(Received for publication, June 24, 1996, and in revised form, September 19, 1996)
From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
A number of studies have demonstrated
that tumor necrosis factor- (TNF-
) is associated with profound
insulin resistance in adipocytes and may also play a critical role in
the insulin resistance of obesity and non-insulin-dependent
diabetes mellitus. Reports on the mechanism of TNF-
action have been
somewhat contradictory. GLUT4 down-regulation has been implicated as a
possible cause of insulin resistance as has been the reduced kinase
function of the insulin receptor. Here we examine the effects of tumor necrosis factor on the protein components thought to be involved in
insulin-stimulated glucose transport in adipocytes, namely the insulin
receptor, its major substrate IRS-1, and the insulin responsive glucose
transporter GLUT4. Prolonged exposure (72-96 h) of 3T3-L1 adipocytes
to TNF-
causes a substantial reduction (>80%) in IRS-1 and GLUT4
mRNA and protein as well as a lesser reduction (>50%) in the
amount of the insulin receptor. Nevertheless, the remaining proteins
appear to be biochemically indistinguishable from those in untreated
adipocytes. Both the insulin receptor and IRS-1 are
tyrosine-phosphorylated to the same extent in response to acute insulin
stimulation following cellular TNF-
exposure. Furthermore, the
ability of the insulin receptor to phosphorylate exogenous substrate in
the test tube is also normal following its isolation from
TNF-
-treated cells. These results are confirmed by the reduced but
obvious level of insulin-dependent glucose transport and
GLUT4 translocation observed in TNF-
-treated adipocytes. We conclude
that the insulin resistance of glucose transport in 3T3-L1 adipocytes
exposed to TNF-
for 72-96 h results from a reduced amount in
requisite proteins involved in insulin action. These results are
consistent with earlier studies indicating that TNF-
reduces the
transcriptional activity of the GLUT4 gene in murine adipocytes, and
reduced mRNA transcription of a number of relevant genes may be the
general mechanism by which TNF-
causes insulin resistance in
adipocytes.
Insulin resistance is defined as the inability of cells or tissues
to respond to physiological levels of insulin and is a characteristic
condition of early stage non-insulin-dependent diabetes
mellitus (1). Insulin resistance has also been described in a number of
disease conditions including cancer, sepsis, endotoxemia, trauma, and
alcoholism. These latter situations are known to cause altered levels
of cytokine expression whose actions, in part, may lead to the
establishment of the pathophysiological state (2). In particular, tumor
necrosis factor- (TNF-
)1 secretion
from activated macrophages has been recognized as one of the initial
responses in these disease states. Evidence from both whole-animal and
cell culture studies indicate that TNF-
alters protein and lipid
metabolism in adipose tissue and in skeletal muscle, the insulin target
tissues whose lack of hormonal response would result in insulin
resistance (2, 3). Studies in cultured cells have demonstrated that
many cytokines can regulate glucose transport (4, 5) and TNF-
, in
particular, causes the decreased expression of the insulin-sensitive
glucose transporter GLUT4 (6). However, it is not completely clear
whether this accounts for organismal insulin resistance, since muscle
GLUT4 levels are normal in insulin-resistant rodents and humans
(7, 8, 9).
On the other hand, numerous recent studies have implicated the
involvement of TNF- in insulin resistance in adipocytes in culture
as well as in whole-animal models. As noted above, TNF-
treatment of
cultured murine adipocytes (3T3-L1s) for several days results in a
significant repression of GLUT4 transcription and expression, resulting
in a condition of insulin resistance without a depletion of lipid
content or a change in other fat-specific genes such as lipoprotein
lipase (6). In vivo studies have demonstrated that the
adipose tissue of obese insulin-resistant rodents (10) and obese humans
(11, 12) has a significant increase in TNF-
production and that
neutralization of TNF-
in insulin-resistant rodents results in an
increase in the peripheral uptake of glucose in response to insulin
(10).
Thus, a role for TNF- in some types of insulin resistance appears
quite likely, and therefore understanding its mechanism of action will
be important. One mechanism that has been suggested for TNF-
-induced
insulin resistance is inhibition of signaling from the insulin receptor
(13, 14). Both a defect in the ability of the insulin receptor to
autophosphorylate and a loss of its ability to phosphorylate, on
tyrosine residues, its major substrate, insulin receptor substrate-1
(IRS-1) has been reported (13, 14). More recently, TNF-
has been
reported to induce serine phosphorylation of IRS-1 which, in turn,
inhibits the insulin receptor from phosphorylating this substrate (15).
