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Departamento de Bioquimica y Biologia Molecular II Instituto de Bioquimica Facultad de Farmacia Universidad Complutense 28040 Madrid, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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activation by
IGF-I. In contrast, IGF-I-induced fetal brown adipocyte proliferation
was PI 3-kinase-independent. Our results show for the first time an
essential requirement of PI 3-kinase in the IGF-I-signaling pathways
leading to fetal brown adipocyte differentiation, but not leading to
mitogenesis. In addition, protein kinase C seems to be a signaling
molecule also involved in the IGF-I differentiation pathways downstream
from PI 3-kinase. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PI 3-kinase is a family of heterodimeric enzymes composed of a p85- regulatory subunit and a p110 catalytic subunit (9, 10). PI 3-kinase is a lipid kinase capable of phosphorylating phosphoinositides at the 3'-position of the inositol ring (11), and these lipids have been postulated as second messengers (11, 12, 13). Although during recent years much information has beed accumulated on the role of PI 3-kinase activities in tyrosine kinase receptor signal transduction or vesicle trafficking, little is known about the possible role of PI 3-kinase in cell differentiation. In this regard, Kaliman et al. (14) have recently implicated PI 3-kinase as an essential positive regulator of terminal differentiation of skeletal muscle cells.
Fetal brown adipocyte primary cultures offer a nonfibroblastic mesenchymal cell model that has proven to be an excellent system in which to study both proliferation and differentiation processes (15, 16, 17, 18, 19) and signal transduction (20). These cells show a high level of IGF-I receptor mRNA expression and bear a high number of high-affinity IGF-I binding sites per cell. In fetal brown adipocyte primary cultures, IGF-I behaved as a mitogen per se in a p21 ras protein content-dependent manner (15, 17, 21). With regard to differentiation, rat brown adipocytes differentiate at the end of fetal life on the basis of two programs: the adipogenic program related to lipid synthesis and the thermogenic program related to heat production associated with the expression of the uncoupling protein (UP) to yield an identifiable and functional tissue at birth (19, 22). The UP expression constitutes a unique molecular marker that distinguishes this cell type from any other mammalian adipose cell. Our previous work has shown that IGF-I, which stimulates PI 3-kinase activity in brown adipocytes (20), is also capable of inducing the expression of both adipogenic (23) and thermogenic (17) genes. However, the signal transduction pathways by which IGF-I is involved in the adipogenic and thermogenic differentiation of fetal brown adipocytes have not yet been clarified.
Accordingly, in the present study we demonstrate that treatment of
brown adipocytes with PI 3-kinase inhibitors (wortmannin/LY294002), but
not with p70S6k inhibitor (rapamycin), impaired the
IGF-I-induced effect on the expression of adipogenic- and
thermogenic-related genes, while brown adipocyte proliferation remained
unaltered. Our results show for the first time that other signaling
molecules such as protein kinase C (PKC) may be involved in
IGF-I-induced brown adipocyte differentiation process.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(upper
panel), there was a 90% inhibition of IGF-I-stimulated PI 3-kinase
activity when cells were pretreated for 15 min with 10 nM
wortmannin, and total inhibition was observed with the dose of 20
nM.
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(lower panel) LY294002 inhibited IGF-I-stimulated PI
3-kinase activity in a dose-dependent manner: half-maximal effect was
elicited at 2.5 µM concentration and its maximal effect
at 10 µM.
To determine whether the effect of PI 3-kinase inhibitors on
IGF-I-stimulated PI 3-kinase activation was due to alterations in
IGF-I-stimulated receptor phosphorylation and/or phosphotransferase
activity, cells were either stimulated for 5 min with 10 nM
IGF-I or pretreated for 15 min with 20 nM wortmannin or 10
µM LY294002 and subsequently stimulated with 10
nM IGF-I for a further 5 min. Then, lysates were subjected
to immunoprecipitation with the Py72 anti-Tyr(P) antibody and assayed
for in vitro protein kinase activity as described in
Materials and Methods. Figure 1B is a representative
autoradiogram showing the tyrosine- phosphorylated proteins in the
immunoprecipitates after separation by SDS-PAGE. The presence of 10
nM IGF-I caused a marked increase in the tyrosine
phosphorylation of the 95-kDa band, which corresponds with the
Mr of the ß-subunit of the IGF-I receptor (20), no
phosphorylation being observed in the control cells. The level of
tyrosine phosphorylation of the 95-kDa band did not change
significantly when cells were pretreated with 10 µM
LY294002, this band being even higher upon 20 nM wortmannin
pretreatment.
