Adenosine Monophosphate-activated Kinase Signaling Research Articles

Small heterodimer partner interacting leucine zipper protein (SMILE) ameliorates autoimmune arthritis via AMPK signaling pathway and the regulation of B cell activation

Rheumatoid arthritis (RA) is an autoimmune disease that causes joint swelling and inflammation and can involve the entire body. RA is characterized by the increase of pro-inflammatory cytokines such as interleukin (IL) and tumor necrosis factor, and the over-activation of T lymphocytes and B lymphocytes, which may lead to severe chronic inflammation of joints. However, despite numerous studies the pathogenesis and treatment of RA remain unresolved. This study investigated the use of small heterodimer partner-interacting leucine zipper protein (SMILE) overexpression to treat a mouse model of RA. SMILE is an insulin-inducible corepressor through adenosine monophosphate-activated kinase (AMPK) signaling pathway. The injection of a SMILE overexpression vector to mice with collagen induced-arthritis resulted in a milder clinical pathology and a reduced incidence of arthritis, less joint tissue damage, and lower levels of Th17 cells and plasma B cells in the spleen. Immunohistochemistry of the joint tissue showed that SMILE decreased B-cell activating factor (BAFF) receptor (BAFF-R), mTOR, and STAT3 expression but increased AMPK expression. In SMILE-overexpressing transgenic mice with collagen antibody-induced arthritis (CAIA), a decrease in the arthritis score and reductions in tissue damage, the number of B cells, and antibody production were observed. The treatment of immune cells in vitro with curcumin, a known SMILE-inducing agent, led to decreases in plasma B cells, germinal center B cells, IL-17-producing B cells, and BAFF-R-positive B cells. Taken together, our findings demonstrate the therapeutic potential of SMILE in RA, based on its inhibition of B cell activation mediated by the AMPK/mTOR and STAT3 signaling pathway and BAFF-R expression.EtcT49u9cQiSZwStZ5UX76Video abstract

The potent AMPK inhibitor BAY-3827 shows strong efficacy in androgen-dependent prostate cancer models.

5' adenosine monophosphate-activated kinase (AMPK) is an essential regulator of cellular energy homeostasis and has been associated with different pathologies, including cancer. Precisely defining the biological role of AMPK necessitates the availability of a potent and selective inhibitor. High-throughput screening and chemical optimization were performed to identify a novel AMPK inhibitor. Cell proliferation and mechanistic assays, as well as gene expression analysis and chromatin immunoprecipitation were used to investigate the cellular impact as well as the crosstalk between lipid metabolism and androgen signaling in prostate cancer models. Also, fatty acid turnover was determined by examining lipid droplet formation. We identified BAY-3827 as a novel and potent AMPK inhibitor with additional activity against ribosomal 6 kinase (RSK) family members. It displays strong anti-proliferative effects in androgen-dependent prostate cancer cell lines. Analysis of genes involved in AMPK signaling revealed that the expression of those encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), fatty acid synthase (FASN) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), all of which are involved in lipid metabolism, was strongly upregulated by androgen in responsive models. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) analysis identified several androgen receptor (AR) binding peaks in the HMGCR and PFKFB2 genes. BAY-3827 strongly down-regulated the expression of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and serine-threonine kinase AKT3 in responsive prostate cancer cell lines. Also, the expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family was inhibited by BAY-3827, and this was paralleled by impaired lipid flux. The availability of the potent inhibitor BAY-3827 will contribute to a better understanding of the role of AMPK signaling in cancer, especially in prostate cancer.