A precedent for this mechanism comes from studies of the phosphatase
inhibitor okadaic acid, which has been shown to cause serine
phosphorylation of IRS-1 and a subsequent inhibition of insulin
receptor signaling (16). This biochemical mechanism makes sense for the
rapid regulation of insulin signaling by TNF-
(17, 18) and okadaic
acid (16), because phosphorylation/dephosphorylation are rapid (seconds
to minutes) events. However, the effect of chronic TNF-
treatment (96-120 h) would seem less likely to utilize such a mechanism. Moreover, although GLUT4 is significantly down-regulated after 96 h of adipocyte TNF-
exposure, there is still some residual insulin-stimulated glucose transport (6) (see Table II).
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Thus, in the present study, we have used insulin-sensitive glucose
uptake as an assay for insulin-dependent signal
transduction to examine the development of TNF--induced insulin
resistance. We see no evidence for any changes in the overall tyrosine
phosphorylation states of the insulin receptor or its major substrate,
IRS-1. On the other hand, TNF-
-induced insulin resistance appears to involve down-regulation of several of the known component proteins required for insulin-stimulated glucose transport. These include GLUT4,
IRS-1, and to a lesser extent the insulin receptor.
Recombinant human TNF- was from Biogen. Murine
TNF-
was purchased from Quality Control Biochemicals. All
experiments were performed with both human and murine TNF-
, and
identical results were obtained with both the murine and human
cytokine. Dulbecco's modified Eagle's medium (DMEM) was purchased
from BioWhittaker, Inc. Bovine and fetal bovine serum were obtained
from Hyclone and Life Technologies, Inc., respectively. Wheat germ
agglutinin-coupled agarose was obtained from E. Y. Laboratories.
[
-32P]ATP and [3H]2-deoxyglucose were
acquired from Dupont NEN. All other chemicals were purchased from
Sigma unless otherwise noted.
Murine 3T3-L1 preadipocytes were cultured,
maintained, and differentiated as described previously (19). Briefly,
cells were plated and grown for 2 days postconfluence in DMEM
supplemented with 10% calf serum. Differentiation was then induced by
changing the medium to DMEM supplemented with 10% fetal bovine serum,
0.5 mM 3-isobutyl-1-methylxanthine, 1 µM
dexamethasone, and 1.7 µM insulin. After 48 h, the
differentiation medium was replaced with maintenance medium containing
DMEM supplemented with 10% fetal bovine serum. The maintenance medium
was changed every 48 h until the cells were utilized for
experimentation. Human or murine TNF- was dissolved in
phosphate-buffered saline containing 0.1% fatty acid-free and growth
factor-depleted bovine serum albumin (Sigma) and was
added to the cell culture media 7 days after the induction of
differentiation when greater than 95% of the cells had the morphological and biochemical properties of adipocytes. In the case of
prolonged TNF-
treatment (>1 day), fully differentiated 3T3-L1
adipocytes were treated every 24 h with the cytokine.
Prior to harvesting,
cells were thoroughly rinsed at 37 °C with DMEM and then incubated
in fresh serum-deprived DMEM for 2-4 h. Untreated and TNF--treated
3T3-L1 adipocytes were rinsed with Buffer A (250 mM
sucrose, 20 mM HEPES, 1 mM EDTA, pH 7.4) at
37 °C and then harvested at 4 °C in Buffer A and homogenized with
a Teflon pestle. Total membranes were pelleted at 250,000 × g for 90 min and resuspended in Buffer B (20 mM
HEPES, 1 mM EDTA, pH 7.4). The cytosolic supernatant was
also saved for further analysis. For some experiments, membranes were
fractionated according to the protocol of Clancy and Czech (20) into
plasma membrane, intracellular membranes, and a nuclear/mitochondrial
fraction as we have previously described (6). Membrane and cytosolic fractions were divided and immediately stored at
70 °C. Both Buffer A and Buffer B contained the following protease and phosphatase inhibitors: 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 50 trypsin inhibitory milliunits of
aprotinin, 1 mM 1,10-phenanthroline, 1 µM
pepstatin, 2.5 mM benzamidine hydrochloride, 100 mM sodium fluoride, 1 mM sodium vanadate, 30 mM sodium pyrophosphate, and 1 mM sodium
molybdate. The protein content for all fractions was determined with a
BCA kit (Pierce) according to the manufacturer's instructions.
