AMP-activated Protein Kinase Knockdown Research Articles

LKB1 suppresses KSHV reactivation and promotes primary effusion lymphoma 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.

Succinate activates UCP2 to suppress neuroinflammation and confer protection following intracerebral hemorrhage.

Succinate, a metabolite in the tricarboxylic acid cycle, is increasingly recognized to play essential roles in inflammation by functioning either as an intracellular or extracellular signaling molecule. However, the role and mechanisms of succinate in inflammation remain elusive. Here, we investigated the mechanism underlying the effects of succinate on neuroinflammation in intracerebral hemorrhage (ICH) models. We unexpectedly found that succinate robustly inhibited neuroinflammation and conferred protection following ICH. Mechanistically, oxidation of succinate by succinate dehydrogenase (SDH) drove reverse electron transport (RET) at mitochondrial complex I, leading to mitochondrial superoxide production in microglia. Complex I-derived superoxide, in turn, activated uncoupling protein 2 (UCP2). By using mice with specific deletion of UCP2 in microglia/macrophage, we showed that UCP2 was needed for succinate to inhibit neuroinflammation, confer protection, and activate downstream AMP-activated protein kinase (AMPK) following ICH. Moreover, knockdown of SDH, complex I or AMPK abolished the therapeutic effects of succinate following ICH. We provide evidence that driving complex I RET to activate UCP2 is a novel mechanism of succinate intracellular signaling and a mechanism underlying the inhibition of neuroinflammation by succinate. succinate; uncoupling protein 2; microglia; neuroinflammation; intracerebral hemorrhage.

Xanthine oxidoreductase inhibition ameliorates high glucose-induced glomerular endothelial injury by activating AMPK through the purine salvage pathway

Xanthine oxidoreductase (XOR) contributes to reactive oxygen species production. We investigated the cytoprotective mechanisms of XOR inhibition against high glucose (HG)-induced glomerular endothelial injury, which involves activation of the AMP-activated protein kinase (AMPK). Human glomerular endothelial cells (GECs) exposed to HG were subjected to febuxostat treatment for 48 h and the expressions of AMPK and its associated signaling pathways were evaluated. HG-treated GECs were increased xanthine oxidase/xanthine dehydrogenase levels and decreased intracellular AMP/ATP ratio, and these effects were reversed by febuxostat treatment. Febuxostat enhanced the phosphorylation of AMPK, the activation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1α and PPAR-α and suppressed the phosphorylation of forkhead box O (FoxO)3a in HG-treated GECs. Febuxostat also decreased nicotinamide adenine dinucleotide phosphate oxidase (Nox)1, Nox2, and Nox4 expressions; enhanced superoxide dismutase activity; and decreased malondialdehyde levels in HG-treated GECs. The knockdown of AMPK inhibited PGC-1α–FoxO3a signaling and negated the antioxidant effects of febuxostat in HG-treated GECs. Despite febuxostat administration, the knockdown of hypoxanthine phosphoribosyl transferase 1 (HPRT1) also inhibited AMPK–PGC-1α–FoxO3a in HG-treated GECs. XOR inhibition alleviates oxidative stress by activating AMPK–PGC-1α–FoxO3a signaling through the HPRT1-dependent purine salvage pathway in GECs exposed to HG conditions.

The Lipophilic Extract from Ginkgo biloba L. Leaves Promotes Glucose Uptake and Alleviates Palmitate-Induced Insulin Resistance in C2C12 Myotubes.

