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0013.7227/96/$03
Endocrinology
00/o
Vol. 137, No. 8
Pm&d
LIZ U.S.A.
Copyright 0 1996 by The Endocrine Society
Expression of a constitutively
(c-Akt) in 3T3-Ll preadipose
Rick Magunl,
and Alexander
Boudewijn
Soriskyl*
activated form of protein kinase B
cells causes spontaneous differentiation
M. Th. Burgeringz,
Paul J. Coffers,
lLoeb Medical Research Institute,
Unwerslty
of Ottawa,
Ottawa,
Canada
CG Utrecht, and 3Hubrecht Laboratory, 3584 CT Utrecht, The Netherlands.
*To whom
correspondence
should
KlY
Dheerja
OG7; ZLaboratory
Pardasanil,
for Physiological
Ying Linl,
Chemistry,
Josee Chabotl,
Uhecht
University,
3584
be addressed.
Abstract
Insulin/IGF-1
is required
for differentiation
of 3T3-Ll adipose cells. Downstream
targets of insulin/IGF-1
that lead to adipocyte
differentiation
appear to include
Ras, phosphatidylinositol
(PI) 3-kinase,
Raf, and mitogen-activated
protein
kinase.
We have
tested whether
protein
kinase B (PKB), a serinelthreonine
kinase activated
by PI 3-kinase,
is sufficient
for 3T3-Ll
preadipose
cell
differentiation.
A plasmid
vector encoding
a version
of PKB that is constitutively
activated
(Gag-PKB)
was expressed
in 3T3-Ll
preadipose
cells (Gag-PKB
cells). Spontaneous
morphological
changes indicative
of adipocyte
differentiation
were observed
in
Gag-PKB
cells. The cells assumed
a spherical
shape and they acquired
characteristic
lipid droplets
that stained
positively
for
Oil Red 0. Northern
blot analysis detected upregulation
of LPL and aP2 mRNA,
specific
indicators
of adipocyte
differentiation.
Our data demonstrate
that constitutive
activation
of PKB is sufficient
to trigger adipocyte
differentiation.
Protein Kinase B (PKB),
also named
Rat, is a 60 kDa
serine/threonine
kinase that has sequence homology
with
protein kinase C (PKC) and protein kinase A (PKA) [1,2].
The same kmase is also recognized
to be the product
of
c-nkt, the cellular
homolog
of the viral oncogene,
v-akt
[3].
PKB possesses a pleckstrin
homology
(PH) domain,
and its
catalytic
domain has a high degree
of similarity
to the
kinase domains
of PKC and PKA. The l’H domain
is
located at the amino-terminus
within
a region responsible
for homodimerization
[4].
F’KB is activated
by insulin and is a newly
identified
downstream
target of phosphatidylinositol
(PI) 3-kinase,
based on inhibition
of PI 3-kinase, either by wortmannin
or
LY294002, or by overexpression
of a deletion mutant
of the
p85 subunit of PI 3-kinase [5,6]. PKB has also recently
been
demonstrated
to activate the p70 S6 kinase [S], an insulinresponsive
enzyme that regulates the translation
of mRNAs
that encode proteins involved
in cell cycle control [8].
The potential role of PKB in insulin-dependent
cellular
responses
is still uncertain.
Differentiation
of 3T3-Ll
preadipose
fibroblast-like
cells into rounded,
lipid-laden
adipose cells is dependent
on insulin/insulin-like
growth
factor-l
(IGF-1)
[9].
Several
insulin/IGF-1
cytosolic
signaling proteins have been implicated
in adipogenesis
by
genetic approaches,
including
Ras, Raf-1, and mitogenactivated protein kinase (MAPK)
[10,11,12]. Other studies,
using
rather
selective
inhibitors,
have
shown
that
PI 3-kinase and p70 56 kinase are also required
for 3T3-Ll
preadipose
cell differentiation
[13,14]. The involvement
of PI 3-kinase and p70 S6 kinase in insulin/IGF-l-induced
adipogenesis
suggests that PKB may link PI 3-kinase to p70
S6 kinase signals. In the present study, we have expressed
a
constitutively
activated
PKB in 3T3-Ll
preadipose
cells,
and show that it is sufficient
to induce spontaneous
differentiation
in the absence of added insulin/IGF-1.