Proteins were separated in 7.5 or 12% polyacrylamide (acrylamide from National Diagnostics) gels containing sodium dodecyl sulfate (SDS) according to Laemmli (21) and transferred to nitrocellulose (Bio-Rad) or Immobilon-P (Millipore) in 25 mM Tris, 192 mM glycine, and 20% methanol. Following transfer, the membrane was blocked in 4% nonfat milk for 1 h at room temperature. Results were visualized with horseradish peroxidase-conjugated secondary antibodies (Sigma) and an enhanced chemoluminescence kit (Pierce).
The antibody used for detection of the insulin receptor (R1064) was generated against the C terminus deduced from the insulin receptor (22). The anti-phosphotyrosine monoclonal antibody, 4G10, was purchased from Upstate Biotechnology, Inc. and used to detect insulin receptor phosphorylation. The anti-phosphotyrosine monoclonal antibody, PY20, was purchased from Leinco and used to detect IRS-1 phosphorylation. A monoclonal antibody for GLUT4 (1F8), was used for detection of the insulin-sensitive glucose transporter (23). Western blots employing a monoclonal antibody were analyzed using goat anti-mouse antibody (Sigma) coupled to horseradish peroxidase followed by chemoluminescence using the Renaissance system (Dupont NEN). Blots using polyclonal antibodies were exposed to goat anti-rabbit (Sigma) coupled to horseradish peroxidase and visualized with chemoluminescence.
Exogenous Kinase Activity AssaysThe exogenous kinase activity of the insulin receptor following its solubilization and partial purification from cell extracts was determined as described previously (24). Briefly, the insulin receptor was purified from total membranes by wheat germ agglutinin-agarose affinity chromatography in the presence of the protease inhibitors and phosphatase inhibitors listed above. Bound receptor was eluted with 0.3 M N-acetylglucosamine in 20 mM HEPES (pH 7.4) containing 0.1% Triton X-100. The receptor was incubated with 50 µM ATP (100 µCi/ml [32P]ATP), 10 mM MgCl2, 8 mM MnCl2, and 2.5 mg/ml of poly(Glu:Tyr) (4:1) as a phosphate acceptor. Following a 10-min incubation, the reaction was stopped by adding EDTA to a final concentration of 67 mM. The reaction mixture was loaded on a 3 × 3-cm piece of 3 M filter paper which was extensively washed with 10% trichloroacetic acid and 100 mM pyrophosphate. Radioactivity incorporated into poly(Glu:Tyr) was determined as Cerenkov radiation in a scintillation counter. For the final results, radioactivity was normalized by the amount of the insulin receptor, which was determined by Western blot as described previously (24).
RNA Isolation and AnalysisTotal RNA was isolated from the
cells by extraction with Trizol (Life Technologies, Inc.) and was
prepared according to the manufacturer's instructions.
Poly(A)+ RNA was selected using a poly(A)+
tract kit (Stratagene). For Northern blot analysis, 5 µg of
poly(A)+ were separated by electrophoresis in 1.3%
agarose, 2.0 M formaldehyde gels and transferred to Gene
Screen Plus (Dupont NEN). After ultraviolet cross-linking, filters were
prehybridized, hybridized, and subjected to analysis as described
previously (19). The cDNAs utilized in these studies were as
follows: GLUT4, the insulin-sensitive glucose transporter (25); IRS-1,
a generous gift from Dr. Morris White (26); and -actin, 1.9-kilobase
HindIII fragment obtained from Dr. D. W. Cleveland
(27).
The assay of [3H]2-deoxyglucose uptake was performed as described previously (19). Prior to the assay, the cells were deprived of serum for 2-4 h. Uptake measurement were made in triplicate under conditions when hexose uptake was linear, and the results were corrected for nonspecific uptake and absorption determined by [3H]2-deoxyglucose uptake in the presence of 5 µM cytochalasin B. Nonspecific uptake and absorption was always less than 10% of the total uptake.
In
hepatocytes, it has been reported that TNF- induces a defect in
insulin signaling in a time frame of less than 1 h (16, 17). Thus,
we examined the effect of relatively low (250 pM) and high
(1 nM) doses of TNF-
treatment on fully differentiated 3T3-L1 adipocytes over a 24-h time course. As shown in Table
I, low doses of TNF-
had essentially no effect on
basal or insulin-stimulated glucose uptake. Furthermore,
insulin-stimulated glucose uptake was unaffected by 1 nM
TNF-
following cytokine treatment for 1 and 6 h (Table I).
