Upstream Kinase Of AMP-activated Protein Kinase Research Articles

Geraniin targeting CaMKK2 inhibits lipid accumulation in 3T3-L1 adipocytes by suppressing lipogenesis

Obesity has become a worldwide burden and is associated with severe medical complications. Geraniin is a polyphenolic compound that has a wide range of bioactive properties. There is also evidence to support its pharmacological effects on improving lipid accumulation and obesity. This research investigates the effect of geraniin on lipid accumulation in adipocytes and the underlying mechanism. Mature adipocytes were differentiated from immature 3T3-L1 cells. Oil Red O staining and a triglyceride content determination were conducted to evaluate the intracellular lipid accumulation. Molecular docking studies were performed to determine the interaction between geraniin and the key proteins. Western blotting was used to detect the expression of lipogenic enzymes and transcription factors. Geraniin dose-dependently inhibited lipid accumulation in adipocytes by reducing the expression of fatty acid synthase and increasing the phosphorylation level of acetyl-coenzyme A carboxylase. Moreover, geraniin promoted the phosphorylation of AMP-activated protein kinase (AMPK) and further reduced the expression of lipogenic transcription factors (peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha). The expression of the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) was increased by the geraniin administration. The molecular docking study demonstrated that geraniin can interact with CaMKK2, which is an upstream kinase of AMPK. A selective CaMKK2 inhibitor reversed the suppressive effect of geraniin on lipogenesis. Geraniin targeted CaMKK2 to inhibit lipid accumulation in 3T3-L1 adipocytes by suppressing lipogenesis, and this supports its potential as a candidate natural anti-obesity drug.

Oral administration of berberine limits post-traumatic osteoarthritis development and associated pain via AMP-activated protein kinase (AMPK) in mice

We investigated the effect of berberine, a natural plant product that can activate AMP-activated protein kinase (AMPK), on Osteoarthritis (OA) development and associated pain in mice. Human primary knee chondrocytes were utilized to investigate how AMPK is activated by berberine. Both global knockout (KO) of AMPKα1 and congenic wild type (WT) mice were subjected to the post-traumatic OA through destabilization of medial meniscus (DMM) surgery. Two weeks after surgery, the mice were randomly divided into two groups with one group receiving berberine chloride daily via drinking water and were sacrificed at 6 and 12 weeks after surgery. OA severity was assessed by histological and histomorphometric analyses of cartilage degradation, synovitis, and osteophyte formation. OA-associated pain behavior was also determined. Immunohistochemistry (IHC) analyses were carried out to examine changes in AMPK signaling. Berberine induced phosphorylation of AMPKα (Thr172) via liver kinase B1 (LKB1), the major upstream kinase of AMPK, in chondrocytes in vitro. Both WT and AMPKα1KO developed OA and associated pain post DMM surgery. However, treatment with berberine significantly reduced severity of OA and associated pain in WT but not AMPKα1KO mice. IHC analysis of WT DMM knee cartilage further revealed that berberine inhibited concomitant loss of expression and phosphorylation of AMPKα and expression of SIRT1 and SIRT3, suggesting an important role of activation of AMPK signaling in mediating beneficial effect of berberine. Berberine acts through AMPK to reduce joint structural damage and pain associated with post-traumatic OA in mice in vivo.

Ghrelin inhibited pressure overload–induced cardiac hypertrophy by promoting autophagy via CaMKK/AMPK signaling pathway

Ghrelin, a novel gut hormone, has been shown to exert protective effects on cardiac dysfunction and remodeling. However, the underlying mechanisms of its protective effects remain unclear. Here, we investigated the effects of ghrelin on cardiac hypertrophy and explored the mechanisms involved. Ghrelin (30 μg.kg−1. day−1) was systemically administered to rats with cardiac hypertrophy induced by abdominal aortic constriction (AAC) by a mini-osmotic pump the next day after surgery continuously for 4 weeks. The AAC treated rats without ghrelin infusion showed decreased ghrelin content and expression of its receptors in the hearts. Exogenous ghrelin greatly attenuated cardiac hypertrophy as shown by heart weight to tibial length (HW/TL), hemodynamics, echocardiography, histological analyses, and expression of hypertrophic markers induced by AAC. This corresponded with decreased cardiac fibrosis and inflammation in the hearts of AAC rats treated with ghrelin. Moreover, ghrelin significantly increased the myocardial expression of autophagy markers, which was further confirmed in cultured cardiomyocytes. Concurrently, cardiomyocyte apoptosis in vivo and in vitro was ameliorated by ghrelin, which was reversed by inhibition of autophagy. The enhancement of autophagy and inhibition of apoptosis by ghrelin were eliminated on pretreatment with compound C, an AMP-activated protein kinase (AMPK) inhibitor. Furthermore, inhibition of Ca2+/Calmodulin-dependent protein kinase kinase (CaMKK), an upstream kinase of AMPK, made ghrelin fail to activate AMPK and simultaneously reversed ghrelin’s promotion of autophagy. In conclusion, ghrelin could exert its cardioprotective effects on cardiac hypertrophy by promoting autophagy, possibly via CaMKK/AMPK signaling pathway.

