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Department of Biochemistry and Molecular Biology, Institute of Biochemistry, and Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Manuel Benito, Department of Biochemistry and Molecular Biology, Institute of Biochemistry, and Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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also induced insulin resistance in cultured adipocytes by
inhibiting insulin receptor autophosphorylation and IRS-1
phosphorylation (12, 13). All these data suggest that alterations in
the early steps of the insulin signaling pathway may play an important
role in several states of insulin resistance. Brown adipose tissue is the main tissue involved in nonshivering thermogenesis in neonates, which is responsible for the heat production associated with the expression of mitocondrial uncoupling protein (14, 15). Brown adipose tissue differentiation also encompasses an adipogenic program related to lipid synthesis, and its accumulation results in a multilocular fat droplet phenotype (15, 16). In addition, it is well known that insulin is the main signal involved in brown adipocyte lipogenesis through its induction of genetic expression of the lipogenic enzymes (16, 17, 18, 19). However, the molecular mechanisms by which insulin actions are initiated in this tissue have not yet been described. Moreover, a deficiency of brown adipose tissue has been shown to result in the development of glucose intolerance and severe insulin resistance (20, 21). Recently, several stable and permanent brown adipocyte cell lines have been obtained by cotransfection with immortalizing simian virus 40 large T antigen (SV40-LTAg) and pMEXneo or mutated H-ras gene (pMEXneo H-raslys12) (22). These cell lines maintained specific properties of brown adipocytes, such as expression of the tissue-specific marker uncoupling protein. In a previous work we have characterized two series of clonal lines: MB4.9.X (achieved by cotransfection with constructs of SV40-LTAg and pMEXneo) and MB1.3.X (achieved by cotransfection with constructs of SV40-LTAg and pMEXneo H-raslys12), which displayed immortalized and transformed phenotypes, respectively (23). Accordingly, in the present paper we have examined the effect of brown adipocyte immortalization (in the representative clone MB4.9.2) or H-ras transformation (in the representative clone MB1.3.19) on the very early events upstream and downstream from Ras in the insulin signaling cascade compared with those events in primary cells. Our results show that the maximal responsiveness to insulin is reduced in immortalized brown adipocytes compared with primary cells. Moreover, mutated H-ras-transformed brown adipocytes developed a marked insulin resistance upstream from Ras as a consequence of a serine-threonine phosphorylation of the receptor; meanwhile, downstream from Ras, the signaling cascade was overstimulated regardless of the presence of the hormone.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p85 mouse monoclonal
antibody were gifts from Dr. E. Rozengurt and J. Sinnet-Smith and Drs.
J. Downward and P. Rodriguez-Viciana, respectively (Imperial Cancer
Research Foundation, London, UK). For IRS-1 immunoprecipitations, a
rabbit polyclonal antibody was the gift of Dr. R. Kahn (Joslin Diabetes
Center, Boston, MA), and another anti-IRS-1 rabbit polyclonal antibody
was purchased from Upstate Biotechnology (Lake Placid, NY). The 4G10
anti-Tyr(P) monoclonal antibody was also obtained from Upstate
Biotechnology. For Raf-1 immunoprecipitations, a rabbit polyclonal anti
Raf-1 antiserum and the recombinant catalytically inactive MEK were
gifts from Dr. S. Gutkind (NIH, Bethesda, MD). The antibodies to
phosphoserine and phosphothreonine were purchased from Zymed (San
Francisco, CA). Wheat-germ lectin coupled to Sepharose (WGA) was
obtained from Pharmacia LKB (Uppsala, Sweden). Immobilized alkaline
phosphatase was prepared by reacting 2 ml Affi-Gel 10 from Bio-Rad
(Richmond, CA) with 5000 U alkaline phosphatase from Sigma Chemical Co.
[
-32P]ATP (3000 Ci/mmol),
[14C]chloramphenicol (54 mCi/mmol), and
[125I]insulin (80 µCi/µg) were obtained from Amersham
(Aylesbury, UK). The calcium phosphate mammalian transfection kit was
purchased from Stratagene (La Jolla, CA). All other reagents used were
of the purest grade available.