Received:
02/16/96
Materials
and Methods
Preadioose
3T3-Ll cells were obtamed
from Amencan
Type Culture
Collection’and
maintained
at low passage. All cell culture
mater&
were
from Gibco BRL A Stratagene mammalian
transfecbon
kit was used
The
enhanced chem~luminescence
(ECL) kit was from Amersham.
Kodak X-AR
fdm was purchased
from Eastman Kodak, Rochester, NY. lnsuhn was from
Boehnnger Mannheim
E,r!a,of~
expresston vectors and DNA probes
Plasmid pMEXneo was a gift from H Hanafusa
Plasmld
pSG5-PKBGag
was constructed
as described
prewously
[5]
Upon
transfectlon
of
mammahan
cells, It generates a fusion protein
of 85 kDa. LPL and aI’2
probes were kmdly provrded
by M C. Scholtz and H. Green, respectn’ely
We thank B.M. Splegelman
for prowding
us with the 3684 probe (151 AnbPKB antibodv
IS dlrected
aaainst a C-termmal
pepbde
of PKB (c-Akt) as
described
[5i.
3T3-Ll preadlpose
cells of low passage number were plated 24 h before
transfecbon
(100,000 tells/60-mm
plate) m Dulbecco’s
Modlfled
Eagle’s
Medium
(DMEM)
supplemented
iO% calf serum and 100 wg/L penicillin
and 100 ma/L streptomycm,
and zrown for 24 h. DNA
was added
as a
coprecipitare
with &i&n
phosp&e
for 4 h. For cotransfection,
2 pg of
pMEXneo was mixed with 10 pg of pSG5-PKBGag
(Gag-PKB cells). Control
transfection
was wth 4pg pMEXneo
alone (Control cells). Total DNA per
plate was 20 pg, with addlhon
of salmon
sperm DNA,
to optmuze
transfecbon
efficiency
The cells were rmsed and Incubated
m DMEM
supplemented
as above. The antlblotlc
G418 was added to the medium
at
400 pg/ml to select for colorues that had stably Integrated the constructs
The cells were selected every 4 days At 18 days representabve
plates were
stained with 011 Red 0
Transfected
cells were washed
wth PBS and f,xed for 2 h m 10%
formalin.
A 0.35% Oil Red 0 ~sopropanol
solution
was diluted
wth equal
volume of water, filtered and added to the fixed cells for 2 h. After washmg,
the cells were mcubated
overnight at 4’C with Giemsa stain. Cells were
washed,
and
stained
triglyceride
droplets
were visualized
and then
photographed.
The differentiation
of 3T3-Ll
preadrpose
cells was performed
under
serum-free condibons
as described by us prewously
1161 Differentlatmn
was
assessed
visually,
then quantltated
by measurement
of glycerol phosphate
dehydrogenase
(GPDH) actiwty, a speclflc marker of terminal dlfferentlabon
m these cells 1171
3590
RAPID COMMUNICATIONS
Fig.1. Spontaneous
cells
(B,
dlfferenuatlon
of Gag-PKB
3TXLI
At 18 days post-transfecrwn.
cells were slauxd
preadlpose
cells uere aansfecred
8s descrked
and SubJecced to GJltl
selcctlon
control
cells(A).
WI~I hp~d-s~eclfic
011 Rod 0 as descrdxd
Representative
fields are shown here (Magnification
Gag-Pm
100x)
WID
3592
Endo.
1996
Vol 137 . No 8
COMMUNICATIONS
-Gag-PKB
28s
18s
- 69
_- PKB
B.
LPL
A
B
aP2
36B4
Fig. ‘2. Immunoblot
analysis
of Gag-PKB
expression.
Total cellular
protein
extracts from control
(lane A) and Gag-PKB
(lane B) cells
(pooled
colonies)
were separated
by 10% SDS PAGE, transferred
to
nitrocellulose
membranes,
and
imrnunoblotted
with
anti-m
antibody
as described.
The positions
of molecular
weight markers,
Gag-PKB and endogenous
PKB are indicated.
demonstrates
equal loading of RNA in all lanes as assessed
by 28s and 18s bands. Fig. 3B shows the Northern
blot after
hybridization
with probes for LPL and aP2 , as indicated.
