(Received for publication, August 10, 1995; and in revised form, November 17, 1995)
From the
Chronic incubation of 3T3-L1 adipocytes with tumor necrosis
factor (TNF) induces a state of insulin resistance characterized by a
diminished ability of insulin to induce phosphorylation of the
subunit of its own receptor and insulin receptor substrate 1 (IRS-1).
When adipocytes are briefly pretreated with TNF and then stimulated
with insulin, tyrosine phosphorylation of IRS-1 increases above the
level induced by insulin alone. By itself, TNF induces the
time-dependent tyrosine phosphorylation of proteins in 3T3-L1
adipocytes. Among these is IRS-1, a docking protein with tyrosine
phosphorylation sites that bind cytoplasmic signaling molecules that
contain Src homology 2 (SH2) domains. TNF stimulation of 3T3-L1
adipocytes also promotes the association of the p85 regulatory subunit
of phosphatidylinositol 3-kinase (PI 3-kinase) with IRS-1 and also its
tyrosine phosphorylation. In murine 3T3-L1 adipocytes, IRS-1 and PI
3-kinase phosphorylation and the association of these proteins are
promoted by murine TNF, which interacts with the type 1 and type 2 TNF
receptors. Human TNF, which binds to the murine type 1 TNF receptor
selectively, also promotes IRS-1 phosphorylation and binding of IRS-1
to PI 3-kinase. This is the first demonstration that a member of the
TNF/nerve growth factor receptor superfamily can use an IRS-1 signaling
system as a component of its cellular response and provides a mechanism
through which TNF receptors may engage downstream elements in signaling
pathways.
Tumor necrosis factor is a multifunctional cytokine that was
first characterized by its ability to induce the regression of cancers
in animals and by the cytotoxic response that it can elicit from
transformed cells(1, 2) . Subsequent investigation
revealed that TNF ()has profound effects on the growth,
differentiation, and metabolism of normal cells and the function of
normal tissues. In addition to its oncolytic activity, TNF promotes
immunity, antiviral responses, inflammation, and in some chronic
diseases the syndrome of wasting and malnutrition known as
cachexia(1, 2) .
TNF has been implicated in the insulin resistance of cancer and non-insulin-dependent diabetes mellitus(1, 2, 3, 4, 5) . The adipose tissues of animal models of obesity and non-insulin-dependent diabetes mellitus produce TNF(3) . Neutralization of TNF in one model, the Zucker (fa/fa) rat, increases insulin-stimulated autophosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1) in muscle and fat, restoring them to near the level observed in lean animals(4) . Chronic treatment of 3T3-L1 adipocytes with TNF inhibits glucose uptake and induces a moderate decrease of insulin-stimulated phosphorylation of the insulin receptor and a more pronounced inhibition of insulin-promoted phosphorylation of IRS-1(5) . Thus, when present chronically, TNF interferes with signaling through the insulin receptor to downstream substrates, such as IRS-1, that mediate responses to insulin(5) .
Here we show that the acute effects of TNF on 3T3-L1 adipocytes are different than those elicited during extended incubations. TNF acutely induces the tyrosine phosphorylation of IRS-1 and its engagement with cytoplasmic signaling molecules, one of which is identified as PI 3-kinase. These observations show that TNF can elicit an acute growth factor or insulin-like effect in cells and provide a mechanism through which TNF receptors can engage signaling processes.
Figure 2: Effect of acute treatment with TNF on insulin-stimulated tyrosine phosphorylation of IRS-1. A, 3T3-L1 adipocytes were treated with 5 nM mTNF at 37 °C for various times, 100 nM insulin (INS) being added during the last 3 min of incubation with the cytokine. Proteins in cell lysates were immunoprecipitated with antiserum to IRS-1, fractionated by SDS-PAGE, and transferred to Immobilon-P. The Western blots were probed with antiserum to phosphotyrosine. B, the Western blot shown in A was scanned with a densitometer, and the results are presented in arbitrary scanning units.
Figure 3: TNF-promoted tyrosine phosphorylations. 3T3-L1 adipocytes were treated with 5 nM mTNF at 37 °C for various times. Phosphorylated proteins were immunoprecipitated from cell lysates using antiserum to phosphotyrosine, fractionated by SDS-PAGE, and transferred to Immobilon-P. The Western blot was incubated with antiserum to phosphotyrosine.
