Insulin signaling in vascular endothelial cells: a key role for heterotrimeric G proteins revealed by siRNA-mediated Gbeta1 knockdown

Activation of insulin receptors stimulates the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in vascular endothelial cells. Heterotrimeric G proteins appear to modulate some of the cellular responses that are initiated by receptor tyrosine kinases, but the roles of specific G protein subunits in signaling are less clearly defined. We found that insulin treatment of cultured bovine aortic endothelial cells (BAEC) activates the alpha isoform of PI3-K (PI3-Kalpha) and discovered that purified G protein Gbeta1gamma2 inhibits PI3-Kalpha enzyme activity. Transfection of BAEC with a duplex siRNA targeting bovine Gbeta1 leads to a 90% knockdown in Gbeta1 protein levels, with no effect on expression of other G protein subunits. siRNA-mediated Gbeta1 knockdown markedly and specifically potentiates insulin-dependent activation of kinase Akt, likely reflecting the removal of the inhibitory effect of Gbetagamma on PI3-Kalpha activity. Insulin-induced tyrosine phosphorylation of insulin receptors is unaffected by Gbeta1 siRNA. By contrast, Gbeta1 knockdown leads to a significant decrease in the level of serine phosphorylation of the insulin receptor substrate IRS-1. We explored the effects of siRNA on several serine/threonine protein kinases that have been implicated in insulin signaling. Gbeta1 siRNA significantly attenuates phosphorylation of the 70 kDa ribosomal protein S6 kinase (p70S6K) in the basal state and following insulin treatment. We also found that IGF-1-initiated activation of Akt is significantly enhanced after siRNA-mediated Gbeta1 knockdown, while IGF-1-induced p70S6K activation is markedly suppressed following transfection of Gbeta1 siRNA. We propose that Gbeta1 participates in the activation of p70S6K, which in turn promotes the serine phosphorylation and inhibition of IRS-1. Taken together, these studies suggest that Gbeta1 plays an important role in insulin and IGF-1 signaling in endothelial cells, both by inhibiting the activity of PI3-Kalpha and by stimulating pathways that lead to activation of protein kinase p70S6K and to the serine phosphorylation of IRS-1.     

Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin.

The ability of insulin to stimulate protein synthesis and cellular growth is mediated through the insulin receptor (IR), which phosphorylates Tyr residues in the insulin receptor substrate-signaling proteins (IRS-1 and IRS-2), Gab-1, and Shc. These phosphorylated substrates directly bind and activate enzymes such as phosphatidylinositol 3'-kinase (PI3K) and the guanine nucleotide exchange factor for p21Ras (GRB-2/SOS), which are in turn required for insulin-stimulated protein synthesis, cell cycle progression, and prevention of apoptosis. We have now shown that one or more members of the atypical protein kinase C group, as exemplified by the zeta isoform (PKC zeta), are downstream of IRS-1 and P13K and mediate the effect of insulin on general protein synthesis. Ectopic expression of constitutively activated PKC zeta eliminates the requirement of IRS-1 for general protein synthesis but not for insulin-stimulated activation of 70-kDa S6 kinase (p70S6K), synthesis of growth-regulated proteins (e.g., c-Myc), or mitogenesis. The fact that PKC zeta stimulates general protein synthesis but not activation of p70S6K indicates that PKC zeta activation does not involve the proto-oncogene Akt, which is also activated by PI3K. Yet insulin is still required for the stimulation of general protein synthesis in the presence of constitutively active PKC zeta and in the absence of IRS-1, suggesting a requirement for the convergence of the IRS-1/PI3K/PKC zeta pathway with one or more additional pathways emanating from the IR, e.g., Shc/SOS/p21Ras/mitogen-activated protein kinase. Thus, PI3K appears to represent a bifurcation in the insulin signaling pathway, one branch leading through PKC zeta to general protein synthesis and one, through Akt and the target of rapamycin (mTOR), to growth-regulated protein synthesis and cell cycle progression.     

Effect of TRB3 on insulin and nutrient-stimulated hepatic p70 S6 kinase activity

Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway.     