In the case of the IGF-I receptor, PI 3-kinase is stimulated by
interaction of the p85-regulatory subunit of PI 3-kinase with
tyrosine-phosphorylated IRS-1. To study whether this interaction is
affected by wortmannin and LY294002, we prepared soluble cell lysates
after incubation of cells with both PI 3-kinase inhibitors and IGF-I as
described above. They were next immunoprecipitated with the 
p85
antibody and analyzed by Western blotting with the anti-Tyr(P) antibody
(4G10) (Fig. 1C). After IGF-I stimulation of brown adipocytes, p85 was
associated with tyrosine-phosphorylated IRS (either IRS-1 and/or
IRS-2). Wortmannin (20 nM) increased and LY294002 (10
µM) did not affect IGF-I-stimulated association of the
p85 subunit of PI 3-kinase with IRS-1/IRS-2. Since what is being
measured in this experiment (Fig. 1C) is both tyrosine phosphorylation
on IRS-1/IRS-2 and its interaction with p85, a possible explanation is
that IRS-1/IRS-2 is phosphorylated to a greater extent in the presence
of wortmannin. This might be associated with the greater tyrosine
kinase activity of the receptor in the presence of wortmannin seen in
Fig. 1B.
Effect of PI 3-Kinase Inhibitors on IGF-I-Stimulated Glucose
Transport in Brown Adipocytes
Recently it has been demonstrated, in newborn brown fat precursor
cells, that PI 3-kinase is involved in the mechanism of insulin-induced
glucose transport (27). The fact that glucose transport is induced in
fetal brown adipocytes upon IGF-I stimulation (20) prompted us to
investigate whether this effect could be blocked by PI 3-kinase
inhibitors in our fetal primary cells. Quiescent cells were treated for
10 min with 10 nM IGF-I and then incubated for a further 5
min in the presence of 2-deoxy-D(1-3H)glucose. At the same
time, another set of cells were pretreated for 15 min with various
doses of wortmannin or LY294002 and subsequently stimulated with IGF-I
as described above. As shown in Fig. 2A, IGF-I-induced
glucose transport was supressed by wortmannin in a dose-dependent
manner at the same doses that inhibited PI 3-kinase activity (Fig. 1A).
This inhibition was statistically significant at 10 and 20
nM concentration of wortmannin, total inhibition being
observed at 20 nM. When cells were pretreated with
LY294002, under the same experimental conditions (Fig. 2B),
IGF-I-induced glucose transport was also blocked at the same doses that
inhibited PI 3-kinase activity (significant inhibition at 5 and 10
µM concentration and total inhibition at 10
µM).
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. In the absence of IGF-I all the mRNA
levels analyzed remained very low, regardless of the presence of PI
3-kinase inhibitors in the culture medium. Upon treatment of cells for
24 h with 10 nM IGF-I, a significant accumulation of
FAS, ME, G3PD, and ACC mRNAs occurred, relative to untreated cells.
Interestingly, when brown adipocytes were cultured in the presence of
IGF-I together with wortmannin or LY294002 (at doses which completely
inhibited PI 3-kinase enzymatic activity), the induction of all the
adipogenic-related mRNAs analyzed was completely prevented, their
expression remaining at the basal levels observed in control cells.
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, upon treatment with IGF-I, a significant increase in the ME protein
content relative to untreated cells (6- to 7-fold increase) occurred,
and this effect was totally precluded in the presence of wortmannin (20
nM) or LY294002 (10 µM) together with IGF-I.
Furthermore, the changes observed in ME protein content in response to
IGF-I and PI 3-kinase inhibitors were parallel to changes in its
enzymatic activity, as shown in Fig. 4B.
