Energy Sensor AMP-activated Protein Kinase Research Articles

AMPK promotes tolerance to Ras pathway inhibition by activating autophagy.

Targeted inhibitors of oncogenic Ras (rat sarcoma viral oncogene)-Raf signaling have shown great promise in the clinic, but resistance remains a major challenge: 30% of tumors with pathway mutations do not respond to targeted inhibitors, and of the 70% that do respond, all eventually develop resistance. Before cancer cells acquire resistance, they respond to initial drug treatment either by undergoing apoptosis ('addiction') or by surviving treatment albeit with reduced growth ('tolerance'). As these drug-tolerant cells serve as a reservoir from which resistant cells eventually emerge, inhibiting the pathways that confer tolerance could potentially delay or even prevent recurrence. Here, we show that melanomas and other cancers acquire tolerance to Ras-Raf pathway inhibitors by activating autophagy, which is mediated by the cellular energy sensor AMP-activated protein kinase (AMPK). Blocking this AMPK-mediated autophagy sensitizes drug-tolerant melanomas to Ras-Raf pathway inhibitors. Conversely, activating AMPK signaling and autophagy enables melanomas that would otherwise be addicted to the Ras-Raf pathway to instead tolerate pathway inhibition. These findings identify a key mechanism of tolerance to Ras-Raf pathway inhibitors and suggest that blocking either AMPK or autophagy in combination with these targeted inhibitors could increase tumor regression and decrease the likelihood of eventual recurrence.

Combined pharmacological activation of AMPK and PPARδ potentiates the effects of exercise in trained mice.

The combined activation of the cellular energy sensor AMP‐activated protein kinase (AMPK) and the nuclear transcription factor peroxisome proliferator‐activated receptor delta (PPAR δ) has been demonstrated to improve endurance and muscle function by mimicking the effects of exercise training. However, their combined pharmacological activation with exercise training has not been explored. Balb/c mice were trained on a treadmill and administered both the AMPK activator AICAR and the PPAR δ agonist GW0742 for 4 weeks. AICAR treatment potentiated endurance, but the combination of AICAR and GW0742 further potentiated endurance and increased all running parameters significantly relative to exercised and nonexercised groups (138–179% and 355% increase in running time, respectively). Despite the lack of change in basal whole‐body metabolism, a significant shift to fat as the main energy source with a decline in carbohydrate utilization was observed upon indirect calorimetry analysis at the period near exhaustion. Increased energy substrates before exercise, and elevated muscle nonesterified fatty acids (NEFA) and elevated muscle glycogen at exhaustion were observed together with increased PDK4 mRNA expression. Citrate synthase activity was elevated in AICAR‐treated groups, while PGC‐1α protein level tended to be increased in GW0742‐treated groups. At exhaustion, Pgc1a was robustly upregulated together with Pdk4, Cd36, and Lpl in the muscle. A robust upregulation of Pgc1a and a downregulation in Chrebp were observed in the liver. Our data show that combined pharmacological activation of AMPK and PPAR δ potentiates endurance in trained mice by transcriptional changes in muscle and liver, increased available energy substrates, delayed hypoglycemia through glycogen sparing accompanied by increased NEFA availability, and improved substrate shift from carbohydrate to fat.

Supplementation of branched-chain amino acids in protein-restricted diets modulates the expression levels of amino acid transporters and energy metabolism associated regulators in the adipose tissue of growing pigs.

