Tumor necrosis factor alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner

Insulin stimulation produced a reliable 3-fold increase in glucose uptake in primary neonatal rat myotubes, which was accompanied by a similar effect on GLUT4 translocation to plasma membrane. Tumor necrosis factor (TNF)-alpha caused insulin resistance on glucose uptake and GLUT4 translocation by impairing insulin stimulation of insulin receptor (IR) and IR substrate (IRS)-1 and IRS-2 tyrosine phosphorylation, IRS-associated phosphatidylinositol 3-kinase activation, and Akt phosphorylation. Because this cytokine produced sustained activation of stress and proinflammatory kinases, we have explored the hypothesis that insulin resistance by TNF-alpha could be mediated by these pathways. In this study we demonstrate that pretreatment with PD169316 or SB203580, inhibitors of p38 MAPK, restored insulin signaling and normalized insulin-induced glucose uptake in the presence of TNF-alpha. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-alpha was still produced. Moreover, TNF-alpha produced inhibitor kappaB kinase (IKK)-beta activation and inhibitor kappaB-beta and -alpha degradation in a p38 MAPK-dependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-alpha produced serine phosphorylation of IR and IRS-1 (total and on Ser(307) residue), and these effects were completely precluded by pretreatment with either PD169316 or salicylate. Consequently, TNF-alpha, through activation of p38 MAPK and IKK, produces serine phosphorylation of IR and IRS-1, impairing its tyrosine phosphorylation by insulin and the corresponding activation of phosphatidylinositol 3-kinase and Akt, leading to insulin resistance on glucose uptake and GLUT4 translocation.     

Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts

We have recently shown that insulin induced myogenesis in the mouse C2C12 skeletal muscle cell line by activation of phosphatidylinositol (PI) 3-kinase/p70S6-kinase and p38-mitogen-activated protein kinase (MAPK) and downregulation of p42/p44-MAPK. This study investigated the insulin-signaling pathways involved in mitogenesis, survival, and membrane ruffling in C2C12 myoblasts, a cellular system that besides IGF-I receptors, expressed a high number of functional insulin receptors. Insulin (10 nM) rapidly stimulated beta-chain insulin receptor and IRS-1 tyrosine phosphorylation, IRS-2 being poorly and SHC not phosphorylated at all. However, an association of SHC with IRS-1 was found under insulin stimulation. Insulin stimulated IRS-1 association with p85alpha leading to the activation of PI3-kinase, and, subsequently AKT and p70S6-kinases. Moreover, both p42/p44- and p38-MAPKs resulted in phosphorylation after insulin stimulation. Insulin treatment for 24 h produced mitogenesis, as demonstrated by the increase in ((3)H)-thymidine incorporation, DNA content, the expression of PCNA and cyclin D1 proteins, and the proportion of cells in S + G2/M phases of the cell cycle. This mitogenic effect of insulin was precluded by inhibition of p70S6-kinase (either by rapamycin or by the PI3-kinase inhibitor LY294002) as well as by inhibition of p44/p42-MAPK with PD098059, but was not affected by inhibition of p38-MAPK. Serum deprivation of C2C12 myoblasts resulted in growth arrest at the GO/G1 phases of the cell cycle and apoptosis, as detected either by DNA laddering or by increase in the percentage of hypodiploid cells. Insulin rescued serum-deprived cells from apoptosis in an AKT-dependent manner, as demonstrated by the inhibition of AKT-activity by the use of LY294002 and ML-9, meanwhile neither inhibition of p70S6-kinase, nor MAPK affected insulin-induced survival. Finally, we evaluated the capacity of insulin to modulate actin cytoskeleton rearrangement. Insulin stimulation of myoblasts produced membrane ruffling and decreased actin stress fibers; this biological response being dependent of p38-MAPK, as demonstrated by the use of the p38-MAPK inhibitors SB203580 or PD169316, but independent of PI3-kinase and p42/p44-MAPK.     

Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle

Recent studies have demonstrated that fatty acids induce insulin resistance in skeletal muscle by blocking insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase). To examine the mechanism by which fatty acids mediate this effect, rats were infused with either a lipid emulsion (consisting mostly of 18:2 fatty acids) or glycerol. Intracellular C18:2 CoA increased in a time-dependent fashion, reaching an approximately 6-fold elevation by 5 h, whereas there was no change in the concentration of any other fatty acyl-CoAs. Diacylglycerol (DAG) also increased transiently after 3-4 h of lipid infusion. In contrast there was no increase in intracellular ceramide or triglyceride concentrations during the lipid infusion. Increases in intracellular C18:2 CoA and DAG concentration were associated with protein kinase C (PKC)-theta activation and a reduction in both insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1 associated PI3-kinase activity, which were associated with an increase in IRS-1 Ser(307) phosphorylation. These data support the hypothesis that an increase in plasma fatty acid concentration results in an increase in intracellular fatty acyl-CoA and DAG concentrations, which results in activation of PKC-theta leading to increased IRS-1 Ser(307) phosphorylation. This in turn leads to decreased IRS-1 tyrosine phosphorylation and decreased activation of IRS-1-associated PI3-kinase activity resulting in decreased insulin-stimulated glucose transport activity.     

Caffeine modulates phosphorylation of insulin receptor substrate-1 and impairs insulin signal transduction in rat skeletal muscle

Caffeine decreases insulin sensitivity and insulin-stimulated glucose transport in skeletal muscle; however, the precise mechanism responsible for this deleterious effect is not understood fully. We investigated the effects of incubation with caffeine on insulin signaling in rat epitrochlearis muscle. Caffeine (≥1 mM, ≥15 min) suppressed insulin-stimulated insulin receptor substrate (IRS)-1 Tyr(612) phosphorylation in a dose- and time-dependent manner. These responses were associated with inhibition of the insulin-stimulated phosphorylation of phosphatidylinositol 3-kinase (PI3K) Tyr(458), Akt Ser(473), and glycogen synthase kinase-3β Ser(9) and with inhibition of insulin-stimulated 3-O-methyl-d-glucose (3MG) transport but not with inhibition of the phosphorylation of insulin receptor-β Tyr(1158/62/63). Furthermore, caffeine enhanced phosphorylation of IRS-1 Ser(307) and an IRS-1 Ser(307) kinase, inhibitor-κB kinase (IKK)-α/β Ser(176/180). Blockade of IKK/IRS-1 Ser(307) by caffeic acid ameliorated the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation and 3MG transport. Caffeine also increased the phosphorylation of IRS-1 Ser(789) and an IRS-1 Ser(789) kinase, 5'-AMP-activated protein kinase (AMPK). However, inhibition of IRS-1 Ser(789) and AMPK phosphorylation by dantrolene did not rescue the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation or 3MG transport. In addition, caffeine suppressed the phosphorylation of insulin-stimulated IRS-1 Ser(636/639) and upstream kinases, including the mammalian target of rapamycin and p70S6 kinase. Intravenous injection of caffeine at a physiological dose (5 mg/kg) in rats inhibited the phosphorylation of insulin-stimulated IRS-1 Tyr(612) and Akt Ser(473) in epitrochlearis muscle. Our results indicate that caffeine inhibits insulin signaling partly through the IKK/IRS-1 Ser(307) pathway, via a Ca(2+)- and AMPK-independent mechanism in skeletal muscle.     

Three mitogen-activated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes

TNFalpha, which activates three different MAPKs [ERK, p38, and jun amino terminal kinase (JNK)], also induces insulin resistance. To better understand the respective roles of these three MAPK pathways in insulin signaling and their contribution to insulin resistance, constitutively active MAPK/ERK kinase (MEK)1, MAPK kinase (MKK6), and MKK7 mutants were overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated transfection procedure. The MEK1 mutant, which activates ERK, markedly down-regulated expression of the insulin receptor (IR) and its major substrates, IRS-1 and IRS-2, mRNA and protein, and in turn reduced tyrosine phosphorylation of IR as well as IRS-1 and IRS-2 and their associated phosphatidyl inositol 3-kinase (PI3K) activity. The MKK6 mutant, which activates p38, moderately inhibited IRS-1 and IRS-2 expressions and IRS-1-associated PI3K activity without exerting a significant effect on the IR. Finally, the MKK7 mutant, which activates JNK, reduced tyrosine phosphorylation of IRS-1 and IRS-2 and IRS-associated PI3K activity without affecting expression of the IR, IRS-1, or IRS-2. In the context of our earlier report showing down-regulation of glucose transporter 4 by MEK1-ERK and MKK6/3-p38, the present findings suggest that chronic activation of ERK, p38, or JNK can induce insulin resistance by affecting glucose transporter expression and insulin signaling, though via distinctly different mechanisms. The contribution of ERK is, however, the strongest.     

Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3-L1 adipocytes

In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1-associated phosphoinositide 3 (PI 3)-kinase activity in response to physiological insulin concentrations. Insulin-induced membrane ruffling, which is dependent on PI 3-kinase activation, was also markedly reduced. These inhibitory effects were associated with an increase in IRS-1 Ser307 phosphorylation. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser307. The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation. In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces the phosphorylation of IRS-1 on Ser307 by an mTOR-dependent pathway. This, in turn, leads to a decrease in early proximal signaling events induced by physiological insulin concentrations. On the other hand, prolonged osmotic stress alters IRS-1 function by inducing its degradation, which could contribute to the down-regulation of insulin action.     

Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) inhibits insulin signal transduction in a variety of cell backgrounds, which might contribute to peripheral insulin resistance. However, because of the large number of potential phosphorylation sites, the mechanism of inhibition has been difficult to determine. One serine residue located near the phosphotyrosine-binding (PTB) domain in IRS-1 (Ser(307) in rat IRS-1 or Ser(312) in human IRS-1) is phosphorylated via several mechanisms, including insulin-stimulated kinases or stress-activated kinases like JNK1. During a yeast tri-hybrid assay, phosphorylation of Ser(307) by JNK1 disrupted the interaction between the catalytic domain of the insulin receptor and the PTB domain of IRS-1. In 32D myeloid progenitor cells, phosphorylation of Ser(307) inhibited insulin stimulation of the phosphatidylinositol 3-kinase and MAPK cascades. These results suggest that inhibition of PTB domain function in IRS-1 by phosphorylation of Ser(307) (Ser(312) in human IRS-1) might be a general mechanism to regulate insulin signaling.     

Enhanced insulin action on glucose transport and insulin signaling in 7-day unweighted rat soleus muscle.

Hindlimb suspension (HS), a model of simulated weightlessness, enhances insulin action on glucose transport in unweighted rat soleus muscle. In the present study, we tested the hypothesis that these changes in glucose transport in 3- and 7-day HS soleus of juvenile, female Sprague-Dawley rats were due to increased functionality of insulin signaling factors, including insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3-kinase), and Akt. Insulin-stimulated (2 mU/ml) glucose transport was significantly (P < 0.05) enhanced in 3- and 7-day HS soleus by 59 and 113%, respectively, compared with weight-bearing controls. Insulin-stimulated tyrosine phosphorylation of IR and Ser(473) phosphorylation of Akt was not altered by unweighting. Despite decreased (34 and 64%) IRS-1 protein in 3- and 7-day HS soleus, absolute insulin-stimulated tyrosine phosphorylation of IRS-1 was not diminished, indicating relative increases in IRS-1 phosphorylation of 62 and 184%, respectively. In the 7-day HS soleus, this was accompanied by increased (47%) insulin-stimulated IRS-1 associated with the p85 subunit of PI3-kinase. Interestingly, the enhanced insulin-stimulated glucose transport in the unweighted soleus was not completely inhibited (89-92%) by wortmannin, a PI3-kinase inhibitor. Finally, protein expression and activation of p38 MAPK, a stress-activated serine/threonine kinase associated with insulin resistance, was decreased by 32 and 18% in 7-day HS soleus. These results indicate that the increased insulin action on glucose transport in the 7-day unweighted soleus is associated with increased insulin signaling through IRS-1 and PI3-kinase and decreased p38 MAPK protein expression. However, PI3-kinase-independent mechanisms must also play a small role in this adaptive response to HS.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Calorie restriction prevents the development of insulin resistance and impaired insulin signaling in skeletal muscle of ovariectomized rats

