Endothelin-1-stimulated glucose uptake is desensitized by tumor necrosis factor-
in 3T3-L1 adipocytes
Nadia Rachdaoui and
Laura E. Nagy
Department of Nutrition, Case Western Reserve University, Cleveland, Ohio
44106
Submitted 11 April 2003
; accepted in final form 19 May 2003
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ABSTRACT
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Tumor necrosis factor-
(TNF-
) is a potent inducer of insulin
resistance, and increased TNF-
expression is associated with impaired
glucose disposal. Although insulin is the primary regulator of glucose
transport in adipose, endothelin-1, a vasoconstrictor peptide that signals
through the heterotrimeric G proteins G
q/11, potently
stimulates glucose uptake in 3T3-L1 adipocytes by a mechanism independent of
phosphatidylinositol (PI) 3-kinase. Here, we report that exposure of 3T3-L1
adipocytes to TNF-
for 48 h dose-dependently decreased
endothelin-1-stimulated glucose uptake and translocation of GLUT4 to the
plasma membrane. TNF-
exposure had no effect on endothelin-1 receptor
number at the cell surface. In contrast, TNF-
treatment reduced the
quantity of G
q/11 and proline-rich tyrosine kinase 2 (PYK2)
and decreased endothelin-1-stimulated PYK2-Tyr402 tyrosine
phosphorylation. Taken together, these results suggest that
TNF-
-induced desensitization of endothelin-1-stimulated GLUT4
translocation and glucose uptake in 3T3-L1 adipocytes is due, at least in
part, to a decreased expression of G
q/11, leading to a
suppression in tyrosine phosphorylation of PYK2.
adipocytes; glucose homeostasis; G protein-coupled receptors; G
q/11; proline-rich tyrosine kinase 2
ALTHOUGH INSULIN IS THE PRIMARY REGULATOR of glucose uptake in
muscle and adipose tissue, endothelin-1, a vasoconstrictor peptide secreted
primarily by endothelial cells
(29), is also a potent
stimulator of GLUT4 translocation and glucose transport in 3T3-L1 adipocytes
(14,
22,
36). In adipocytes,
endothelin-1 action is mediated by binding to the ETA receptor, a G
protein-coupled receptor expressed in many tissues and cultured cells
including 3T3-L1 adipocytes (4,
36). Although the signal
transduction cascade leading from the ETA receptor to the
activation of glucose transport is not completely understood, work from a
number of laboratories has identified a critical role for
G
q/11 in the endothelin-1 pathway
(14,
17,
22).
G
q/11-stimulated glucose transport is independent of
phosphatidylinositol (PI) 3-kinase
(3,
18,
35,
36). Instead, activation of
ETA receptor or G
q/11 stimulates GLUT4
translocation via a mechanism that requires tyrosine phosphorylation
(6,
36) and involves ADP
ribosylation factor-6, as well as F-actin polymerization
(3,
19).
Endothelin-1/G
q/11 activates the src family
tyrosine kinase Yes (13), as
well as proline-rich tyrosine kinase 2 (PYK2)
(22), in 3T3-L1 adipocytes.
Dominant inhibitory constructs of PYK2 prevent endothelin-1 stimulation of
F-actin polymerization and GLUT4 translocation
(22).
Tumor necrosis factor-
(TNF-
), a cytokine secreted by
activated macrophages as well as adipose tissue, is an important mediator of
insulin resistance (11).
TNF-
production by adipose tissue increases during the development of
insulin resistance and obesity in animal models
(11), and adipose tissue is
one of the major and immediate targets of TNF-
(26). Although it is clear
that TNF-
interferes with glucose homeostasis and lipid metabolism,
contributing to the development of insulin resistance in obesity and type 2
diabetes (11), its mechanism
of action is not completely understood. Exposure to TNF-
leads to
serine phosphorylation of insulin receptor substrate (IRS)-1, which inhibits
the tyrosine kinase activity of the
-subunit of the insulin receptor and
impairs insulin-stimulated signal transduction
(12,
27). Increasing evidence
suggests that serine/threonine kinases, including protein kinase C and p42/44
MAP kinases, mediate this inhibitory effect of TNF-
on insulin
signaling (8,
20). Additional mechanisms of
TNF-
-induced insulin resistance may include decreased GLUT4 expression
(30), impaired myoinositol
incorporation, and Ca2+ mobilization
(37), as well as the induction
of specific proteins by TNF-
such as suppressors of cytokine
signaling-1, -3, and -6, which can negatively modulate insulin signaling
(7,
21).
