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

The expression of active caspase-3, p-AMPK and p-mTOR were analyzed by Western blotting. AICAR and metformin were purchased from Toronto Research Chemicals (ON, Canada) and Sigma-Aldrich (MO, https://colegioaquinta.com/trenbolone-a-comprehensive-guide/ USA) respectively. Compound C and JNK-IN-8 were from Millipore (MA, USA) and Selleck (China) respectively.

We also determined that the areas for the subpolysomal and polysomal portions of the traces were significantly less in the obese (OC and OA) mice (Table 2). The subpolysomal-to-polysomal ratio was higher in OC and OA than LC and LA mice (Table 2) and higher in LA than LC mice. We observed higher 80S peak areas in the lean (LC and LA) than obese (OC and OA) mice, and the 80S peak area did not increase with AICAR treatment in OA compared with OC mice (Table 2). 5-Aminoimidazole-4 -carboxamide-1-β-D-ribofuranoside (AICAR) is an activator of AMPK that also decreases mTORC1 signaling; it displays anticancer, antihypertensive, anti-diabetic, and anti-inflammatory activities. AICAR enhances the efficacy of rapamycin in several cancer cells and suppresses signaling by HER2 and EGFR2 in breast cancer cells.

Should AICAR regulate the activity of all the active enzymes involved in sperm motility, its presentation may have the potential to provide a profound positive effect on fertilization. AICAR represses the MUC1 activity in EGFR-mutant lung cancer, disrupting protein–protein interactions between MUC1-CT and JAK1 and EGFR. The human colorectal carcinoma cell lines HCT116, RKO and HT29 were purchased from ATCC (LGC Standards SLU, Barcelona, Spain).

The mixture was incubated for 15 min at room temperature in the dark and then analyzed by FACSCalibur Flow Cytometer (BD Biosystems, Heidelberg, Germany). For experiments, cells were incubated in the presence of 0.25 mM palmitate (palmitate-challenged condition) or in the absence of palmitate (standard culture condition) for indicated time. Antibodies against p53 phosphorylated at serine 15 was obtained from New England Biolabs, UK. Whole cellular protein extracts were resolved in reducing and denaturing conditions by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Is AICAR safe for human use?

Both human and murine lymphocytes (CD3+CD45+) were characterised as T helper (% CD4+ of CD3+CD45+) and cytotoxic T cells (% CD8+ of CD3+CD45+). In murine tissues, F4/80+ macrophages (CD45+F480+) were subcategorised as inflammatory M1 macrophages (% CD11c+ of CD45+F480+) or M2 macrophages (% CD206+ of CD45+F480+). In human WAT, the mononuclear/macrophage population (CD45+) was identified as M1 macrophages (% CD11c+ of CD45+), M2a macrophages (% CD206+ of CD45+), M1/M2b macrophages (% CD86+ of CD45+) or M2a/M2c macrophages (% CD163+ of CD45+). Gating was determined using Fluorescence-Minus-One controls (see ESM Methods for further details). The aims of this study were to evaluate the therapeutic potential of AICAR for the promotion of metabolic health and reduction of liver and kidney disease in mice fed a high-fat diet (HFD) and to determine if such protection was dependent on adiponectin. Since white adipose tissue (WAT) inflammation is a key driver of obesity-related pathophysiology 25,26,27,28,29, this was characterised in detail.

• Flow cytometry analysis showed that AICAR-treated cells had the highest while NAM-treated cells had the lowest level of ANNEXIN-positive cells, even less than a third of the AICAR-treated MSCs.
• Colorectal cancer (CRC) is still the third most common cancer and the second most common causes of cancer-related death around the world.
• Despite these limitations, our discovery in AICAR paves a new way to block lung tumour growth by blocking MUC1 and its interacting proteins including JAK1 and EGFR.
• AICAR did not affect the total number of renal macrophages in the mouse model used, but rather shifted their phenotype towards resolution by reducing the percentage of CD11c+ M1 macrophages.

Upon confluence, serum was depleted for 24 hours and then cells were treated accordingly. In summary, to delay the senescence, increasing autophagy by selective inhibition of mTORC1 seems to be an efficient approach to employ. Additionally, mTORC1 inhibition keeps the cells sensitive to growth factors and preserves their proliferative capacity while in culture; this will help us have a higher number of young cells after in vitro expansion.

Availability of data and materials

In conclusion, the present study demonstrated that different regulations of AICAR and metformin on INS-1E cell apoptosis were caused by differences in culture conditions and downstream mediators. Our work should be informative for future investigations focusing on the effects of AMPK activators on apoptosis in other β-cell lines or primary β-cells. Given that JNK activation contributed to AICAR-induced INS-1E cell apoptosis under standard culture condition, AICAR should have synergized with palmitate to cause apoptosis. However, AICAR-mediated reversal of TG overload, activation of Akt and inhibition of p38 MAPK under palmitate-challenged condition were sufficient to compensate for the impairment caused by JNK activation. Nevertheless, TG was not overloaded under standard culture condition (without exposure of palmitate), and thus the protective part contributed by reversion of palmitate-induced TG accumulation was gone.

In this stage, the cells were grown in osteogenic induction medium alone (control group) or osteogenic induction medium 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). To evaluate the morphology of the MSCs on the tissue culture dish at passage 10, we used ImageJ software (NIH, MD, USA). Briefly, a line was drawn over the scale bar, using the “Straight free hand line,” and under the “Analyze” tab, “Set Scale” was selected. In the “Set Scale” window, the length of the known distance of the scale bar and units of measurement (500 μm in our study) was typed in the appropriate boxes. Then, under the “Analyze” tab, “Measure” was selected to calculate the cross-sectional surface area of the selected cell. This procedure was performed for at least 100 cells per study group (AICAR, NAM, AICAR+NAM, and control).

Supplemental Data

Thus, we speculate that Nrf2 and NLRP3 inflammasome pathway may mediate essential parts in the protective roles of AICAR against oxidative stress and inflammation in sodium taurocholate-induced PALI rats. In this study, our data has demonstrated that extrinsic AICAR treatment induces apoptosis and increases DNA damage in EGFR-mutant lung cancer cell lines. AICAR reduces JAK-STAT signalling by blocking physical protein–protein interactions between MUC1-CT and JAK1.

MSCs treated with AICAR, NAM, or both displayed an increase in proliferation and osteogenic differentiation, which was augmented in the group receiving both. Treatment with AICAR or NAM led to decreased expression of β-galactosidase, reduced accumulation of dysfunctional lysosomes, and characteristic morphologic features of young MSCs. Furthermore, while NAM administration could significantly reduce the total cellular ROS in aged MSCs, AICAR treatment did not. Moreover, AICAR-treated cells possess a high proliferation capacity; however, they also show the highest level of cellular apoptosis. The observed effects of AICAR and NAM were in light of the attenuated mTORC1 activity and increased AMPK activity and autophagy. We also tested the hypothesis that acetylation of OSCP on the K139 residue is a marker of cellular energy stress, and that exercised trained muscle would be less susceptible to exercise-induced changes in K139 acetylation.