It's well and truly time to stop stating that AMPK regulates glucose uptake and fat oxidation during exercise.

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Repeated ethanol exposure influences key enzymes in cholesterol and lipid homeostasis via the AMPK pathway in the rat prefrontal cortex.

Cholesterol homeostasis has been proposed to be implicated in the development of addiction. However, the effects of ethanol on cholesterol homeostasis within the brain are not well understood. One of the most important regulators of cholesterol homeostasis is HMG-CoA reductase (HMG-CoAR), the rate-limiting enzyme of cholesterol biosynthesis. We examined the phosphorylation of HMG-CoAR and the other key regulator of lipid synthesis, acetyl-CoA carboxylase (ACC), following acute or chronic treatment with ethanol (0.5, 1, or 2g/kg) in the rat prefrontal cortex. The phosphorylation of AMP-activated protein kinase (AMPK), which regulates the HMG-CoAR activity, and its well-known upstream regulators, was also studied. The phosphorylation of HMG-CoAR and ACC were transiently increased by ethanol treatment only in animals previously treated chronically with ethanol. Acute administration to naïve animals did not induce the phosphorylation, regardless of dosage. Similarly, the phosphorylation of AMPK and the upstream regulators, LKB1 and CaMK4, were transiently increased only in chronically ethanol-treated animals. In naïve animals, a high dose (2g/kg) of ethanol decreased phosphorylation. The phosphorylation of TAK1, another upstream kinase of AMPK, was increased only from 30min to 24h after the chronic treatment with ethanol. Together, these results indicate that repeated exposure is required for the activating effect of ethanol on HMG-CoAR and ACC. This effect seems to be mediated by the AMPK system, and may contribute to the long-lasting neuroadaptation involved in the development of alcohol dependence.

AMPK Alters Detrusor Contractility During Emptying in Normal Bladder and Hypertrophied Bladder with Partial Bladder Outlet Obstruction via CaMKKβ.

AMP-activated protein kinase (AMPK) has been implicated in contractility changes in bladders with partial bladder outlet obstruction (PBOO), but the role of AMPK in the contractile response of normal bladder remains unclear. We investigated the phosphorylation of AMPKα and expression of the involved upstream AMPK kinases (AMPKKs) in a model of bladders with PBOO and sought to determine whether the pharmacological inhibition of these two factors affected detrusor contractility in normal bladders, using female Sprague–Dawley rats. Cystometry and Western blot analysis were performed in rats that were subjected to PBOO induction or a sham operation. Cystometry was performed in normal rats that received selective inhibitors of AMPKα and Ca2+/calmodulin-dependent protein kinase kinase (CaMKKβ) (compound C and STO-609, respectively) at doses determined in the experiments. In the PBOO bladders, bladder weight and micturition pressure (MP) were higher and AMPKα phosphorylation (T172) and CaMKKβ expression was significantly reduced. Compound C and STO-609 increased MP. The increased contractile response in bladders with PBOO-induced hypertrophy was related to decreased CaMKKβ/AMPK signaling activity, and the pharmacological inhibition of this pathway in normal bladders increased detrusor contractility, implying a role of CaMKKβ/AMPK signaling in the bladder in the regulation of detrusor contractility.

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-β (TGF-β)-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.