The aim of this study was to determine whether chronic TNF-
exposure
inhibits endothelin-1-stimulated glucose transport and to define the
components of the endothelin-1-signaling pathway involved in the response to
TNF-
exposure. We found that chronic exposure of 3T3-L1 adipocytes to
TNF-
decreased endothelin-1-stimulated glucose uptake. TNF-
exposure decreased the expression of G
q/11 and PYK2,
resulting in a reduced endothelin-1-stimulated PYK2-Tyr402 tyrosine
phosphorylation and a subsequent decrease in glucose uptake. These results
suggest that impaired endothelin-1 signaling may contribute to the development
of impaired glucose homeostasis after chronic exposure to TNF-
.
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MATERIALS AND METHODS
|
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Materials. The murine 3T3-L1 fibroblast cell line was obtained
from the American Type Culture Collection (ATCC, Rockville, MD). Cell culture
reagents were purchased from GIBCO (Grand Island, NY). Antibodies were
obtained from the following sources: rabbit polyclonal
anti-G
q/11 (carboxy-terminal) (Calbiochem, La Jolla, CA),
rabbit polyclonal anti-GLUT4 (Biogenesis, Sandown, NH), rabbit polyclonal
anti-GLUT1 (Chemicon International, Temecula, CA), anti-PYK2 (BD Transduction
Labs, San Diego, CA, and Santa Cruz Biotechnology, Santa Cruz, CA),
phosphospecific PYK2 antibodies (Biosource International, Camarillo, CA), and
anti-cmyc (9E10; Developmental Hybridoma Studies Bank). Goat
anti-rabbit and anti-mouse IgG coupled to horseradish peroxidase (HRP) were
from Roche (Indianapolis, IN). 2-Deoxy-[3H]glucose and
125I-labeled endothelin-1 were from Amersham (Arlington Heights,
IL). Porcine insulin and endothelin-1 were purchased from Sigma (St. Louis,
MO). The c-myc-GLUT4 expression plasmid, containing a 14-amino acid
sequence of the human c-myc epitope inserted into the first exofacial
loop of GLUT4 (16), was from
Drs. G. Hermann and R. Kelly (University of California at San Fransisco).
Endotoxin-free plasmid kits were from Qiagen (Valencia, CA).
Cell culture and treatments. 3T3-L1 fibroblasts were cultured in
Dulbecco's modified Eagle's medium (high glucose) with 10% fetal bovine serum
(DMEM-FBS) at 37°C in a 10% CO2 atmosphere for 4 days. 3T3-L1
cells were then cultured in DMEM-FBS containing 10 µg/ml insulin, 0.25
µM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine for 4 days to
initiate differentiation. Medium was then changed to DMEM-FBS with insulin
alone, cultured for 3 more days, and then changed to DMEM-FBS for 2 or 3 days
before treatment. Under these conditions, >95% of the cell population
exhibited the morphological characteristics of adipocytes. TNF-
treatments were initiated 9-10 days after the start of differentiation, at
which time 3T3-L1 adipocytes were cultured for 48 h with 0-10 ng/ml
TNF-
in DMEM-FBS.
Uptake of 2-deoxyglucose. Uptake of 2-deoxy-[3H]glucose
was measured in 3T3-L1 adipocytes differentiated in 24-well plates. After
culture with or without TNF-
for 48 h, cells were washed once with
serum-free DMEM and incubated in 1 ml of serum-free DMEM for 1 h at 37°C.
Cells were then washed twice in PBS containing 0.1 mM CaCl2, 0.1 mM
MgCl2, and 25 mM HEPES, pH 7.4, and then incubated in the same
buffer in the presence or absence of insulin or endothelin-1 for 30 min at
37°C. In some experiments, 100 nM wortmannin was added 15 min before the
addition of insulin or endothelin-1. After agonist treatment, uptake of 10
µM 2-deoxy-[3H]glucose was measured over 10 min. Reactions were
terminated by rapidly washing the cells twice with ice-cold PBS. Cells were
then extracted in 0.1 N KOH, and aliquots from the cell extracts were counted
by liquid scintillation and used to determine protein concentration.
