AMPK activates the Nrf2-Keap1 pathway to govern dendrite pruning via the insulin pathway in Drosophila.

During Drosophila metamorphosis, the ddaC dendritic arborisation sensory neurons selectively prune their larval dendrites in response to steroid hormone ecdysone signalling. The Nrf2-Keap1 pathway acts downstream of ecdysone signalling to promote proteasomal degradation and thereby dendrite pruning. However, how the Nrf2-Keap1 pathway is activated remains largely unclear. Here, we demonstrate that the metabolic regulator AMP-activated protein kinase (AMPK) plays a cell-autonomous role in dendrite pruning. Importantly, AMPK is required for Mical and Headcase expression and for activation of the Nrf2-Keap1 pathway. We reveal that AMPK promotes the Nrf2-Keap1 pathway and dendrite pruning partly via inhibition of the insulin pathway. Moreover, the AMPK-insulin pathway is required for ecdysone signalling to activate the Nrf2-Keap1 pathway during dendrite pruning. Overall, this study reveals an important mechanism whereby ecdysone signalling activates the Nrf2-Keap1 pathway via the AMPK-insulin pathway to promote dendrite pruning, and further suggests that during the nonfeeding prepupal stage metabolic alterations lead to activation of the Nrf2-Keap1 pathway and dendrite pruning.

LncRNA LncHrt preserves cardiac metabolic homeostasis and heart function by modulating the LKB1-AMPK signaling pathway.

Metabolic modulation is a promising therapeutic approach to prevent adverse remodeling of the ischemic heart. Because little is known about the involvement of long non-coding RNAs (lncRNAs) in regulating cardiac metabolism, we used unbiased transcriptome profiling in a mouse model of myocardial infarction (MI). We identified a novel cardiomyocyte-enriched lncRNA, called LncHrt, which regulates metabolism and the pathophysiological processes that lead to heart failure. AAV-based LncHrt overexpression protects the heart from MI as demonstrated by improved contractile function, preserved metabolic homeostasis, and attenuated maladaptive remodeling responses. RNA-pull down followed by mass spectrometry and RNA immunoprecipitation (RIP) identified SIRT2 as a LncHrt-interacting protein involved in cardiac metabolic regulation. Mechanistically, we established that LncHrt interacts with SIRT2 to preserve SIRT2 deacetylase activity by interfering with the CDK5 and SIRT2 interaction. This increases downstream LKB1-AMPK kinase signaling, which ameliorates functional and metabolic deficits. Importantly, we found the expression of the human homolog of mouse LncHrt was decreased in patients with dilated cardiomyopathy. Together, these studies identify LncHrt as a cardiac metabolic regulator that plays an essential role in preserving heart function by regulating downstream metabolic signaling pathways. Consequently, LncHrt is a potentially novel RNA-based therapeutic target for ischemic heart disease.

Is TAK1 a Direct Upstream Kinase of AMPK?

Alongside Liver kinase B1 (LKB1) and Ca2+/Calmodulin-dependent protein kinase kinase 2 (CaMKK2), Transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) has been suggested as a direct upstream kinase of AMP-activated protein kinase (AMPK). Several subsequent studies have reported on the TAK1-AMPK relationship, but the interpretation of the respective data has led to conflicting views. Therefore, to date the acceptance of TAK1 as a genuine AMPK kinase is lagging behind. This review provides with argumentation, whether or not TAK1 functions as a direct upstream kinase of AMPK. Several specific open questions that may have precluded the consensus are discussed based on available data. In brief, TAK1 can function as direct AMPK upstream kinase in specific contexts and in response to a subset of TAK1 activating stimuli. Further research is needed to define the intricate signals that are conditional for TAK1 to phosphorylate and activate AMPKα at T172.

Transforming growth factor-ß-activated kinase 1 regulates angiogenesis via AMP-activated protein kinase-α1 and redox balance in endothelial cells.