Figure 4: TNF-promoted phosphorylation of IRS-1. A, 3T3-L1 adipocytes were treated with 5 nM mTNF at 37 °C for various times. Proteins in cell lysates were immunoprecipitated with antiserum to IRS-1, fractionated by SDS-PAGE, and transferred to Immobilon-P. The Western blot was incubated with antiserum to phosphotyrosine. B, the Western blot was stripped and reprobed with antiserum to IRS-1. C, the Western blot was scanned with a densitometer, and the effect of TNF on IRS-1 phosphorylation is reported in arbitrary scanning units.
Figure 5: Top, effect of mTNF on PI 3-kinase. A, association of IRS-1 and PI 3-kinase. 3T3-L1 adipocytes were incubated with 5 nM mTNF or 100 nM insulin (INS) for 15 min at 37 °C. Proteins in cell lysates were immunoprecipitated with IRS-1 antiserum, fractionated by SDS-PAGE, and transferred to Immobilon-P, which was probed with antiserum to the regulatory subunit of PI 3-kinase. Also, proteins in a cell lysate were immunoprecipitated with antiserum to PI 3-kinase, fractionated by SDS-PAGE, and blotted to Immobilon-P, which was probed with antiserum to PI 3-kinase, which definitively identified the regulatory subunit of the enzyme. B, mTNF-promoted phosphorylation of PI 3-kinase. 3T3-L1 adipocytes were incubated in the absence or presence of 5 nM mTNF for 15 min at 37 °C. Proteins in cell lysates were immunoprecipitated with antiserum to PI 3-kinase, fractionated by SDS-PAGE, and transferred to Immobilon-P, which was probed with antiserum to phosphotyrosine. Bottom, phosphorylations induced by hTNF. Cells were incubated with 5 nM hTNF or 100 nM insulin for 15 min at 37 °C. C, association of IRS-1 and PI 3-kinase was examined as described in the legend to A. D, tyrosine phosphorylation of IRS-1 was evaluated as described in the legend to Fig. 4.
Chronic incubation of 3T3-L1 adipocytes with mTNF has been
reported to inhibit the ability of insulin to induce tyrosine
phosphorylation of its receptor and IRS-1(5) . We have
confirmed that TNF was capable of inducing a state of insulin
resistance in the 3T3-L1 adipocytes employed in the present study. To
accomplish this, 3T3-L1 adipocytes incubated for 5 days in the absence
or presence of 0.1 nM mTNF were tested for responsiveness to
insulin, based on the ability of insulin to induce tyrosine
phosphorylation of the subunit of its own receptor and of IRS-1.
Since high concentrations of TNF induce dedifferentiation of
adipocytes(3, 7) , experiments testing for chronic
effects of TNF on insulin signaling were conducted with a low
concentration of the cytokine (0.1 nM TNF) that causes no
general reduction of mRNAs associated with the adipocyte
phenotype(3) . As shown in Fig. 1, mTNF inhibited the
ability of insulin to promote the tyrosine phosphorylation of its own
receptor and of IRS-1 (Fig. 1A), without affecting
expression of either the insulin receptor or IRS-1 (Fig. 1B). Quantitative analysis using a densitometer
showed that, relative to control, TNF diminished the ability of insulin
to promote phosphorylation of its receptor and IRS-1 by 46 and 61%,
respectively.
Figure 1: Effect of chronic treatment with TNF on insulin-stimulated phosphorylations. A, 3T3-L1 adipocytes were incubated in the absence or presence of 0.1 nM mTNF for 5 days and then stimulated with 100 nM insulin (INS) for 3 min. Proteins in cell lysates were immunoprecipitated with antiserum to the insulin receptor (IR) or IRS-1, fractionated by SDS-PAGE, and transferred to Immobilon-P. The Western blot was then probed with antiserum to phosphotyrosine. B, the Western blot was stripped and reprobed with antiserum to the insulin receptor or IRS-1.
Acute incubation of 3T3-L1 adipocytes with mTNF did not inhibit the ability of insulin to promote IRS-1 phosphorylation; rather, when adipocytes were briefly treated with mTNF for various times and then stimulated with insulin, tyrosine phosphorylation of IRS-1 and a group of proteins that immunoprecipitate with IRS-1 increased above the level induced by insulin alone (Fig. 2A). Quantitative analysis (Fig. 2B) showed that the maximal effect on IRS-1 phosphorylation was observed when cells were treated with mTNF for 15 min, insulin being present during the last 3 min of incubation. One explanation for this observation is that mTNF may induce the tyrosine phosphorylation of proteins in 3T3-L1 adipocytes, including IRS-1.