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, and at the end of the culture period Nile Red fluorescence was
analyzed in the Flow cytometer and quantitated in arbitrary units as
shown in Fig. 5. The presence of 10 nM IGF-I
in the culture medium for 24 h significantly doubled the lipid
content detected in control cells. However, when cells were cultured
with IGF-I together with 20 nM wortmannin or 10
µM LY294002, lipid content reached a level similar to
that observed in control cells.
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. Upon treatment with IGF-I, a significant accumulation
in UP mRNA relative to control cells (cultured in the absence of IGF-I)
occurred. However, UP mRNA expression induced by IGF-I was totally
prevented when either wortmannin or LY294002 was added to the culture
medium.
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is a
representative Northern blot of brown adipocytes cultured for 24 h
with 10 nM IGF-I in both the absence and presence of 25
ng/ml rapamycin, which has been proved to completely block
p70S6K activity. As shown in Fig. 7, the expression of both
adipogenic (FAS, ME, G3PD, ACC) and thermogenic (UP) markers was partly
precluded in the presence of rapamycin compared with IGF-I-treated
brown adipocytes.
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Activity Is Inhibited by Wortmannin and
LY294002 in Brown Adipocytes, which has recently found
to be involved in adipogenesis (35), is activated by
phosphatidylinositol 3,4,5-trisphosphate (PIP3) (36).
Furthermore PKC is expressed in brown adipocytes, and its activity
is induced by IGF-I (20). These previous findings prompted us to
investigate whether the blockade of PI 3-kinase could interfere with
IGF-I-induced PKC activity in brown adipocytes. Quiescent cells (20
h serum-starved) were either stimulated with IGF-I (10 nM)
for 5 min or pretreated for 15 min with 20 nM wortmannin or
10 µM LY294002 before the addition of IGF-I for a further
5 min. Then, cells were lysed and assayed for PKC activity in the
immune complexes as described in Materials and Methods. As
shown in Fig. 8A, myelin basic protein (MBP)
phosphorylation induced in the presence of IGF-I was totally prevented
by both wortmannin and LY294002 pretreatments and reached the basal
levels observed in control cells.
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activity was
affected by the blockade of p70S6K activity. Quiescent
cells were either stimulated with IGF-I (10 nM) for 5 min
or pretreated for 15 min with 25 ng/ml rapamycin before the addition of
IGF-I for a further 5 min. Then, cells were lysed and assayed for
PKC activity in the immune complexes. As shown in Fig. 8B, when
fetal brown adipocytes were pretreated with the immunosuppressant
rapamycin before the addition of IGF-I, PKC activity remained
unchanged relative to that observed in IGF-I-induced cells.
PI 3-Kinase Inhibitors Did Not Block IGF-I-Induced Mitogenesis or
Proliferating Cellular Nuclear Antigen (PCNA) Expression in Fetal Brown
Adipocytes
Based on the fact that IGF-I is a complete mitogen in fetal
brown adipocyte primary cultures (15, 17), we proceeded finally to
investigate whether PI 3-kinase inhibitors could also block
IGF-I-induced mitogenesis in our cells. Quiescent (20 h serum-starved)
brown adipocytes were cultured for 24 h with 1.4 nM
IGF-I (which maximally stimulates brown adipocyte growth) in both the
absence and presence of 20 nM wortmannin and 10
µM LY204002, and [3H]thymidine
incorporation was measured during the last 4 h of culture. As
shown in Fig. 9A, fetal brown adipocytes cultured in the
presence of 1.4 nM IGF-I increased
[3H]thymidine incorporation by 3-fold relative to control
cells, as previously described (21). The presence of wortmannin or
LY294002 in the culture medium, together with IGF-I, did not
significantly modify the levels of [3H]thymidine
incorporation relative to those observed in IGF-I-treated
cells. Furthermore, no significant effect of PI 3-kinase inhibitors was
observed in the percentage of cells in S+G2+M phases of the cell cycle
as compared with cells stimulated with IGF-I alone (Fig. 9B).