This experiment was conducted to investigate the effects of branched-chain amino acids (BCAA) supplemented in protein-restricted diets on the growth performance and the expression profile of amino acid transporters and energy metabolism related regulators in the white adipose tissue (WAT) of different regional depots including dorsal subcutaneous adipose (DSA) and abdominal subcutaneous adipose (ASA). A total of 24 crossbred barrows (7.40 ± 0.70 kg) were randomly divided into 4 groups and were fed the following isocaloric diets for 33 days: 1) a recommended adequate protein diet (AP, 20% CP, as a positive control); 2) a low protein diet (LP, 17% CP); 3) the LP diet supplemented with BCAA (LP + B, 17% CP) to reach the same level of the AP diet group; 4) the LP diet supplemented with 2 times the amount of BCAA (LP + 2B, 17% CP). The daily gain and daily feed intake of the LP diet group were the lowest among all the treatments (P < 0.01). The feed conversion was improved markedly in the group of LP + B compared with the LP diet group (P < 0.05). No significant difference was noted for the serum biochemical parameter concentrations of glucose, triglyceride, nonesterified fatty acid and insulin among the groups (P > 0.05). Moreover, BCAA supplementation down-regulated the expression levels of amino acid transporters including L-type amino acid transporter 1 and sodium-coupled neutral amino acid transporter 2 in DSA, but up-regulated the expression level of L-type amino acid transporter 4 in ASA (P < 0.05). Meanwhile, the energy sensor AMP-activated protein kinase α was activated in the DSA of pigs fed LP diet and in the ASA of the pigs fed AP or LP + 2B diets (P < 0.05). The mRNA expression profile of the selected mitochondrial component and mitochondrial biogenesis associated regulators in DSA and ASA also responded differently to dietary BCAA supplementation. These results suggested that the growth performance of growing pigs fed protein restricted diets supplemented with BCAA could catch up to that of the pigs fed AP diets. The results also partly demonstrated that the regulation mechanisms of BCAA are different in the adipose tissues of different depots.

Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

AMPK in Pathogens.

During host-pathogen interactions, a complex web of events is crucial for the outcome of infection. Pathogen recognition triggers powerful cellular signaling events that is translated into the induction and maintenance of innate and adaptive host immunity against infection. In opposition, pathogens employ active mechanisms to manipulate host cell regulatory pathways toward their proliferation and survival. Among these, subversion of host cell energy metabolism by pathogens is currently recognized to play an important role in microbial growth and persistence. Extensive studies have documented the role of AMP-activated protein kinase (AMPK) signaling, a central cellular hub involved in the regulation of energy homeostasis, in host-pathogen interactions. Here, we highlight the most recent advances detailing how pathogens hijack cellular metabolism by suppressing or increasing the activity of the host energy sensor AMPK. We also address the role of lower eukaryote AMPK orthologues in the adaptive process to the host microenvironment and their contribution for pathogen survival, differentiation, and growth. Finally, we review the effects of pharmacological or genetic AMPK modulation on pathogen growth and persistence.

Abstract 13103: A Novel Anchor Protein TUG Mediates GLUT4 Translocation by AMP-activated Protein Kinase in the Ischemic Heart

Introduction: Ischemic heart disease is a leading cause of death, and it is caused by reduced blood flow to the ischemic area. Thus, an increasing nutrient uptake is a key approach to increase cardiomyocyte survival rate during the ischemia and reperfusion (I/R) period. TUG (tether containing a UBX domain, for GLUT4, 60 KDa) is a regulator of GLUT4 trafficking, it can be cleaved to mobilize GLUT4 from intracellular membranes to the cell surface after insulin stimulation in skeletal muscle. The energy sensor AMP-activated protein kinase (AMPK) is known to play an important cardioprotective role during myocardial I/R by regulating GLUT4 translocation and glucose uptake. Hypothesis: TUG is one of the downstream targets of AMPK, which can be phosphorylated by hypoxia/ischemia induced AMPK activation. Phosphorylation of TUG accelerates its cleavage and increases GLUT4 translocation during ischemia/reperfusion in the heart. Methods: In vitro hypoxia chamber and ex vivo isolated mouse heart perfusion Langendorff system were used to test the hypothesis. Antithrombin (AT) is an endogenous AMPK agonist in the heart, which was used to define the role of TUG in regulating GLUT4 trafficking during ischemia and reperfusion in the heart. Results: The ex vivo heart perfusion data demonstrated that AT triggered AMPK activation and significantly increase glucose uptake and GLUT4 translocation during ischemia and reperfusion (p&lt;0.05 vs. vehicle). Intriguingly, GLUT4 immunoprecipitation data showed that AT treatment caused a dissociation of TUG from GLUT4. Moreover, AT treatment increased abundance of a TUG cleavage product (42 KDa) in response to I/R. All of these glucose transporter trafficking events are blunted in the AMPK kinase dead (KD) transgenic hearts. In HL-1 cardiomyocytes, TUG proteins were phosphorylated by activated AMPK during hypoxia. Moreover, TUG siRNA knockdown the TUG of HL-1 cells caused significantly increased cell surface GLUT4 and glucose uptake. Conclusions: Cardiac AMPK activation stimulates TUG cleavage and causes the dissociation between TUG and GLUT4 in the intracellular vesicles. TUG is a critical mediator that modulates cardiac GLUT4 translocation to cell surface and enhances glucose uptake by AMPK signaling pathway.

