Akt stands at the crossroads of several intracellular signaling pathways and plays important functions in regulating cellular survival, proliferation, transcription, and metabolism 
. Using the F344BN aging rat model 
, we observed a hyper-phosphorylation of Akt (Ser473 and Thr308) in aging skeletal muscle. Paradoxically, this increased Akt phosphorylation is associated with lower Akt kinase activity, diminished response to insulin, higher levels of myocyte apoptosis, lower abundance of contractile proteins myosin and actin, and a smaller muscle fiber cross-sectional area (atrophy). Further, we demonstrate that acetaminophen administration functions to prevent age-associated Akt hyper-phosphorylation and dysfunction in aging muscle by decreasing S-nitrosylated Akt and restoring PTEN protein levels.
Activation of Akt requires the phosphorylation of Ser473 within the carboxyl-terminal hydrophobic motif, which primarily facilitates the phosphorylation of Thr308 in the activation loop to fully activate Akt kinase activity 
. In the present study, phosphorylation of Ser473 and Thr308 were found to be higher in the soleus of very aged rats, which theoretically indicates a higher Akt kinase activity. Interestingly, these data do not appear to predict down-stream Akt signaling since the phosphorylation level of mTOR (Ser2448) 
(), tuberous sclerosis complex-2 (TSC2) and forkhead box O1 (FoxO1) (data not shown) was lower in the very aged muscle. Similarly, the abundance of upstream Akt molecules, IR-β and PI3K 
, were also lower in the very aged muscle. Additionally, PDK1 total protein, a kinase that is thought to phosphorylate Akt at Thr308 
, and the phosphorylated (active) form of PDK1(Ser241) 
, were also lower in the very aged muscle. These results indicate that hyper-phosphorylation of Akt in aged muscle may be not directly related to the levels or phosphorylation of its putative upstream regulators. In an effort to understand the potential physiological significance of these findings, two important experiments, one in vitro
, the other ex vivo
, were performed. Using the in vitro
Akt kinase activity assay we demonstrated that less GSK-3 fusion protein was phosphorylated by per unit of immunoprecipitated Akt obtained from very aged muscle. This finding demonstrates that Akt kinase activity is decreased with aging (). Interestingly, this decrease in activity occurs even though the amount of Akt phosphorylation (Ser473 and Thr308) is actually increased in the aged muscle. Using an ex vivo
approach resulted in a similar conclusion as the aged control muscles required higher levels of Akt phosphorylation (both Ser474 and Thr308) to phosphorylate similar amounts of GSK3α and GSK3β compared to the acetaminophen-treated rats (). Taken together, these two different sets of experiments both suggest that aging in the F344BN soleus is associated with a mismatch between Akt phosphorylation and Akt kinase activity.
As a key molecule promoting anabolism and cellular survival, it is likely that age-associated Akt dysregulation may directly contribute to the deterioration of cellular metabolism and physiological function. In our study, the abundance of the contractile proteins, myosin and actin, were significantly decreased with aging (). Changes in actin and myosin, were in turn, associated with a decrease in mTOR, a key regulator of protein translation 
. It is possible that these decreases in myosin and actin contribute to the impairment of muscle function commonly observed with aging 
. In addition to these losses in contractile proteins, we also observed a loss of myocyte size, a dramatic increase in the abundance of Bax, a shift in the Bax/Bcl-2 ratio and an increase in the number of myocytes undergoing apopotosis (, ). Interestingly, the restoration of Akt kinase activity by chronic acetaminophen intervention was associated with increases in the amount of contractile proteins and myocyte size, and a decrease in the incidence of myocyte apoptosis ( and ). Taken together these data suggest that an impairment in Akt signaling might be related to the development of age-related metabolic and functional disorders in skeletal muscle and importantly, that the re-establishment of Akt functionality may be a useful strategy for diminishing the deleterious effects of aging on muscle structure and function.
