1. 1
May
4th,
2010
Natasha
A.
Mahadeo
Genes
and
Development-­‐
2010
Cell
Development
&
Biology
©
Cold
Spring
Harbor
Laboratory
Press
2010
Dr.
J.
Morris;
T
9:00-­‐12:45
24:543-­‐548
www.genesdev.org
Primary
Literature
Analysis
Pregnancy
Restores
the
Regenerative
Capacity
of
the
Aged
Liver
via
Activation
of
an
mTORC1-­‐Controlled
Hyperplasia/Hypertrophy
Switch
YUVAL
GIELICHINSKY
1,
2
,
NERI
LAUFER
2
,
EFI
WEITMAN
3
,
RINAT
ABRAMOVITCH
4
,
ZVI
GRANOT
1
,
YEHUDIT
BERGMAN1, 6
and
ELI
PIKARSKY
3,
5
1Department
of
Developmental
Biology
and
Cancer
Research,
Institute
for
Medical
Research
Israel-­‐
Canada
Hebrew
University
Hadassah
Medical
School,
Ein
Kerem,
Jerusalem
91120,
Israel;
2Department
of
Obstetrics
and
Gynecology,
Hadassah-­‐Hebrew
University
Medical
Center,
Jerusalem
91120,
Israel;
3
Department
of
Pathology
and
the
Lautenberg
Center
for
Immunology,
Institute
for
Medical
Research
Israel-­‐Canada,
Hebrew
University
Hadassah
Medical
School,
Ein
Kerem,
Jerusalem
91120,
Israel;
4The
Goldyne
Savad
Institute
for
Gene
Therapy,
Hadassah-­‐Hebrew
University
Medical
Center,
Jerusalem
91120
Israel
Abstract
The
regenerative
capacity
of
cells
is
progressively
lost
as
the
body
ages
(Labat-­‐Robert
2004;
Kirkwood
2005;
Beausejour
et
al.
2006;
Rossi
et
al.
2008).
This
is
most
likely
due
to
changes
in
cell
growth
factors,
extracellular
matrix
components,
DNA
damage,
increased
amounts
of
oxidative
species
and
the
decline
in
progenitor
cell
populations.
This
study
found
that
the
induction
of
the
Akt/mTORC1
pathway
stimulated
cell
hypertrophy,
thereby
improving
the
regenerative
capacity
of
hepatic
cells
in
mice.
The
inhibition
of
this
pathway
increased
cell
proliferation
alone.
The
induction
of
this
pathway
demonstrated
cell
hypertrophy
that
mimics
cell
behavior
during
pregnancy.
This
model
for
cell
growth
holds
potential
as
a
therapeutic
strategy
for
resolving
age
related
cell
deterioration
in
hepatocytes
(Gielchinsky
et
al.
2010).
Introduction
The
process
of
cellular
aging
is
observed
in
the
cell
types
of
all
organisms
including
liver,
skin,
bone,
hematopoietic,
nerve,
and
muscle
cells.
This
is
due
mainly
to
the
altered
function
of
many
of
the
cells
biological
processes
imparting
from
age
related
dysfunction.
Liver
regeneration
is
a
process
of
cell
growth
that
rapidly
increases
the
number
and
size
of
cells.
Loss
of
liver
parenchyma
is
a
growing
problem

2. 2
in
the
elderly
population
due
to
age
related
dysfunctions
such
as
fulminant
hepatitis
and
liver
tumors.
The
best
treatment
option
for
this
problem
is
currently
surgical
resection,
where
two-­‐thirds
of
the
liver
is
removed
in
a
partial
hepatecomy
procedure.
From
2008,
the
mortality
rate
of
elderly
patients
who
had
undergone
surgical
resectioning
increased
by
2%
per
year
(Asiyanbola
et
al.
2008).
This
demands
an
improved
clinical
model
for
regenerating
liver
tissue
to
combat
the
rise
of
mortality
rates
within
the
geriatric
population
as
a
consequence
of
age-­‐related
liver
dysfunction
(Gielchinsky
et
al.
2010).
It
was
observed
in
earlier
studies
that
heterochronic
parabiosis
could
improve
the
regenerative
capacity
of
striated
muscle
in
old
mice
and
increase
the
standard
rate
of
cell
proliferation
in
aged
livers
(Conboy
et
al.
