Protein Kinase C θ (PKCθ)-dependent Phosphorylation of PDK1 at Ser504 and Ser532 Contributes to Palmitate-induced Insulin Resistance*

Clinical, epidemiological, and biochemical studies have highlighted the role of obesity-induced insulin resistance in various metabolic diseases. However, the underlying molecular mechanisms remain to be established. In the present study, we show that palmitate-induced serine phosphorylation of phosphoinositide-dependent protein kinase-1 (PDK1) negatively regulates insulin signaling. PDK1-mediated Akt phosphorylation at Thr308 in the activation loop is reduced in C2C12 myotubes treated with palmitate or overexpressing protein kinase C θ (PKCθ), a kinase that has been implicated in hyperlipidemia-induced insulin resistance. Palmitate treatment also inhibited platelet-derived growth factor-stimulated Akt phosphorylation, suggesting that the inhibition could occur at a site independent of IRS1/2. The inhibitory effect of palmitate on PDK1 and Akt was diminished in PKCθ-deficient mouse embryonic fibroblasts (MEFs) by treating C2C12 myotubes with PKCθ pseudosubstrates. In vivo labeling studies revealed that PDK1 undergoes palmitate-induced phosphorylation at two novel sites, Ser504 and Ser532. Replacing Ser504/532 with alanine disrupted PKCθ-catalyzed PDK1 phosphorylation in vitro and palmitate-induced PDK1 phosphorylation in cells. PDK1-deficient MEFs transiently expressing PDK1S504A/S532A but not PDK1S504E/S532D showed increased basal and insulin-stimulated Akt phosphorylation at Thr308 when compared with MEFs expressing wild-type PDK1. Taken together, our results identify PDK1 as a novel target in free fatty acid-induced insulin resistance and PKCθ as the kinase mediating the negative regulation.

A reduced capacity for insulin to stimulate glucose uptake and metabolism in target tissues such as skeletal muscle and adipose tissues is a common feature of obesity and diabetes.
Chronic elevation of free fatty acid (FFA) 2 levels in plasma has been found to be closely associated with impaired insulin-mediated glucose uptake (1,2) and often coexists with obesity and type 2 diabetes (3). However, although the association between hyperlipidemia and insulin resistance is well established, the molecular mechanisms remain to be established.
Insulin regulates circulating blood glucose levels and whole body energy homeostasis by interacting with its membrane receptor in cells of target tissues such as liver, skeletal muscle, and adipose. The binding of insulin to its receptor activates the phosphatidylinositol (PI) 3-kinase signaling pathway, which is essential for insulin-mediated biological events such as glucose uptake in insulin target cells. Components of the PI 3-kinase pathway include members of the protein kinase A/protein kinase G/protein kinase C family such as protein kinase B (PKB/ Akt) and some protein kinase C (PKC) isoforms. Activation of these kinases requires phosphorylation in their activation loop, which is mediated by 3Ј-phosphoinositide-dependent kinase-1 (PDK1). Thus, PDK1 functions as a master kinase to regulate various downstream biological events such as insulin-stimulated glucose uptake and protein synthesis.
PDK1 is a 63-kDa Ser/Thr kinase with a catalytic domain near its N terminus and a pleckstrin homology domain at its C terminus. The pleckstrin homology domain is necessary for targeting PDK1 to the plasma membrane to interact with and phosphorylate its substrates such as Akt (4,5). We and others have shown that autophosphorylation of PDK1 in the activation loop (Ser 241 /Ser 244 for human and mouse PDK1, respectively) is essential for PDK1 activity (6,7). In addition, we found that phosphorylation of endogenous PDK1 at this site could be stimulated by insulin in certain cell types (8), suggesting that the activity and function of endogenous PDK1 is regulated.
Recent studies suggest that PDK1 may be subject to phosphorylation-mediated negative regulation. Kondo and Kahn (8) showed that insulin-stimulated PDK1 phosphorylation at Ser 244 in the activation loop is greatly reduced in the liver of ob/ob mice. Reduced PDK1 phosphorylation in the activation loop was also found in cells treated with the proinflammatory cytokine tumor necrosis factor ␣ (9). These findings suggest a novel mechanism by which hyperlipidemia induces insulin resistance in vivo. However, how obesity leads to down-regulation of PDK1 activity and function remains unknown. Obesity has been shown to activate several serine kinases such as the c-Jun N-terminal kinase (JNK) (10,11), inhibitor B kinase (12), and PKC (13,14) that act on IRS-1. Thus, the reduced PDK1 activity could be due to reduced tyrosine phosphorylation of IRS-1 and its downstream events. On the other hand, hyperlipidemia-induced negative regulation may target directly at PDK1.
