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Insulin promotes survival of amyloid-beta oligomers neuroblastoma damaged cells via caspase 9 inhibition and Hsp70 upregulation.

Di Carlo M, Picone P, Carrotta R, Giacomazza D, San Biagio PL - J. Biomed. Biotechnol. (2010)

Bottom Line: By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation.Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program.Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Biomedicina e Immunologia Molecolare A. Monroy, Consiglio Nazionale delle Ricerche, 90146 Palermo, Italy. di-carlo@ibim.cnr.it

ABSTRACT
Alzheimer's disease (AD) and type 2 diabetes are connected in a way that is still not completely understood, but insulin resistance has been implicated as a risk factor for developing AD. Here we show an evidence that insulin is capable of reducing cytotoxicity induced by Amyloid-beta peptides (A-beta) in its oligomeric form in a dose-dependent manner. By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation. A protective role of insulin on mitochondrial damage is also shown by using Mito-red vital dye. Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program. Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD.

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Related in: MedlinePlus

Effects of insulin against rAβ42 oligomers on caspase 9 and 3 activation. LAN5 were untreated (control), treated with rAβ42 oligomers (40 μM) for 1 h (rAβ42), rAβ42 oligomers (40 μM) for 1 h and insulin (100 μM) for 4 h  (rAβ42 + insulin), insulin alone (100 μM) for 4 h  (insulin), 50 μM Vinblastine for 4 h  (activator)  and submitted to caspase 8 (a), caspase 9 (b), and caspase 3 (c) luminescent assays. Data expressed as percentage of the corresponding control. The dashed line shown in the histogram corresponds to a control culture. The activation of caspase 8 (a) is significantly high with activator *P < .02, whereas it is not significantly high in rAβ42 versus control. The activation of caspase 9 (b) is significantly high in rAβ42 *P < .01 and in the activator *P < .05, whereas it is not significantly high in rAβ42 + insulin versus control. The activation of caspase 3 (c) is significantly high in rAβ42 *P < .01 and in the activator *P < .01, whereas it is not significantly high in rAβ42 + insulin versus control.  Values are referred to control, the data are the mean ±  SD of three separate experiments.
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fig3: Effects of insulin against rAβ42 oligomers on caspase 9 and 3 activation. LAN5 were untreated (control), treated with rAβ42 oligomers (40 μM) for 1 h (rAβ42), rAβ42 oligomers (40 μM) for 1 h and insulin (100 μM) for 4 h (rAβ42 + insulin), insulin alone (100 μM) for 4 h (insulin), 50 μM Vinblastine for 4 h (activator) and submitted to caspase 8 (a), caspase 9 (b), and caspase 3 (c) luminescent assays. Data expressed as percentage of the corresponding control. The dashed line shown in the histogram corresponds to a control culture. The activation of caspase 8 (a) is significantly high with activator *P < .02, whereas it is not significantly high in rAβ42 versus control. The activation of caspase 9 (b) is significantly high in rAβ42 *P < .01 and in the activator *P < .05, whereas it is not significantly high in rAβ42 + insulin versus control. The activation of caspase 3 (c) is significantly high in rAβ42 *P < .01 and in the activator *P < .01, whereas it is not significantly high in rAβ42 + insulin versus control. Values are referred to control, the data are the mean ±  SD of three separate experiments.

Mentions: The major executioners in the apoptotic program are proteases known as caspases. In some forms of apoptosis, the extrinsic apoptotic pathway is initiated by activation of caspase 8 after death receptor binding; in other forms, activation of the intrinsic apoptotic pathway is initiated by signaling molecules, recruited by mitochondria. They lead a release of cytochrome C from mitochondrial matrix to cytoplasm where it binds to Apaf-1 protein to form the apoptosome that activates caspase 9 [16]. Both these pathways are able to activate the executrix caspase-3 involved in the final part of death process. To identify in which rAβ42-oligomer induced apoptotic pathway insulin interferes, we performed caspase 8 and caspase 9 luminometric assays. A caspase activator was employed as a control. According to previous results, no activation of capsase 8 by oligomers stimulus occurred [15] and the same result for insulin stimulus was obtained (Figure 3(a)). Instead, as can be seen in Figure 3(b), A-beta oligomers activate caspase 9 and this activation is noticeably reduced by the presence of insulin, indicating, once again, that insulin is able to produce a positive protective effect. In order to confirm this result, LAN5 cells treated as described above were put through to caspase 3 assay, the typical executrix caspase. As expected, in the LAN5 cells treated with oligomers and insulin a reduction in the caspase 3 activation with respect to the A-beta oligomer treated cells was detected (Figure 3(c)). These results confirm that A-beta oligomers induce intrinsic apoptotic pathway and the insulin is able to down-regulate caspases 9 and 3 activation.


