Mechanism of Activation of AMPK by Cordycepin

Cordycepin (3′-deoxyadenosine) is a major bioactive agent in Cordyceps militaris, a fungus used in traditional Chinese medicine. It has been proposed to have many beneficial metabolic effects by activating AMP-activated protein kinase (AMPK), but the mechanism of activation remained uncertain. We report that cordycepin enters cells via adenosine transporters and is converted by cellular metabolism into mono-, di-, and triphosphates, which at high cordycepin concentrations can almost replace cellular adenine nucleotides. AMPK activation by cordycepin in intact cells correlates with the content of cordycepin monophosphate and not other cordycepin or adenine nucleotides. Genetic knockout of AMPK sensitizes cells to the cytotoxic effects of cordycepin. In cell-free assays, cordycepin monophosphate mimics all three effects of AMP on AMPK, while activation in cells is blocked by a γ-subunit mutation that prevents activation by AMP. Thus, cordycepin is a pro-drug that activates AMPK by being converted by cellular metabolism into the AMP analog cordycepin monophosphate.</p

Restoration of SMN in Schwann cells reverses myelination defects and improves neuromuscular function in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, primarily affecting lower motor neurons. Recent evidence from SMA and related conditions suggests that glial cells can influence disease severity. Here, we investigated the role of glial cells in the peripheral nervous system by creating SMA mice selectively overexpressing SMN in myelinating Schwann cells (Smn(−/−);SMN2(tg/0);SMN1(SC)). Restoration of SMN protein levels restricted solely to Schwann cells reversed myelination defects, significantly improved neuromuscular function and ameliorated neuromuscular junction pathology in SMA mice. However, restoration of SMN in Schwann cells had no impact on motor neuron soma loss from the spinal cord or ongoing systemic and peripheral pathology. This study provides evidence for a defined, intrinsic contribution of glial cells to SMA disease pathogenesis and suggests that therapies designed to include Schwann cells in their target tissues are likely to be required in order to rescue myelination defects and associated disease symptoms

AMPK Causes Cell Cycle Arrest in LKB1-deficient Cells via Activation of CAMKK2

The AMP-activated protein kinase (AMPK) is activated by phosphorylation at Thr172, either by the tumor suppressor kinase LKB1 or by an alternate pathway involving the Ca(2+)/calmodulin-dependent kinase, CAMKK2. Increases in AMP:ATP and ADP:ATP ratios, signifying energy deficit, promote allosteric activation and net Thr172 phosphorylation mediated by LKB1, so that the LKB1-AMPK pathway acts as an energy sensor. Many tumor cells carry loss-of-function mutations in the STK11 gene encoding LKB1, but LKB1 re-expression in these cells causes cell cycle arrest. Therefore, it was investigated as to whether arrest by LKB1 is caused by activation of AMPK or of one of the AMPK-related kinases, which are also dependent on LKB1 but are not activated by CAMKK2. In three LKB1-null tumor cell lines, treatment with the Ca(2+) ionophore A23187 caused a G1-arrest that correlated with AMPK activation and Thr172 phosphorylation. In G361 cells, expression of a truncated, CAMKK2 mutant also caused G1-arrest similar to that caused by expression of LKB1, while expression of a dominant negative AMPK mutant, or a double knockout of both AMPK-α subunits, also prevented the cell cycle arrest caused by A23187. These mechanistic findings confirm that AMPK activation triggers cell cycle arrest, and also suggest that the rapid proliferation of LKB1-null tumor cells is due to lack of the restraining influence of AMPK. However, cell cycle arrest can be restored by re-expressing LKB1 or a constitutively active CAMKK2, or by pharmacological agents that increase intracellular Ca(2+) and thus activate endogenous CAMKK2. IMPLICATIONS: Evidence here reveals that the rapid growth and proliferation of cancer cells lacking the tumor suppressor LKB1 is due to reduced activity of AMPK, and suggests a therapeutic approach by which this block might be circumvented