Constitutive cyclophilin-D ablation in mice increases exercise and cognitive-behavioral performance under normoxic and hypoxic conditions

We recently reported that constitutive ablation of cyclophilin-D (Cyp-D) in mice reduces oxygen consumption (VO2) while paradoxically increasing exercise endurance, thereby demonstrating increased O2 utilization efficiency. This response was associated with augmented glucose uptake and glucose utilization, in part mediated through adenosine monophosphate-activated kinase signaling. We now hypothesized that Cyp-D knock-out (KO) mice might also exhibit improved cognitive-behavioral performance and that these favorable adaptive responses may persist under hypoxic conditions. We therefore assessed under normoxic (20.9% O2, simulating ground O2 levels) and hypoxic (8% O2, simulating 7600 m altitude O2 levels) conditions exercise capacity and cognitive-behavioral performance. We used a treadmill test to assess exercise capacity, a pole-test to assess agility, an elevated-plus-maze test to assess anti-anxiety, and a passive avoidance test to assess learning and memory retention. Compared to wild type, Cyp-D KO mice showed comparable treadmill work under normoxia (48 ± 12 vs 47 ± 9 Joules) but increased treadmill work (12 ± 1 vs 8 ± 1 Joules; p = 0.02) under hypoxia. Cyp-D KO mice displayed increased pole-descending time (17 ± 3 vs 8 ± 2 s; p ≤ 0.05) under normoxia but shorter pole-descending time (21 ± 3 vs 37 ± 4 s; p ≤ 0.01) under hypoxia. In addition, the Cyp-D KO mice demonstrated increased elevated plus-maze open arm time (91 ± 31 vs 23 ± 12 s; p ≤ 0.05) under hypoxia and increased latency to enter dark chamber (261 ± 23 vs 185 ± 42 s; p ≤ 0.05) under normoxia. Thus, our experiments showed that under normoxia Cyp-D KO mice displayed anti-anxiety behavior and improved learning and memory retention. Under hypoxia, Cyp-D KO mice displayed increased exercise capacity, increased agility, and increased anti-anxiety consistent with our previously reported findings of increased O2 utilization efficiency. Identifying interventions to elicit these effects could be beneficial in a myriad of physiological and clinical conditions in which increasing O2 utilization efficiency would be advantageous.

Abstract 559: AMP-Activated Protein Kinase And Estrogen-Dependent Mechanisms Underlying Increased Susceptibility To Cardiovascular Disease During Menopause

According to the CDC (2017), more women than men have died from heart disease over the last 20-25 years. Yet, premenopausal women are protected against heart and cardiovascular disease (CVD) compared to age-matched men. The cellular and molecular mechanisms underlying the transition from premenopause/perimenopause (CVD-resistance) to postmenopause (CVD-susceptible) in women is unknown and is the focus of this project. The critical barrier impeding translational progress is the lack of appropriate models to study menopause. Most studies have used surgical removal of ovaries (ovariectomy) as a model of menopause; yet this technique poorly recapitulates the natural, physiological transition to menopause that 90% of women experience. We overcome this barrier with administration of the chemical, 4-vinylcyclohexene diepoxide (VCD), where we can mirror progressive ovarian failure and preserve the critical “perimenopause” transitional period, identical to humans. Using this model, we demonstrate that perimenopausal , like cycling (premenopausal) females, are protected from pathological angiotensin II (Ang II)-induced cardiac remodeling, while menopausal females are not. Our novel finding that perimenopausal females remain protected, despite irregular cycling (prior to complete loss of estrogen), underscores the importance of studying the role of estrogen in CVD, across the transition from perimenopause to menopause. Multiple molecular, genetic and cellular mechanisms have been suggested to underlie protection against CVD in non-cycling females, many of which put estrogen as the key mediator. We discovered that one of these pathways, the adenosine monophosphate-activated kinase (AMPK) signaling axis, is activated by estrogen through direct binding of estrogen receptors to the α-catalytic subunit of AMPK. Taken together with our published and preliminary data, we hypothesis that loss of AMPK signaling is responsible for the exacerbated pathological cardiac remodeling in menopause (see Fig.). To date, there are no studies that have tested the ability of AMPK activation to mitigate menopausal susceptibility to CVD. Aim: Determine if AMPK is necessary and/or sufficient to impart protection against pathological cardiac remodeling in menopausal females. Rationale: We discovered that AMPK signaling is severely disabled during menopause where females are more susceptible to CVD. Using the VCD mouse model of menopause, we will determine if protection against Ang II-induced cardiac remodeling during menopause is AMPK-dependent. Summary: We plan to determine the cellular and molecular mechanisms in premenopausal and perimenopausal females that prevent associated cardiovascular morbidities; and determine the underlying shift that gives rise to increased susceptibility to CVD in menopausal females.

