were
added. RR-SRC serves as a substrate for phosphorylation by the activated tyrosine
kinase receptor. The pellet of membranes was broken up by gently flicking the
bottom of the Eppendorf tubes. Various concentrations of insulin or metformin were
added to the PMC to activate the receptor. Fifteen min after addition of 10 pi 32P-
ATP solution (made by combining 100 pi of 15 mM cold ATP stock, 50 pi 32P-ATP,
400 pi Modified Sadler Buffer), the reaction was stopped with 1% Triton-X. After
centrifugation (15,000 g) for 30 sec, the supernatant was transferred to a Pierce
phosphocellulose bucket and centrifuged for 30 sec. The phosphocellulose (which
binds protein such as RR-SRC, not ATP) was washed with 500 pi of a 74 mM
phosphoric acid stock and again centrifuged for 30 sec. Buckets with
phosphocellulose were transferred to clean Pierce eppendorf tubes and again washed
with 500 pi phosphoric acid and centrifuged for 30 sec. Lastly the buckets were
placed in scintillation vials with water. 32P activity was measured by a scintillation
counter.
17


CHAPTER 3
RESULTS
The results from all in vivo experiments (whole cells) are presented first: this is
followed by a brief discussion of the in vitro (PMC) results.
In vivo Experiments
Dose Response
After polyacrylamide electrophoretic separation and transfer to PVDF paper,
the amount of tyrosine phosphorylated proteins was quantified. After insulin
addition, there was a distinctive increase in the intensity of the 95 kDa P-subunit band
(as evaluated by SigmaGel), and this has been used as a measure of the activation of
IGF-l/insulin receptor (Figure 3.1). Peak responsiveness was at 0.01 nM and 1.0 nM
insulin. A decrease in kinase activity is apparent at 10 nM. Metformin addition to
whole oocytes also increased the intensity of this band. A metformin concentration
of 0.1 pg/ml was not sufficient to induce a strong response. However, 10 pg/ml
metformin produced a 7-fold increase above baseline levels (Figure 3.2). This
18


250
Figure 3.1: Insulin stimulation of receptor tyrosine kinase (n=2). Insulin was added
to oocytes to a concentration of 0 (control), 0.001, 0.01,0.1,1.0,10 nM.
The intensity of the purple band at 95 kDa (the IGF-l/insulin receptor)
was used as a measure of receptor tyrosine kinase activity (Y-axis) and
expressed as a percent of control activity.
19


800
Metformin Concentration (pg/mL)
Figure 3.2: Metformin stimulation of receptor tyrosine kinase (n=2). Metformin
was added to oocytes to a concentration of 0 (control), 0.1,1.0, or 10.0
pg/ml. The intensity of the purple band at 95 kDa (the IGF-l/insulin
receptor) was used as a measure of receptor tyrosine kinase activity (Y-
axis) and expressed as a percent of control activity.
20


concentration of metformin is similar to therapeutic concentrations given to diabetic
patients (21).
Time Course
Metformin (10 |ig/ml) stimulation of receptor autophosphorylation peaks at
60-90 min (Figure 3.3). At 120 min the level of receptor activity begins to slowly
drop off. Therefore, metformin requires a 60 min incubation before it fully activates
the receptor tyrosine kinase.
In Figure 3.4,1 pM insulin produces a characteristic rapid increase in tyrosine
kinase activity within 5 min and peaks at 30 min. At 1 h, activity returns to near
baseline level. As compared with metformin, insulin exerts its effects rapidly.
In vitro Experiments
Dose Response
A large increase in tyrosine kinase activity was obtained with the addition of
10 pg/ml metformin to PMC (Figure 3.5).
Receptor tyrosine kinase activity peaked at 0.1 nM insulin, and remained
steady at 10 nM (Figure 3.6). A sharp decrease in kinase activity follows at 200 nM
insulin.
21


Minutes post-treatment
Figure 3.3: Time course of metformin stimulation of receptor tyrosine kinase using
whole cells (n=3). Kinase activity was measured at 15, 30, 60, 90, and
120 min post-treatment.
22


600
Figure 3.4: Time course of insulin stimulation of receptor tyrosine kinase using
whole cells (n=3). Kinase activity was measured at 5,15, 30, 60, and
120 min post-treatment.
23


cpm
(% Control)
\
Figure 3.5: Metformin stimulation of receptor tyrosine kinase (n=l). Metformin
was added to plasma membrane cortices to a concentration of 0
(control), 0.01, 0.1,1.0, 10.0 pg/ml. 32P activity was used as a measure
of receptor tyrosine kinase activity (Y-axis) and expressed as a percent
of control activity.
24


