Arachidonic Acid Inhibits the Insulin Induction of Glucose-6-phosphate Dehydrogenase via p38 MAP Kinase*

Polyunsaturated fatty acids are potent inhibitors of lipogenic gene expression in liver. The lipogenic enzyme glucose-6-phosphate dehydrogenase (G6PD) is unique in this gene family, in that fatty acids inhibit at a post-transcriptional step. In this study, we have provided evidence for a signaling pathway for the arachidonic acid inhibition of G6PD mRNA abundance. Arachidonic acid decreases the insulin induction of G6PD expression; by itself, arachidonic acid does not inhibit basal G6PD mRNA accumulation. The insulin stimulation of G6PD involves the phosphoinositide 3-kinase (PI 3-kinase) pathway (Wagle, A., Jivraj, S., Garlock, G. L., and Stapleton, S. R. (1998) J. Biol. Chem. 273, 14968-14974). Incubation of hepatocytes with arachidonic acid blocks the activation of PI 3-kinase by insulin as observed by a decrease in Ser473 phosphorylation of Akt, the downstream effector of PI 3-kinase. The decrease in PI 3-kinase activity was associated with an increase in Ser307 phosphorylation of IRS-1. Western analysis demonstrated increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) in arachidonic acid-treated cells, whereas extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activity was not changed. Incubating the hepatocytes with the p38 MAPK inhibitor, SB203580, blocked the arachidonic acid inhibition of G6PD mRNA accumulation. Furthermore, SB203580 decreased the arachidonic acid-mediated Ser307 phosphorylation of IRS-1 and rescued Akt activation that was otherwise decreased by arachidonic acid. Thus, arachidonic acid inhibits the insulin stimulation of G6PD mRNA accumulation by stimulating the p38 MAPK pathway, thereby inhibiting insulin signal transduction.

Insulin is the central hormone required for the activation of lipogenic genes in the liver.Feeding animals a high carbohydrate diet enhances the expression of the lipogenic genes.This effect involves the stimulatory actions of both dietary glucose and insulin (1,2).In contrast, dietary polyunsaturated fats attenuate the stimulatory effect of feeding a high carbohydrate diet (3,4).We have used glucose-6-phosphate dehydrogenase (G6PD), 2 a member of the lipogenic gene family, as a model system for studying the mechanism of action of fatty acids.The advantage of this model is that insulin is the primary inducer of G6PD expression, and fatty acids such as arachidonic acid are the primary inhibitors of G6PD expression; this regulation is independent of other hormonal requirements (5,6).The intracellular mechanisms by which polyunsat-urated fats inhibit G6PD or other lipogenic genes are not completely understood.Inhibition by polyunsaturated fatty acid may represent a direct action of fatty acids on factors involved in gene expression.Alternatively, fatty acids may act indirectly via the inhibition of stimulatory signal transduction pathways of glucose or insulin.We hypothesized that fatty acids inhibit G6PD expression by inhibition of the insulin induction.
Insulin transduces its signal upon binding to the insulin receptor.Transduction of this signal in liver involves phosphorylation of two intracellular substrates, insulin receptor substrate (IRS)-1 and IRS-2 (7).These proteins play complementary roles in insulin signaling (8).Activation of phosphoinositide (PI) 3-kinase is associated with the stimulatory effects of insulin on metabolic pathways, including lipogenesis (9 -11).
The IRS proteins can be phosphorylated on both tyrosines and serines.A known mechanism for the inhibition of IRS-1 activation is by phosphorylation at serines 307, 612, and 632 (12).These serine residues, when phosphorylated might interfere with the interaction between IRS-1 and the insulin receptor or PI 3-kinase (13,14).Among the factors known to cause serine phosphorylation of IRS-1 are the mitogen-activated protein (MAP) kinases.Activation of the MAP kinases extracellular regulated kinase (ERK) (15,16), c-Jun NH 2 -terminal kinase (JNK) (17)(18)(19), or p38 MAP kinase (MAPK) (17,20) is associated with the development of insulin resistance in muscle and adipose tissue.
