Metformin is the most commonly prescribed oral anti-diabetic agent worldwide. unable to be phosphorylated by PKA, increased both AMPK activation and the suppression of glucose production in primary hepatocytes treated with metformin. Intriguingly, salicylate/aspirin prevents the phosphorylation of AMPK at Ser-485, blocks cAMP-PKA negative regulation of AMPK, and improves metformin resistance. We propose that aspirin/salicylate may augment metformin’s hepatic action to suppress glucose production. test and ANOVA test. Significance was accepted at the level of < 0.05. Results The cAMP-PKA Pathway Negatively Regulates AMPK Activation Previously, we found that pretreatment of primary hepatocytes with metformin led to greater suppression of glucose production (10). Since metformin suppresses hepatic glucose production through the activation of AMPK (10, 27), we examined AMPK activation by measuring the phosphorylation of the -subunit at Thr-172, a crucial phosphorylation site in the activation of AMPK (28), in primary hepatocytes pre-treated with metformin. As shown in Fig. 1= and (and assembly assay. Purified FLAG-tagged AMPK1 and 2-fold greater concentrations of purified 1 and 1 were incubated with different concentrations (0, 0.01, 0.1, 0.5, and 1 mm) of salicylate for 1 h, then AMPK1 subunits was immunoprecipitated using anti-AMPK1 subunit specific antibody. Unlike metformin, salicylate had no effect on promoting the formation of the AMPK heterotrimeric complex (Fig. 5phosphorylation assay using LKB1-STRAD-MO25 (referred to as LKB1 kinase), and found that salicylate had a minimal effect on LKB1-mediated AMPK1 phosphorylation at Thr-172 (Fig. 5phosphorylation assay. As shown in Fig. 5and and ?and4),4), this suggests that PKA is unlikely a direct target of salicylate. However, salicylate is able to bind to an AMPK subunit (20), it is possible that salicylate binding masks the phosphorylation of AMPK at Ser-485, consequently increasing phosphorylation at Thr-172 and the formation Myricetin (Cannabiscetin) of the AMPK heterotrimeric complex. FIGURE 5. Salicylate blocks the phosphorylation of AMPK at Ser-485. animal experiment in which mice were treated with metformin and/or aspirin. This experiment was designed to test the combined effect of metformin and aspirin treatment, using therapeutic doses of metformin. We found that fasting glucose levels (6 h fasting) were significantly lower in high-fat-diet-fed mice treated with both metformin (25 mg/kg) and aspirin (10 mg/kg) compared with high-fat-diet-fed control mice (Fig. 6assays, low Myricetin (Cannabiscetin) metformin concentrations did indeed increase the assembly of the AMPK heterotrimeric complex and the phosphorylation of the subunit at Thr-172 by LKB1(23). This effect was lost when the 1 subunit was absent in the reaction, suggesting that the AMPK subunit plays an essential role in metformin-stimulated phosphorylation of the 1 subunit at Thr-172 Myricetin (Cannabiscetin) by LKB1(23). This result is in good agreement with a previous study shown that cells expressing a mutant AMPK subunit (R531G) were resistant to metformin-mediated AMPK activation (31). This evidence also indicates that AMPK subunits Rabbit Polyclonal to MSK2. are metformin’s direct targets and that metformin binding to an AMPK subunit promotes the formation of the AMPK heterotrimeric complex, making the phosphorylation site of the subunit at Thr-172 more accessible to LKB1. In the clinic, over one-third of diabetic patients exhibit either a lack of response or a delayed response to metformin treatment (3,C6) and become less sensitive to metformin over time (4). We found that PKA is able to directly phosphorylate AMPK subunit at Ser-485 (10), and several studies have documented that phosphorylation of AMPK by PKA reduces subunit phosphorylation at Thr-172 and decreases AMPK enzymatic activity (17,C19). Therefore, we hypothesized the elevated serum glucagon levels or glucagon to insulin ratios often observed in diabetes would lead to the increase in PKA-mediated AMPK subunit phosphorylation at Ser-485, and therefore, cause metformin resistance/insensitivity (11,C14). Indeed, in a pyruvate tolerance test, mice with constitutively activated PKA exhibited metformin resistance when compared with WT mice (Fig. 2, and administration of either agent alone (Figs. 6 and ?and7).7). Given that diabetic patients often have hyperglucagonemia, the combination of salicylate and metformin in a low dose might enhance AMPK activation and subsequent treatment efficacy without producing unwanted toxicity associated with high doses of these drugs. Of note, activation of AMPK by metformin and salicylate increases ACC phosphorylation (Fig. 6R00DK085142R01DK107641R01DK63349. *This work was supported in part by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health: R00DK085142 (to L. H.), R01DK107641 (to L. H.), and R01DK063349 (to F. E. W.). The authors declare that they have no conflicts of interest with the contents of this article. The content is Myricetin (Cannabiscetin) solely the responsibility of.