Quantification of AICAR and study of metabolic markers after administration PMC
Quantification of AICAR and study of metabolic markers after administration PMC
Indeed, inactivation of the RCAN2 gene in mice also ameliorates age- and diet-induced obesity by causing a reduction in food intake (Sun et al. 2011). Besides AICAR targeting MUC1, our data suggest that AICAR also blocks JAK1 phosphorylation. Besides JAK1, mutant EGFR mediates MUC1-CT expression in the transgenic lung cancer mouse model.
- We initially tested this low dose AICAR injection on lean mice fed a low fat chow diet to determine the potential effects on body weight.
- As shown in Figure 5, AICAR exhibited synergistic effect with docetaxel treatment in prostate cancer cells.
- At this point, MSCs were grown in AdipoMAX™ Differentiation Media alone (control group) or AdipoMAX™ differentiation Media supplemented with AICAR at 1 mM or NAM at 5 mM or in the presence of simultaneous AICAR and NAM (1 mM and 5 mM, respectively).
- For ITT, mice were injected intraperitoneally with 1–1.8 unit/kg of human insulin (Humulin R, Eli Lilly, Indiana, IN) after a 6-hr food removal, and glucose levels were measured at different time points (0, 15, 30, 60, 120 minutes).
AICAR and Compound C suppress T cell activation in an AMPK-independent manner
We demonstrate that in skeletal muscle, insulin, AICAR, and contraction directly regulate TBC1D1 phosphorylation. We also report the de novo identification of phosphorylation sites on endogenous TBC1D1 from mouse skeletal muscle and their comparative regulation by insulin and AICAR. However, AXIN2 was expressed at a readily detectable level in these cells, compared to MEFs, HEK293 cells, and the liver (Supplementary information, Fig.S1h, i), implying that in HEK293T cells AXIN2 was compensating for AXIN1 when the latter was depleted. Moreover, knockdown of AXIN1 in HEK293T cells elevated the levels of AXIN2 (Supplementary information, Fig. S1h). We also knocked down AXIN1 in AXIN2−/− HEK293T cells and found that the activation of AMPK by glucose starvation was indeed largely abrogated (Fig. 1h). Similarly, introduction of AXIN2 into AXIN1−/− MEFs (in which AXIN2 was almost undetectable) led to a significant activation of AMPK upon glucose starvation (Fig. 1i).
AICAR treatment blocks 3D organoid growth from EGFR-mutant patient-derived xenograft and transgenic mouse lung tumour tissues. steroids in USA Combinational treatment with AICAR and EGFR and JAK inhibitors further decreases organoid formation. Collectively, our results highlight the new molecular mechanisms of AICAR that target MUC1-CT and its interactions with EGFR and JAK1 and provide a new therapeutic approach against EGFR-mutant lung cancer.
Consistently, AICAR treatment increased late apoptosis and cell death in EGFR-mutant PC9 cells (Fig. 1f). However, cell apoptosis and death did not change significantly after AICAR treatment in EGFR wild-type cell lines, A549 and H23 (Fig. 1g, h). These data suggest that AICAR induces cytotoxicity by increasing cell apoptosis in EGFR-mutant lung cancer cells.
Regulation of AMPK activity by upstream kinases
According to this possibility, increases in the expression and activity of different kinases have been observed in a variety of muscle pathologies 62, 90–92. This study provided novel evidence that SAP caused a drastic reduction in hepatic expression levels of phosphorylated AMPK in sodium taurocholate- and L-arginine-induced rodent SAP models, suggesting that AMPK may be involved in the pathogenesis of PALI (Figure 1A, Figures 8A,C). On the one hand, administration of the direct AMPK activator AICAR led to a dramatic elevation in hepatic expression levels of phosphorylated AMPK and prevented PALI in sodium taurocholate- and L-arginine-induced rodent SAP models (Figures 1A–G, Figures 7A–E, Figures 8A,C).
Intracellular staining was performed to analyze the phosphorylation of p-S6S235/236B., p-S6S240/244C. The values of MFI are shown above the histograms and representtreatments of no P/I control, P/I+DMSO, P/I+CC and P/I+AICAR from left to right, respectively. In summary, our data indicate that AICAR, a direct AMPK activator, exhibits significant therapeutic effects against PALI in sodium taurocholate- and L-arginine-induced rodent models by promoting AMPK phosphorylation by effectively inhibiting hepatic oxidative stress and inflammation. Thus, as a cell permeable nucleoside, AICAR has high therapeutic value for the treatment of PALI. Importantly, this study provides new insight into the mechanisms underlying the improvement of hepatic oxidative stress and inflammation in PALI by AICAR.
AICAR-stimulated PAS-160 Phosphorylation—AICAR-stimulated activation of the AMPK also causes PAS-160 phosphorylation (11, 12, 14). We determined the time course of AICAR-stimulated PAS phosphorylation and found that PAS-160 and AMPK Thr-172 phosphorylation in both soleus (Fig. 3A) and tibialis anterior (Fig. 3B) muscles were maximal at 30 min. Using lysates from the 30-min time point, we determined that AICAR-stimulated PAS-160 phosphorylation was greatest in tibialis anterior muscle not soleus muscle (Fig. 3C). AMPK Thr-172 phosphorylation was greater in soleus than tibialis anterior muscle, suggesting that the lower level of PAS-160 phosphorylation in soleus was not due to less activation of AMPK. Thus, AS160 expression and PAS-160 phosphorylation with both insulin and AICAR stimulation were completely dissociated.
Accumulation of cellular ROS was measured at P5 and the end of P10, after incubation with AICAR, NAM, and both AICAR and NAM. The level of cytoplasmic ROS was evaluated with reactive oxygen species detection assay kit (Abcam, Cambridge, MA, USA). Following the manufacturer’s instruction, the cells were washed with PBS, suspended, and stained in a conical test tube with 20 μM 2′,7′-dichlorofluorescin diacetate (DCFDA) in the supplemented buffer (10% FBS in the buffer 1X) and incubated for 30 min at 37 °C in the dark.