Known activators of MAP kinases include tumor necrosis factor ␣ (TNF␣) and very high fat diets.TNF␣, a potent mediator of insulin resistance, activates all three of the MAP kinases.Phosphorylation and activation of p38 MAPK by TNF␣ correlates with IRS-1 serine phosphorylation and a decrease in PI 3-kinase activity (17,20).In muscle and adipose tissue, this results in the decrease in glucose uptake associated with insulin resistance.Likewise, diets containing 40% or more of the energy content as fat also decrease PI 3-kinase activation and result in an insulin-resistant phenotype in intact animals (21)(22)(23).This may involve activation of MAP kinases (18,19).In contrast to the pathological effects of large amounts of dietary fat, the addition of smaller amounts of polyunsaturated fat (Ͻ20% of energy) to a high carbohydrate diet has a potent but reversible inhibitory effect on lipogenic gene expression (3).The question remains whether modulation of insulin signaling by low levels of dietary fat is involved in the non-pathological regulation of metabolic pathways and, in particular, the de novo synthesis of fatty acids.
In this paper, we have reported a signaling pathway by which polyunsaturated fatty acids inhibit G6PD expression.We have demonstrated that arachidonic acid inhibits the insulin-mediated induction of G6PD mRNA abundance in primary rat hepatocytes.This inhibition of insulin action occurs by Ser 307 phosphorylation of IRS-1.Incubation of hepatocytes with arachidonic acid results in an activation of p38 MAPK, thereby inducing IRS-1 serine phosphorylation and reducing PI 3-kinase activation.Inhibition of p38 MAPK results in a loss of the arachidonic acid-mediated inhibition of G6PD expression.This is the first report of p38 MAPK involvement in the regulation of a lipogenic gene by polyunsaturated fatty acids.

EXPERIMENTAL PROCEDURES
Animal Care and Cell Culture-Male Sprague-Dawley rats (175-225 g) were starved 16 h prior to surgery.Hepatocytes were isolated by a modification of the technique of Seglen (24) as previously described (6).Hepatocytes (3.1 ϫ 10 6 ) were placed in 60-mm dishes coated with rat tail collagen and incubated in Hi/Wo/Ba medium (Waymouth MB752/1 plus 20 mM HEPES, pH 7.4, 0.5 mM serine, 0.5 mM alanine, 0.2% bovine serum albumin).The medium containing the treatments indicated in the figure legends was added after 24 h in culture.Fatty acids (Nu-Check Prep) were added as a complex bound to bovine serum albumin (25).The fatty acid (4 mM) albumin (1 mM) stocks contained butylated hydroxytoluene (0.1%), and the medium contained ␣-tocopherol phosphate, disodium (10 g/liter) to minimize oxidation of fatty acids.The concentration of arachidonic acid used (175 M) resulted in the maximum inhibition of G6PD expression (6,26).Hepatocytes not receiving arachidonic acid were treated with an equivalent amount of albumin and butylated hydroxytoluene.Because arachidonic acid is metabolized rapidly by the hepatocytes, the medium was replenished every 12 h.The ERK inhibitor (PD98059) was purchased from Cell Signaling Technology; the inhibitors of PI 3-kinase (LY294002) and p38 MAPK (SB203580) were purchased from Calbiochem.
Isolation of Total RNA and Ribonuclease Protection Assay-Total RNA from 2-3 plates/treatment was isolated by the method of Chomczynski and Sacchi (27).Quantitation of RNA was performed using RNase protection assays (Ambion, Inc.).Rat G6PD exon 13 template was synthesized as described previously (26).The templates for the rat ␤-actin and 18S probes were purchased from Ambion, Inc. Probe synthesis, hybridizations, and RNase digestion were as previously described (28).The resulting hybridization products protected from RNase digestion were separated in a 5% denaturing polyacrylamide gel.Images were visualized by storage phosphor technology and quantified using Image-Quant software by Amersham Biosciences.