Insulin promotes survival of amyloid-beta oligomers neuroblastoma damaged cells via caspase 9 inhibition and Hsp70 upregulation.

Di Carlo M, Picone P, Carrotta R, Giacomazza D, San Biagio PL - J. Biomed. Biotechnol. (2010)

Effects of insulin against rAβ42 oligomers on caspase 9 and 3 activation. LAN5 were untreated (control), treated with rAβ42 oligomers (40 μM) for 1 h (rAβ42), rAβ42 oligomers (40 μM) for 1 h and insulin (100 μM) for 4 h  (rAβ42 + insulin), insulin alone (100 μM) for 4 h  (insulin), 50 μM Vinblastine for 4 h  (activator)  and submitted to caspase 8 (a), caspase 9 (b), and caspase 3 (c) luminescent assays. Data expressed as percentage of the corresponding control. The dashed line shown in the histogram corresponds to a control culture. The activation of caspase 8 (a) is significantly high with activator *P < .02, whereas it is not significantly high in rAβ42 versus control. The activation of caspase 9 (b) is significantly high in rAβ42 *P < .01 and in the activator *P < .05, whereas it is not significantly high in rAβ42 + insulin versus control. The activation of caspase 3 (c) is significantly high in rAβ42 *P < .01 and in the activator *P < .01, whereas it is not significantly high in rAβ42 + insulin versus control.  Values are referred to control, the data are the mean ±  SD of three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Effects of insulin against rAβ42 oligomers on caspase 9 and 3 activation. LAN5 were untreated (control), treated with rAβ42 oligomers (40 μM) for 1 h (rAβ42), rAβ42 oligomers (40 μM) for 1 h and insulin (100 μM) for 4 h (rAβ42 + insulin), insulin alone (100 μM) for 4 h (insulin), 50 μM Vinblastine for 4 h (activator) and submitted to caspase 8 (a), caspase 9 (b), and caspase 3 (c) luminescent assays. Data expressed as percentage of the corresponding control. The dashed line shown in the histogram corresponds to a control culture. The activation of caspase 8 (a) is significantly high with activator *P < .02, whereas it is not significantly high in rAβ42 versus control. The activation of caspase 9 (b) is significantly high in rAβ42 *P < .01 and in the activator *P < .05, whereas it is not significantly high in rAβ42 + insulin versus control. The activation of caspase 3 (c) is significantly high in rAβ42 *P < .01 and in the activator *P < .01, whereas it is not significantly high in rAβ42 + insulin versus control. Values are referred to control, the data are the mean ±  SD of three separate experiments.
Mentions: The major executioners in the apoptotic program are proteases known as caspases. In some forms of apoptosis, the extrinsic apoptotic pathway is initiated by activation of caspase 8 after death receptor binding; in other forms, activation of the intrinsic apoptotic pathway is initiated by signaling molecules, recruited by mitochondria. They lead a release of cytochrome C from mitochondrial matrix to cytoplasm where it binds to Apaf-1 protein to form the apoptosome that activates caspase 9 [16]. Both these pathways are able to activate the executrix caspase-3 involved in the final part of death process. To identify in which rAβ42-oligomer induced apoptotic pathway insulin interferes, we performed caspase 8 and caspase 9 luminometric assays. A caspase activator was employed as a control. According to previous results, no activation of capsase 8 by oligomers stimulus occurred [15] and the same result for insulin stimulus was obtained (Figure 3(a)). Instead, as can be seen in Figure 3(b), A-beta oligomers activate caspase 9 and this activation is noticeably reduced by the presence of insulin, indicating, once again, that insulin is able to produce a positive protective effect. In order to confirm this result, LAN5 cells treated as described above were put through to caspase 3 assay, the typical executrix caspase. As expected, in the LAN5 cells treated with oligomers and insulin a reduction in the caspase 3 activation with respect to the A-beta oligomer treated cells was detected (Figure 3(c)). These results confirm that A-beta oligomers induce intrinsic apoptotic pathway and the insulin is able to down-regulate caspases 9 and 3 activation.

Bottom Line: By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation.Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program.Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Biomedicina e Immunologia Molecolare A. Monroy, Consiglio Nazionale delle Ricerche, 90146 Palermo, Italy. di-carlo@ibim.cnr.it

ABSTRACT
Alzheimer's disease (AD) and type 2 diabetes are connected in a way that is still not completely understood, but insulin resistance has been implicated as a risk factor for developing AD. Here we show an evidence that insulin is capable of reducing cytotoxicity induced by Amyloid-beta peptides (A-beta) in its oligomeric form in a dose-dependent manner. By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation. A protective role of insulin on mitochondrial damage is also shown by using Mito-red vital dye. Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program. Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD.

Show MeSH
Related in: MedlinePlus