MicroRNA-33-dependent regulation of macrophage metabolism directs immune cell polarization in atherosclerosis.

Cellular metabolism is increasingly recognized as a controller of immune cell fate and function. MicroRNA-33 (miR-33) regulates cellular lipid metabolism and represses genes involved in cholesterol efflux, HDL biogenesis, and fatty acid oxidation. Here, we determined that miR-33-mediated disruption of the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Macrophage-specific Mir33 deletion increased oxidative respiration, enhanced spare respiratory capacity, and induced an M2 macrophage polarization-associated gene profile. Furthermore, miR-33-mediated M2 polarization required miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK), but not cholesterol efflux. Notably, miR-33 inhibition increased macrophage expression of the retinoic acid-producing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1A2) and retinal dehydrogenase activity both in vitro and in a mouse model. Consistent with the ability of retinoic acid to foster inducible Tregs, miR-33-depleted macrophages had an enhanced capacity to induce forkhead box P3 (FOXP3) expression in naive CD4(+) T cells. Finally, treatment of hypercholesterolemic mice with miR-33 inhibitors for 8 weeks resulted in accumulation of inflammation-suppressing M2 macrophages and FOXP3(+) Tregs in plaques and reduced atherosclerosis progression. Collectively, these results reveal that miR-33 regulates macrophage inflammation and demonstrate that miR-33 antagonism is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and Treg induction.

The protective effect of trimetazidine on myocardial ischemia/reperfusion injury through activating AMPK and ERK signaling pathway

IntroductionTrimetazidine (TMZ) is an anti-anginal drug that has been widely used in Europe and Asia. The TMZ can optimize energy metabolism via inhibition of long-chain 3-ketoacyl CoA thiolase (3-KAT) in the heart, with subsequent decrease in fatty acid oxidation and stimulation of glucose oxidation. However, the mechanism by which TMZ aids in cardioprotection against ischemic injury has not been characterized. AMP-activated protein kinase (AMPK) is an energy sensor that controls ATP supply from substrate metabolism and protects heart from energy stress. TMZ changes the cardiac AMP/ATP ratio by modulating fatty acid oxidation, thereby triggering AMPK signaling cascade that contributes to the protection of the heart from ischemia/reperfusion (I/R) injury. MethodsThe mouse model of in vivo regional ischemia and reperfusion by the ligation of the left anterior descending coronary artery (LAD) was used for determination of myocardial infarction. The infarct size was compared between C57BL/6J WT mice and AMPK kinase dead (KD) transgenic mice with or without TMZ treatment. The ex vivo working heart perfusion system was used to monitor the effect of TMZ on glucose oxidation and fatty acid oxidation in the heart. ResultsTMZ treatment significantly stimulates cardiac AMPK and extracellular signal-regulated kinase (ERK) signaling pathways (p<0.05 vs. vehicle group). The administration of TMZ reduces myocardial infarction size in WT C57BL/6J hearts, the reduction of myocardial infarction size by TMZ in AMPK KD hearts was significantly impaired versus WT hearts (p<0.05). Intriguingly, the administration of ERK inhibitor, PD98059, to AMPK KD mice abolished the cardioprotection of TMZ against I/R injury. The ex vivo working heart perfusion data demonstrated that TMZ treatment significantly activates AMPK signaling and modulating the substrate metabolism by shifting fatty acid oxidation to glucose oxidation during reperfusion, leading to reduction of oxidative stress in the I/R hearts. Therefore, both AMPK and ERK signaling pathways mediate the cardioprotection of TMZ against ischemic injury. The metabolic benefits of TMZ for angina patients could be due to the activation of energy sensor AMPK in the heart by TMZ administration.

