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Insulin Rescues Retinal Neurons from Apoptosis by a Phosphatidylinositol 3-Kinase/Akt-mediated Mechanism That Reduces the Activation of Caspase-3*

Open AccessPublished:August 01, 2001DOI:https://doi.org/10.1074/jbc.M104738200
      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 ∼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-κB nuclear translocation, blocked the rescue effect of insulin, but neither insulin or serum deprivation induced phosphorylation of IκB. 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-κB or p53. These data suggest that insulin provides trophic support for retinal neurons through a PI 3-kinase/Akt-dependent pathway.
      IGF
      insulin-like growth factor
      HA
      hemagglutinin
      PI
      phosphatidylinositol
      PBS
      phosphate-buffered saline
      Insulin is an important survival factor for primary cerebellar neurons because it can supply the trophic needs for these cells in culture (
      • Huck S.
      ). Less is known about neurons from the retina, but it is clear that the retina expresses abundant insulin receptors, which are at the highest concentration in the inner plexiform layer, where many neuronal projections are located (
      • Naeser P.
      ,
      • Rodrigues M.
      • Waldbillig R.J.
      • Rajagopalan S.
      • Hackett J.
      • LeRoith D.
      • Chader G.J.
      ). Barres and co-workers (
      • Meyer-Franke A.
      • Kaplan M.R.
      • Pfrieger F.W.
      • Barres B.A.
      ) have shown that insulin is among the growth factors that promote survival and growth of primary retinal ganglion cells in culture.
      The insulin receptor has multiple downstream targets that are conserved in many cell types. It is generally thought that its antiapoptotic mechanism signals primarily through PI 3- kinase to activate the serine-threonine kinase, Akt (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ). Akt inhibits many proapoptotic targets by phosphorylating caspase-9, glycogen synthase kinase, BCL-2-associated death promoter, and members of the forkhead family (
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ,
      • Hetman M.
      • Cavanaugh J.E.
      • Kimelman D.
      • Xia Z.G.
      ,
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Kops G.J.
      • de Ruiter N.D.
      • De Vries-Smits A.M.
      • Powell D.R.
      • Bos J.L.
      • Burgering B.M.
      ,
      • Nakae J.
      • Park B.C.
      • Accili D.
      ,
      • Rena G.
      • Guo S.
      • Cichy S.C.
      • Unterman T.G.
      • Cohen P.
      ). Insulin also inhibits the release of cytochromec from mitochondria and IκB, which regulates NF-κB translocation (
      • Kennedy S.G.
      • Kandel E.S.
      • Cross T.K.
      • Hay N.
      ,
      • Benoliel A.M.
      • Kahn-Peries B.
      • Imbert J.
      • Verrando P.
      ,
      • Bertrand F.
      • Philippe C.
      • Antoine P.J.
      • Baud L.
      • Groyer A.
      • Capeau J.
      • Cherqui G.
      ,
      • Zhou G.
      • Kuo M.T.
      ), similar to the IGF-11 receptor-stimulated pathway (
      • Blakesley V.A.
      • Scrimgeour A.
      • Esposito D.
      • Le Roith D.
      ,
      • Baserga R.
      • Resnicoff M.
      • D'Ambrosio C.
      • Valentinis B.
      ).
      NF-κB is a transcription factor that is translocated from the cytoplasm to the nucleus and has been implicated in neuronal survival (
      • Mattson M.P.
      • Culmsee C., Yu, Z.
      • Camandola S.
      ). NF-κB may also be involved in insulin signaling to prevent apoptosis. Insulin activates NF-κB in Chinese hamster ovary cells transfected with the insulin receptor, and the anti-apoptotic signal of insulin is blocked by overexpression of a dominant negative IκB-α, which is the inhibitory peptide for NF-κB (
      • Bertrand F.
      • Atfi A.
      • Cadoret A.
      • L'Allemain G.
      • Robin H.
      • Lascols O.
      • Capeau J.
      • Cherqui G.
      ). It is possible that the anti-apoptotic effect mediated by NF-κB is due to increased transcription of manganese-superoxide dismutase (
      • Bertrand F.
      • Desbois-Mouthon C.
      • Cadoret A.
      • Prunier C.
      • Robin H.
      • Capeau J.
      • Atfi A.
      • Cherqui G.
      ). Overexpression of the NF-κB c-rel subunit in neurons blocked apoptosis as potently as IGF-1, whereas overexpression of a dominant-negative IκB-α enhanced apoptosis, suggesting that the function of NF-κB in neurons is primarily neuroprotective (
      • Heck S.
      • Lezoualc'h F.
      • Engert S.
