AMP-activated protein kinase contributes to zinc-induced neuronal death via activation by LKB1 and induction of Bim in mouse cortical cultures

AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine kinase involved in metabolic regulation. It is phosphorylated by the upstream liver kinase B1 (LKB1) or calcium/calmodulin-dependent kinase kinase 2 (CaMKKβ). In cultured cells, AMPK activation correlates with LKB1 activity. The phosphorylation activates AMPK, shifting metabolism toward catabolism and promoting mitogenesis. In muscles, inactivity reduces AMPK activation, shifting the phenotype of oxidative muscles toward a more glycolytic profile. Here, we compared the basal level of AMPK activation in glycolytic and oxidative muscles and analyzed whether this relates to LKB1 or CaMKKβ. Using Western blotting, we assessed AMPK expression and phosphorylation in soleus, gastrocnemius (GAST), extensor digitorum longus (EDL), and heart from C57BL6J mice. We also assessed LKB1 and CaMKKβ expression, and CaMKKβ activity in tissue homogenates. AMPK activation was higher in oxidative (soleus and heart) than in glycolytic muscles (gastrocnemius and EDL). This correlated with AMPK α1-isoform expression, but not LKB1 and CaMKKβ. LKB1 expression was sex dependent and lower in male than female muscles. CaMKKβ expression was very low in skeletal muscles and did not phosphorylate AMPK in muscle lysates. The higher AMPK activation in oxidative muscles is in line with the fact that activated AMPK maintains an oxidative phenotype. However, this could not be explained by LKB1 and CaMKKβ. These results suggest that the regulation of AMPK activation is more complex in muscle than in cultured cells. As AMPK has been proposed as a therapeutic target for several diseases, future research should consider AMPK isoform expression and localization, and energetic compartmentalization.NEW & NOTEWORTHY It is important to understand how AMP-activated kinase, AMPK, is regulated, as it is a potential therapeutic target for several diseases. AMPK is activated by liver kinase B1, LKB1, and calcium/calmodulin-dependent kinase kinase 2, CaMKKβ. In cultured cells, AMPK activation correlates with LKB1 expression. In contrast, we show that AMPK-activation was higher in oxidative than glycolytic muscle, without correlating with LKB1 or CaMKKβ expression. Thus, AMPK regulation is more complex in highly compartmentalized muscle cells.

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK α2 protein and activity were reduced by 45%. In β(1,2,3)-adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr(172) phosphorylation. In contrast, acute α- or β-adrenergic blockade in WT mice resulted in increased AMPK α Thr(172) phosphorylation and AMPK α1 and α2 activity in BAT. But short term blockade of α-adrenergic signaling in β(1,2,3)-adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser(485/491), a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser(485/491) phosphorylation resulting from α-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of α-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr(172) phosphorylation and chronically through changes in the α catalytic subunit protein levels. Furthermore, AMPK α Ser(485/491) phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects.

We recently reported that AMP-activated protein kinase (AMPK) contributes to zinc-induced neuronal death by inducing Bim, a pro-apoptotic Bcl-2 homology domain 3-only protein, in a liver kinase B1 (LKB1)-dependent manner. Current data suggest AMPK plays key roles in excitotoxicity and ischemic brain injury, with zinc neurotoxicity representing at least one mechanism of ischemic neuronal death. Inhibition of AMPK could be a viable therapeutic strategy to prevent ischemic brain injury following stroke. This prompted our search for novel inhibitors of AMPK activity and zinc-induced neuronal death using cultured mouse cortex and a rat model of brain injury after middle cerebral artery occlusion (MCAO). In structure-based virtual screening, 118 compounds were predicted to bind the active site of AMPK α2, and 40 showed in vitro AMPK α2 inhibitory activity comparable to compound C (a well-known, potent AMPK inhibitor). In mouse cortical neuronal cultures, 7 of 40 compound reduced zinc-induced neuronal death at levels comparable to compound C. Ultimately, only agents 2G11 and 1H10 significantly attenuated various types of neuronal death, including oxidative stress, excitotoxicity, and apoptosis. When administered as intracerebroventricular injections prior to permanent MCAO in rats, 2G11 and 1H10 reduced brain infarct volumes, whereas compound C did not. Therefore, these novel AMPK inhibitors could be drug development candidates to treat stroke.

