| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Causes Insulin Receptor Substrate-2-Mediated Insulin Resistance and Inhibits Insulin-Induced Adipogenesis in Fetal Brown Adipocytes1
Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain
Address all correspondence and requests for reprints to: Margarita Lorenzo, Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain. E-mail: mlorenzo{at}eucmax.sim.ucm.es
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
for 24 h resulted in a partial
impairment in the expression of fatty acid synthase,
glycerol-3-phosphate dehydrogenase, and glucose transporter (GLUT)-4
messenger RNAs (mRNAs), as well as in the enhancement in the
cytoplasmic lipid content in response to insulin. However, the
expression of the tissue-specific gene, uncoupling protein 1, is
increased by the presence of TNF-. The antiadipogenic effect of
TNF- was accompanied by a down-regulation of CCAAT/enhancer-binding
protein- and ß mRNAs and up-regulation of CCAAT/enhancer-binding
protein-
, with the expression of peroxisome proliferator-activated
receptor-
remaining essentially unmodified. Moreover, TNF- caused
an insulin resistance on the insulin-induced glucose uptake in brown
adipocytes. Pretreatment with TNF- resulted in hypophosphorylation
of the insulin receptor in response to insulin, without affecting the
number of insulin receptors per cell or its molecular mass. However,
insulin receptor substrate (IRS)-1 and IRS-2 signaling in response to
insulin showed functional differences. Thus, TNF- pretreatment
induced a hypophosphorylation of IRS-2 but not of IRS-1. This effect
leads to an impairment in the IRS-2-associated phosphatidylinositol
(PI) 3-kinase activation due to a decreased association of -p85
regulatory subunit of PI 3-kinase with IRS-2 but not in the
IRS-1-associated PI 3-kinase activation in response to insulin. Our
results indicate that TNF- induced an IRS-2- but not IRS-1-mediated
insulin resistance on glucose transport and lipid synthesis in fetal
brown adipocytes. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Tumor necrosis factor (TNF)- is a potent cytokine with pleiotropic
biological effects that elicits lipolytic and antilipogenic effects in
adipose tissue and produces the dedifferentiation of 3T3-L1 adipocytes
(9). Long-term treatment of 3T3-F442A adipocytes with TNF- led to
down-regulation of GLUT4 and adipsin messenger RNAs (mRNAs) (10, 11, 12).
TNF- also prevents the expression of G3PD and fatty acid-binding
protein aP2 genes, completely abolishing differentiation and reverting
fully differentiated fat cells into fibroblasts (11, 13, 14). These
effects of TNF- are produced by preventing the expression of the
peroxisome proliferator-activated receptor (PPAR)) and the
CCAAT/enhancer-binding protein (C/EBP)- transcription factors (11, 13, 15) that seem to be necessary for the induction of fully
differentiated adipocytic phenotype (as reviewed in Ref.16). Moreover,
TNF- induces insulin resistance, a smaller than normal response to a
given amount of insulin, in cultured cells or in whole animals (17).
Thus, TNF- produced opposing physiological effects to insulin on
3T3-L1 cells, inhibiting insulin-stimulated glucose uptake and
insulin-induced adipocyte differentiation (9, 18, 19). So far, the
effects of TNF- on insulin-induced differentiation of fetal brown
adipocytes remains unexplored.
Regarding insulin signaling, chronic exposure of 3T3-F442A adipocytes
to TNF- strongly impaired insulin-stimulated glucose uptake by
inhibiting insulin receptor autophosphorylation and IRS-1
phosphorylation (17). In this cell type, TNF- was shown to induce
serine phosphorylation of IRS-1 and convert IRS-1 into an inhibitor of
the insulin receptor tyrosine kinase activity in vitro (20).
A rapid inhibition of insulin receptor autophosphorylation by TNF-
has been described in NIH 3T3 cells overexpressing the human insulin
receptor, as well as in hepatoma cells (21, 22). Whether or not TNF-
could cause insulin resistance in brown adipocytes and its potential
underlying molecular mechanism remains to be established. In this work,
we investigated the effects of pretreatment for 24 h with TNF-
on insulin-induced adipogenic and thermogenic gene expression, on the
expression of transcription factors such as C/EBPs and PPAR, and on
insulin-induced glucose uptake and lipid accumulation. Furthermore, we
explored whether or not TNF- affects the early events in insulin
signaling by examining insulin receptor autophosphorylation, IRS-1 and
IRS-2 phosphorylation, and its association with PI 3-kinase. Our
results show that TNF- causes insulin resistance by decreasing IRS-2
phosphorylation and IRS-2-associated PI 3-kinase activity, with IRS-1
signaling being unaffected.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and collagenase were
from Boehringer Mannheim (Mannheim, Germany). RNazol B was from Biotecx
Lab. (Dallas, TX). Nylon membranes were GeneScreen (NEN Research
Products, Boston, MA). Autoradiographic films were Kodak X-O-MAT/AR
(Eastman Kodak Co., Rochester, NY).