OBJECTIVE: Transforming growth factor-ß-activated kinase 1 (TAK1) is a mitogen-activated protein 3-kinase and an AMP-activated protein kinase (AMPK) kinase in some cell types. Although TAK1(-/-) mice display defects in developmental vasculogenesis, the role of TAK1 in endothelial cells has not been investigated in detail. APPROACH AND RESULTS: TAK1 downregulation (small interfering RNA) in human endothelial cells attenuated proliferation without inducing apoptosis and diminished endothelial cell migration, as well as tube formation. Cytokine- and vascular endothelial growth factor (VEGF)-induced endothelial cell sprouting in a modified spheroid assay were abrogated by TAK1 downregulation. Moreover, VEGF-induced endothelial sprouting was impaired in aortic rings from mice lacking TAK1 in endothelial cells (TAK(ΔEC)). TAK1 inhibition and downregulation also inhibited VEGF-stimulated phosphorylation of several kinases, including AMPK. Proteomic analyses revealed that superoxide dismutase 2 (SOD2) expression was reduced in TAK1-deficient endothelial cells, resulting in attenuated hydrogen peroxide production but increased mitochondrial superoxide production. Endothelial cell SOD2 expression was also attenuated by AMPK inhibition and in endothelial cells from AMPKα1(-/-) mice but was unaffected by inhibitors of c-Jun N-terminal kinase, p38, extracellular signal-regulated kinase 1/2, or phosphatidylinositol 3-kinase/Akt. Moreover, the impaired endothelial sprouting from TAK(ΔEC) aortic rings was abrogated in the presence of polyethylene glycol-SOD, and tube formation was normalized by the overexpression of SOD2. A similar rescue of angiogenesis was observed in polyethylene glycol-SOD-treated aortic rings from mice with endothelial cell-specific deletion of the AMPKα1. CONCLUSIONS: These results establish TAK1 as an AMPKα1 kinase that regulates vascular endothelial growth factor-induced and cytokine-induced angiogenesis by modulating SOD2 expression and the superoxide anion:hydrogen peroxide balance.

Lipids and Inflammation: Novel Molecular Targets and Therapeutic Implications.

Atherosclerotic cardiovascular disease represents the most common cause of death worldwide. Altered cholesterol metabolism and inflammation are major cardiovascular risk factors that underpin atherosclerotic plaque growth and destabilization. While initial evidence considered dyslipidemia and inflammation as independent atherogenic actors, growing evidence has revealed that several molecular mechanisms implicated in cholesterol metabolism participate in multiple inflammatory signalling pathways. In particular, proprotein convertase subtilisin/kexin type 9, adenosine monophosphate-activated protein kinase pathway, oxidized low-density lipoproteins, and lipoprotein (a) have been demonstrated to share concurrent atherogenic and inflammatory properties. Novel lipid-lowering therapies targeting these molecular pathways have been implemented. Mechanistic and clinical studies have addressed their hypolipidemic potential and explored their role in atherosclerosis-related vascular inflammation, and ongoing randomized clinical trials are investigating their prognostic role. The purpose of this review was to dive into the signalling pathways linking cholesterol metabolism and inflammation and outline the current evidence on the anti-inflammatory activities of the novel lipid-lowering drugs.

Pathogenesis-related protein 1 suppresses oomycete pathogen by targeting against AMPK kinase complex.

INTRODUCTION: During the arms race between plants and pathogens, pathogenesis-related proteins (PR) in host plants play a crucial role in disease resistance, especially PR1. PR1 constitute a secretory peptide family, and their role in plant defense has been widely demonstrated in both hosts and in vitro. However, the mechanisms by which they control host-pathogen interactions and the nature of their targets within the pathogen remain poorly understood. OBJECTIVES: The present study was aimed to investigate the anti-oomycete activity of secretory PR1 proteins and elaborate their underlying mechanisms. METHODS: This study was conducted in the potato-Phytophthora infestans pathosystem. After being induced by the pathogen infection, the cross-kingdom translocation of secretory PR1 was demonstrated by histochemical assays and western blot, and their targets in P. infestans were identified by yeast-two-hybrid assays, bimolecular fluorescence complementation assays, and co-immunoprecipitation assay. RESULTS: The results showed that the expression of secretory PR1-encoding genes was induced during pathogen infection, and the host could deliver PR1 into P. infestans to inhibit its vegetative growth and pathogenicity. The translocated secretory PR1 targeted the subunits of the AMPK kinase complex in P. infestans, thus affecting the AMPK-driven phosphorylation of downstream target proteins, preventing ROS homeostasis, and down-regulating the expression of RxLR effectors. CONCLUSION: The results provide novel insights into the molecular function of PR1 in protecting plants against pathogen infection, and uncover a potential target for preventing pre- and post-harvest late blight.