2005).
The
state
of
heterochromic
parabiosis
is
the
connection
of
circulation
between
a
young
and
old
organism,
namely
during
pregnancy
where
organisms
partly
share
blood
systems
in
which
the
mother
is
exposed
to
a
very
young
fetus.
Studies
on
pregnancy
in
mice
have
shown
to
increase
the
baseline
proliferation
of
pancreatic
β-­‐cells
and
re-­‐myelination
of
neurons
after
cell
necrosis.
This
data
suggested
pregnancy
might
induce
the
increased
rate
of
cell
proliferation
in
the
elderly
(Shingo
et
al.
2003;
Gregg
et
al.
2007;
Karnik
et
al.
2007).
In
addition,
this
data
may
also
suggest
that
pregnancy
may
also
attenuate
to
the
age-­‐related
decline
of
liver
cell
regeneration.
This
study
focused
on
how
pregnancy
significantly
improved
liver
regeneration
in
older
mice
that
were
treated
with
chemical
inducers
to
activate
the
Akt/mTORC1
pathway,
which
were
further
analyzed
to
determine
how
the
chemical
and
signaling
components
of
this
pathway
were
affected
(Gielchinsky
et
al.
2010).
To
better
stimulate
cell
regeneration
in
aged
organisms,
researchers
mimicked
the
pregnancy
state
in
old
and
young
mice
to
study
the
effects
of
hypertrophy
stimulated
liver
cell
growth.
Hypertrophy
is
a
temporary
period
during
pregnancy
where
the
volume
of
an
organ
increases
as
the
size
of
its’
cellular
components
enlarge
(Gielchinsky
et
al.
2010).
The
Akt/mTORC1
pathway
is
a
key
mediator
in
cell
growth
of
several
organs
and
systems,
including
the
liver
(Manning
and
Cantley
2007;
Mullany
et
al.
2007;
Haga
et
al.
2009).
Since
the
Akt/mTORC1
pathway
has
shown
to
be
necessary
for
cell
regeneration,
the
inhibition
of
this
pathway
blocks
hypertrophy
but
still
increases
overall
rate
of
cell

3. 3
proliferation
(hyperplasia).
The
liver
generally
has
a
high
capacity
to
regenerate
after
injury
and
also
adjust
its’
size
to
the
host.
After
a
partial
hepatecomy
procedure,
the
liver
parenchyma
has
been
observed
to
regenerate
as
quickly
as
10-­‐12
hours
post-­‐
surgery,
terminating
the
growth
process
after
three
days
(Michalopoulos
et
al.
1997).
Previous
studies
have
demonstrated
that
older
mice
have
a
lower
rate
of
cell
proliferation
than
that
of
younger
mice
(Asiyanbola
et
al.
2008).
Researchers
mimicked
pregnancy
in
mice
after
a
partial
hepatecomy
procedure
that
activated
the
Akt/mTORC1
pathway,
which
sufficed
to
induce
a
hypertrophic
state
in
liver
cells.
This
resulted
in
improved
cell
regeneration
capacity
and
lifespan
in
the
aged
mice,
ultimately
reducing
their
mortality
rate
for
their
age
group.
This
switch
in
the
Akt/mTORC1
signal
pathway
from
hyperplasic
to
hypertrophic
activity
can
be
utilized
as
a
potential
therapy
for
patients
experiencing
a
loss
in
cell
efficiency
and
growth,
more
specifically
in
the
elderly
who
have
lost
their
regenerative
capacity
because
of
age-­‐related
disease.
This
module
of
inductive
hypertrophy
is
ideal,
as
it
stands
relatively
resistant
to
the
effects
of
aging
(Gielchinsky
et
al.
2010).
Materials
and
Methods
The
study
examined
how
pregnancy
induces
hypertrophy
in
aged
liver
tissue
by
switching
the
hyperplasmic
proliferation
of
cells
to
a
hypertrophic
state
observed
during
pregnancy.
It
was
hypothesized
that
cell
growth
rates
would
increase
after
partial
hepatecomy
in
older
pregnant
mice
as
much
as
(or
more
than)
the
rate
of
cell
regeneration
observed
in
younger
pregnant
mice
if
cell
regeneration
favored
a
hypertrophic
model
(Gielchinsky
et
al.