Nonspecific uptake was measured in the presence of 10 mM phloretin, an
inhibitor of equilibrative glucose transport.
Transient transfection of 3T3-L1 adipocytes and c-myc-GLUT4 cell
surface assay. 3T3-L1 adipocytes differentiated in six-well plates were
transfected with 2 µg of the c-myc-GLUT4 expression vector using
TransFast at a 3:1 ratio according to the manufacturer's instructions
(Promega, Madison, WI) for 1 h. The transfection reagent was then removed, and
cells were cultured in 2 ml DMEM-FBS for 2 h. Cells were then washed with 1 ml
of warm PBS, incubated with 2 mg/ml collagenase and 0.25% trypsin for 5 min to
detach cells, and then replated in 96-well plates at 33,000 cells/well. Cells
were allowed to attach for 2 h before treatment with or without TNF-
for 48 h.
After 48-h treatment with TNF-
, cells were serum starved for 1 h and
then stimulated with or without 100 nM insulin or 10 nM endothelin-1 for 30
min. Cell surface-accessible c-myc-GLUT4 were measured using a cell
surface ELISA, as described by Wang et al.
(34). Briefly, cells were
fixed in 1% paraformaldehyde for 30 min and washed twice for 1 min in 25 mM
glycine and twice in PBS. Wells were blocked for 90 min in PBS containing 2%
BSA and 1% fish gelatin. Cells were then incubated for 2-3 h with antibody to
c-myc (9E10, 2.4 µg/ml) in blocking buffer, washed, and incubated
for 1 h in anti-mouse IgG HRP (1:1,000). Cells were then washed and incubated
with peroxidase substrate (Sigma) for 30 min, and the optical density at 450
µm was measured. Nonspecific binding was measured in wells incubated
without primary antibody.
Isolation of total membranes and subcellular fractions. 3T3-L1
adipocytes differentiated in 100-mm plates were cultured with 0 or 10 ng/ml
TNF-
for 48 h. The cells were then serum starved for 1 h and then
stimulated with or without 10 nM insulin or endothelin-1 for 30 min. Reactions
were terminated by washing the 3T3-L1 adipocytes with ice-cold PBS. Plasma
membrane fractions were then isolated as described previously
(33) with slight
modifications. Briefly, cells were homogenized in a buffer containing 20 mM
Tris, pH 7.4, 1 mM EDTA, 255 mM sucrose, and protease inhibitors (Complete;
Boehringer Mannheim). After a 20-min centrifugation at 16,000 g, the
pellet was resuspended in the same buffer, layered on a 1.12 M sucrose pad,
and centrifuged at 100,000 g for 70 min. The interface was then
collected and centrifuged at 16,000 g for 15 min to pull down the
plasma membrane fraction. In some experiments, total membranes were isolated
by centrifuging the homogenate at 200,000 g for 60 min. Equal amounts
of proteins (25-50 µg/lane) were solubilized for 15 min at 37°C in
Laemmli sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and
transferred to polyvinylidene difluoride membranes. Western blotting was
carried out as described previously
(23).
PYK2 tyrosine phosphorylation. After TNF-
treatments,
3T3-L1 adipocytes differentiated in six-well plates were stimulated with 10 nM
endothelin-1 for 2 or 5 min, washed twice with ice-cold PBS, and then
solubilized in 25 mM HEPES, pH 7.4, 1% Nonidet P-40, 5 mM NaCl, 2% glycerol, 5
mM sodium fluoride, 1 mM EDTA, 1 mM sodium vanadate, 1 mM sodium
pyrophosphate, and protease inhibitors for 20 min on ice and sheared four
times through a 22-gauge needle. The lysates were centrifuged at 12,000
g for 15 min to remove insoluble material. Samples (60 µg of
proteins) were then boiled in Laemmli buffer for 5 min and resolved by
SDS-polyacrylamide gel electrophoresis.