Insulin resistance of skeletal muscle glucose transport due to prolonged loss of ovarian function in ovariectomized (OVX) rats is accompanied by other features of the metabolic syndrome and may be confounded by increased calorie consumption. In this study, we investigated the role of calorie consumption in the development of insulin resistance in OVX rats. In addition, we examined the cellular mechanisms underlying skeletal muscle insulin resistance in OVX rats. Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (SHAM). OVX rats either had free access to food, pair feeding (PF) with SHAM or received a 35% reduction in food intake (calorie restriction; CR) for 12weeks. Compared with SHAM, ovariectomy induced skeletal muscle insulin resistance, which was associated with decreases (32-70%) in tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), IRS-1 associated p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), and Akt Ser(473) phosphorylation whereas insulin-stimulated phosphorylation of IRS-1 Ser(307), SAPK/JNK Thr(183)/Tyr(185), and p38 mitogen-activated protein kinase (MAPK) Thr(180)/Tyr(182) was increased (24-62%). PF improved the serum lipid profile but did not restore insulin-stimulated glucose transport, indicating that insulin resistance in OVX rats is a consequence of ovarian hormone deprivation. In contrast, impaired insulin sensitivity and defective insulin signaling were not observed in the skeletal muscle of OVX+CR rats. Therefore, we provide evidence for the first time that CR effectively prevents the development of insulin resistance and impaired insulin signaling in the skeletal muscle of OVX rats.     

Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in tumor necrosis factor-treated cells through targeting multiple serine kinases

The hypoglycemic effects of high dose salicylates in the treatment of diabetes were documented before the advent of insulin. However, the molecular mechanisms by which salicylates exert these anti-diabetic effects are not well understood. In this study, we analyzed the effects of aspirin (acetylsalicylic acid) on serine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells treated with tumor necrosis factor (TNF)-alpha. Phosphorylation of IRS-1 at Ser307, Ser267, and Ser612 was monitored by immunoblotting with phospho-specific IRS-1 antibodies. In 3T3-L1 and Hep G2 cells, phosphorylation of IRS-1 at Ser307 in response to TNF-alpha treatment correlated with phosphorylation of JNK, c-Jun, and degradation of IkappaBalpha. Moreover, phosphorylation of IRS-1 at Ser307 in embryo fibroblasts derived from either JNK or IKK knockout mice was reduced when compared with that in the wild-type controls. Taken together, these data suggest that serine phosphorylation of IRS-1 in response to TNF-alpha is mediated, in part, by JNK and IKK. Interestingly, aspirin treatment inhibited the phosphorylation of IRS-1 at Ser307 as well as the phosphorylation of JNK, c-Jun, and degradation of IkappaBalpha. Furthermore, other serine kinases including Akt, extracellular regulated kinase, mammalian target of rapamycin, and PKCzeta were also activated by TNF-alpha (as assessed by phospho-specific antibodies). Phosphorylation of IRS-1 at Ser267 and Ser612 correlated with the activation of these kinases. Phosphorylation of Akt and the mammalian target of rapamycin (but not extracellular regulated kinase or PKCzeta) in response to TNF-alpha was inhibited by aspirin treatment. Finally, aspirin rescued insulin-induced glucose uptake in 3T3-L1 adipocytes pretreated with TNF-alpha. We conclude that aspirin may enhance insulin sensitivity by protecting IRS proteins from serine phosphorylation catalyzed by multiple kinases.     

A role for AMPK in the inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids

Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as β-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser³⁰⁷ phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.     

Insulin receptor substrate-2-dependent interleukin-4 signaling in macrophages is impaired in two models of type 2 diabetes mellitus