Two separate
pools of Gag-PKB
colonies
demonstrate
induction
of LPL( lane 12). aP2 induction
is seen in one of
the two pools (lane 2). No such induction
of LPL or aP2
was observed
for control cells (lane 3). Control
cells were,
however,
completely
capable of undergoing
adipogenesis
when
exposed
to a differentiation
medium
containing
insulin (data not shown).
Since PI 3-kinase has been proposed
to be an upstream
modulator
of PKB, we determined
whether
inhibition
of
PI bkinase
by wortmannin
would block adipogenesis.
We
used a defined,
serum-free
differentiation
medium,
in
which insulin was the sole growth
factor. As was found in
previous
work studying
wortmannin
inhibition
of adipose
cell differentiation
using a serum-supplemented
medium
[13], wortmannin
inhibited
differentiation;
in or~~s;~z
approximately
50%. Differentiation
was assesse
logically,
and quantitated
by measurement
of GPDH.
GPDH
increased
from
29units/mg
protein
to
2114 units/mg
protein in differentiated
cells. The GPDH in
preadipose
cells undergoing
the same differentiation
protocol, but in the presence of 100 nM wortmannin,
was
protein.
The
insulin-stimulated
1062
units/mg
accumulation
of PI(3,4,5)P3,
the growth
factor-responsive
3-phosphorylated
inositol phospholipid
in preadipose
cells
[201, was inhibited
in parallel
fashion.
After
confluent
3T3-Ll preadipose
cells were exposed to 5 min of 100 nM
insulin,
PI(3,4,5)P3
increased
4-fold,
but pre-incubation
with
100 nM wortmannin
for 5 min prior to insulin
stimulation
completely
abrogated
this response.
These data,
together
with those previously
reported
1131, show that
PI 3-kinase
is required
for differentiation.
They provide
indirect support
for PKB in the Induction
of adipogenesis,
given that PI bkmase
has been reported
to activate PKB.
Fig. 3. Upregulation
of LPL and al?? mRNA
in Gag-PKB
cells. Total
cellular
RNA was isolated
from control
and Gag-PKB
cells @&d
colonies)
after 18 days of G418 selection.
0 17 pg of RNA was
resolved
on a formaldehyde
gel as described.
Lane 1,2 - RNA from
two separate
pools of Gag-PKB
cells, lane 3 - RNA from pooled
control
cells. Equivalent
amounts
of 28s and 185 RNA appear
in
each lane. Cs) RNA was then transferred
to Hybond
nylon
membrane,
and hybridized
with 32P-labelled
probes
for aP2, LPL,
and 36B4 (a further
control
for amount
of RNA),
as described.
Lanes 1,2, and 3 designated
as above.
Discussion
Our data demonstrate
that expression
of a constitutively
activated
form of PKB in 3T3-Ll cells induces spontaneous
adipose cell conversion.
Differentiation
of these cells was
assessed by positive Oil Red 0 staining and by upregulation
of differentiation-specific
mRNAs for LPL and aP2. These
results suggest that the programmed
induction
of gene
expression leading to adipogenesis
is subject to regulation
by
PKB.
Both within
as well as between
individual
Gag-PKB
colonies, the pattern
of adipose
conversion
was somewhat
heterogeneous,
in that each cell did not acquire
lipid to the
exact same extent.
The differences
in the degree
of
differentiation
between
Gag-PKB
colonies
might
be
accounted
for by variations
in the expression
level ot the
transfected
cDNA.
The heterogeneity
of adipogenesis
amongst the cells of a single Gag-PKB
colony could be due
to particularities
of the immediate
cellular environment,
such as whether
the cell is at the growing
edge of the
colony as opposed to near the center in a zone of relative
confluence.
WID
Differentiation
of 3T3-Ll preadipose
cells into the mature
phenotype
ensues after
confluence
is reached
(growth
arrest),
and requires the presence
of insulin/IGF-1.
These
factors stimulate
a limited round of post-confluent
mitoses,
defined
as the clonal expansion
phase of differentiation.
During
this
time,
the
interaction
of
trans-acting
differentiation
factors
with
regulatory
regions
of
differentiation-specific
genes is facilitated,
and a defined
temporal
sequence
of gene expression
ensues 1201. This
induction
of lipid
transport
proteins
and synthesizing
enzymes
results in the formation
of the characteristic
lipid
droplet.
The identities
of some of the downstream
targets
activated
by insulm/IGF-1
that produce
this differentiation
response are slowly emerging.