Preparation of Cell Extract and Western Blot Analysis-The hepatocytes were lysed in buffer containing 10 mM Tris, pH 7.4, and 1% SDS, and total protein was quantified using a BCA protein assay (Pierce).Equal amounts of denatured protein (40 g/lane) were loaded onto a 7.5% polyacrylamide gel and transferred to Immun-Blot TM polyvinylidene difluoride membrane (Bio-Rad) at 100 V for 1.5 h.The membranes were blocked in 5% nonfat dry milk for 1 h and incubated with primary antibody diluted in 5% bovine serum albumin overnight at 4 °C.The primary antibodies against phosphorylated Akt (Ser 473 ), phosphorylated IRS-1 (Ser 307 and Ser 612 ), phosphorylated p38 MAPK (Thr 180 / Tyr 182 ), phosphorylated ERK (Tyr 183/185 ), phosphorylated JNK (Thr 183 / Tyr 185 ) and total Akt, IRS-1, p38 MAPK, ERK, and JNK were obtained from Cell Signaling Technology.Anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling Technology) was used as the secondary antibody, and the immunocomplexes were detected by enhanced chemiluminescence (Pierce).Images were visualized with film (Pierce) and quantified by densitometry using ImageQuant software (Amersham Biosciences).

Insulin Induces the Expression of G6PD mRNA, whereas Arachidonic
Acid Attenuates This Effect-Primary rat hepatocytes were used for these studies, because they are the only cell culture system that mimics the effects of dietary polyunsaturated fat on the regulation hepatic FIGURE 1. Arachidonic acid inhibits the insulin-mediated induction of G6PD mRNA in primary rat hepatocytes.Rat hepatocytes were plated onto collagen-coated tissue culture dishes in Hi/Wo/Ba medium plus 5% newborn calf serum.After 3-4 h, the medium was changed to serum-free medium and after 10 h Matrigel® was added.24 h post-isolation, the cells (3 plates/treatment) were treated with or without 0.04 M insulin Ϯ 175 M arachidonic acid or with arachidonic acid alone.After 24 h of treatment, the cells were harvested, and total RNA was isolated.The top panel is a representative RNase protection assay.The lower panel shows the results of two experiments (separate hepatocyte isolations) quantified using phosphorimaging and ImageQuant software.G6PD mRNA was normalized to the amount of ␤-actin mRNA, and the results are expressed relative to the amount of mRNA in no addition.NA, no addition; FA, arachidonic acid; Ins, insulin.FIGURE 2. Arachidonic acid inhibits Akt activation and induces Ser 307 phosphorylation of IRS-1.Hepatocytes were treated with or without arachidonic acid (175 M) 24 h prior to the addition of insulin (0.04 M) and isolated 5, 10, 30, or 60 min after the addition of insulin.A, Western blot analysis was performed against total and phosphorylated IRS-1 and Akt.The antibodies specifically detected IRS-1 phosphorylated at Ser 307 or Ser 612  and Akt phosphorylated at Ser 473 .The asterisk denotes a nonspecific band detected with the IRS-1 Ser 307 antibody.B, the amount of phosphorylated Akt in n ϭ 4 experiments for the 5-, 10-, and 30-min time points, and n ϭ 3 experiments for the 60 min.Each bar is the mean Ϯ S.E. for the indicated number of experiments, and the values are expressed as the fold increase in phosphorylated Akt relative to the amount in hepatocytes not treated with insulin or arachidonic acid.C, the amount of phosphorylated IRS-1 (Ser 307 ) for n ϭ 4 experiments for the 5-, 10-, and 30-min time points, and n ϭ 2 experiments for 60 min.The values are expressed relative to the amount of phosphorylated protein in hepatocytes not treated with insulin or arachidonic acid.P-Akt, phosphorylated Akt; P-IRS-1, phosphorylated IRS-1; FA, arachidonic acid; I, insulin.
