Salicylic Acid Reverses Phorbol 12-Myristate-13-Acetate (PMA)- and Tumor Necrosis Factor alpha  (TNFalpha )-induced Insulin Receptor Substrate 1 (IRS1) Serine 307 Phosphorylation and Insulin Resistance in Human Embryonic Kidney 293 (HEK293) Cells*

Guoqiang JiangDagger, Qing Dallas-Yang, Franklin Liu, David E. Moller, and Bei B. ZhangDagger

From the Department of Molecular Endocrinology-Diabetes, Merck Research Laboratories, Rahway, New Jersey 07065

Received for publication, June 5, 2002, and in revised form, October 21, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Salicylates, including aspirin, have been shown to improve insulin sensitivity both in human and animal models. Although it has been suggested that salicylates sensitize insulin action by inhibiting Ikappa B kinase beta  (IKKbeta ), the detailed mechanisms remain unclear. Protein kinase C isoforms and tumor necrosis factor alpha  (TNFalpha ) signaling pathways are well described mediators of insulin resistance; they are implicated in the activation of IKKbeta and the subsequent inhibition of proximal insulin signaling via insulin receptor substrate 1 (IRS1) and Akt. This study investigated the effect of salicylic acid on phorbol 12-myristate 13-acetate (PMA)- and TNFalpha -induced insulin resistance in a human embryonic kidney 293 (HEK293) cell line stably expressing recombinant human IRS1. The results showed that both PMA and TNFalpha inhibited insulin-induced Akt phosphorylation and promoted IRS1 phosphorylation on Ser-307. Salicylic acid pretreatment completely reversed the effects of PMA and TNFalpha on both Akt and IRS1. Whereas PMA activated protein kinase C isoforms and IKKbeta , TNFalpha activated neither. On the other hand, both PMA and TNFalpha activated the c-Jun N-terminal kinase (JNK), which has been reported to directly phosphorylate IRS1 Ser-307. SP600125, a JNK inhibitor, prevented PMA and TNFalpha -induced IRS1 Ser-307 phosphorylation. Finally, salicylic acid inhibited JNK activation induced by both PMA and TNFalpha . Taken together, these observations suggest that salicylic acid can reverse the inhibitory effects of TNFalpha on insulin signaling via an IKKbeta -independent mechanism(s), potentially involving the inhibition of JNK activation. The role of JNK in salicylic acid-mediated insulin sensitization, however, requires further validation because the JNK inhibitor SP600125 appears to have other nonspecific activity in addition to inhibiting JNK activity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ikappa B kinase beta  (IKKbeta ),1 a serine/threonine kinase, is a component of the larger IKK signalosome, which also consists of IKKalpha and IKKgamma . Whereas IKKalpha is also a serine/threonine kinase and is highly homologous to IKKbeta , IKKgamma is a scaffold protein with no enzymatic activity. Upon exposure to stimuli including stress or cytokines, both IKKalpha and IKKbeta undergo phosphorylation and activation. The activated IKK kinases phosphorylate serine residues in Ikappa B, which subsequently becomes degraded via the proteasome pathway. Prior to its degradation, Ikappa B binds to NFkappa B and prevents its translocation into the nucleus. Thus, IKKs potentiate NFkappa B-mediated gene transcription (as occurs in the inflammatory states). Interestingly, IKKbeta but not IKKalpha deficiency appears to result in immunodeficiency in mice, suggesting that IKKbeta but not IKKalpha plays a dominant role in immunoresponses (1, 2).

Salicylates, including aspirin, have been shown to be antidiabetic in both animal models and man in both long term (i.e. 3-4 week) and short term (i.e. overnight) treatments (3-5). Several lines of evidence suggest that the antidiabetic effects of salicylates are mediated via direct inhibition of IKKbeta . First, salicylates were reported to specifically inhibit the kinase activity of recombinant IKKbeta but not IKKalpha proteins in vitro and to down-regulate NFkappa B-mediated transcription regulation in cultured cells (6-8). Second, IKKbeta heterozygous mice have improved insulin sensitivity and reduced plasma glucose levels (9). Third, IKKbeta heterozygosity in ob/ob background also improves insulin sensitivity in the mice (9). Finally, IKKbeta heterozygosity and salicylate treatment prevents lipid-induced insulin resistance in animal models (5).