2010).
Aged
and
young
mice
were
spilt
into
two
groups
by
age:
mice
(aged)
10-­‐
12
months
old
and
pregnant
or
near-­‐pregnant
(young)
mice
about
3
months
old.
They
were
further
subdivided
into
pregnant
and
non-­‐
pregnant
mice,
totaling
four
groups
of
mice.
The
genetic
background
for
all
mice
was
C57
Black.
Mating
females
with
males
induced
a
pseudo
–
pregnancy
state
in
which
the
presence
of
vaginal
plugs
affirmed
if
the
pregnancy
state
was
successfully
mimicked
in
the
female
(Gielchinsky
et
al.
2010).

4. 4
Immunohistochemical
analysis
of
liver
cross-­‐sections
(5
µm
thick)
taken
from
mice
that
were
injected
with
BrdU
in
order
to
study
the
rate
of
liver
regeneration
over
the
course
of
four
days.
The
average
cell
size,
cell
proliferation,
and
survival
rate
were
assayed
in
varying
groups
of
mice
(Gielchinsky
et
al.
2010).
The
Akt/mTORC1
pathway
is
a
key
mediator
of
cell
growth
in
many
systems,
so
researchers
wanted
to
analyze
the
components
of
this
pathway
using
Western
blots
of
liver
cross
sections
from
pregnant
and
non-­‐pregnant
mice.
Liver
tissue
samples
were
collected
on
Days
1,
2,
and
4
following
a
partial
hepatecomy
procedure.
To
determine
the
significance
of
this
pathway,
mice
were
treated
with
rapamycin,
an
inhibitor,
which
blocks
the
mTORC1
pathway
by
binding
to
its
intracellular
receptor
preventing
cell
growth,
proliferation
and
motility
(Huang
et
al.
2003).
To
induce
the
hypertrophy
model
along
this
pathway,
mice
were
also
injected
with
bpV/phen
so
the
pathway
would
potentially
favor
cell
hypertrophy
instead
of
cell
hyperplasia.
Results
Recent
studies
have
shown
that
heterochromic
parabiosis
can
restore
the
regenerative
capacity
of
muscle
tissue
in
older
mice
and
increase
the
basic
rate
of
cell
proliferation
in
liver
tissue
(Conboy
et
al.
2005).
The
state
of
heterochromic
parabiosis
exists
during
pregnancy
when
there
is
a
connection
between
the
circulations
of
an
old
and
young
organism.
To
expand
on
conclusions
from
this
previous
study,
researchers
wanted
to
examine
how
pregnancy
influenced
the
decline
in
age-­‐related
regeneration
capacity
of
the
liver.

5. 5
Figure
1:
A-­‐C
Serial
magnetic
resonance
imaging
(MRI)
was
used
to
assay
the
process
of
liver
regeneration
post-­‐hepatecomy
in
non-­‐pregnant
and
near
term
pregnant
mice
(3mths)(see
Fig.
1A).
The
application
of
MRI
analysis
has
shown
to
be
the
best
technique
at
measuring
liver
volume
than
CT
imaging.
MRI’s
produce
a
higher
image
quality,
mainly
due
to
the
developments
of
fast
breath
hold-­‐
sequences,
a
modified
scanning
technique
to
produce
a
consistent
image
quality,
and
liver
specific
contrast
agents
(Inderbitzin
et
al.
2004;
Prokop
2001).
This
enabled
scientists
to
study
individual
mice
along
the
regeneration
process.
The
hatched
lines
denote
the
liver
contours
in
the
two
different
groups
of
aged
pregnant
and
non-­‐pregnant
mice,
which
showed
that
liver
regeneration
is
more
prominent
in
aged
pregnant
mice
because
of
the
visible
increase
in
liver
volume
overall
in
the
cross
sections
observed
(Fig.
1A)
(Gielchinsky
et
al.
2010).
The
group
of
non-­‐pregnant
mice
post-­‐surgery
showed
an
82%+-­‐8%
increase
in
liver
volume
two
days
following.
The
aged
mice
only
showed
a
46%
increase
in
liver
regeneration.
