(Received for publication, June 23, 1995; and in revised form, July 31, 1995)
From the
Tumor necrosis factor- (TNF) has been suggested to be the
mediator of insulin resistance in infection, tumor cachexia, and
obesity. We have previously shown that TNF diminishes insulin-induced
tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1). The
current work examines potential mechanisms that mediate this event. TNF
effect on IRS-1 in Fao hepatoma cells was not associated with a
significant reduction in insulin receptor tyrosine kinase activity as
measured in vitro but impaired the association of IRS-1 with
phosphatidylinositol 3-kinase, localizing TNF impact to IRS-1. TNF did
not increase protein-tyrosine phosphatase activity and protein-tyrosine
phosphatase inhibition by vanadate did not change TNF effect on IRS-1
tyrosine phosphorylation, suggesting that protein-tyrosine phosphatases
are not involved in this TNF effect. In contrast, TNF increased IRS-1
phosphorylation on serine residues, leading to a decrease in its
electrophoretic mobility. TNF effect on IRS-1 tyrosine phosphorylation
was not abolished by inhibiting protein kinase C using staurosporine,
while inactivation of Ser/Thr phosphatases by calyculin A and okadaic
acid mimicked it. Our data suggest that TNF induces serine
phosphorylation of IRS-1 through inhibition of serine phosphatases or
activation of serine kinases other than protein kinase C. This
increased serine phosphorylation interferes with insulin-induced
tyrosine phosphorylation of IRS-1 and impairs insulin action.
Tumor necrosis factor- (TNF), (
)a cytokine
produced primarily by activated macrophages, mediates various
detrimental manifestations seen in sepsis and
cancer(1, 2) . In addition to other TNF effects, it is
involved in inducing insulin resistance in these disease
states(3, 4, 5, 6) . Recently,
accumulating data suggest that TNF may be the cause and link of
obesity-induced insulin resistance(7, 8, 9) .
We have shown that in Fao hepatoma cells TNF leads to a decrease in
insulin-stimulated tyrosine phosphorylation of the insulin receptor
(IR) and its major substrate IRS-1(10) . In a rodent model of
obesity, blocking TNF effects led to increased insulin sensitivity (7, 11) in parallel with correction of the impaired
insulin-induced tyrosine phosphorylation of IRS-1(11) . In
addition, insulin sensitizing agents of the thiazolidinediones family
were able to reverse TNF-induced decrease in tyrosine phosphorylation
of IRS-1 (12) . These observations underline the role of
impaired IRS-1 tyrosine phosphorylation in TNF impact on insulin
action.
TNF acts through specific membrane receptors whose mechanism of signal transduction has not yet been fully elucidated(13, 14, 15) . Several pathways activated by TNF may influence insulin-induced tyrosine phosphorylation. For example, TNF-induced activation of multiple Ser/Thr protein kinases(16, 17, 18, 19, 20, 21, 22, 23) or inactivation of Ser/Thr phosphatases(24, 25) , may inhibit kinase activity of IR and/or the tyrosine phosphorylation of IRS-1 through multisite phosphorylation on Ser/Thr residues(26, 27, 28, 29) . Alternatively, TNF inhibitory effect may be mediated through the activation of specific tyrosine phosphatases(30) . This study tries to elucidate whether these mechanisms are involved in the TNF effect on insulin signaling pathways.
Figure 1:
Effect of TNF on insulin-stimulated
protein tyrosine phosphorylation in intact Fao cells. Fao cells were
incubated with 5 nM TNF for 1 h and stimulated with 100 nM insulin for 1 min at 37 °C. Cell extracts were subjected to
immunoprecipitation with antibodies to phosphotyrosine (pTyr
Ab), to IRS-1 (IRS-1 Ab), or to the IR -subunit (IR-Ab). Total cell extracts and immune pellets were analyzed
by SDS-PAGE. Proteins were transferred to nitrocellulose papers, and
phosphotyrosine-containing proteins were probed with Tyr(P) antibodies.
These proteins were visualized using a chemiluminescent peroxidase
substrate and autoradiography.
Figure 2: Time course of TNF effect on insulin-stimulated tyrosine phosphorylation of IRS-1. Fao cells were incubated with 5 nM TNF for the indicated times followed by a 1-min stimulation with 100 nM insulin at 37 °C. Cell extracts were analyzed and probed as described in the legend to Fig. 1.