Serine phosphorylation of insulin receptor substrate proteins (IRSs) such as IRS1 has been strongly implicated as a mechanism of insulin resistance (10-13). Upon serine phosphorylation, IRS1 proteins have a reduced ability to interact with the insulin receptor, to be tyrosine phosphorylated by the insulin receptor, and to bind phosphatidylinositol 3-kinase (PI3K) (10, 14-17). Such negative phosphorylation in IRS1 has been mapped to several serine residues (13, 14, 18-21). It was reported recently that IRS1 Ser-307 (refers to the rat IRS1 protein or Ser-312 in human IRS1) is a key regulatory site (17, 22). Moreover, phosphorylation at this site can be mediated by the c-Jun N-terminal kinase (JNK) and may be an important contributor to the inhibitory effects of tumor necrosis factor alpha  (TNFalpha ) on insulin signaling. It was further proposed that salicylate treatment or IKKbeta deficiency can improve insulin sensitivity by reducing the serine phosphorylation of IRS proteins (i.e. IRS1) (5, 9).

Salicylates inhibit the kinase activity of recombinant IKKbeta in vitro and the biological function of endogenous IKKbeta in cultured cells with an EC50 of 50-100 µM (6). On the other hand, in in vivo experiments in which antidiabetic effects were demonstrated, salicylates reached millimolar concentrations in the plasma (5). Such a discrepancy between in vitro and in vivo potency may be ascribed to, for example, potentially lower salicylate levels in the intracellular environment than in the plasma. On the other hand, it is also possible that at such a high concentration in vivo, salicylates may manifest antidiabetic effects by regulating other targets in addition to or other than IKKbeta . In fact, at high concentrations salicylates have been shown to have additional effects such as inhibition of mitochondria function (23, 24) and inhibition of phosphorylation and nuclear translocation of signal transducers and activators of transcription (STATs) (25). Furthermore, it was reported that treatment of intact cells with salicylate inhibited TNFalpha -induced but not interleukin-1-induced IKK activity and that this inhibition was prevented by a p38 MAPK inhibitor, suggesting that salicylate does not directly bind to and inhibit IKKbeta in intact cells (26). These observations suggest that the antidiabetic effects of salicylates may not be mediated exclusively by the inhibition of IKKbeta .

The current study was initiated to investigate the effects of salicylic acid on phorbol 12-myristate-13-acetate (PMA)- and TNFalpha -induced insulin resistance in a HEK293 cell line stably expressing recombinant IRS1. Results from this study suggest the following. 1) PMA and TNFalpha inhibit insulin-stimulated Akt phosphorylation and promote inhibitory IRS1 serine 307 phosphorylation. 2) Salicylic acid reverses the effects of PMA and TNFalpha on Akt and IRS1 phosphorylation. 3) Salicylic acid attenuates the inhibitory effects of TNFalpha on insulin signaling through an IKKbeta -independent mechanism. 4) JNK kinase is activated by and may be important for both PMA and TNFalpha -induced insulin resistance in these cells. 5) Salicylic acid partially inhibits PMA and TNFalpha -induced JNK activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Insulin, TNFalpha , PMA, and salicylic acid were purchased from Sigma. Salicylic acid was prepared as 1 M stock solution in 1 M Tris-HCl, pH7.5, aliquoted, and stored at -20 °C. Rabbit polyclonal antibodies against total insulin receptor beta  subunit, total IRS1, IRS1 phosphorylated at Ser-307 (IRS1-pS307), and total PKCdelta were purchased from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal antibody (4G10) against tyrosine-phosphorylated proteins was also purchased from Upstate Biotechnology. Rabbit polyclonal antibodies against total Akt, Akt phosphorylated at threonine 308 (Akt-pT308), total IKKalpha , total IKKbeta , both IKKalpha phosphorylated at serine 180 and IKKbeta phosphorylated at serine 181, phosphorylated PKC isoforms (PKC-pan), PKCalpha /beta II phosphorylated at threonine 638 and 641, PKCdelta phosphorylated at threonine 505, PKCdelta phosphorylated at serine 643, PKCµ phosphorylated at serine 744/748, PKCµ phosphorylated at serine 916, PKCtheta phosphorylated at threonine 538, PKCxi /lambda phosphorylated at threonine 410 and 403, total PKCµ, total c-Jun, and c-Jun phosphorylated at serine 73 were purchased from Cell Signaling (Beverly, MA). Rabbit polyclonal antibodies against total p38 MAP kinase, p38 MAPK phosphorylated at threonine 180 and tyrosine 182, total p42/44 MAP kinase, p42/44 MAP kinase phosphorylated at threonine 202 and tyrosine 204, total JNK, and JNK phosphorylated at Thr-183, and Tyr-185 were purchased from New England Biolabs (Beverly, MA). Mouse monoclonal antibody against total PKCtheta was purchased from Santa Cruz Biotechnology. SP600125, a JNK inhibitor, was purchased from Biomol (Plymouth Meeting, PA).