Abstract B32: Pro-apoptotic functions of AMPK revealed by Myc

Abstract Oncogenic transcription factor Myc deregulates the cell cycle and simultaneously reprograms cellular metabolism to meet the biosynthetic and bioenergetic needs of proliferation. Myc also sensitizes cells to mitochondria-dependent apoptosis. Although metabolic reprogramming has been circumstantially connected to vulnerability to apoptosis, the connecting molecular pathways have remained poorly defined. Our recent studies revealed that Myc-dependent ATP depletion activates the energy sensor AMP-activated protein kinase (AMPK), which induces stabilizing phosphorylation of p53 at Ser15. AMPK-stabilized tumor suppressor protein p53 then accumulates in the mitochondria and interacts with the protein complex comprised of Bak and Bcl-xL, to sensitize cells to apoptosis. These results reveal an unexpected pro-apoptotic function of AMPK in context of Myc transformed cells. We have explored possibilities to therapeutically exploit the Myc-AMPK facilitated apoptosis pathway. We show that a BH3-mimetic small molecule ABT-737, which neutralizes Bcl-2 and Bcl-xL, sensitizes to Myc-dependent apoptosis ex vivo and in vivo WAP-Myc tumours. ABT-737 also reduced WAP-Myc driven primary tumor growth and lung metastases in transgenic and syngrafted mice. Nevertheless, ABT-737 only delayed but did not prevent the formation of Wap-Myc tumors. In a drug combination screen designed to identify small molecules that potentiate the efficacy of ABT-737, we found several compounds influencing the status of AMPK activity. The synergistic apoptotic action between ABT-737 and AMPK modulating agents has been validated in explant 3D cultures from patient derived Myc positive breast cancers. The mechanisms behind the proapoptotic AMPK function will be discussed. Our results provide evidence that AMPK has a context-dependent pro-apoptotic function that can be therapeutically exploited as a tumour cell-specific vulnerability. Citation Format: Heidi M. Haikala, Elsa Marques, Vilja Eskelinen, Anni Viheriäranta-Nieminen, Sirkku Saarikoski, Liina Nevalaita, Panu Kovanen, Päivi Heikkilä, Marjut Leidenius, Daniel Laajala, Tero Aittokallio, Pauliina Munne, Juha Klefström. Pro-apoptotic functions of AMPK revealed by Myc. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B32.

Abstract IA22: Regulation of metabolic plasticity in effector T cells

Abstract T lymphocytes are central effectors of the adaptive immune response, and their proper function is critical for long-lasting immunity to foreign pathogens and tumor immunosurveillance. One of the fundamental programs triggered by T cell activation is the reprogramming of cellular metabolism, which is essential to support the bioenergetic and biosynthetic demands of effector T (Teff) cell function. While often considered a “housekeeping” function, cellular metabolism has recently been linked to proper Teff cell differentiation and effector function in vivo, raising the idea that nutrient availability can be immunomodulatory. However, how nutrient availability shapes T cell metabolism and function remains poorly understood. Here we present evidence suggesting that effector T cells possess “metabolic plasticity” and are capable of altering their metabolic programs in response to nutrient availability. We show that T cells possess a glucose-sensitive metabolic checkpoint controlled by the energy sensor AMP-activated protein kinase (AMPK) that regulates cellular bioenergetics and T cell viability. Effector T cells lacking AMPKα1 display reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation in vitro or pathogenic challenge in vivo. Finally, we find that AMPKα1 is essential for robust CD4+ and CD8+ T cell responses to viral and bacterial infections in vivo. Our data highlight AMPK-dependent regulation of metabolic homeostasis as a key regulator of T cell-mediated adaptive immunity. These results and the use AMPK agonists to regulate T cell function will be discussed. Citation Format: Russell Graham Jones. Regulation of metabolic plasticity in effector T cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA22.