The AMP-activated protein kinase (AMPK) is activated by a fall in the ATP:AMP ratio within the cell in response to metabolic stresses. Once activated, it phosphorylates and inhibits key enzymes in energy-consuming biosynthetic pathways, thereby conserving cellular ATP. The creatine kinase-phosphocreatine system plays a key role in the control of ATP levels in tissues that have a high and rapidly fluctuating energy requirement. In this study, we provide direct evidence that these two energy-regulating systems are linked in skeletal muscle. We show that the AMPK inhibits creatine kinase by phosphorylation in vitro and in differentiated muscle cells. AMPK is itself regulated by a novel mechanism involving phosphocreatine, creatine and pH. Our findings provide an explanation for the high expression, yet apparently low activity, of AMPK in skeletal muscle, and reveal a potential mechanism for the co-ordinated regulation of energy metabolism in this tissue. Previous evidence suggests that AMPK activates fatty acid oxidation, which provides a source of ATP, following continued muscle contraction. The novel regulation of AMPK described here provides a mechanism by which energy supply can meet energy demand following the utilization of the immediate energy reserve provided by the creatine kinase-phosphocreatine system.

IntroductionAMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and performed translational studies of AMPK pharmacologic activation.MethodsWe assessed activity (phosphorylation) of LKB1 and AMPKα in mouse knee OA cartilage, in aging mouse cartilage (6 to 24 months), and in chondrocytes after mechanical injury by dynamic compression, via immunohistochemistry or western blot. We knocked down LKB1 by siRNA transfection. Nitric oxide, matrix metalloproteinase (MMP)-3, and MMP-13 release were measured by Griess reaction and ELISA, respectively.ResultsKnockdown of LKB1 attenuated chondrocyte AMPK activity, and increased nitric oxide, MMP-3 and MMP-13 release (P <0.05) in response to IL-1β and TNFα. Both LKB1 and AMPK activity were decreased in mouse knee OA and aged knee cartilage, and in bovine chondrocytes after biomechanical injury. Pretreatment of bovine chondrocytes with AMPK activators AICAR and A-769662 inhibited both AMPKα dephosphorylation and catabolic responses after biomechanical injury.ConclusionLKB1 is required for chondrocyte AMPK activity, thereby inhibiting matrix catabolic responses to inflammatory cytokines. Concurrent loss of LKB1 and AMPK activity in articular chondrocytes is associated with OA, aging and biomechanical injury. Conversely, pharmacologic AMPK activation attenuates catabolic responses to biomechanical injury, suggesting a potentially novel approach to inhibit OA development and progression.