Cell culture
Brown adipocyte primary cells were obtained from interscapular
brown adipose tissue of 20-day-old Wistar rat fetuses and isolated by
collagenase dispersion as previously described (17). Cells were plated
at 3.5 x 106 cells/100-mm tissue culture plate 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 in 10% FCS-MEM until 80% confluence was
reached. At this time cells were cultured for an additional 20 h
in serum-free medium and subsequently stimulated for 5 min with various
doses of insulin or in the absence of insulin as a control for cellular
quiescence.
Brown adipocyte cell lines were established by means of cotransfection of brown adipocyte primary cells with constitutive constructs containing a neomycin resistance marker (pMEXneo) along with immortalizing SV40 LTAg (clones MB4.9.2, MB4.9.3, and MB4.9.6) or with transforming H-ras gene (pMEXneo H-raslys12) in cooperation with SV40-LTAg (clones MB1.3.19, MB1.3.12, and MB1.3.25) (22, 23). All of the cell lines were plated at 1 x 106 cells/100-mm plates and grown in 10% FCS-DMEM with antibiotics and G418 (250 µg/ml) for selection of the neo resistance marker to 80% confluence. Then cells were starved for 20 h in serum-free DMEM and subsequently stimulated for 5 min with various doses of insulin as indicated in Results and in the figure legends. Control cells were maintained in DMEM in the absence of insulin.
[125I]Insulin binding
Cells cultured for 20 h in a serum-free medium were
incubated for 3 h at 20 C with 0.03 nM
[125I]insulin in 1 ml binding buffer containing 25
mM HEPES-PBS and 1 mg/ml BSA in the absence or presence of
graded concentrations of unlabeled insulin. Triplicate dishes were used
for each data point. At the end of incubation, monolayers were rinsed
with either ice-cold PBS-BSA or ice-cold 0.3 M sodium
acetate, pH 4.5 (containing 0.15 M NaCl), then rinsed twice
with PBS-BSA and dissolved in 0.1 N NaOH-1% SDS-2%
Na2CO3, as previously described (24).
Radioactivity was counted in a Packard -counter (Downers Grove, IL).
The radioactivity associated with the cells submitted to an acid wash
representing internalized [125I]ligand was negligible.
Total binding in the absence of competing ligand was approximately 5%
of the radioactivity added in all cell types studied. Nonspecific
binding was defined as the radioactivity that remained bound in the
presence of 1000 nM unlabeled ligand and represented
approximately 10% of the total binding. Bound vs. free
plots, molecular masses, and number of binding sites per cell
(calculated from the Scatchard plots) were derived from three separated
experiments.
Immunoprecipitations
After 20 h of serum starvation, primary fetal brown
adipocytes or brown adipocyte cell lines were treated with various
doses of insulin for 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
phenylmethylsulfonylfluoride, 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
determination of protein content, equal amount of protein (600 µg for
primary brown adipocytes and 600 µg to 3 mg for brown adipocyte cell
lines) were immunoprecipitated at 4 C with the monoclonal antibodies
antiinsulin receptor (Ab-3), anti-Tyr(P) (Py72), and anti-p85 or
with a polyclonal antibody against a C-terminal peptide of IRS-1. The
immune complexes were collected on antimouse IgG-agarose beads or, in
the case of IRS-1 antibody, on protein A-agarose beads.
Immunoprecipitates were washed three times with lysis buffer, 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, and 10% glycerol, pH 6.8), and analyzed by SDS-PAGE
as described in Results and in the figure legends.
Western blotting
After SDS-PAGE, proteins were transferred to Immobilon
membranes. Membranes 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% nonfat dried milk in 10 mM Tris-HCl, and 150
mM NaCl, pH 7.5. Immunoreactive bands were visualized using
the ECL Western blotting protocol (Amersham, Arlington Heights,
IL).