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shows that PCNA levels induced by IGF-I
remained unchanged regardless of the presence of PI 3-kinase inhibitors
in the culture medium. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In previous reports, we have suggested that IGF-I might have a role leading brown adipose tissue to adipogenic and thermogenic differentiation before birth (19, 23). Furthermore, fetal brown adipose cells bear a high number of high-affinity IGF-I receptors (17), and this fact allowed us to characterize the very early events of the brown adipocyte IGF-I-signaling cascade from the receptor toward the nucleus (20). Among all of these events, PI 3-kinase enzymatic activity was significantly activated subsequent to its association with phosphorylated IRS-1. The results presented here demonstrate that wortmannin, at nanomolar concentrations, dramatically inhibits IGF-I-stimulated PI 3-kinase activity in brown adipocytes. Furthermore, the PI 3-kinase inhibitor LY294002, which is structurally unrelated to wortmannin, also inhibited IGF-I-stimulated PI 3-kinase activity in a concentration-dependent manner. However, neither of these two inhibitors disrupted other cellular events of the IGF-I-signaling cascade upstream from PI 3-kinase activation, such as ß-chain receptor autophosphorylation, IRS-1 tyrosine phosphorylation, and its association with the p85 subunit of the PI 3-kinase.
Recent findings indicate that PI 3-kinase is required for the movement
of glucose transporters to the cell membrane in both white and brown
adipose tissues and in muscle cells (27, 38, 39). Moreover,
overexpression of the catalytic subunit p110 of PI 3-kinase
increases glucose transport with translocation of glucose transporters
in 3T3L1 adipocytes (40, 41). Although in fetal brown adipocyte primary
cultures we have previously described an induction of Glut4 mRNA levels
following 24 h of treatment with insulin and IGF-I (23), in this
paper we found that glucose transport increased significantly after 10
min treatment with IGF-I (20, 23), probably due to Glut4 translocation.
Both wortmannin and LY294002 impaired IGF-I-induced glucose transport
at the same concentrations at which they inhibited PI 3-kinase
activity. These results provide evidence that IGF-I-induced glucose
transport in brown adipocytes during fetal development is dependent on
PI 3-kinase activation.
It is known that IGF-I plays a dual role by inducing both mitogenesis and differentiation in fetal brown adipocytes (17, 18, 19, 23). With regard to differentiation, our results show that the expression of several adipogenic genes induced by IGF-I is completely prevented by treatment of brown adipocytes with PI 3-kinase inhibitors. The induction of ME activity was also impaired according to its protein content. As a result, the fat droplet content of brown adipocytes newly synthesized in the presence of IGF-I measured by Nile red fluorescence was significantly reduced. Because the expression of the thermogenic differentiation marker UP is also inhibited in the presence of wortmannin/LY294002, the results presented here implicate PI 3-kinase activation as a crucial step in the brown adipocyte adipogenic and thermogenic differentiation-signaling pathways.
Previous findings in confluent 3T3L1 mouse fibroblasts undergoing insulin-induced adipogenic differentiation demonstrate that insulin increases the ras.GTP/ras.GDP ratio (42). In addition, transforming ras transfection induced 3T3L1 adipogenic differentiation in the absence of IGF-I (43). On the other hand, insulin-stimulated glucose uptake has been shown by a number of groups to be dependent on activation of PI 3-kinase (39, 44, 45). It is not known to what extent IRS-1/PI 3-kinase and ras.GTP reside in distinct signaling branches or whether significant cross-talk occurs. Recently, a reciprocal relationship between PI 3-kinase and p21-ras has been demonstrated to exist (46, 47, 48). The fact that in brown adipocytes IGF-I increased the expression of differentiation-related genes in parallel with an increase in the amount of p21-ras.GTP active form (18), together with the data presented here, gives rise to the possibility that IRS-1-associated PI 3-kinase in conjunction with ras.GTP is required for the signaling involved in inducing and/or maintaining the differentiation state of brown adipocytes before birth. However, the relative contribution of these two pathways leading to PI 3-kinase activation and hence brown adipocyte differentiation in response to IGF-I deserves further experimental work.
