Akt is another upstream kinase known to phosphorylate
Akt is another upstream kinase known to phosphorylate AMPK Ser489 in response to high insulin levels , . HepG2 Cinobufagin show an increase in phosphorylation of AMPK at Ser485 when incubated with either insulin or troglitazone individually (Fig. 5A and C). Despite this concurrent increase, only cells treated with insulin demonstrated an increase in p-Akt, indicating again that this is likely not the agent responsible for Ser485 phosphorylation in this scenario (Fig. 5A and B).
As mentioned previously, we expanded our search for an upstream kinase to include other known AMPKSer485/491 kinases; PKCs ι, λ, δ, and θ as well as IKKβ and p70s6k, however, no significant changes in response to troglitazone treatments were observed (data not shown). Finally, protein kinase A (PKA) has also been reported to phosphorylate this site in INS-1 cells in response to forskolin or GIP stimulation and could be a potential target of investigation for future experiments . Inhibition of AMPK through this mechanism by multiple kinases suggests a biological need to maintain control over AMPK activity. Whether more, unknown kinases also phosphorylate AMPK at Ser485/491 to modulate its activity and biological functions remain to be seen.
When working with TZDs it is important to understand that troglitazone is only one of three, the others being rosiglitazone and pioglitazone. Although used primarily as anti-diabetic agents, the TZDs are known to have slightly differing effects and thus should all be considered when investigating the effects of TZDs. In our current study we found that, at equal concentrations (50 μM) and length of incubation (1 h), troglitazone is significantly more effective than either rosiglitazone or pioglitazone both in activating the AMPK molecule, and in phosphorylating AMPK at Ser485 (Fig. 5). Prior studies have shown rosiglitazone and pioglitazone to increase AMPK Thr172 phosphorylation in various cell types; however the concentrations and time-courses in which these changes occur vary vastly. Pioglitazone has been found to increase AMPK Thr172 at concentrations of 200 μM for 10 min in platelets  and at 30 μM for up to 48 h in vascular smooth muscle . Our data contribute to the idea that the TZDs act differently depending on their incubation and indicate that troglitazone has the most potent effect on AMPK in HepG2 cells at our measured dose and time. In order to expand on these results, more extensive dose-response and time-course experiments are recommended on HepG2 cells in the future, as variations in cellular response to different drugs may be vital when deciding which specific TZD to use in a clinical setting.
New mechanisms of AMPK Ser485 phosphorylation and their effects on the activity of AMPK are an exciting and important aspect in understanding the function of AMPK and its role in T2D and other metabolic diseases. Understanding the effects of TZDs as well as other drugs on AMPK is also an important area of future research, particularly due to the increasing levels of the disease both in the United States and worldwide . The more we understand the mechanisms of action of both the disease and the drugs used to treat it, and the further we are able to pinpoint the molecular basis of their positive and negative effects, the better we will become at providing safer and more effective medications.
Acknowledgements This work was supported by grants from the National Institutes of Health, USA (DK19514and DK67509 to NBR and AKS).
Introduction Ultra-violet (UV) radiation (UVR) to human retinas can induce injury to the resident retinal pigment epithelium (RPE) cells [, , ]. UVR induces reactive oxygen species (ROS) production and oxidative stress, causing lipid peroxidation and DNA damage, and eventually RPE cell death and apoptosis [, , , , , , ]. Reversely, ROS scavenging and oxidative stress inhibition shall protect RPE cells and other retinal cells from UVR [5,, , , , , ]. Our previous study has shown that MIND4-17, a nuclear-factor-E2-related factor 2 (Nrf2)-targeting thiazole-containing compound, can protect RPE cells from UVR via inhibiting oxidative stress .