Cell Culture, Compound Treatment, and Protein Extraction-- HEK293 cells stably expressing recombinant human IRS1 (HEK293.IRS1 cells) were a gift from Dr. Richard Roth. The cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37 °C in 10% CO2. For the experiments, the cells were first incubated in serum-free Dulbecco's modified Eagle's medium overnight. The cells were then treated with vehicle, salicylic acid, or SP600125 for 2 h at 37 °C. PMA or TNFalpha were then added to the media. After incubation for another 30 min, the cells were exposed to insulin for 15 min, rinsed with ice-cold phosphate-buffered saline, and then lysed in ice-cold lysis buffer. The lysis buffer contains 20 mM HEPES, pH7.4, 1% Triton X-100, 20 mM beta -glycerophosphate, 150 mM sodium chloride, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 1× concentration of a protease inhibitor mixture (Roche Diagnostics). Cell lysates were cleared by centrifugation. Protein concentrations were determined using Bradford reagent (Bio-Rad).

Western Blot Analysis-- Cell lysates were resuspended in SDS-loading buffer (Invitrogen) and separated in precast 4-20% gradient NuPAGE SDS-PAGE gels (Invitrogen). The proteins were then transferred to polyvinylidene difluoride membrane and probed with primary antibodies. Detection was performed with ECF Western blotting Kit (Amersham Biosciences) by scanning with a Storm® gel and blot imaging system (Amersham Biosciences) per the manufacturer's recommendation.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Salicylic Acid Reverses PMA and TNFalpha -mediated Inhibition on Insulin-stimulated Akt Phosphorylation in the HEK293.IRS1 Cells-- Both PMA (19, 27, 28) and TNFalpha (14, 18, 29) have been shown to inhibit insulin signaling in cultured cells. Akt phosphorylation (and activation) is a key event in insulin signaling and has been commonly used as a surrogate marker for assessing the level of activation of the proximal insulin signaling pathway (30). We therefore determined whether PMA and/or TNFalpha inhibit insulin-stimulated Akt phosphorylation in the HEK293.IRS1 cells. If so, we sought to see if such inhibition could be reversed by salicylic acid. As shown in Fig. 1A, insulin stimulation resulted in an apparent increase in Akt phosphorylation at threonine 308 (Akt-pT308) in the HEK293.IRS1 cells (Fig. 1A, lane 2 versus 1). PMA treatment reduced insulin-stimulated Akt phosphorylation (Fig. 1A, lane 3 versus 2). Although salicylic acid pre-treatment by itself did not affect insulin-stimulated Akt phosphorylation (Fig. 1A, lane 5 versus 2), it reversed the negative effects by PMA (Fig. 1A, lane 4 versus 3). Similar observations were made on cells treated with TNFalpha (Fig. 1B). Taken together, these results suggest that PMA and TNFalpha negatively affect insulin signaling in HEK293.IRS1 cells and that such negative regulation can be reversed by salicylic acid.


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Fig. 1.   Salicylic acid reverses PMA and TNFalpha -induced inhibition of insulin-stimulated Akt phosphorylation in the HEK293.IRS1 cells. Cells were serum-starved overnight and treated with vehicle (V), 5 mM salicylic acid (SA), 2 µM PMA, 6 nM TNFalpha , and 100 nM insulin (Ins) as described under "Experimental Procedures." Equivalent amounts of crude proteins were loaded in each of the lanes. Western blots were performed using antibodies against total Akt (Akt-t) or Akt phosphorylated at threonine 308 (Akt-pT308). Similar results were obtained in at least three experiments for both A and B. Similar abbreviations are used in all figures.