Insulin promotes rat retinal neuronal cell survival in a p70S6K-dependent manner

The purpose of this study was to examine the role of the ribosomal protein S6 protein kinase (p70S6K), a protein synthesis regulator, in promoting retinal neuronal cell survival. Differentiated R28 rat retinal neuronal cells were used as an experimental model. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, and during the period of experimentation were exposed either to the absence or presence of 10 nm insulin. Insulin treatment induced p70S6K, mTOR, and Akt phosphorylation, effects that were completely prevented by the PI3K inhibitor, LY294002. Insulin-induced phosphorylation of p70S6K and mTOR was prevented by the mTOR inhibitor, rapamycin. Apoptosis, induced by serum deprivation and evaluated by Hoechst staining, was inhibited by insulin treatment in R28 cells, but not in L6 muscle cells. This effect of insulin was also largely prevented by rapamycin. Inhibition of p70S6K activity by exogenous expression of a dominant negative mutant of p70S6K prevented insulin-induced cell survival, whereas, overexpression of wild type p70S6K or expression of a rapamycin resistant form of the kinase enhanced the effect of insulin on survival. Enhanced cell survival under the latter condition was accompanied by increased p70S6K activity and phosphorylation. Rapamycin did not inhibit insulin induced p70S6K phosphorylation and activity in cells transfected with the rapamycin-resistant mutant. Together, these results suggest that p70S6K plays a key role in insulin stimulated retinal neuronal cell survival.     

Liver-specific p70 S6 kinase depletion protects against hepatic steatosis and systemic insulin resistance

Obesity-associated hepatic steatosis is a manifestation of selective insulin resistance whereby lipogenesis remains sensitive to insulin but the ability of insulin to suppress glucose production is impaired. We created a mouse model of liver-specific knockdown of p70 S6 kinase (S6K) (L-S6K-KD) by systemic delivery of an adeno-associated virus carrying a shRNA for S6K and examined the effects on steatosis and insulin resistance. High fat diet (HFD) fed L-S6K-KD mice showed improved glucose tolerance and systemic insulin sensitivity compared with controls, with no changes in food intake or body weight. The induction of lipogenic gene expression was attenuated in the L-S6K-KD mice with decreased sterol regulatory element-binding protein (SREBP)-1c expression and mature SREBP-1c protein, as well as decreased steatosis on HFD. Our results demonstrate the importance of S6K: 1) as a modulator of the hepatic response to fasting/refeeding, 2) in the development of steatosis, and 3) as a key node in selective hepatic insulin resistance in obese mice.     

Prevention of TGF-β-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-β (TGF-β). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-β-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-β-induced apoptosis.     

Basal activation of p70S6K results in adipose-specific insulin resistance in protein-tyrosine phosphatase 1B -/- mice

Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adipose tissue from PTP-1B-/- mice does not show enhanced insulin-stimulated insulin receptor phosphorylation. Investigation of glucose uptake in isolated adipocytes revealed that the adipocytes from PTP-1B-/- mice have a significantly attenuated insulin response as compared with PTP-1B+/+ adipocytes. This insulin resistance manifests in PTP-1B-/- animals older than 16 weeks of age and could be partially rescued by adenoviral expression of PTP-1B in null adipocytes. Examination of adipose signaling pathways found that the basal p70S6K activity was at least 50% higher in adipose from PTP-1B-/- mice compared with wild type animals. The increased basal activity of p70S6K in PTP-1B-/- adipose correlated with decreases in IR substrate-1 protein levels and insulin-stimulated Akt/protein kinase B activity, explaining the decrease in insulin sensitivity even as insulin receptor phosphorylation was unaffected. The insulin resistance of the of the PTP-1B-/- adipocytes could also be rescued by treatment with rapamycin, suggesting that in adipose the loss of PTP-1B results in basal activation of mTOR (mammalian target of rapamycin) complex 1 leading to a tissue-specific insulin resistance.     