We have shown previously that hyperinsulinemia inhibits interferon-alpha-dependent activation of phosphatidylinositol 3-kinase (PI3-kinase) through mammalian target of rapamycin (mTOR)-induced serine phosphorylation of insulin receptor substrate (IRS)-1. Here we report that chronic insulin and high glucose synergistically inhibit interleukin (IL)-4-dependent activation of PI3-kinase in macrophages via the mTOR pathway. Resident peritoneal macrophages (PerMPhis) from diabetic (db/db) mice showed a 44% reduction in IRS-2-associated PI3-kinase activity stimulated by IL-4 compared with PerMPhis from heterozygote (db/+) control mice. IRS-2 from db/db mouse PerMPhis also showed a 78% increase in Ser/Thr-Pro motif phosphorylation without a difference in IRS-2 mass. To investigate the mechanism of this PI3-kinase inhibition, 12-O-tetradecanoylphorbol-13-acetate-matured U937 cells were treated chronically with insulin (1 nm, 18 h) and high glucose (4.5 g/liter, 48 h). In these cells, IL-4-stimulated IRS-2-associated PI3-kinase activity was reduced by 37.5%. Importantly, chronic insulin or high glucose alone did not impact IL-4-activated IRS-2-associated PI3-kinase. Chronic insulin + high glucose did reduce IL-4-dependent IRS-2 tyrosine phosphorylation and p85 association by 54 and 37%, respectively, but did not effect IL-4-activated JAK/STAT signaling. When IRS-2 Ser/Thr-Pro motif phosphorylation was examined, chronic insulin + high glucose resulted in a 92% increase in IRS-2 Ser/Thr-Pro motif phosphorylation without a change in IRS-2 mass. Pretreatment of matured U937 cells with rapamycin blocked chronic insulin + high glucose-dependent IRS-2 Ser/Thr-Pro motif phosphorylation and restored IL-4-dependent IRS-2-associated PI3-kinase activity. Taken together these results indicate that IRS-2-dependent IL-4 signaling in macrophages is impaired in models of type 2 diabetes mellitus through a mechanism that relies on insulin/glucose-dependent Ser/Thr-Pro motif serine phosphorylation mediated by the mTOR pathway.     

Insulin-induced stimulation of JNK and the PI 3-kinase/mTOR pathway leads to phosphorylation of serine 318 of IRS-1 in C2C12 myotubes

Increased serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is associated with cellular insulin resistance. We have recently identified serine 318 (Ser318) as a novel protein kinase C-zeta (PKC-zeta)-dependent phosphorylation site within IRS-1. As other kinases may phosphorylate at this serine residue as well, we aimed to identify such kinases in the present study. In C2C12 myotubes, exposure to insulin or phorbol ester markedly increased Ser318 phosphorylation. In contrast, high glucose, tumor necrosis factor-alpha, and free fatty acids did not provoke Ser318 phosphorylation. JNK and the PI 3-kinase/mTOR pathway were found to be implicated in insulin-induced Ser318 phosphorylation, but not in TPA-stimulated phosphorylation that was, at least partly, mediated by classical or novel PKC. In conclusion, with JNK and the PI 3-kinase/mTOR pathway as mediators of insulin-induced Ser318 phosphorylation, we have identified kinases that have previously been reported to play key roles in phosphorylation of other serine residues in IRS-1.     

RhoA/Rho kinase blocks muscle differentiation via serine phosphorylation of insulin receptor substrate-1 and -2

Although the RhoA/Rho kinase (RhoA/ROK) pathway has been extensively investigated, its roles and downstream signaling pathways are still not well understood in myogenic processes. Therefore, we examined the effects of RhoA/ROK on myogenic processes and their signaling molecules using H9c2 and C2C12 cells. Increases in RhoA/ROK activities and serine phosphorylation levels of insulin receptor substrate (IRS)-1 (Ser307 and Ser636/639) and IRS-2 were found in proliferating myoblasts, whereas IRS-1/2 tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity increased during the differentiation process. ROK strongly bound to IRS-1/2 in proliferation medium but dissociated from them in differentiation medium (DM). ROK inactivation by a ROK inhibitor, Y27632, or a dominant-negative ROK, decreased IRS-1/2 serine phosphorylation with increases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity, which led to muscle differentiation even in proliferation medium. Inhibition of ROK also enhanced differentiation in DM. ROK activation by a constitutive active ROK blocked muscle differentiation with the increased IRS-1/2 serine phosphorylation, followed by decreases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity in DM. Interestingly, fibroblast growth factor-2 added to DM also blocked muscle differentiation through RhoA/ROK activation. Fibroblast growth factor-2 blockage of muscle differentiation was reversed by Y27632. Collectively, these results suggest that the RhoA/ROK pathway blocks muscle differentiation by phosphorylating IRS proteins at serine residues, resulting in the decreased IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity. The absence of the inhibitory effects of RhoA/ROK in DM due to low concentrations of myogenic inhibitory growth factors seems to allow IRS-1/2 tyrosine phosphorylation, which stimulates muscle differentiation via transducing normal myogenic signaling.     

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.     

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.