Ginkgo biloba L. (ginkgo) is a widely used medicinal plant around the world. Its leaves, which have been used as a traditional Chinese medicine, are rich in various bioactive components. However, most of the research and applications of ginkgo leaves have focused on terpene trilactones and flavonol glycosides, thereby overlooking the other active components. In this study, a lipophilic extract (GL) was isolated from ginkgo leaves. This extract is abundant in lipids and lipid-like molecules. Then, its effect and potential mechanism on glucose uptake and insulin resistance in C2C12 myotubes were investigated. The results showed that GL significantly enhanced the translocation of GLUT4 to the plasma membrane, which subsequently promoted glucose uptake. Meanwhile, it increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream targets. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor compound C reversed these effects. Additionally, GL ameliorated palmitate-induced insulin resistance by enhancing insulin-stimulated glucose uptake, increasing the phosphorylation of protein kinase B (PKB/AKT), and restoring the translocation of GLUT4 from the cytoplasm to the membrane. However, pretreatment with compound C abolished these beneficial effects of GL. In conclusion, GL enhances basal glucose uptake in C2C12 myotubes and improves insulin sensitivity in palmitate-induced insulin resistant myotubes through the AMPK pathway.

Low-dose hypomethylating agents cooperate with ferroptosis inducers to enhance ferroptosis by regulating the DNA methylation-mediated MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway in acute myeloid leukemia

BackgroundFerroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells, especially relapsed and refractory (R/R)-AML, present high GPX4 levels and enzyme activities, pharmacological inhibition of GPX4 alone has limited application in AML. Thus, whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown.MethodsLipid reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC), ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3), or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally, we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo.ResultsWe first assessed GPX4 expression and found that GPX4 levels were higher in AML cells, especially those with MLL rearrangements, than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects, low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis, and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically, DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK, suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition, DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally, DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models.ConclusionsOur study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients, especially for R/R-AML.

Icariin exerts anti-tumor activity by inducing autophagy via AMPK/mTOR/ULK1 pathway in triple-negative breast cancer

BackgroundBreast cancer is the most prevalent female tumor, of which triple-negative breast cancer (TNBC) accounts for about 15%. Characterized by its aggressive nature and limited treatment options, TNBC currently stands as a significant clinical challenge. This study aimed to investigate the effects of icariin (ICA) on TNBC and explore the underlying molecular mechanism.MethodsCell viability was assessed using CCK-8 assay, whereas the impact of ICA on cell proliferation was determined using colony formation assay and detection of proliferating cell nuclear antigen protein. Wound healing and transwell assays were used to evaluate the effects of ICA on cell migration and invasion, respectively. Flow cytometry was used to analyze cell cycle distribution and apoptosis. Transmission electron microscopy and monodansylcaverine staining were performed to detect the induction of autophagy, whereas molecular docking was conducted to predict the potential targets associated with autophagy. The in vivo anti-tumor effects of ICA were evaluated using a TNBC 4T1 xenograft mouse model. Protein expression levels were examined using immunoblotting and immunohistochemistry.ResultsIn vitro, ICA effectively suppressed the viability, proliferation, migration, and invasion of TNBC cells and induced G0/G1 phase cell cycle arrest, apoptosis, and autophagy in TNBC cells by regulating the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) signaling pathway. The knockdown of AMPK and inhibition of autophagy with 3-methyladenine reversed the effects of ICA, highlighting the importance of AMPK and autophagy in the anti-cancer mechanism of ICA. In vivo, ICA significantly inhibited TNBC growth, promoted autophagy, and regulated AMPK/mTOR/ULK1 pathway.ConclusionsOur findings demonstrated that ICA exerts anti-cancer effects against TNBC and the associated molecular mechanisms. This study will help to facilitate further preclinical and clinical investigations for the treatment of TNBC.

Enhancing β-cell function and identity in type 2 diabetes: The protective role of Coptis deltoidea C. Y. Cheng et Hsiao via glucose metabolism modulation and AMPK signaling activation