p70S6K has been identified as a molecular downstream target
of PI 3-kinase (30, 31, 32, 33). Our results demonstrate that inhibition of the
phosphorylation and activation of p70S6K with the
immunosuppressant rapamycin partly, but not totally, inhibited
IGF-I-induced adipogenic and thermogenic related gene expression. We
can therefore suggest that in addition to p70S6K, other
molecules that are activated downstream from PI 3-kinase might
participate in the molecular cascade leading to the nucleus where gene
expression is regulated. In this regard, PKC has been shown to be
activated by the PI 3-kinase product PIP3 (36), and its
activity is induced in brown adipocytes upon IGF-I stimulation, in
parallel with cell proliferation (20). The fact that in our cells
IGF-I-induced PKC activation is wortmannin/LY294002-sensitive
indicate that this PKC isoenzyme could be involved in the protein
network downstream from PI 3-kinase, which leads or maintains the onset
of brown adipocyte differentiation.
In addition to its role in inducing differentiation, IGF-I is a complete mitogen in fetal brown adipocyte primary cultures by inducing DNA synthesis, cell number increase, the entry of cells into the cell cycle, and PCNA expression (15, 17, 21). Finally, we have demonstrated that IGF-I-induced brown adipocyte proliferation is not inhibited by the presence of wortmannin/LY294002 in the medium. The exact role of PI 3-kinase in regulating cell proliferation has been the subject of controversy. It has been reported that microinjection of neutralizing antibodies against PI 3-kinase blocks the ability of a number of growth factors to induce DNA synthesis in fibroblasts (49). In 3T3L1 adipocytes, PI 3-kinase inhibits GTPase-activating protein, allowing insulin to fully activate p21-ras (50). On the other hand, experiments using wortmannin in Chinese hamster ovary (CHO) cells indicated that PI 3-kinase activity is not required for ras activation (45). In our fetal cells IGF-I-induced mitogenesis, which has been shown to be p21 ras-dependent (20, 21), seems to be PI 3-kinase-independent. Interestingly, it has been reported that SHC (SRC homology domain and collagen-like) is the predominant signaling molecule that activates ras in the insulin-signaling cascade (51, 52). SHC is tyrosine phosphorylated following IGF-I stimulation of brown adipocytes (20). Thus, the exact contribution of IRS-1/IRS-2, SHC, and perhaps other docking proteins to IGF-I-induced proliferation and/or differentiation remains to be established.
In conclusion, our results indicate that PI 3-kinase is a requirement
for the IGF-I-induced adipogenic and thermogenic differentiation
signaling pathways in fetal brown adipocytes, partly diverging through
p70S6k. However, the IGF-I-induced mitogenesis-signaling
pathway is PI 3-kinase-independent. In addition, PKC seems to be a
signaling molecule also involved in the IGF-I-induced differentiation
pathways downstream from PI 3-kinase.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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p85 subunit of PI 3-kinase mouse
monoclonal antibodies were the generous gifts of Dr. E. Rozengurt and
J. Sinnet-Smith and Drs. J. Downward and P. Rodriguez-Viciana (Imperial
Cancer Research Fundation, London), respectively. For IRS-1
immunoprecipitations, a rabbit polyclonal antibody was the generous
gift of Dr. R. Kahn (Joslin Diabetes Center, Boston, MA). The 4G10
anti-Tyr(P) monoclonal antibody was purchased from Upstate
Biotechnology (Lake Placid, NY). The anti-PCNA mouse monoclonal
antibody was purchased from Boehringer. The anti-ME polyclonal antibody
was obtained as previously described (16). For PKC
immunoprecipitations the rabbit polyclonal antiserum used was a gift of
Dr. J. Moscat (Centro de Biologia Molecular, Madrid).
[
32P]ATP (3000 Ci/mmol), [32P]dCTP (3000
Ci/mmol), 2-deoxy-D[1-3H]glucose (11.0
Ci/mmol), and [3H]thymidine (0.2 mCi/ml) were purchased
from Amersham (Aylesbury, UK). All other reagents were of the purest
grade available.