G6PD expression.In hepatocytes, insulin increases G6PD mRNA abundance, and arachidonic acid inhibits G6PD mRNA amount, similar to the changes in G6PD expression observed in intact liver, in animals fed a high carbohydrate diet, and a diet supplemented with polyunsaturated fat (6% safflower oil), respectively (6,29).Furthermore, regulation of G6PD expression occurs at a nuclear post-transcriptional step in both intact liver and primary hepatocyte cultures (6,28,29).We first asked whether arachidonic acid per se regulated G6PD expression.Because G6PD activity is essential for cell viability, the amount of G6PD mRNA remains readily detectable in the absence of insulin (Fig. 1).The addition of arachidonic acid alone did not decrease the expression of G6PD mRNA.In contrast, insulin induced G6PD mRNA expression 7-fold, and arachidonic acid inhibited this induction by 50% or more.Thus, arachidonic acid appears to inhibit the insulin-mediated induction of G6PD expression.
Arachidonic Acid Inhibits Akt Phosphorylation and Induces Ser 307 Phosphorylation of IRS-1-Because the arachidonate inhibition is only observed in cells with intact insulin action, we hypothesized that arachidonic acid interferes with the PI 3-kinase pathway and thus prevents the induction of G6PD mRNA by insulin.The induction of G6PD mRNA by insulin has been demonstrated to require the PI 3-kinase signal transduction pathway (Ref.30 and data not shown).The form or metabolite of arachidonic acid that inhibits gene expression is not known.Thus, the hepatocytes were pre-incubated with arachidonic acid to allow time for incorporation of this fatty acid into membrane phospholipid and/or generation of the relevant intracellular metabolite.The rapid signal transduction by the insulin receptor could otherwise have preceded the generation of the relevant inhibitory form of arachidonic acid.Insulin induced Akt phosphorylation 24 -75-fold across time points (Fig. 2, A  and B).This induction was rapid and persisted through 60 min.Preincubation of hepatocytes with arachidonic acid attenuated the insulinmediated induction of Akt phosphorylation by 50 -72% across the time points (Fig. 2, A and B).The amount of total Akt was not affected by these treatments (Fig. 2A).Similarly, arachidonic acid inhibited PI 3-kinase activity by 50%, consistent with it being an upstream activator of Akt (data not shown).
The decrease in Akt phosphorylation in cells incubated with arachidonic acid suggests that arachidonic acid is inhibiting a step at or prior to PI 3-kinase activation.Because phosphorylation of IRS-1 at serines can interfere with its interaction with either the insulin receptor or PI 3-kinase and thereby inhibits insulin signal transduction (12), we measured changes in the amount of IRS-1 phosphorylation on two different serine residues.Incubation of hepatocytes with arachidonic acid prior to and during the addition of insulin increased IRS-1 Ser 307 phosphorylation 4 -6-fold at each time point (Fig. 2, A and C).Insulin alone caused a smaller but reproducible increase in Ser 307 phosphorylation.Arachidonic acid does not cause an increase in the phosphorylation of all regulatory serines in IRS-1.In this regard, arachidonic acid did not stimulate an increase in phosphorylation at Ser 612 of IRS-1 (Fig. 2A).Although phosphorylation of Ser 612 can also abolish tyrosine phosphorylation of IRS-1 (7), arachidonic acid inhibition of insulin signaling in hepatocytes does not appear to involve this serine residue.These results suggest that arachidonic acid increases Ser 307 phosphorylation of IRS-1; this in turn interferes with the PI 3-kinase activity and Akt phosphorylation, which are required for the insulin induction of G6PD mRNA.