Effects of PI3K catalytic subunit and Akt isoform deficiency on mTOR and p70S6K activation in myoblasts

The PI3K/Akt/mTOR signaling pathway is critical for cellular growth and survival in skeletal muscle, and is activated in response to growth factors such as insulin-like growth factor-I (IGF-I). We found that in C2C12 myoblasts, deficiency of PI3K p110 catalytic subunits or Akt isoforms had distinct effects on phosphorylation of mTOR and p70S6K. siRNA-mediated knockdown of PI3K p110α, p110β, and simultaneous knockdown of p110α and p110β resulted in increased basal and IGF-I-stimulated phosphorylation of mTOR S2448 and p70S6K T389; however, phosphorylation of S6 was reduced in p110β-deficient cells, possibly due to reductions in total S6 protein. We found that IGF-I-stimulated Akt1 activity was enhanced in Akt2- or Akt3-deficient cells, and that knockdown of individual Akt isoforms increased mTOR/p70S6K activation in an isoform-specific fashion. Conversely, levels of IGF-I-stimulated p70S6K phosphorylation in cells simultaneously deficient in both Akt1 and Akt3 were increased beyond those seen with loss of any single Akt isoform, suggesting an alternate, Akt-independent mechanism that activates mTOR/p70S6K. Our results collectively suggest that mTOR/p70S6K is activated in a PI3K/Akt-dependent manner, but that in the absence of p110α or Akt, alternate pathway(s) may mediate activation of mTOR/p70S6K in C2C12 myoblasts.     

Resistance exercise decreases eIF2Bepsilon phosphorylation and potentiates the feeding-induced stimulation of p70S6K1 and rpS6 in young men

We investigated the effect of resistance exercise and feeding on the activation of signaling proteins involved in translation initiation. Nine young men (23.7+/-0.41 yr; BMI=25.5+/-1.0 kg/m2; means+/-SE) were tested twice after they performed a strenuous bout of unilateral resistance exercise, such that their contralateral leg acted as a nonexercised comparator, in either the fasted and fed [1,000 kJ, each 90 min (3 doses): 10 g protein, 41 g carbohydrate, 4 g fat] states. Muscle biopsies were obtained 6 h postexercise from both legs, resulting in four experimental conditions: rest-fasted, rest-fed, exercise-fasted, and exercise-fed. Feeding increased PKB/Akt (Ser473) phosphorylation (P<0.05), while exercise increased the phosphorylation of Akt and the downstream 70 kDa S6 protein kinase (p70S6K1, Thr389) and ribosomal protein S6 (rpS6, Ser235/236, Ser240/244; all P<0.05). The combination of resistance exercise and feeding increased the phosphorylation of p70S6K1 (Thr389) and rpS6 (Ser240/244) above exercise alone (P<0.05). Exercise also reduced phosphorylation of the catalytic epsilon subunit of eukaryotic initiation factor 2B (eIF2Bepsilon, Ser540; P<0.05). Mammalian target of rapamycin (mTOR, Ser2448), glycogen synthase kinase-3beta (GSK-3beta, Ser9), and focal adhesion kinase (FAK, Tyr576/577) phosphorylation were unaffected by either feeding or resistance exercise (all P>0.14). In summary, feeding resulted in phosphorylation of Akt, while resistance exercise stimulated phosphorylation of Akt, p70S6K1, rpS6, and dephosphorylation eIF2Bepsilon with a synergistic effect of feeding and exercise on p70(S6K1) and its downstream target rpS6. We conclude that resistance exercise potentiates the effect of feeding on the phosphorylation and presumably activation of critical proteins involved in the regulation of muscle protein synthesis in young men.     

Hepatic overexpression of a dominant negative form of raptor enhances Akt phosphorylation and restores insulin sensitivity in K/KAy mice

Several serine/threonine kinases reportedly phosphorylate serine residues of IRS-1 and thereby induce insulin resistance. In this study, to investigate the effect of mTOR/raptor on insulin signaling and metabolism in K/KAy mice with genetic obesity-associated insulin resistance, a dominant negative raptor, COOH-terminally deleted raptor (raptor-DeltaC(T)), was overexpressed in the liver via injection of its adenovirus into the circulation. Hepatic raptor-DeltaC(T) expression levels were 1.5- to 4-fold that of endogenously expressed raptor. Glucose tolerance in raptor-DeltaC(T)-overexpressing mice improved significantly compared with that of LacZ-overexpressing mice. Insulin-induced activation of p70S6 kinase (p70(S6k)) was significantly suppressed in the livers of raptor-DeltaC(T) overexpressing mice. In addition, insulin-induced IRS-1, Ser(307), and Ser(636/639) phosphorylations were significantly suppressed in the raptor-DeltaC(T)-overexpressing liver, whereas tyrosine phosphorylation of IRS-1 was increased. PI 3-kinase activation in response to insulin stimulation was increased approximately twofold, and Akt phosphorylation was clearly enhanced under both basal and insulin-stimulated conditions in the livers of raptor-DeltaC(T) mice. Thus, our data indicate that suppression of the mTOR/p70(S6k) pathway leads to improved glucose tolerance in K/KAy mice. These observations may contribute to the development of novel antidiabetic agents.     