Insulin receptor autophosphorylation assay
Insulin receptor autophosphorylation was measured as previously
described (25). The anti-Tyr(P) or anti-insulin receptor immune
complexes were incubated in 20 µl buffer containing 20 mM
HEPES, 3 mM MnCl2, 10 mM
MgCl2, and 20 µCi [-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 PI as described (26). Fetal brown adipocytes and
brown adipocyte cell lines were incubated in the absence or presence of
insulin as indicated in the figure legends. After washing with ice-cold
PBS, cells were solubilized in lysis buffer containing 10 µg/ml
leupeptin, 10 µg/ml aprotinin, and 1 mM
phenylmethylsulfonylfluoride. Lysates were clarified by centrifugation
at 15,000 x g for 10 min at 4 C, and proteins were
immunoprecipitated with anti-IRS-1 polyclonal antibody. The
immunoprecipitates were washed successively in PBS containing 1%
Triton X-100 and 100 µM Na3VO4
(twice); in 100 mM Tris (pH 7.5) containing 0.5
M LiCl, 1 mM EDTA, and 100 µM
Na3VO4 (twice); and in 25 mM Tris
(pH 7.5) containing 100 mM NaCl and 1 mM EDTA
(twice). To each pellet were added 25 µl 1 mg/ml
L--PI/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 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 silica gel TLC plate (Merck, Rahway, NJ). TLC plates were
developed in propanol-1 and acetic acid (2 N; 65:35,
vol/vol), dried, visualized by autoradiography, and quantitated by
scanning laser densitometry.
Raf kinase activity
The activation of Raf-1 was measured by the phosphorylation of
recombinant, catalytically inactive MEK in immunoprecipitates of Raf-1
antibodies, as described (27). After stimulation, cells were washed
twice with ice-cold PBS and lysed in 500 µl lysis buffer containing
20 mM HEPES (pH 7.5), 10 mM EGTA, 2.5
mM MgCl2, 40 mM ß-glycerol
phosphate, 1% Nonidet P-40, 2 mM
Na3VO4, 1 mM
phenylmethylsulfonylfluoride, and 1 mM dithiothreitol.
Proteins from equal quantities of cell lysates were immunoprecipitated
with 10 µl Raf-1 polyclonal antiserum for 2 h, and protein
A-agarose beads were added for the last 45 min. Immune complexes were
washed three times with ice-cold PBS containing 1% Nonidet P-40; once
with 100 mM Tris (pH 7.4) and 0.5 M LiCl; and
once in kinase buffer containing 12.5 mM MOPS
(3-[N-morpholino]propane-sulfonic acid) (pH 7.5), 12.5
mM ß-glycerol phosphate, 7.5 mM
MgCl2, 10 mM MnCl2, 0.5
mM EGTA, 0.5 mM NaF, and 0.5 mM
Na3VO4. The kinase reaction was performed in 30
µl kinase buffer supplemented with 10 µCi of
[-32P]ATP, 50 µM ATP, and 1 µg
recombinant, catalytically inactive MEK for 25 min at 30 C and was
terminated by the addition of 4 x SDS-PAGE sample buffer followed
by boiling at 95 C for 5 min. Samples were resolved in 10% SDS-PAGE,
and gels were dried and subjected to autoradiography.
GTP/GDP binding to p21-ras
Serum-deprived cells were incubated for 4 h in
phosphate-free DMEM and then labeled overnight in the same medium with
100 µCi/ml [32P]orthophosphate. After stimulation,
cells were washed with ice-cold PBS and then lysed and scraped in 1 ml
of a lysis buffer containing 50 mM Tris-HCl, 100
mM NaCl, 1 mM EGTA, 5 mM
MgCl2, 1 mM dithiothreitol, 1% Triton X-114, 1
mM phenylmethylsulfonylfluoride, and 10 µg/ml
leupeptin/aprotinin, pH 7.5. Mature p21-ras was separated
from nonprocessed p21-ras by a Triton X-114 phase split as
described (28). p21-ras was immunoprecipitated from the
detergent phase with the monoclonal antibody Y13–259. Bound
nucleotides were eluted from immunoprecipitates and analyzed by
ascending TLC. Radioactivity corresponding to GDP and GTP was
quantitated by phosphorimaging (Bio-Rad Laboratories, Richmond, CA).
The percentage of GTP in relation to the total nucleotide pool (GTP
plus GDP) was calculated, taking into account the different phosphorus
contents of GTP and GDP, with the formula, % GTP = GTP x
100/GDP x 1.5 + GTP.