Viruses normally reprogram the host cell metabolic pathways as well as metabolic sensors to facilitate their persistence. The serine-threonine liver kinase B1 (LKB1) is a master upstream kinase of 5'-AMP-activated protein kinase (AMPK) that senses the energy status and therefore regulates the intracellular metabolic homeostasis. Previous studies showed that AMPK restricts Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in endothelial cells during primary infection and promotes primary effusion lymphoma (PEL) cell survival. However, the role of LKB1 in KSHV lytic reactivation and KSHV-associated malignancies is unclear. In this study, we found that LKB1 is phosphorylated or activated in KSHV-positive PEL cells. Mechanistically, KSHV-encoded vCyclin mediated LKB1 activation in PEL cells, as vCyclin knockout ablated, while vCyclin overexpression enhanced LKB1 activation. Furthermore, knockdown of LKB1 inactivated AMPK and induced KSHV reactivation, as indicated by the increased expression of viral lytic genes and the increased virions in supernatants. Accordingly, AMPK inhibition by functional knockdown or a pharmacologic inhibitor, Compound C, promoted KSHV reactivation in PEL cells. Furthermore, inhibition of either LKB1 or AMPKα1 efficiently induced cell death by apoptosis of PEL cells both in vitro and in vivo. Together, these results identify LKB1 as a vulnerable target for PEL, which could be potentially exploited for treating other virus-associated diseases.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human cancers, such as primary effusion lymphoma (PEL). Here, we showed that serine-threonine liver kinase B1 (LKB1), upstream of 5' AMP-activated protein kinase (AMPK), is activated by KSHV-encoded vCyclin and maintains KSHV latency in PEL cells. Inhibition of either LKB1 or AMPK enhances KSHV lytic replication from latency, which at least partially accounts for PEL cell death by apoptosis. Compound C, a potent AMPK inhibitor, induced KSHV reactivation and efficiently inhibited PEL progression in vivo. Thus, our work revealed that LKB1 is a potential therapeutic target for KSHV-associated cancers.

Fyn-deficient mice display increased AMP-activated Protein Kinase (AMPK) activity as a result of Fyn-dependent regulation of Liver Kinase B1 (LKB1) in skeletal muscle. Mutation of Fyn-specific tyrosine sites in LKB1 results in LKB1 export into the cytoplasm and increased AMPK activation site phosphorylation. This study characterizes the structural elements responsible for the physical interaction between Fyn and LKB1. Effects of point mutations in the Fyn SH2/SH3 domains and in the LKB1 proline-rich motif on 1) Fyn and LKB1 binding, 2) LKB1 subcellular localization and 3) AMPK phosphorylation were investigated in C2C12 muscle cells. Additionally, novel LKB1 proline-rich motif mimicking cell permeable peptides were generated to disrupt Fyn/LKB1 binding and investigate the consequences on AMPK activity in both C2C12 cells and mouse skeletal muscle. Mutation of either Fyn SH3 domain or the proline-rich motif of LKB1 resulted in the disruption of Fyn/LKB1 binding, re-localization of 70% of LKB1 signal in the cytoplasm and a 2-fold increase in AMPK phosphorylation. In vivo disruption of the Fyn/LKB1 interaction using LKB1 proline-rich motif mimicking cell permeable peptides recapitulated Fyn pharmacological inhibition. We have pinpointed the structural elements within Fyn and LKB1 that are responsible for their binding, demonstrating the functionality of this interaction in regulating AMPK activity.

None for Perspective.