To determine whether tyrosine phosphorylation of cellular proteins
is induced by TNF, phosphoproteins from control and mTNF-stimulated
3T3-L1 adipocytes were immunoprecipitated with antiserum to
phosphotyrosine, fractionated by SDS-PAGE, and analyzed by Western
blotting using antiphosphotyrosine antibodies. As illustrated in Fig. 3, mTNF promoted the tyrosine phosphorylation of a group of
cellular proteins. Phosphorylation of some proteins was evident within
5 min, reached a maximal level at 15 min, and then diminished. The M values of several of the tyrosine-phosphorylated
proteins were consistent with those of signaling molecules implicated
in the actions of some growth factors. Among these were 180-kDa IRS-1
and the 85-kDa subunit of PI-3 kinase.
To test whether IRS-1 is
involved in TNF signaling, 3T3-L1 adipocytes were incubated with 5
nM mTNF for various times at 37 °C. IRS-1 in cell lysates
was then immunoprecipitated and separated from other proteins by
SDS-PAGE. The phosphorylation of IRS-1 isolated from control and
mTNF-treated cells was then evaluated by Western blotting using
antiserum to phosphotyrosine. As illustrated in Fig. 4A, phosphorylation of IRS-1 and proteins that
associate with IRS-1 was stimulated by mTNF without any effect on the
cellular content of IRS-1 (Fig. 4B). Analysis of
autoradiographs with a densitometer showed that IRS-1 phosphorylation
induced by mTNF reached a maximal level after 15 min and then
diminished (Fig. 4C). TNF also appeared to promote a
very modest increase in the tyrosine phosphorylation of the
subunit of the insulin receptor (data not shown).
We next tested whether the phosphorylation induced by mTNF led to coupling of IRS-1 with cytoplasmic signaling molecules that contain SH2 domains, which bind phosphotyrosine(8, 9) . One such is PI 3-kinase, a heterodimer composed of 85-kDa regulatory and 110-kDa catalytic subunits(10) . To determine whether IRS-1 associates with PI 3-kinase during stimulation with mTNF, IRS-1 was immunoprecipitated from control 3T3-L1 adipocytes or adipocytes stimulated with TNF or insulin for 15 min. After SDS-PAGE, Western blots were probed with antiserum to the 85-kDa regulatory subunit of PI 3-kinase. As shown in Fig. 5A, PI 3-kinase coimmunoprecipitated with IRS-1 from mTNF- and insulin-treated cells.
Since the physical association
of PI 3-kinase with activated tyrosine kinase systems may render it a
preferred substrate for tyrosine phosphorylation(11) , we
determined whether mTNF induces this process. Thus, 3T3-L1 adipocytes
were treated with mTNF for 15 min after which PI 3-kinase was
immunoprecipitated from cell lysates. After SDS-PAGE, Western blots
were probed with antiserum to phosphotyrosine, which revealed that PI
3-kinase and two proteins of M 170-190 were
highly phosphorylated in cytokine-treated but not in control cells (Fig. 5B). The high M
proteins
that co-immunoprecipitated with PI 3-kinase are probably IRS-1 and a
functionally related protein with a molecular weight and function
similar to that of IRS-1 called 4PS or
IRS-2(12, 13, 14) .
TNF binds to either of two receptors, the 55-kDa type 1 receptor (TNFR-1) and the 75-kDa type 2 receptor (TNFR-2)(15) . Murine TNF receptors display a species specificity toward TNFs; whereas mTNF binds to both receptors, hTNF is selectively recognized by TNFR-1(16) . Human TNF promoted the association of IRS-1 with PI 3-kinase (Fig. 5C) and also tyrosine phosphorylation of IRS-1 (Fig. 5D). These observations demonstrate that TNFR-1 promotes these events without precluding the possibility that TNFR-2 can also induce these responses.
