Basal Autophagy Induction Without AMP-Activated Protein Kinase Under Low Glucose Conditions

When ATP levels in a cell decrease, various homeostatic intracellular mechanisms initiate attempts to restore ATP levels. As a prominent energy sensor, AMP-activated protein kinase (AMPK) represents one molecular gauge that links energy levels to regulation of anabolic and catabolic processes to restore energy balance. Although pharmacological studies have suggested that an AMPK activator, AIC AR (5-aminoimidazole-4-carboxamide ribonucleoside) may link AMPK activation to autophagy, a process that can provide short-term energy within the cell, AICAR can have AMPK-independent effects. Therefore, using a genetic-based approach we investigated the role of AMPK in cellular energy balance. We demonstrate that genetically altered cells, mouse embryonic fibroblasts (MEFs), lacking functional AMPK, display altered energy balance under basal conditions and die prematurely under low glucose-serum starvation challenge. These AMPK mutant cells appear to be abnormally reliant on autophagy under low glucose basal conditions, and therefore cannot rely further on autophagy like wild-type cells during further energetic stress and instead undergo apoptosis. This data suggests that AMPK helps regulate basal energy levels under low glucose. Further, AMPK mutant cells show increased basal phosphorylation of p53 at serine 15, a residue phosphorylated under glucose deprivation. We propose that cells lacking AMPK function have altered p53 activity that may help sensitize these cells to apoptosis under energetic stress

Past strategies and future directions for identifying AMP-activated protein kinase (AMPK) modulators

AMP-activated protein kinase (AMPK) is a promising therapeutic target for cancer, type II diabetes, and other illnesses characterized by abnormal energy utilization. During the last decade, numerous labs have published a range of methods for identifying novel AMPK modulators. The current understanding of AMPK structure and regulation, however, has propelled a paradigm shift in which many researchers now consider ADP to be an additional regulatory nucleotide of AMPK. How can the AMPK community apply this new understanding of AMPK signaling to translational research? Recent insights into AMPK structure, regulation, and holoenzyme-sensitive signaling may provide the hindsight needed to clearly evaluate the strengths and weaknesses of past AMPK drug discovery efforts. Improving future strategies for AMPK drug discovery will require pairing the current understanding of AMPK signaling with improved experimental designs

LKB1 and AMPK maintain epithelial cell polarity under energetic stress

LKB1 is mutated in both familial and spontaneous tumors, and acts as a master kinase that activates the PAR-1 polarity kinase and the adenosine 5′monophosphate–activated kinase (AMPK). This has led to the hypothesis that LKB1 acts as a tumor suppressor because it is required to maintain cell polarity and growth control through PAR-1 and AMPK, respectively. However, the genetic analysis of LKB1–AMPK signaling in vertebrates has been complicated by the existence of multiple redundant AMPK subunits. We describe the identification of mutations in the single Drosophila melanogaster AMPK catalytic subunit AMPKα. Surprisingly, ampkα mutant epithelial cells lose their polarity and overproliferate under energetic stress. LKB1 is required in vivo for AMPK activation, and lkb1 mutations cause similar energetic stress–dependent phenotypes to ampkα mutations. Furthermore, lkb1 phenotypes are rescued by a phosphomimetic version of AMPKα. Thus, LKB1 signals through AMPK to coordinate epithelial polarity and proliferation with cellular energy status, and this might underlie the tumor suppressor function of LKB1

Carbonic anhydrase III (Car3) is not required for fatty acid synthesis and does not protect against high-fat diet induced obesity in mice

Carbonic anhydrases are a family of enzymes that catalyze the reversible condensation of water and carbon dioxide to carbonic acid, which spontaneously dissociates to bicarbonate. Carbonic anhydrase III (Car3) is nutritionally regulated at both the mRNA and protein level. It is highly enriched in tissues that synthesize and/or store fat: liver, white adipose tissue, brown adipose tissue, and skeletal muscle. Previous characterization of Car3 knockout mice focused on mice fed standard diets, not high-fat diets that significantly alter the tissues that highly express Car3. We observed lower protein levels of Car3 in high-fat diet fed mice treated with niclosamide, a drug published to improve fatty liver symptoms in mice. However, it is unknown if Car3 is simply a biomarker reflecting lipid accumulation or whether it has a functional role in regulating lipid metabolism. We focused our in vitro studies toward metabolic pathways that require bicarbonate. To further determine the role of Car3 in metabolism, we measured de novo fatty acid synthesis with in vitro radiolabeled experiments and examined metabolic biomarkers in Car3 knockout and wild type mice fed high-fat diet. Specifically, we analyzed body weight, body composition, metabolic rate, insulin resistance, serum and tissue triglycerides. Our results indicate that Car3 is not required for de novo lipogenesis, and Car3 knockout mice fed high-fat diet do not have significant differences in responses to various diets to wild type mice

AMPK Directly Inhibits NDPK Through a Phosphoserine Switch to Maintain Cellular Homeostasis

