(Received for publication, November 6, 1995; and in revised form, February 23, 1996)
From the
The sphingomyelin pathway is a newly described signal
transduction pathway mediating the action of several cytokines
including tumor necrosis factor- (TNF). TNF was recently shown to
interfere with insulin-induced tyrosine phosphorylation of the insulin
receptor substrate-1 (IRS-1). In this work we examined the possible
effect of direct activation of the sphingomyelin pathway on
insulin-induced tyrosine phosphorylation of IRS-1. Incubation of the
insulin-sensitive rat hepatoma Fao cells with bacterial
sphingomyelinase (SMase) that causes membrane hydrolysis of
sphingomyelin led to a time- and dose-dependent decrease in
insulin-induced tyrosine phosphorylation of IRS-1. The effect was
apparent after 10 min of incubation and with a dose of 10 milliunits/ml
SMase. It was not associated with a decrease in insulin receptor
autophosphorylation. In addition, SMase treatment interrupted the
association of the 85-kDa catalytic subunit of phosphatidylinositol
3-kinase with IRS-1. A similar impact on IRS-1 tyrosine phosphorylation
was observed after addition of cell-permeable ceramide analogs (C2 and
C6). Comparable changes in IRS-1 tyrosine phosphorylation and
electrophoretic mobility were found after exposure of cells to either
TNF, SMase, or ceramide. Our findings suggest that TNF may utilize the
sphingomyelin pathway in its effect on the insulin-stimulated tyrosine
phosphorylation of IRS-1.
The sphingomyelin pathway is a new signal transduction pathway
mediating the action of the cytokines, tumor necrosis factor-
(TNF), (
)interleukin-1, and interferon-
(reviewed in (1, 2, 3) ). This pathway is initiated by
activation of a neutral sphingomyelinase (SMase), which hydrolyzes
membrane sphingomyelin to ceramide and phosphocholine. In addition,
ceramide can be generated through the action of an acidic SMase
activated by 1,2-diacylglycerol as a result of stimulation of a
ligand-responsive phosphatidylcholine-specific phospholipase C
(PC-PLC). Ceramide functions as a second messenger and can stimulate a
specific proline-directed Ser/Thr protein kinase as well as a specific
protein phosphatase(1, 2, 3) . The role of
the sphingomyelin pathway in TNF action has been documented in several
cell lines including
hepatocytes(4, 5, 6, 7, 8) .
TNF led to rapid breakdown of sphingomyelin by SMases, while addition
of neutral SMase or cell-permeable analogs of ceramide to cells
mimicked TNF actions.
We and others have recently demonstrated that TNF leads to suppression of early events in insulin signal transduction(9, 10, 11) . Specifically, it inhibited the insulin-induced tyrosine phosphorylation of the major insulin receptor substrate, IRS-1, and impaired the association of IRS-1 with phosphatidylinositol (PI) 3-kinase(11) . In the current work we examined the involvement of the sphingomyelin pathway in this cross-talk between TNF and insulin transmembrane signaling mechanisms.
Figure 1: Effect of SMase on insulin-stimulated tyrosine phosphorylation in intact Fao cells. Upper panel, Fao cells were incubated with 100 milliunits (mU)/ml SMase for 20 min in serum-free medium and stimulated with 100 nM insulin for 1 min at 37 °C. Total cell extracts were subjected to SDS-PAGE under reducing conditions or to immunoprecipitation with the insulin receptor antibody (IR-Ab) prior to SDS-PAGE. Proteins were transferred to nitrocellulose papers, and phosphotyrosine-containing proteins were probed with phosphotyrosine antibodies. These proteins were visualized using a chemiluminescent peroxidase substrate and autoradiography. Lower panel, Fao cells were incubated with increasing doses of SMase for 20 min prior to insulin stimulation and analyzed as in the upper left panel.
Figure 2: SMase effects on insulin-stimulated tyrosine phosphorylation of IRS-1 and its association with PI 3-kinase. Confluent monolayers of Fao cells were treated as described in Fig. 1with 100 milliunits/ml SMase for the times indicated prior to stimulation with insulin and analyzed in parallel in three different ways. Panel A, tyrosine-containing proteins in total cell extracts were detected as described in Fig. 1. Panel B, cell extracts were subjected to immunoprecipitation with IRS-1 antibodies prior to SDS-PAGE, and tyrosine-containing proteins were detected in immunoprecipitates as described in Fig. 1. Panel C, cell extracts were subjected to immunoprecipitation with antibodies to IRS-1 and analyzed by SDS-PAGE. The p85 subunit of PI 3-kinase that coprecipitated with IRS-1 was detected by immunoblotting with a specific antibody.