2-Deoxy-D[1-3H]-glucose (11.0 Ci/mmol),
[125I]insulin (80 µCi/µg),
[-32P]deoxycytidine triphosphate (3000 Ci/mmol),
[32P]-ATP (3000 Ci/mmol), and the multiprimer
DNA-labeling system kit were purchased from Amersham (Aylesbury, UK).
All other reagents used were of the purest grade available. The
complementary DNAs (cDNAs) used as probes were FAS (23), G3PD (24),
GLUT4 (25), UCP1 (26), ß-actin (27), C/EBPs (28), and PPAR (29).
Antiinsulin receptor monoclonal antibody-3 (Ab-3) was purchased from
Oncogene Science (Uniondale, NY). The Py20 anti-Tyr(P) monoclonal
antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Antimouse
IgG-agarose was from Sigma Chemical Co. Protein A-agarose was from
Boehringer Mannheim. The Py72 anti-Tyr(P) mouse monoclonal antibody was
the generous gift of Drs. E. Rozengurt and J. Sinnet-Smith (Imperial
Cancer Research Foundation, London). For IRS-1 and IRS-2
immunoprecipitations, glutathione S-transferase (GST)-fusion protein
rabbit polyclonal antibodies were the generous gift of Dr. M. White
(Joslin Diabetes Center, Boston, MA). The anti--p85 mouse monoclonal
antibody was purchased from Upstate Biotechnology (Lake Placid, NY).
Wheat germ lectin coupled to Sepharose (WGA) was from LKB-Pharmacia
(Uppsala, Sweden).
Cell culture
Brown adipocytes were obtained from interscapular brown adipose
tissue of 20-day Wistar rat fetuses and isolated by collagenase
dispersion as described (6). Cells were plated at 1.5 x
106 cells/60-mm tissue culture dishes in 2.5 ml MEM with
Earle’s salts supplemented with 10% FCS. After 4–6 h of culture at
37 C, cells were rinsed twice with PBS, with 70% of the initial cells
attached to the dish forming a monolayer. Cells were maintained for
20 h in a serum-free medium supplemented with 0.2% (wt/vol) BSA
as described (6) and were further cultured for 24 h in the absence
or in the presence of 0.6 nM TNF- with or without
insulin (1, 10, and 100 nM, as indicated in each case). In
other experiments, 20-h, serum-deprived cells were pretreated with
TNF- for 24 h and then stimulated with insulin for 5 min to
1 h, as indicated in each case.
Flow cytometric analysis of Nile Red fluorescence
Cytoplasmic 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) (30). Results represent mean
intensities of fluorescence (obtained from the histograms of number 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 lysed directly with RNazol B, following the protocol
supplied by the manufacturer for total RNA isolation (31). 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 membranes using a
VacuGene blotting apparatus (LKB-Pharmacia), and cross-linked to the
membranes by UV light. Hybridization was in 0.25 mM NaHP04,
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 40 h at 42 C, as
previously described (32). cDNA labeling was carried out with
[32P]deoxycytidine triphosphate to a specific activity
of 109 cpm/µg 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.
Membranes were subjected to autoradiography, and relative densities of
the hybridization signals were determined by densitometric scanning of
the autoradiograms in a laser densitometer (Molecular Dynamics,
Sunnyvale, CA). Each Northern blot analysis was performed in duplicate
samples from three independent experiments.
Measurement of the 2-deoxyglucose transport
Cells were serum-deprived for 20 h and cultured for a
further 24 h in the absence or presence of TNF-. After culture,
cells were washed three times with ice-cold Krebs-Ringer-phosphate
buffer (KRP) (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 or without insulin for 10 min at 37 C.
2-Deoxy-D[1-3H]glucose (500 nCi/ml) was added to this
solution, 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, as previously described (33). The radioactivity of a
200-µl aliquot was determined in a scintillation counter. Glucose
transport was determined in triplicate dishes from three independent
experiments.
[125I]insulin binding
Cells cultured for 20 h in a serum-free medium in either
the absence or presence of TNF- were incubated for 3 h at 20 C
with 0.03 nM [125I]insulin, 1 ml of 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 the
incubation, monolayers were rinsed either with ice-cold PBS-BSA or with
ice-cold 0.3 M sodium acetate, pH 4.5 (containing 0.15
M NaCl), then two more times with PBS-BSA, and then
dissolved in 0.1N NaOH-1% SDS-2%Na2CO3, as
previously described (4, 6). 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 the cell types studied. Nonspecific binding was defined as the
radioactivity that remained bound in the presence of 1000
nM of 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
At the end of the culture time, cells were 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 (600 µg) were
immunoprecipitated at 4 C with monoclonal antibodies anti-Tyr(P) (Py72)
and antiinsulin receptor (Ab-3), or with polyclonal antibodies against
p85 regulatory subunit of PI 3-kinase, IRS-1 and IRS-2. The immune
complexes were collected on antimouse IgG-agarose beads or on protein
A-agarose beads. Immunoprecipitates were washed three times with lysis
buffer and extracted for 10 min at 95 C in 2 times 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
as described in the figure legends.
Western blotting
After SDS-PAGE, proteins were transferred to Immobilon membranes
(Millipore Corp., Bedford, MA), were blocked using 5% nonfat dried
milk in TBS (10 mM Tris/HCl, 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 ml TBS. Immunoreactive
bands were visualized using the enhanced chemiluminiscence (ECL)
Western blotting protocol (Amersham).