AMPK and Estrogen‐dependent mechanisms underlying hypersensitivity to Cardiovascular Disease during menopause

According to the CDC (2017), more women than men have died from heart disease over the last 20–25 years. Yet, premenopausal women are protected against heart and cardiovascular disease (CVD) compared to age‐matched men. The cellular and molecular mechanisms underlying the transition from premenopause/perimenopause (CVD‐resistance) to postmenopause (CVD‐susceptible) in women is unknown and is the focus of this project. The critical barrier impeding translational progress is the lack of appropriate models to study menopause. Most studies have used surgical removal of ovaries (ovariectomy) as a model of menopause; yet this technique poorly recapitulates the natural, physiological transition to menopause that 90% of women experience. We overcome this barrier with administration of the chemical, 4‐vinylcyclohexene diepoxide (VCD), where we can mirror progressive ovarian failure and preserve the critical “perimenopause” transitional period, identical to humans. Using this model, we demonstrate that perimenopausal, like cycling (premenopausal) females, are protected from pathological angiotensin II (Ang II)‐induced cardiac remodeling, while menopausal females are not. Our novel finding that perimenopausal females remain protected, despite irregular cycling (prior to complete loss of estrogen), underscores the importance of studying the role of estrogen in CVD, across the transition from perimenopause to menopause. Multiple molecular, genetic and cellular mechanisms have been suggested to underlie protection against CVD in non‐cycling females, many of which put estrogen as the key mediator. We discovered that one of these pathways, the adenosine monophosphate‐activated kinase (AMPK) signaling axis, is activated by estrogen through direct binding of estrogen receptors to the α‐catalytic subunit of AMPK. Taken together with our published and preliminary data, we hypothesis that loss of AMPK signaling is responsible for the exacerbated pathological cardiac remodeling in menopause(see Fig.). To date, there are no studies that have tested the ability of AMPK activation to mitigate menopausal susceptibility to CVD.AimDetermine if AMPK is necessary and/or sufficient to impart protection against pathological cardiac remodeling in menopausal females.RationaleWe discovered that AMPK signaling is severely disabled during menopause where females are more susceptible to CVD. Using the VCD mouse model of menopause, we will determine if protection against Ang II‐induced cardiac remodeling during menopause is AMPK‐dependent.SummaryWe plan to determine the cellular and molecular mechanisms in premenopausal and perimenopausal females that prevent associated cardiovascular morbidities; and determine the underlying shift that gives rise to increased susceptibility to CVD in menopausal females.Support or Funding InformationIMSD: R25GM062584 BME CVTG (NIH‐T32): 5T32HL007955‐15AHA: 16GRNT31390006This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dengue Virus Activates the AMP Kinase-mTOR Axis To Stimulate a Proviral Lipophagy.

Robust dengue virus (DENV) replication requires lipophagy, a selective autophagy that targets lipid droplets. The autophagic mobilization of lipids leads to increased β-oxidation in DENV-infected cells. The mechanism by which DENV induces lipophagy is unknown. Here, we show that infection with DENV activates the metabolic regulator 5' adenosine-monophosphate activated kinase (AMPK), and that the silencing or pharmacological inhibition of AMPK activity decreases DENV replication and the induction of lipophagy. The activity of the mechanistic target of rapamycin complex 1 (mTORC1) decreases in DENV-infected cells and is inversely correlated with lipophagy induction. Constitutive activation of mTORC1 by depletion of tuberous sclerosis complex 2 (TSC2) inhibits lipophagy induction in DENV-infected cells and decreases viral replication. While AMPK normally stimulates TSC2-dependent inactivation of mTORC1 signaling, mTORC1 inactivation is independent of AMPK activation during DENV infection. Thus, DENV stimulates and requires AMPK signaling as well as AMPK-independent suppression of mTORC1 activity for proviral lipophagy.IMPORTANCE Dengue virus alters host cell lipid metabolism to promote its infection. One mechanism for altered metabolism is the induction of a selective autophagy that targets lipid droplets, termed lipophagy. Lipophagy mobilizes lipid stores, resulting in enhanced β-oxidation and viral replication. We show here that DENV infection activates and requires the central metabolic regulator AMPK for its replication and the induction of lipophagy. This is required for the induction of lipophagy, but not basal autophagy, in DENV-infected cells.