Competitive 125I-endothelin-1 binding. 3T3-L1
adipocytes differentiated in 24-well plates were cultured with or without 10
ng/ml TNF-
for 48 h. Specific 125I-endothelin-1 binding was
determined as described previously, with slight modifications
(36). Briefly, the cells were
placed on ice, washed, and preincubated in binding buffer (Hanks' buffered
saline containing 25 mM HEPES, pH 7.4, 0.2% BSA and 0.1% glucose) with 20 pM
125I-endothelin-1 with increasing concentrations of unlabeled
endothelin-1 for 3 h at 4°C. 3T3-L1 adipocytes were then washed twice with
ice-cold PBS to remove unbound radioligand and solubilized in 1 N NaOH. Bound
125I-endothelin-1 was then counted by
-spectrometry.
Nonspecific binding was determined in the presence of 1 µM
endothelin-1.
Statistical analysis. Data are presented as means ± SE and
are compared by Student's t-test or analysis of variance. Analysis of
variance was carried out using the general linear model procedure on SAS for
personal computers. Differences between treatment groups were then determined
by least square means analysis. All data were tested for normal
distribution.
 |
RESULTS
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TNF-
decreases endothelin-1-stimulated 2-deoxyglucose
uptake in 3T3-L1 adipocytes. Although many studies have demonstrated that
TNF-
exposure decreases insulin-stimulated glucose transport
(11), here we investigated
whether chronic TNF-
interferes with endothelin-1-stimulated glucose
uptake in 3T3-L1 adipocytes. In control 3T3-L1 adipocytes, endothelin-1
increased 2-deoxyglucose uptake 4.4-fold over basal
(Fig. 1) compared with a
7.4-fold increase with insulin. Culture of 3T3-L1 adipocytes with 2, 5, or 10
ng/ml TNF-
for 48 h dose-dependently decreased both insulin- and
endothelin-1-stimulated 2-deoxyglucose uptake
(Fig. 1).
GLUT4 transporter quantity can be rate limiting for glucose uptake in
adipocytes (31). We first
asked whether TNF-
supressed endothelin-1-stimulated glucose uptake by
decreasing GLUT4 expression. However, chronic exposure of 3T3-L1 adipocytes to
10 ng/ml TNF-
for 48 h had no effect on total GLUT4 quantity
(Fig. 2A). 3T3-L1
adipocytes also express GLUT1 transporter, and several reports have shown that
TNF-
and other inflammatory cytokines increase GLUT1 expression
(5). After treatment of 3T3-L1
adipocytes with 10 ng/ml TNF-
for 48 h, total GLUT1 quantity was
increased 2.6-fold over control cells (Fig.
2A).
Endothelin-1 stimulates glucose uptake by promoting GLUT4 vesicle
translocation to the plasma membrane. Therefore, we investigated whether the
decrease in endothelin-1-stimulated glucose uptake after TNF-
treatment
was associated with impaired GLUT4 translocation. In control cells,
stimulation of 3T3-L1 adipocytes with 10 nM insulin or endothelin-1 increased
GLUT4 protein at the plasma membrane 1.9- and 1.6-fold, respectively
(Fig. 2B). However,
after chronic exposure of 3T3-L1 adipocytes to 10 ng/ml TNF-
, GLUT4
translocation to the plasma membrane in response to insulin or endothelin-1
stimulation was reduced by 30-40% (Fig.
2B). GLUT1 translocation to the plasma membrane can also
be increased by insulin in 3T3 adipocytes and contribute to insulin-stimulated
glucose uptake (1). In control
3T3-L1 adipocytes, insulin increased GLUT1 quantity at the plasma membrane
1.5-fold, whereas endothelin-1 had no effect on GLUT1 translocation. Chronic
treatment with TNF-
increased GLUT1 quantity at the plasma membrane at
baseline, and insulin no longer stimulated GLUT1 translocation to the plasma
membrane (Fig.
2B).
Chronic TNF-
exposure also decreased endothelin-1-dependent
translocation of c-myc-tagged GLUT4 to the cell surface. The
c-myc tag is inserted into the first exofacial loop of the GLUT4
peptide (16); immunological
detection of the c-myc epitope at the cell surface is a measure of
GLUT4 translocation (34).
Stimulation of 3T3-L1 adipocytes with 100 nM insulin or 10 nM endothelin-1
increased surface-accessible c-myc-GLUT4 in control cells 2.6- and
3.3-fold, respectively (Fig.