As an important signaling molecule involved in many physiological processes, nitric oxide regulates protein structure and function by interacting with cysteine sulfhydryls and inhibiting the formation of disulfide bonds 
. Increases in the amount of cellular S-nitrosylated proteins have been shown to be associated with the disruption of protein structure along with increases in tissue damage and mortality 
. S-nitrosylated proteins, including Akt, are involved in pathogenesis of insulin resistance 
. We found that both the abundance of S-nitrosylated Akt and ratio of S-nitrosylated Akt/total Akt were substantially increased with aging (). These increases occurred concomitant to increases in the amount of iNOS, an enzyme that catalyzes the production of nitric oxide needed for the S-nitrosylation reaction 
. Although it is unclear why aging might increase iNOS levels, other reports have suggested that increased extracellular glucose levels can induce iNOS expression 
. A previous study by our laboratory demonstrated that age-associated hyperglycemia and decreases in muscle glucose transporter-4 (Glut4) can be reversed by acetaminophen intervention 
, while other work using cultured cell has shown that acetaminophen can directly inhibit NO production and iNOS expression through its ability to diminish NF-kappaB binding to the iNOS gene promoter 
. Consistent with these findings, we show that age-associated increases in iNOS expression were reversed after acetaminophen treatment, and further that this decrease in iNOS expression coincided with decreases in the amount of S-nitrosylated Akt (). As expected, this decrease in S-nitrosylated Akt was found to parallel the normalization of Akt phosphorylation and increases in Akt kinase activity. As such, it is likely that the S-nitrosylation of Akt is involved in contributing to age-associated Akt dysfunction. Unlike that observed for iNOS, acetaminophen did not appear to affect the regulation of nNOS or eNOS expression () suggesting perhaps that the normalization of Akt S-nitrosylation by acetaminophen may be dependent on the reduced expression of iNOS. Why acetaminophen may target iNOS instead of other NOS isoforms is currently unclear. Further studies designed to examine the effects of aging on individual cysteine residues within the Akt protein will no doubt be useful in furthering our understanding on how S-nitrosylation might compromise Akt kinase activity.
The phosphatidylinositol 3′-phosphatase PTEN is considered a key negative regulator of Akt signaling 
. PTEN catalyzes phosphatidylinositol 3,4,5-trisphosphate (PIP3), a key mediator of PI3K activity, into phosphatidylinositol 4,5-trisphosphate (PIP2), resulting in the attenuation of phosphorylation (activation) of Akt 
. It has been reported that the phosphorylation of Thr308 of Akt, but not Ser473, is regulated by PTEN in adipocytes 
. In present study, the abundance of pAkt-Ser473 was higher in muscles obtained from both the 33-month control and acetaminophen-treated rats, while pAkt-Thr308 was dramatically increased in control rats. These results are consistent with the alterations in PTEN protein levels and suggest that PTEN may regulate the phosphorylation of Akt Thr308 in skeletal muscle using a mechanism similar to that previously observed in adipocytes 
. Interestingly, 6 months of acetaminophen intervention restored PTEN protein levels similar to that found in 6- and 27-month rats. This increase in PTEN protein appeared to parallel decreases in the amount of Akt-Thr308 phosphorylation which support the notion that a loss of PTEN protein with aging may contribute to the hyper-phosphorylation of Akt, and that acetaminophen intervention may function in reducing Akt phosphorylation by increasing PTEN levels.
Why aging decreases PTEN protein levels is not clear. It is thought that high glucose levels can result in decreased PTEN expression and decreases in PTEN phosphatase activity 
. Our previous study found that the aging-associated hyperglycemia can be reversed by acetaminophen intervention 
. It is possible that the normalization of blood glucose by acetaminophen may contribute to the increased PTEN expression. It has also been documented that phosphorylation of the C-terminal tail of PTEN decreases the degradation of PTEN protein by increasing its stability 
. Our data also show that pPTEN levels were decreased with aging and that acetaminophen treatment restored pPTEN comparable to that observed in 6- and 27-month rats (). Therefore, aging-associated hyperglycemia and decreases in pPTEN level may result in decreased PTEN protein levels, which could act to increase the phosphorylation of Akt at Thr308 in aging muscle. Whether changes in PTEN expression alone or if the presence of other factors is required to explain the effects of aging on Akt expression and phosphorylation will require further investigation.
In summary, age-associated decreases in muscle Akt kinase function may be related to increased muscle apoptosis and atrophy, and decreases in myosin and actin expression (). Chronic acetaminophen treatment at a therapeutic dosage is able to restore the kinase activity of Akt in advancing age, which may be due to decreases in iNOS and diminished S-nitrosylation of Akt. The data of the present study show that a loss of PTEN protein in aging may contribute to the increased phosphorylation of Akt, and that acetaminophen intervention can restore both PTEN and pPTEN levels. This report provides evidence that Akt may be a key target molecule for interventions designed to attenuate the effects of aging on skeletal muscle and that acetaminophen may be useful for the treatment of age-related muscle disorders.
Proposed mechanism of age-associated Akt dysfunction and the effects of acetaminophen intervention.