Methotrexate promotes glucose uptake and lipid oxidation in skeletal muscle via AMPK activation

Methotrexate (MTX) is a widely used anticancer and antirheumatic drug that has been postulated to protect against metabolic risk factors associated with type 2 diabetes, although the mechanism remains unknown. MTX inhibits 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) and thereby slows the metabolism of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl-5′-monophosphate (ZMP) and its precursor AICAR, which is a pharmacological AMPK activator. We explored whether MTX promotes AMPK activation in cultured myotubes and isolated skeletal muscle. We found MTX markedly reduced the threshold for AICAR-induced AMPK activation and potentiated glucose uptake and lipid oxidation. Gene silencing of the MTX target ATIC activated AMPK and stimulated lipid oxidation in cultured myotubes. Furthermore, MTX activated AMPK in wild-type HEK-293 cells. These effects were abolished in skeletal muscle lacking the muscle-specific, ZMP-sensitive AMPK-γ3 subunit and in HEK-293 cells expressing a ZMP-insensitive mutant AMPK-γ2 subunit. Collectively, our findings underscore a role for AMPK as a direct molecular link between MTX and energy metabolism in skeletal muscle. Cotherapy with AICAR and MTX could represent a novel strategy to treat metabolic disorders and overcome current limitations of AICAR monotherapy.</jats:p

The LKB1-AMPK-α1 signaling pathway triggers hypoxic pulmonary vasoconstriction downstream of mitochondria

International audienceHypoxic pulmonary vasoconstriction (HPV), which aids ventilation-perfusion matching in the lungs, is triggered by mechanisms intrinsic to pulmonary arterial smooth muscles. The unique sensitivity of these muscles to hypoxia is conferred by mitochondrial cytochrome c oxidase subunit 4 isoform 2, the inhibition of which has been proposed to trigger HPV through increased generation of mitochondrial reactive oxygen species. Contrary to this model, we have shown that the LKB1-AMPK-α1 signaling pathway is critical to HPV. Spectral Doppler ultrasound revealed that deletion of the AMPK-α1 catalytic subunit blocked HPV in mice during mild (8% O2) and severe (5% O2) hypoxia, whereas AMPK-α2 deletion attenuated HPV only during severe hypoxia. By contrast, neither of these genetic manipulations affected serotonin-induced reductions in pulmonary vascular flow. HPV was also attenuated by reduced expression of LKB1, a kinase that activates AMPK during energy stress, but not after deletion of CaMKK2, a kinase that activates AMPK in response to increases in cytoplasmic Ca2+ Fluorescence imaging of acutely isolated pulmonary arterial myocytes revealed that AMPK-α1 or AMPK-α2 deletion did not affect mitochondrial membrane potential during normoxia or hypoxia. However, deletion of AMPK-α1, but not of AMPK-α2, blocked hypoxia from inhibiting KV1.5, the classical "oxygen-sensing" K+ channel in pulmonary arterial myocytes. We conclude that LKB1-AMPK-α1 signaling pathways downstream of mitochondria are critical for the induction of HPV, in a manner also supported by AMPK-α2 during severe hypoxia

Phosphorylation by Akt within the ST loop of AMPK-α1 down-regulates its activation in tumour cells

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser(487) in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr(172). Surprisingly, the equivalent site on AMPK-α2, Ser(491), is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr(172) phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca(2+) ionophore A23187, effects we show to be dependent on Akt activation and Ser(487) phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr(172) phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)–AMPK pathway, which would otherwise inhibit cell growth and proliferation

AMP-activated protein kinase:a cellular energy sensor that comes in twelve flavours