Transient transfections
Each 100-mm culture dish was transiently transfected using the
calcium phosphate precipitation technique with 10 µg of a fusion
plasmid containing the chloramphenicol acetyltransferase (CAT) reporter
gene under the control of five activating protein-1 (AP-1) binding
sites arranged in tandem (29) or with 20 µg transforming Ras DNA
cloned in an eukaryotic expression vector (pMEXneo
H-raslys12) (22). After 4 h of incubation,
cells were shocked with 3 ml 15% glycerol for 2 min, washed, and then
incubated 24 h in serum-free medium, as previously described (23).
Cells were then lysed as described above for receptor purification or
stimulated with various doses of insulin for an additional 24 h.
Finally, cells were washed with cold PBS, CAT activity was determined
by incubating 50 µl cell extracts with 0.25 µCi
[14C]chloramphenicol-0.5 mM acetyl coenzyme A
in 0.25 M Tris (pH 7.8) at 37 C for 12 h, and then
samples were submitted to ascending TLC and subjected to
autoradiography.
Purification and phosphorylation of the insulin receptor
Serum-deprived cells were solubilized in lysis buffer as
described above. Solubilized receptors were added to 1 ml WGA-Sepharose
and rotated end over end for 2 h at room temperature. After
extensive washing with a buffer containing 50 mM Tris (pH
7.4), 0.05% Triton X-100, 100 mM NaCl, 2.5 mM
KCl, and 1 mM CaCl2, receptors were eluted with
300 µl of the same buffer supplemented with 0.3 M
N-acetylglucosamine.
WGA-purified receptors (10 µg protein) were incubated for 1 h at 4 C with 500 U immobilized alkaline phosphatase to remove phosphate before performing phosphorylation reactions. The supernatant was separated from the phosphatase beads by centrifugation and subsequently used in phosphorylation assays as previously described (30). Parallel samples were prepared by incubation of the purified receptors with alkaline phosphatase, which was inactivated by boiling for 30 min.
Portions containing 10 µg WGA-purified receptors (pretreated with active or inactive alkaline phosphatase) were preincubated for 1 h at room temperature with 100 nM insulin. The phosphorylation reaction was performed as indicated. After 15 min on ice, phosphorylation was stopped, and receptors were immunoprecipitated with the antiinsulin receptor antibody or the py72 anti-Tyr(P) antibody and separated 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).
Protein determination
Protein determination was performed by the Bradford dye method
(31), using the Bio-Rad reagent (Bio-Rad) and BSA as the standard.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The level of tyrosine
phosphorylation of the 95-kDa band was maximal at 1 nM
insulin and did not change significantly when higher concentrations of
insulin were added (10 or 100 nM; Fig. 1B). To investigate
the pattern of tyrosine-phosphorylated proteins in immortalized or
H-raslys12-transformed brown adipocytes, clones
MB4.9.2 (representative of the MB4.9.X cell lines) and MB1.3.19
(representative of the MB1.3.X cell lines) were grown in the presence
of 10% FCS until 80% confluence was reached. Then, cells were
cultured for an additional 20 h in a serum-free medium and
subsequently stimulated with various doses of insulin for 5 min at 37
C, after which whole cell lysates (600 µg protein) were assayed as
described above. As shown in Fig. 1 (A and B), immortalized brown
adipocytes (clone MB4.9.2) reached maximal insulin receptor ß-chain
tyrosine autophosphorylation at 1 nM insulin, although
these cells showed a reduced maximal responsiveness to insulin compared
to that of primary cells. In addition,
H-raslys12-transformed brown adipocytes (clone
MB1.3.19) lacked the insulin-induced tyrosine phosphorylation of the
insulin receptor ß-chain at 1 nM insulin, and only a
minimal amount of the 95-kDa phosphotyrosine band was detectable in
these cells upon treatment with 100 nM insulin.