In the present study, we investigated the molecular mechanism underlying FFA-induced insulin resistance. Our data show that PDK1 undergoes palmitate stimulation and PKCdependent phosphorylation at Ser 504 and Ser 532 . Replacing Ser 504/532 with alanine alleviates the inhibitory effect of palmitate on Akt phosphorylation, suggesting that phosphorylation of PDK1 at this site plays a negative role in PDK1 activity and function. The identification of PDK1 as a novel target in FFAinduced inhibition of insulin signaling should provide a better understanding of the molecular mechanism of insulin resistance, which is one of the major causes of type 2 diabetes.
Preparing Palmitate Solution-BSA-bound palmitate was made according to the procedure as described previously with some modifications (13,15). In brief, palmitate was dissolved in ethanol to a concentration of 0.75 M, which was then diluted with 20% FFA-free BSA to a working concentration of 7.5 mM (the molar ratio of palmitate to BSA is ϳ2.5). This stock solution was filtered and stored at Ϫ20°C and was used within 2 weeks. The same concentration of ethanol mixed with 20% of BSA was used as control.
Cell Culture, Immunoprecipitation, and Western Blot-Chinese hamster ovary cells stably expressing the insulin receptor (CHO/IR) cells were grown in Ham's F-12 medium (Invitrogen) supplemented with 10% newborn calf serum and 1% penicillin/ streptomycin. C2C12 cells (ATCC) were cultured in DMEM (ATCC catalog number  supplemented with 10% newborn calf serum and 1% penicillin/streptomycin. Differentiation of C2C12 was performed by incubating cells in fresh medium containing DMEM, 1% penicillin/streptomycin, 0.1% fetal bovine serum, and 50 nM insulin for 4 days. Differentiation was observed when 80 -100% of the cells formed myotubes. Wild-type and PDK1-deficient mouse embryonic fibroblasts (MEFs) (gifts of Dr. Wataru Ogawa, Kobe University, Kobe, Japan) were cultured in DMEM (Invitrogen) supplemented with 10% newborn calf serum and 1% penicillin/streptomycin. The PKC-deficient MEFs were prepared from 13-day embryos of PKC Ϫ/Ϫ knock-out mice (16). The embryo carcasses were minced and digested with trypsin after removal of the limbs, internal organs, and brain. After digestion at 37°C for 10 min, the cell suspension was collected as MEFs and washed with DMEM supplemented with 10% newborn calf serum. The cells were plated in a 100-mm cell culture plate in serum-containing medium, and the medium was changed 24 h later. After one passage, the cells were collected as MEFs. Transfections were performed using Lipofectamine reagent according to the manufacturer's protocol (Invitrogen). For immunoprecipitation, cells were lysed in 300 l of buffer A (50 mM Hepes, pH 7.6, 150 mM NaCl, 1% Triton X-100, 10 mM NaF, 20 mM sodium pyrophosphate, 20 mM ␤-glycerol phosphate, 1 mM sodium orthovanadate, 10 g/ml leupeptin, 10 g/ml aprotinin, and 1 Serum-starved C2C12 myotubes were treated with or without 0.75 mM palmitate for the indicated times and then with or without insulin (10 nM) for 10 min. Phosphorylation of endogenous Akt (A) or PDK1 (B) was determined by Western blot using a phosphospecific antibody to Akt Thr308 (P-ATK T308 ) or PDK1 Ser244 (P-PDK1 S244 ), respectively. Results are representative of three independent experiments with similar findings. mM phenylmethylsulfonyl fluoride). Cell lysates were centrifuged at 14,000 g for 10 min at 4°C, and the supernatants were incubated with specific antibodies bound to protein G beads overnight at 4°C. After incubation, immunoprecipitates were washed extensively with ice-cold buffer B (50 mM Hepes, pH 7.6, 150 mM NaCl, and 0.1% Triton X-100). Proteins bound to the beads were eluted by heating at 95°C for 4 min in SDS-PAGE sample loading buffer. The eluted proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected with specific antibodies.