In vitro kinase assay
The anti-Tyr(P) 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, as previously
described (34). The complexes were washed twice with ice-cold PBS and
then resuspended in 2x SDS-PAGE sample buffer and analyzed by
SDS-PAGE. The separated proteins were dried in the gel and the
incorporation [32P]phosphate into protein was visualized
by autoradiography and quantitated by scanning laser densitometry.
Purification and phosphorylation of the insulin receptor
Serum-deprived cells pretreated for 24 h with 0.6
nM TNF- 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.
Portions containing 10 µg WGA-purified receptors were preincubated
for 1 h at room temperature with 10 nM insulin. The
phosphorylation reaction was performed as indicated. After 15 min on
ice, phosphorylation was stopped, and receptors were immunoprecipitated
with 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.
PI 3-kinase activity
PI 3-kinase activity was measured by in vitro
phosphorylation of phosphatidylinositol as previously described (35).
At the end of the culture time, cells were washed with ice-cold PBS,
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 or the anti-IRS-2 antibodies. 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 25 µl 1 mg/ml
L--phosphatidylinositol/L--phosphatidyl-L-serine sonicated in 25
mM HEPES (pH 7.5) and 1 mM EDTA were added.
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 (2N; 65:35 vol/vol), dried,
visualized by autoradiography, and quantitated by scanning laser
densitometry.
Protein determination
Protein determination was performed by the Bradford dye method
(36) using the Bio-Rad reagent (Bio-Rad, Richmond, CA) and BSA as the
standard.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
down-regulates expression of insulin-induced adipogenic
genes in fetal brown adipocyte primary cultures produced opposing effects to insulin in 3T3-L1 cells,
inhibiting insulin-induced adipogenic differentiation (9, 18, 19). We
first characterized the effect of TNF- treatment on insulin-induced
fetal brown adipocyte gene expression, exploring the expression of
genes encoding enzymes directly involved in lipid synthesis such as FAS
and G3PD, and genes indirectly involved in adipogenesis such as the
insulin-regulated GLUT4 and the tissue-specific thermogenic marker
UCP1. Cells (after 4 h of attachment in FCS medium) were cultured
for 20 h in a serum-free medium and further cultured for 24 h
either in the absence or presence of 0.6 nM TNF- either
in combination or not with different doses of insulin (1 and 10
nM). The expression of the adipogenic and thermogenic genes
was studied by Northern blot, as depicted in a representative
experiment that is shown in Fig. 1
; the
resulting densitometric analysis is shown in Table 1. Treatment with TNF- by itself
decreased the basal expression of FAS and GLUT4 mRNAs but had no
significant effect on the basal expression of G3PD. Insulin
treatment produced a dose-dependent induction of adipogenic genes, as
well as a dramatic accumulation of GLUT4 mRNA, in agreement with the
data previously described (4). However, the treatment with TNF-
completely abolished the 1 nM insulin effect on the
expression of adipogenic genes and decreased FAS mRNA levels by 75%
and G3PD mRNA levels by 55% at 10 nM insulin. Moreover,
TNF- caused down-regulation on the expression of GLUT4 induced by
insulin (Fig. 1A and Table 1). However, ß-actin mRNA content remained
essentially unmodified throughout the different treatments (Fig. 1A).
Regarding UCP1 mRNA expression, TNF- up-regulated UCP1 gene
expression by itself, although to a lower extent than insulin 10
nM, whereas the combined presence of insulin and TNF-
produced an additive effect on UCP1 mRNA accumulation (Fig. 1B and
Table 1).
|
|
inhibits insulin-induced cytoplasmic lipid content in fetal
brown adipocytes down-regulated the expression of adipogenic genes
induced by insulin, we studied Nile Red fluorescence [a sensitive
detector of cytoplasmic lipid content (37)], to assess the role of
TNF- in the overall adipogenic program. Brown adipocytes were
cultured as described in Fig. 1
, 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 a representative experiment
in Table 2. Treatment for 24 h with
0.6 nM TNF- by itself significantly decreased the basal
lipid content by a 25%. The presence of 10 nM insulin in
the culture medium for 24 h increased by 3-fold the lipid content
detected in control cells. However, when cells were treated with 0.6
nM TNF- together with 10 nM insulin, their
lipid content decreased by 50%, in agreement with the partial
inhibitory effect observed above in the accumulation of adipogenic
mRNAs under the same experimental conditions.
|
down-regulates C/EBP and -ß and up-regulates C/EBP
in fetal brown adipocytes; PPAR expression remains essentially
unmodified and the C/EBPs seems to be necessary in the induction of fully
differentiated adipocyte phenotype (16). Furthermore, both families of
transcription factors seem also to be involved in thermogenic
differentiation, because C/EBP and -ß and PPAR have been
proposed as transcriptional activators of the UCP1 gene promoter (38, 39). Thus, we studied the effect of TNF- treatment of brown
adipocytes on C/EBP mRNA accumulation by Northern blot as shown in a
representative experiment in Fig. 2A; the
resulting densitometric analysis is shown in Table 1 in cells cultured
under the same experimental conditions seen above. Brown adipocytes
cultured under basal conditions expressed the three isoforms of C/EBP
transcription factors, i.e. , ß, and . TNF-
per se decreased basal expression of C/EBP and ß,
and indeed increased C/EBP mRNA accumulation. Insulin
treatment induced C/EBP and ß mRNA content at 10 nM,
with this effect being totally precluded by the treatment with TNF-.
