The most significant results of this study suggest that a profound loss of constitutive Akt activity occurs in aging hepatocytes. Akt phosphorylation state, which is largely reflective of Akt activity, appears to be the primary lesion responsible for this marked decline. Taken to its logical extent, the data presented in this study further suggests that there is a link between Akt activity, age, and perhaps also oxidative stress resistance. These changes would be expected to render hepatocytes susceptible to a variety of stresses, which otherwise would be successfully repulsed. Thus, our data extends the concept of Akt as a significant regulator of cellular stress response to show that its loss of function with age may portend a decline in the capacity to resist a variety of oxidative, xenobiotic, and environmental insults.
The systematic loss of Akt activity is somewhat surprising, given that Akt activation regulates permissiveness toward apoptosis, which could therefore lead to depletion of cells especially in post-mitotic organs. However, downregulation of Akt activity may be balanced by greater FOXO-mediated transcription that would eventually lead to lower metabolism and upregulation of cellular antioxidants (Morris 2005
), even in cells that are not undergoing continuous mitosis. This period of cell cycle arrest may have effects similar to caloric restriction (currently the best-known method of achieving longevity), and the dauer hibernation-like state that takes place in C. elegans
(Kenyon et al. 1993
). Thus, Akt may have a dual role in stress response and aging. Lower Akt activity may predispose the cell to death from oxidative or toxic stress, but in so doing, may also limit other more onerous risks to survival. For example, the prevalence of cancer increases with the 4th power of age (Ames et al. 1993
), suggesting that lowered Akt activity could balance cell death with prevention of neoplasia, a compromise that is imperative for survival of the individual. Under advantageous conditions, cells from the aged animal survive, but under stress, apoptosis may prevail.
Regardless of the consequences, the underlying mechanism(s) responsible for diminished Akt activity with age lies in its clearly lower constitutive phosphorylation state. Phosphorylation loss had a very age-specific pattern. There was no age difference in Thr308 phosphorylation, but phosphorylation at the Ser473 residue in hepatocytes from old animals was significantly lower than that seen in cells from young rats. This is in accord with Li et al. (2003)
, who showed an age-related decrease in Akt phosphorylation in mouse skeletal muscle. Further analysis of our data revealed that the decline we observed was not due to hyper-activation of either of two relevant protein phosphatases, as there were no age-related differences in either PP2A or PTEN activities in hepatocytes. Because phosphorylation at the Thr308 site was not affected with age, we argue that the aging lesion must reside in signaling processes that ultimately phosphorylate the Ser473 site of Akt. However, the precise mechanism involved was not explored in the present study, as multiple kinases and signaling pathways recognize and contribute to Ser473 phosphorylation. Despite the often conflicting and controversial literature on the governance of Akt phosphorylation at Ser473, it must be recognized that phosphorylation at this site can be blocked by LY294002, a PI3K inhibitor, and so it was in our study. Thus, any signals generated through IR-mediated signal transduction pathways or those that trigger PI3K phosphorylation, may contribute to age-specific loss of Akt phosphorylation. The precise signaling mechanism(s) associated with loss of Ser473 phosphorylation represent a significant future focus in identifying the loss of Akt-dependent survival mechanisms with age.
In relation to the general control that PI3K affords to Akt phosphorylation, we found that LA reintroduces Akt phosphorylation and its activity specifically through the PI3K pathway (). Indeed, phosphorylation of Akt on Ser473 in hepatocytes from old rats is the same as observed in young animals after treatment with relatively small concentrations of LA (50 μM) (). These results are in accord with our previous work that showed a similar improvement in Akt activity in endothelial cells (Smith and Hagen 2003
), and also work done in isolated monocytes (Zhang et al. 2007
). Though the insulin pathway that stimulates Akt phosphorylation on both sites appears to be intact in hepatocytes from both young and old animals (), LA did not act as an insulin mimetic in the absence of insulin (). Interestingly, LA alone stimulated Akt phosphorylation to a greater degree in cells from old animals, suggesting that there is indeed an age-related lesion that is remediated by LA. However, none of these studies revealed the precise mechanism involved in LA-dependent Akt activation.