In Vitro Phosphorylation of PDK1 by PKC-Wild-type PDK1 and its mutants were purified by immunoprecipitation from CHO/IR cells transiently expressing these proteins. Immunoprecipitated PDK1 proteins were incubated in 30 l of buffer C (50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1 mM Na 3 VO 4 , 1 mM sodium pyrophosphate, 1 mM NaF, and 1 mM phenylmethylsulfonyl fluoride) containing purified PKC and 2 Ci of [␥-32 P]ATP. In vitro phosphorylation reaction was carried out for 30 min at 30°C, and in vitro phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography.
In Vivo 32 P Labeling, Phosphoamino Acid Analysis, and Phosphopeptide Mapping of PDK1-The experiments were carried out as described in our previous studies (17)(18)(19).
Statistical Analysis-Quantification of the relative increase in insulin-stimulated protein phosphorylation was performed by analyzing Western blots using the NIH Scion Image software and was normalized with the amount of protein expression in each experiment. Results are expressed as the mean Ϯ S.E. Differences between the groups were examined for statistical significance using analysis of variance.

PDK1 Phosphorylation at Ser 244 in the Activation Loop Is Negatively
Regulated by Free Fatty Acid Treatment-To study the molecular mechanism underlying hyperlipidemia-induced insulin resistance, we investigated PDK1 phosphorylation in C2C12 myotubes treated with or without palmitate, which has been shown to cause insulin resistance in these cells (13). Insulin greatly stimulated Akt phosphorylation at Thr 308 , a site targeted by PDK1 (Fig. 1A). The insulin-stimulated Akt phosphorylation in C2C12 myotubes was greatly inhibited by palmitate treatment in a time-dependent manner (Fig. 1A). Palmitate treatment also markedly inhibited PDK1 phosphorylation at Ser 244 in the activation loop (Fig. 1B), which is consistent with the previous finding that insulin-stimulated PDK1 A, serum-starved C2C12 myotubes were incubated with or without 0.75 mM of palmitate for 18 h and then treated with or without 10 nM insulin or 100 ng/ml of PDGF for 10 min. Activation of Akt or PDK1 was determined by Western blot using a phosphospecific antibody to Akt Thr308 (P-ATK T308 ) or PDK1 Ser244 (P-PDK1 S244 ), respectively. B, the phosphorylation of PDK1 and Akt shown in A was quantified by the NIH Scion Image program. Results are expressed as the mean Ϯ S.E. (n ϭ 4). Differences between the groups were examined for statistical significance using analysis of variance. **, p Ͻ 0.01. C, serumstarved wild-type (WT) and PKC-deficient MEFs were cultured in the presence or absence of 0.75 mM palmitate for 18 h. Cells were then treated with or without 100 nM insulin for 10 min. Phosphorylation of PDK1 at Ser 244 or Akt at Thr 308 was determined by Western blot using a phosphospecific antibody. D, 5-week-old PKC Ϫ/Ϫ mice and wild-type littermates were sacrificed, and fat tissues were homogenized. The phosphorylation and protein levels of PDK1 and/or PKC in tissue homogenates were determined by Western blot with specific antibodies as indicated. E, serum-starved C2C12 myotubes were treated with or without 10 M of PKC pseudosubstrate (PKC PS) for 1 h and then incubated with or without 0.75 mM palmitate for 18 h. The cells were then treated with or without 10 nM insulin for 10 min. Phosphorylation of PDK1 at Ser 244 or Akt at Thr 308 was determined by Western blot using a phosphospecific antibody. The data are representative of at least three experiments with similar results. F, serum-starved C2C12 myotubes were treated with or without 10 M of PKC pseudosubstrate for 1 h and then incubated with or without 0.75 mM palmitate for 18 h. The cells were further treated with or without 200 nM insulin for 30 min. Glucose uptake was determined as described previously (20). phosphorylation at Ser 244 is greatly reduced in the liver of ob/ob mice (8).