Metformin: (future) best friend of the radiation oncologist?

Several molecules are being investigated for their ability to enhance the anti-tumor effect of radiotherapy. The widely prescribed antidiabetic drug metformin has been suggested to possess anti-cancer activity; data indicate that metformin could also enhance radiation sensitivity. The purpose of this review is to summarize current knowledge on the specific effect of metformin in the field of RT, while also discussing the many unknowns that persist. Preclinical models point to multiple mechanisms involved in the radiosensitizing effects of metformin that are mainly linked to mitochondrial complex I inhibition and AMP-activated protein kinase. Transposition of results from bench to bedside will be discussed through the lens of the drug concentration, its potential limits in human settings, and possible alternatives. Clinical data suggest metformin improves progression-free and overall survival in patients for many different cancers treated with RT; nevertheless, the results are not always consistent. The main limitations of the reviewed literature are the retrospective nature of studies, and most of the time, a lack of information on MTF treatment duration and the administered dosages. Despite these limitations, the possible mechanisms of the role of metformin and its utility in enhancing radiotherapy treatments are analyzed. Ongoing clinical trials are also discussed.

Theaflavin TF3 Relieves Hepatocyte Lipid Deposition through Activating an AMPK Signaling Pathway by targeting Plasma Kallikrein.

Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming the leading cause of chronic liver diseases throughout the world. The deficit of pharmacotherapy for NAFLD calls for an urgent need for a new drug discovery and lifestyle management. Black tea is the most popular and functional drink consumed worldwide. Its main bioactive constituent theaflavin helps to prevent obesity-a major risk factor for NAFLD. To find new targets for the development of effective and safe therapeutic drugs from natural plants for NAFLD, we found a theaflavin monomer theaflavin-3,3'-digallate (TF3), which significantly reduced lipid droplet accumulation in hepatocytes, and directly bound and inhibited the activation of plasma kallikrein (PK), which was further proved to stimulate adenosine monophosphate activated protein kinase (AMPK) and its downstream targets. Taken together, we proposed that the TF3-PK-AMPK regulatory axis is a novel mechanism of lipid deposition mitigation, and PK could be a new target for NAFLD treatment.

The Spectrum of Neurological and White Matter Changes and Premutation Status Categories of Older Male Carriers of the FMR1 Alleles Are Linked to Genetic (CGG and FMR1 mRNA) and Cellular Stress (AMPK) Markers.

The fragile X premutation (PM) allele contains a CGG expansion of 55-200 repeats in the FMR1 gene's promoter. Male PM carriers have an elevated risk of developing neurological and psychiatric changes, including an approximately 50% risk of the fragile X-associated tremor/ataxia syndrome (FXTAS). The aim of this study was to assess the relationships of regional white matter hyperintensities (wmhs) semi-quantitative scores, clinical status, motor (UPDRS, ICARS, Tremor) scales, and cognitive impairments, with FMR1-specific genetic changes, in a sample of 32 unselected male PM carriers aged 39-81 years. Half of these individuals were affected with FXTAS, while the non-FXTAS group comprised subcategories of non-affected individuals and individuals affected with non-syndromic changes. The dynamics of pathological processes at the cellular level relevant to the clinical status of PM carriers was investigated using the enzyme AMP-activated protein kinase (AMPK), which is a highly sensitive cellular stress-sensing alarm protein. This enzyme, as well as genetic markers - CGG repeat number and the levels of the FMR1 mRNA - were assessed in blood lymphoblasts. The results showed that the repeat distribution for FXTAS individuals peaked at 85-90 CGGs; non-FXTAS carriers were distributed within the lowest end of the PM repeat range, and non-syndromic carriers assumed an intermediate position. The size of the CGG expansion was significantly correlated, across all three categories, with infratentorial and total wmhs and with all motor scores, and the FMR1 mRNA levels with all the wmh scores, whilst AMPK activity showed considerable elevation in the non-FXTAS combined group, decreasing in the FXTAS group, proportionally to increasing severity of the wmhs and tremor/ataxia. We conclude that the size of the CGG expansion relates to the risk for FXTAS, to severity of infratentorial wmhs lesions, and to all three motor scale scores. FMR1 mRNA shows a strong association with the extent of wmhs, which is the most sensitive marker of the pathological process. However, the AMPK activity findings - suggestive of a role of this enzyme in the risk of FXTAS - need to be verified and expanded in future studies using larger samples and longitudinal assessment.