The expression of C/EBP remained essentially unmodified under
insulin treatment, increasing, however, in the combined presence of 1
nM insulin and TNF- (Fig. 2A and Table 1).
|
was studied in cells cultured for 24 h
in the absence or presence of 0.6 nM TNF- and insulin
(10 nM), as shown in a representative experiment (Fig. 2B)
and in Table 1. Brown adipocytes cultured under basal conditions
expressed PPAR mRNA. Nevertheless, no significantly changes on
PPAR mRNA expression were observed under treatment with either
TNF- or insulin or both combined compared with control cells (Fig. 2B).
TNF- inhibits insulin-stimulated glucose uptake in fetal brown
adipocytes
TNF- inhibits insulin-stimulated glucose uptake in 3T3-L1 cells
(18, 19). The fact that glucose transport is induced in fetal brown
adipocytes on insulin stimulation (4), prompted us to investigate
whether this effect could be blocked by TNF- in those cells. Cells
were serum deprived for 20 h and further cultured for 24 h in
the presence or absence of 0.6 nM TNF- before
stimulation for 15 min with insulin at the doses indicated (Table 3). Glucose uptake was measured during
the last 5 min of culture as described in Materials and
Methods. Results from three independent experiments are expressed
as disintegrations per minute per microgram of protein. Insulin
stimulated basal glucose-uptake by 3-fold, with the effect being
observed at all the doses tested. Glucose uptake was significantly
higher (34%) in cells pretreated with TNF- for 24 h compared
with untreated cells. However, 1 nM insulin did not
significantly induced glucose uptake under pretreatment with TNF-,
and only at higher doses (10 and 100 nM) produced a
1.5-fold increase in the glucose uptake, an effect 50% lower than that
observed in TNF- untreated cells (Table 3).
|
inhibits insulin-induced insulin-receptor ß-subunit
tyrosine autophosphorylation in fetal brown
adipocytes, we decided to investigate the early events in the insulin
signaling pathway. Serum-starved brown adipocytes were pretreated for
24 h with or without 0.6 nM TNF- and then incubated
in the absence or presence of insulin for 1 h or 5 min, and
insulin-receptor ß-chain autophosphorylation analyzed by several
methods (Fig. 3). First, we determined
the level of the tyrosine phosphorylation in purified insulin
receptors. WGA-purified proteins from cells pretreated with or without
TNF- were stimulated with or without 10 nM insulin for
1 h, and then the in vitro kinase assay was performed
as described in Materials and Methods, and the insulin
receptors were immunoprecipitated with the Py72 anti-Tyr(P) antibody
(Fig. 3A). Insulin-treated brown adipocytes showed a marked increase in
the insulin-receptor ß-subunit tyrosine autophosphorylation as
compared with nontreated cells. This insulin receptor ß-chain
autophosphorylation was greatly impaired by TNF- pretreatment. No
insulin receptor tyrosine phosphorylation band was observed in control
cells.
|
, and then cells were stimulated either in the
absence of presence of 10 nM insulin for 5 min, after which
whole-cell lysates (600 µg protein) were subjected to
immunoprecipitation with the Py72 anti-Tyr(P) antibody. The
immunoprecipitates were submitted to an in vitro kinase
assay, as described in Materials and Methods (Fig. 3B). The
presence of 10 nM insulin in the culture medium caused
a marked increase in the insulin receptor 32 ß-subunit tyrosine
phosphorylation as compared with nontreated cells. In the cells
pretreated with TNF-, the insulin receptor ß-subunit was
hypophosphorylated on insulin stimulation (Fig. 3B).
Alternatively, the autophosphorylation of the insulin receptor was
studied by direct immunoprecipitation with the antiinsulin receptor
ß-subunit antibody (Fig. 3C). Thus, primary fetal brown adipocytes
pretreated for 24 h with TNF- were stimulated with/without 10
nM insulin for 5 min at 37 C, extracted as described in
Materials and Methods, immunoprecipitated with the
antiinsulin receptor monoclonal antibody, and then the resulting immune
complexes were analyzed by Western blotting with the Py20 anti-Tyr(P)
antibody. As shown in Fig. 3C, the insulin-receptor tyrosine
phosphorylation induced by insulin was greatly impaired in TNF-
pretreated cells. These results were consistent with those found in the
in vitro kinase assays seen above.
As an additional step, we determined whether the alterations in the
insulin receptor autophosphorylation described above could be due to a
reduced number of insulin receptors after pretreatment for 24 h
with 0.6 nM TNF-. However, quantitation of the insulin
receptors by radioligand binding using [125I]insulin
revealed an equivalent number of specific insulin binding sites per
cell (36,000 ± 8,000) with a similar affinity (kDa =
18.8 ± 4.7 nM) on the surface of primary brown
adipocytes under pretreatment with TNF- than in primary brown
adipocytes under basal culture conditions (35,000 ± 9,500 binding
sites per cell; kDa = 16.9 ± 6.3 nM).