The tyrosine phosphorylation of growth factor receptors leads to their interaction with cytoplasmic signaling molecules that contain a conserved motif of about 100 amino acids called the SH2 domain(8, 9) . The SH2 domain binds phosphotyrosine and promotes interactions of phosphorylated receptors with signaling molecules. Some receptors, such as those for insulin and IGF-1, do not interact strongly with most of the SH2 proteins known to be components of their signaling pathways (17) . These receptors stimulate tyrosine phosphorylation of an accessory protein, IRS-1, which serves as an intermediary and interacts with signaling proteins that contain SH2 domains and are associated with the production of cellular responses(17, 18, 20) . Recently, IRS-1 has been shown to be a component of the signaling system used by members of the cytokine/hematopoietin receptor family(21, 22, 23, 24) . These latter observations provide insight into how non-tyrosine kinase receptors interact with cytoplasmic proteins that contain SH2 domains and play a role in promoting cellular responses.
The present study is the first to demonstrate that members of the TNF/nerve growth factor receptor superfamily utilize a signaling mechanism employing IRS-1. TNF acutely induces tyrosine phosphorylation of IRS-1 and its association with other proteins. One of the proteins with which IRS-1 binds in response to TNF stimulation is PI 3-kinase, a dual specificity lipid and serine kinase consisting of an 85-kDa regulatory subunit that contains two SH2 domains and a 110-kDa catalytic subunit(10, 11) . Activation of PI 3-kinase is believed to be initiated by association of its regulatory subunit with phosphotyrosines in activated mitogen receptors or in accessory proteins such as IRS-1(11, 25) . These interactions promote relocalization of PI 3-kinase to the plasma membrane, bringing the enzyme closer to its substrates, which may account for activation of the enzyme(11) . Once activated, PI 3-kinase catalyzes production of phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate(26) . The functions of these lipid second messengers are not known; however, they may be important to intracellular protein trafficking and activation of specific isoforms of protein kinase C(27, 28) . Activation of PI 3-kinase is associated with insulin-induced glucose transport and with the growth stimulatory activity of mitogens(29, 30) . PI 3-kinase also promotes the serine phosphorylation of IRS-1 (31) ; however, it is not known whether this activity relates to the ability of TNF to induce insulin resistance during long term incubations with 3T3-L1 adipocytes. In any event, our observations show that TNF induces an acute insulin- or growth factor-like response in cells. This observation may, in part, explain the proliferative response induced by TNF in fibroblasts and some transformed cells (32, 33, 34) .
The responses induced by TNF through IRS-1 need not be identical to those of other factors that utilize this docking protein. IRS-1 contains at least 20 potential tyrosine phosphorylation sites, at least eight of which are phosphorylated in response to insulin(35) . Differences in the specificity of the as yet unidentified tyrosine kinase activated by TNF, compared with those utilized by insulin and other growth factors and cytokines, may result in phosphorylation of distinct sites in IRS-1, binding to a unique array of signaling proteins and induction of distinct responses.
TNF initiates its actions by binding to either of two receptors(15) . The extracellular domains of the receptors share homologies with one another and with other members of the TNF/nerve growth factor receptor superfamily(36) . The intracellular domains do not display sequence similarities, which accounts for the distinct responses induced by the receptors; TNFR-1 induces cytotoxicity, fibroblast proliferation, antiviral responses, and the host defense against pathogens, whereas TNFR-2 plays a role in cytotoxicity that is still being defined, inhibits early hematopoiesis, and is involved in the proliferation of monocytes and T cells(2, 37, 38, 39, 40, 41, 42) . Neither receptor contains protein tyrosine kinase activity or any motif, which suggests a mechanism through which signals can be transmitted into the cell(15, 36) . The present study demonstrates that TNFR-1 activates a tyrosine kinase that promotes phosphorylation of IRS-1 and its engagement with cytoplasmic signaling molecules; we cannot exclude the possibility that TNFR-2 also uses this mechanism.
Elaboration of TNF is associated with the insulin resistance of cancer, infection, and non-insulin-dependent diabetes mellitus (19) . The ability of TNF to impair signaling through the insulin receptor and IRS-1 during chronic treatments suggests sites at which metabolic abnormalities may initiate. Acutely, TNF activates tyrosine kinase activity and induces phosphorylation of IRS-1 and its interaction with PI 3-kinase. These observations suggest a mechanism through which TNF receptors can couple with cytoplasmic signaling molecules, thereby forming complexes that may promote responses to TNF.