2C). Chronic treatment of 3T3-L1 adipocytes with 10 ng/ml
TNF-
decreased surface-accessible c-myc-GLUT4 in response to
both insulin and endothelin-1 stimulation
(Fig. 2C), consistent
with the TNF-
-induced decrease in endothelin-1-stimulated
2-deoxyglucose uptake and GLUT4 translocation.
Chronic exposure to TNF-
did not change the
ETA receptor number or affinity in 3T3-L1
adipocytes. Endothelin-1 stimulates GLUT4 translocation and glucose
transport via the ETA receptor, which is the only endothelin-1
receptor isoform expressed in 3T3-L1 adipocytes
(14,
35,
36). Therefore, we next asked
whether suppression of endothelin-1-stimulated glucose uptake in 3T3-L1
adipocytes in response to TNF-
was due to a decrease in the number of
binding sites for endothelin-1 at the cell surface. Competition binding
experiments were carried out to determine the levels of ETA
receptor density after treatment of 3T3-L1 adipocytes for 48 h with or without
10 ng/ml TNF-
. Neither the Bmax (99.65 ± 0.08% of
control) nor the Kd (3.47 ± 0.46 nM in control
cells; 2.57 ± 0.22 nM in TNF-
-treated cells) for
125I-endothelin-1 binding was affected by chronic exposure of
3T3-L1 adipocytes to TNF-
(Fig.
3).
Endothelin-1 stimulation of glucose uptake in 3T3-L1 adipocytes is
independent of PI 3-kinase. It is well established that insulin
stimulation of GLUT4 translocation and glucose transport requires the
activation of PI 3-kinase. TNF-
impairs insulin-stimulated glucose
transport by inducing a serine phosphorylation of IRS-1, which impairs the
ability of insulin to activate PI 3-kinase
(12,
27). Endothelin-1-stimulated
glucose uptake is independent of PI 3-kinase
(17,
36). However, because one
group (14) has reported that
PI 3-kinase activity is required for endothelin-1 stimulation of glucose
transport, we also examined whether PI 3-kinase is involved in the
endothelin-1 stimulation of 2-deoxyglucose uptake in 3T3-L1 adipocytes.
Pretreatment of 3T3-L1 adipocytes with 100 nM wortmannin for 15 min reduced
insulin-stimulated 2-deoxyglucose uptake to basal levels
(Fig. 4). In contrast,
wortmannin had no effect on endothelin-1-stimulated 2-deoxyglucose uptake in
3T3-L1 adipocytes, consistent with other reports
(17,
36) demonstrating that
endothelin-1-stimulated glucose uptake in 3T3-L1 adipocytes is PI 3-kinase
independent. These data suggest that the mechanisms by which TNF-
desensitizes endothelin-1-stimulated glucose uptake are independent of the
effects of TNF-
on the IRS-1/PI 3-kinase pathway.

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Fig. 4. Wortmannin inhibits insulin-stimulated, but not endothelin
(ET)-1-stimulated, 2-deoxyglucose uptake in 3T3-L1 adipocytes. Serum-starved
3T3-L1 adipocytes were treated with or without 100 nM wortmannin for 15 min at
37°C and then stimulated with or without 10 nM insulin or endothelin-1 for
30 min. 2-Deoxy-[3H]glucose uptake was then measured over 10 min.
Values represent means ± SE; n = 5. *P
< 0.05 compared with cells not pretreated with wortmannin.
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ETA receptor couples to the G
q/11 family of
heterotrimeric G proteins (4).
G
q/11 is a necessary signaling intermediate in
endothelin-1-induced GLUT4 translocation and glucose transport
(3,
14). Because
endothelin-1-stimulated glucose transport is independent of PI 3-kinase, we
postulated that TNF-
-induced desensitization of endothelin-1-stimulated
glucose uptake might result from decreased expression of
G
q/11. 3T3-L1 adipocytes were cultured with or without
TNF-
for 48 h, and G
q/11 quantity was meaured by
Western blot analysis. 3T3-L1 adipocytes expressed both G
q
and G
11 polypeptides
(Fig. 5). Chronic exposure of
3T3-L1 adipocytes to 2 or 10 ng/ml TNF-
decreased G
q
and G
11 quantity by 50 and 70%, respectively
(Fig. 5).