Sitagliptin attenuates myocardial apoptosis via activating LKB-1/AMPK/Akt pathway and suppressing the activity of GSK-3β and p38α/MAPK in a rat model of diabetic cardiomyopathy

The present study aimed to investigate the protective effect of sitagliptin, a dipeptidyl peptidase-4 inhibitor, on diabetic cardiomyopathy (DCM)-associated apoptosis and if this effect is mediated via modulating the activity of the survival kinases; AMP-activated protein kinase (AMPK) and Akt & the apoptotic kinases; glycogen synthase kinase-3 β (GSK-3β) and p38 mitogen-activated protein kinase (p38MAPK). Diabetes was induced by a single intraperitoneal injection of streptozotocin (55 mg/kg). Diabetic rats were treated with sitagliptin (10 mg/kg/day, p.o.) and metformin (200 mg/kg/day, p.o. as positive control) for six weeks. Chronic hyperglycemia resulted in elevation of serum cardiac biomarkers reflecting cardiac damage which was supported by H&E stain. The mRNA levels of collagen types I and III were augmented reflecting cardiac fibrosis and hypertrophy which was supported by Masson trichome stain and enhanced phosphorylation of p38MAPK. Cardiac protein levels of cleaved casapse-3, BAX were elevated, whereas, the levels of Bcl-2 and p-BAD were reduced indicating cardiac apoptosis which could be attributed to the diabetes-induced reduced phosphorylation of Akt and AMPK with concomitant augmented activation of GSK-3β and p38MAPK. Protein levels of liver kinase B-1, the upstream kinase of AMPK were also supressed. Sitagliptin administration alleviated the decreased phosphorylation of AMPK and Akt, inactivated the GSK-3β and p38 AMPK, therefore, attenuating the apoptosis and hypertrophy induced by hyperglycemia in the diabetic heart. In conclusion, sitagliptin exhibits valuable therapeutic potential in the management of DCM by attenuating apoptosis. The underlying mechanism may involve the modulating activity of AMPK, Akt, GSK-3β and p38MAPK.

Gliclazide, a KATP channel blocker, inhibits vascular smooth muscle cell proliferation through the CaMKKβ–AMPK pathway

Gliclazide, a sulfonylurea that is widely used to treat type II-diabetes, specifically blocks KATP channels and recombinant smooth muscle (SUR2B/Kir6.1) KATP channels with high potency. Furthermore, it exerts antioxidant properties and inhibits tumor cell proliferation. In this study, we investigated the inhibitory effect of gliclazide on vascular smooth muscle cell (VSMC) proliferation and tried to identify the underlying signaling pathway. We first investigated the effect of gliclazide-induced AMP-activated protein kinase (AMPK) activation on the proliferation of VSMCs. Gliclazide induced phosphorylation of AMPK in a dose- and time-dependent manner and inhibited VSMC proliferation following stimulation by platelet-derived growth factor (PDGF). However, KATP channel openers and Kir6.1 siRNA prevented gliclazide-mediated inhibition of VSMC proliferation. Gliclazide also increased the levels of Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), an upstream kinase of AMPK. These findings suggested that the effects of KATP channels on AMPK activity were mediated by the regulation of intracellular Ca2+ levels. Oral administration of 2mg/kg gliclazide resulted in the activation of CaMKKβ and AMPK in vivo, suggesting that gliclazide suppressed VSMC proliferation via the CaMKKβ–AMPK signaling pathway. Taken together, our observations indicated that gliclazide-induced AMPK activation may act to prevent diabetes-associated atherosclerosis.

SIRT2 Acts as a Cardioprotective Deacetylase in Pathological Cardiac Hypertrophy.