      • Behl C.
      ).
      The tumor suppressor gene p53 is also strongly implicated in apoptosis of neurons (
      • Hughes P.E.
      • Alexi T.
      • Schreiber S.S.
      ,
      • Inamura N.
      • Araki T.
      • Enokido Y.
      • Nishio C.
      • Aizawa S.
      • Hatanaka H.
      ). Although p53 is known to induce growth arrest, it is likely that it induces apoptosis by an independent mechanism (
      • Bennett M.R.
      ). Overexpression of p53 in postmitotic neurons leads to apoptosis (
      • Slack R.S.
      • Belliveau D.J.
      • Rosenberg M.
      • Atwal J.
      • Lochmuller H.
      • Aloyz R.
      • Haghighi A.
      • Lach B.
      • Seth P.
      • Cooper E.
      • Miller F.D.
      ) and involves activation of BAX and caspase-3 (
      • Cregan S.P.
      • MacLaurin J.G.
      • Craig C.G.
      • Robertson G.S.
      • Nicholson D.W.
      • Park D.S.
      • Slack R.S.
      ). p53 also up-regulates glyceraldehyde-3-phosphate dehydrogenase in cerebellar granule neurons during apoptosis (
      • Chen R.W.
      • Saunders P.A.
      • Wei H.
      • Li Z.
      • Seth P.
      • Chuang D.M.
      ). However, p53 may not be involved in apoptosis induced by factors that do not lead directly to DNA damage, such as low potassium (
      • Enokido Y.
      • Araki T.
      • Tanaka K.
      • Aizawa S.
      • Hatanaka H.
      ,
      • Paterson I.A.
      • Zhang D.
      • Warrington R.C.
      • Boulton A.A.
      ). The involvement of p53 in apoptosis of retinal neurons is unclear, but it is elevated after retinal ischemia (
      • Joo C.K.
      • Choi J.S.
      • Ko H.W.
      • Park K.Y.
      • Sohn S.
      • Chun M.H.
      • Oh Y.J.
      • Gwag B.J.
      ).
      Here, we tested the hypothesis that physiological concentrations of insulin may act as a survival factor in R28 cells, which are a subclone of E1A-NR.3 transfected rat retina cells (
      • Seigel G.M.
      • Liu L.
      ,
      • Seigel G.M.
      ). R28 cells were originally isolated from neonatal (P12) rat retina as a mixed cell type and transfected with an E1A construct (
      • Seigel G.M.
      • Mutchler A.L.
      • Imperato E.L.
      ). They have been described as having both neuronal and glial characteristics, expressing the neuron-specific antigen Thy 1.1 and some glial cell markers (
      • Seigel G.M.
      • Mutchler A.L.
      • Imperato E.L.
      ,
      • Seigel G.M.
      ). We first show that a combination of laminin substrate and cAMP induces a neuronal phenotype in these cells (
      • Davis G.E.
      • Varon S.
      • Engvall E.
      • Manthorpe M.
      ). The ability of insulin to rescue R28 cells from apoptosis induced by serum deprivation was investigated. The data demonstrate that the PI 3-kinase/Akt pathway mediates the survival effect of insulin and leads to inhibition of caspase-3.

      DISCUSSION

      The data presented here demonstrate that physiological concentrations of insulin can protect retinal neurons from apoptosis induced by serum deprivation in culture. This concept has been demonstrated in in vitro models of neurons from other parts of the nervous system, including sensory nerves (
      • Fernyhough P.
      • Willars G.B.
      • Lindsay R.M.
      • Tomlinson D.R.
      ), cortex (
      • Ryu B.R.
      • Ko H.W.
      • Jou I.
      • Noh J.S.
      • Gwag B.J.
      ), cerebellar granule cells (
      • Foulstone E.J.
      • Tavare J.M.
      • Gunn-Moore F.J.
      ), spinal motor neurons (
      • Ang L.C.
      • Bhaumick B.
      • Munoz D.G.
      • Sass J.
      • Juurlink B.H.
      ), and PC12 cells (
      • Estevez A.G.
      • Radi R.
      • Barbeito L.
      • Shin J.T.
      • Thompson J.A.
      • Beckman J.S.
      ). In this study, we used differentiated R28 cells to model retinal neurons, and the results further support previous observations that both insulin and IGF-1 are trophic factors for the survival of retinal neurons (
      • Meyer-Franke A.
      • Kaplan M.R.
      • Pfrieger F.W.
      • Barres B.A.
      ). To the best of our knowledge, this is the first study to examine the signaling mechanisms for retinal neurons in response to insulin. The potency of insulin to act as a survival factor in vitro has led to the suggestion that insulin alone is sufficient for neuronal survival, at least at high concentration (
      • Huck S.