TNF- inhibits insulin-induced IRS-2 phosphorylation in fetal
brown adipocytes
The next step was to compare the effect of acute insulin treatment
on inducing tyrosine phosphorylation of IRS-1 and IRS-2 in fetal brown
adipocytes. After 20 h of serum starvation, cells pretreated with
or without TNF- were incubated in the absence or presence of insulin
for 5 min at 37 C. Cell lysates (600 µg protein) were then
immunoprecipitated with anti-IRS-1 or anti-IRS-2 polyclonal antibodies
as described in Materials and Methods, and the immune
complexes were subjected to Western blotting analysis with Py20
anti-Tyr(P) antibody. As shown in Fig. 4A, no tyrosine phosphorylation of either
IRS-1 or IRS-2 was found in cells pretreated with or without TNF- in
the absence of insulin. However, there was a huge increase (>20-fold)
in the tyrosine phosphorylation of the 180-kDa band corresponding to
IRS-1 on insulin stimulation (Fig. 4A, left panel). Moreover, a marked
increase (16-fold) in the tyrosine phosphorylation of the 190-kDa band
corresponding to IRS-2 was observed on insulin treatment (Fig. 4A, right panel). In cells pretreated with TNF-, IRS-1 tyrosine
phosphorylation was increased by 2-fold on insulin stimulation compared
with the phosphorylation observed under insulin stimulation alone.
However, IRS-2 tyrosine phosphorylation was strongly impaired (>60%
of inhibition) in cells pretreated with TNF- on insulin stimulation
compared with insulin stimulation alone. A possible explanation for the
altered IRS phosphorylation in the presence of TNF- would be changes
at the level of IRS-1/IRS-2 expression. To assess this, equals amount
of protein from cells either untreated or 24 h pretreated with
TNF- were submitted to direct Western blot analysis with the
anti-IRS-1/IRS-2 antibodies. As depicted in Fig. 4B, both IRS-1 and
IRS-2 protein levels are similar in control cells. However,
pretreatment for 24 h with TNF- doubled the amount of IRS-1,
without affecting the expression of IRS-2 compared with untreated
cells. The observed increased IRS-1 tyrosine phosphorylation in cells
pretreated with TNF- on acute insulin stimulation could be due to
the higher IRS-1 protein content found under this experimental
condition.
|
inhibits insulin-induced IRS-2-associated PI 3-kinase
activity in fetal brown adipocytes on insulin stimulation. A representative TLC
experiment is shown in Fig. 5A (upper
panel), meanwhile means ± SEM of PI 3-kinase
activities from four independent experiments are shown in Fig. 5A
(lower panel). Cells pretreated with or without TNF- were
immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibodies under
identical conditions as described in Fig. 4. The resulting immune
complexes were assayed for PI 3-kinase activity. As shown in Fig. 5A, primary brown adipocytes showed no detectable PI 3-kinase activity from
IRS-1 immunoprecipitates under control conditions, regardless the
presence or absence of TNF-. On treatment with 10 nM
insulin, PI 3-kinase activity markedly increased in IRS-1
immunoprecipitates, with this activity increasing under pretreatment
with TNF-. PI 3-kinase activity from IRS-2 immunoprecipitates under
control conditions or TNF- pretreatment was not detectable, although
on acute treatment with 10 nM insulin, PI 3-kinase activity
markedly increased from IRS-2 immunoprecipitates to a similar extent
than from IRS-1 immunoprecipitates. In contrast, TNF- pretreatment
resulted in a marked decrease (50% of inhibition) of IRS-2-associated
PI 3-kinase activity on insulin stimulation (Fig. 5A). Finally, we
investigated whether these changes in PI 3-kinase activity associated
with either IRS-1 or IRS-2 under TNF- pretreatment were due to
altered association of the -p85 regulatory subunit of PI 3-kinase
with IRSs (Fig. 5B). Cells treated as described above were
immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibodies and
submitted to Western blot analysis with the anti--p85 monoclonal
antibody. No p85 association to either IRS-1 or IRS-2 was found in
control cells in the absence or presence of TNF-. Nevertheless,
under insulin stimulation, -p85 was associated to both IRS-1 and
IRS-2 to a similar extent. However, TNF- pretreatment before the
stimulation with insulin increased the association of p85 to IRS-1 and
decreased the association of p85 to IRS-2 compared with insulin
effects. These results on p85 association to IRSs are in agreement with
the changes observed in PI 3-kinase associated activity.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
led to a severe insulin resistance on the expression of
adipogenic genes and GLUT4 gene and also on the lipid synthesis, with
this effect being partially ameliorated at higher concentrations of
insulin. However, TNF- did not impair the insulin effect on UCP1
gene expression. In fact, TNF- produced a small accumulation in UCP1
mRNA either in the absence or presence of insulin. Our results clearly
indicate that TNF- specifically regulates gene expression in brown
adipocytes, down-regulating adipogenic genes and GLUT4 gene in a