300
250 -
200 -
to 150
o O
100 -
50 -
control 0.1 1.0 10.0
Insulin Concentration (nM)
200.0
Figure 3.6: Insulin stimulation of receptor tyrosine kinase (n=l). Insulin was added
to plasma membrane cortices to a concentration of 0 (control), 0.1,1.0,
10, or 200 nM. 32P activity was used as a measure of receptor tyrosine
kinase activity (Y-axis) and expressed as a percent of control activity.
25


Time Course
Data from the metformin dose response indicated that 10 pg/ml elicits the
strongest response of the IGF-1/insulin receptors in membrane cortical preparations
and should be utilized for all time course incubations (Figure 3.7). A significant peak
appears at 5 min that is 3-fold above baseline levels. Maximal IGF-1/insulin receptor
activation peaks at 15 min with a 6-fold increase over basal level. At 30 min and 60
min, a decrease in kinase activity is noted. Using PMC, metformin is able to activate
receptor tyrosine kinase activity within 5 min yet metformin action with whole cells
requires 60 min. This suggests that metformin may need to cross the cell membrane
before exerting its effect on the receptor tyrosine kinase.
Insulin (1 pM) stimulates an immediate increase in IGF-l/insulin receptor
kinase activity in the PMC (Figure 3.8). By 15 min, maximal levels of activity were
reached (over 5-fold increase from baseline levels). A decrease is seen at 30 min and
eventually reaches baseline levels by 60 min.
26


700
Minutes post-treatment
Figure 3.7: Time course of metformin stimulation of receptor tyrosine kinase using
plasma membrane cortices (n=l). Kinase activity was measured at 1, 5,
15, 30, and 60 min post-treatment.
27


0 10 20 30 40 50 60
Minutes post-treatment
Figure 3.8: Time course of insulin stimulation of receptor tyrosine kinase using
plasma membrane cortices (n=l). Kinase activity was measured at 1,
15,30, and 60 min post-treatment.
28


CHAPTER 4
DISCUSSION
Dose Response Comparison
By comparing in vivo and in vitro data, it can be seen that metformin
stimulates tyrosine kinase activity and these data support the suggestion that
metformin does act at an intracellular site. Based on the literature, metformin does
not affect IGF-1/ insulin receptor number or affinity (14). If the drug bound directly
to the a-subunit of the receptor as insulin does, one might expect a change in the
affinity of insulin. Also metformin would act as swiftly as insulin. Since metformin
was much slower than insulin, this led us to believe that its target of action may be at
a location other than the extracellular hormone binding region. We hypothesize that
metformin functions initially by stimulating the tyrosine kinase activity by binding to
the intracellular portion of the P-subunit of the IGF-1/insulin receptor.
Results of the in vivo and in vitro dose response relationships for metformin
were similar. Maximal receptor tyrosine kinase activity occurred at 10 (ig/ml which
is similar to therapeutic concentrations given to diabetic patients (21). Since
metformin concentration is established data, this experiment further solidifies the idea
that the Xenopus oocyte model as highly efficient for our studies. It also shows that it
29


is possible to measure kinase activity with a great level of accuracy. However, there
has been a controversy over whether the IGF-l/insulin receptor is affected by
metformin. In support of metformins stimulation of increasing kinase activity, one
group found that metformin increased tyrosine kinase activity of the insulin receptors
from rat muscle cells (25). However, Matthaei et al. (21) and Jacobs et al. (14) found
that metformin did not stimulate the receptor tyrosine kinase in rat adipocytes. The
inability of some investigators to record stimulation of the receptor tyrosine kinase by
metformin may be due to tissue differences or the inability to record small increases
in the density of autoradiogram bands. In addition, our system offers the advantage
that it does not involve removal of the receptor from the membrane with detergent.
After insulin addition, both in vivo and in vitro, similar results were produced.
In vivo and in vitro results showed a peak between 0.01 nM and 0.1 nM insulin.
These results are contrary to other studies where Xenopus oocytes required 1 pM
insulin in order to stimulate a response (11).
A progressive decrease in kinase activity occurred at higher insulin
concentrations under both in vivo and in vitro conditions. This could be explained by
a type of desensitization. When target cells are exposed to high levels of insulin, they
lose the ability to respond to the stimulus with their original sensitivity (1). The
process involves reversibly adjusting its sensitivity to the level of the stimulus by
responding to changes in the concentration of a signaling ligand. This has been found
to occur with insulin receptors (24).
30


