AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK ␣1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in ␣ subunits, we screened a chemical library with inactive human ␣1 394 (␣1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK ␣1 394 , ␣1 335 , ␣2 398 , and even heterotrimer ␣11␥1. Based on PT1-docked AMPK ␣1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 without an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.The AMP-activated protein kinase (AMPK) 3 is a highly conserved serine/threonine protein kinase that is widely expressed in higher eukaryotes, yeast, and plants and plays a unique and central role in the responses of cells to metabolic stresses such as nutrient starvation, heat shock, ischemia/hypoxia, and vigorous muscular exercise by depleting cellular ATP and elevating AMP levels (1, 2). Once activated, AMPK prevents depletion of ATP by increasing the rate of ATP generation, triggering changes in the rates of glucose transport, fatty acid oxidation, lipogenesis, sterol synthesis, and gluconeogenesis through direct regulation of key metabolic enzymes and transcriptional control of specific genes (1-4). There is mounting evidence of the involvement of AMPK in human physiological and pathological processes, especially type 2 diabetes and obesity. Previous studies indicate that several of the beneficial effects of rosiglitazone and metformin, two widely used antidiabetic drugs, are mediated by indirect activation of AMPK, suggesting the potential role of the AMPK pathway in the treatment of type 2 diabetes (5, 7). Two adipocyte-derived hormones, leptin and adiponectin, stimulate fatty acid oxidation and glucose uptake in peripheral tissues such as skeletal muscle and liver, whic...
Abstract. To identify the major serum biomarkers predicting the response to methotrexate (MTX) treatment in patients with early rheumatoid arthritis (RA), we evaluated the relationships between the individual response to MTX and various associated factors utilizing the 1 H nuclear magnetic resonance ( 1 H NMR)-based metabolomic method. Thirty-eight early RA patients were enrolled in this cohort study, and they received MTX (10 mg/week) orally as monotherapy for 24 weeks. According to the American College of Rheumatology criteria for improvement, clinical evaluation following MTX treatment was carried out at baseline and at the end of 24 weeks. Furthermore, collected serum samples were analyzed using 600 M 1 H NMR for spectral binning. The obtained data were processed by both the unsupervised principal component analysis (PCA) and the supervised partial least squares discriminant analysis (PLS-DA). Lastly, multivariate analyses were performed to recognize the spectral pattern of endogenous metabolites related to MTX treatment. Differential clustering of 1 H NMR spectra identified by PCA was found between the effective (n=25) and non-effective (n=13) group of RA patients receiving MTX treatment. Multivariate statistical analysis showed a difference in metabolic profiles between the two groups using PLS-DA (R 2 =0.802, Q 2 =0.643). In targeted profiling, 11 endogenous metabolites of the effective group showed a significant difference when compared with those of the non-effective group (p<0.05). Serum metabolites correlated with MTX treatment in patients with early RA were identified, which may be the major predictive factors for evaluating the response to MTX treatment in patients with early RA. Furthermore, our results highlight the usefulness of 1 H NMR-based metabolomics as a feasible and efficient prognostic tool for predicting therapeutic efficacy to MTX treatment. IntroductionRheumatoid arthritis (RA) is a systemic chronic inflammatory joint disease, which is characterized by persistent synovitis, systemic inflammation and autoantibodies (1). Methotrexate (MTX) is the most widely used and is regarded as the anchor drug in the treatment of RA. Despite the advent of newer biologic therapies, MTX retains its central role since it is relatively inexpensive, broad experience with its use exists, and it is widely used in combination regimens with other disease-modifying antirheumatic drugs (DMARDs) (2). In the US and European countries, the recommended general dose of MTX is 15-20 mg/week, but individual optimal dose is in the range of 5-25 mg/week. In China, the conventional dose of MTX is 10 mg/week, but in practice 5-20 mg/week is prescribed based on individual sensitivity to and tolerance of MTX (3,4). Although well proven, it is recognized that there are large individual differences in the optimal dose of MTX for RA patients (5). The reasons for those individual differences are thought to be different concentrations of intracellular MTX-polyglutamates (MTX-PGs) and different enzyme activities at MTX-active sit...
Neural stem cell differentiation and the determination of lineage decision between neuronal and glial fates have important implications in the study of developmental, pathological, and regenerative processes. Although small molecule chemicals with the ability to control neural stem cell fate are considered extremely useful tools in this field, few were reported. AICAR is an adenosine analog and extensively used to activate AMP-activated protein kinase (AMPK), a metabolic "fuel gauge" of the biological system. In the present study, we found an unrecognized astrogliogenic activity of AICAR on not only immortalized neural stem cell line C17.2 (C17.2-NSC), but also primary neural stem cells (NSCs) derived from post-natal (P0) rat hippocampus (P0-NSC) and embryonic day 14 (E14) rat embryonic cortex (E14-NSC). However, another AMPK activator, Metformin, did not alter either the C17.2-NSC or E14-NSC undifferentiated state although both Metformin and AICAR can activate the AMPK pathway in NSC. Furthermore, overexpression of dominant-negative mutants of AMPK in C17.2-NSC was unable to block the gliogenic effects of AICAR. We also found AICAR could activate the Janus kinase (JAK) STAT3 pathway in both C17.2-NSC and E14-NSC but Metformin fails. JAK inhibitor I abolished the gliogenic effects of AICAR. Taken together, these results suggest that the astroglial differentiation effect of AICAR on neural stem cells was acting independently of AMPK and that the JAK-STAT3 pathway is essential for the gliogenic effect of AICAR.Neural stem cell differentiation is controlled by intrinsic regulators and the extracellular environment. In many cases, these factors act in concert to confer potent change in cell lineages.
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