Salicylic Acid Reverses PMA and TNFalpha -induced IRS1 Ser-307 Phosphorylation in HEK293.IRS1 Cells-- Both PMA and TNFalpha have been shown to inhibit insulin signaling by promoting IRS1 phosphorylation at multiple serine residues (13, 14, 18-21). Furthermore, it was reported recently that IRS1 Ser-307 phosphorylation mediates the inhibitory effects of TNFalpha on insulin signaling (17, 22). We therefore investigated whether PMA and/or TNFalpha induce IRS Ser-307 phosphorylation in the HEK293.IRS1 cells. We further determined whether salicylic acid could attenuate the effect of PMA or TNFalpha on IRS Ser-307 phosphorylation. As shown in Fig. 2A, PMA treatment resulted in ~4-fold increase in IRS1 Ser-307 phosphorylation. Such phosphorylation was reversed by pre-treatment with salicylic acid. Similar observations were made using cells treated with TNFalpha (Fig. 2B). Taken together, these results suggest that PMA and TNFalpha induce IRS1 Ser-307 phosphorylation and that such phosphorylation can be effectively blocked by salicylic acid treatment in the HEK293.IRS1 cells.


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Fig. 2.   Salicylic acid reverses PMA and TNFalpha -induced IRS1 phosphorylation at Ser-307 in the HEK293.IRS1 cells. Cells were maintained and treated as described in the Fig. 1 legend. Western blots were performed using antibody against total IRS1 (IRS1-t) or IRS1 phosphorylated at Ser-307 (IRS1-pS307). Quantification was done using phosphorimaging. Similar results were obtained in at least three experiments for both A and B. V, vehicle; SA, salicylic acid.

Salicylic Acid Does Not Affect PMA-induced Activation of Multiple Isoforms of PKCs in the HEK293.IRS1 Cells-- PMA is known to activate multiple isoforms of classical and novel PKCs (31, 32). We performed experiments to identify the PKC isoforms that were activated by PMA in the HEK293.IRS1 cells and determine whether the activation of any of the PKC isoforms was reversed by salicylic acid. To this end, Western blots were performed using antibodies against PKC isoforms that were phosphorylated at their activation loops, autophosphorylation sites, or at hydrophobic sites within the C terminus of the enzyme. Such phosphorylation is known to correlate to the activation and catalytic activity of the PKC isoforms (33, 34). As expected, PMA treatment did not activate PKCxi /lambda , the atypical PKC isoforms, in the HEK293.IRS1 cells (Fig. 3, bottom panel). Although PMA did not appear to activate classical PKCalpha /beta II (Fig. 3, second panel from top), it activated novel PKCµ, PKCdelta , and PKCtheta (Fig. 3, third through ninth panels from top). Pre-treatment with salicylic acid did not appear to affect PMA-mediated activation of any of these PKC isoforms. Taken together, these results suggest that salicylic acid did not affect PMA-mediated PKC activation.


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Fig. 3.   Salicylic acid does not affect PMA-mediated activation of multiple isoforms of PKCs in the HEK293.IRS1 cells. Cells were maintained and treated as described in the Fig. 1 legend. Western blots were performed using antibodies against total (t) or phosphorylated (p) PKC isoforms. It was noticed that PMA treatment increased both total and phosphorylated PKCtheta . Similar results were obtained in at least three experiments. V, vehicle.

TNFalpha Does Not Activate PKC Isoforms, IKKalpha , or IKKbeta in the HEK293.IRS1 Cells-- Some of the PMA-responsive PKC isoforms (i.e. PKCtheta and PKCzeta ) have been implicated in the activation of IKKbeta (35-37). It has also been reported that TNFalpha activates IKKbeta (2). We decided to investigate whether PMA and/or TNFalpha activate IKKbeta in the HEK293.IRS1 cells and, if so, whether such activation could be reversed by salicylic acid. To this end, we determined the activation of IKKalpha and IKKbeta using an antibody that specifically recognizes both IKKalpha phosphorylated at serine 180 and IKKbeta phosphorylated at serine 181. Phosphorylation of serine 176 and 180 in IKKalpha (177 and 181 in IKKbeta ) are required for activation of the kinases (38). In fact, for IKKbeta there is full correspondence between its kinase activity and its Ser-177 and Ser-181 phosphorylation (39).