TNF-
reduced total PYK2 and endothelin-1-stimulated
PYK2-Tyr402 phosphorylation in 3T3-L1 adipocytes. Tyrosine
phosphorylation is required for endothelin-1 stimulation of GLUT4
translocation and glucose transport. PYK2, a cytoplasmic tyrosine kinase known
to be required for endothelin-1-stimulated glucose uptake
(22), is tyrosine
phosphorylated in response to endothelin-1, but not insulin, stimulation.
Because chronic exposure of 3T3-L1 adipocytes to TNF-
decreased
G
q/11 expression, we hypothesized that endothelin-1
stimulation of PYK2 tyrosine phosphorylation would be decreased in
TNF-
-treated adipocytes compared with controls. Although PYK2 can be
tyrosine phosphorylated on four residues, activation of PYK2 is dependent on
tyrosine phosphorylation on Tyr402
(2). Using a
PYK2-Tyr402-specific antibody, we found that endothelin-1 increased
PYK2-Tyr402 tyrosine phosphorylation in control cells, with a
maximum phosphorylation observed after 5-min stimulation with endothelin-1
(data not shown). Chronic exposure of 3T3-L1 adipocytes to 10 ng/ml
TNF-
decreased total PYK2 expression by 50%
(Fig. 6). Endothelin-1
stimulated the phosphorylation of PYK2-Tyr402 1.6-fold over
baseline in control adipocytes (Fig.
6). However, after chronic exposure to TNF-
,
endothelin-1-stimulated tyrosine phosphorylation of PYK2-Tyr402 was
decreased to only 60-70% of the phosphorylation observed in controls
(Fig. 6).
 |
DISCUSSION
|
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The key finding of the present investigation is the observation that
chronic treatment of 3T3-L1 adipocytes with TNF-
induced a marked
inhibition of endothelin-1-stimulated glucose uptake. Although TNF-
has
potent inhibitory effects on insulin signaling, the extent to which this
important cytokine can affect endothelin-1-stimulated glucose uptake in
adipocytes has not been previously investigated. Here, we report that
TNF
exposure decreases endothelin-1-stimulated glucose uptake by
impairing GLUT4 translocation to the plasma membrane. Although the signal
transduction pathways leading from endothelin-1 receptor activation to GLUT4
translocation are not completely understood, it is clear that
G
q/11 and PYK2 are critical intermediates in this process
(3,
14,
22). After chronic exposure to
TNF-
, the expression of both G
q/11 and PYK2 was
decreased in 3T3-L1 adipocytes. This reduction was associated with an impaired
ability of endothelin-1 to stimulate the phosphorylation of
PYK2-Tyr402, demonstrating that TNF-
suppresses
endothelin-1-stimulated glucose uptake by interfering with the signal
transduction cascade required to activate GLUT4 translocation in 3T3-L1
adipocytes.
Prolonged exposure to TNF-
, especially at periods longer than 72 h,
induces dedifferentiation of fully differentiated adipocytes, including a
decrease in GLUT4 expression
(8). Several reports have
demonstrated that TNF-
-induced insulin resistance is associated with
decreased GLUT4 expression
(30,
32). However, under the
conditions of TNF-
exposure used in our model system, no changes in the
3T3-L1 adipocyte morphology and lipid content were observed (data not shown).
Furthermore, suppression of endothelin-1-stimulated glucose transport by
TNF-
was not associated with a decrease in total GLUT4 protein
quantity. Instead, decreased insulin and endothelin-1-stimulated glucose
uptake after TNF-
exposure was associated with impaired GLUT4
translocation to the plasma membrane. Although chronic TNF-
exposure
for 48 h had no effect on GLUT4 expression, the quantity of GLUT1 was
increased. Cytokines, including TNF-
and inter-leukin-1, have been
shown to upregulate GLUT1 and increase basal glucose uptake through GLUT1
(5). However, this increase in
GLUT1 quantity in response to TNF-
exposure was not sufficient to
compensate for the loss of insulin- and endothelin-1-stimulated glucose
uptake.
3T3-L1 adipocytes contain a single class of endothelin-1-binding sites
identified as the ETA receptor sub-type
(4,
36). One possible consequence
of chronic exposure to TNF-
could be a decrease in endothelin-1
receptor number and affinity leading to less endothelin-1 stimulation of
glucose transport. However, competition binding experiments showed that the
Bmax and Kd for binding of endothelin-1 to the
ETA receptor were not changed by chronic exposure of 3T3-L1
adipocytes to TNF-
, suggesting that TNF-
exposure affected
endothelin-1-dependent signaling downstream of the ETA
receptor.