Pathological cardiac hypertrophy induced by stresses such as aging and neurohumoral activation is an independent risk factor for heart failure and is considered a target for the treatment of heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. We aimed to investigate the roles of SIRT2 in aging-related and angiotensin II (Ang II)-induced pathological cardiac hypertrophy. Male C57BL/6J wild-type and Sirt2 knockout mice were subjected to the investigation of aging-related cardiac hypertrophy. Cardiac hypertrophy was also induced by Ang II (1.3 mg/kg/d for 4 weeks) in male C57BL/6J Sirt2 knockout mice, cardiac-specific SIRT2 transgenic (SIRT2-Tg) mice, and their respective littermates (8 to ≈12 weeks old). Metformin (200 mg/kg/d) was used to treat wild-type and Sirt2 knockout mice infused with Ang II. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice. SIRT2 protein expression levels were downregulated in hypertrophic hearts from mice. Sirt2 knockout markedly exaggerated cardiac hypertrophy and fibrosis and decreased cardiac ejection fraction and fractional shortening in aged (24-month-old) mice and Ang II-infused mice. Conversely, cardiac-specific SIRT2 overexpression protected the hearts against Ang II-induced cardiac hypertrophy and fibrosis and rescued cardiac function. Mechanistically, SIRT2 maintained the activity of AMP-activated protein kinase (AMPK) in aged and Ang II-induced hypertrophic hearts in vivo as well as in cardiomyocytes in vitro. We identified the liver kinase B1 (LKB1), the major upstream kinase of AMPK, as the direct target of SIRT2. SIRT2 bound to LKB1 and deacetylated it at lysine 48, which promoted the phosphorylation of LKB1 and the subsequent activation of LKB1-AMPK signaling. Remarkably, the loss of SIRT2 blunted the response of AMPK to metformin treatment in mice infused with Ang II and repressed the metformin-mediated reduction of cardiac hypertrophy and protection of cardiac function. SIRT2 promotes AMPK activation by deacetylating the kinase LKB1. Loss of SIRT2 reduces AMPK activation, promotes aging-related and Ang II-induced cardiac hypertrophy, and blunts metformin-mediated cardioprotective effects. These findings indicate that SIRT2 will be a potential target for therapeutic interventions in aging- and stress-induced cardiac hypertrophy.

20S-Protopanaxadiol, an aglycosylated ginsenoside metabolite, induces hepatic stellate cell apoptosis through liver kinase B1–AMP-activated protein kinase activation

BackgroundPreviously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved. MethodsUsing LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. H2O2 productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection. Results and conclusionOf the 11 ginsenosides tested, 20S-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20S-PPD was blocked by N-acetyl-l-cysteine pretreatment. In addition, 20S-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20S-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20S-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20S-PPD. Thus, 20S-PPD appears to induce HSC apoptosis by activating LKB1–AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.

Noise-Induced Loss of Hair Cells and Cochlear Synaptopathy Are Mediated by the Activation of AMPK.

Noise-induced hearing loss (NIHL) is a major unresolved public health problem. Here, we investigate pathomechanisms of sensory hair cell death and suggest a novel target for protective intervention. Cellular survival depends upon maintenance of energy homeostasis, largely by AMP-activated protein kinase (AMPK). In response to a noise exposure in CBA/J mice, the levels of phosphorylated AMPKα increased in hair cells in a noise intensity-dependent manner. Inhibition of AMPK via siRNA or the pharmacological inhibitor compound C attenuated noise-induced loss of outer hair cells (OHCs) and synaptic ribbons, and preserved auditory function. Additionally, noise exposure increased the activity of the upstream AMPK kinase liver kinase B1 (LKB1) in cochlear tissues. The inhibition of LKB1 by siRNA attenuated the noise-increased phosphorylation of AMPKα in OHCs, reduced the loss of inner hair cell synaptic ribbons and OHCs, and protected against NIHL. These results indicate that noise exposure induces hair cell death and synaptopathy by activating AMPK via LKB1-mediated pathways. Targeting these pathways may provide a novel route to prevent NIHL. Our results demonstrate for the first time that the activation of AMP-activated protein kinase (AMPK) α in sensory hair cells is noise intensity dependent and contributes to noise-induced hearing loss by mediating the loss of inner hair cell synaptic ribbons and outer hair cells. Noise induces the phosphorylation of AMPKα1 by liver kinase B1 (LKB1), triggered by changes in intracellular ATP levels. The inhibition of AMPK activation by silencing AMPK or LKB1, or with the pharmacological inhibitor compound C, reduced outer hair cell and synaptic ribbon loss as well as noise-induced hearing loss. This study provides new insights into mechanisms of noise-induced hearing loss and suggests novel interventions for the prevention of the loss of sensory hair cells and cochlear synaptopathy.

Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation

The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells.

Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic β-Cells

In pancreatic β-cells, voltage-gated potassium 2.1 (Kv2.1) channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β-cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase (AMPK). Activation of AMPK mimics whereas inhibition of AMPK occludes the effect of leptin. Inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase β, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase (PKA) is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, whereas stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization, and pharmacologically induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (KATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface KATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. This study supports a model in which leptin exerts concerted trafficking regulation of KATP and Kv2.1 channels to coordinately inhibit insulin secretion.

