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  • br Material and methods br Results br

    2024-02-19


    Material and methods
    Results
    Discussion In order to find new and improved ways of treating type 2 diabetes (T2D) and other related conditions it is vital that investigators gain a more in-depth understanding of the effects that anti-diabetic treatments, such as TZDs, have on molecules implicated in the disease. In 2014, Saha et al. emphasized the importance of AMPK as a potential therapeutic target and, although TZDs have been widely studied, the mechanisms that link these drugs to certain target molecule are not yet fully understood [4]. Although it has long been presumed that troglitazone's effect on AMPK was directly a result of Thr172 phosphorylation [20] we have shown that the reality is not as simple. In this study we first sought to discover whether troglitazone treatments increase AMPK phosphorylation at Thr172 in HepG2 olopatadine hcl as it has been shown to in other cell types. We found that, not only does AMPK phosphorylation at this location increase significantly over time, the trending increase is seen as early as 5 min, and it says elevated for up to 12 h incubation (Fig. 1A and B). These data support the growing body of research describing the increased Thr172 phosphorylation of AMPK after with TZD treatments on both an acute (minutes) and extended (hours) time scale [14], [19]. Phosphorylation of ACC at Ser79, a site directly phosphorylated by AMPK, was found to increase in a time-dependent manner similar to the increase p-AMPK Thr172, with the earliest increase seen also at 5 min (Fig. 1A and D). These data support the aforementioned idea that TZDs improve AMPK activity levels in tissues including HepG2 cells. It is, however, important to note that, although ACC phosphorylation remained elevated through an extended time course, up to 6 h, there was an observable decrease at by 12 h (Fig. 1A and D). This suggests the possibility that other factors involved in AMPK regulation come into play over a longer time-scale and provides an area of future investigation into what these factors may be. The lesser-known site of AMPK phosphorylation at Ser485 has more recently been implicated as a potential target for T2D treatment, and a large number of studies are showing that phosphorylation at this site can inhibit AMPK, regardless of whether the molecule is phosphorylated at Thr172[8], [14], [16]. Due to the increasing emphasis in the literature, we sought to discover whether troglitazone affect this site in HepG2 cells. To our knowledge, the effects of TZD treatments on p-AMPK Ser485 have not been examined prior to this study. We observed a marked increase in phosphorylation of AMPK at Ser485 which peaked at 2 h yet lasted for up to 12 h (Fig. 1A and C). As with Thr172, an upward trend of p-AMPK Ser485 phosphorylation can be seen prior to one hour incubation beginning as early as 5 min, although the results were not statistically significant potentially a result of the relatively small sample size (Fig. 1C). These results provide multiple points of interest. To begin with, this is the first study, to our knowledge, that shows a concurrent increase in phosphorylation at the Thr172 and Ser485 sites on AMPK. Previous studies have found these two sites to be phosphorylated either in an opposing manner (with one increasing and the other decreasing) [7], or in a manner when one is affected while the other is not [8], [9]. The results from this study provide even more insight into the potentially disparate mechanisms of phosphorylation, showing that phosphorylation at the two sites is neither time- nor dose-related, nor dependent on the level of phosphorylation at the other site. Interestingly, despite this increase at both sites, the net result was found to increase AMPK activation, a reasonable, assumption given that the increase in Thr172 phosphorylation was greater than or equal to that of Ser485 at almost every time point and dose measured (Fig. 1, Fig. 2). After determining that troglitazone treatment not only phosphorylates Thr172 but also Ser485 on AMPK, the next step was to consider the potential upstream kinase(s) responsible for these actions. Several upstream kinases have been identified that phosphorylate AMPK at Ser485/491 and inhibit its activity in various tissues. We first decided to focus on our most recent research that found PKD1 (a member of the PKC family of kinases) is a novel upstream kinase of Ser485 in C2C12 skeletal muscle cells and mouse EDL muscle [16]. In the previous study, PKD1 was activated by treatment with PMA, a mimetic of DAG and known PKC activator [16]. In this study the same conditions were used with HepG2 cells in order to determine whether this newly elucidated mechanism occurred in tissue other than skeletal muscle. Our data support the results of the previous study, finding that a 30 min incubation with 50 nM of PMA did, as before, effectively increase levels of p-PKD1 Ser916 as well as levels of p-PKD1 Ser485 and decrease levels of p-AMPK Thr172 (Fig. 4A–D). In order to establish overall AMPK activation p-ACC Ser79 was measured and found to significantly decrease after PMA treatments, supporting our previous assertion that AMPK activity can be diminished via PKD1 phosphorylation at Ser485 (Fig. 4A and E) [16]. A concurrent increase in p-AMPK Ser485 was found in HepG2 cells treated with troglitazone, however a lack of p-PKD1 indicates that there must be a different upstream kinase eliciting the same effect after treatment with troglitazone.