Insulin and endothelin-1 have additive effects on glucose uptake in
adipocytes (25,
36). Endothelin-1-stimulated
glucose uptake in 3T3-L1 adipocytes is independent of PI 3-kinase. Instead,
numerous studies have demonstrated a critical role of G
q/11
proteins in the endothelin-1-stimulated
(14), as well as
insulin-stimulated (15), GLUT4
vesicle translocation and glucose transport
(17). Overexpression of
constitutively active forms of G
q (Q209L) or
G
11 (Q209L) in 3T3-L1 adipocytes stimulates F-actin
polymerization, GLUT4 translocation, and glucose uptake
(3,
18). Conversely,
microinjection of RGS2 (regulator of G protein signaling 2), a protein that
inhibits G
q/11 activity, or microinjection of antibodies
against G
q/11 inhibits GLUT4 translocation
(14). Prolonged exposure to
TNF-
has been reported to modulate the expression of heterotrimeric G
proteins in different cell types
(9,
10,
24,
28). In the present study, we
show that chronic exposure of 3T3-L1 adipocytes to TNF-
decreased
G
q/11 expression. This decrease in G
q/11
quantity likely contributes to the TNF-
desensitization of
endothelin-1-stimulated GLUT4 translocation and glucose uptake in 3T3-L1
adipocytes. Moreover, because G
q/11 has also been implicated
in insulin-stimulated GLUT4 translocation, TNF-
-induced decrease in
G
q/11 expression may also contribute, in conjunction with
impaired insulin receptor signaling
(11), to the effects of
TNF-
on insulin-stimulated glucose uptake in 3T3-L1 adipocytes.
Insulin and endothelin-1/G
q/11 utilize distinct signaling
pathways to stimulate GLUT4 translocation and F-actin polymerization
(22). PYK2, a
Ca2+-sensitive protein tyrosine kinase, is a downstream
effector in the endothelin-1/G
q/11, but not the insulin,
stimulation of GLUT4 vesicle translocation to the plasma membrane
(22). Dominant inhibitory
constructs of PYK2 (CADTK-related nonkinase, CRNK) inhibited endothelin-1 but
not insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes
(22). Because
TNF-
-induced desensitization of insulin-stimulated glucose uptake is
primarily due to impaired insulin signaling via the PI 3-kinase pathway
(11), we hypothesized that the
effects of TNF-
on endothelin-1-stimulated glucose uptake would reveal
an additional signaling pathway susceptible to regulation by TNF-
in
3T3-L1 adipocytes. Here, we show that endothelin-1 stimulates the
phosphorylation of PYK2-Tyr402 in 3T3-L1 adipocytes. Importantly,
when 3T3-L1 adipocytes were exposed to 10 ng/ml TNF-
for 48 h,
expression of total PYK2 was decreased and endothelin-1 stimulation of
PYK2-Tyr402 phosphorylation was impaired. These data identify PYK2
expression and phosphorylation as new targets for regulation by TNF-
and suggest that TNF-
-induced decreases in
G
q/11-mediated signaling, in combination with decreased PYK2
protein quantity, together contribute to TNF-
-induced desensitization
of endothelin-1-stimulated glucose transport.
 |
DISCLOSURES
|
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This work was supported by US Public Health Service National Institute on
Alcohol Abuse and Alcoholism Grant R01 AA-11876. The antibody to
c-myc (9E10), developed by J. M. Bishop, was from the Developmental
Studies Hybridoma Bank, maintained by the Department of Biological Sciences,
University of Iowa (Iowa City, IA) under contract NO1-HD-7-3263.
 |
ACKNOWLEDGMENTS
|
---|
We thank Gary Herman and Regis Kelly (UCSF) for the c-myc GLUT 4 expression
vector.
 |
FOOTNOTES
|
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Address for reprint requests and other correspondence: L. E. Nagy, Dept. of
Nutrition, Case Western Reserve University, 2123 Abington Rd., Rm. 201,
Cleveland, OH 44106-4906 (E-mail:
Len2{at}po.cwru.edu).
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