However,
there
was
a
dramatic
increase
in
the
group
of
aged
pregnant
mice,
which
showed
a
96%+-­‐
increase
in
liver
volume
restored
within
two
days
after
surgery
(Fig.
1C).
The
total
liver
volume
was
compared
to
average
liver
volume
restored
to
the
original
size
after
the
partial
hepatecomy
(mean+-­‐SEM).
Two
days
after,
photographs
representative
of
the
liver
removed
from
aged
pregnant
and
non-­‐pregnant
mice
after
surgery
showed
an
increase
in

6. 6
liver
size
of
the
pregnant
mice.
Regardless
of
how
liver
regeneration
improves
after
surgical
resectioning,
even
in
the
window
of
Days
2-­‐3
when
the
highest
rate
of
regenerative
activity
occurs
in
liver
tissue
(Michalopoulos
et
al.
1997),
the
older
mice
that
were
pregnant
showed
a
substantial
increase
in
organ
size
as
opposed
to
those
mice
that
were
not
pregnant
that
underwent
the
same
procedure.
(Fig.
1B)(Gielchinsky
et
al.
2010).
The
percent
of
restored
liver
volume
was
assayed
on
Days
0,
1,
2,
and
5
days
after
surgery
in
four
different
groups
of
old
and
young,
pregnant
and
non-­‐pregnant
mice
after
the
hepatecomy.
The
portion
of
the
liver
volume
that
was
regenerated
after
surgery
was
recorded
earlier
by
MRI
imaging
(Fig.
1A)
then
compared
as
a
percentage
of
the
total
liver
volume
that
was
present
before
the
hepatecomy
procedure
(Fig.
1C).
The
P
values
were
calculated
for
aged
pregnant
mice
(n=5)
and
compared
to
values
obtained
for
aged
non-­‐pregnant
mice
(n=5)
using
a
Students
T-­‐test.
The
values
obtained
after
Day
2
show
the
percent
of
liver
volume
restored
after
hepatecomy
in
the
aged
pregnant
mice
was
statistically
significant
(P<0.001),
therefore
it
can
be
concluded
the
state
of
pregnancy
attributes
to
the
process
of
liver
regeneration
in
aged
mice
(Fig.
1C)(Gielchinsky
et
al.
2010).
The
aged
pregnant
mice
showed
the
highest
rate
of
liver
volume
regenerated
over
the
5
days
after
the
surgery,
while
the
aged
non-­‐
pregnant
mice
had
the
lowest
percent
of
liver
volume
regenerated.
The
young
pregnant
and
non-­‐pregnant
mice
showed
similar
rates
of
liver
volume
restored
after
the
partial
hepatecomy
procedure
and
regeneration
of
liver
tissue
was
consistent
over
the
course
of
5
days.
Liver
regeneration
normally
begins
with
a
priming
phase
followed
by
a
regenerative
spurt
where
hepatocytes
enter
the
cell
cycle
(Taub
2004;
Michalopoulos
2007).
To
explain
the
increase
in
liver
regeneration
in
aged
pregnant
mice,
researchers
hypothesized
that
pregnancy
enhances
liver
regeneration
by
shortening
the
priming
phase
prior
to
the
cell
cycle
or
by
recruiting
a
large
number
of
hepatocytes
into
the
cell
cycle
(Gielchinsky
et
al.
2010).
To
test
this
hypothesis,
aged
pregnant
and
non-­‐pregnant
mice
were
injected
with
BrdU
(thymidine
analog
5-­‐bromo-­‐2-­‐deoxyuridine)
at

7. 7
several
time
points
after
the
partial
hepatecomy
(Gielchinsky
et
al.
2010).
The
analog
BrdU
is
a
synthetic
nucleotide
that
is
commonly
used
in
the
detection
of
proliferating
cells
in
living
tissue.
It
is
incorporated
into
the
S
phase
of
the
cell
cycle
of
newly
synthesized
DNA
in
replicating
cells.
Antibodies
specific
for
BrdU
are
used
to
detect
this
chemical
using
immunochemistry
techniques,
thereby
indicating
which
cells
are
actively
replicating
their
DNA
(Chien
2008).
Figure
2:
A-­‐E
The
uptake
of
BrdU
by
the
cells
was
assayed
using
immunochemistry.