The “adaptive responses” of low concentrations of HBCD in L02 cells and the underlying molecular mechanisms

This study aimed to investigate the "adaptive responses" of hexabromocyclododecanes (HBCD) at environmentally relevant concentrations in human hepatocytes L02. L02 cells were pre-treated with low concentrations of HBCD (10(-13)-10(-11) M), followed by treatment with high concentrations of HBCD, α-hexachlorocyclohexane (α-HCH), polychlorinated biphenyls (PCBs), or polybrominated diphenyl ether-47 (BDE47). The results showed that the pre-treatment with low concentrations of HBCD induced "adaptive responses" to high concentrations of HBCD/α-HCH exposure (but not to PCBs and BDE47), as evidenced by attenuation of survival inhibition, reactive oxygen species (ROS) over-production, and deoxyribonucleic acid (DNA) damage induction. The "adaptive responses" induced by low concentrations of HBCD, which depended on the activation of the phosphatidylinositide 3-kinase/protein kinase B (PI3K/Akt) pathway, reduced the phosphorylation of adenosine monophosphate-activated kinase (AMPK) and enhanced the phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). The observations were further confirmed by the experiments with inhibitors. Moreover, the evaluation on the changes of metabolic enzymes revealed that HBCD and α-HCH shared a similar pattern of cytochrome P450 induction (CYP2B6), which was different from those of PCBs and BDE47 (CYP1A1 and CYP2B6). These results indicated that low concentrations of HBCD could induce "adaptive responses" to the subsequent treatment with high concentrations of HBCD/α-HCH in L02 cells, which was associated with the PI3K/Akt pathway, and AMPK and p38 MAPK signaling. The "adaptive responses" seemed to be dependent on the types of chemicals in terms of the metabolic patterns and chemical structures.

The Role of AMPK Pathway in Neuroprotection.

Adenosine monophosphate-activated kinase (AMPK) is a highly conserved protein kinase found in all eukaryotic genomes. It exists as heterotrimeric protein consisting of α, β, and γ subunits. AMPK is activated by elevated levels of adenosine mono-phosphate (AMP), which is produced during conditions of low ATP production and perhaps mitochondrial dysfunction. Activation of AMPK has been shown to regulate a large number of downstream pathways. These will either increase energy production such as increase oxidation of fatty acids and glucose, or decrease energy utilization such as inhibiting synthesis of glycogen, fatty acid synthesis, and protein synthesis. In addition, being a key regulator of physiological energy dynamics, AMPK has been demonstrated to play roles in regulating various cellular processes such as mitochondrial biogenesis (Jager etal. Proc Natl Acad Sci U S A 104:12017-12022, 2007), autophagy (Hyttinen etal. Rejuven Res 14:651-660, 2011) and inflammation and immune responses (Giri etal. 2004). Retinal neurons have a high energy demand but have a poor energy storage capacity. Because of this, it is likely that the AMPK signaling pathway plays an important role in maintaining energy balance, and therefore may be a therapeutic target to prevent or delay retinal degeneration.