      ). However, high concentrations of insulin can also activate the IGF-1 receptor. In the present study, lower concentrations of insulin were used. Insulin had a maximal effect in reducing apoptosis at a concentration of 10 nm, which is within the physiological range for plasma insulin and does not stimulate the IGF-1 receptor. Therefore, we conclude that the protective effect of insulin was specific for the insulin receptor.
      Although it is not likely that neurons of the retina are directly exposed to plasma when the blood-retinal barrier is intact, the concentration of insulin in the ocular fluids—vitreous and aqueous humors—has been estimated to be in the physiological range (
      • Feman S.S.
      • Turinsky J.
      • Lam K.W.
      ,
      • Dorn A.
      • Bernstein H.G.
      • Rinne A.
      • Hahn H.J.
      • Ziegler M.
      ). Insulin may be transported into the retina across the blood-retinal barrier similar to the blood-brain barrier (
      • Banks W.A.
      • Jaspan J.B.
      • Kastin A.J.
      ,
      • Banks W.A.
      • Jaspan J.B.
      • Huang W.
      • Kastin A.J.
      ) or may arise byde novo synthesis within the retina, as indicated by the presence of preproinsulin mRNA in retina (
      • Budd G.C.
      • Pansky B.
      • Glatzer L.
      ). Although the source of insulin is not known, it is clear that both insulin and its receptor are abundant in the retina, especially in the inner plexiform layer, which is mostly formed by neuronal projections (
      • Rodrigues M.
      • Waldbillig R.J.
      • Rajagopalan S.
      • Hackett J.
      • LeRoith D.
      • Chader G.J.
      ,
      • Rosenzweig S.A.
      • Zetterstrom C.
      • Benjamin A.
      ,
      • Hyndman A.G.
      ). There is strong evidence that insulin has biological activity in the central nervous system. The insulin receptor is expressed in the brain (
      • Gupta G.
      • Azam M.
      • Baquer N.Z.
      ). Insulin-like peptides may also be released from cultured neuronal cells, synaptosomes, and astrocytes (
      • Clarke D.W.
      • Mudd L.
      • Boyd Jr., F.T.
      • Fields M.
      • Raizada M.K.
      ,
      • Wei L.T.
      • Matsumoto H.
      • Rhoads D.E.
      ,
      • Kadle R.
      • Suksang C.
      • Roberson E.D.
      • Fellows R.E.
      ). Therefore, it is reasonable to suggest that insulin signaling in retinal neurons is active in vivo with canonical physiological consequences.
      The R28 cells used here as a model of retinal neurons were derived from mixed retinal cells but have been used successfully in other studies of neuronal apoptosis (
      • Seigel G.M.
      • Mutchler A.L.
      • Imperato E.L.
      ,
      • Tezel G.
      • Wax M.B.
      ). Under the culture conditions employed here, these cells had a neuronal phenotype with no evidence to suggest glial or endothelial cell characteristics. When laminin is used as a substrate for other neuronal cell lines, such as PC12 cells, it causes them to develop extensive neurite outgrowth (
      • Weeks B.S.
      • Wilson P.J.
      • Heffernan C.C.
      • Ahmad A.
      • Mahadeo K.
      ). This effect may be modulated by protein kinase C, but a direct regulatory role has not been established. The role of cAMP in further differentiating R28 cells is also important to consider. cAMP elevation promotes survival of spinal motor neurons in vitro (
      • Hanson Jr., M.G.
      • Shen S.
      • Wiemelt A.P.
      • McMorris F.A.
      • Barres B.A.
      ), possibly by elevating the recruitment of the neurotrophin TrkB receptor to the plasma membrane (
      • Barres B.A.
      • Schmid R.
      • Sendnter M.
      • Raff M.C.
      ). It is not clear whether cAMP has a similar effect on R28 cells. Although the E1A transfection of R28 cells causes their persistent proliferation, we have shown that the addition of laminin and cAMP provides a more stable model of retinal neurons.
      The characteristics of the insulin receptor in the retina differ from those seen in peripheral tissues such as liver, muscle, and fat. The predominant insulin receptor in the central nervous system is less glycosylated on both α and β subunits (
      • Waldbillig R.J.
      • Fletcher R.T.
      • Chader G.J.
      • Rajagopalan S.
      • Rodrigues M.
      • LeRoith D.
