Several studies in animals and humans have clearly demonstrated the capacity of IPC to protect organs against ischemic injury (see reviews (Dirnagl et al., 2003
; Yenari et al., 2008
)). Various pathways have been implicated including activation of protein kinase C (PKC), ATP-sensitive K+
channels, PI3-Kinase, adenosine receptors, and glutamate receptors (see reviews (Dirnagl et al., 2003
; Gidday, 2006
)). One of the ultimate goals of understanding the pathways involved in IPC is to design novel therapies to mimic IPC pharmacologically. A number of different chemicals mimic IPC, such as sulphonylurea, volatile anaesthetics, levosimendan, erythropoietin, opioids, and estrogen, among others (Dirnagl et al., 2003
; Gidday, 2006
; Yenari et al., 2008
Biologically active trans-resveratrol protects a variety of tissues such as heart, brain, and kidney from ischemia-reperfusion injury (Raval et al., 2008
). Here we have demonstrated that in vivo
resveratrol pre-treatment induces tolerance against an otherwise lethal episode of ischemia and that this neuroprotection depends on SIRT1 activation. We showed that pre-treating rats with low doses of resveratrol 48 hours before induction of lethal ischemia can induce neuroprotection by activating pathways similar to IPC. The resveratrol-induced neuroprotection was blocked by sirtinol, a SIRT1 specific inhibitor, demonstrating the pivotal role of SIRT1 activation (). Earlier we demonstrated that sirtinol also blocks IPC induced neuroprotection in vitro (Raval et al., 2006
Previous studies in pancreatic β-cells have established that SIRT1 activity modulates levels of the mitochondrial uncoupling protein, UCP2 (Bordone et al., 2006
). UCP2 is a member of a family of inner mitochondrial membrane proteins capable of driving the ATP synthase pathway via regulation of the proton electrochemical gradient (Esteves and Brand, 2005
). However, the exact role of UCP2 in mitochondrial function is not fully understood. UCP2 has been implicated in thermogenesis, diabetes, aging, and diseases of the immunological, circulatory and neurological systems (Brand and Esteves, 2005
Resveratrol pretreatment induced a decrease in UCP2 levels in hippocampal mitochondria when measured at 48 hours. The decrease in UCP2 was blocked by sirtinol (). Thus, in vivo
resveratrol pretreatment regulation of UCP2 levels requires SIRT1 activation. Despite lower UCP2 levels in hippocampal mitochondria in RPC-treated rats, we did not detect any change in ΔΨm
. This could be due to different compensatory mechanisms present in the mitochondrial membrane (Scheffler, 1999
). In contrast to the lack of change in ΔΨm
we observed a significant increase in ADP/O ratio (), which is an indicator of increased mitochondrial ATP synthesis efficiency. Brain ATP levels may be maintained for a longer duration when subjected to ischemia due to increased mitochondrial ATP synthesizing capacity. Our data are in agreement with previous studies in cardiomyocytes and in the liver in which increased ATP levels were reported to occur when UCP2 levels were decreased (Bodyak et al., 2007
; Evans et al., 2007
). These data suggest that resveratrol-induced downregulation of UCP2 protects against lethal ischemia, at least in part, by increasing mitochondrial ATP production efficiency ().
The neuroprotective role of UCP2 against cerebral ischemia is not clearly defined. On one hand, higher levels of UCP2 following IPC were shown to play a role in neuroprotection following cardiac and cerebral ischemia (Mattiasson et al., 2003
; McLeod et al., 2005
). In contrast, de Bilbao et al. observed that ischemic brain damage was lower in UCP2 knockout mice after focal cerebral ischemia (de Bilbao et al., 2004
). In addition, a recent study showed that overexpression of UCP2 protected thalamic neurons against global cerebral ischemia but not other brain regions such as hippocampus (Deierborg et al., 2008
). Moreover, another study demonstrated that UCP2 overexpression results in increased cell death after hypoxiareoxygenation in adult rat cardiomyocytes (Bodyak et al., 2007
). UCP2 overexpression led to decreased ROS generation and promoted a shift in hydrogen peroxide release from an intramitochondrial to an extramitochondrial site (de Bilbao et al., 2004
). Thus, UCP2 overexpression altered cellular redox signalling (Arsenijevic et al., 2000
; Mattiasson et al., 2003
). In contrast, the UCP2-depleted condition led to increased ROS production and an increase in cellular ROS buffering capacity by means of increased reduced glutathione (GSH) and mitochondrial manganese superoxide dismutase levels (de Bilbao et al., 2004
). The common denominator in both UCP2-overexpressed and -depleted conditions is decreased ROS. Our results suggest that besides increased ROS buffering capacity, UCP2-depleted conditions also increase mitochondrial ATP production capacity.
The importance of ROS in the induction of IPC and damage following cerebral ischemia has been extensively studied (Perez-Pinzon et al., 2005
). The increased cellular ROS buffering capacity is likely to result in lower ROS induced damage to protein, DNA and lipids during the ischemia/reperfusion. Moreover, our ADP/O ratio results indicate more efficient mitochondrial ATP production. From these results we propose that lower levels of UCP2 following RPC leads to increased mitochondrial ATP production efficiency, which in turn protects cells from ischemia.
As we described in our previous study (Raval et al., 2006
), although both RPC and IPC were neuroprotective against ischemia, the time course for the activation of SIRT1 was different between the two paradigms. As expected, resveratrol rapidly and transiently increased SIRT1 activity, whereas IPC-induced increase of SIRT1 activity was only observed before induction of ACA. However, at this stage we do not know the duration of the resveratrol-induced SIRT1 activity increase. Our data only indicate that this increase in activity returns to baseline by 48 hours. Early activation of SIRT1 following RPC resulted in a decrease in UCP2 protein levels at 48 hours, with not significant change in mRNA levels. Previous studies showed that the induction of UCP2 expression in vivo is regulated at translational level and not accompanied by a change in UCP2 mRNA (Pecqueur et al., 2001
). The half-life of UCP2 is not clearly defined and varies greatly in different tissues (Pecqueur et al., 2001
; Rousset et al., 2007
). We conjecture that the observed delay in UCP2 down-regulation reflects a longer half-life of UCP2 in brain. Overall, the resveratrol results suggest that the transient increase in SIRT1 activity initiates a neuroprotective state lasting at least 48 hours. Loss of neuroprotection due to inhibition of SIRT1 activation during 48 hours of reperfusion after RPC confirms the fact that SIRT1 activation is critical for the induction of a neuroprotective state. Further studies are needed to further define the mechanisms by which SIRT1 activation promotes UCP2 protein levels to decrease. In addition, additional studies are required to define the time range whereby resveratrol promotes neuroprotection after a single administration.
To summarize, in vivo pre-treatment with intraperitoneal injection of 10 or 50 mg/Kg resveratrol mimicked ischemic preconditioning: resveratrol protected the CA1 region of the hippocampus against global cerebral ischemia. This neuroprotection depended on the activation of SIRT1, an NAD+ deacetylase linked with life-span extension by caloric restriction. We also demonstrated that resveratrol preconditioning afforded neuroprotection by decreasing UCP2 levels and increasing mitochondrial ATP synthesizing efficiency. Studies under way are aimed at defining other mechanisms involved in resveratrol-induced neuroprotection against ischemic injury. A better understanding of common mechanisms of neuroprotection shared by IPC and resveratrol could help us develop new strategies against cerebral ischemia. Our results provide a foundation for a new pharmacological approach to neuroprotection in patients with a high probability for stroke via improved mitochondrial metabolism.