Arachidonic Acid Induces p38 MAPK Phosphorylation-To explore the mechanism for the increase in IRS-1 Ser 307 phosphorylation, we determined whether arachidonic acid in the presence of insulin activates serine/threonine kinases involved in serine phosphorylation of IRS-1.Members of the MAP kinase family are known to stimulate serine phosphorylation of IRS-1 (15)(16)(17)(18)(19)(20).The amounts of phosphorylated and activated as well as the total amounts of the three MAP kinases, ERK, JNK, and p38, were measured by Western blot analyses (Fig. 3, A and B).Insulin stimulated a small but consistent increase in p38 MAPK phosphorylation.A similar increase was detected with arachidonic acid alone (Fig. 3C).The greatest increase in the phosphorylation of p38 MAPK (4 -6-fold) was observed with arachidonic acid in the presence of insulin as compared with no additions.Furthermore, the increase in p38 MAPK phosphorylation occurred within the same time frame as the changes in Akt and IRS-1 Ser 307 phosphorylation.The total amount of p38 MAPK was not regulated by these treatments (Fig. 3C).In contrast, arachidonic acid did not increase the amounts of activated ERK and JNK.Fluctuations in ERK and JNK activation were observed across treatments and likely reflect stimulation due to the manipulation of the cells for the addition of the treatments.There was no consistent effect of the treatments per se on ERK or JNK activation.This result suggests that arachidonic acid stimulates Ser 307 phosphorylation of IRS-1 by the activation of p38 MAPK.
Inhibition of the p38 MAPK Pathway Decreases the Arachidonic Acidmediated Inhibition of G6PD mRNA Expression-We next tested whether the activation of p38 MAPK by arachidonic acid is responsible for the inhibition of G6PD mRNA accumulation.The p38 MAP kinase pathway was inhibited by SB203580.SB203580 had little or no effect on the insulin-mediated induction of G6PD mRNA abundance but abrogated the inhibition by arachidonic acid (Fig. 4).In contrast, inhibition of ERK signaling with the inhibitor PD98059 had no detectable effect on G6PD regulation by insulin or arachidonic acid (data not shown), consistent with the lack of ERK activation by arachidonic acid.To verify that activation of p38 MAPK was involved in the Ser 307 phosphorylation of IRS-1, the effects of arachidonic acid on Ser 307 phosphorylation of IRS-1 and Akt phosphorylation were measured in hepatocytes incubated with SB203580.Arachidonic acid in the presence of the p38 MAPK inhibitor did not increase the Ser 307 phosphorylation of IRS-1 as compared with insulin-and SB203580-treated hepatocytes (Fig. 5, compare lanes 4 and 5 versus 2 and 3 for P-IRS1).At the same time, Akt phosphorylation in hepatocytes incubated with arachidonic acid and SB203580 did not decrease with respect to hepatocytes incubated with insulin and SB203580 (Fig. 5, lanes 4 and 5 versus 2 and 3 for P-Akt).These results are consistent with the idea that activation of p38 MAP kinase by arachidonic acid impairs the activation of the PI 3-kinase by inducing Ser 307 phosphorylation of IRS-1, which in turn inhibits the induction of G6PD mRNA by insulin.
Multiple Unsaturated Fatty Acids Inhibit Insulin Signal Transduction-Polyunsaturated fatty acids of both the 6 and 3 families inhibit G6PD expression (6).We asked whether other polyunsaturated fatty acids also inhibit insulin signal transduction.Eicosapentaenoic acid (20:53) was as effective as arachidonic acid (20:46) at both decreasing Akt phosphorylation and increasing p38 MAPK phosphorylation (TABLE ONE).The eighteen carbon precursors of these fatty acids, especially linoleate (18:2), caused similar changes in Akt and p38 MAPK phosphorylation but were somewhat less effective, similar to the lower inhibitory effect of these fatty acids on G6PD mRNA accumulation both historically (6) and in this experiment (data not shown).