Bioinformatics Approach to Identify Novel AMPK Targets.

In silico analysis of Big Data is a useful tool to identify putative kinase targets as well as nodes of signaling cascades that are difficult to discover by traditional single molecule experimentation. System approaches that use a multi-tiered investigational methodology have been instrumental in advancing our understanding of cellular mechanisms that result in phenotypic changes. Here, we present a bioinformatics approach to identify AMP-activated protein kinase (AMPK) target proteins on a proteome-wide scale and an in vitro method for preliminary validation of these targets. This approach offers an initial screening for the identification of AMPK targets that can be further validated using mutagenesis and molecular biology techniques.

Regulation of Kv1.4 potassium channels by PKC and AMPK kinases.

Over the last years extensive kinase-mediated regulation of a number of voltage-gated potassium (Kv) channels important in cardiac electrophysiology has been reported. This includes regulation of Kv1.5, Kv7.1 and Kv11.1 cell surface expression, where the kinase-mediated regulation appears to center around the ubiquitin ligase Nedd4-2. In the present study we examined whether Kv1.4, constituting the cardiac Ito,s current, is subject to similar regulation. In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK). In contrast, manipulating the activities of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serum and glucocorticoid-regulated kinase 1 (SGK1) were without effect on channel localization. The PKC and AMPK-mediated downregulation of Kv1.4 membrane surface localization was confirmed by two-electrode voltage clamp in Xenopus laevis oocytes, where pharmacological activation of PKC and AMPK reduced Kv1.4 current levels. We further demonstrate that unlike related Kv channels, Kv1.4 current levels in Xenopus laevis oocytes are not reduced by co-expression of Nedd4-2, or the related Nedd4-1 ubiquitin ligase. In conclusion, we demonstrate that the surface expression of Kv1.4 is downregulated by the two kinases AMPK and PKC, but is unaffected by PI3K-SGK1 signaling, as well as Nedd4-1/Nedd4-2 activity. In the light of previous reports, our results demonstrate an impressive heterogeneity in the molecular pathways controlling the surface expression of highly related potassium channel subunits.

The activation of AMPK in cardiomyocytes at the very early stage of hypoxia relies on an adenine nucleotide-independent mechanism.

The energy status of a cell plays a key role in its survival, and the exposure of eukaryotic cells to the hypoxia that accompanies the depletion of intracellular ATP triggers specific systemic adaptive responses. AMP-activated protein kinase (AMPK) has emerged as a key regulator of energy metabolism in the heart and plays a critical role in inducing these responses. However, the specific mechanism responsible for AMPK activation in cardiomyocytes at very early stages of hypoxia remain unclear. The goals of this study were to assess the relative contribution to AMPK activation of phosphorylation by AMPK kinase (AMPKK) and of positive allosterism due to AMP:ATP ratios in the early stages of hypoxia. Our results demonstrated that, compared with normoxic controls, neither intracellular AMP concentrations nor AMP:ATP ratios significantly increased within 1h of hypoxia onset. In contrast, a SAMS peptide phosphorylation assay and an immunoblot analysis revealed significant increases in both AMPK activity and ACC phosphorylation within 5min of hypoxic treatment. Furthermore, exposure of cardiomyocytes to hypoxia significantly increased AMPK phosphorylation within 5min, by 3- to 4-fold compared with controls (P<0.01), while overall levels of AMPKα protein did not differ between aerobic and anoxic cardiomyocytes. We also observed increased AMPKK activity in anoxic cardiomyocytes, through use of an α(312) substrate. Taken together, our findings demonstrate that in the early stage of hypoxia in cardiomyocytes, increases in AMPK activity occur prior to and independently of increases in AMP concentration or in the AMP:ATP ratio. Instead, under these circumstances, AMPK is primarily activated by phosphorylation of the conserved Thr-172 residue in its activation loop by its upstream kinase AMPKK.