As shown in Fig. 4, PMA treatment led to phosphorylation (and activation) of both IKKalpha and IKKbeta (Fig. 4, top three panels, lane 3 versus 1). In contrast, TNFalpha stimulation did not result in the phosphorylation of either IKKalpha or IKKbeta (Fig. 4, top three panels, lane 2 versus 1). In addition, TNFalpha treatment did not activate PKC isoforms based on Western blot using an antibody against phosphorylated pan-PKC (Fig. 4, bottom panel, lane 2 versus 1) or antibodies against phosphorylated individual PKC isoforms (data not shown). The effect of PMA to induce IKKalpha and IKKbeta phosphorylation implies that these two IKKs are functional in these cells. Because the same concentration of TNFalpha that was sufficient to impair insulin signaling did not activate either IKKalpha or IKKbeta , it appears that IKKbeta is not involved in TNFalpha -mediated intracellular signaling in these cells. Furthermore, these results exclude the possibility that the salicylic acid-elicited blockade of TNFalpha -induced insulin resistance is mediated via IKKbeta in these cells.


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Fig. 4.   PMA but not TNFalpha treatments activate IKKbeta in the HEK293.IRS1 cells. Cells were maintained and treated as described in Fig. 1. Western blots were performed using antibodies against total IKKalpha (IKKalpha -t), total IKKbeta (IKKbeta -t), both IKKalpha and IKKbeta that were serine phosphorylated (IKKalpha /beta -p), and phosphorylated PKC isoforms (pan-PKC-p). Similar results were obtained in at least three independent experiments. V, vehicle.

JNK Is Activated by PMA and TNFalpha and May Be Important for PMA and TNFalpha -induced IRS1 Ser-307 Phosphorylation in the HEK293.IRS1 Cells-- JNK has been reported to phosphorylate IRS1 at serine 307 and inhibit insulin signaling (17, 22). We performed experiments to address whether JNK was activated by PMA and/or TNFalpha in the HEK293.IRS1 cells. As shown in Fig. 5A, PMA treatment leads to phosphorylation (and activation) of the 54- and 46-kDa isoforms of JNK. A similar observation was made about TNFalpha -treated cells (Fig. 5B). Taken together, these results demonstrated that both PMA and TNFalpha treatments in the HEK293.IRS1 cells activate JNK.


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Fig. 5.   Salicylic acid reverses PMA and TNFalpha -induced JNK activation in the HEK293.IRS1 cells. Cells were maintained and treated as described in the Fig. 1 legend. Western blots were performed using antibodies against total JNK (JNK-t) and phosphorylated and activated JNK (JNK-p) (A) and total p42/44 MAPK kinase (p42/44-t) and phosphorylated and activated p42/44 (p42/44-p) (B). Quantification was done using phosphorimaging. Similar results were obtained in two experiments. V, vehicle; SA, salicylic acid.

Next we assessed whether JNK is critical or required for PMA and TNFalpha -mediated effects on insulin signaling. To address this question, we determined the effect of SP600125, a JNK small molecular inhibitor (40), on PMA- and TNFalpha -induced IRS1 Ser-307 phosphorylation. It was reported that, at 5µM, SP600125 inhibits JNK activity in intact hemopoietic cells (40). We first determined whether SP600125 inhibits JNK activity in the HEK293.IRS1 cells. As shown in Fig. 6, SP600125 did not affect JNK phosphorylation (and activation) upon both TNFalpha (Fig. 6B, third panel from top, lanes 5 and 6 versus 3 and 4) and PMA treatment (data not shown). On the other hand, SP600125 inhibited phosphorylation of the transcription factor c-Jun, a physiological substrate of JNK, upon PMA treatment (Fig. 6A, third panel from top, lanes 5 and 6 versus 3 and 4). We had difficulty detecting TNFalpha -induced c-Jun phosphorylation (data not shown). Taken together, these results confirm that SP600125 indeed inhibits the kinase activity but not the activation process of JNK in the HEK293.IRS1 cells.