There may be a slight decrease in insulin-stimulated glucose uptake in
adipocytes exposed to the high doses of TNF-
for 24 h (7.8-fold
stimulation versus 9.2-fold, untreated). However, at this
time, GLUT4 protein is slightly diminished (data not shown), and the
basal uptake is slightly elevated (Table I). Thus, we observe no
significant compromise in insulin-stimulated glucose transport in
adipocytes exposed to relatively short treatments of TNF-
and
therefore, no lesion in insulin-dependent signal transduction.
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We also performed a longer time course of TNF- treatment under
conditions previously shown to cause insulin-resistant glucose uptake
in fully differentiated 3T3-L1 adipocytes (28). As in the cited study,
maximal inhibition of insulin-sensitive glucose uptake was not achieved
until after 96 h of TNF-
treatment (Table II).
Only after 48 h of TNF-
exposure do the adipocytes exhibit a
significant decrease in insulin-stimulated glucose uptake as compared
to untreated cells (Table II). Furthermore, there is minimal
synergistic action of TNF-
and insulin when serum is supplemented
with a high insulin concentration (Table II). Exposure to TNF-
for
96 h resulted in an increase in basal glucose uptake, but more
importantly, there remained a 2-3-fold increase in insulin-stimulated glucose transport even after 96 h of TNF-
exposure, thus
indicating the likelihood that insulin-dependent signal
transduction pathway(s) remain intact at this time. TNF-
treatment
for more than 96 h did not result in a further decline of
insulin-sensitive glucose uptake (data not shown).
We next examined the effects of TNF- on
the level of the insulin receptor, IRS-1, and GLUT4 proteins (Fig.
1) as well as on mRNA levels for the latter two
(Fig. 2). Fully differentiated 3T3-L1 adipocytes were
treated for 0, 24, 72, or 96 h with 250 pM TNF-
,
and total membranes were probed by Western blotting with the
appropriate antibody. Cytosolic fractions or whole-cell extracts were
examined for IRS-1 content, and the results were identical for both
procedures (data not shown). Fig. 1 shows that TNF-
treatment
resulted in a slight decrease (~30%) in insulin receptor protein
after 96 h. However, there is a very significant decrease in both
GLUT4 and IRS-1 levels over this time. Both of these protein decreased
by 50-70% after 72 h and were only 20% of controls after
96 h of TNF-
exposure. The quantitative analysis of these data
was normalized against the content of secretory component-associated
membrane proteins, a set of proteins known to be present in GLUT4
vesicles (29, 30) which are not regulated by TNF-
in 3T3-L1
adipocytes (Fig. 1). Fig. 2 is a representative Northern blot of IRS-1,
GLUT4, and
-actin mRNA expression in untreated adipocytes and in
cells treated with 250 pM TNF-
for 96 h. As
expected and as normalized against
-actin mRNA expression, IRS-1
and GLUT4 mRNA from TNF-
-treated adipocytes are less than 20%
of the levels in untreated adipocytes.
The Effect of TNF-
It has been suggested that prolonged TNF-
treatment (96 h or more) results in diminished insulin receptor
phosphorylation and kinase activity in adipocytes (13, 14).
Accordingly, we examined insulin receptor amount and its ability to
undergo autophosphorylation in cells following 96 h of adipocyte
exposure to various TNF-
doses. Fig. 3 shows that the
insulin receptor content diminishes slightly with exposure to 250 pM TNF (see also Fig. 1) and is more dramatically
down-regulated at higher cytokine doses (Fig. 3, bottom).
However, the tyrosine phosphorylation state of the insulin receptor
from TNF-
-treated cells was essentially unchanged (Fig. 3,
top). There is no insulin receptor autophosphorylation unless cells are exposed to insulin, and when they are, they respond completely normally as determined by phosphotyrosine blotting (Fig. 3,
top). The autophosphorylation signal precisely corresponds to the amount of receptor, and they are diminished in parallel in
response to TNF-
treatment.
Fig. 4 depicts another experiment in which insulin
receptors were isolated from untreated and TNF--treated adipocytes
by wheat germ agglutinin chromatography (see under "Experimental Procedures") and then examined for insulin receptor content and phosphorylation state (top) by Western blotting. We observed
a small decrease in both receptor amount and phosphorylation state (top) from the TNF-
-treated cells (see also Fig. 3). We
used the partially purified receptor to phosphorylate substrate
(bottom) in the test tube, and because slightly less
receptor was isolated from the TNF-
-treated cells (top),
slightly lower substrate phosphorylation was seen (bottom).