It
was
observed
that
a
large
increase
in
cell
proliferation
occurred
after
injection
in
the
non-­‐pregnant
group
48
to
96
hours
following
the
hepatecomy.
Each
data
point
represents
a
single
mouse
for
each
group.
The
horizontal
lines
show
average
value
for
each
group
over
the
course
of
four
days
after
the
partial
hepatecomy
procedure.
The
non-­‐pregnant
mice
showed
a
higher
affinity
for
the
uptake
of
BrdU
in
each
of
the
four
groups.
The
pregnant
mice
showed
a
very
low
uptake
of
the
BrdU
labeled
hepatocytes
at
any
of
the
time
points
measured
(Fig.
2A)(Gielchinsky
et
al.
2010).

8. 8
To
ensure
that
researchers
did
not
miss
a
specific
time
point
crucial
to
BrdU
uptake,
the
analog
was
placed
into
the
drinking
water
4
days
after
the
procedure
to
make
sure
that
the
cells
entering
S
phase
would
uptake
the
BrdU.
The
immunostaining
analysis
showed
that
pregnant
mice
had
few
hepatocytes
labeled
with
BrdU
as
compared
to
non-­‐pregnant
mice
that
showed
a
more
efficient
uptake
of
82%+-­‐7%
(Fig
2B).
Pregnancy
also
affected
the
liver
regeneration
in
young
mice,
in
which
BrdU
uptake
was
observed
as
92%+-­‐1%
in
non-­‐pregnant
mice
and
5%+-­‐1%
in
pregnant
mice
(Gielchinsky
et
al.
2010).
E-­‐cadherin
stained
images
(Fig.
2C)
were
used
to
observe
changes
in
cell
size
between
pregnant
and
non-­‐pregnant
mice
after
the
hepatecomy.
E-­‐
cadherin
is
a
glycoprotein
necessary
for
cell-­‐to-­‐cell
adhesion
and
epithelial
junction
formation.
Using
immunochemistry
to
stain
E
cadherin
junctions
will
clearly
show
the
expansion
in
cell
size
and
number
of
epithelial
cells
(Charafe-­‐Jauffret
et
al.
2004).
On
Day
0,
the
pregnant
mice
showed
a
relative
increase
in
cell
size
of
424+-­‐13
µ2when
compared
to
the
pregnant
mice
who
had
an
average
cell
size
of
307+-­‐
29µ2.
At
day
four,
the
cell
size
of
pregnant
mice
increased
significantly
after
partial
hepactomy
when
compared
to
the
cell
size
of
non-­‐pregnant
mice
(Gielchinsky
et
al.
2010).
These
observations
suggested
that
the
increase
in
liver
regeneration
of
aged
pregnant
mice
was
a
function
of
cell
growth
rather
than
cell
proliferation.
The
aged
pregnant
mice
showed
a
66%
increase
in
liver
regeneration
in
the
cross-­‐section
area,
while
the
aged
non-­‐pregnant
mice
only
showed
a
13%
increase
in
liver
regeneration
due
to
the
enlargement
in
cell
size
(Fig.
2D
and
2E).
Figure
2D
shows
the
distribution
of
cell
size
changing
over
the
course
of
four
days
post-­‐hepatecomy
(Gielchinsky
et
al.
2010).
The
pregnant
mice
had
a
significant
increase
in
the
average
cell
size
per
day
as
opposed
to
the
non-­‐pregnant
mice.
The
non-­‐pregnant
mice
had
an
increase
in
average
cell
size
most
notably
on
Day
2
but
declined
in
size
when
observed
at
Day
4.
Each
point
on
the
graph
represents
a
group
of
2-­‐3
mice
with
four
liver
cross
sections
analyzed.
Each
point
harbors
a
count
of
at
least
100
cells
for
each
mouse
(Gielchinsky
et
al.
2010).
Figure
2E
represents
the
percent
of
cell
size
in
livers
after
the
hepatecomy
in
pregnant
and
non-­‐pregnant
mice.
The
results
were
gathered
on
Days
0,
1,
2
and
4
following
the
procedure.
On
Day
0
and
1,

9. 9
about
32%
percent
of
cells
in
non-­‐pregnant
mice
were
on
average
between
200-­‐300µ2
in
size.