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Fig. 6.   SP600125, a JNK inhibitor, reverses PMA and TNFalpha -induced phosphorylation of IRS1 at serine 307 in the HEK293.IRS1cell. A and B, cells were maintained and treated with vehicle (V), 5 µM SP600125 (SP), PMA, and TNFalpha as described in the Fig. 1 legend. Western blots were performed using antibodies against total and/or phosphorylated IRS1, JNK, c-Jun, p42/44 MAPK, p38 MAPK, IKKalpha , and IKKbeta . The level of total c-Jun, p42/44, p38 MAPK, IKKalpha , and IKKbeta were similar across all treatments (data not shown). The levels of total c-Jun, p42/44, p38 MAPK, IKKalpha , and IKKbeta were similar across all treatments (data not shown). C, cells were treated with vehicle (V) alone (left lane), 100 nM insulin alone (center lane), or pretreated with 5 µM SP600125 (SP) and then stimulated with 100 mM insulin (right lane). Western blots were performed using antibodies against total IRS-1, total Akt, and phosphorylated Akt as described in the Figs. 1 and 2 legends. Western blots were also performed to determine total insulin receptor (IR) using a rabbit polyclonal antibody against the insulin receptor beta  subunit and determine the tyrosine-phosphorylated insulin receptor (IR-pTyr) and IRS1 (IRS1-pTyr) using the mouse monoclonal antibody 4G10. The level of total Akt, IRS1, and insulin receptor were similar across all treatments (data not shown). Similar results were obtained in at least two experiments for A, B, and C.

It was reported that, although SP600125 potently inhibits the kinase activity of recombinant JNK enzymes (IC50 of 40-90 nM), it is rather inactive against the kinase activity of recombinant p42/44, p38, IKKalpha , IKKbeta , and PKC isoforms (including PKCtheta ) (IC50 of more than 10 µM), indicating a high degree of JNK selectivity for SP600125 (40). Furthermore, it was also reported that at 5 µM or less the effect of SP600125 on JNK activity is specific because SP600125 does not affect the activity or activation of other serine/threonine kinases in the intact hemopoietic cells (40). We decided to confirm such specificity in the HEK293.IRS1 cells. Indeed, SP600125 did not appear to affect PMA-induced activating phosphorylation of IKKalpha and IKKbeta (Fig. 6A, bottom panel, lanes 5 and 6 versus 3 and 4). SP600125 treatment also did not affect PMA- (data not shown) and TNFalpha -induced activation of both p42/44 MAPK kinases (Fig. 6B, fourth panel from top, lanes 5 and 6 versus 3 and 4) and p38 MAPK (Fig. 6B, bottom panel, lanes 5 and 6 versus 3 and 4). Taken together, these results are consistent with the notion that at 5 µM SP600125 specifically inhibits JNK activity in the HEK293.IRS1 cells (see more in Fig. 6C and the corresponding discussion for additional nonspecific activity).

Finally, we determined whether JNK enzymes are important in mediating PMA- and TNFalpha -induced IRS1 Ser-307 phosphorylation. The results indicate that SP600125 treatment prevented IRS1 Ser-307 phosphorylation induced by both PMA (Fig. 6A, top two panels, lanes 5 and 6 versus 3 and 4) and TNFalpha (Fig. 6B, top two panels, lanes 5 and 6 versus 3 and 4). Taken together, these results suggest that JNK is activated by PMA and TNFalpha and may be important for PMA and TNFalpha -induced IRS1 Ser-307 phosphorylation.

Salicylic Acid Inhibits PMA and TNFalpha -- mediated JNK Activation---The above results suggest that JNK was required for PMA- and TNFalpha -mediated IRS1 Ser-307 phosphorylation and insulin resistance. A key question was whether the effects of salicylic acid on insulin signaling, including IRS1 Ser-307 phosphorylation, were mediated by the inhibition of JNK. Thus, we determined whether salicylic acid affected JNK activation by PMA and TNFalpha . As shown in Fig. 5, salicylic acid significantly inhibited the activating phosphorylation of both the 54- and 46-kDa JNK induced by both PMA (Fig. 5A) and TNFalpha (Fig. 5B). Furthermore, the inhibition of JNK phosphorylation by salicylic acid appeared to be specific because salicylic acid did not inhibit the activating phosphorylation of p42/44 MAP kinases induced by both PMA (Fig. 5A, bottom two panels, lanes 5 and 6 versus 3 and 4) and TNFalpha (data not shown). Salicylate acid also did not affect the activating phosphorylation of the p38 MAPK kinases by PMA and TNFalpha (data not shown). Taken together, these results suggest that salicylic acid specifically inhibits JNK activation by PMA and TNFalpha .

