In this study we demonstrated that rapid preconditioning protects against focal cerebral ischemia as a function of ischemic severity in rats. That is, the less severe the ischemia, the stronger the protection. Rapid preconditioning enhanced Akt activity after stroke, and a specific PI3K/Akt inhibitor abolished the protective effect of preconditioning, suggesting that Akt activity contributes to its protection. Nevertheless, Akt phosphorylation (Ser 473) did not correlate with the protective effect of rapid preconditioning, therefore, Akt phosphorylation is not a meaningful marker for neuroprotection.
Our results suggest that Akt phosphorylation (ser 473) may not be used as the “gold standard” marker for Akt activity as it has been used in most previous studies (reviewed by [31
]). For example, many studies consistently reported that Akt phosphorylation is transiently increased after cerebral ischemia, leading to the conclusion that Akt activity is increased after stroke [27
]. However, together with this current study, we recently demonstrated that P-Akt level does not always represent its true activity reflected by the Akt kinase assay [8
]. First, we showed that P-Akt is transiently increased after stroke, while Akt activity is actually decreased at the same time points [8
]. Second, intra-ischemic moderate hypothermia inhibits Akt phosphorylation, while it increases Akt activity compared with normothermia [32
]. Third, our current study shows that rapid preconditioning did not increase the level of P-Akt at 5h, but it enhances Akt activity compared with control ischemia. As we have discussed before [31
], such a discrepancy may be due to the fact that Akt activity is regulated by two phosphorylation sites (Ser 473 and Thr 308), and P-Akt (Ser473) alone is insufficient to stimulate its activity [1
]. However, the phosphorylation level of P-Akt (Thr 308) is not an ideal marker either for Akt activity after stroke, as we have shown that its levels were not changed by stroke [32
]. Furthermore, tyrosine phosphorylation is essential for Akt activation [3
]. Taken together, P-Akt levels (ser 473) alone don’t necessarily explain the beneficial effects of Akt activity in mediating neuroprotection.
Whether Akt contributes to the protective effect of delayed preconditioning is controversial in previous reports. In studies concluding that Akt does not contribute to ischemic preconditioning, the authors note that the early P-Akt (Ser473) peak is inhibited by delayed preconditioning [10
]. In other studies demonstrating that Akt contributes to preconditioning, the authors usually emphasized that P-Akt (Ser473) level is improved by preconditioning at later time points, without interpreting the inhibited early peak of P-Akt (Ser473) after preconditioning [27
]. Moreover, these studies show that inhibiting Akt activity by the PI3K inhibitor Wortmannin or LY 294002 blocks the protective effect of delayed preconditioning [12
]. Therefore, such controversies are partly derived from the fact that Akt kinase assay was not performed in those studies.
We further showed that total protein levels of Akt, PTEN, PDK1, GSK 3β, and β-catenin were reduced after stroke, and rapid preconditioning had no effect on total proteins of Akt and GSK 3β, but attenuated reductions in PTEN, PDK1 and β-catenin at certain time points after stroke. Previous studies have focused on Akt phosphorylation after stroke, but total Akt protein levels were usually not quantitated. From our current study, total Akt protein doesn’t correlate with the final pathological outcomes. Nevertheless, the total protein levels of PTEN, PDK1 and β-catenin correspond to the reduction in infarction size in rats receiving preconditioning. Although our current study cannot answer whether the preservation of these protein levels is simply the effect of protection or the cause of protection, it is an indication that merely studying their phosphorylation is insufficient.