similar fashion as in white fat and 3T3-L1 cells (9, 10, 11, 12, 18, 19) but
up-regulating the tissue-specific thermogenic marker UCP1. Because the
antiadipogenic effects of TNF- have been related to a negative
regulation of C/EBP and PPAR transcription factors in adipocytic
differentiated fibroblasts, we previously investigated the expression
of these transcription factors in brown adipocytes (11, 13, 15). The
three canonical C/EBP isoforms are expressed in fetal brown adipocytes,
with C/EBP and ß being positively modulated by insulin. In this
study, treatment with TNF- down-regulated the endogenous expression
of C/EBP and ß mRNA. In addition, TNF- impaired the insulin
effect on C/EBP-ß gene expression. These results are in agreement
with those reported in TA1 and 3T3-L1 adipocytes (11, 13). However,
C/EBP mRNA accumulation is regulated in a reciprocal fashion to
C/EBP and -ß by TNF-. PPAR gene is highly expressed in brown
adipocytes, with its expression not being modulated by the presence of
insulin or TNF- in the long term in fetal brown adipocytes. Our
results differ from those observed by Zhang et al. (15) in
3T3-L1 cells, who found the maximal anti-PPAR effect of TNF- at
24 h. Our results clearly point out that the adipogenic effect of
insulin as well as the antiadipogenic effect induced by TNF- on
insulin action are mediated by the modulation of C/EBP and ß
expression but not by PPAR expression in fetal brown adipocytes.
Besides impairing long-term insulin effects on adipogenesis, TNF-
induced an insulin resistance on the early events of insulin signaling
and also on the insulin-induced glucose uptake in fetal brown
adipocytes. As demonstrated by several approaches, pretreatment with
TNF- strongly inhibits insulin-induced tyrosine autophosphorylation
of the ß-chain insulin receptor in brown adipocytes in a similar
fashion as in adipocytic cell lines, fibroblasts, and hepatoma cells
(17, 21, 22). This effect cannot be accounted for the presence of a
lower number of insulin binding sites or altered insulin receptor
affinity under treatment with TNF-. Moreover, we explored the effect
of TNF- on insulin-induced phosphorylation of IRS-1 and IRS-2. In a
previous work (5), we described that insulin induced tyrosine
phosphorylation of IRS-1 in fetal brown adipocytes in culture. In this
study, we show for the first time that in fetal brown adipocytes, both
IRS-1 and IRS-2 turned out to be tyrosine phosphorylated in a similar
extent on insulin stimulation, suggesting that both insulin substrates
are involved in the insulin signaling pathways. Indeed, brown
adipocytes constitute a different scenario as compared with primary
white adipocytes and differentiated 3T3-L1 fibroblasts, in which IRS-2
tyrosine phosphorylation and IRS-2-associated PI 3-kinase activation on
insulin stimulation was shown to be almost undetectable (40). More
importantly, pretreatment with TNF- blunted the IRS-2 signaling in
response to insulin without affecting IRS-2 protein expression.
However, IRS-1 signaling was fully active in response to insulin under
pretreatment with TNF-. Moreover, this pretreatment for 1 day with
TNF- causes up-regulation of IRS-1 expression in fetal brown
adipocytes. Several potential mechanisms have been proposed to explain
the inhibition of insulin signaling induced by TNF- in different
cell types. A decreased insulin-induced IRS-1 tyrosine phosphorylation
has been observed in differentiated 3T3-L1 cells chronically treated
(3–5 days) with TNF-, proposing that TNF- in murine cultured
adipocytes induce serine phosphorylation of IRS-1 and convert IRS-1
into an inhibitor of the insulin receptor tyrosine kinase activity
in vitro (20). Moreover, this chronic treatment causes
down-regulation of IRS-1 expression (12). Acute TNF- treatment
induces serine phosphorylation of IRS-1 through inhibition of serine
phosphatases in rat hepatoma cells; this increased serine
phosphorylation interferes with insulin-induced tyrosine
phosphorylation of IRS-1 and impairs insulin effect (41). Moreover, in
this cell model, TNF- increased abundance of SH2-phosphotyrosine
phosphatases (42). All these effects of TNF- inhibiting the insulin
signaling seem to be mediated through the p55 TNF receptor by
activating the sphingomyelinase. The addition of exogenous
sphingomyelinase and ceramides to IR/IRS-1-reconstituted myeloid cells
mimicked the inhibitory effect of TNF- on insulin signaling,
although IR/IRS-2-reconstituted 32D cells were resistant to this
inhibition (43). However, a recent study involved IRS-2 and not IRS-1
in the impairment of IGF-I action in bovine fibroblasts under
pretreatment with insulin and interleukin-4 (44). Our results strongly
suggest functional differences between IRS-1 and IRS-2 in a cell system
in which both substrates compete for the insulin receptor on insulin
stimulation. Whether or not IRS-2 could be of critical importance to
the understanding of insulin-resistance states produced by TNF-
remains to be established.