Sensing and responding to energetic stress: The role of the AMPK-PGC1α-NRF1 axis in control of mitochondrial biogenesis in fish

Remodeling the muscle metabolic machinery in mammals in response to energetic challenges depends on the energy sensor AMP-activated protein kinase (AMPK) and its ability to phosphorylate PPAR γ coactivator 1 α (PGC1α), which in turn coactivates metabolic genes through direct and indirect association with DNA-binding proteins such as the nuclear respiratory factor 1 (NRF1) (Wu et al., 1999). The integrity of this axis in fish is uncertain because PGC1α i) lacks the critical Thr177 targeted by AMPK and ii) has mutations that may preclude binding NRF1. In this study we found no evidence that AMPK regulates mitochondrial gene expression through PGC1α in zebrafish and goldfish. AICAR treatment of zebrafish blastula cells increased phosphorylation of AMPK and led to changes in transcript levels of the AMPK targets mTOR and hexokinase 2. However, we saw no increases in mRNA levels for genes associated with mitochondrial biogenesis, including PGC1α, NRF1, and COX7C, a cytochrome c oxidase subunit. Further, AMPK phosphorylated mammalian peptides of PGC1α but not the corresponding region of zebrafish or goldfish in vitro. In vivo cold acclimation of goldfish caused an increase in mitochondrial enzymes, AMP and ADP levels, however AMPK phosphorylation decreased. In fish, the NRF1-PGC1α axis may be disrupted due to insertions in fish PGC1α orthologs within the region that serves as NRF1 binding domain in mammals. Immunocopurification showed that recombinant NRF1 protein binds mammalian but not fish PGC1α. Collectively, our studies suggest that fish have a disruption in the AMPK-PGC1α-NRF1 pathway due to structural differences between fish and mammalian PGC1α.

Differential control of Yorkie activity by LKB1/AMPK and the Hippo/Warts cascade in the central nervous system

The Hippo (Hpo) pathway is a highly conserved tumor suppressor network that restricts developmental tissue growth and regulates stem cell proliferation and differentiation. At the heart of the Hpo pathway is the progrowth transcriptional coactivator Yorkie [Yki-Yes-activated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) in mammals]. Yki activity is restricted through phosphorylation by the Hpo/Warts core kinase cascade, but increasing evidence indicates that core kinase-independent modes of regulation also play an important role. Here, we examine Yki regulation in the Drosophila larval central nervous system and uncover a Hpo/Warts-independent function for the tumor suppressor kinase liver kinase B1 (LKB1) and its downstream effector, the energy sensor AMP-activated protein kinase (AMPK), in repressing Yki activity in the central brain/ventral nerve cord. Although the Hpo/Warts core cascade restrains Yki in the optic lobe, it is dispensable for Yki target gene repression in the late larval central brain/ventral nerve cord. Thus, we demonstrate a dramatically different wiring of Hpo signaling in neighboring cell populations of distinct developmental origins in the central nervous system.

A Critical SUMO1 Modification of LKB1 Regulates AMPK Activity during Energy Stress.

SUMOylation has been implicated in cellular stress adaptation, but its role in regulating liver kinase B1 (LKB1), a major upstream kinase of the energy sensor AMP-activated protein kinase (AMPK), is unknown. Here, we show that energy stress triggers an increase in SUMO1 modification of LKB1, despite a global reduction in both SUMO1 and SUMO2/3 conjugates. During metabolic stress, SUMO1 modification of LKB1 lysine 178 is essential in promoting its interaction with AMPK via a SUMO-interacting motif (SIM) essential for AMPK activation. The LKB1 K178R SUMO mutant had defective AMPK signaling and mitochondrial function, inducing death in energy-deprived cells. These results provide additional insight into how LKB1-AMPK signaling is regulated during energy stress, and they highlight the critical role of SUMOylation in maintaining the cell's energy equilibrium.

