We investigated pioglitazone's potential role in reducing peripheral and central markers in a well-characterized animal model of cognitive decline with aging. We chose the antidiabetic drug because of its favorable permeability into the CNS (~10%, 
), positive effects in the treatment of type 2 diabetes mellitus (T2DM), and its reported success in the treatment of neurodegenerative disease in several animal models. Choice of animal age, dose, and delivery method were selected to mimic clinical conditions associated with the treatment of late-onset diabetes. In prior studies on the impact of the TZD in the brain, higher doses have been used, and often in more pathological models, including Alzheimer's, Parkinson's disease, and spinal cord or brain injury. These models are characterized by pathological processes that are likely present in normal aging, but to a lesser extent. As such, and because of the reported beneficial use of TZDs in alleviating cognitive decline in elderly patients with or without diabetes, we felt it important to test PIO's role in an animal model of normal aging. We focused on several well-established brain biomarkers of aging including learning and memory and inflammatory processes.
Several lines of evidence show that specific TZDs can have direct effects on Ca2+
homeostasis, particularly L-type voltage-gated Ca2+
channels in cardiovascular tissues 
, as well as in neurons 
. Given that Ca2+
dysregulation is considered a hallmark of brain aging 
, is present in animal models of diabetes 
, and appears to contribute to insulin resistance in the periphery 
, we tested the hypothesis that PIO might reduce Ca2+
-dependent brain biomarkers of aging in vivo
and thereby provide some relief from the cognitive decline seen with aging. At the dose and duration tested, PIO did not alleviate age-dependent memory decline (), nor did it improve LTP maintenance (). This was surprising given PIO's effect on the Ca2+
-dependent slow after-hyperpolarizing potential (). Indeed, it is well-recognized that reducing the AHP increases cellular excitability and can improve cognitive performance (reviewed in 
). In prior in vitro
studies, we have shown that PIO can reduce NMDA-mediated currents and associated intracellular Ca2+
, and it is possible that the beneficial effects of reducing the AHP with age in the current study were masked by concomitant decreases in NMDA-mediated signaling. However, because the effects of PIO on NMDA currents were determined in neurons in vitro
, future studies focusing on whether long-term in vivo
PIO can have inhibitory effects on NMDA currents in hippocampal slices will help to address this issue. With respect to the effect of PIO on the AHP, one possibility is that it is mediated by an indirect action on glucocorticoid regulation. In fact, in AD models, TZDs have been shown to improve cognition via alterations in glucocorticoid signaling 
, and there is good evidence that the AHP is sensitive to glucocorticoids 
We measured insulin levels as well as insulin signaling in the brain and the periphery of young and aged animals. Our results show that insulin and glucose levels were not elevated with age, but insulin levels were significantly reduced by PIO in both age groups (). Given that our animals were not diabetic, we predicted that a decrease in insulin levels would result in a concomitant reduction in insulin receptor signaling, at least in the periphery and perhaps also in the brain. ELISA assays on liver (peripheral) and cortical (central) tissues showed that while the ratio of phosphorylated to total insulin receptor did not change, PIO did cause a significant reduction in phosphorylated insulin receptor levels (). These results suggest that insulin receptor levels exist in a dynamic insulin-sensing equilibrium both in the brain and the periphery. It is not clear, however, whether the fewer total receptors remaining might be more efficient at translating insulin's action through differential regulation of downstream targets of the insulin receptor, including IRS-1, Pi3K, Akt, and SH2 
. Further, it is also likely that other insulin sensitive tissues (e.g., muscle or fat) could show enhanced sensitivity to PIO's effects on insulin, resulting in more robust changes in insulin receptor signaling. Because insulin levels were decreased in both young and aged animals treated with PIO, and AHP reductions were only seen in the aged group, we do not believe insulin levels directly influenced the AHP under the condition tested. There is, however, evidence supporting a decrease in insulin sensitivity in the brain of AD patients and in AD models 
, and we are currently testing this hypothesis in our aging model. However, it is unclear whether the aging F344 rat is a good model of T2DM, and responds to high fat diets with signs of insulin resistance in both the periphery and the brain 
. Here, therefore, we believe that PIO's effects on insulin signaling were somewhat blunted because animals were healthy and non-diabetic. On the other hand, one would predict that under conditions more representative of human aging, where accumulated exposure to high fat diets and a sedentary life style contribute to T2DM, PIO might significantly reduce insulin levels 
, increase insulin sensitivity, and likely increase insulin signaling.