Telmisartan prevention of LPS-induced microglia activation involves M2 microglia polarization via CaMKKβ-dependent AMPK activation

Brain inflammation plays an important role in the pathophysiology of many psychiatric and neurological diseases. During brain inflammation, microglia cells are activated, producing neurotoxic molecules and neurotrophic factors depending on their pro-inflammatory M1 and anti-inflammatory M2 phenotypes. It has been demonstrated that Angiotensin II type 1 receptor blockers (ARBs) ameliorate brain inflammation and reduce M1 microglia activation. The ARB telmisartan suppresses glutamate-induced upregulation of inflammatory genes in cultured primary neurons. We wished to clarify whether telmisartan, in addition, prevents microglia activation through polarization to an anti-inflammatory M2 phenotype. We found that telmisartan promoted M2 polarization and reduced M1 polarization in LPS-stimulated BV2 and primary microglia cells, effects partially dependent on PPARγ activation. The promoting effects of telmisartan on M2 polarization, were attenuated by an AMP-activated protein kinase (AMPK) inhibitor or AMPK knockdown, indicating that AMPK activation participates on telmisartan effects. Moreover, in LPS-stimulated BV2 cells, telmisartan enhancement of M2 gene expression was prevented by the inhibitor STO-609 and siRNA of calmodulin-dependent protein kinase kinase β (CaMKKβ), an upstream kinase of AMPK. Furthermore, telmisartan enhanced brain AMPK activation and M2 gene expression in a mouse model of LPS-induced neuroinflammation. In addition, telmisartan reduced the LPS-induced sickness behavior in this in vivo model, and this effect was prevented by prior administration of an AMPK inhibitor. Our results indicate that telmisartan can be considered as a novel AMPK activator, suppressing microglia activation by promoting M2 polarization. Telmisartan may provide a novel, safe therapeutic approach to treat brain disorders associated with enhanced inflammation.

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.

The CamKKβ Inhibitor STO609 Causes Artefacts in Calcium Imaging and Selectively Inhibits BKCa in Mouse Carotid Body Type I Cells.

It has previously been reported that AMP-activated protein kinase (AMPK) may be critical for hypoxic chemotransduction in carotid body type I cells. This study sought to determine the importance of the regulatory upstream kinase of AMPK, CamKKβ, in the acute response to hypoxia in isolated mouse type I cells.Initial data indicated several previously unreported artefacts associated with using the CamKKβ inhibitor STO609 and Ca(2+) imaging techniques. Most importantly Fura-2 and X-Rhod1 imaging revealed that STO609 quenched emission fluorescence even in the absence of intracellular Ca(2+) ([Ca(2+)](I)). Furthermore, STO609 (100 μM) rapidly inhibited outward macroscopic currents and this inhibition was abolished in the presence of the selective BK(Ca) inhibitor paxilline.Taken together these data suggest that ST0609 should be used with caution during Ca(2+) imaging studies as it can directly interact with Ca(2+) binding dyes. The rapid inhibitory effect of STO609 on BK(Ca) was unexpected as the majority of studies using this compound required an incubation of approximately 10 min to inhibit the kinase. Furthermore, as AMPK activation inhibits BK(Ca), inhibiting AMPK's upstream kinases would, if anything, be predicted to have the opposite effect on BK(Ca). Future work will determine if the inhibition of BK(Ca) is via CamKKβ or via an off target action of STO609 on the channel itself.

AMP-activated protein kinase (AMPK) activity negatively regulates chondrogenic differentiation

Chondrocytes are derived from mesenchymal stem cells, and play an important role in cartilage formation. Sex determining region Y box (Sox) family transcription factors are essential for chondrogenic differentiation, whereas the intracellular signal pathways of Sox activation have not been clearly elucidated. AMP-activated protein kinase (AMPK) is a serine–threonine kinase generally regarded as a key regulator of cellular energy homeostasis. It is known that the catalytic alpha subunit of AMPK is activated by upstream AMPK kinases (AMPKKs) including liver kinase B1 (LKB1). We have previously reported that AMPK is a negative regulator of osteoblastic differentiation. Here, we have explored the role of AMPK in chondrogenic differentiation using in vitro culture models. The phosphorylation level of the catalytic AMPK alpha subunit significantly decreased during chondrogenic differentiation of primary chondrocyte precursors as well as ATDC-5, a well-characterized chondrogenic cell line. Treatment with metformin, an activator of AMPK, significantly reduced cartilage matrix formation and inhibited gene expression of sox6, sox9, col2a1 and aggrecan core protein (acp). Thus, chondrocyte differentiation is functionally associated with decreased AMPK activity.