Inhibitory mechanisms of tea polyphenols on the ultraviolet B-activated phosphatidylinositol 3-kinase-dependent pathway

In this study, we investigated the effect of tea polyphenols, (-)-epigallocatechin-3-gallate or theaflavins, on UVB-induced phosphatidylinositol 3-kinase (PI3K) activation in mouse epidermal JB6 Cl 41 cells. Pretreatment of cells with these polyphenols inhibited UVB-induced PI3K activation. Furthermore, UVB-induced activation of Akt and ribosomal p70 S6 kinase (p70 S6-K), PI3K downstream effectors, were also attenuated by the polyphenols. In addition to LY294002, a PI3K inhibitor, pretreatment with a specific mitogen-activated protein/extracellular signal-regulated protein kinases (Erks) kinase 1 inhibitor, U0126, or a specific p38 kinase inhibitor, SB202190, blocked UVB-induced activation of both Akt and p70 S6-K. Pretreatment with LY294002 restrained UVB-induced phosphorylation of Erks, suggesting that in UVB signaling, the Erk pathway is mediated by PI3K. Moreover, pretreatment with rapamycin, an inhibitor of p70 S6-K, inhibited UVB-induced activation of p70 S6-K, but UVB-induced activation of Akt did not change. Interestingly, UVB-induced p70 S6-K activation was directly blocked by the addition of (-)-epigallocatechin-3-gallate or theaflavins, whereas these polyphenols showed only a weak inhibition on UVB-induced Akt activation. Because PI3K is an important factor in carcinogenesis, the inhibitory effect of these polyphenols on activation of PI3K and its downstream effects may further explain the anti-tumor promotion action of these tea constituents.     

Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt

Activation of p70 S6 kinase (p70(S6K)) by growth factors requires multiple signal inputs involving phosphoinositide 3-kinase (PI3K), its effector Akt, and an unidentified kinase that phosphorylates Ser/Thr residues (Ser(411), Ser(418), Ser(424), and Thr(421)) clustered at its autoinhibitory domain. However, the mechanism by which G protein-coupled receptors activate p70(S6K) remains largely uncertain. By using vascular smooth muscle cells in which we have demonstrated Ras/extracellular signal-regulated kinase (ERK) activation through Ca(2+)-dependent, epidermal growth factor (EGF) receptor transactivation by G(q)-coupled angiotensin II (Ang II) receptor, we present a unique cross-talk required for Ser(411) phosphorylation of p70(S6K) by Ang II. Both p70(S6K) Ser(411) and Akt Ser(473) phosphorylation by Ang II appear to involve EGF receptor transactivation and were inhibited by dominant-negative Ras, whereas the phosphorylation of p70(S6K) and ERK but not Akt was sensitive to the MEK inhibitor. By contrast, the phosphorylation of p70(S6K) and Akt but not ERK was sensitive to PI3K inhibitors. Similar inhibitory pattern on these phosphorylation sites by EGF but not insulin was observed. Taken together with the inhibition of Ang II-induced p70(S6K) activation by dominant-negative Ras and the MEK inhibitor, we conclude that Ang II-initiated activation of p70(S6K) requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.     