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). Thus, primary fetal brown adipocytes
and clones MB4.9.2 and MB1.3.19 were stimulated with various doses of
insulin for 5 min at 37 C, extracted as described in Materials
and Methods and immunoprecipitated with the antiinsulin receptor
monoclonal antibody, and then the resulting immune complexes were
analyzed by Western blotting with the 4G10 anti-Tyr(P) antibody. As
shown in Fig. 2, constitutive tyrosine phosphorylation of the ß-chain
was not observed in either primary cells or immortalized or
H-raslys12-transformed cell lines. However,
5-fold more protein loading (3 mg) than that needed for primary cells
was required for immunodetection of the tyrosine-phosphorylated insulin
receptor in response to insulin in both cell lines. Thus, immortalized
cells reached the maximal insulin receptor ß-subunit tyrosine
autophosphorylation at 1 nM, and this tyrosine
phosphorylation in response to insulin was lower than that found in
primary cells. These results were consistent with those of in the
in vitro kinase assay (Fig. 1
). However, transformed cells
suppressed the insulin receptor ß-subunit tyrosine phosphorylation in
response to 1 nM insulin; this lack of response was
slightly ameliorated in response to higher insulin concentrations in a
dose-dependent manner (Fig. 2).
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).
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p85 monoclonal antibody as
described in Materials and Methods, and the immune complexes
were subjected to Western blotting analysis with the 4G10 anti-Tyr(P)
antibody. As shown in Fig. 4 (left panel),
there was a significant increase in the tyrosine phosphorylation of the
160- to 185-kDa band compared with that in control cells, corresponding
to the IRS-1 associated with p85-PI 3-kinase upon addition of 1
nM insulin to quiescent fetal brown adipocytes. The level
of tyrosine phosphorylation of this band did not change significantly
when higher doses of insulin were added (10 and 100 nM).
Interestingly, in fetal brown adipocytes, p85-PI 3-kinase was not
phosphorylated in tyrosine residues in response to insulin.
Furthermore, we could not detect the 95-kDa band corresponding to the
tyrosine-phosphorylated insulin receptor ß-subunit in these anti-p85
immuno-precipitates. As we could not detect the 160- to 185-kDa band
corresponding to phosphorylated IRS-1 in the immunoprecipitates from
clones MB4.9.2 and MB1.3.19 after loading 600 µg protein (data not
shown), we immunoprecipitated a 5-fold higher protein content (3 mg).
As shown in Fig. 4 (right panel), we observed a maximal
tyrosine phosphorylation of IRS-1 induced by insulin at 1
nM in immortalized brown adipocytes (clone MB4.9.2),
although this effect was much lower than that observed in primary
cells. However, in H-ras-transformed brown adipocytes (clone
MB1.3.19), IRS-1 tyrosine phosphorylation was suppressed by 1
nM insulin; this lack of effect was ameliorated at higher
insulin concentrations in a dose-dependent manner. Thus, a half-maximal
effect was elicited at 10 nM, and a maximal effect occurred
at 100 nM. Furthermore, none of these cell lines showed the
p85-PI 3-kinase tyrosine-phosphorylated band in response to
insulin.
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, IRS-1 levels were
similar in the three cell types studied.
Effect of insulin on IRS-1-associated PI 3-kinase activity in
primary, immortalized, and H-ras-transformed brown adipocytes
We also investigated whether PI kinase activity was associated
with the IRS-1 immunoprecipitates in response to insulin in primary and
immortalized or transformed brown adipocytes. After serum starvation,
cells were stimulated with various doses of insulin for 5 min and
extracted as described in Materials and Methods, and 600
µg protein (from both primary brown adipocytes and cell lines) were
immunoprecipitated with an anti-IRS-1 antibody. The resulting immune
complexes were assayed for PI 3-kinase activity. As shown in Fig. 5, primary brown adipocytes displayed almost
undetectable PI 3-kinase activity in IRS-1 immunoprecipitates under
control conditions. Upon treatment with 1 nM insulin, the
basal PI 3-kinase activity increased by 8-fold. No further increase was
observed at higher concentrations of insulin (10 and 100
nM). MB4.9.2 or MB1.3.19 cell lines also displayed a very
low basal PI 3-kinase activity (Fig. 5). When clone MB4.9.2 was
stimulated with 1 nM insulin, there was a 3-fold increase
in PI 3-kinase activity, which was significantly lower that than
observed in primary cells with the same dose of the hormone.
