Phosphatidylinositol 3‐kinase‐mediated regulation of neuronal apoptosis and necrosis by insulin and IGF‐I

We examined effects of two insulin‐like growth factors, insulin and insulin‐like growth factor‐I (IGF‐I), against apoptosis, excitotoxicity, and free radical neurotoxicity in cortical cell cultures. Like IGF‐I, insulin attenuated serum deprivation‐induced neuronal apoptosis in a dose‐dependent manner at 10–100 ng/mL. The anti‐apoptosis effect of insulin against serum deprivation disappeared by addition of a broad protein kinase inhibitor, staurosporine, but not by calphostin C, a selective protein kinase C inhibitor. Addition of PD98059, a mitogen‐activated protein kinase kinase (MAPKK) inhibitor, blocked insulin‐induced activation of extracellular signal‐regulated protein kinases (ERK1/2) without altering the neuroprotective effect of insulin. Cortical neurons underwent activation of phosphatidylinositol (PI) 3‐kinase as early as 1 min after exposure to insulin. Inclusion of wortmannin or LY294002, selective inhibitors of PI 3‐K, reversed the insulin effect against apoptosis. In contrast to the anti‐apoptosis effect, neither insulin nor IGF‐I protected excitotoxic neuronal necrosis following continuous exposure to 15 μM N‐methyl‐d ‐aspartate or 40 μM kainate for 24 h. Surprisingly, concurrent inclusion of 50 ng/mL insulin or IGF‐I aggravated free radical‐induced neuronal necrosis over 24 h following continuous exposure to 10 μM Fe²⁺ or 100 μM buthionine sulfoximine. Wortmannin or LY294002 also reversed this potentiation effect of insulin. These results suggest that insulin‐ like growth factors act as anti‐apoptosis factor and pro‐oxidant depending uon the activation of PI 3‐kinase. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 536–546, 1999     

Insulin-induced oxidative neuronal injury in cortical culture: mediation by induced N-methyl-D-aspartate receptors

While effectively attenuating neuronal apoptosis in mouse cortical culture, insulin paradoxically induced neuronal necrosis with 48 h of exposure. The insulin neurotoxicity was blocked by an antioxidant but not by caspase inhibitors. Exposure to insulin led to tyrosine phosphorylation of the insulin receptor and the insulin-like growth factor-1 (IGF-1) receptor and activation of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3-kinase). Inhibitors of tyrosine kinase and PKC, but not PI3-kinase, attenuated the insulin neurotoxicity. Conversely, the inhibitor of PI3-kinase but not PKC reversed the antiapoptotic effect of insulin. Suggesting that the gene activity-dependent emergence of excitotoxicity contributed to insulin neurotoxicity, macromolecule synthesis inhibitors and N-methyl-D-aspartate (NMDA) antagonists blocked it. Consistently, exposure to insulin increased the level of the NR2A subunit of the NMDA receptor without much altering NR1 or NR2B levels. The present study suggests that insulin can be both neuroprotective and neurotoxic in the same cell system but by way of different signaling cascades.     

Insulin-like growth factor-I promotes resistance of bovine preimplantation embryos to heat shock through actions independent of its anti-apoptotic actions requiring PI3K signaling

For the bovine preimplantation embryo, insulin-like growth factor-I (IGF-I) is a survival factor that blocks the induction of apoptosis and reduces the decrease in development caused by heat shock. The first objective was to determine the signaling pathways whereby IGF-I acts to increase embryo cell number while inhibiting heat-shock induced apoptosis. Exposure of embryos to heat shock reduced cell number and increased percent apoptosis, but IGF-I increased cell number and blocked induction of apoptosis caused by heat shock. Actions of IGF-I to increase cell number were blocked by treatment with the mitogen activated protein kinase kinase (MAPKK) inhibitor PD 98059 whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002 had no effect. Conversely, LY 294002 but not PD 98059 blocked actions of IGF-I to inhibit induction of apoptosis caused by heat shock. The second objective was to determine whether IGF-I blocks effects of heat shock on development to the blastocyst stage by preventing apoptosis. Culture of embryos with IGF-I was effective in blocking the reduction in blastocyst development caused by heat shock-this action occurred even in the presence of LY 294002. Addition of another inhibitor of apoptosis, the caspase-3 inhibitor z-DEVD-fmk, did not mimic the protective effects of IGF-I on blastocyst development. Surprisingly, IGF-I was not effective in blocking the reduction in blastocyst development caused by heat shock when cultured with z-DEVD-fmk. In conclusion, the anti-apoptotic actions of IGF-I require PI3K signaling while actions to promote proliferation require MAPKK signaling. Moreover, actions of IGF-I to allow heat-shocked embryos to continue development to the blastocyst stage are independent of its anti-apoptotic effects.     

Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.     

Somatostatin inhibits hepatic growth hormone receptor and insulin-like growth factor I mRNA expression by activating the ERK and PI3K signaling pathways

Previously, we reported that somatostatins (SS) inhibit organismal growth by reducing hepatic growth hormone (GH) sensitivity and by inhibiting insulin-like growth factor I (IGF-I) production. In this study, we used hepatocytes isolated from rainbow trout to elucidate the mechanism(s) associated with the extrapituitary growth-inhibiting actions of SS. SS-14, a predominant SS isoform, stimulated tyrosine phosphorylation of several endogenous proteins, including extracellular signal-regulated kinase (ERK), a member the mitogen-activated protein kinase (MAPK) family, and protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K). SS-14 specifically stimulated the phosphorylation of both ERK 1/2 and Akt in a concentration-dependent fashion. This activation occurred within 5-15 min, then subsided after 1 h. The ERK inhibitor U0126 retarded SS-14-stimulated phosphorylation of ERK 1/2, whereas the PI3K inhibitor LY294002 blocked SS-14-stimulated phosphorylation of Akt. SS-14-inhibited expression of GH receptor (GHR) mRNA was blocked by U0126 but not by LY294002. By contrast, U1026 had no effect on SS-14 inhibition of GH-stimulated IGF-I mRNA expression, whereas LY294002 partially blocked the inhibition of GH-stimulated IGF-I mRNA expression by SS-14. These results indicate that SS-14-inhibited GHR expression is mediated by the ERK signaling pathway and that the PI3K/Akt pathway mediates, at least in part, SS-14 inhibition of GH-stimulated IGF-I expression.     

Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase 3 and apoptosis of cardiac muscle cells

Apoptosis of cardiac muscle cells contributes to the development of cardiomyopathy. Recent studies showed that insulin-like growth factor I (IGF-I) inhibits apoptosis of cardiac muscle cells and improves myocardial function in experimental heart failure. This study was carried out to elucidate the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the anti-apoptotic actions of IGF-I in cardiomyocytes and to explore whether expression of constitutively active PI 3-kinase can inhibit apoptosis in cardiomyocytes. Apoptosis of primary cardiomyocytes was induced by doxorubicin treatment and serum withdrawal. Transduction of cardiomyocytes with constitutively active PI 3-kinase specifically lead to serine phosphorylation of Akt, whereas phosphorylation of IGF-I receptor, IRS1/2 and p44/42 mitogen-activated protein kinase were not increased. In the cardiomyocytes transduced with constitutively active PI 3-kinase, activation of the pro-apoptotic caspase 3 was attenuated and fragmentation of DNA was reduced. Preincubating cells with PI 3-kinase inhibitor LY294002 was associated with loss of anti-apoptotic actions of IGF-I and PI 3-kinase. Neither IGF-I nor constitutively active PI 3-kinase lead to serine phosphorylation of Bad, suggesting that the anti-apoptotic effects of PI 3-kinase are not mediated through Bad phosphorylation in cardiac muscle cells. To determine whether activation of caspase 3 is sufficient to induce apoptosis in cardiomyocytes, an engineered TAT-caspase 3 protein was introduced to cardiomyocytes. Significant reduction of cell viability occurred in the cardiomyocytes transduced with active caspase 3, indicating that activation of caspase 3 is sufficient to cause cardiomyocyte death. These findings indicate the existence of an IGF-I receptor-PI 3-kinase-caspase 3 pathway in cardiomyocytes that plays an important role in the anti-apoptotic actions of IGF-I in heart. Moreover, these data suggest that modulation of PI 3-kinase activities may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in cardiomyopathy.     

Inhibition of phosphatidylinositol-3 kinase/Akt or mitogen-activated protein kinase signaling sensitizes endothelial cells to TNF-alpha cytotoxicity.