      ). The differentiated R28 cells express both the 125- and 115-kDa species of the α-subunit of the insulin receptor. The β-subunits of the insulin receptors were tyrosine-phosphorylated by 10 nm insulin, but the IGF-1 receptor was not. Thus, differentiated R28 cells respond to insulin stimulation with receptor tyrosine phosphorylation. This is in agreement with other data showing that exogenous physiological concentrations of insulin also cause receptor tyrosine phosphorylation in intact retinas (
      • Gardner T.W.
      • Reiter C.E.N.
      • Antonetti D.A.
      ).
      Insulin reduced the activation of caspase-3 in R28 retinal neurons, and this effect was blocked by the PI 3-kinase inhibitors. These data imply that insulin regulates the activation of caspase-3 through the PI 3-kinase/Akt pathway. Akt can directly phosphorylate caspase-9, which is at the head of the caspase protease cascade (
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ). Therefore, caspase-3 activation may be blocked by insulin receptor signaling in retinal neurons through Akt phosphorylation of caspase-9. This implies that reduction of insulin stimulation could lead to apoptosis mediated by caspase-3 activation in retinal neurons.
      Previous reports have suggested that the PI 3-kinase pathway activates NF-κB in response to insulin, to promote neuronal survival. We used four approaches to test this hypothesis in R28 cells. First, the peptide inhibitor of p65 nuclear translocation, SN50, blocked the protective effect of insulin. These data suggested that translocation of NF-κB to the nucleus is required for insulin to rescue the R28 cells. However, additional experiments did not support this initial interpretation. Transfection of a dominant negative IκB mutant blocked the apoptosis induced by serum deprivation, rendering this approach inoperable. Therefore, effects of insulin on IκB phosphorylation were examined. Serum deprivation or insulin did not induce detectable IκB phosphorylation despite a relative abundance of IκB protein compared with HeLa cells. Because phosphorylation of IκB is generally considered to be required for NF-κB translocation, it appears that this may not be an important insulin signaling mechanism in R28 cells. Finally, immunohistochemistry revealed no nuclear translocation of the p65 subunit in response to insulin. Although SN50 augmented apoptosis, it may not be specific for NF-κB, because it also blocks other transcription factors with nuclear localization sequences by competing for binding to components of the nuclear transport machinery (
      • Lin Y.-Z.
      • Yao S.
      • Veach R.A.
      • Torgerson T.R.
      • Hawiger J.
      ,
      • Torgerson T.R.
      • Colosia A.D.
      • Donahue J.P.
      • Lin Y.Z.
      • Hawiger J.
      ). Taken together, these data suggest that another transcription factor requiring nuclear translocation may be involved. Therefore, the rescue effect of insulin in R28 cells deprived of serum may not occur by activation of NF-κB.
      In conclusion, our data show that insulin can act as a survival factor for R28 cells by stimulating the PI 3-kinase, Akt/protein kinase B pathway and inhibiting the activation of caspase-3. Although p53 is transiently invoked during this form of cell death, the survival effect of insulin does not alter p53 expression, implying that it modulates downstream effectors of p53. Insulin may regulate multiple systems in retinal tissue, and it is likely that part of its function is to act as a survival signal for retinal neurons. Recent evidence shows that experimental diabetes induces retinal neurodegeneration by increasing apoptosis of inner retinal neurons (
      • Barber A.J.
      • Lieth E.
      • Khin S.A.
      • Antonetti D.A.
      • Buchanan A.G.
      • Gardner T.W.
      ). Apoptosis increases soon after the onset of streptozotocin diabetes and is reversed by exogenous insulin. This cell death gives rise to a chronic neurodegeneration in which ∼10% of retinal ganglion cells is lost after 7.5 months. Insulin also increases glutamine synthetase activity in the retinas of diabetic rats (
      • Lieth E.
      • Gardner T.W.
      • Barber A.J.
      • Antonetti D.A.
      ,
      • Lieth E.
      • LaNoue K.F.
      • Antonetti D.A.
      • Ratz M.
      ,
      • Lieth E.
      • Barber A.J.
      • Xu B.
      • Dice C.
      • Ratz M.J.
      • Tanase D.
      • Strother J.M.
      ). Acute insulin treatment also reverses the effects of diabetes on the expression of glial fibrillary acidic protein and the tight junction protein, occludin, in diabetic rats (
      • Barber A.J.
      • Antonetti D.A.
      • Gardner T.W.
      ). Taken together these findings raise the possibility that defective insulin signaling could contribute to retinal neurodegeneration in diabetes.

      Acknowledgments

      We thank Dr. Lois E. H. Smith for the gift of JB3 and Dr. Shao-Cong Sun for the gift of IκB and NFκB antibodies and dominant negative IκB constructs, along with his invaluable advice concerning the transfection experiments.

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