After tyrosine phosphorylation, IRS-1 binds to several SH2 proteins including the 85-kDa regulatory subunit of PI 3-kinase(14, 15) . To evaluate the SMase effect on this interaction, IRS-1 immunoprecipitates were immunoblotted with antibodies directed against the 85-kDa subunit of PI 3-kinase. Preincubation with SMase, prior to insulin stimulation, led to a decrease in the amount of the 85-kDa subunit of PI 3-kinase that coprecipitated with IRS-1 (Fig. 2C, lanes b-e). This decrease did not result from a reduction in IRS-1 content, probed by immunoblotting with IRS-1 antibodies (not shown), but rather from a decrease in its tyrosine phosphorylation (Fig. 2B). While incubation with SMase for 30 min, without insulin stimulation, did not induce tyrosine phosphorylation of IRS-1 or association with PI 3-kinase, it caused a dose-dependent tyrosine phosphorylation of an unidentified 200-210-kDa band (Fig. 2, lane f, ppTyr).
Figure 3: Effect of C2-ceramide analog on insulin-stimulated tyrosine phosphorylation in intact Fao cells. Fao cells were incubated with 5 µM C2-ceramide (C2-Cer) analog for the indicated times in serum-free medium and stimulated without or with 100 nM insulin for 1 min at 37 °C. Total cell extracts were analyzed as in Fig. 1.
Figure 4: Parallel effects of TNF, SMase, and C6-ceramide analog on insulin-stimulated tyrosine phosphorylation of IRS-1. Fao cells were incubated with 5 nM TNF for 1 h or 100 milliunits/ml SMase (SMa) for 20 min or 3 µM C6-ceramide analog for 30 min in serum-free medium and stimulated with 100 nM insulin for 1 min at 37 °C. Total cell extracts were analyzed in parallel as in Fig. 1.
As TNF is known to activate SMase and to interfere with insulin-induced tyrosine phosphorylation, cells were exposed in parallel to TNF, ceramide, and SMase. A high degree of similarity between the effect of all three agents on insulin-induced IRS-1 tyrosine phosphorylation and electrophoretic mobility was observed (Fig. 4), though consistently the effects of SMase were the most pronounced.
In the current work we supply evidence that activation of the
sphingomyelin pathway interferes with the early events of insulin
transmembrane signaling. Addition of SMase that hydrolyzes
sphingomyelin in the plasma membrane to ceramide and phosphocholine, or
of cell-permeable ceramide analogs to Fao cells, both led to a decrease
in insulin-induced tyrosine phosphorylation of IRS-1. This was not
associated with inhibition of the insulin receptor intrinsic kinase
activity as insulin receptor autophosphorylation of its -subunit
was not reduced. IRS-1 is the major substrate for the insulin receptor,
and upon tyrosine phosphorylation it transduces insulin effects through
association with multiple SH2-containing
molecules(14, 15) . This downstream step was also
disrupted by activation of the sphingomyelin pathway as demonstrated
with PI 3-kinase.
Similar effects were previously reported by us in
the same system exposed to TNF, which utilizes the sphingomyelin
pathway for signal transduction(9, 11) . The
similarity between the TNF effect on insulin induction of IRS-1
tyrosine phosphorylation and that achieved by directly activating the
sphingomyelin pathway suggests that TNF may employ this pathway in its
interference with IRS-1 tyrosine phosphorylation. This observation may
fit into a working model explaining TNF action in this system. TNF
increases IRS-1 phosphorylation on serine residues leading to a
decrease in its electrophoretic mobility(11) . TNF-induced
serine phosphorylation of IRS-1 may interfere with insulin-stimulated
tyrosine phosphorylation of the substrate and impair insulin action.
Ceramide acts as the second messenger in the sphingomyelin pathway and
activates proline-directed Ser/Thr kinases including a specific
membrane-associated protein kinase (18, 19) ,
mitogen-activated protein kinases(6, 20) , and
stress-activated protein kinases(8, 21, 22) .
IRS-1 has multiple X-Ser/Thr-Pro-X motifs and is a
substrate for the mitogen-activated protein
kinase(14, 15) . Thus, activation by the sphingomyelin
pathway of these proline-directed Ser/Thr kinases may mediate the TNF
effect on IRS-1, leading both to a delay in its electrophoretic
mobility as well as impaired tyrosine phosphorylation. A similar
scenario where TNF activates neutral SMase, which in turn activates a
cascade of Ser/Thr phosphorylations, has been described in other cell
lines(2, 3, 6, 22, 23) . It
is noteworthy that other cytokines, namely interleukin-1 and
interferon-, which activate the sphingomyelin pathway(1) ,
have also been shown to decrease insulin-induced tyrosine
phosphorylation of IRS-1(10) .
Combined with previous reports the current data help portray a more detailed picture of the possible cross-talk between cytokines and insulin transmembrane signaling pathways. The clinical relevance of this cross-talk is underscored by the ample information supporting the possible role of TNF as a the link and cause of insulin resistance in sepsis, cancer, and obesity(24) .