Adrenalectomy enhances the insulin sensitivity of muscle protein synthesis

After confirming that adrenalectomy per se does not affect skeletal muscle protein synthesis rates, we examined whether endogenously produced glucocorticoids modulate the effect of physiological insulin concentrations on protein synthesis in overnight-fasted rats 4 days after either a bilateral adrenalectomy (ADX), ADX with dexamethasone treatment (ADX + DEX), or a sham operation (Sham; n = 6 each). Rats received a 3-h euglycemic insulin clamp (3 mU. min(-1). kg(-1)). Rectus muscle protein synthesis was measured at the end of the clamp, and the phosphorylation states of protein kinase B (Akt), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and ribosomal protein S6 kinase (p70(S6K)) were quantitated before and after the insulin clamp. The basal phosphorylation states of Akt, 4E-BP1, and p70(S6K) were similar between ADX and Sham rats. Insulin significantly enhanced the phosphorylation of Akt (P < 0.03), 4E-BP1 (P = 0.003), and p70(S6K) (P < 0.002) in ADX but not in Sham rats. Protein synthesis was significantly greater after insulin infusion in ADX than in Sham rats (P = 0.01). Glucocorticoid replacement blunted the effect of insulin on Akt, 4E-BP1, and p70(S6K) phosphorylation and protein synthesis. In conclusion, glucocorticoid deficiency enhances the insulin sensitivity of muscle protein synthesis, which is mediated by increased phosphorylation of translation initiation-regulatory proteins.     

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.     

IGF-I elicits growth of human intestinal smooth muscle cells by activation of PI3K,PDK-1, and p70S6 kinase

Endogenous IGF-I regulates growth of human intestinal smooth muscle cells by jointly activating phosphatidylinositol 3-kinase (PI3K) and ERK1/2. The 70-kDa ribosomal S6 kinase (p70S6 kinase) is a key regulator of cell growth activated by several independently regulated kinases. The present study characterized the role of p70S6 kinase in IGF-I-induced growth of human intestinal smooth muscle cells and identified the mechanisms of p70S6 kinase activation. IGF-I-induced growth elicited via either the PI3K or ERK1/2 pathway required activation of p70S6 kinase. IGF-I elicited concentration-dependent activation of PI3K, 3-phosphoinositide-dependent kinase-1 (PDK-1), and p70S6 kinase that was sequential and followed similar time courses. IGF-I caused time-dependent and concentration-dependent phosphorylation of p70S6 kinase on Thr(421)/Ser(424), Thr(389), and Thr(229) that paralleled p70S6 kinase activation. p70S6 kinase(Thr(421)/Ser(424)) phosphorylation was PI3K dependent and PDK-1 independent, whereas p70S6 kinase(Thr(389)) and p70S6 kinase(Thr(229)) phosphorylation and p70S6 kinase activation were PI3K dependent and PDK-1 dependent. IGF-I elicited sequential Akt(Ser(308)), Akt(Ser(473)), and mammalian target of rapamycin(Ser(2448)) phosphorylation; however, transfection of muscle cells with kinase-inactive Akt1(K179M) showed that these events were not required for IGF-I to activate p70S6 kinase and stimulate proliferation of human intestinal muscle cells.     

EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway

The role of epidermal growth factor receptor (EGFR) tyrosine kinase and its downstream targets in the regulation of the transition from the G0/G1 phase into DNA synthesis in response to ANG II has not been previously investigated in intestinal epithelial IEC-18 cells. ANG II induced a rapid and striking EGFR tyrosine phosphorylation, which was prevented by selective inhibitors of EGFR tyrosine kinase activity (e.g., AG-1478) or by broad-spectrum matrix metalloproteinase (MMP) inhibitor GM-6001. Pretreatment of these cells with either AG-1478 or GM-6001 reduced ANG II-stimulated DNA synthesis by approximately 50%. To elucidate the downstream targets of EGFR, we demonstrated that ANG II stimulated phosphorylation of Akt at Ser473, mTOR at Ser2448, p70S6K1 at Thr389, and S6 ribosomal protein at Ser(235/236). Pretreatment with AG-1478 inhibited Akt, p70S6K1, and S6 ribosomal protein phosphorylation. Inhibition of phosphatidylinositol (PI)3-kinase with LY-294002 or mTOR/p70S6K1 with rapamycin reduced [3H]thymidine incorporation by 50%, i.e., to levels comparable to those achieved by addition of either AG-1478 or GM-6001. Utilizing Akt small-interfering RNA targeted to Akt1 and Akt2, Akt protein knockdown dramatically inhibited p70S6K1 and S6 ribosomal protein phosphorylation. In contrast, AG-1478 or Akt gene silencing exerted no detectable inhibitory effect on ANG II-induced extracellular signal-regulated kinase 1/2 phosphorylation in IEC-18 cells. Taken together, our results demonstrate that EGFR transactivation mediates ANG II-stimulated mitogenesis through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway in IEC-18 cells.     