Figure 3:
TNF effect on insulin-stimulated receptor
tyrosine kinase activity in Fao cells. Cells were incubated with 5
nM TNF for the indicated times, and insulin was added (100
nM final concentration) to the indicated plates for the final
10 min. Cells were lysed and immunoprecipitated with antibodies to the
IR -subunit, and IR tyrosine kinase activity was determined after
addition of poly(Glu,Tyr) (4:1) and
[
-
P]ATP. Data are presented as the means
± S.E. of the percentage of receptor tyrosine kinase activity in
cells treated with insulin alone.
Figure 4: TNF effect on insulin-induced interaction of IRS-1 and PI 3-kinase. Cells were incubated with 5 nM TNF for 1 h and stimulated with 100 nM insulin for 1 min at 37 °C. Cell extracts were subjected to immunoprecipitation with antibodies to IRS-1 and analyzed by sequential immunoblotting with antibodies to the p85 subunit of PI 3-kinase (rightpanel), Tyr(P) Ab (middlepanel), and IRS-1 Ab (leftpanel), as described in Fig. 1.
Figure 5: Effect of TNF on insulin-stimulated protein tyrosine phosphorylation in intact Fao cells in the presence of vanadate. After incubation for 16 h without (sixleftlanes) or with 50 µM vanadate (sixrightlanes) Fao cells were incubated with 5 nM TNF for 1 h and stimulated with 100 nM insulin for 1 min at 37 °C. Total cell extracts were analyzed by immunoblotting with Tyr(P) Ab as described in Fig. 1.
Figure 6:
Effect of TNF on protein-tyrosine
phosphatases. Particulate and cytosolic fractions from Fao cells
treated with 5 nM TNF for the indicated times were added to
immunopurified P-tyrosine-phosphorylated IR. After a
20-min incubation at 30 °C the receptor was separated on SDS-PAGE
and visualized by autoradiography. The degree of dephosphorylation was
evaluated by densitometry and expressed as density of the IR
-subunit band after incubation with buffer alone or with the
appropriate cellular fraction.
Figure 7:
Effect of TNF and calyculin A on IRS-1
phosphorylation. Upper left panel, P-labeled Fao
cells were incubated at 37 °C for 20 min without (Con) or
with 5 nM TNF or 100 nM calyculin A (CAL)
and subjected to immunoprecipitation with IRS-1 Ab prior to SDS-PAGE
and autoradiography. Upper right panel,
P-labeled
Fao cells were incubated at 37 °C for 20 min without (Con)
or with 5 nM TNF or for 1 min with 100 nM insulin (Ins). Cell extracts were subjected to immunoprecipitation
with pTyr-Ab prior to SDS-PAGE and autoradiography. Lower
panel, phosphoamino acid analysis of immunoprecipitated IRS-1.
P-Labeled Fao cells were incubated for 20 min without (Con) or with 5 nM TNF and subjected to
immunoprecipitation with IRS-1 Ab prior to SDS-PAGE and blotting. IRS-1
bands, excised from the membrane, were acid-hydrolyzed, and the
phosphoamino acids were analyzed by one-dimensional high voltage
electrophoresis on thin layer plate. The positions of standard
phosphoamino acids identified by ninhydrin staining are
indicated.
Figure 8: Comparison of TNF and Ser/Thr phosphatase inhibitors effect on insulin-induced tyrosine phosphorylation. Fao cells were stimulated with 100 nM insulin for 1 min at 37 °C before (lanea) and after (laneb) incubation for 30 min with 5 nM TNF or 100 nM okadaic acid (lanec). Alternatively, cells were pretreated with increasing doses of calyculin A for 15 (lanesd and e) or 30 min (lanes f-h). Total cell extracts were evaluated by immunoblotting with Tyr(P) antibodies as described in Fig. 1.
Figure 9: Effect of staurosporine on TNF inhibition of insulin-stimulated tyrosine phosphorylation of IRS-1. After incubation for 30 min without or with 50 nM or 10 nM staurosporine (STS), Fao cells were incubated without or with 5 nM TNF for 1 h and stimulated with 100 nM insulin for 1 min at 37 °C. Total cell extracts were analyzed by immunoblotting with Tyr(P) Ab as described in Fig. 1.
Kinase activity of the IR is regulated by multisite
phosphorylation(34) . IR phosphorylation on Ser/Thr sites has
an inhibitory regulatory effect on the insulin-induced
autophosphorylation of the
-subunit(26, 27, 34) . Likewise, IRS-1
contains multiple potential Ser/Thr phosphorylation
sites(34, 35) , and phosphorylation on these residues
reduces its ability to undergo tyrosine phosphorylation by the
IR(28, 29) . In our study we supplied ample evidence
to suggest that TNF leads to a reduction in insulin-induced tyrosine
phosphorylation of IRS-1 through increased serine phosphorylation of
the substrate.