TNF␣ Induces p38 MAP Kinase, Inhibits Activation of Akt, and Also Diminishes Induction of G6PD by Insulin-To corroborate that arachidonic acid activation of p38 MAPK was involved in its inhibition of G6PD expression, we tested the effect of another activator of p38 MAPK.TNF␣ inhibits insulin-mediated Akt phosphorylation in 3T3-L1 adipocyte myotubes (17,20).In addition, TNF␣ activates p38 and other MAP kinases in various cell lines, resulting in inhibition of the Hepatocytes were isolated and plated as described in the legend to Fig. 1. Cell lysates were prepared after 2, 5, 10, 30, and 60 min from the untreated, insulin (0.04 M)-treated, or insulin ϩ arachidonic acid (175 M)-treated cells.A, Western blot analysis was performed against the phosphorylated and total forms of ERK, JNK, and p38 MAP kinases.The antibodies for the phosphorylated MAP kinases recognize phosphates at Tyr 183/185 , Thr 183 /Tyr 185 , and Thr 180 /Tyr 182 , respectively.A representative blot of at least four separate experiments is shown.B, quantitative representation of the fold induction of phosphorylated p38 MAPK in hepatocytes treated with insulin alone or insulin ϩ arachidonic acid.Each bar represents the mean Ϯ S.E., and the values are expressed relative to the absence of treatment.Repetition was as follows: n ϭ 4 for 2 min, n ϭ 6 for 5 min, n ϭ 8 for 10 min, n ϭ 4 for 30 min, and n ϭ 2 for 60 min.C, hepatocytes were incubated with insulin (0.4 M) alone, arachidonic acid (175 M) alone, or insulin ϩ arachidonic acid.Cell lysates were prepared 5, 10, or 30 min later, and Western blot analysis was performed against phosphorylated and total p38 MAPK.The blot is representative of n ϭ 3 separate experiments.FA, arachidonic acid; P-p38, phosphorylated p38; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK; I, insulin.
PI 3-kinase pathway (20).Incubation of rat hepatocytes with insulin and TNF␣ resulted in a concomitant 5-fold increase in phosphorylated p38 MAPK and a 53-76% decrease in phosphorylated Akt across time points (Fig. 6, A and B, respectively).These changes in p38 MAPK and Akt phosphorylation occurred within the same time frame as the changes caused by arachidonic acid.In addition, TNF␣ also decreased the insulin induction of G6PD mRNA by 50% (Fig. 6C).Thus, activation of MAP kinases by multiple effectors is associated with a decrease in Akt activation and a decrease in the accumulation of G6PD mRNA.

DISCUSSION
In previous work, we have defined a unique post-transcriptional mechanism whereby polyunsaturated fatty acids inhibit G6PD expression, a decrease in pre-mRNA splicing (26,31).In the present study, we sought to determine the signaling pathway involved in this inhibition.We demonstrated that the polyunsaturated fatty acid inhibition of G6PD expression involves inhibition of the insulin-mediated induction of G6PD (Fig. 1).This inhibition was a consequence of arachidonic acid inhibition of insulin signal transduction via PI 3-kinase (Fig. 2).Incubation of hepatocytes with insulin and arachidonic acid activated p38 MAPK, resulting in Ser 307 phosphorylation of IRS-1 (Figs. 2 and 3), a modification that inhibits insulin activation of PI 3-kinase (7).In confirmation of this, inhibition of p38 MAPK activity blocks the arachidonic acid inhibition of G6PD expression, decreases Ser 307 phosphorylation of IRS-1, and increases PI 3-kinase activity as measured by Akt phosphorylation (Figs. 4 and 5).This is the first report of inhibition of a lipogenic gene in liver via activation of the p38 MAPK pathway.
Polyunsaturated fatty acids have been demonstrated to activate MAP kinases in various cell types (32)(33)(34).In primary rat hepatocytes, the action of arachidonic acid is specific for p38 MAPK; extracellular signal-regulated and c-Jun kinases are not activated by arachidonic acid (Fig. 3).Activation of p38 MAPK is generally thought to be part of the stress-activated signal transduction pathway (35,36), and in this regard, arachidonic acid is a known generator of toxic peroxides (37,38).Several lines of evidence suggest that oxidative stress is not the mechanism by which arachidonic acid in primary rat hepatocytes activates p38 MAPK.First, the extent of p38 MAPK activation was the same with either arachidonic acid or insulin alone (Fig. 3C).In addition, arachidonic acid in the absence of insulin did not inhibit G6PD expression, despite readily detectable levels of G6PD mRNA (Fig. 1).Second, oxidation of arachidonic acid in the culture system was minimized by the preparation of fatty acid stocks with butylated hydroxytoluene, and the culture medium was supplemented with additional ␣-tocopherol.Although these will not completely eliminate oxidation, peroxidation is not detectable using a thiobarbituric acid assay (6).More importantly, enhanced oxidation induces G6PD expression (39,40).Thus, if arachidonic acid was causing oxidative stress, an increase in G6PD expression would have been observed.