Time Course Comparisons
The most notable difference in results between in vivo and in vitro conditions
occurs with the metformin time course. In Xenopus oocytes, the full effect of
metformin can only be seen after 1 h to 1.5 h of incubation. In agreement with our
findings, Jacobs, et al. (14) and Matthaei, et al. (21) treated rat adipocytes with
metformin alone and noted that long (hours) incubation was necessary for a
significant increase in cell responsiveness. Metformin takes a longer period of time
to move across membranes (17).
On the other hand, in the in vitro model (which uses PMC), both external and
internal faces of plasma membrane are exposed. With the addition of metformin to
this PMC, increased tyrosine kinase activity was seen within 5 min. This is much
faster than with whole cells. Since the internal portion of the membrane is exposed,
the internal tyrosine kinase portion of the IGF-l/insulin receptors are exposed as well.
The drug does not have to cross the membrane in order to exert its effects and can
directly move to its target. Khan et al. (17) found that Sepharose-coupled-metformin
did not stimulate insulin-like action in the oocyte. In addition, the cells incubated
with free metformin accumulated the drug, whereas the cells incubated with
sepharose-coupled-metformin failed to take up this biguanide molecule. This
supports the model that the target of metformin in stimulating insulin action is
situated intracellularly and that the movement of metformin into oocytes seems to be
an important factor in stimulating the insulin actions (14,17). Fischer et al. (7)
31


suggested insulin and metformin do not act upon the same signaling path since insulin
required minutes to stimulate glucose uptake into rat cardiomyocytes whereas
relatively weak stimulation by metformin occurred only after a 90 min lag period.
Since they demonstrated that this lag period was not due to protein synthesis, and in
light of our data, we suggest that the lag period is required for entry of metformin
into the cell.
Further support of the hypothesis that metformin may act intracellularly is the
results of the insulin time course. Under both in vivo and in vitro conditions, insulin
increases kinase activity with maximal levels within 30 min. This is consistent with
the mechanism in which insulin binds to the outside of the cell. Insulin acts on the
extracellular surface of a cell and binds to the a-subunit of the IGF-1/insulin receptor
(25). By this mechanism, it should not make a difference in the time it takes for
insulin to exert its effects, either with a whole cell or the plasma membrane cortex
preparation.
Conclusion
Our results suggest that the IGF-l/insulin receptor protein tyrosine kinase is a
potential mediator of metformins intracellular action. We have found that metformin
stimulates the IGF-l/insulin receptor tyrosine kinase activity and suggest that this is
how metformin is beneficial to diabetic patients.
32


The molecule is small but very hydrophilic due to an easily protnated primary
amine and it does not readily cross the cell membrane and this would account for its
slow action. Metformin mimics insulin but acts independently of insulin to work
inside the cell to increase the tyrosine kinase activity. The slow effect of metformin
further supports our hypothesis that metformin exerts its effects at an intracellular
site. In addition, work by others shows that metformin does not alter insulin binding.
Insulin resistance in NIDDM is predominantly due to a depletion of cellular glucose
transporters with consequently fewer transporters available for insulin-induced
translocation to the plasma membrane (21). In the diabetic patient, it is possible that
metformin increases the conversion-turnover of the translocatable glucose-transporter
isoform to the insulin-sensitive glucose-transporter isoform (21). It is suggested that
metformin accomplishes this task by increasing tyrosine activity of the IGF-1/insulin
receptor which stimulates the translocation of glucose transporters.
33


APPENDIX A
RAW DATA FROM IN VIVO EXPERIMENTS:
DENSITY OF 95 kDa BAND ON WESTERN BLOTS
time course
Expt I Expt 2 Expt 3
Area % Ctrl Area % Ctrl Area % Ctrl Mean St Dev
216.0 100.0 234.0 100.0 370.0 100.0 100.0 0.0
315.0 145.8 138.0 59.0 777.0 210.0 138.3 75.8
412.0 190.7 409.0 174.8 621.0 167.8 177.8 11.7
489.0 226.4 857.0 366.2 1383.0 373.8 322.1 83.0
641.0 296.8 876.0 374.4 1389.0 375.4 348.8 45.1
229.0 106.0 390.0 166.7 1063.0 287.3 186.7 92.3
5 course
Expt 1 Expt 2 Expt 3
Area % Ctrl Area. % Ctrl Area % Ctrl Mean St Dev
1154 100.0 294 100.0 897 100.0 100.0 0.0
2015 174.6 574 195.2 2058 229.4 199.8 27.7
2431 210.7 1167 396.9 1724 192.2 266.6 113.3
2693 233.4 3698 1257.8 1534 171.0 554.1 610.3
1790 155.1 1237 420.7 1515 168:9 248.3 149.5
716 62.0 1357 461.6 899 100.2 207.9 220.5
dose response
Expt 1 Expt 2
Area % Ctrl Area % Ctrl Mean St Dev
395 100.0 292 100.0 100.0 0.0
557 141.0 513 175.7 158.3 24.5
1583 400.8 647 221.6 311.2 126.7
4437 1123.3 940 321.9 722.6 566.7
e response
Expt 1 Expt 2
Area % Ctrl Area % Ctrl Mean St Dev
842 100.0 1718 100.0 100.0 0.0
1218 144.7 1731 100.8 122.7 31.0
2138 253.9 2477 144.2 199.0 77.6
2203 261.6 2465 143.5 202.6 83.6
2114 1015 251.1 120.5 2201 128.1 189.6 120.5 86.9
34