Palmitate Inhibits Akt Phosphorylation Independent of IRS1/2-FFAs have been shown to inhibit insulin-stimulated IRS-1 tyrosine phosphorylation, leading to reduced PI 3-kinase signaling (10,12,14). To test whether palmitate could inhibit PI 3-kinase signaling in an IRS-1/2-indpendent manner, we examined the effect of palmitate on PDGF-stimulated phosphorylation of Akt and PDK1. Unlike insulin, PDGF activates PI 3-kinase by promoting the interaction between PDGF receptor and p85 subunit of PI 3-kinase, independent of IRS1/2 (21). Treating C2C12 myotubes with either insulin or PDGF led to a marked increase in Akt phosphorylation at Thr 308 , and this phosphorylation was markedly inhibited by palmitate (Fig. 2, A  and B). These results suggest that palmitate-induced inhibition of Akt could occur at a step(s) other than IRS-1/2 along the PI 3-kinase signaling pathway. Consistent with the idea that PDK1 may be the site of negative regulation, palmitate treatment inhibited PDGF-stimulated PDK1 phosphorylation at Ser 244 (Fig. 2, A and B).
Palmitate-induced PDK1 Phosphorylation Is Mediated by PKC-Activation of PKC has been implicated in obesity-induced insulin resistance (22,23). To determine whether PKC is involved in palmitate-induced PDK1 phosphorylation, we examined the effect of palmitate on insulin-stimulated phosphorylation of PDK1 at Ser 244 in wild-type and PKC Ϫ/Ϫ MEFs. Insulin stimulated PDK1 phosphorylation at Ser 244 and Akt phosphorylation at Thr 308 in wild-type MEFs, and these phosphorylation events were markedly inhibited by palmitate treatment (Fig.  2C). The basal and insulin-stimulated PDK1 Ser 244 phosphorylation was greatly increased in the PKC-deficient MEFs, suggesting a negative role of PKC in PDK1 activity. Insulin-stimulated Akt phosphorylation at Thr 308 was also greatly increased in the PKCdeficient MEFs, and knocking out PKC disrupted the inhibitory effect of palmitate on insulin-stimulated PDK1 and Akt phosphorylation in the activation loop (Fig. 2C). Consistent with a negative role of PKC in PDK1 activation, PDK1 phosphorylation at Ser 244 in the activation loop is greatly increased in the fat of PKC knockout mice (Fig. 2D). To further explore the role of PKC in PDK1/ Akt signaling, we examined whether inhibiting PKC relieves the negative effect of palmitate on insulin-stimulated Akt and PDK1 phosphorylation. We found that treating cells with the PKC pseudosubstrates greatly diminished the inhibitory effect of palmitate on insulin-stimulated PDK1 and Akt phosphorylation in the activation loop (Fig. 2E). In addition, treating C2C12 myotubes with the PKC pseudosubstrates partially relieved the inhibitory effect of palmitate on insulin-stimulated glucose uptake (Fig. 2F). Taken together, these results provide strong evidence on the involvement of PKC in palmitate-induced negative regulation of PDK1/Akt signaling. The findings that inhibition of PKC by the pseudosubstrates only partially restored insulin-stimulated glucose uptake is consistent with a very recent finding that FFA-induced insulin resistance could result from defects downstream of IRS and even downstream of Akt (24).
To determine whether PKC negatively regulates PDK1/Akt by directly phosphorylating PDK1, we generated a double mutant of PDK1 in which the critical ATP binding site (Lys 114 ) and the autophosphorylation site (Ser 244 ) were mutated to glycine and alanine, respectively (PDK1 K114G/S244A ). PDK1 K114G/S244A is not autophosphorylated in cells, which facilitates our study of heterogeneous kinase-mediated phosphorylation of PDK1. C2C12 myoblasts transiently expressing PDK1 K114G/S244A were in vivo labeled with 32 P and treated with palmitate in the presence of several protein kinase inhibitors. As shown in Fig. 3A, treating cells with palmitate greatly increased PDK1 K114G/S244A phosphorylation. The palmitate-induced PDK1 K114G/S244A phosphorylation was suppressed by the PKC pseudosubstrate or the broad spectrum PKC inhibitor GÖ 6983, but not by the PKC⑀ pseudosubstrate or the JNK inhibitor SP600125. To determine whether PKC is able to directly phosphorylate PDK1, we carried out an in vitro kinase assay using purified PDK1 mutants and PKC. We found that both PDK1 K114G and PDK1 K114G/S244A mutants were directly phosphorylated by PKC (Fig. 3B). These results reveal that PDK1 is a direct target of PKC and that the phosphorylation occurred at a site independent of Ser 244 in PDK1.