The mechanism by which arachidonic acid activates p38 MAPK is not    (41,42).Still, other reports suggest that protein kinase C is an important upstream kinase.Preliminary studies using the inhibitor of protein kinase C, GF109203X, demonstrated that inhibition of protein kinase C does not block the inhibition of G6PD expression by arachidonic acid. 3lternatively, polyunsaturated fatty acids can also activate AMP-activated protein kinase in rat liver and hepatocytes (43,44).Activation of AMPactivated protein kinase is associated with an increase in p38 MAPK phosphorylation in skeletal and cardiac muscles, and this increase is associated with an increase in glucose uptake (45,46).Although p38 MAPK has not been shown to be a substrate for AMP-activated protein kinase, these results are consistent with AMP-activated protein kinase being upstream in the p38 MAPK signaling pathway.A, hepatocytes were treated with or without insulin (0.04 M) in the presence or absence of TNF␣ (10 ng/ml).Total cell lysates were isolated 5, 10, and 30 min after the addition of the treatments.Western blot analysis was performed against the phosphorylated and total forms of ERK, JNK, and p38 MAP kinases.The antibodies for the phosphorylated MAP kinases recognize phosphates at Tyr 183/185 , Thr 183 /Tyr 185 , and Thr 180 /Tyr 182 , respectively.B, hepatocytes were incubated with or without TNF␣ (10 ng/ml) for 24 h prior to the addition of insulin (0.04 M).Total cell lysates were isolated 5, 10, and 30 min following the addition of insulin.Western blot analysis was performed with the antibody against the phosphorylated Akt (Ser 473 ).The 5-min samples were from the same experiment but on a separate gel from the other time points and were exposed on the same film.C, hepatocytes were treated with or without insulin (0.04 M) and TNF␣ (10 ng/ml) as shown.After 24 h, total RNA was isolated, and the amounts of G6PD and ␤-actin mRNA were measured by RNase protection assay and imaged used phosphorimaging.All of the figures are representative blots of n ϭ 3 experiments.FA, arachidonic acid; P-Akt, phosphorylated Akt; P-p38, phosphorylated p38; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK.

FIGURE 3 .
FIGURE 3. Arachidonic acid activates p38 MAPK.Hepatocytes were isolated and plated as described in the legend to Fig.1.Cell lysates were prepared after 2, 5, 10, 30, and 60 min from the untreated, insulin (0.04 M)-treated, or insulin ϩ arachidonic acid (175 M)-treated cells.A, Western blot analysis was performed against the phosphorylated and total forms of ERK, JNK, and p38 MAP kinases.The antibodies for the phosphorylated MAP kinases recognize phosphates at Tyr 183/185 , Thr 183 /Tyr 185 , and Thr 180 /Tyr 182 , respectively.A representative blot of at least four separate experiments is shown.B, quantitative representation of the fold induction of phosphorylated p38 MAPK in hepatocytes treated with insulin alone or insulin ϩ arachidonic acid.Each bar represents the mean Ϯ S.E., and the values are expressed relative to the absence of treatment.Repetition was as follows: n ϭ 4 for 2 min, n ϭ 6 for 5 min, n ϭ 8 for 10 min, n ϭ 4 for 30 min, and n ϭ 2 for 60 min.C, hepatocytes were incubated with insulin (0.4 M) alone, arachidonic acid (175 M) alone, or insulin ϩ arachidonic acid.Cell lysates were prepared 5, 10, or 30 min later, and Western blot analysis was performed against phosphorylated and total p38 MAPK.The blot is representative of n ϭ 3 separate experiments.FA, arachidonic acid; P-p38, phosphorylated p38; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK; I, insulin.

FIGURE 4 .