Fatty acid infusion selectively impairs insulin action on Akt1 and protein kinase C lambda /zeta but not on glycogen synthase kinase-3

To determine the mechanism(s) for insulin resistance induced by fatty acids, we measured the ability of insulin to activate phosphoinositide 3-kinase (PI3K) and multiple distal pathways in rats. Following a 5-h infusion of lipid or glycerol (control), rats underwent a euglycemic hyperinsulinemic clamp. Insulin stimulated IRS-1-associated PI3K activity in muscle of glycerol-infused rats 2.4-fold but had no effect in lipid-infused rats. IRS-2- and phosphotyrosine-associated PI3K activity were increased 3.5- and 4.8-fold, respectively, by insulin in glycerol-infused rats but only 1.6- and 2.3-fold in lipid-infused rats. Insulin increased Akt1 activity 3.9-fold in glycerol-infused rats, and this was impaired 41% in lipid-infused rats. Insulin action on Akt2 and p70S6K were not impaired, whereas activation of protein kinase C lambda/zeta activity was reduced 47%. Insulin inhibited glycogen synthase kinase 3alpha (GSK-3alpha) activity by 30% and GSK-3beta activity by approximately 65% and increased protein phosphatase-1 activity by 40-47% in both glycerol- and lipid-infused rats. Insulin stimulated glycogen synthase activity 2.0-fold in glycerol-infused rats but only 1.4-fold in lipid-infused rats. Thus, 1) elevation of fatty acids differentially affects insulin action on pathways distal to PI3K, impairing activation of Akt1 and protein kinase C lambda/zeta and 2) insulin action on glycogen synthase can be regulated independent of effects on GSK-3 and protein phosphatase-1 activity in vivo.     

Burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle

The molecular bases underlying burn- or critical illness-induced insulin resistance still remain unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We therefore examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared with sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous substrate of Akt/PKB, glycogen synthase kinase-3beta (GSK-3beta), was significantly impaired in burned rats. Furthermore, insulin consistently failed to increase the phosphorylation of p70 S6 kinase, another downstream effector of Akt/PKB, in rats with burn injury, whereas phosphorylation of p70 S6 kinase was increased by insulin in controls. The protein expression of Akt/PKB, GSK-3beta, and p70 S6 kinase was unaltered by burn injury. However, insulin-stimulated activation of ERK, a signaling pathway parallel to Akt/PKB, was not affected by burn injury. These results demonstrate that burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle and suggest that attenuated Akt/PKB activation may be involved in deranged metabolism and muscle wasting observed after burn injury.     

Regulation of IGF-1/PI3K/Akt signalling by the phosphoinositide phosphatase pharbin

Pharbin, a 5-phosphatase that induces arborization, is one of the phosphoinositide 5-phosphatases that is highly mutated in patients with Joubert syndrome. Pharbin can hydrolyse PI(4,5)P(2) and PI(3,4,5)P(3) and has the same substrate specificity as SHIP2 and SKIP, which negatively regulate PI3K signalling. Here, we investigated the role of pharbin in IGF-1/PI3K signalling. Ectopic expression of pharbin markedly suppressed the IGF-1-induced activation of Akt without affecting p42/44 MAP kinase phosphorylation. In contrast, pharbin silencing by RNA interference increased the IGF-1-induced phosphorylation of Akt, suggesting that pharbin negatively regulates PI3K/Akt signalling. Pharbin expression also inhibited the phosphorylation of p70 S6 kinase and 4E-BP1 as well as the subsequent protein synthesis in response to IGF-1 treatment. Taken together, these results indicate that pharbin is an important negative regulator of IGF-1/PI3K/Akt signalling and protein synthesis.