Cell Culture
Brown adipocytes were obtained from interscapular brown adipose
tissue of 20-day Wistar rat fetuses and isolated by collagenase
dispersion as previously described (17, 23). Cells were plated at
5 x 106 cells/100 mm or 1–1.2 x
106 cells/60-mm tissue culture plates in MEM supplemented
with 10% FCS to allow cell attachment to the plastic surface of the
plates. After 4–6 h of culture at 37 C, cells were rinsed twice with
PBS, and 80% of the initial cells were attached. Cells were maintained
for 20 h in a serum-free medium supplemented with 0.2% (wt/vol)
BSA. At this time, cells were treated for 5 min with IGF-I (10
nM) or preincubated for 15 min with several doses of PI
3-kinase inhibitors (wortmannin or LY294002) and subsequently
stimulated with IGF-I for a further 5 min. Both inhibitors were
initially disolved in dimethyl sulfoxide and in all experimental series
control cells were treated with the corresponding volumes of dimethyl
sulfoxide.
To analyze the effect of PI 3-kinase inhibitors on IGF-I-induced brown adipocyte differentiation, 20 h serum-deprived cells were cultured for a further 24 h in the presence of IGF-I either in the absence or presence of wortmannin or LY294002 at the doses indicated in Results and in the figure legends. Due to the instability of wortmannin in aqueous solutions, the medium from cells treated with wortmannin was replaced every 6 h.
Immunoprecipitations
Quiescent fetal brown adipocytes (5 x 106
cells/100-mm tissue culture dish) were treated with IGF-I for 5 min or
preincubated for 15 min with wortmannin and LY294002 and subsequently
stimulated with IGF-I for a further 5 min as indicated, and lysed at
4°C in 1 ml of a solution containing 10 mM Tris-HCl, 5
mM EDTA, 50 mM NaCl, 30 mM sodium
pyrophosphate, 50 mM NaF, 100 µM
Na3VO4, 1% Triton X-100, and 1 mM
phenylmethylsulfonyl fluoride, pH 7.6 (lysis buffer). Lysates were
clarified by centrifugation at 15,000 x g for 10 min,
and the supernatants were transferred to a fresh tube. After protein
content determination, equal amounts of protein were immunoprecipitated
at 4°C either with the monoclonal antibodies anti-Tyr(P) (Py72) and
p85, or with a polyclonal antibody against IRS-I. The immune
complexes were collected on anti-mouse IgG-agarose beads or, in the
case of the IRS-I antibody, on Protein A-agarose beads.
Immunoprecipitates were washed three times with lysis buffer and
extracted for 10 min at 95 C in 2 x SDS-PAGE sample buffer (200
mM Tris-HCl, 6% SDS, 2 mM EDTA, 4%
2-mercaptoethanol, 10% glycerol, pH 6.8) and analyzed by SDS-PAGE and
as described in Results and in the figure legends.
Western Blotting
After SDS-PAGE, proteins were transferred to Immobilon membranes
and were blocked using 5% nonfat dried milk in 10 mM
Tris-HCl and 150 mM NaCl, pH 7.5, and incubated overnight
with several antibodies as indicated in 0.05% Tween-20, 1% non-fat
dried milk in 10 mM Tris-HCl, and 150 mM NaCl,
pH 7.5. Immunoreactive bands were visualized using the enhanced
chemiluminescence (ECL) Western blotting protocol (Amersham).
In Vitro Kinase Assay
The protein kinase activity of the immunoprecipitates was
measured as described (53). The immune complexes were incubated in 20
µl of buffer containing 20 mM HEPES, 3 mM
MnCl2, 10 mM MgCl2, and 20 µCi of
[32P]ATP (in a final concentration of 5
µM) for 15 min at room temperature. The complexes were
washed twice with cold PBS and then resuspended in 2 x SDS-PAGE
sample buffer and analyzed by SDS-PAGE. The separated proteins were
dried in the gel, and the incorporation of [32P]phosphate
into protein was visualized by autoradiography and quantitated by
scanning laser densitometry (Molecular Dynamics densitometer,
Sunnyvale, CA).