Prior studies in CNS and peripheral cell types modeling trauma and insult (e.g., LPS, PMA) demonstrate that PPAR-γ activators play a critical role in reducing inflammatory cytokines (interleukin-6, interleukin 1-β and TNF-α), including activation of inducible nitric oxide synthase (iNOS) 
. Similarly, in animal models of ischemia, stroke, hypertension, stress, and Parkinson's disease, which are also characterized as pro-inflammatory conditions, PPAR-γ agonists provide significant neuroprotection 
. In AD animal models also, PPAR-γ agonists appear to reduce baseline inflammatory levels 
. Only one prior animal study examined the effects of TZDs under non-pathological aging conditions. The authors reported that the increase in proinflammatory cytokine levels (IL-1β) in the hippocampus of aged F344 rats was not sensitive to the actions of the TZD rosiglitazone (10 mg/Kg/day) yet the drug caused significant improvement in contextual fear conditioning 
. Similarly, a prior publication using 20 mg/Kg/day PIO for four months in transgenic mouse models of AD (Tg2576) revealed very little anti-inflammatory effects of PIO (based on microglial activation, soluble Aβ levels, and plaque burden) when compared to ibuprofen treatment 
. The same group, however, convincingly showed that a 40 mg/Kg/day PIO dose could significantly reduced brain inflammation in the APPV717I transgenic mouse 
. Here, therefore, we examined inflammatory cytokines in the serum, and inflammatory signaling in the brain using hippocampal microarray analyses in the context of normal aging, and at low doses of PIO. While a clear inflammatory signature was present in the brain as previously reported in microarray studies of aging 
, PIO did not significantly reduce inflammatory markers in the hippocampus. In the periphery, no robust age-dependent change in inflammatory cytokine levels was seen (although a trend in increased IL-6 levels was noted in aged animals), precluding an effect of PIO. Importantly, reductions in peripheral insulin and lipids indicate the target therapeutic window for PIO was reached. Under these conditions, thus, it is unclear that PIO was able to reduce central and peripheral inflammatory markers in an animal model of aging, and together, these results suggest that higher PIO doses might be necessary to reduce inflammatory pathways and exert beneficial cognitive effects.
Using an unbiased microarray analysis approach on hippocampal tissue, our study compared the effects of a brain permeant TZD treatment in younger and older animals, and showed that while age-dependent gene signatures were clearly present at both the gene () and pathway () levels, PIO effects on gene expression were virtually absent. Possible reasons for this include: low statistical detection power, insufficient drug exposure, or lack of influence of this treatment regimen on hippocampal transcription. Low statistical power is a possibility; however, the treatment main-effect histogram dips well below chance at small p values (), and it seems unlikely that increasing the number of subjects would allow us to detect significant transcriptional effects of PIO. Regarding drug exposure, PIO reduced peripheral blood chemistry measures significantly and in the direction predicted by prior work (see discussion
above and ), suggesting that treatment levels were appropriate. Thus, it seems reasonable to conclude that PIO did not exert a detectable transcriptional effect on hippocampal gene transcription, and that this lack of central influence may be due to either reduced blood-brain barrier penetration or a frank lack of response from hippocampal tissue. Nevertheless, because PIO has established effects involving insulin and inflammatory processes, we also directly investigated genes associated with these processes and tested their sensitivity to PIO in the brain. Although it is not possible to evaluate the biological importance of the 6 genes that were identified without functional genetic manipulation (e.g., knock-in), our resampling analysis revealed that these targeted pathways were not statistically significant. It is interesting to note, however, that of the six genes identified, one of them (Tlr4) recently was found to be sensitive to PIO in monocytes and macrophages 
, reinforcing the role of PIO as an anti-inflammatory agent, and suggesting Tlr4 might be a common target of PIO in peripheral and central tissues.
Compared to prior aging studies, our overlap analysis ( inset) suggests that, irrespective of where along the dorsal-ventral axis it is sampled, the hippocampus shows increased inflammatory markers with age, and validates the use of hippocampal extremities in future microarray studies. Interestingly, upregulated inflammatory categories, historically the most consistent and largest magnitude of the aging brain transcriptional signatures, remain largely unperturbed by PIO administration.
To our knowledge, this is the first study testing long-term PIO treatment in the F344 rat with age, specifically investigating whether a commonly prescribed drug may have off-target cognition enhancing effects in a rat model of aging. The study was designed using a clinically-relevant dose and delivery method. As expected, several signatures of aging were present in older animals, characterized by weak peripheral and robust central inflammatory increases, reduced spatial memory and LTP maintenance, and increased Ca2+-dependent AHPs. Peripheral insulin levels, phosphorylated insulin receptors in the CNS and the periphery, and the AHP were significantly reduced by PIO. While the mechanism through which PIO may mediate its central effects (AHP reduction, reduced phosphorylated insulin receptor) is not clear, it does not appear to occur via a transcriptional process. Given the increased incidence in metabolic syndrome and T2DM seen in the aging population, together with the high numbers of prescriptions written for TZDs, our study has direct therapeutic relevance and suggests future experiments testing the use of these agents at clinically-relevant doses for the treatment of neurological or cognitive conditions are needed. Nevertheless, the results of our study do not preclude the beneficial effects of TZDs in the elderly where metabolic dysregulation and diabetes are often reported.