AMP-activated protein kinase (AMPK) consists of a catalytic α subunit and regulatory β and γ subunits, and is activated in response to an increase in the AMP/ATP ratio upon metabolic stresses. Two AMPK kinases have been identified to mediate activating phosphorylation of AMPKα at Thr172, namely liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) [reviewed in 1]. Increased AMP:ATP can activate AMPK allosterically by binding to the AMPKγ subunit and inhibiting dephosphorylation of Thr172 due to constitutive LKB1 activity. In cells expressing CaMKKβ, increased intracellular Ca2+ concentrations activate AMPK independent of changes in AMP:ATP. Once activated, AMPK phosphorylates multiple downstream catabolic targets that promote ATP generation, whereas anabolic processes including fatty acid synthesis and protein translation tend to be suppressed.1 During tumorigenesis and related depletion of cellular energy, AMPK activation is thought to favor energy-producing catabolic processes. The role of AMPK in prostate cancer (PC) remains to be fully characterized, with significant discrepancies in the literature. We have recently observed that AMPK Thr172 phosphorylation was significantly elevated in human prostate cancer (PC) cells and clinical PC samples. In clinical PC, while not statistically significant, there was a trend for increasing phospho-AMPK Thr172 with increasing Gleason sum score (or increasingly less differentiated disease). The increased AMPK Thr172 phosphoryation was associated with phosphorylation of the AMPK substrate, acetyl CoA-carboxylase (ACC; p = 0.0023), suggesting enhanced AMPK and inhibited ACC activities in PC.2 These findings support earlier reports of increased phospho-ACC levels in clinical PC.3 Functional significance of the observed upregulation of AMPK/ACC status in PC progression remains unclear. In DU145 cells, an androgen receptor (AR) negative human PC line with activated Akt and upregulated glycolysis, 5-aminoimidazole-4-carboxamide riboside (AICAR)-mediated AMPK activation has been reported to suppress proliferation without evidence of increased apoptosis.4 Our recent comparative analysis of the paired PC3 and PC3M cells as models of progressively invasive (androgen-independent) PC also supported the notion of tumor suppressing effects upon activation of AMPK (Fig. 1). Treatment with AICAR or an alternative direct AMPK activator, A-769662 suppressed proliferation, migration and invasion in both cell lines, with down-regulation of mTOR and P70S6Ki levels regardless of the Akt status. Involvement of AMPK was confirmed with the AMPK inhibitor, Compound C and siRNA-mediated AMPK silencing.2 Figure 1. A schematic representation of signaling network involving AMPK and other key signaling nodes in prostate carcinogenesis. The AMPK-mediated anti-tumor effects (proliferative/migratory/invasive) are considered downstream of fatty acid and protein synthesis. ... Despite similar functional responses in PC3 and PC3M cells, AMPK activation also resulted in sustained phospho-Akt activation in PC3M cells, but not PC3 cells. However, combining LY-294002 with AICAR to simultaneously suppress phosphatidylinositol 3′-kinase (PI3K) and activate AMPK did not produce any additional anti-proliferative effects in PC3M cells. This suggests that any efforts to exploit AMPK as a potential therapeutic target may be considered independent of PI3K/Akt signaling. This is a surprising finding as there is significant evidence for cross talk between AMPK and AKT/mTOR pathways. Indeed, the PI3K signaling network exhibits extensive cross-regulatory interactions with the intermediate metabolism network, including AMPK, to fuel resistance to stress and uncontrolled growth.5 Akt has also been reported to dramatically reduce the AMP/ATP ratio and suppress AMPK activity in cells overexpressing a constitutive Akt mutant.6 Hence, while the effects of AMPK activation was not influenced by a PI3K inhibitor in our study, this may be context and cell type specific, and a detailed understanding of AMPK/PI3K/mTOR crosstalk will have important implications in cancer therapies. Popovics et al.7 have recently hypothesized that AMPK activators used in pre-clinical studies are likely to promote autophagy and apoptosis since they mediate their effects, at least in part, by an inhibitory action on the mTORC1 complex. They proposed that modulators of the CaMKKβ-AMPK pathway may be used in combination with mTOR-specific inhibitors, such as the rapamycin analogs, to maximise anti-tumor effects, thus avoiding the the potential of stimulating diverse pro-tumor processes in parallel with antitumor effects when mTOR inhibitor is used alone. Ongoing research into the role of AMPK in the followings research priorities will provide important insight into its potential for targeted therapy: (1) The in vivo impact of AMPK-mediated signaling in the context of primary and metastatic diseases; (2) The complex interplay between CAMKKβ, androgen receptor and AMPK (Fig. 1); (3) Factors that modulate AMPK-induced effects on cell fate (including survival, apoptosis and autophagy) and motility; and (4) Factors influencing the metabolic consequences of AMPK functions, including glucose and lipid metabolism.