The
tissue
observed
in
the
pregnant
mice
had
a
majority
of
34%
regenerated
cells
sized
between
300-­‐400µ2
from
Day
2
to
Day
4,
showing
the
majority
of
hepatocytes
in
the
cross
sections
had
increased
in
size
and
the
overall
distribution
of
cell
size
remained
consistent
after
Day
2
following
surgery.
Non-­‐pregnant
mice
showed
a
significant
spike
in
cell
size
on
Day
1
following
the
procedure.
The
majority
of
cells
undergoing
the
process
of
cell
hypertrophy
increased
in
size
within
the
range
of
500-­‐600µ2,
from
22%
to
about
35%
between
Day
0
to
1.
From
Day
2
to
Day
4,
the
cells
of
pregnant
mice
had
a
substantial
increase
in
hypertrophy,
with
a
majority
of
the
cell
population
sized
in
the
range
of
700-­‐800µ2.
When
comparing
the
extent
of
proliferation
and
hypertrophy
of
non-­‐
pregnant,
mid-­‐pregnant,
and
late
pregnant
mice,
results
showed
the
hypertrophy
model
is
induced
and
gradually
takes
dominance
during
the
pregnancy
state
(Table
1).
Together
this
data
shows
that
liver
regeneration
in
aged
pregnant
mice
5
days
after
partial
hepatecomy
results
from
cell
hypertrophy.
The
percent
of
cell
size
was
compared
in
varying
stages
of
pregnancy
along
with
the
uptake
of
BrdU.
The
uptake
of
BrdU
was
more
prominent
in
the
non-­‐pregnant
mice,
but
also
showed
the
cell
size
increase
five
days
after
surgery
was
relatively
low.
The
late
pregnant
and
pseudo-­‐
pregnant
mice
had
the
lowest
rates
of
BrdU
uptake
yet
they
also
showed
the
most
dramatic
increase
in
cell
size
(Table
1).
Previous
studies
indicated
that
pregnancy
induced
slight
liver
growth
due
to
cell
hypertrophy
(Kennedy
et
al.
1958;
Hollister
et
al.
1987).
Restoration
of
liver
mass
was
also
shown
to
occur
in
situation
where
treatments
with
dexmethasone
or
5-­‐florouracil
(Nagy
et
al.
2001)
to
induce
liver
hyperplasia,
where
cells
proliferate
as
opposed
to
increase
in
size.
This

10. 10
collective
data
indicates
that
hyperplasia
and
hypertrophy
are
two
potential
models
for
liver
regeneration.
The
results
from
this
study
indicate
that
the
physiological
state
of
pregnancy
causes
a
switch
from
liver
regeneration
based
on
cell
proliferation
to
liver
regeneration
mediated
by
cell
growth.
To
study
the
fate
of
hypertrophied
hepatocytes,
late
pregnant
mice
were
subjected
to
a
partial
hepatecomy.
This
time,
BrdU
was
placed
into
the
drinking
water
only
after
delivery
five
days
post-­‐hepectomy.
The
control
mice
were
aged
pregnant
mice
as
well
but
they
did
not
undergo
the
surgical
procedure.
It
was
found
that
the
hypertrophic
hepatocytes
that
are
generated
in
pregnant
hepatectomized
mice
returned
to
increased
cell
proliferation
activity
after
delivery
(Fig.
S5-­‐see
attached).
Immunochemistry
was
used
to
determine
the
incorporation
of
BrdU
in
hepatocytes.
This
suggests
that
pregnancy
related
hypertrophy
is
modulated
by
a
hormone
or
substance
throughout
pregnancy,
yet
returns
to
normal
levels
(as
seen
in
the
non-­‐regnant
mice)
after
delivery
(Gielchinsky
et
al.
2010).
Liver
sections
were
immunostained
for
cell
cycle
regulators
p53,
p21,
and
p27.
Levels
of
p27
did
not
differentiate
between
non-­‐pregnant
and
pregnant
mice.
The
level
of
p21
and
p53
were
up
regulated
after
the
partial
hepatecomy
in
non-­‐pregnant
mice,
but
was
not
seen
in
the
pregnant
mice
(Fig.
S6-­‐see
attached).
This
showed
that
the
up
regulation
of
these
two
cell
regulators
respond
to
hepatocyte
proliferation,
which
was
not
observed
in
pregnant
mice
(Fig.
