AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD

JB was supported by a Research and Professional Development Grant by Gettysburg College. Luminescence, fluorescence and absorbance measurements were performed with a Synergy HT microplate reader (Bio-Tek instruments, Vinoosky VT, USA). Fibroblasts were maintained in DMEM (Biological Industries, Kibbutz Beit Haemek, Israel) medium containing 4.5 g glucose per liter and supplemented with 10% fetal calf serum, 50 µg/ml uridine, and 110 µg/ml pyruvate (GLU, permissive medium) at 37°C, 5%CO2. Representative of this type are 5-Aminoimidazole-4-carboxamide ribotide (AICAR), oltipraz, bezafibrate and sodium phenylbutyrate.

24 h after the addition of the agents, the effect of metformin on colorectal cancer cell survival was performed a cell viability assay (MTT). (B) Photographs of soft agar colonies of HCT116, RKO and HT29 cells 2 weeks after the treatment with 5 mM metformin and 5 mM AICAR. (3) Cancer cell lines were treated with different concentrations of metformin (1, 5 and 10 mM) for various time, then, the expression of active caspase-3 and LC3-II/I ratio was assessed by western blotting.

Acadesine is added to K562 cell lines or primary cells (103 CD34+ cells/mL) growing in semisolid methyl cellulose medium. MethoCult H4100 or H4236 are used for cell lines and primary CD34+ cells respectively. Colonies are detected after 10 days of culture by adding 1 mg/mL of the MTT reagent and are scored by Image J quantification software. By boosting glucose uptake and enhancing glycolysis, AICAR effectively increases cellular energy levels.

For example, AICAR (5-Aminoimidazole-4-carboxamide ribonucleoside) is the first identified AMPK agonist, which is commonly used to activate AMPK in many in vitro and in vivo studies 11, 12. Mechanistically, AICAR is converted intracellularly to ZMP, an intermediate in the late steps of de novo purine biosynthesis, which mimics AMP and activates AMPK regardless of cellular energy status 11, 13. AICAR is being used clinically to protect against cardiac ischemic injury and to improve myocardial protection in coronary artery bypass grafting 14-16. Moreover, AICAR is also a promising drug for the treatment of B-cell neoplasms and chronic lymphocytic leukemia 17-19. In contrast, Compound C (6-4-(2-Piperidin-1-ylethoxy) phenyl-3-pyridin-4-ylpyrazolo 1,5-apyrimidine) (CC), is well-known for its potent inhibitory effect on AMPK activation. In combination with AMPK agonists (e.g. AICAR), Compound C is often used as an AMPK antagonist to study AMPK-dependent cellular events 5, 20, 21.

• Our study was the first to describe the intrinsic metabolite AICAR physically binding and targeting MUC1 to induce lung tumour cell apoptosis.
• Studies on the pathogenesis of AMD indicate that inflammation is a fundamental component of the disease process, and that the alternative pathway (AP) of complement plays a critical role in driving the inflammatory response.
• Moreover, suppression of both JNK and p38 MAPK phosphorylation by metformin was also reversed by application of compound C (Fig 4C).
• However, in embryonic stem cells, AICAr increased the cell population at both G1 and non-cycling S phases 85.

Protein preparation of KGN and Western immunoblotting analysis (ECL-WB)

The excess inflammatory change is thought to impair the intrafollicular circumstances. AICAR (treated 48 hours) inhibited cell proliferation of mantle cell lymphoma (MCL) cell lines, REC-1, JEKO-1, UPN-1, JVM-2, MAVER-1 and Z-138, with IC50s of 0.28, 0.59, 0.64, 0.98, 0.50, and 0.14 Mm respectively 4. AICAR inhibited the growth and depletion of pyrimidine nucleotide pools in fibroblasts 5, accelerated repletion of purine nucleotide pools in heart 6, inhibition of fatty acid, sterol synthesis, and gluconeogenesis in hepatocytes, and increase in glucose uptake in muscle 7.

AICAR efficiently increased the AMPK Thr172 phosphorylation and decreased the mTORC1 activity; this led to an increase in autophagy. This preserved the proliferation capacity and reduced the cell size in the tested MSCs. Moreover, solely the NAM treatment significantly increased autophagy and AMPK activity and decreased the mTORC1 activity, but not as well as AICAR-alone treatment.

AICAR promotes, but Compound C inhibits, AMPK activation in T cells

Unexpectedly, 60 min of high-intensity acute exercise caused further increases in SIRT3 protein abundance in both previously sedentary and trained mice (Figure 8A). This increase in protein content was not accompanied by a parallel increase in SIRT3 mRNA in these samples relative to samples from any of the other groups (data not shown). Contrary to the AMPK α2 KD/WT exercise training study described above (Figures 2E,F), SIRT1 and catalase protein levels increased in response to exercise training. Furthermore, MnSOD protein levels increased with exercise training (Figure 8D) to a similar degree as WT mice in the AMPK α2 KD/WT training study (Figure 8D vs. Figure 2C). The fibroblasts for this study were chosen to represent CI deficiency, the most common OXPHOS defect. The cells were derived from patients with different CI defects, in order to assess individual responses to different compounds.

Figure 1.

However, mounting evidence indicates AMPK-independent effects for these chemicals and how they regulate immune cell functions remains largely unknown. Herein, using T cells from AMPK conditional knockout mice and their wild type littermates, we demonstrate that AICAR and Compound C can, indeed, activate or inhibit AMPK activity in T cells, respectively. Specifically, AICAR inhibits, but Compound C promotes, Ca2+-induced T cell death in an AMPK-dependent manner. In contrast, our data also demonstrate that AICAR and Compound C inhibit T cell activation and cytokine production in an AMPK-independent manner. Moreover, we find that the AMPK-independent activity of AICAR and steroids buy online Compound Cis mediated via the mTOR signaling pathway in activated T cells. Our results not only reveal the critical role of AMPK in regulating T cell survival and function, but also demonstrate AMPK-dependent and independent rolesof AICAR/Compound C in regulating T cell responses, thus suggesting a context-dependent effect of these “AMPK regulators”.