In this regard, it is interesting to note that the age-related improvement in Akt activity also agrees with a larger body of work showing orally supplied LA stimulates insulin receptor (IR) activation, thus initiating insulin dependent signal transduction pathways and cell survival systems (Yaworsky et al. 2000
). In fact, LA has been used as a diabetes therapy in Germany for 50 years because of its insulin mimetic effects on the insulin/IGF-1 pathway and ability to improve glucose transport (Evans and Goldfine 2000
). Recently, one group has shown a putative binding pocket for LA on the IR (Diesel et al. 2007
), the importance of which is yet to be elucidated. Thus, LA may work in part, via a direct action on upstream kinases from Akt.
In addition to its activation of PI3K-mediated Akt phosphorylation, there is increasing evidence that LA inhibits specific phosphatases, including those that have direct relevance to Akt Ser473 phosphorylation. Cho et al. showed that LA inactivates the tyrosine phosphatase, PTP1B, in vitro (Cho et al. 2003
), which halts insulin-generated signals by dephosphorylating proteins of the insulin pathway. We previously showed that LA lowers PP2A activity in isolated aortic endothelial cells by inhibiting neutral sphing-omyelinases (nSMase) and subsequent ceramide-induced activation of this particular phosphatase (Smith et al. 2006
). It has also been shown that expression of wild type PTEN in rhabdomyosarcoma and myoblast cell lines reduces Akt phosphorylation at Ser473 but not Thr308 (Wan and Helman 2003
), and the authors suggest a role for regulation of the putative PDK2 proteins by PTEN, which would target Ser473 Akt specifically. Overall, this prior evidence fits well with our present results showing that LA improves Akt phosphorylation state and activity by simultaneously
increasing PI3K-dependent signaling and inhibiting PTEN and PP2A phosphatase action (). In fact, the data showing LA-induced gain of PP2A phosphorylation on Tyr307 and loss of phosphorylation of PTEN on Ser380, both of which are inhibitory, indicates that the observed phosphorylation of Ser473 Akt in response to LA may be in part a function of its action on phosphatases. The relatively slow timing of LA-induced Akt phosphorylation in insulin-replete medium () also supports this contention.
The marked improvement of Akt activity and cell survival by LA treatment in hepatocytes from aged rats adds weight to the growing body of evidence that LA supplementation may be a useful adjunct to maintaining stress resistance mechanisms in aging. Showing the involvement of Akt in LA-mediated cell survival and reduced susceptibility to oxidative stress helps to establish a mechanism for this phenomenon. It is notable that LA does not induce Akt phosphorylation in isolated cells from young animals at the low concentration used, which is a level likely to be achieved in the blood plasma following oral supplementation (Petersen Shay et al. 2008
). However, there is a noticeable increase in Ser473 Akt phosphorylation in liver tissue from young animals () and the difference may be that the isolated cells are cultured in insulin-replete medium. We have also found that substantially higher and potentially supraphysiological levels of LA do stimulate Akt activity in vitro for cultured cells from young animals (data not shown). Thus, LA appears to induce a compensation for the constitutive decline in Akt activity, preserving this enzyme’s critical function which otherwise declines with age.
The reversible rather than inducible nature of LA action is an important concept in two ways, namely, (i) LA action must stimulate pathways distinct from the aging lesion, thereby compensating for the age-related loss of Akt phosphorylation, i.e., PTEN and PP2A; and (ii) LA improves resistance to Akt-driven stress response mechanisms without associated problems that may arise from chronic overstimulation of Akt activity. Thus, the risk for neoplasia that is associated with Akt overexpression would be minimized. The net effect of LA on Akt phosphorylation in cells from old animals is a reversal of loss, and is age-specific. This study thus presents a rationale for future work to examine the benefits of LA and other allied micronutrients to maintain Akt-driven stress response mechanisms in the elderly.