Furthermore, no changes in PI 3-kinase activity were observed when
higher doses of insulin were added (10 and 100 nM).
Transformed cells (clone MB1.3.19) showed a very low IRS-1-associated
PI 3-kinase activity in response to 1 nM insulin; this
effect was ameliorated by higher insulin concentrations in a
dose-dependent manner. Thus, a half-maximal effect (1.5-fold) was
elicited at 10 nM, and a maximal effect (3-fold) occurred
at 100 nM insulin (Fig. 5).
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, after 20 h of serum starvation, primary
fetal brown adipocytes showed 20% of the Ras.GTP-active form.
Moreover, the percentage of Ras.GTP increased by 3-fold after 5-min
treatment with 1 nM insulin. Immortalized brown adipocytes
(clone MB4.9.2) constitutively showed 30% of the Ras.GTP-active form,
and this percentage doubled after 5-min treatment with 1 nM
insulin. However, in H-raslys12-transformed
brown adipocytes we found a higher percentage of constitutive
p21-Ras.GTP-active form (66%) than in primary or immortalized brown
adipocytes, and this percentage remained unchanged after insulin
stimulation at either 1 nM (Table 1) or higher doses
(results not shown).
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showed a marked
increase (6-fold) in MEK phosphorylation in primary brown adipocytes
treated for 5 min with insulin (1 nM) with respect to
control cells without any further increase at higher insulin doses.
Immortalized brown adipocytes (MB4.8.2) showed a maximal 2-fold
increase in Raf-1 kinase activity upon treatment with the same dose of
insulin (1 nM) compared with control cells. However, in
transformed cell lines (MB1.3.19), a higher constitutive MEK
phosphorylation was found, and this activity was insulin
independent.
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, insulin treatment for 24 h
stimulated CAT activity by 2-fold in immortalized brown adipocytes
(clone MB4.9.2); this effect was maximal at 1 nM. However,
H-raslys12-transformed brown adipocytes (clone
MB1.3.19) showed a higher constitutive CAT activity than immortalized
cells, and this activity was insulin independent.
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, H-raslys12-transformed brown
adipocytes showed a marked insulin receptor ß-chain phosphorylation
in the basal state, which was only detected by immunoprecipitation with
the antiinsulin receptor antibody, whereas no tyrosine phosphorylation
of the ß-subunit occurred after immunoprecipitation of the insulin
receptor with the Py72 anti-Tyr(P) antibody. When WGA-purified proteins
were preincubated with immobilized active alkaline phosphatase before
performing the in vitro kinase reaction, phosphorylation of
the insulin receptor (previously detected by immunoprecipitation with
the antiinsulin receptor antibody) was totally abolished. Then,
WGA-purified proteins from serum-deprived MB1.3.19 cells were
stimulated with 100 nM insulin, and the in vitro
kinase assay was performed. When WGA-purified proteins were
immunoprecipitated with the antiinsulin receptor antibody, a band
corresponding to the phosphorylated insulin receptor was observed. This
band was similar to that observed in unstimulated cells. However, when
the immunoprecipitation was performed with the Py72 anti-Tyr(P)
antibody, the band corresponding to the tyrosine-phosphorylated
receptor after insulin stimulation was almost undetected, as shown in
the experiments performed with whole cell lysates (Figs. 1 and 2).
Finally, WGA-purified proteins were incubated with immobilized alkaline
phosphatase before stimulation with 100 nM insulin and
in vitro kinase assay. As shown in Fig. 8A, there was a
3-fold increase in tyrosine phosphorylation [detected by
immunoprecipitation with the Py72 anti-Tyr(P) antibody] of insulin
receptors preincubated with active alkaline phosphatase, suggesting
that removal of the basal serine-threonine-bound phosphates restores
tyrosine phosphorylation of the insulin receptor ß-subunit upon
stimulation with insulin.