Tyrosine-phosphorylated insulin receptor substrates bind and activate
several signaling molecules, including the 85-kDa regulatory subunit of
the PI 3-kinase (45, 46, 47). The NH2-terminal SH2 domain of p85 can bind
to both IRS-1 and IRS-2, and this interaction results in the activation
of the PI 3-kinase (40). Recent findings indicate that PI 3-kinase is
required for the movement of glucose transporter to the cell membrane
in both white and brown adipose tissues and muscle cells, as well as
the importance of PI 3-kinase activity for IGF-I-induced
differentiation of brown adipocytes and myoblasts (5, 48, 49, 50, 51). In this
study, we found that insulin induced IRS-1- and IRS-2-associated PI
3-kinase activity. Cells pretreated with TNF- showed an impairment
in IRS-2-associated PI 3-kinase activation on insulin stimulation. This
effect was due to a diminished p85 association to IRS-2 under
pretreatment with TNF- and insulin stimulation. However,
IRS-1-associated PI 3-kinase activation in response to insulin was
fully activated, as well as IRS-1 association to p85. These results
correlated with those seen above on IRS-1 and IRS-2 phosphorylation in
response to insulin in brown adipocytes pretreated with TNF-, and
provide further support to the functional differences between IRS-1 and
IRS-2 in the insulin signaling pathways suggested above. Moreover, this
selective impairment of IRS-2-associated PI 3-kinase signaling, but not
IRS-1-associated PI 3-kinase signaling, may account for the partial
inhibitory effect on glucose transport and also on adipogenic-related
gene expression in response to insulin observed in brown adipocytes
pretreated with TNF-. In fact, we recently reported that the insulin
effect on glucose transport and also on lipid synthesis were completely
blunted in brown adipocytes pretreated with PI 3-kinase inhibitors (5).
Whether or not IRS-2 could play a predominant role in glucose uptake
and adipogenesis as opposed to IRS-1 in fetal brown adipocytes, merits
further investigations.
In conclusion, our results show that TNF- caused an impairment in
the IRS-2-associated PI 3-kinase signaling in response to insulin, with
IRS-1-associated PI 3-kinase signaling remaining unaltered. This effect
led to an insulin resistance on glucose transport and also on the
adipogenic-related gene expression in fetal brown adipocytes.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Equal contributors to the experimental work.
3 Recipient of a fellowship from the Comunidad Autonoma de
Madrid, Spain.
4 Recipient of a fellowship from the Ministerio de Educacion y
Ciencia.
Received October 10, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in insulin-like
growth factor I-induced brown adipocyte mitogenic signaling cascades:
inhibition by cyclic adenosine 3,5-monophosphate. Endocrinology 137:3832–3841[Abstract]
: direct role in
obesity-linked insulin resistance. Science 259:87–91 and GLUT4 genes in 3T3–L1 adipocytes by
tumor necrosis factor-. J Biol Chem 267:13580–13584-induced insulin resistance in 3T3–L1 adipocytes is
accompanied by a loss of insulin receptor substrate-1 and GLUT4
expression without a loss of insulin-receptor mediated signal
transduction. J Biol Chem 272:971–976 gene expression contributes to the
antiadipogenic effects of tumour necrosis factor . Mol Endocrinol 10:1457–1466 inhibits signaling from the insulin
receptor. Proc Natl Acad Sci USA 91:4854–4858 on differentiation, insulin-stimulated glucose uptake, and gene
expression in 3T3–L1 cells. Endocrinology 136:1474–1481[Abstract]
and obesity-induced
insulin resistance. Science 271:665–668[Abstract]
- and hyperglycemia-induced insulin resistance.
J Clin Invest 97:1471–1477[Medline]
suppresses insulin-induced
tyrosine phosphorylation of insulin receptor and its substrates. J
Biol Chem 268:26055–26058- and ß- tubulin and cytoplasmatic ß- and -actin genes
using specific cloned cDNA probes. Cell 20:95–105[CrossRef][Medline]
. Gene 162:297–302[CrossRef][Medline]
and p85ß
isoforms upon T cell activation. J Biol Chem 268:10780–10788 and ß are transcriptional
activators of the brown fat uncoupling protein gene promoter. Biochem
Biophys Res Commun 198:653–659[CrossRef][Medline]
promotes brown adipocyte
differentiation. J Biol Chem 271:29909–29914This article has been cited by other articles:
 |
|
 |
|