Bovine carotid artery endothelial (BAE) cells are resistant to tumor necrosis factor-alpha (TNF), like most other cells. We examined if mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI3) kinase/Akt pathways are involved in this effect. In BAE cells, TNF activates MAP kinase in a MAP kinase kinase 1 (MEK1) manner and Akt in PI3-kinase-dependent manner. Pretreatment with either the MEK1 inhibitor U0126 or PI3-kinase inhibitor LY294002 sensitized BAE cells to TNF-induced apoptosis. Neither U0126 nor LY294002 pretreatment affected TNF-induced activation of NF-kappaB, suggesting that the MAP kinase or PI3-kinase/Akt-mediated anti-apoptotic effect induced by TNF was not relevant to NF-kappaB activation. Both MAP kinase and PI3-kinase/Akt -mediated signaling could prevent cytochrome c release and mitochondrial transmembrane potential (Deltapsi) decrease. PI3-kinase/Akt signaling attenuated caspase-8 activity, whereas MAP kinase signaling impaired caspase-9 activity. These results suggest that TNF-induced MAP kinase and PI3-kinase/Akt signaling play important roles in protecting BAE cells from TNF cytotoxicity.     

Ethanol Induces Apoptosis in Cerebellar Granule Neurons by Inhibiting Insulin-Like Growth Factor 1 Signaling

Abstract: The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 m M K⁺ to one containing 5 m M K⁺. IGF-1 protected granule neurons from apoptosis in medium containing 5 m M K⁺. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K⁺, and failed to potentiate cell death in the presence of 5 m M K⁺. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15–20%) was observed at 1 m M and was half-maximal at 45 m M . The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.     

GTPases and phosphatidylinositol 3-kinase are critical for insulin-like growth factor-I-mediated Schwann cell motility

Previously, we reported insulin-like growth factor-I (IGF-I) promotes motility and focal adhesion kinase (FAK) activation in neuronal cells. In the current study, we examined the role of IGF-I in Schwann cell (SC) motility. IGF-I increases SC process extension and motility. In parallel, IGF-I activates IGF-I receptor, insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3 (PI-3)-kinase, and FAK. LY294002, a PI-3 kinase inhibitor, blocks IGF-I-induced motility and FAK phosphorylation. The Rho family of GTPases is important in the regulation of the cytoskeleton. Overexpression of constitutively active Leu-61 Cdc42 and Val-12 Rac1 enhances SC motility which is unaffected by LY294002. In parallel, stable transfection of SC with dominant negative Asn-17 Rac1 blocks IGF-I-mediated SC motility and FAK phosphorylation, implying Rac is an upstream regulator of FAK. Collectively our results suggest that IGF-I regulates SC motility by reorganization of the actin cytoskeleton via the downstream activation of a PI-3 kinase, small GTPase, and FAK pathway.     

Involvement of the phosphoinositide 3-kinase/protein kinase B signaling pathway in insulin/IGF-I-induced chondrogenesis of the mouse embryonal carcinoma-derived cell line ATDC5

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin/insulin-like growth factor-I (IGF-I) stimulation. In the present study, we examined whether insulin/IGF-I stimulation caused activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway in ATDC5 cells. We also determined whether the insulin-stimulated differentiation of ATDC5 cells into chondrocytes could be mimicked by activation of the PKB pathway alone. ATDC5 cells produced phosphatidylinositol 3,4,5-trisphosphate and the pleckstrin homology domain of PKB was recruited to the plasma membrane in response to insulin stimulation. This was probably a result of activation of PI3K because the PI3K inhibitors, wortmannin and LY294002, inhibited both responses, although the effective concentrations were as high as 10 microM. Insulin stimulation caused the chondrogenic differentiation of ATDC5 cells as assessed by chondrogenic nodule staining with alcian blue. The addition of wortmannin or LY294002, PI3K inhibitors, suppressed the staining, and the suppression was reversible, indicating the effect of the inhibitors is not toxic. Finally, we exogenously expressed a constitutively-activated from of PKB (myristoylated PKB, myr-PKB) in ATDC5 cells, and found the chondrogenic differentiation of ATDC5 cells to form nodules occurred in the absence of insulin stimulation. The kinase-negative mutant of myr-PKB did not caused differentiation, indicating that kinase activity is required. These results support the hypothesis that the PI3K/PKB signaling pathway is involved in the chondrogenic differentiation of ATDC5 cells in response to insulin/IGF-I stimulation. This is the first report that demonstrates the involvement of phosphoinositide signaling in the induction of chondrogenesis from undifferentiated cells.     