FIGURE 4. A p38 MAPK inhibitor, SB203580, reverses the arachidonic acid-mediated decrease of G6PD mRNA.Hepatocytes were treated with or without 10 M SB203580 for 1 h.After 1 h, arachidonic acid (175 M) was added, and 2 h later, insulin (0.04 M) was added.After 12 h, the medium was replenished with one of the same composition.Total RNA was isolated after 24 h with insulin.The isolated RNA was analyzed by RNase protection assay and quantified using phosphorimaging and ImageQuant software.The values are the amount of G6PD mRNA normalized to the amount of ␤-actin mRNA and are plotted relative to the amount of RNA in the absence of treatments or inhibitor.Each bar represents the mean Ϯ S.E. of n ϭ 3 experiments.I, insulin; FA, arachidonic acid.

FIGURE 5 .
FIGURE 5. Inhibition of p38 MAPK blocks the decrease in Akt activation and the increase in IRS-1 Ser 307 phosphorylation by arachidonic acid.Hepatocytes were treated with or without arachidonic acid (175 M) for 23 h.The cells were then incubated with or without 10 M SB203580 for 1 h, prior to the addition of insulin (0.04 M).Total cell lysates were isolated 5 min after the addition of insulin.Western blot analysis was performed with antibodies against total and phosphorylated Akt and IRS-1.The antibody for phosphorylated IRS-1 recognizes phosphate at Ser 307 , and the antibody for phosphorylated Akt recognizes phosphate at Ser 473 .FA, arachidonic acid; P-IRS-1, phosphorylated IRS-1; P-Akt, phosphorylated Akt.

FIGURE 6 .
FIGURE 6. TNF␣ inhibits activation of Akt, activates p38 MAPK, and inhibits induction of G6PD mRNA expression by insulin.A, hepatocytes were treated with or without insulin (0.04 M) in the presence or absence of TNF␣ (10 ng/ml).Total cell lysates were isolated 5, 10, and 30 min after the addition of the treatments.Western blot analysis was performed against the phosphorylated and total forms of ERK, JNK, and p38 MAP kinases.The antibodies for the phosphorylated MAP kinases recognize phosphates at Tyr 183/185 , Thr 183 /Tyr 185 , and Thr 180 /Tyr 182 , respectively.B, hepatocytes were incubated with or without TNF␣ (10 ng/ml) for 24 h prior to the addition of insulin (0.04 M).Total cell lysates were isolated 5, 10, and 30 min following the addition of insulin.Western blot analysis was performed with the antibody against the phosphorylated Akt (Ser 473 ).The 5-min samples were from the same experiment but on a separate gel from the other time points and were exposed on the same film.C, hepatocytes were treated with or without insulin (0.04 M) and TNF␣ (10 ng/ml) as shown.After 24 h, total RNA was isolated, and the amounts of G6PD and ␤-actin mRNA were measured by RNase protection assay and imaged used phosphorimaging.All of the figures are representative blots of n ϭ 3 experiments.FA, arachidonic acid; P-Akt, phosphorylated Akt; P-p38, phosphorylated p38; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK.

TABLE ONE Effect of various polyunsaturated fatty acids on Akt and p38 MAPK phosphorylation
Rat hepatocytes were either pre-incubated with fatty acid for 24 h prior to the addition of insulin (Akt-phosphorylation) or treated simultaneously with insulin and 175 M fatty acid (p38 MAPK phosphorylation).Total cell lysates were prepared 10 and 30 min after the addition of insulin.Total and phosphorylated Akt and p38 MAPK were determined by Western blot analysis.The values are the ratio of phosphorylated to total Akt or p38 MAPK and are expressed relative to insulin alone.The data are representative of n ϭ 2 experiments that showed the same results.18:2, linoleic acid; 18:3, linolenic acid; DECEMBER 9, 2005 • VOLUME 280 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 40665 clear.Multiple steps exist upstream of p38 MAPK and regulate its phosphorylation.Stress and cytokines generally induce p38 MAPK via Tak1 or Ask1