Upstream to IRS-1, no significant reduction in IR
kinase activity was observed in purified receptors from cells treated
with TNF. This is similar to the findings in protein kinase
C-transfected Chinese hamster ovary cells, where
12-O-tetradecanoylphorbol-13-acetate inhibition of insulin
action was shown to occur through an increase in IRS-1 Ser/Thr
phosphorylation, leading to a decrease in its tyrosine phosphorylation.
Like in our system it was not associated with a decline in the IR
kinase activity as measured in vitro(28) . Downstream
to IRS-1, TNF exposure led to decreased PI 3-kinase association with
the substrate, suggesting that the IRS-1 impairment is central to TNF
induction of insulin resistance.
Of special interest is the close mimicry between TNF effects and those achieved using the Ser/Thr phosphatase inhibitors, okadaic acid and calyculin A. All three reagents inhibit insulin metabolic effects (37, 38, 39) and induce a similar cellular protein phosphorylation pattern (24, 25) . Tanti et al.(29) demonstrated that in 3T3-L1 adipocytes treatment with okadaic acid repressed insulin effect on glucose uptake in parallel to reducing insulin-induced tyrosine phosphorylation of IRS-1. Moreover, this was linked to a decrease in IRS-1 electrophoretic mobility due to phosphorylation on Ser/Thr residues(29) . Our similar data in Fao cells confirm the uniquely parallel pattern induced by TNF and Ser/Thr phosphatase inhibitors on IRS-1 phosphorylation and suggest that TNF-induced increase in serine phosphorylation may occur through inactivation of these phosphatases.
An alternative pathway
leading to increased Ser/Thr content of IRS-1 may be TNF activation of
serine kinases. Indeed, TNF has been shown to activate multiple serine
kinases including protein kinase C, protein kinase A, -casein
kinase, and mitogen-activated protein (MAP)
kinases(16, 17, 18, 19, 20, 21, 22, 23) .
Protein kinase C activation and/or overexpression has been shown to
hamper insulin-induced tyrosine phosphorylation as well as insulin
action in intact cells(26, 27, 28) . In
addition, TNF activates protein kinase C in several cell
lines(22, 23) , making it a candidate for conveying
the TNF effect. The possibility to down-regulate protein kinase C was
not utilized in our system, since phorbol esters can potentially lead
to shedding of TNF receptors(40) . Use of staurosporine, a
potent nonspecific protein kinase C inhibitor, augmented IRS-1 tyrosine
phosphorylation but did not alter TNF effect. It is noteworthy that in
FS-4 fibroblasts TNF induction of heat shock protein 28 phosphorylation
was also not inhibited by staurosporine and other protein kinase C
inhibitors, and the increased serine phosphorylation of this protein
was attributed to inhibition of serine phosphatases(41) . Other
well characterized kinases whose activity was shown to be increased by
TNF in several cells including hepatocytes are the MAP kinase
isoforms(17, 18, 19, 20) . Recently,
IRS-1 phosphorylation by MAP kinase was suggested, making it a
plausible candidate to be involved in TNF-induced phosphorylation of
IRS-1(34, 35) . Moreover, in 3T3-L1 adipocytes
Kletzien et al.(12) underlined the complex potential
interactions between the multiple MAP kinase species, TNF and insulin.
While TNF obliterated the insulin-induced phosphorylation and
activation of p44 MAP kinase, it stimulated a p38 MAP kinase.
Serine
phosphorylation of IRS-1 may not be the only mechanism mediating TNF
inhibition of insulin-induced tyrosine phosphorylation. In this study
obliteration of protein-tyrosine phosphatase activity by vanadate did
not reverse TNF inhibition, implying that protein-tyrosine phosphatases
are not involved in this effect. Moreover, TNF did not alter
protein-tyrosine phosphatase activity in Fao cells when measured in
vitro using the phosphorylated IR -subunit as a substrate.
In summary, the phenomenon investigated in this work constitutes an interesting example for the complex cross-talk between cytokines and growth factors that operate through receptors with intrinsic tyrosine kinase activity. This may represent one of the delicate physiological regulatory mechanisms of insulin action that when exaggerated may lead to insulin resistance. The immediate clinical significance of this interaction was recently underlined by Hotamisligil and Spiegelman (10) , who suggested that TNF is the cause and link in obesity-induced insulin resistance. According to their hypothesis, TNF overproduced by adipose tissue inhibits insulin action and is connected to the development of non-insulin-dependent diabetes mellitus. Better understanding of the cascade of events involved in obesity-linked non-insulin-dependent diabetes mellitus may form the basis for reversal of this prevalent deleterious disease.