Expression
of an activated
version
of Ras or Raf-1
induces
spontaneous
3T3-Ll
preadipose
cell adipogenesis
[lO,ll],
in an identical fashion to
what we have shown here with Gag-PKB expression.
Insulin
activates Ras and Raf-1, and a dominant
negative
mutant
of
Ras that prevents
Ras activation
also inhibits Raf-1 activation
and cell differentiation.
These authors also noted that Raf-1
appeared
to induce
differentiation
by a pathway
distinct
from p42 MAPK or p90 S6 kinase [ll]. Another
group, using
a
phosphorothioate-oliogonucleotide-based
antisense
strategy, showed that differentiation
was dependent
on the
presence of p42/p44
MAPK
[12]. The role of p70 56 kinase
was not considered
in these studies.
PI 3-kinase also appears to play a role in differentiation
of
3T3-Ll preadipose
cells. Inhibition
of differentiation
of 3T3Ll preadipose
cells in serum-supplemented
medium
by
wortmannin,
corresponding
to
a decrease
in
the
accumulation
of PI(3,4,5)P3,
has been reported
[13]. Our
own work has shown
that the differentiation-inducing
insulin/IGF-1
increase
levels
of
PIf3,4,5)P3
factors
specifically,
with no response of PI(3,4)P2.
I’DGF , a growth
factor which does not induce differentiation,
raises levels of
both P1(3,4)P2 and PI(3,4,5)P3
[16]. This difference
in the
pattern
of PI 3-kinase
products
suggests
one possible
contributing
mechanism
to the differentiation
effect
of
insulin/IGF-1
versus
PDGF.
In this study,
we
have
confirmed
the effect of wortmannin
on differentiation,
using
a serum-free
differentiation
protocol,
and the effect
on
PI(3,4,5)P3
accumulation.
Insulin-stimulated
accumulation
of PI(3,4,5)P3 was completely
blocked by wortmarmin.
Since
inhibition
of PI 3.kinase reduced adipose cell conversion,
this
provides
indirect support that its downstream
target, PKB, is
involved
in this process.
More
work
to provide
direct
evidence of the role of endogenous
PKB in adipogenesis
will
be needed.
p70 S6 kinase,
also activated
by insulin,
has been
implicated
in the differentiation
of 3T3-Ll preadipose
cells,
based on the observation
that rapamycin
blocks adipogenic
differentiation
[14]. Rapamycin
is a specific inhibitor
of p70
56 kinase, and is thought
to act on an intracellular
receptor
immediately
upstream
of p70 S6 kinase [Zl]. Emerging
data
indicate that stimulation
of p70 S6 kinase by insulin involves
the sequential
upstream
activation
of PI 3-kmase
and PKB.
Inhibition
of
PI 3-kinase by selective
inhibitors,
or by
overexpression
of a deletion mutant
of the PI 3-kinase ~85
subunit, abrogates activation
of PKB [5,6,7] and of p70 S6
kinase [5]. An activated
form of I’KB can stimulate
p70 S6
kinase, even in the presence
of the PI 3-kinase
inhibitors,
placing PKB downstream
of PI 3-kinase, and upstream
of
p70 S6 kinase [5]. One proposed
mechanism
by which
PI
3-kinase
activates
PKB
is through
production
of
3-phosphorylated
inositol phospholipids,
based on in vitro
activation
of PKB by PI(3)I’
[6]. PKB activation
of p70 56
kinase appears to be mediated
by phosphorylation,
although
it is not clearly established
that p70 56 kinase is a direct
substrate of PKB [5,8].
We have found
that expression
of a constitutively
COMMUNICATIONS
3593
activated
PKB is sufficient
to induce adipogenesis.
Based on
the rapamycin-mediated
inhibition
of differentiation
of
preadipose
cells [14], the most
likely
proximal
target
of PKB is p70 S6 kmase. Other possibilities,
however,
cannot
be completely
excluded.
For example,
PKB has also been
reported
to phosphorylate
and inactivate
glycogen synthase
kinase-3 [22]. Further work will be directed at identifying
the
role of endogenous
PKB and p70 56 kinase in adipogenesis.
Acknowledgments
This work was supported
Medical
Research Council
Medical
Research Council
A.S.).
by an operating
grant from the
of Canada (MA-129321,
and by a
of Canada Scholarship
award (to
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