AMPK Stimulates Urea Permeability and Urine Osmolality

Vasopressin is a key regulator of urine concentration through its actions on water and urea transporters, but what happens when vasopressin signaling breaks down‾ We investigated adenosine monophosphate activated kinase (AMPK) as a possible alternate, vasopressin‐independent regulatory pathway for urea and water transport. Mice lacking a vasopressin receptor (V2R‐KO) are polyuric. Injection of metformin to stimulate AMPK significantly increased urine osmolality from 196 (control) to 399 mOsM at 1h, and 586 mOsM at 5h post treatment in the V2R‐KO mice. We further investigated the role of AMPK using isolated perfused rat inner medullary collecting ducts (IMCDs). We showed that metformin and AICAR, AMPK activators, significantly increased urea permeability (control:metformin 16.4 ± 2.3 to 22.3 ± 3.5 x10‐5 cm/s; control:AICAR 19.3 ± 2.4 to 23.3 ± 3.0 x10‐5 cm/s). Hypertonicity stimulates urea transport though PKC. To determine if AMPK is additive to hypertonic stimulation, 400µM metformin was added to the tubule bath either before or after subjecting the IMCDs to hypertonic stimulation. Increasing the osmolality of perfusate and bath from 290 to 690 mOsM increased urea permeability from 18.9 ± 1.4 to 48.4 ± 3.1 x10‐5 cm/s. Addition of metformin further increased urea permeability from 48.4 ± 3.1 to 57.0 ± 4.1 x10‐5 cm/s. Initial stimulation with metformin raised urea permeability from 23.6 ± 1.9 to 32.2 ± 2.5 x10‐5 cm/s. Subsequent increase of the osmolality from 290 to 690 mOsM in the presence of metformin significantly increased urea permeability from 32.2 ± 2.5 to 68.0 ± 7.9 x10‐5 cm/s. We conclude that AMPK signaling may provide a means to increase urea permeability in addition to, or in place of, PKA and PKC. AMPK activation may be useful to increase urine concentrating ability when vasopressin signaling breaks down.

Lkb1 regulates organogenesis and early oncogenesis along AMPK-dependent and -independent pathways

The tumor suppressor Lkb1/STK11/Par-4 is a key regulator of cellular energy, proliferation, and polarity, yet its mechanisms of action remain poorly defined. We generated mice harboring a mutant Lkb1 knockin allele that allows for rapid inhibition of Lkb1 kinase. Culturing embryonic tissues, we show that acute loss of kinase activity perturbs epithelial morphogenesis without affecting cell polarity. In pancreas, cystic structures developed rapidly after Lkb1 inhibition. In lung, inhibition resulted in cell-autonomous branching defects. Although the lung phenotype was rescued by an activator of the Lkb1 target adenosine monophosphate-activated kinase (AMPK), pancreatic cyst development was independent of AMPK signaling. Remarkably, the pancreatic phenotype evolved to resemble precancerous lesions, demonstrating that loss of Lkb1 was sufficient to drive the initial steps of carcinogenesis ex vivo. A similar phenotype was induced by expression of mutant K-Ras with p16/p19 deletion. Combining culture of embryonic tissues with genetic manipulation and chemical genetics thus provides a powerful approach to unraveling developmental programs and understanding cancer initiation.

Alpha-lipoic acid attenuates cardiac fibrosis in Otsuka Long-Evans Tokushima Fatty rats

BackgroundHyperglycemia leads to cardiac oxidative stress and an imbalance in glucose homeostasis. Diabetic cardiomyopathy is characterised by cardiac hypertrophy and fibrosis. However, the underlying mechanisms of diabetic cardiomyopathy are not fully understood. This study aimed to investigate the effects of alpha-lipoic acid (ALA) on cardiac energy metabolism, antioxidant effect, and fibrosis in the hearts of Otsuka Long-Evans Tokushima fatty (OLETF) rats.MethodsAnimals were separated into non-diabetic Long-Evans Tokushima Otsuka (LETO) rats and diabetes-prone OLETF rats with or without ALA (200 mg/kg/day) administration for 16 weeks. Diabetic cardiomyopathy was assessed by staining with Sirius Red. The effect of ALA on AMPK signalling, antioxidant enzymes, and fibrosis-related genes in the heart of OLETF rats were performed by Western blot analysis or immunohistochemistry.ResultsWestern blot analysis showed that cardiac adenosine monophosphate-activated kinase (AMPK) signalling was lower in OLETF rats than in LETO rats, and that ALA treatment increased the signalling in OLETF rats. Furthermore, the low antioxidant activity in OLETF rats was increased by ALA treatment. In addition to increased Sirius red staining of collagen deposits, transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) were expressed at higher levels in OLETF rat hearts than in LETO rat hearts, and the levels of these factors were decreased by ALA.ConclusionsALA enhances AMPK signalling, antioxidant, and antifibrogenic effect. Theses findings suggest that ALA may have beneficial effects in the treatment of diabetic cardiomyopathy.