NDRG2 overexpression enhances glucose deprivation-mediated apoptosis in breast cancer cells via inhibition of the LKB1-AMPK pathway.

The newly identified tumor suppressor, N-myc downstream-regulated gene 2 (NDRG2), has been studied in various cancers because of its anticancer and antimetastasis effects. In this study, we examined the effect of NDRG2 expression on cell viability in MDA-MB-231 human breast cancer cells under conditions that are similar to the microenvironment of solid tumors, which include glucose deprivation. NDRG2 overexpression enhanced the pro-apoptotic effects of glucose deprivation. Glucose deprivation also induced the activation of AMP-activated protein kinase (AMPK), which plays a role in protecting tumor cells from metabolic stresses. NDRG2 overexpression strongly reduced glucose deprivation-induced AMPK phosphorylation and increased the cleavage of poly (ADP-ribose) polymerase (PARP), which indicated the induction of apoptosis. The expression of a constitutively active form of AMPK effectively blocked glucose deprivation-induced apoptosis in NDRG2-overexpressing MDA-MB-231 cells. Moreover, NDRG2 overexpression also enhanced the pro-apoptotic effects of 2-deoxyglucose (2-DG) or hypoxia, an inducer of metabolic stresses. Finally, we showed that LKB1 is an upstream kinase of AMPK that is involved in the inhibition of glucose deprivation-induced AMPK activity in NDRG2-overexpressing cells. Our findings collectively suggest that NDRG2 is a negative regulator of AMPK activity and functions as a sensitizer of glucose deprivation.

The Interaction of AMP-Activated Protein Kinase and its Upstream Activator, Lkb1/Mo25/Strad, Modifies Contractile Function in Rat Cardiac Trabeculae

AMP-activated protein kinase (AMPK) is an energetic regulator that is activated by alterations in cellular AMP. I, as well as others, have shown that AMPK modifies both metabolic and contractile function of the heart. However, to become fully activated, it is necessary for AMPK to be phosphorylated by the upstream AMPK kinase complex, LKB1/MO25/STRAD. Yet, the relationship between this upstream kinase complex, AMPK activation, and myofilament contractile function is not known. We hypothesize that a high LKB1/MO25/STRAD to AMPK ratio will have a different impact on myofilament function than a low LKB1/MO25/STRAD to AMPK ratio. Accordingly, demembranated rat cardiac trabeculae were pre-incubated with varying ratios of LKB1/MO25/STRAD to AMPK and contractile function was measured. Surprisingly, at sarcomere length of 2.2 μm, cardiac fibers pre-incubated with the LKB1/MO25/STRAD complex alone are desensitized to Ca2+ (EC50 4.16 ±0.12 μM [n=6] vs 2.56±0.11 μM [n=13] p<0.05) and have lowered maximum tension (20.02±2.68 mN/mm2 [n=6] vs 39.18± 4.27mN/mm2 [n=13] p<0.05).A high LKB1/MO25/STRAD:AMPK (2.5 mol LKB1/MO25/STRAD: 1 mol AMPK ) decreases Ca2+-sensitivity of tension (EC50 3.71±0.17 μM [n=8] vs. 2.56±0.11 μM [n=13] p<0.05). Tension generation is also somewhat compromised (24.02±1.82 mN/mm2 [n=6] vs 39.18± 4.27mN/mm2 [n=13] p<0.05). An even lower ratio (1 mol LKB1/MO25/STRAD: 2 mol AMPK) results in an increase in Ca2+-sensitivity of tension (EC50 1.36±0.06 μM [n=8] vs. 2.56±0.11 μM [n=13] p<0.05). Tension generation also is restored to control values. Length dependent activation, cooperativity, and rate of tension redevelopment remain unaltered regardless of the presence of AMPK or LKB1/MO25/STRAD.In conclusion, the LKB1/MO25/STRAD complex has been identified as a potential novel regulator of myofilament function. The interaction between LKB1/MO25/STRAD and its target kinase AMPK can also alter contractile function.