When normalized to receptor amount, there is no difference in the
in vitro kinase activity or autophosphorylation state of
receptors isolated from untreated and TNF-
-treated adipocytes. We
also performed an experiment using receptors purified from untreated
and TNF-
-treated (96 h) adipocytes that had not been exposed to
insulin. Subsequent measurement of autophosphorylation and exogenous
kinase activity gave identical results for receptors from
TNF-
-treated and untreated cells (data not shown).
The Effect of TNF-
From
the previous figures, only the amount of protein and mRNA for the
various components of insulin-stimulated glucose uptake were shown to
diminish in response to TNF-. However, the function of IRS-1 has
also been suggested to be altered in TNF-
-induced insulin resistance
in adipocytes (13, 14, 15). Therefore, we examined the
insulin-dependent tyrosine phosphorylation of IRS-1 in
chronically TNF-
-treated adipocytes (250 pM for 96 h). As also shown in Fig. 1, Fig. 5 shows that TNF-
treatment results in significant loss of IRS-1 protein (>80%,
top). IRS-1 tyrosine phosphorylation is diminished to the
same extent (bottom). Thus, as was the case for insulin
receptor autophosphorylation (Fig. 3), TNF-
diminishes the amount of
IRS-1, yet the remaining IRS-1 protein is tyrosine-phosphorylated in a
normal fashion in response to insulin.
Effect of TNF-
The prior data
(Figs. 1, 2, 3, 4, 5) coupled with the transport data of Table II suggests no
obvious lesion in insulin signaling in adipocytes due to TNF-
exposure except for a decreased amount of the component proteins of the
signaling pathway. As a further verification of this, we performed a
GLUT4 translocation assay on adipocyte membrane fractions (Fig.
6) on insulin-sensitive (untreated) and
insulin-resistant (96 h of TNF-
exposure) 3T3-L1 adipocytes. The
cells were exposed or not to insulin stimulation (100 nM
for 8 min) and then fractionated into membrane compartments. In
insulin-treated adipocytes, there is an increase in GLUT4 at the plasma
membrane (PM), and a corresponding decrease in internal membrane (IM) GLUT4, a result indicative of transporter
translocation to the plasma membrane (31). The level of GLUT4 protein
present in the plasma membrane of untreated cells is higher than
expected from the transport data of Table II but is consistent with
many published studies that indicate membrane fractionation of 3T3-L1 adipocytes is less precise than that of rat adipocytes (e.g.
see Ref. 20). In any case, following 96 h of TNF-
exposure,
GLUT4 levels were markedly diminished in both plasma membrane and
internal membrane membrane fractions (top) as would be
expected from Figs. 1 and 2 and from previous studies (6, 10, 28).
Nevertheless, when enough protein was loaded onto SDS-polyacrylamide
gels and immunoblotted for GLUT4 protein (bottom), an
increase in the transporter at the plasma membrane is caused by insulin
exposure of TNF-
-treated adipocytes as is a depletion of GLUT4 from
the internal membranes. Thus, GLUT4 translocation to the plasma
membrane appears normal even after prolonged exposure of fat cells to
TNF-
, a result consistent with the glucose uptake data (Table
II).
In general, the biochemical changes in cells brought about by
cytokines such as TNF- are thought to occur as result of altered gene expression, often due to activation of the STAT
(
ignal
ransducers and
ctivators
of
ranscription) family of transcription factors (32, 33).
Thus, the recent results from several groups indicating that TNF-
may cause rapid (17, 18) or slow (13, 14, 15) alterations in the ability of
insulin to phosphorylate IRS-1 in hepatocytes (17, 18) and adipocytes
(13, 14, 15) prompted us to re-examine this process in 3T3-L1 adipocytes.
Previous studies with these cells indicated that TNF-
results in
insulin resistance after prolonged exposure to TNF-
(6, 10, 13, 14,
28) either as a result of GLUT4 down-regulation (6, 10, 28) or from
diminished insulin receptor signaling, probably at the level of IRS-1
tyrosine phosphorylation (13, 14, 15). On the other hand, our current
studies show no defect of the insulin receptor in its ability to
autophosphorylate in TNF-
-treated adipocytes (Fig. 3) or in
vitro after isolation from identically treated adipocytes (Fig.
4). We also see no defect in IRS-1 tyrosine phosphorylation in
TNF-
-exposed adipocytes (Fig. 5) nor is the ability of the insulin
receptor to phosphorylate a synthetic substrate compromised after
isolation from chronically TNF-
-treated (96 h) adipocytes (Fig. 4).