  A. D. Maurer, Q. Chen, C. McPherson, and R. A. Reimer Changes in satiety hormones and expression of genes involved in glucose and lipid metabolism in rats weaned onto diets high in fibre or protein reflect susceptibility to increased fat mass in adulthood J. Physiol., February 1, 2009; 587(3): 679 - 691. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lorenzo, S. Fernandez-Veledo, R. Vila-Bedmar, L. Garcia-Guerra, C. De Alvaro, and I. Nieto-Vazquez Insulin resistance induced by tumor necrosis factor-{alpha} in myocytes and brown adipocytes J Anim Sci, April 1, 2008; 86(14_suppl): E94 - E104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. L. Ding, Y. Song, V. S. Malik, and S. Liu Sex Differences of Endogenous Sex Hormones and Risk of Type 2 Diabetes: A Systematic Review and Meta-analysis JAMA, March 15, 2006; 295(11): 1288 - 1299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Rahman, A. Dobrzyn, S.-H. Lee, P. Dobrzyn, M. Miyazaki, and J. M. Ntambi Stearoyl-CoA desaturase 1 deficiency increases insulin signaling and glycogen accumulation in brown adipose tissue Am J Physiol Endocrinol Metab, February 1, 2005; 288(2): E381 - E387. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. CANNON and J. NEDERGAARD Brown Adipose Tissue: Function and Physiological Significance Physiol Rev, January 1, 2004; 84(1): 277 - 359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Arribas, A. M. Valverde, and M. Benito Role of IRS-3 in the Insulin Signaling of IRS-1-deficient Brown Adipocytes J. Biol. Chem., November 14, 2003; 278(46): 45189 - 45199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Valverde, M. Arribas, C. Mur, P. Navarro, S. Pons, A.-M. Cassard-Doulcier, C. R. Kahn, and M. Benito Insulin-induced Up-regulated Uncoupling Protein-1 Expression Is Mediated by Insulin Receptor Substrate 1 through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway in Fetal Brown Adipocytes J. Biol. Chem., March 14, 2003; 278(12): 10221 - 10231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Mur, M. Arribas, M. Benito, and A. M. Valverde Essential Role of Insulin-Like Growth Factor I Receptor in Insulin-Induced Fetal Brown Adipocyte Differentiation Endocrinology, February 1, 2003; 144(2): 581 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Cohade, M. Osman, H. K. Pannu, and R. L. Wahl Uptake in Supraclavicular Area Fat ("USA-Fat"): Description on 18F-FDG PET/CT J. Nucl. Med., February 1, 2003; 44(2): 170 - 176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ma, P. Wang, J. F. Kuebler, I. H. Chaudry, and J. L. Messina Hemorrhage induces the rapid development of hepatic insulin resistance Am J Physiol Gastrointest Liver Physiol, January 1, 2003; 284(1): G107 - G115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. N. Finck and R. W. Johnson Anti-inflammatory agents inhibit the induction of leptin by tumor necrosis factor-alpha Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2002; 282(5): R1429 - R1435. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Teruel, R. Hernandez, and M. Lorenzo Ceramide Mediates Insulin Resistance by Tumor Necrosis Factor-{alpha} in Brown Adipocytes by Maintaining Akt in an Inactive Dephosphorylated State Diabetes, November 1, 2001; 50(11): 2563 - 2571. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Li, J. F. Youngren, V. P. Manchem, M. Kozlowski, B. B. Zhang, B. A. Maddux, and I. D. Goldfine Small Molecule Insulin Receptor Activators Potentiate Insulin Action in Insulin-Resistant Cells Diabetes, October 1, 2001; 50(10): 2323 - 2328. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. Meng, J. Zhou, H. Sasano, T. Suzuki, K. M. Zeitoun, and S. E. Bulun Tumor Necrosis Factor {{alpha}} and Interleukin 11 Secreted by Malignant Breast Epithelial Cells Inhibit Adipocyte Differentiation by Selectively Down-Regulating CCAAT/Enhancer Binding Protein {{alpha}} and Peroxisome Proliferator-activated Receptor {{gamma}}: Mechanism of Desmoplastic Reaction Cancer Res., March 1, 2001; 61(5): 2250 - 2255. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. ROSSMEISL, I. SYROVY, F. BAUMRUK, P. FLACHS, P. JANOVSKÁ, and J. KOPECKY Decreased fatty acid synthesis due to mitochondrial uncoupling in adipose tissue FASEB J, September 1, 2000; 14(12): 1793 - 1800. [Abstract] [Full Text]
|
|
|
|
|
|
J. Klein, M. Fasshauer, M. Ito, B. B. Lowell, M. Benito, and C. R. Kahn beta 3-Adrenergic Stimulation Differentially Inhibits Insulin Signaling and Decreases Insulin-induced Glucose Uptake in Brown Adipocytes J. Biol. Chem., December 3, 1999; 274(49): 34795 - 34802. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Desvergne and W. Wahli Peroxisome Proliferator-Activated Receptors: Nuclear Control of Metabolism Endocr. Rev., October 1, 1999; 20(5): 649 - 688. [Abstract] [Full Text]
|
|
|
|
 |
|
 H. D. Venters, Q. Tang, Q. Liu, R. W. VanHoy, R. Dantzer, and K. W. Kelley A new mechanism of neurodegeneration: A proinflammatory cytokine inhibits receptor signaling by a survival peptide PNAS, August 17, 1999; 96(17): 9879 - 9884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Poretsky, N. A. Cataldo, Z. Rosenwaks, and L. C. Giudice The Insulin-Related Ovarian Regulatory System in Health and Disease Endocr. Rev., August 1, 1999; 20(4): 535 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Khanna, S. Roy, L. Packer, and C. K. Sen Cytokine-induced glucose uptake in skeletal muscle: redox regulation and the role of alpha -lipoic acid Am J Physiol Regulatory Integrative Comp Physiol, May 1, 1999; 276(5): R1327 - R1333. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