PI 3-Kinase Activity
PI 3-Kinase activity was measured by in vitro
phosphorylation of phosphatidylinositol as described (54). Fetal brown
adipocytes were incubated with IGF-I in the absence or presence of PI
3-kinase inhibitors as indicated in the figure legends. After washing
with ice-cold PBS, cells were solubilized in lysis buffer containing
leupeptin (10 µg/ml), aprotinin (10 µg/ml), and 1 mM
phenylmethylsulfonyl fluoride. Lysates were clarified by centrifugation
at 15,000 x g for 10 min at 4 C, and proteins were
immunoprecipitated with the anti-IRS-1 polyclonal antibody. The
immunoprecipitates were washed successively in PBS containing 1%
Triton X-100 and 100 µM Na3VO4
(twice), 100 mM Tris (pH 7.5) containing 0.5 M
LiCl, 1 mM EDTA and 100 µM
Na3VO4 (two times), and 25 mM Tris
(pH 7.5) containing 100 mM NaCl and 1 mM EDTA
(twice). To each pellet were added 25 µl of 1 mg/ml
L--phosphatidylinositol/L--phosphatidyl-L-serine
sonicated in 25 mM HEPES (pH 7.5) and 1 mM
EDTA.
The PI 3-kinase reaction was started by the addition of 100
nM [32P]ATP (10 µCi) and 300
µM ATP in 25 µl of 25 mM HEPES, pH 7.4, 10
mM MgCl2, and 0.5 mM EGTA. After 15
min at room temperature, the reaction was stopped by the addition of
500 µl CHCl3-methanol (1:2) in a 1% concentration of HCl
plus 125 µl chloroform and 125 µl HCl (10 mM). The
samples were centrifuged, and the lower organic phase was removed and
washed once with 480 µl methanol-100 mM HCl plus 2
mM EDTA (1:1). The organic phase was extracted, dried
in vacuo, and resuspended in chloroform. Samples were
applied to a a silica gel TLC plate (Whatman, Clifton, NJ). TLC plates
were developed in propanol-1-acetic acid (2 N; 65:35
vol/vol), dried, visualized by autoradiography, and quantitated by
scanning laser densitometry (Molecular Dynamics personal
densitometer).
PKC Activity
Fetal brown adipocytes either untreated or stimulated were
extracted with lysis buffer (50 mM Tris, pH 7.5, 150
mM NaCl, 1% Triton X-100, 2 mM EDTA, 1
mM EGTA, 1 mM phenylmethylsulfonyl
fluoride, 25 µg/ml leupeptin, and 25 µg/ml aprotinin) and
immunoprecipitated with an anti-PKC antibody as previously described
(20). Immune complexes were washed seven times with ice-cold lysis
buffer with 0.5 M NaCl and twice with kinase buffer (35
mM Tris, pH 7.5, 10 mM MgCl2, 0.5
mM EGTA, and 1 mM
Na3VO4). The kinase reaction was performed in
20 µl kinase buffer containing 1 µCi [32P]ATP, 60
µM ATP, and 1 µg MBP as a substrate for 30 min at 30 C
and was terminated by the addition of 4 x SDS-PAGE sample buffer
followed by boiling for 5 min at 95 C. Samples were resolved in 12%
SDS-PAGE, and gels were dried out and subjected to autoradiography.
Measurement of the 2-Deoxyglucose
Transport 2-Deoxyglucose transport was measured as described
(55). After culture, quiescent brown adipocytes (1–1.2 x
106 cells/60-mm plate) were washed three times with
Krebs-Ringer-phosphate buffer (KRP) containing 135 mM NaCl,
5.4 mM KCl, 1.4 mM CaCl2, 1.4
mM MgSO4, and 10 mM sodium
pyrophosphate, pH 7.4, and then incubated with 1 ml KRP buffer with
IGF-I for 10 min at 37 C or preincubated with PI 3-kinase inhibitors
for 15 min and subsequently stimulated with IGF-I for another 10 min.
2-Deoxy-D[1-3H]glucose was added to this
solution to a final concentration of 0.1 mM and 250 nCi/ml,
and the incubation was continued for 5 min at 37 C. The cells were then
washed three times with ice-cold KRP buffer and solubilized in 1 ml 1%
SDS. The radioactivity of a 200-µl aliquot was determined in a
scintillation counter.