Abstract IA20: Targeting AMPK signaling in melanoma

Abstract Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated anti-tumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. We have previously reported that AMPK signalling is attenuated by BRAFV600E in melanoma through phosphorylation of LKB1, a major upstream kinase of AMPK. More recently, we have also discovered that BRAF is directly phosphorylated by AMPK, leading to impaired BRAF-KSR1 association and MEK-ERK signalling (Mol Cell, 2013, 52:161). Based on this crosstalk between the AMPK and BRAF signaling pathways, we investigated the anti-tumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Co-treatment of BRAF mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared to the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells. In contrast, PLX4720 selectively targeted JARID1B negative cells, which represents the majority of cells in the tumors, and spared the JAR1D1B-positive slowing cycling population. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAFV600E/PTENnull-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma. Citation Format: Bin Zheng. Targeting AMPK signaling in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA20.

Mitochondrial dysfunction and Parkinson disease: a Parkin-AMPK alliance in neuroprotection.

Although a subject of intense research, the etiology of Parkinson disease (PD) remains poorly understood. However, a wide range of studies conducted over the past few decades have collectively implicated aberrant mitochondrial homeostasis as a key contributor to the development of PD. Particularly strong support for this came from the recent demonstration that parkin, a familial PD-linked gene, is a critical regulator of mitochondrial quality control. Indeed, Parkin appears to be involved in all stages of the mitochondrial life cycle (i.e., from biogenesis to its exit from the cell (via mitophagy). Interestingly, the role of Parkin in the biogenesis and clearance of mitochondria is akin to that performed by the energy sensor AMP-activated protein kinase (AMPK), suggesting that the two proteins might act in a functionally converging manner to maintain the quality of cellular mitochondria. In this review, we discuss the contribution of mitochondrial dysfunction to PD pathogenesis and the role of Parkin and AMPK in preserving neuronal mitochondrial homeostasis. Alongside this, we will also articulate our thoughts on the potential alliance between Parkin and AMPK in offering neuroprotection through their ability to maintain energy balance in the brain.

Abstract 422: Metabolic Regulation by miR-33 in Macrophages Controls Immune Effector Responses

Cellular metabolism is increasingly recognized to control immune cell fate and functions. MicroRNA-33 (miR-33) is a central regulator of cellular lipid metabolism that represses genes involved in cholesterol efflux and HDL biogenesis (Abca1, Abcg1) and fatty acid oxidation (Cpt1a, Crot, Ampk). Here we show that by altering the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation, miR-33 inhibition instructs macrophage polarization to an M2 phenotype and shapes innate and adaptive immune responses. Targeted deletion of miR-33 in macrophages increases oxidative phosphorylation, enhances spare respiratory capacity, and induces the expression of genes that define M2 macrophage polarization (Arg1, Fizz1, Cd206, Ym1). Furthermore, inhibition of miR-33 in Abca1-/- macrophages showed that these changes are independent of effects on cholesterol efflux, but instead require miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK). Notably, inhibition of miR-33 markedly increased macrophage expression of the retinoic acid-producing enzyme Aldh1a2 and retinal dehydrogenase activity both in vitro and in vivo. Consistent with the ability of retinoic acid to foster inducible regulatory T cells, these macrophages had an enhanced capacity to induce FoxP3 expression in naïve CD4+ T cells. Finally, treatment of western diet-fed Ldlr-/- mice with miR-33 inhibitors for 8 weeks (conditions that do not alter HDL cholesterol levels) reduced atherosclerosis progression by 40%, and promoted the accumulation of M2 macrophages and FoxP3+ T regulatory cells in plaques. Collectively, these results identify a novel role for miR-33 in the regulation of macrophage inflammation and show that antagonism of miR-33 is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and regulatory T cell induction.

Lemon verbena (Lippia citriodora) polyphenols alleviate obesity-related disturbances in hypertrophic adipocytes through AMPK-dependent mechanisms