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, we did not find
phosphorylation of the ß-chain in the parenteral cells after
performing the in vitro kinase assay and immunoprecipitation
under the experimental conditions described in Fig. 8A. Indeed,
pretreatment of the purified receptors from primary cells with active
alkaline phosphatase did not suppress the high tyrosine phosphorylation
of the receptor observed in the presence of insulin, regardless of the
antibody used for the immunoprecipitation. Similar results were
obtained with WGA-purified receptors from MB4.9.2 immortalized cells
(results not shown).
To further rule out possible effects of clonal selection, we
transiently transfected the immortalized brown adipocyte cell line
(MB4.9.2) with the pMEXneoH-raslys12 construct.
WGA-purified receptors from MB4.9.2 and
MB4.9.2H-raslys12 cells were stimulated with 100
nM insulin. Then, the in vitro kinase assay was
performed, and the insulin receptors were immunoprecipitated with the
Py72 anti-Tyr(P) antibody. As shown in Fig. 8C, the presence of insulin
induced tyrosine autophosphorylation of the insulin receptor of the
immortalized cells; this effect was much less than that in the primary
cells (as shown in Figs. 1 and 2). Interestingly, this band was
suppressed when these cells were transiently transfected with the
pMEXneoH-raslys12 construct. Furthermore,
pretreatment of the WGA-purified receptors from
MB4.9.2H-raslys12 cells with alkaline
phosphatase before insulin stimulation partly restored the tyrosine
phosphorylation of the insulin receptor that was lost in
H-raslys12-transfected cells (Fig. 8C).
Finally, to show direct evidence of the constitutive insulin receptor
serine-threonine phosphorylation induced by H-ras
transformation of brown adipocytes, we performed an
antiphosphoserine/threonine Western blot analysis of insulin receptor
immunoprecipitates from the three cell types studied. As shown in Fig. 8D, after immunoprecipitation of serum-deprived primary, immortalized,
and H-raslys12-transformed brown adipocytes
followed by immunodetection with antiphosphoserine/threonine
antibodies, we could only detect a single band corresponding to the
insulin receptor ß-chain in the lane corresponding to
H-raslys12-transformed cells (MB1.3.19).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As an additional step, we have examined the insulin signaling pathway in a representative SV40-LTAg immortalized brown adipocyte cell line (clone MB4.9.2) compared with that in primary brown adipocytes. After immortalization, brown adipocytes respond to insulin at the same doses as parenteral cells, but the maximal responsiveness was significantly lower in all steps studied, including insulin receptor ß-chain tyrosine autophosphorylation, p85-PI 3-kinase-associated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, and Raf-1 kinase activation in immortalized cells. As the number of insulin receptors per cell, its molecular mass, and the insulin sensitivity remain unaltered compared with those of parenteral cells, all of these results indicate only a reduced maximal responsiveness to insulin either upstream or downstream from Ras in immortalized brown adipocyte cell lines.
We also compared the insulin signaling pathway in a H-raslys12-transformed brown adipocyte cell line (MB1.3.19) with that in primary or immortalized brown adipocytes (MB4.9.2). Mutated H-raslys12-transformed brown adipocytes suppressed tyrosine autophosphorylation of the insulin receptor in response to low concentrations of insulin (1 nM) compared with primary or immortalized brown adipocytes; this impairment of insulin responsiveness was slightly ameliorated at higher insulin concentrations in a dose-dependent manner. Consistently, the p85-PI 3-kinase-associated tyrosine phosphorylations of IRS-1 and PI 3-kinase activation were almost suppressed in response to low concentrations of insulin. This effect was also ameliorated by stimulation at higher insulin concentrations, in a dose-dependent manner. These results provide additional evidence of the severe insulin resistance induced by Ras transformation in brown adipocytes. The dramatic inhibition of insulin receptor autophosphorylation in response to insulin cannot be accounted for by a constitutive tyrosine-phosphorylated state of the insulin receptor, the presence of a lower number of insulin-binding sites, or altered insulin receptor affinity. Thus, mutant p21-Ras by itself or through constitutive activated proteins downstream from Ras, as discussed below, could be inhibiting insulin receptor ß-chain tyrosine autophosphorylation in response to insulin by a feedback mechanism.