S6-­‐see
attached)
(Gielchinsky
et
al.
2010).
The
Akt/mTORC1
pathway
has
proven
to
be
necessary
for
cell
growth
in
many
systems
(Manning
and
Cantley
2007).
The
researchers
wanted
to
assay
the
significance
of
Akt/mTORC1
signaling
in
liver
cell
regeneration
of
aged
pregnant
mice.

11. 11
Figure
3:
A-­‐B
Western
blotting
of
liver
extracts
were
taken
on
Days
1,
2,
and
4
showed
that
the
rate
of
phosphorylation
of
Akt,
S6
kinase,
and
4E-­‐BP1
(Fig.
3A),
all
of
which
are
essential
signaling
proteins
of
the
mTORC1
pathway.
The
S6
kinase
is
a
protein
involved
in
signal
transduction;
4E-­‐BP1
mediates
the
regulation
of
protein
translation
by
hormones,
growth
factors
and
other
stimuli
that
signal
through
the
mTORC1
pathway
(Pause
et
al.
2004);
Akt
is
a
protein
kinase
that
plays
a
role
in
the
cell
cycle.
When
Akt
is
activated,
the
protein
can
overcome
cell
cycle
arrest
in
the
G1
and
G2
phase
and
allow
for
the
proliferation
and
survival
of
cells
(Kandel
et
al.
2002;
Ramaswamy
et
al.
1999).
The
technique
of
Western
blotting
is
useful
to
detect
one
protein
in
a
mixture
of
many
proteins
while
also
giving
information
about
the
size
of
a
protein
accumulation
in
cells.
Western
blots
are
dependent
on
the
quality
of
antibody
used
to
probe
for
the
protein
of
interest
as
well
as
how
specific
an
antibody
is
for
a
particular
protein.
Prior
to
hepactomy,
mice
were
treated
with
the
phosphatase
and
tensin
PTEN
inhibitor
bpV/phen,
which
has
been
shown
to
activate
the
Akt/mTORC1
pathway
(Fig.
3A).
BpV/phen
is
a
protein
phospotase
inhibitor
and
an
insulin
kinase
receptor
activator.
For
this
study,
it
was
used
because
it
arrests
proliferation
of
cells
that
are
transitioning
into
the
G2/M
phase
of
the
cell
cycle
(Yale
et
al.
1995;
Posner
et
al.
1994).

12. 12
Results
showed
that
levels
of
S6,
Akt,
and
4E-­‐BP1
proteins
were
expressed
significantly
more
in
the
cells
of
pregnant
mice.
Day
2
was
the
time
frame
in
which
the
cells
underwent
the
most
protein
activity
in
both
pregnant
and
non-­‐pregnant
mice
until
Day
4,
where
protein
expression
began
to
decline
slightly.
The
V
segment
of
the
blot
represents
the
marker
ladder
that
shows
the
expression
of
proteins
specific
to
the
Akt/mTORC1
pathway.
This
group
consisted
of
young
mice
that
were
treated
with
bvP/phen
before
partial
hepatecomy.
It
shows
the
standard
level
of
proteins
involved
in
activating
the
Akt/mTORC1
pathway
(Fig.
3A).
The
increase
of
protein
expression
in
cells
demonstrated
that
bvP/phen
is
sufficient
to
activate
the
Akt/mTORC1
pathway.
Images
were
taken
of
liver
cross
sections
and
stained
using
immunofluorescent
dyes
to
mark
E-­‐cadherin,
BrdU,
and
4E-­‐BP1
(Fig
3B).
The
majority
of
cells
in
the
liver
cross
section
of
pregnant
mice
showed
a
visible
increase
in
the
phosphorylation
of
4E-­‐BP1.
The
staining
of
E-­‐
cadherin
junctions
showed
the
dramatic
expansion
in
cell
size
between
Days
3
and
4.
The
low
uptake
of
BrdU
was
seen
in
only
the
pregnant
group
of
mice.
Earlier
tests
showed
that
BrdU
uptake
was
higher
in
non-­‐
pregnant
mice
and
accompanied
by
an
increase
in
cell
proliferation.