Rather, we observe a global down-regulation of some of the known
component proteins of insulin-stimulated glucose transport but not of
several control proteins (Figs. 1, 2, 3), in agreement with prior results
examining TNF-
-induced gene regulation in adipocytes (6). The
remaining component proteins of the insulin-stimulated glucose
transport apparatus appear to function normally at the level of both
transport activation (Table II) and GLUT4 translocation (Fig. 6).
We are at a loss to reconcile our current results with those from very
similar studies employing the same or similar cultured cell lines of
murine adipocytes (14, 15). It is possible that variations in the
clonal nature of 3T3-L1 cells could give different results, and in
addition, one of the published studies used a different adipocyte cell
line, 3T3-F442A cells, where a loss of insulin sensitivity was detected
prior to a loss in GLUT4 (14). One methodological difference between
our studies and those cited above (14, 15) is that, except for the
experiment of Table II, all of the figures we show here were from
experiments in which fat cells were exposed to TNF- in the absence
of insulin, whereas Hotamisligil and colleagues performed their
experiments in the presence of this hormone (14, 15). The reason that
we generally omitted insulin from our studies is that chronic exposure
of 3T3-L1 adipocytes to insulin has long been known to down-regulate
insulin receptors (34). More recently, it has been shown that long term exposure of 3T3-L1 fat cells to low doses of insulin causes a significant loss in the cellular GLUT4 content (35) as well as a 70%
decrease in IRS-1 protein which, nevertheless, is still tyrosine-phosphorylated in response to insulin (36). As shown in Table
II, the presence of insulin during TNF-
exposure leads to a slight
decrease in insulin-stimulated glucose transport as compared to TNF
alone as early as 48 h after treatment. Similarly, we showed that
the effects of cytokine plus insulin on insulin receptor expression was
greater than cytokine alone, but receptor autophosphorylation and IRS-1
phosphorylation dropped in parallel (data not shown), as they do for
the TNF only condition (Figs. 3 and 5). Since these results (Table II
and data not shown) are in complete agreement with previously published
studies of the effect of insulin alone on its receptor, GLUT4, and IRS1
(34, 35, 36), we chose to present the novel observation that TNF-
alone
has a similar effect to insulin in down-regulating several components
of insulin-stimulated glucose transport in cultured fat cells.
On the other hand, we have no information concerning the data from
studies on hepatocytes in which TNF- was shown to affect insulin
receptor function in a matter of minutes (17, 18). In adipocytes, acute
TNF-
treatment (15 min) or pretreatment of TNF-
followed by
insulin stimulation results in an enhancement of IRS-1 tyrosine
phosphorylation and promotes its association with phosphatidylinositol
3-kinase (37). In the course of our studies, we have also observed a
very small enhancement of IRS-1 tyrosine phosphorylation in adipocytes
exposed to prolonged TNF-
treatment (data not shown). However, it
has not been demonstrated that IRS-1 is a mediator of TNF-
signal
transduction in any cell type.
Another question raised by our data concerns the nature of the TNF
receptor that mediates the effects we observe. There are two cell
surface TNF- receptors, a type I receptor of
Mr 55,000 and a type II species of
Mr 75,000, that are present in all cell types
(38). Most biological actions of TNF-
are mediated by the type I
receptor (39), which in the case of murine cells is the only receptor
isotype that binds human TNF-
(40). As indicated under
"Experimental Procedures," we performed all our experiments with
TNF-
from both species, and we obtained identical results, thus
indicating that the type I receptor is mediating the responses we
observed in adipocytes. Very recently, Peraldi et al.
reached the same conclusion based on the same protocol of comparing
human and murine TNF-
actions in 3T3-L1 cells (41).
In summary, we conclude that TNF--induced insulin resistance in
3T3-L1 adipocytes is not accompanied by a defect in insulin receptor
phosphorylation, IRS-1 tyrosine phosphorylation, or the ability of the
insulin receptor to recruit GLUT4 to the plasma membrane. Our data
indicate that the primary mechanism of TNF-
action is likely to be
at the level of regulation of gene expression, the general mode of
action for cytokines (32, 33). This hypothesis is supported by previous
studies which demonstrate that short (2 h) exposure of 3T3-L1
adipocytes to TNF-
results in a >90% inhibition of GLUT4
transcription in the absence of protein synthesis (28). These
conclusions differ dramatically from other investigators (13, 14, 15, 17,
18), and further studies are warranted to resolve both the current
discrepancies and the mechanism of action of TNF in adipocytes.