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is expressed in essentially all eukaryotic cells, suggesting that it arose during early eukaryotic evolution. It occurs universally as heterotrimeric complexes containing catalytic α subunits and regulatory β and γ subunits. Although Drosophila melanogaster contains single genes encoding each subunit, in mammals each subunit exists as multiple isoforms encoded by distinct genes, giving rise to up to 12 heterotrimeric combinations. The multiple isoforms of each subunit are 2R-ohnologues generated by the two rounds of whole genome duplication that occurred at the evolutionary origin of the vertebrates. Although the differential roles of these isoform combinations remain only partly understood, there are indications that they may have different subcellular locations, different inputs and outputs, and different functions. The multiple isoforms are of particular interest with respect to the roles of AMPK in cancer, because genes encoding some isoforms, such as PRKAA1 and PRKAB2 (encoding α1 and β2) are quite frequently amplified in tumour cells, while genes encoding others, such as PRKAA2 (encoding α2) tend instead to be mutated, which may in some but not all cases cause a loss of function. Thus, although AMPK acts downstream of the tumour suppressor LKB1, and some of its isoform combinations may act as tumour suppressors that restrain growth and proliferation of tumour cells, other isoform combinations may paradoxically act as oncogenes, perhaps by aiding the survival of tumour cells that are undergoing environmental stresses such as hypoxia or nutrient deprivation. This article is protected by copyright. All rights reserved.</p

Bitter Melon (Momordica charantia) Extract Inhibits Tumorigenicity and Overcomes Cisplatin-Resistance in Ovarian Cancer Cells Through Targeting AMPK Signaling Cascade

OBJECTIVE: Acquired chemoresistance is a major obstacle in the clinical management of ovarian cancer. Therefore, searching for alternative therapeutic modalities is urgently needed. Bitter melon (Momordica charantia) is a traditional dietary fruit, but its extract also shows potential medicinal values in human diabetes and cancers. Here, we sought to investigate the extract of bitter melon (BME) in antitumorigenic and cisplatin-induced cytotoxicity in ovarian cancer cells. METHODS: Three varieties of bitter melon were used to prepare the BME. Ovarian cancer cell lines, human immortalized epithelial ovarian cells (HOSEs), and nude mice were used to evaluate the cell cytotoxicity, cisplatin resistance, and tumor inhibitory effect of BME. The molecular mechanism of BME was examined by Western blotting. RESULTS: Cotreatment with BME and cisplatin markedly attenuated tumor growth in vitro and in vivo in a mouse xenograft model, whereas there was no observable toxicity in HOSEs or in nude mice in vivo. Interestingly, the antitumorigenic effects of BME varied with different varieties of bitter melon, suggesting that the amount of antitumorigenic substances may vary. Studies of the molecular mechanism demonstrated that BME activates AMP-activated protein kinase (AMPK) in an AMP-independent but CaMKK (Ca2+/calmodulin-dependent protein kinase)-dependent manner, exerting anticancer effects through activation of AMPK and suppression of the mTOR/p70S6K and/or the AKT/ERK/FOXM1 (Forkhead Box M1) signaling cascade. CONCLUSION: BME functions as a natural AMPK activator in the inhibition of ovarian cancer cell growth and might be useful as a supplement to improve the efficacy of cisplatin-based chemotherapy in ovarian cancer.published_or_final_versio

The Reflection Component from Cygnus X-1 in the Soft State Measured by NuSTAR and Suzaku

The black hole binary Cygnus X-1 was observed in late 2012 with the Nuclear Spectroscopic Telescope Array (NuSTAR) and Suzaku, providing spectral coverage over the ~1-300 keV range. The source was in the soft state with a multi-temperature blackbody, power law, and reflection components along with absorption from highly ionized material in the system. The high throughput of NuSTAR allows for a very high quality measurement of the complex iron line region as well as the rest of the reflection component. The iron line is clearly broadened and is well described by a relativistic blurring model, providing an opportunity to constrain the black hole spin. Although the spin constraint depends somewhat on which continuum model is used, we obtain ɑ_* > 0.83 for all models that provide a good description of the spectrum. However, none of our spectral fits give a disk inclination that is consistent with the most recently reported binary values for Cyg X-1. This may indicate that there is a >13° misalignment between the orbital plane and the inner accretion disk (i.e., a warped accretion disk) or that there is missing physics in the spectral models