AMP-activated protein kinase (AMPK) is a key energy sensor that regulates metabolism to maintain cellular energy balance. AMPK activation has also been proposed to mimic benefits of caloric restriction and exercise. Therefore, identifying downstream AMPK targets could elucidate new mechanisms for maintaining cellular energy homeostasis. We identified the phosphotransferase nucleoside diphosphate kinase (NDPK), which maintains pools of nucleotides, as a direct AMPK target through the use of two-dimensional differential in-gel electrophoresis. Furthermore, we mapped the AMPK/NDPK phosphorylation site (serine 120) as a functionally potent enzymatic “off switch” both in vivo and in vitro. Because ATP is usually the most abundant cellular nucleotide, NDPK would normally consume ATP, whereas AMPK would inhibit NDPK to conserve energy. It is intriguing that serine 120 is mutated in advanced neuroblastoma, which suggests a mechanism by which NDPK in neuroblastoma can no longer be inhibited by AMPK-mediated phosphorylation. This novel placement of AMPK upstream and directly regulating NDPK activity has widespread implications for cellular energy/nucleotide balance, and we demonstrate in vivo that increased NDPK activity leads to susceptibility to energy deprivation–induced death

Calcium/Calmodulin-Dependent Protein Kinase II Alters Structural Plasticity and Cytoskeletal Dynamics in Drosophila

Drosophila dendritic arborization (da) neurons contain subclasses of neurons with distinct dendritic morphologies. We investigated calcium/calmodulin-dependent protein kinase II (CaMKII) regulation of dendritic structure and dynamics in vivo using optically transparent Drosophila larvae. CaMKII increases the dynamic nature and formation of dendritic filopodia throughout larval development but only affects neurons that normally contain dendritic filopodia. In parallel, we examined the effects of Rac1 activity on dendritic structure to explore signaling specificity. In contrast to CaMKII activity, Rac1 does not alter filopodia stability but instead causes de novo filopodia formation on all da neurons. Although both mediators increase cytoskeletal turnover, measured by fluorescence recovery after photobleaching experiments, only CaMKII increases the dynamic nature of dendritic filopodia. CaMKII signaling thus appears to use mechanisms and machinery distinct from Rac1 signaling. This study illustrates a molecular means of uncoupling cytoskeletal regulation from morphological regulation. Our results suggest that Drosophila dendritic filopodia may share some cytoskeletal regulatory mechanisms with mammalian dendritic filopodia. Furthermore, general dendrite cytoskeletal compartmentalization is conserved in multipolar neurons

Basal autophagy induction without AMP-activated protein kinase under low glucose conditions

When ATP levels in a cell decrease, various homeostatic intracellular mechanisms initiate attempts to restore ATP levels. As a prominent energy sensor, AMP-activated protein kinase (AMPK) represents one molecular gauge that links energy levels to regulation of anabolic and catabolic processes to restore energy balance. Although pharmacological studies have suggested that an AMPK activator, AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) may link AMPK activation to autophagy, a process that can provide short-term energy within the cell, AICAR can have AMPK-independent effects. Therefore, using a genetic-based approach we investigated the role of AMPK in cellular energy balance. We demonstrate that genetically altered cells, mouse embryonic fibroblasts (MEFs), lacking functional AMPK, display altered energy balance under basal conditions and die prematurely under low glucose-serum starvation challenge. These AMPK mutant cells appear to be abnormally reliant on autophagy under low glucose basal conditions, and therefore cannot rely further on autophagy like wild-type cells during further energetic stress and instead undergo apoptosis. This data suggests that AMPK helps regulate basal energy levels under low glucose. Further, AMPK mutant cells show increased basal phosphorylation of p53 at serine 15, a residue phosphorylated under glucose deprivation. We propose that cells lacking AMPK function have altered p53 activity that may help sensitize these cells to apoptosis under energetic stress

Energy-Dependent Modulation of Glucagon-Like Signaling in Drosophila via the AMP-Activated Protein Kinase

Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks

AMP-activated protein kinase (AMPK) activity is not required for neuronal development but regulates axogenesis during metabolic stress

Mammalian brain connectivity requires the coordinated production and migration of billions of neurons and the formation of axons and dendrites. The LKB1/Par4 kinase is required for axon formation during cortical development in vivo partially through its ability to activate SAD-A/B kinases. LKB1 is a master kinase phosphorylating and activating at least 11 other serine/threonine kinases including the metabolic sensor AMP-activated protein kinase (AMPK), which defines this branch of the kinome. A recent study using a gene-trap allele of the β1 regulatory subunit of AMPK suggested that AMPK catalytic activity is required for proper brain development including neurogenesis and neuronal survival. We used a genetic loss-of-function approach producing AMPKα1/α2-null cortical neurons to demonstrate that AMPK catalytic activity is not required for cortical neurogenesis, neuronal migration, polarization, or survival. However, we found that application of metformin or AICAR, potent AMPK activators, inhibit axogenesis and axon growth in an AMPK-dependent manner. We show that inhibition of axon growth mediated by AMPK overactivation requires TSC1/2-mediated inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. Our results demonstrate that AMPK catalytic activity is not required for early neural development in vivo but its overactivation during metabolic stress impairs neuronal polarization in a mTOR-dependent manner