Determination of ME Activity
At the end of the culture period, ME activity was measured in
the cytosolic supernatants as previosly described (16). Enzyme activity
was expressed as milliunits/mg of protein. A milliunit is nanomoles
NADPH formed/min.
Flow Cytometric Analysis of Nile Red Fluorescence
Cytoplasmatic lipid content was determined by Nile Red
fluorescence emission 530 (BP 530/30 nm) in a FACScan flow cytometer
(Becton-Dickinson, San Jose, CA). Cells were detached from dishes by
addition of 0.05% trypsin-0.02% EDTA, and lipid content was
determined in aliquots of 2 x 105 cells after the
addition of Nile Red (0.1 µg/ml) (56). Results represent mean
intensities of fluorescence (obtained from the histograms of numbers of
cells vs. intensity of fluorescence) and are expressed in
arbitrary units.
RNA Extraction and Analysis
At the end of the culture time, cells were washed twice with
ice-cold PBS, and RNA was isolated with RNazol B (Biotecx Lab, Dallas,
TX) following the protocol supplied by the manufacturer for total RNA
isolation (57). Total cellular RNA (10 µg) was submitted to Northern
blot analysis, i.e . electrophoresed on 0.9% agarose gels
containing 0.66 M formaldehyde, transferred to GeneScreen
(NEN Research Products, Boston, MA) membranes using a VacuGene blotting
apparatus (LKB-Pharmacia, Upsala, Sweden). Hybridization was in 0.25
mM NaHPO4, pH 7.2, 0.25 M NaCl, 100
µg/ml denatured salmon sperm DNA, 7% SDS, and 50% deionized
formamide, containing denatured 32P-labeled cDNA
(106 cpm/ml) for 24 h at 42 C. Complementary DNA
labeling was carried out with [32P]dCTP to a specific
activity of 109 cpm/µg of DNA by using multiprimer
DNA-labeling system kit. For serial hybridization with different
probes, the blots were stripped and rehybridized subsequently as needed
in each case. The cDNAs used as probes were FAS (58), ME (59), G3PD
(60), ACC (61), UP (62), and ß-actin (63). Membranes were subjected
to autoradiography, and relative densities of the hybridization signals
were determined by densitometric scanning of the autoradiograms.
Determination of [3H]Thymidine
Incorporation into Acid-Insoluble Material
DNA synthesis was determined after 24 h of cell culture in
the presence of IGF-I (1.4 nM) in the absence or presence
of PI 3-kinase inhibitors by [3H]thymidine incorporation
(0.2 mCi/ml) into acid-insoluble material during the last 4 h of
culture (15). Results are expressed as disintegrations per
min/dish.
Cell Cycle Analysis by Flow Cytometry
After culture of cells for 24 h in the presence of IGF-I
without or with PI 3-kinase inhibitors, cells were detached from plates
by addition of 0.05% trypsin-0.02% EDTA. Trypsinization was stopped
by addition of 10% FCS to the culture medium. The percentages of cells
in G0/G1 and in S+G2+M phases of the cell cycle were determined after
nuclei were stained with propidium iodine by using the Cycle test DNA
reagent kit (Becton-Dickinson, San Jose, CA), measured in a Double
Discriminator Module and computer analyzed. All measurements were
performed in a FACScan flow cytometer (Becton-Dickinson).
Protein Determination
Protein determination was performed by the Bradford dye method
(64), using the Bio-Rad reagent (Bio-Rad, Richmond, CA) and BSA as the
standard.
Experimental Animals
The animals used for the required experiments in this report
were treated in accord with the "Guidelines for Care and Use of
Experimental Animals."
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by a SAF 96/0115 Grant from the Comision Interministerial de Ciencia y Tecnologia, Spain. P. Navarro was a recipient of a fellowship from the Ministerio de Educacion y Ciencia, Spain.
Received for publication December 5, 1996. Revision received February 6, 1997. Accepted for publication February 12, 1997.
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