The
pregnant
mice
had
a
lower
uptake
of
BrdU,
but
also
showed
a
higher
percent
of
liver
volume
regenerated
due
to
hypertrophy
(Gielchinsky
et
al.
2010).
Together
these
results
suggest
that
pregnancy
increases
liver
regeneration
by
cell
hypertrophy
that
is
mediated
by
the
Akt/mTORC1
pathway
(Gielchinsky
et
al.
2010).
The
mice
were
treated
with
rapamycin,
an
inhibitor
that
induces
a
strong
anti-­‐proliferation
response
in
liver
cells,
as
seen
in
earlier
studies
(Sanders
et
al.
2008).
Blocking
cell
proliferation
in
the
Akt/mTORC1
pathway
was
hypothesized
to
favor
the
process
of
cell
hypertrophy
more.
Young
non-­‐pregnant
mice
were
treated
with
bpV⏐phen
hormones
to
activate
the
Akt
-­‐pathway
through
hypertrophy
(Gielchinsky
et
al.
2010).
The
rates
of
cell
proliferation
were
compared
pre
and
post
hepatecomy
in
mice
treated
with
bpV|phen
alone,
rapamycin
alone,
or
combined
treatment
using
both
mediators.

13. 13
Figure
4:
A-­‐F
Immunohistochemical
analysis
of
liver
cross-­‐
sections
(5
µm
thick)
taken
from
mice
treated
with
bvP/phen
showed
a
low
cell
proliferation
rate
although
increased
cell
growth
was
observed
in
the
cross
sectional
area
by
115%(Fig
4C).
The
rapamycin
treatment
alone
was
proven
to
reduce
post-­‐hepatecomy
proliferation
in
cells
(Sanders
et
al.
2008).
Cell
proliferation
was
being
inhibited
by
the
rapamycin
treatment,
thus
tissue
regeneration
was
attributed
to
hypertrophic
cell
activity
(Haga
et
al.
2009;
Gielchinsky
et
al.
2010).
Mice
treated
with
rapamycin
and
bpV/phen
showed
cell
growth,
but
the
presence
of
bpV
induced
the
cell
to
switch
from
hyperplasia
to
hypertrophy
(Fig.
4D).
Figure
4A
shows
an
immunochemical
stain
in
aged
pregnant
mice
two
days
after
a
partial
hepectomy.
BrdU
was
used
to
stain
cells
that
proliferated
following
the
procedure.
The
second
group
of
pregnant
mice
treated
with
rapamycin
showed
increased
proliferation
even
though
they
were
administered
an
anti-­‐proliferative
drug.
Immunofluorescent
staining
of
E-­‐cadherin
showed
that
cell
size
was
larger
in
pregnant
mice
without
rapamycin
than
those
who
were
given
the
drug,
explaining
the
paradox
observed
in
tissue
that
underwent
cell
growth
in
the
presence
of
an
inhibitor.

14. 14
Figure
4B
is
an
analysis
of
the
data
from
the
stain
that
shows
the
distribution
of
cell
sizes
contributing
to
the
total
volume
of
regenerated
liver
tissue.
The
pregnant
mice
had
a
higher
index
of
cells
ranging
in
different
sizes.
The
majority
of
liver
volume
in
pregnant
mice
were
attributed
to
larger
cells
ranging
between
600-­‐700µ2
in
size.
Pregnant
mice
treated
with
rapamycin
showed
a
similar
increase
in
cell
regeneration,
but
the
majority
of
cells
were
sized
between
500-­‐600µ2.
The
non-­‐pregnant
mice
also
showed
an
increase
in
cell
regeneration,
but
the
size
of
cells
observed
in
pregnant
mice
confirmed
that
this
was
due
to
cell
proliferation
rather
than
cell
hypertrophy.
BrdU
was
used
to
immunoassay
liver
cross
sections
taken
from
pregnant
and
non-­‐pregnant
mice.
One
group
of
non-­‐pregnant
mice
was
given
bpV/phen
and
the
other
was
left
untreated.
The
results
suggested
that
bpV/phen
activity
is
regulated
via
Akt/mTORC1
pathway
and
is
sufficient
to
activate
hypertrophy
in
non-­‐pregnant
mice
(Fig.
4C;
Fig.
S8)(Gielchinsky
et
al.
2010).