Phosphatidylinositol 3'-kinase, but not S6-kinase, is required for insulin-like growth factor-I and IL-4 to maintain expression of Bcl-2 and promote survival of myeloid progenitors

Phosphatidylinositol 3'-kinase (PI 3-kinase) catalyzes the formation of 3' phosphoinositides and has been implicated in an intracellular signaling pathway that inhibits apoptosis in both neuronal and hemopoietic cells. Here, we investigated two potential downstream mediators of PI 3-kinase, the serine/threonine p70 S6-kinase (S6-kinase) and the antiapoptotic protein B cell lymphoma-2 (Bcl-2). Stimulation of factor-dependent cell progenitor (FDCP) cells with either IL-4 or insulin-like growth factor (IGF)-I induced a 10-fold increase in the activity of both PI 3-kinase and S6-kinase. Rapamycin blocked 90% of the S6-kinase activity but did not affect PI 3-kinase, whereas wortmannin and LY294002 inhibited the activity of both S6-kinase and PI 3-kinase. However, wortmannin and LY294002, but not rapamycin, blocked the ability of IL-4 and IGF-I to promote cell survival. We next established that IL-3, IL-4, and IGF-I increase expression of Bcl-2 by >3-fold. Pretreatment with inhibitors of PI 3-kinase, but not rapamycin, abrogated expression of Bcl-2 caused by IL-4 and IGF-I, but not by IL-3. None of the cytokines affected expression of the proapoptotic protein Bax, suggesting that all three cytokines were specific for Bcl-2. These data establish that inhibition of PI 3-kinase, but not S6-kinase, blocks the ability of IL-4 and IGF-I to increase expression of Bcl-2 and protect promyeloid cells from apoptosis. The requirement for PI 3-kinase to maintain Bcl-2 expression depends upon the ligand that activates the cell survival pathway.     

Inhibition of phosphatidylinositol 3-kinase and p70S6 kinase blocks osteogenic protein-1 induction of alkaline phosphatase activity in fetal rat calvaria cells

Published studies reveal that Osteogenic Protein-1 (OP-1) and insulin-like growth factor-I (IGF-I) synergistically stimulate alkaline phosphatase (AP) activity and bone nodule formation in fetal rat calvaria (FRC) cells. In the present study, we examined whether there are interactions between the signal transduction pathways activated by these two growth factors. OP-1 did not significantly affect the levels of IRS-1, IRS-2, the p85alpha subunit of phosphatidylinositol 3-kinase (PI 3-kinase) or the extracellular signal-regulated kinase (ERK)-2, but stimulated ERK-1 protein by twofold. OP-1 also induced phosphorylation of ERK-1 and -2, but not of Akt/protein kinase B (PKB), a protein kinase that is downstream of PI 3-kinase. By comparison, IGF-I increased the levels of the phosphorylated forms of ERK-1 and -2, and Akt/PKB. Inhibition of ERK activation by PD98059 did not significantly alter the stimulation of AP activity by OP-1 or OP-1 in combination with IGF-I. In contrast, inhibition of PI 3-kinase activity by LY294002 blocked the induction of AP activity by OP-1 and OP-1 plus IGF-I. Treatment of cells with rapamycin, an inhibitor of the mammalian target of mTOR, resulted in a 47% and a 53% decrease in the AP activity induced by OP-1 alone and by OP-1 plus IGF-I, respectively. These studies suggest that PI 3-kinase and mTOR contribute to the induction of AP activity by OP-1 and the synergistic effect of OP-1 and IGF-I on AP activity in FRC cells.     

Interleukin-6 inhibits transforming growth factor-beta-induced apoptosis through the phosphatidylinositol 3-kinase/Akt and signal transducers and activators of transcription 3 pathways.

The multifunctional cytokine interleukin-6 (IL-6) regulates growth and differentiation of many cell types and induces production of acute-phase proteins in hepatocytes. Here we report that IL-6 protects hepatoma cells from apoptosis induced by transforming growth factor-beta (TGF-beta), a well known apoptotic inducer in liver cells. Addition of IL-6 blocked TGF-beta-induced activation of caspase-3 while showing no effect on the induction of plasminogen activator inhibitor-1 and p15(INK4B) genes, indicating that IL-6 interferes with only a subset of TGF-beta activities. To further elucidate the mechanism of this anti-apoptotic effect of IL-6, we investigated which signaling pathway transduced by IL-6 is responsible for this effect. IL-6 stimulation of hepatoma cells induced a rapid tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and its kinase activity followed by the activation of Akt. Inhibition of PI 3-kinase by wortmannin or LY294002 abolished the protection of IL-6 against TGF-beta-induced apoptosis. A dominant-negative Akt also abrogated this anti-apoptotic effect. Dominant-negative inhibition of STAT3, however, only weakly attenuated the IL-6-induced protection. Finally, inhibition of both STAT3 and PI 3-kinase by treating cells overexpressing the dominant-negative STAT3 with LY294002 completely blocked IL-6-induced survival signal. Thus, concomitant activation of the PI 3-kinase/Akt and the STAT3 pathways mediates the anti-apoptotic effect of IL-6 against TGF-beta, with the former likely playing a major role in this anti-apoptosis.     

Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: the phosphatidylinositol 3-kinase/Akt and Raf/ERK pathways transmit opposing signals

The matrix metalloproteinase (MMP)-2 has been recognized as a major mediator of basement membrane degradation, angiogenesis, tumor invasion, and metastasis. The factors that regulate its expression have not, however, been fully elucidated. We previously identified the type I insulin-like growth factor (IGF-I) receptor as a regulator of MMP-2 synthesis. The objective of the present study was to investigate the signal transduction pathway(s) mediating this regulation. We show here that in Lewis lung carcinoma subline H-59 cells treated with IGF-I (10 ng/ml), the PI 3-kinase (phosphatidylinositol 3'-kinase) /protein kinase B (Akt) and C-Raf/ERK pathways were activated, and MMP-2 promoter activity, mRNA, and protein synthesis were induced. MMP-2 induction was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin, by overexpression of a dominant-negative Akt or wild-type PTEN (phosphatase and tensin homologue deleted on chromosome 10), and by rapamycin. In contrast, a MEK inhibitor PD98059 failed to reduce MMP-2 promoter activation and actually increased MMP-2 mRNA and protein synthesis by up to 30%. Interestingly, suppression of PI 3-kinase signaling by a dominant-negative Akt enhanced ERK activity in cells stimulated with 10 ng/ml but not with 100 ng/ml IGF-I. Furthermore, at the higher (100 ng/ml) IGF-I concentration, C-Raf and ERK, but not PI 3-kinase activation, was enhanced, and this resulted in down-regulation of MMP-2 synthesis. This effect was reversed in cells expressing a dominant-negative ERK mutant. The results suggest that IGF-I can up-regulate MMP-2 synthesis via PI 3-kinase/Akt/mTOR (the mammalian target of rapamycin) signaling while concomitantly transmitting a negative regulatory signal via the Raf/ERK pathway. The outcome of IGF-IR (the receptor for IGF-I) activation may ultimately depend on factors, such as ligand bioavailability, that can shift the balance preferentially toward one pathway or the other.     

Insulin-like growth factor I prevents the development of sensitivity to kainate neurotoxicity in cerebellar granule cells

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.     

Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal

Insulin-like growth factor-I (IGF-I) is emerging as an important growth factor able to modulate the programmed cell death (PCD) pathway mediated by the cysteine-dependent aspartate proteases (caspases); however, little is known about the effect of IGF-I after nerve growth factor (NGF) withdrawal in neurons. To begin to understand the neuronal death-sparing effect of IGF-I under NGF-free conditions, we tested whether embryonic sensory dorsal root ganglion neurons (DRG) were able to survive in defined serum-free medium in the presence of IGF-I. We further studied the role of IGF-I signaling and caspase inhibition after NGF withdrawal. NGF withdrawal produced histological changes of apoptosis including chromatin condensation, shrinkage of the perikaryon and nucleus, retention of the plasma membrane, and deletion of single cells. Both IGF-I and Boc-aspartyl (OMe)-fluoromethylketone (BAF), a caspase inhibitor, equally reduced apoptosis after NGF withdrawal. The antiapoptotic effect of IGF-I was completely blocked by LY294002, an inhibitor of PI 3-kinase signaling, but not by the mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) activated protein kinase inhibitor PD98059. Functional IGF-I receptors were extensively expressed both in rat and human DRG neurons, although they were most abundant in the neuronal growth cone. Collectively, these findings indicate that IGF-I, signaling though the PI-3 kinase pathway, is important in modulating PCD in cultured DRG neurons after NGF withdrawal, and IGF-I may be important in DRG embryogenesis. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 455–467, 1998     

Insulin, insulin-like growth factor-I, and platelet-derived growth factor activate extracellular signal-regulated kinase by distinct pathways in muscle cells