One of the mechanisms proposed to cause inhibition of the tyrosine
kinase activity of the insulin receptor is the serine-threonine
phosphorylation of the insulin receptor ß-subunit. In this regard,
covalent modification of the insulin receptor by protein kinase A or C
has been shown to reduce the intrinsic tyrosine kinase activity of the
insulin receptor in response to insulin, resulting in insulin
resistance (37, 38, 39). Treatment of WGA-purified receptors with alkaline
phosphatase suggests that in the basal state (20 h of serum
starvation), there is a serine-threonine phosphorylation of the insulin
receptor in the H-raslys12-transformed brown
adipocyte cell line. Importantly, removal of serine-threonine-bound
phosphate before insulin stimulation and in vitro
phosphorylation increased the tyrosine kinase activity of the insulin
receptor in response to insulin, restoring the tyrosine kinase activity
virtually lost in the Ras-transformed cells. This effect of Ras has
been reproduced in immortalized cells by transient transfection with
mutated H-raslys12 construct, ruling out
possible effects of clonal selection. These results demonstrate that
the constitutive serine-threonine phosphorylation of the insulin
receptor only seen in the MB1.3.19 cell line is specifically induced by
mutated H-ras. A possible candidate that might mediate this
effect could be a cytosolic serine-threonine kinase directly activated
downstream from Ras. In this regard, protein kinase C
, which has
been described as a target of Ras (40), is highly expressed in MB1.3.19
cells (data not shown). However, we cannot exclude the possibility that
a serine-threonine kinase associated with the insulin receptor could be
involved. Further experimental work will be required to address this
important issue.
Attenuations in insulin receptor protein tyrosine kinase activity due to increased serine phosphorylation have been recently described in patients with polycystic ovary syndrome (41). Indeed, women with polycystic ovary syndrome developed insulin resistance and glucose intolerance. Thus, H-ras-transformed brown adipocytes inoculated into nude mice might provide a new model in which to study the insulin resistance linked to alterations in the early events of insulin postreceptor signaling caused by serine-threonine phosphorylation of the insulin receptor in malignant cells.
As expected, H-ras-transformed brown adipocytes expressed a very high percentage of the Ras.GTP-active form compared with primary or immortalized brown adipocytes. This percentage was insulin independent. Consistently, an event downstream from Ras, such as Raf 1-kinase activity, was constitutively stimulated in the transformed brown adipocytes in an insulin-independent manner. These results provide additional support to our previous statement that constitutive activated proteins downstream from Ras may be inducing a serine-threonine phosphorylation on the insulin receptor by a feedback mechanism, resulting in severe insulin resistance. Finally, we have extended our investigations regarding the alterations in the insulin signaling caused by H-ras transformation toward to the nucleus. Previous experiments performed in a differentiated adipocyte cell line have established AP-1 transcription factors, which bind to the TRE found in enhancers of various genes, as mediators for insulin-regulated gene expression (42). In this respect, our data show that insulin significantly trans-activates the expression of the TRE-CAT fusion gene transiently transfected in immortalized brown adipocytes. However, in H-raslys12-transformed brown adipocytes, we observed a higher constitutive TRE-CAT activity, and this activity remained unchanged upon insulin treatment. These results confirm that the constitutive activation of events downstream from Ras in the cytosol by mutated H-ras transformation also occurs at the nuclear level. Thus, we found that overstimulation downstream from Ras results in mutated H-ras transformation regardless of the presence of insulin.
In conclusion, our results show that immortalization of brown adipocytes causes a decrease in the maximal responsiveness to insulin compared with that of parenteral cells. In addition, transformation of brown adipocytes by mutated H-ras gives rise to severe insulin resistance (at the early postreceptor events upstream from Ras) in the insulin signaling pathway. However, downstream from Ras, several events are overstimulated, suggesting a possible feedback mechanism causing an impairment of the insulin receptor by serine-threonine phosphorylation, resulting in severe insulin resistance upstream from Ras. Accordingly, Ras-transformed brown adipocytes provide a new insight into the insulin signal transduction linked to an insulin receptor defect caused by its serine-threonine phosphorylation.
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Acknowledgments |
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Footnotes |
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2 Recipient of a fellowship from the Comunidad Autonoma de
Madrid.
Received December 4, 1996.
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