AICAR ameliorates high-fat diet-associated pathophysiology in mouse and ex vivo models, independent of adiponectin

Additionally, it has been demonstrated that while there is no difference between the aged and young levels of Thr172 in other cells, the responsiveness and sensitivity of this phosphorylation site drop drastically in aged animals, regardless of the tissue or cell type. This decreased sensitivity and phosphorylation level is attributed either to the increased action of the corresponding phosphatases or to the decreased activity or access of upstream kinases on this residue 49, 51, 58, 63. One explanation would be the age-related mitochondrial defects, leading to a gradual disturbance of energy balance and AMP level 48, 49. In turn, AMPK cannot be efficiently activated anymore, causing a gradual decline in autophagy. An additional reduction of autophagy leads to the accumulation of dysfunctional mitochondria, which also increases the cellular ROS level.

• Methotrexate administration had no effect on the therapeutic activity of AICAR in the study.
• Our results provide the first direct evidence of the beneficial effects of pharmacological activation of AMPK by AICAR against the progression of PALI, including reduced redox stress and decreased NLRP3 inflammasome activation.
• By contrast, ACC2 appears to be phosphorylated only under conditions of more severe nutrient stress (lack of both glucose and glutamine), when fatty acid oxidation may need to be facilitated as an alternate energy source.
• Overall, our data indicate that the treatment of MSCs with AICAR and NAM maintained and improved their multi-lineage differentiation capacity, which otherwise deteriorated after a long-term in vitro culture.

In these studies a 2-wk interim between AICAR administration and behavioral tests was implemented to determine if changes may last beyond acute administration. In the present experiments brief administration (3 d) also enhanced performance in young mice in the water maze when training commenced 2 wk later. Interestingly, in aged mice, 14 d rather than 3 d of treatment were needed to observe cognitive benefits. The ROC analysis was conducted for several substances with significant up- and down-regulation of differential metabolites in group A2 and group B, and the AUC results were almost 1. 15 shows that these substances can effectively distinguish between samples taken after 16 hours of AICAR administration and pre-administration samples.

Are there health risks to using AICAR?

In group 4, treated with HFD + AC 1, on the 35th day of the study (week 5), food intake was significantly higher compared to animals from group 3 (HFD + vehicle). In group 5 (HFD + AC 7), food intake was higher compared to group 3 (HFD + vehicle) on the steroids 49th day (seventh week) (Table 4). Additionally, intragroup differences were observed in all the groups relative to the 7th and 21st days of the study, except for group 4 (HFD + AC 1) (Table 2). Starting from the fifth (groups 1, 2, 3, 6) week or from the seventh (group 2) week, the animals showed an increase in food consumption relative to the first week of the study.

4. AICAR via Activating AMPK Inhibits the NF-κB Pathway to Attenuate Liver Injury and Fibrosis in BDL Rats

Polyethylenimine (Polysciences, Inc., Cat. #23966) at a final concentration of 10 μM was used to transfect HEK293T cells. Lentiviruses for infection of the MEFs were packaged in HEK293T cells using Lipofectamine 2000 transfection. LAMTOR1F/For AXINF/F MEFs were established by introducing SV40 T antigen into primary cultured embryonic cells from a mouse litter. LAMTOR1−/− or AXIN−/− MEFs were generated by infecting LAMTOR1F/F or AXINF/F MEFs with adenovirus expressing Cre recombinase for 12 h. The infected cells were then incubated in fresh DMEM for another 8–10 h before further treatments.

Annexin V flow cytometry assay

The results show that the concentration of AICAR significantly increases within 16 hours after taking the medicine, which can be used as a reference time point for drug metabolism clearance rate (Fig. 9). Accuracy and precision of quality control (QC) level should be within ±15–20% (20% only for the lowest concentration point of the standard). No carry-over effect was observed by analyzing the reconstituted solvent injected after the highest calibration standard sample concentration ( ng mL−1). The autosampler and column temperatures were set to 4 °C and 40 °C, respectively, with an injection volume of 4 μL. The samples were run using a stratified method, with samples from different subjects placed in a random order.

Here, we show that, depending on the degree of elevation of cellular AMP, distinct compartmentalized pools of AMPK are activated, phosphorylating different sets of targets. Low glucose activates AMPK exclusively through the AMP-independent, AXIN-based pathway in lysosomes to phosphorylate targets such as ACC1 and SREBP1c, exerting early anti-anabolic and pro-catabolic roles. Moderate increases in AMP expand this to activate cytosolic AMPK also in an AXIN-dependent manner. In contrast, high concentrations of AMP, arising from severe nutrient stress, activate all pools of AMPK independently of AXIN. Surprisingly, mitochondrion-localized AMPK is activated to phosphorylate ACC2 and mitochondrial fission factor (MFF) only during severe nutrient stress. Our findings reveal a spatiotemporal basis for hierarchical activation of different pools of AMPK during differing degrees of stress severity.

Our data suggest that relative to insulin, AICAR increased phosphorylation of Ser-231, Ser-660, and Ser-700 and that Thr-253 phosphorylation may be greater in insulin-treated samples. However, in contrast to the other phosphorylation sites we identified, Thr-253 is not conserved in humans. Thr-499 phosphorylation was only detected with AICAR stimulation, and Ser-621 phosphorylation was similar between AICAR and insulin. Although combined amino acid coverage between AICAR- and insulin-stimulated TBC1D1 exceeded 97%, we did not initially achieve coverage of the TBC1D1 PAS site, Thr-590, perhaps due to modification of the epitope by the PAS antibody.

Mice were euthanized by cervical dislocation immediately after the cessation of contraction, and tibialis anterior muscles were immediately dissected and snap-frozen in liquid nitrogen. The hydroxyproline assay was performed after 4 weeks of modelling in all three groups to determine collagen deposition in the liver (Hydroxyproline Assay Kit; Sigma-Aldrich). The hydroxyproline concentration was determined by comparing the absorbance of the samples to the standard curve at 560nm. HEK293T, HEK293 and MEFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, cat. 11965) supplemented with 10% fetal bovine serum (FBS), 100 IU penicillin, 100 mg/ml streptomycin at 37 °C in a humidified incubator containing 5% CO2.

A metabolic pathway for the synthesis of glucose from precursor substrates such as lactate and amino acids. A very common disease in humans in which there is an excessive accumulation of fat in the liver (steatosis) in individuals who are not alcoholic. Bone marrow (BM) was flushed from the femur and tibia, dispersed, and cultured in DMEM containing 10% FBS and 30% L929 conditional medium for 8 days. Peritoneal macrophages were isolated by lavage 4 days after intraperitoneal injection of 3% thioglycollate (2 ml; Difco, BD Biosciences, San Jose, CA). The cells were plated at a density of 1.2×106 cells/well in 6-well plates and cultured in RPMI 1640 medium containing 10% heat-inactived FBS. For treatment with Fatty acids to induce ER stress, stearate (Sigma-Aldrich, St. Louis, MO) was conjugated with BSA at a 4∶1 molar ratio.