We have investigated the signaling pathways initiated by insulin, insulin-like growth factor-1 (IGF-I), and platelet-derived growth factor (PDGF) leading to activation of the extracellular signal-regulated kinase (ERK) in L6 myotubes. Insulin but not IGF-I or PDGF-induced ERK activation was abrogated by Ras inhibition, either by treatment with the farnesyl transferase inhibitor FTP III, or by actin disassembly by cytochalasin D, previously shown to inhibit Ras activation. The protein kinase C (PKC) inhibitor bisindolylmaleimide abolished PDGF but not IGF-I or insulin-induced ERK activation. ERK activation by insulin, IGF-I, or PDGF was unaffected by the phosphatidylinositol 3-kinase inhibitor wortmannin but was abolished by the MEK inhibitor PD98059. In contrast, activation of the pathway involving phosphatidylinositol 3-kinase (PI3k), protein kinase B, and glycogen synthase kinase 3 (GSK3) was mediated similarly by all three receptors, through a PI 3-kinase-dependent but Ras- and actin-independent pathway. We conclude that ERK activation is mediated by distinct pathways including: (i) a cytoskeleton- and Ras-dependent, PKC-independent, pathway utilized by insulin, (ii) a PKC-dependent, cytoskeleton- and Ras-independent pathway used by PDGF, and (iii) a cytoskeleton-, Ras-, and PKC-independent pathway utilized by IGF-I.     

Insulin rescues retinal neurons from apoptosis by a phosphatidylinositol 3-kinase/Akt-mediated mechanism that reduces the activation of caspase-3.

The ability of insulin to protect neurons from apoptosis was examined in differentiated R28 cells, a neural cell line derived from the neonatal rat retina. Apoptosis was induced by serum deprivation, and the number of pyknotic cells was counted. p53 and Akt were examined by immunoblotting after serum deprivation and insulin treatment, and caspase-3 activation was examined by immunocytochemistry. Serum deprivation for 24 h caused approximately 20% of R28 cells to undergo apoptosis, detected by both pyknosis and activation of caspase-3. 10 nm insulin maximally reduced the amount of apoptosis with a similar potency as 1.3 nm (10 ng/ml) insulin-like growth factor 1, which acted as a positive control. Insulin induced serine phosphorylation of Akt, through the phosphatidylinositol (PI) 3-kinase pathway. Inhibition of PI 3-kinase with wortmannin or LY294002 blocked the ability of insulin to rescue the cells from apoptosis. SN50, a peptide inhibitor of NF-kappaB nuclear translocation, blocked the rescue effect of insulin, but neither insulin or serum deprivation induced phosphorylation of IkappaB. These results suggest that insulin is a survival factor for retinal neurons by activating the PI 3-kinase/Akt pathway and by reducing caspase-3 activation. The rescue effect of insulin does not appear to be mediated by NF-kappaB or p53. These data suggest that insulin provides trophic support for retinal neurons through a PI 3-kinase/Akt-dependent pathway.     

Activation of Akt Kinase Inhibits Apoptosis and Changes in Bcl-2 and Bax Expression Induced by Nitric Oxide in Primary Hippocampal Neurons

Emerging data indicate that growth factors such as insulin-like growth factor-1 (IGF-1) prevent neuronal death due to nitric oxide (NO) toxicity. On the other hand, growth factors can promote cell survival by acting on phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, serine-threonine kinase Akt, in various types of cells. Here, we examined the mechanism by which IGF-1 inhibits neuronal apoptosis induced by NO in primary hippocampal neurons. IGF-1 was capable of preventing apoptosis and caspase-3-like activation induced by a NO donor, sodium nitroprusside or 3-morpholin-osydnonimine. Incubation of neurons with a P13-kinase inhibitor, wortmannin or LY294002, blocked the effects of IGF-1 on NO-induced neurotoxicity and caspase-3-like activation. In addition, the P13-kinase inhibitors blocked the effect of IGF-1 on down-regulation in Bcl-2 and upregulation in Bax expression induced by NO. Adenovirus-mediated overexpression of the activated form of Akt significantly inhibited NO-induced cell death, caspase-3-like activation, and changes in Bcl-2 and Bax expression. Moreover, expression of the kinase-defective form of Akt almost completely blocked the effects of IGF-1. These findings suggest that activation of Akt is necessary and sufficient for the effect of IGF-1 and is capable of preventing NO-induced apoptosis by modulating the NO-induced changes in Bcl-2 and Bax expression.