Despite its importance in terms of energy homeostasis, the role of AMP-activated protein kinase in adipose tissue remains controversial. Initial studies have described an anti-lipolytic role for AMP-activated protein kinase, whereas more recent studies have suggested the converse. Thus we have addressed the role of AMP-activated protein kinase in adipose tissue by modulating AMP-activated protein kinase activity in primary rodent adipocytes using pharmacological activators or by adenoviral expression of dominant negative or constitutively active forms of the kinase. We then studied the effects of AMP-activated protein kinase activity modulation on lipolytic mechanisms. Finally, we analyzed the consequences of a genetic deletion of AMP-activated protein kinase in mouse adipocytes. AMP-activated protein kinase activity in adipocytes is represented mainly by the alpha(1) isoform and is induced by all of the stimuli that increase cAMP in adipocytes, including fasting. When AMP-activated protein kinase activity is increased by 5-aminoimidazole-4-carboxamide-riboside, phenformin, or by the expression of a constitutively active form, isoproterenol-induced lipolysis is strongly reduced. Conversely, when AMP-activated protein kinase activity is decreased either by a dominant negative form or in AMP-activated protein kinase alpha(1) knock-out mice, lipolysis is increased. We present data suggesting that AMP-activated protein kinase acts on hormone-sensitive lipase by blocking its translocation to the lipid droplet. We conclude that, in mature adipocytes, AMP-activated protein kinase activation has a clear anti-lipolytic effect.

AMP-activated protein kinase (AMPK) is a multifunctional kinase that negatively regulates the mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) signaling, two signaling pathways linked to pain promotion after injury, such as surgical incision. AMPK can be activated directly using positive allosteric modulators, as well as indirectly through the upregulation of upstream kinases, such as liver kinase B1 (LKB1), which is a mechanism of action of metformin. Metformin’s antihyperalgesic effects occur only in male mice, raising questions about how metformin regulates pain sensitivity. We used metformin and other structurally distinct AMPK activators narciclasine (NCLS), ZLN-024, and MK8722, to treat incision-induced mechanical hypersensitivity and hyperalgesic priming in male and female mice. Metformin was the only AMPK activator to have sex-specific effects. We also found that indirect AMPK activators metformin and NCLS were able to reduce mechanical hypersensitivity and block hyperalgesic priming, whereas direct AMPK activators ZLN-024 and MK8722 only blocked priming. Direct and indirect AMPK activators stimulated AMPK in dorsal root ganglion (DRG) neuron cultures to a similar degree; however, incision decreased phosphorylated AMPK (p-AMPK) in DRG. Because AMPK phosphorylation is required for kinase activity, we interpret our findings as evidence that indirect AMPK activators are more effective for treating pain hypersensitivity after incision because they can drive increased p-AMPK through upstream kinases like LKB1. These findings have important implications for the development of AMPK-targeting therapeutics for pain treatment.SIGNIFICANCE STATEMENTNonopioid treatments for postsurgical pain are needed. Our work focused on whether direct or indirect AMP-activated protein kinase (AMPK) activators would show greater efficacy for inhibiting incisional pain, and we also tested for potential sex differences. We conclude that indirect AMPK activators are likely to be more effective as potential therapeutics for postsurgical pain because they inhibit acute pain caused by incision and prevent the long-term neuronal plasticity that is involved in persistent postsurgical pain. Our work points to the natural product narciclasine, an indirect AMPK activator, as an excellent starting point for development of therapeutics.