Identification of Ser 504 and Ser 532 as the PKC-stimulated Phosphorylation Site in PDK1-To map the palmitate-stimulated phosphorylation, we generated plasmids encoding fulllength and truncation mutants of PDK1 fused to YFP (Fig. 4A). In vivo labeling experiments revealed that deletion of the C terminus of PDK1 (amino acids 402-559) greatly reduced palmitate-stimulated PDK1 phosphorylation (Fig. 4B), suggesting that the phosphorylation site(s) is/are localized at or near the C terminus. To determine whether PKC directly phosphorylates PDK1 at the C terminus, we performed in vitro kinase assays using autophosphorylation-defective (S244A) full-length and truncation mutants of PDK1. We found that deletion of the C terminus of PDK1 greatly reduced PKC-catalyzed PDK1 phosphorylation in vitro (Fig. 4C), confirming that the PKC-mediated phosphorylation sites are located at the C terminus.
To determine the nature of palmitate-stimulated PDK1 phosphorylation, we analyzed in vivo labeled PDK1 S244A by phosphoamino acids analysis. We found that palmitatestimulated PDK1 S244A phosphorylation occurred almost exclusively at serine residues (Fig. 4D). There are three serine residues at the C terminus of mouse PDK1, including Ser 504 , Ser 532 , and Ser 552 (17). To map the site(s) of phosphorylation, we generated PDK1 K114G/S244A/S504A , PDK1 K114G/S244A/S532A , and PDK1 K114G/S244A/S552A triple mutants. In vivo labeling experiments showed that replacing Ser 552 with alanine had no significant effect on palmitate-stimulated PDK1 phosphorylation (data not shown). However, replacing Ser 504 or Ser 532 with alanine greatly diminished palmitateinduced PDK1 phosphorylation (Fig.  4E). In vitro kinase assays showed that PKC efficiently phosphorylated PDK1 K114G/S244A and that the phosphorylation was greatly reduced when Ser 504 or Ser 532 was mutated to alanine (Fig. 4F), further confirming that Ser 504 and Ser 532 are the major PKC-mediated phosphorylation sites on PDK1.
Phosphorylation at Ser 532 /Ser 504 Negatively Regulates the Ability of PDK1 to Phosphorylate Akt-To investigate the functional role of PDK1 phosphorylation, we replace Ser 504 and Ser 532 of PDK1 with negatively charged amino acid residues (PDK1 S504E/S532D ) to mimic phosphorylation. We also mutated Ser 504 and Ser 532 of PDK1 to alanine (PDK1 S504A/S532A ) to disrupt the potential phosphorylation sites. To avoid the potential masking effect of endogenous PDK1 on PDK1 S504E/S532D and PDK1 S504A/S532A , wild type or mutants of PDK1 were transiently expressed in PDK1-deficient MEFs that are defective in insulin-stimulated Akt phosphorylation (25). Cells expressing wild type or mutants of PDK1 were serumstarved and treated with palmitate followed by insulin. As shown in Fig.  5, A and B, overexpression of wildtype PDK1 rescued insulin-stimulated Akt phosphorylation in the PDK1 Ϫ/Ϫ MEFs, and the stimulatory effect of insulin was inhibited by palmitate treatment. The stimulatory effect of insulin was notably increased in cells expressing PDK1 S504A/S532A , probably due to removal of a feedback inhibition caused by Ser 504/S532 phosphorylation. However, the insulin-stimulated Akt phosphorylation was still partially suppressed by palmitate, suggesting that in addition to negative regulation of PDK1, palmitate could induce insulin resistance by targeting other step(s) along the PI 3-kinase signaling pathway. The stimulatory effect of insulin on Akt phosphorylation was reduced in PDK1 Ϫ/Ϫ MEFs expressing PDK1 S504E/S532D when compared with cells expressing wild type or PDK1 S504A/S532A , suggesting that the negatively charged amino acid residues mimics palmitate-induced phosphorylation to negatively regulate PDK1 function.

DISCUSSION
The role of obesity in insulin resistance has been well recognized, but the underlying molecular mechanisms remain to be fully elucidated. Obesity has been shown to activate several serine kinases such as the JNK (10, 11), inhibitor B kinase (12), and PKC (13,14) that negatively regulates insulin signaling by serine phosphorylation of IRS-1/2. However, it is unclear whether hyperlipidemia-induced insulin resistance could occur at other sites along the insulin-signaling pathway.