SP600125 Inhibits Insulin-stimulated Tyrosine Phosphorylation of Akt, IRS1, and Insulin Receptor beta  Subunit-- To further demonstrate that JNK plays a negative role in insulin signaling and that its activation is required for PMA and TNFalpha -mediated insulin resistance, we decided to determine whether SP600125 can reverse PMA and TNFalpha -mediated inhibition on insulin signaling. Rather surprisingly, however, we found that SP600125 by itself completely inhibited insulin-stimulated threonine phosphorylation of Akt (Fig. 6C, top panel) as well as tyrosine phosphorylation of IRS-1 (middle panel) and the insulin receptor beta  subunit (bottom panel). These results suggest that SP600125 has some nonspecific activity that has not been reported previously (40). Unfortunately, such nonspecific activity excludes us from determining whether SP600126 treatments can reverse PMA- and TNFalpha -mediated inhibition of insulin signaling.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the current study, several observations have been made using a cellular model system for insulin signaling, i.e. a HEK293 cell line stably expressing recombinant human IRS1. First, both PMA and TNFalpha inhibited insulin-stimulated activating phosphorylation of Akt and promoted IRS1 Ser-307 phosphorylation. Second, the effects of PMA and TNFalpha on Akt phosphorylation and IRS1 serine phosphorylation were reversed by salicylic acid pretreatment. Third, PMA treatment led to activation of multiple serine/threonine kinases including PKC isoforms and IKKbeta ; however, such activation was not affected by salicylic acid. Fourth, TNFalpha treatment did not active either PKC isoforms or IKKbeta . Fifth, JNK was activated by both TNFalpha and PMA, and its inhibition by a small molecule inhibitor prevented TNFalpha - and PMA-induced IRS1 Ser-307 phosphorylation. The small molecular inhibitor, however, has some additional nonspecific activity. Finally, salicylic acid also inhibited TNFalpha - and PMA-induced activating phosphorylation of JNK.

The goal of this study was to further elucidate the role of IKKbeta and salicylic acid in inhibiting or augmenting insulin signaling, respectively. In our experimental system, PMA and TNFalpha were chosen to induce insulin resistance for several reasons. First, the negative effects of PMA and TNFalpha on insulin signaling are well documented, at least in cultured cells in vitro (11, 14, 18, 19, 27-29, 41). Second, IKKbeta is a well established downstream kinase for TNFalpha (2). It has also been shown that IKKbeta can be activated by PKC isoforms (i.e. PKCtheta and PKCzeta ) (35-37). In fact, it was recently reported that PKCzeta deficiency in mice impaired IKKbeta activation (37).

The results from the current study indicate that PMA activates multiple isoforms of PKC and IKK enzymes in the HEK293.IRS1 cells (Fig. 3). In addition to activating IKKbeta (2), TNFalpha has also been shown to activate PKC in multiple cell types (42-46). It was therefore rather surprising that TNFalpha did not activate IKKalpha , IKKbeta , and the multiple PKC isoforms examined in the HEK293.IRS1 cells (Fig. 4). Based on these observations, we conclude that in the HEK293.IRS1 cells IKKbeta is intact but is not involved in TNFalpha signaling. Therefore, the lack of IKKbeta activation by TNFalpha in the HEK293.IRS1 cells provided us with a unique setting to determine whether IKKbeta activation and its inhibition are involved and/or required for salicylate-mediated insulin sensitization. It then follows that the effect of TNFalpha on insulin signaling in the HEK293.IRS1 cells is independent of IKKbeta activation. Because salicylate was readily able to reverse TNFalpha -induced effects on insulin signaling, it also appears that these effects of the drug must be also IKKbeta -independent. In contrast, the current study does not provide sufficient evidence to assess whether the action of salicylic acid to reverse the effects of PMA on insulin action is mediated via IKKbeta inhibition.

How does salicylic acid reverse insulin resistance in these cells if not via inhibition of IKKbeta ? We considered that salicylic acid might improve insulin sensitivity by inhibiting kinase(s) other than IKKbeta , which were responsive to both PMA and TNFalpha . To this end, we examined the serine/threonine kinase JNK. JNK has been shown to be activated by both PMA (47-49) and TNFalpha (50-52). Most interestingly, TNFalpha was recently shown to inhibit insulin signaling by activating JNK, which subsequently becomes associated with Ser-307 in IRS1 and phosphorylates it (22).