Background &amp; Hypothesis: The serine-threonine liver kinase B1 (LKB1) activates AMP-activated protein kinase (AMPK) and negatively regulates aerobic glycoloysis (Warburg effect). LKB1 and AMPK have long been established as tumor suppressors, leading to clinical trials that test the efficacy of AMPK activators as cancer therapeutics. Paradoxically, we found that high expression levels of LKB1 and subunits of AMPK at diagnosis correlate with poor clinical outcome in patients with high risk B precursor acute lymphoblastic leukemia (ALL) (n = 207). These findings seem to contradict the historical notion of LKB1-AMPK as a tumor suppressor pathway, suggesting that the functions of LKB1-AMPK pathway may depend on cellular and genetic contexts. Results: Here, we focus on the role of LKB1 in BCR-ABL1-driven leukemia - chronic myeloid leukemia (CML) and B cell lineage Ph+ ALL. To do so, genetic mouse models for 4-hydroxytamoxifen (4-OHT)-inducible deletion of Lkb1 in BCR-ABL1-transformed hematopoietic stem and progenitor cells (CML-like) and B cell progenitors (Ph+ ALL) were developed. In agreement with previous findings in solid tumors, Cre-mediated Lkb1 deletion in CML-like cells resulted in enhanced proliferation. Unexpectedly, deletion of Lkb1 in Ph+ ALL cells led to apoptosis and cell cycle arrest. Moreover, Lkb1deletion delayed the onset of Ph+ ALL development as well as prolonged overall survival of transplant recipient mice in vivo. Consistent with the above observations, Arf, p53 and p27 levels were reduced in Lkb1-deficient CML cells, while Lkb1 deletion in Ph+ ALL cells up-regulated Arf, p53 and p27 levels. Decreases in glucose consumption and lactate production were also observed in Lkb1-deificient Ph+ ALL cells; however, increases in the levels of glucose consumed and lactate produced were detected in CML cells following Lkb1 deletion. Importantly, inhibition of AMPK using Compound C (an ATP-competitive inhibitor) resulted in apoptosis in patient-derived Ph+ ALL cells, while Compound C had no significant effects on the viability of a panel of lymphoma and multiple myeloma cell lines tested. Furthermore, patient-derived Ph+ ALL cells were resistant to treatment with various AMPK activators (metformin, phenformin and AICAR). Finally, Compound C showed synergistic responses in combination with Imatinib and different PI3K/AKT inhibitors (BKM120, AZD5363 and GSK690693) in Ph+ ALL. In vivo, Compound C in combination with BKM120, a PI3K inhibitor, exerted significantly more potent inhibitory effect on leukemia progression than each agent alone, prolonging the overall survival of recipient mice. Conclusions: Taken together, our findings demonstrate that LKB1 plays divergent roles in myeloid lineage CML and B cell lineage Ph+ ALL. While AMPK activators were shown to be effective against CML cells in previous studies, inhibiting the LKB1-AMPK pathway may provide a better therapeutic avenue for treatment of Ph+ ALL. Citation Format: Lai N. Chan, Seyedmehdi Shojaee, Christian Hurtz, Huimin Geng, Carina Ng, Behzad Kharabi, Markus Müschen. Lineage-specific metabolic reprogramming in BCR-ABL1-driven leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2447. doi:10.1158/1538-7445.AM2014-2447

AMP-activated protein kinase (AMPK) is a cellular energy sensor that is conserved in eukaryotes. Although AMPK is traditionally thought to play a major role in the regulation of cellular lipid and protein metabolism, recent discoveries reveal that AMPK inhibits mammalian target of rapamycin (mTOR) signaling and connects with several tumor suppressors such as liver kinase B1 (LKB1), p53, and tuberous sclerosis complex 2 (TSC2), indicating that AMPK may be a potential target for cancer prevention and treatment. For the first time, we demonstrated that apigenin, a naturally occurring nonmutagenic flavonoid, induced AMPK activation in human keratinocytes (both cultured HaCaT cell line and primary normal human epidermal keratinocytes). Through experiments with over-expression of constitutively active Akt and knockdown of LKB1 expression by siRNAs, we further found that the activation of AMPK by apigenin was not dependent on its inhibition of Akt, and was independent of the activation of upstream kinase LKB1. Instead, another upstream kinase of AMPK, calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ), was required for apigenin-induced AMPK activation. We have demonstrated that knockdown of CaMKKβ expression by siRNA or inhibition of CaMKKβ activity by either CaMKK inhibitor STO-609 or BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; a chelator of intracellular Ca(2+)) prevented apigenin-induced AMPK activation. Apigenin-induced AMPK activation inhibited mTOR signaling and further induced autophagy in human keratinocytes. These results suggest that one of the mechanisms by which apigenin exerts its chemopreventive action may be through activation of AMPK and induction of autophagy in human keratinocytes.