Abstract P196: The Impact of MicroRNA195 on the Lkb1/AMPK Signaling Axis and Hypertrophic Cardiomyopathy

Objectives: MicroRNAs (miRs) have been identified as chief post-transcriptional regulators of cardiac disease progression. In addition, a critical role of the adenosine monophosphate-activated kinase (AMPK) pathway in the development of myocardial hypertrophy has been revealed. Yet, regulation of the AMPK pathway by miRs in the heart has not been addressed. We hypothesized that components of the AMPK pathway are targeted by miRs and alter AMPK signaling in a mouse model of hypertrophic cardiomyopathy (HCM). Methods and results: Using real-time PCR, a candidate miR screen that included 22 miRs implicated in pathological cardiac disease and/or metabolic dysregulation was performed on hearts from 60-, 120-, and 240-day-old transgenic HCM male mice harboring an R403Q mutation in the myosin heavy gene. Among early (60 day) elevated miRs were miR-195 and -451. Both miR-195 and -451 have conserved target sites in the 3′ UTR of CAB39 (MO25), a central component of the MO25/STRAD/LKB1 complex that acts as an upstream kinase for AMPK and its subsequent activation. We further confirmed the elevation of miR-195 and -451 by Northern blotting. Next, we demonstrated specific expression and a similar distribution pattern of miR-195 and -451 in cardiomyocytes of R403Q HCM hearts by in situ hybridization. To determine whether the conserved sites in MO25 3′ UTR acted as functional targets, either the miR-195 or miR-451 target sequence was cloned into a luciferase expression vector. MiR-195 but not miR-451 suppressed luciferase activity compared to the missense sequence control vector in C2C12 cells. In addition, over-expression of miR195 in C2C12 cells knocked down MO25 expression levels and downstream AMPK signaling (phosphorylation of Acetyl CoA carboxylase). Finally, parallel changes were measured in 60 day R403Q HCM male hearts that included reduced MO25 expression and lowered phosphorlation of AMPK and Acetyl CoA carboxylase. Conclusion: Our findings indicate that miR-195 targets the LKB1/AMPK signaling axis and suggest a functional role for miR-195 elevation in R403Q HCM disease progression.

Arsenic Inhibits Neurite Outgrowth by Inhibiting the LKB1–AMPK Signaling Pathway

BackgroundArsenic (As) is an environmental pollutant that induces numerous pathological effects, including neurodevelopmental disorders.Objectives and MethodsWe evaluated the role of the LKB1–AMPK pathway in As-induced developmental neurotoxicity using Neuro-2a (N2a) neuroblastoma cells as a model of developing neurons.ResultsThe addition of low concentrations of As (≤ 5 μM) during differentiation caused an inhibitory effect on the neurite outgrowth in N2a cells in the absence of cell death. Activation of adenosine monophosphate–activated kinase (AMPK) induced by retinoic acid in differentiating cells was blocked by As. Pretreatment with the AMPK-specific activator 5-aminoimidazole-4-carboxamide riboside or overexpression of a constitutively active AMPK-α1 plasmid reversed As-induced inhibition of neurite outgrowth. The activation of LKB1 (serine/threonine kinase 11), a major AMPK kinase, was also suppressed by As by inhibiting both the phosphorylation and the translocation of LKB1 from nucleus to cytoplasm. Antioxidants, such as N-acetyl cysteine and superoxide dismutase, but not catalase, protected against As-induced inactivation of the LKB1–AMPK pathway and reversed the inhibitory effect of As on neurite outgrowth.ConclusionsReduced neurite outgrowth induced by As results from deficient activation of AMPK as a consequence of a lack of activation of LKB1. Oxidative stress induced by As, especially excessive superoxide, plays a critical role in blocking the LKB1–AMPK pathway. Our studies provide insight into the mechanisms underlying As-induced developmental neurotoxicity, which is important for designing a new strategy for protecting children against this neurotoxic substance.