In the present study, we show that palmitate inhibited both insulin-stimulated and PDGF-stimulated PDK1 and Akt phosphorylation in the activation loop ( Fig. 2A). Because PDGF activates Akt independent of IRS-1/2 (21), these results suggest that FFA-activated protein kinases could target components in the PI 3-kinase signaling pathway other than IRS-1/2. In agreement with this view, we found that PDK1 undergoes palmitatestimulated and PKC-dependent phosphorylation at Ser 504 and Ser 532 in C2C12 cells, and replacing these residues with alanine improves PDK1 kinase activity toward Akt in intact cells (Fig.  5). These results provide evidence for the first time that FFA could induce insulin resistance by targeting multiple components downstream of the insulin receptor. Our result is consistent with previous findings that insulin-stimulated PDK1 phosphorylation at Ser 244 is greatly reduced in the liver of ob/ob mice (8) and in cells treated with tumor necrosis factor ␣ (9). Thus, serine phosphorylation of PDK1 at Ser 504 and Ser 532 may provide a mechanism underlying obesity-induced insulin resistance in vivo.
Our results showed that treating cells with inhibitors of PKC⑀ or JNK, which are two kinases that have been implicated in obesity-induced insulin resistance (11,26), had only a minor effect on palmitate-induced PDK1 phosphorylation. On the other hand, suppressing the expression levels or activity of PKC greatly reduced palmitate-stimulated PDK1 phosphorylation (Fig. 3A). These results indicate that PKC is the major kinase that mediates palmitate-induced PDK1 phosphorylation. Our findings are consistent with numerous recent studies showing that PKC plays a critical role in hyperlipidemia-induced insulin resistance both in insulin-sensitive cells (13, 14, 26 -30) and in the skeletal muscle of diabetic humans and animals (26,(31)(32)(33). However, a recent study showed that PKCknock-out mice gained more body weight and suffered more severe insulin resistance on high fat diet than wild-type littermates (16). This result suggests that under normal physiological conditions, basal PKC activity is important for regulation of energy homeostasis.
A question that remains to be established is how PKC-mediated phosphorylation at Ser 504/532 negatively regulates PDK1 function. We previously found that PDK1 activity is induced by dimerization and trans-phosphorylation (19). Thus, serine phosphorylation of PDK1 at Ser 504/532 may reduce PDK1 activity by inhibiting its dimerization and trans-phosphorylation. However, we found that palmitate treatment had little effect on PDK1 dimerization (data not shown). Another possibility is that palmitate-stimulated PDK1 Ser 504/532 phosphorylation may induce a conformational change that inhibits PDK1 autophosphorylation in the activation loop. Serine phosphorylation at Ser 504/532 may also affect the ability of PDK1 to activate Akt by inhibiting its membrane translocation, which has been shown to be important for PDK1 activity toward Akt in intact cells (4). Further studies will be needed to test these possibilities.
Recent studies suggest that hyperlipidemia and overproduction of proinflammatory cytokines contribute significantly to obesity-induced insulin resistance (34,35). In the present study, we show that palmitate-induced serine phosphorylation of PDK1 inhibits insulin signaling. This new finding provides a novel mechanism by which obesity leads to insulin resistance. Characterization of the mechanism of FFA-induced PDK1 phosphorylation and its physiological consequences should FIGURE 5. The effects of mutating PDK1 Ser 504 and Ser 532 on the activity or function of PDK1. A, PDK1 Ϫ/Ϫ MEFs transiently expressing PDK1, PDK1 S504A/S532A , and PDK1 S504E/S532D were serum-starved, treated with or without 0.75 mM palmitate (PA) for 18 h, and then with or without 100 nM insulin for 10 min. Phosphorylation of PDK1 or Akt was determined by Western blot using a phosphospecific antibody to PDK1 Ser244 (P-PDK1 S244 ) or Akt Thr308 (P-AKT t308 ), respectively. The data are representative of at least three independent experiments with similar results. The phosphorylation of PDK1 (B) and Akt (C) shown in A was quantified by the NIH Scion Image program. Results are expressed as the mean Ϯ S.E. (n ϭ 3). Differences between the groups were examined for statistical significance using analysis of variance. *, p Ͻ 0.05; **, p Ͻ 0.01. WT, wild type. provide important information on our understanding of the pathophysiology of insulin resistance and type 2 diabetes.