Endocannabinoids and ghrelin are potent appetite stimulators and are known to interact at a hypothalamic level. However, both also have important peripheral actions, including beneficial effects on the ischemic heart and increasing adipose tissue deposition, while ghrelin has direct effects on carbohydrate metabolism. The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that functions as a fuel sensor to regulate energy balance at both cellular and whole body levels, and it may mediate the action of anti-diabetic drugs such as metformin and peroxisome proliferator-activated receptor gamma agonists. Here we show that both cannabinoids and ghrelin stimulate AMPK activity in the hypothalamus and the heart, while inhibiting AMPK in liver and adipose tissue. These novel effects of cannabinoids on AMPK provide a mechanism for a number of their known actions, such as the reduction in infarct size in the myocardium, an increase in adipose tissue, and stimulation of appetite. The beneficial effects of ghrelin on heart function, including reduction of myocyte apoptosis, and its effects on lipogenesis and carbohydrate metabolism, can also be explained by its ability to activate AMPK. Our data demonstrate that AMPK not only links the orexigenic effects of endocannabinoids and ghrelin in the hypothalamus but also their effects on the metabolism of peripheral tissues.

Introduction: Obesity is an independent risk factor for the development of breast cancer. Molecular effects of obesity are mediated via perturbations in the adipocytokine profile. Adiponectin is widely considered as an adipocytokine with therapeutic potential as it inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effects of adiponectin are still elusive. The present study was designed to systematically elucidate the underlying mechanisms by which adiponectin inhibits growth of breast cancer cells. Methods: Characteristic of autophagy were evaluated using transmission electron microscopy, acridine orange staining and western blot analysis of key effector molecules of autophagy. RT-PCR, western blot and immunofluorescence analysis were used to examine SIRT1 (Sirtuin 1), liver kinase B1 (LKB1), and AMP-activated protein kinase (AMPK) axes. Functional importance of AMPK activation and LKB1 overexpression in the biologic effects of adiponectin was examined by using AMPK-null and AMPK-wild type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. LKB1 immunoprecipitates were used to examine LKB1 deacetylation in response to adiponectin. Results: We provide strong evidence that adiponectin causes autophagy in breast cancer cells. Adiponectin treated breast cancer cells exhibit autophagosomes and major autophagosomal protein level changes including ATG1 (autophagy related 1) activation, cleavage of LC3 and suppression of p62 (p62/SQSTM1) expression. Adiponectin-induced autophagy leads to breast cancer cell death as evident by increased levels of cleaved-caspase 9, cleaved-PARP, and Tunel positivity. Adiponectin-mediated autophagy-induction as well as autophagic cell death is attenuated in the presence of autophagy inhibitors. Analysis of the underlying molecular mechanisms reveals that adiponectin treatment increases AMPK activation that is required for adiponectin-mediated ATG1 overexpression and phosphorylation. Intriguingly, we discover that adiponectin increases SIRT1 expression which deacetylates LKB1 leading to its activation. LKB1 knock-down inhibits adiponectin mediated autophagy induction as evidenced by modulations in major autophagosomal protein levels including ATG1. Analysis of breast tumors treated with adiponectin reveals significant increase in the levels of SIRT1, LKB1, phospho AMPK, phospho ATG1 as well as decreased p62. Conclusions: These data uncover a novel role of adiponectin as an inducer of autophagic cell death and provide the first in vitro and in vivo evidence of the integral role of the SIRT1-LKB1-ATG1 axis in adiponectin mediated autophagic cell death of breast cancer cells. Citation Format: Seung J. Chung, Neeraj Saxena, Dipali Sharma. Adiponectin induces autophagic cell death in breast cancer cells through SIRT1 mediated deacetylation of LKB1 leading to ATG1 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1673. doi:10.1158/1538-7445.AM2013-1673