BackgroundThere is growing evidence that natural products, mostly plant-derived polyphenols, are important in the relationship between nutrients and health in humans. PurposeWe aimed to investigate if verbascoside (VB) and other lemon verbena polyphenols could ameliorate obesity-induced metabolic disturbances, as well as their putative mechanism. Study designWe used an insulin-resistant hypertrophic 3T3-L1-adipocyte model to test the effects of VB or lemon verbena extract on triglyceride accumulation, inflammation and oxidative stress and a murine model of diet-induced obesity to assess the in vivo metabolic response. ResultsPolyphenols decreased triglyceride accumulation, the generation of reactive oxygen species (ROS) and restored mitochondrial membrane potential in adipocytes. The underlying mechanisms seemed to occur via ROS-mediated downregulation of nuclear factor kappa-B transcription factor (NF-κB) and peroxisome proliferator-activated receptor gamma (PPAR-γ)-dependent transcriptional upregulation of adiponectin. We also observed a potent activation of AMP-activated protein kinase (AMPK), the mRNA expression upregulation of PPAR-α and the mRNA expression downregulation of fatty acid synthase. Experiments in mice suggested a significant improvement in fat metabolism. ConclusionDecreased lipogenesis, enhanced fatty acid oxidation and the activation of the energy sensor AMPK, probably through activating transcriptional factors, are involved in the observed beneficial effects. VB effects were less potent than those observed with the extract, so a potential synergistic, multi-targeted action is proposed. The polypharmacological effects of plant-derived polyphenols from lemon verbena may have the potential for clinical applications in obesity.

5′-AMP-activated protein kinase alpha regulates stress granule biogenesis

Stress granule (SG) assembly represents a conserved eukaryotic defense strategy against various insults. Although essential for the ability to cope with deleterious conditions, the signaling pathways controlling SG formation are not fully understood. The energy sensor AMP-activated protein kinase (AMPK) is critical for the cellular stress response. Human cells produce two AMPK catalytic α-subunits with not only partially overlapping, but also unique functions. Here, we provide direct support for structural and functional links between AMPK-α isoforms and SGs. As such, several stressors promote SG association of AMPK-α2, but not AMPK-α1. Multiple lines of evidence link AMPK activity to SG biogenesis. First, pharmacological kinase inhibition interfered with SG formation. Second, AMPK-α knockdown combined with in-depth quantitative SG analysis revealed isoform-specific changes of SG characteristics. Third, overexpression of mutant α-subunits further substantiated that AMPK regulates SG parameters. Finally, we identified the SG-nucleating protein G3BP1 as an AMPK-α2 binding partner. This interaction is stimulated by stress and notably occurs in SGs. Collectively, our data define the master metabolic regulator AMPK as a novel SG constituent that also controls their biogenesis.

Estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression

Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen-ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen-ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP3), which opens EnR IP3R calcium channels, rapidly depleting EnR Ca(2+) stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca(2+) levels, but the open EnR IP3R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI's novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration.

Computational analysis of an autophagy/translation switch based on mutual inhibition of MTORC1 and ULK1.

We constructed a mechanistic, computational model for regulation of (macro)autophagy and protein synthesis (at the level of translation). The model was formulated to study the system-level consequences of interactions among the following proteins: two key components of MTOR complex 1 (MTORC1), namely the protein kinase MTOR (mechanistic target of rapamycin) and the scaffold protein RPTOR; the autophagy-initiating protein kinase ULK1; and the multimeric energy-sensing AMP-activated protein kinase (AMPK). Inputs of the model include intrinsic AMPK kinase activity, which is taken as an adjustable surrogate parameter for cellular energy level or AMP:ATP ratio, and rapamycin dose, which controls MTORC1 activity. Outputs of the model include the phosphorylation level of the translational repressor EIF4EBP1, a substrate of MTORC1, and the phosphorylation level of AMBRA1 (activating molecule in BECN1-regulated autophagy), a substrate of ULK1 critical for autophagosome formation. The model incorporates reciprocal regulation of mTORC1 and ULK1 by AMPK, mutual inhibition of MTORC1 and ULK1, and ULK1-mediated negative feedback regulation of AMPK. Through analysis of the model, we find that these processes may be responsible, depending on conditions, for graded responses to stress inputs, for bistable switching between autophagy and protein synthesis, or relaxation oscillations, comprising alternating periods of autophagy and protein synthesis. A sensitivity analysis indicates that the prediction of oscillatory behavior is robust to changes of the parameter values of the model. The model provides testable predictions about the behavior of the AMPK-MTORC1-ULK1 network, which plays a central role in maintaining cellular energy and nutrient homeostasis.