APPENDIX B
CHEMICALS, SOLUTIONS AND EQUIPMENT
Chemicals
Albumin, bovine -(Sigma)
Albumin, fatty acid free -(Sigma)
Ammonium Persulfate -(BioRad)
Anti-phosphotyrosine antibody -(Transduction Labs)
32P-ATP (cold) -(Boehringer Mannheim)
32P-ATP (hot) -(New England Nuclear)
P-Mercaptoethanol -(Sigma)
BCA kit -(Sigma)
BCIP/NBT -(Sigma)
Bromophenol Blue -(Sigma)
CaCl2 -(J.T. Baker Chemical Comp)
EDTA -(Sigma)
EGTA -(Sigma)
filter paper -(Whatman)
Freon -(Sigma)( 1,1,2-trichloro-1,2,2-trifluoroethane)
35


Glycerol -(Sigma)
Gycine -(Sigma)
HEPES -(Research Organics)
Insulin -(Sigma)
Methanol -(VWR Scientific)
Metformin -(Lipha Labs)
MgCl2 -(J.T. Baker Chemical Comp)
NaCl -(Sigma)
Phosphocellulose Units -(Pierce)
Phosphoric Acid -(J.T. Baker Chemical Comp)
Polyacrylamide, 4% -(AMRESCO)
Polyacrylamide, 8% -(AMRESCO)
PMSF -(Research Organics)
PMSG -(Sigma)
PVDF -(Sigma)
Lauryl Sulfate -(Sigma)
RR.-SRC -(Biomol)
TEMED -(Sigma)(N,N,N',N'-tetramethylethylenediamine)
Tris-base -(J.T. Baker and Sigma)
Tris-HCl -(Sigma)
Triton-X -(Sigma)
36


Tween 20
-(Sigma)
Solutions
Blocking buffer 200 ml wash buffer 2% BSA
Electrode buffer 12.4 mM Tris-base 100 mM Glycine 1.7 mM SDS
Homogenizing solution 15 mM Tris, pH 7.0 at room temp. 200 mM PMSF lOmMEGTA
Insulin 140 (iM stock, [final] = 1 pM
Metformin 0.988 (ig/ml stock, [final] = 1 pg/ml, 10 pg/ml
Modified Sadler's buffer 200 ml Regular Sadler's buffer 5 mM MgCl2
Oocyte ringer's solution (OR2) 83 mM NaCl 0.5 mM CaCl, 1 mM MgCl2 lOmMHEPES pH 7.9
PMSG Pregnant Mare Serum Gonadotropin 116.67 U/ml
Regular Sadler's buffer 10 mM NaCl 10 mM HEPES pH 7.2
RR-SRC 1.315 ml Modified Sadler's buffer add to 1 mg lyophilized RR-SRC, [final]
- 0.5 mM
37


Sample buffer 0.5 M Tris-HCl, pH 6.8 10% Glycerol 0.36 mM SDS 0.2 mM bromophenol blue 25% P-mercaptoethanol
Standard buffer 25 mM Tris-base 192 mM Glycine 20% MeOH
Wash buffer 10 mM Tris lOOmMNaCl 0.1% Tween 20
Shaker Equipment -(Stoval Life Science, Greensboro, NC)
Electrophoresis chamber -(Bio Rad Labs, Hurcules, CA)
Genie apparatus -(Idea Scientific, Corvallis, OR)
Microfiiges -(Beckman Instruments, Fullerton, CA)
Power supply model EC702 -(EC Apparatuc Corp, St. Petersburg, FL)
Power supply model DX5 -(Schauer Manufacturing Corp., Cinncinati, OH)
Scintillation counter -(LKB-Wallace, Pharmacia, Gaithersburg, MD)
Shaking water bath -(Precision Scientific, Chicago, IL)
38


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