To elucidate the role of JNK in PMA- and TNFalpha -induced insulin resistance and its reversal by salicylic acid, we performed a separate series of experiments and made several interesting observations. First, by demonstrating that both PMA and TNFalpha treatment induced activating phosphorylation of JNK (Fig. 5), we confirmed that JNK is involved in signaling in response to both PMA and TNFalpha in the HEK 293 cells. Second, by showing that SP600125 inhibits PMA- and TNFalpha -induced IRS1 Ser-307 phosphorylation, we propose that JNK activity may be important for the effects of PMA and TNFalpha on insulin signaling in these cells (see more discussion later on the nonspecific activity of SP600125). Finally, we showed that salicylic acid inhibits PMA- and TNFalpha -induced JNK activating phosphorylation (Fig. 6). Thus, these observations suggest that JNK is activated by (and its activity may be important for) PMA- and TNFalpha -induced insulin resistance. Most interestingly, the results also suggest that salicylic acid improves insulin sensitivity potentially by inhibiting JNK activation.

Fig. 7 summarizes the working hypothesis on the effects of salicylic acid on PMA- and TNFalpha -induced insulin resistance in the HEK293.IRS1 cells. In short, we propose that JNK may be directly responsible for IRS1 Ser-307 phosphorylation. IRS1 phosphorylated at Ser-307 has an impaired ability to undergo tyrosine phosphorylation by insulin receptor, leading to insulin resistance as reflected in reduced Akt phosphorylation (and activation). Salicylic acid reversed the effects of PMA and TNFalpha in association with reduced JNK activation. Although the detailed mechanism by which salicylic acid inhibits JNK activation in these cells remains unknown, it is most likely to be IKKbeta -independent. Although both PMA and TNFalpha treatment lead to JNK activation, the signaling pathways were apparently different. PMA activates PKC isoforms as well IKK kinases. TNFalpha did not activate either. Nevertheless, salicylic acid appears to inhibit JNK activation by both PMA and TNFalpha .


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Fig. 7.   Salicylic acid reverses TNFalpha -induced insulin resistance by an IKKbeta -independent fashion, probably by inhibiting JNK activation. Shown is a schematic that summarizes the results from the current experiments and depicts our proposed mechanism(s) by which salicylic acid promotes insulin signaling. IRS1-pY, tyrosine phosphorylated IRS1. Solid arrow, direct interaction. Dotted line, interaction with known or unknown intermediate steps. The thickness of an arrow represents the rate of signaling events.

In summary, the results from the current study suggest that IKKbeta inhibition is not necessarily critical to the mechanism(s) for salicylate-mediated insulin sensitization in the TNFalpha treatment paradigm. One approach to further validate our results would be to determine whether PMA and TNFalpha can induce IRS1 Ser-307 phosphorylation in IKKbeta -deficient cells and, if so, whether the effects by PMA and TNFalpha can be reversed by salicylic acid. However, it also remains to be established that the antidiabetic effects of salicylate in vivo are directly tied to inhibition of IKKbeta . Nevertheless, the apparent existence of IKKbeta -independent alternative pathway(s) underscores the need for additional knowledge regarding the mechanism(s) of salicylate-mediated insulin sensitization

Finally, although the current study suggests that JNK activation plays an important role in PMA and TNFalpha -mediated insulin resistance and that its inhibition may be responsible for salicylic acid-mediated insulin sensitization, such a proposal needs further validation because some of the results were derived from the use of the JNK inhibitor SP600125. We found that although SP600125 does not affect PMA- or TNFalpha -induced activation of several kinases (Fig. 6, A and B), it inhibits insulin signaling (Fig. 6C), indicating that the inhibitor may not be as specific as originally reported (40). One approach to further test the current theory would be to determine whether PMA and TNFalpha induce IRS1 Ser-307 phosphorylation and inhibit insulin signaling in JNK-deficient cells.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence may be addressed: Metabolic Disorders-Diabetes, Merck Research Laboratories, RY80N-C31, P.O. Box 2000, Rahway, NJ 07065. Tel.: 732-594-2176; Fax: 732-594-3925; E-mail: guoqiang_jiang@merck.com or bei_zhang{at}merck.com.

Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M205565200

    ABBREVIATIONS

The abbreviations used are: IKK, Ikappa B kinase; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; TNFalpha , tumor necrosis factor alpha ; MAP, mitogen-activated protein; MAPK, MAP kinase; PMA, phorbol 12-myristate 13-acetate; HEK293, human embryonic kidney 293; PKC, protein kinase C.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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