BackgroundAbnormalities in glucose metabolism may be the underlying cause of β-cell dysfunction and identity impairment resulting from high glucose exposure. In China, Coptis deltoidea C. Y. Cheng et Hsiao (YL) has demonstrated remarkable hypoglycemic effects. Hypothesis/purposeTo investigate the hypoglycemic effect of YL and determine the mechanism of YL in treating diabetes. MethodsA type 2 diabetes mouse model was used to investigate the pharmacodynamics of YL. YL was administrated once daily for 8 weeks. The hypoglycemic effect of YL was assessed by fasting blood glucose, an oral glucose tolerance test, insulin levels, and other indexes. The underlying mechanism of YL was examined by targeting glucose metabolomics, western blotting, and qRT-PCR. Subsequently, the binding capacity between predicted AMP-activated protein kinase (AMPK) and important components of YL (Cop, Ber, and Epi) were validated by molecular docking and surface plasmon resonance. Then, in AMPK knockdown MIN6 cells, the mechanisms of Cop, Ber, and Epi were inversely confirmed through evaluations encompassing glucose-stimulated insulin secretion, markers indicative of β-cell identity, and the examination of glycolytic genes and products. ResultsYL (0.9 g/kg) treatment exerted notable hypoglycemic effects and protected the structural integrity and identity of pancreatic β-cells. Metabolomic analysis revealed that YL inhibited the hyperactivated glycolysis pathway in diabetic mice, thereby regulating the products of the tricarboxylic acid cycle. KEGG enrichment revealed the intimate relationship of this process with the AMPK signaling pathway. Cop, Ber, and Epi in YL displayed high binding affinities for AMPK protein. These compounds played a pivotal role in preserving the identity of pancreatic β-cells and amplifying insulin secretion. The mechanism underlying this process involved inhibition of glucose uptake, lowering intracellular lactate levels, and elevating acetyl coenzyme A and ATP levels through AMPK signaling. The use of a glycolytic inhibitor corroborated that attenuation of glycolysis restored β-cell identity and function. ConclusionYL demonstrates significant hypoglycemic efficacy. We elucidated the potential mechanisms underlying the protective effects of YL and its active constituents on β-cell function and identity by observing glucose metabolism processes in pancreatic tissue and cells. In this intricate process, AMPK plays a pivotal regulatory role.

AMPK activator-treated human cardiac spheres enhance maturation and enable pathological modeling

BackgroundCardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation.MethodshiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli.ResultsTreatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features—including enhanced Ca2+ transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells.ConclusionAMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases.

Vitamin D plays a protective role in osteoarthritis by regulating AMPK/mTOR signalling pathway to activate chondrocyte autophagy.

The deletion of chondrocyte autophagy seems to play a key role in the pathogenesis of osteoarthritis (OA). Patients with OA often have vitamin D (VD) deficiency, and VD supplementation can improve pain and alleviate the progression of joint structures in patients. In this study, we aimed to investigate whether VD could enhance autophagy by activating the adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling pathway and protect against OA. In this study, the levels of target proteins and genes were examined by western blot and qRT-PCR. Apoptotic cells were detected using TUNEL staining. Characteristics of autophagy were observed by LysoTracker red staining, mRFP-GFP-LC3 adenovirus transfection, and transmission electron microscopy. siRNA-mediated AMPK and mTOR knockdown were used to investigate the role of the AMPK/ mTOR signalling pathway in VD-induced autophagy. Haematoxylin and eosin and safranin-O/fast green staining were used detect cartilage alterations. We suggested that VD significantly reduced chondrocyte death and alleviated extracellular matrix degradation. Further studies showed that VD promoted the expression of the autophagy-related protein LC3II through the AMPK/mTOR signalling pathway in chondrocytes, activated lysosome activity, promoted the formation of autophagy-associated lysosomes, which played a crucial role in the degradation of intracellular organelles and maintained homeostasis. The anti-apoptotic effect of VD on chondrocytes was associated with the activation of autophagy. The group of AMPK-normal and mTOR-knockdown in the presence of VD inhibited chondrocyte apoptosis by promoting autophagy. This study highlights that VD can activate chondrocyte autophagy through the AMPK/mTOR signalling pathway.

Kaempferol attenuates nonalcoholic fatty liver disease in type 2 diabetic mice via the Sirt1/AMPK signaling pathway

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases with limited treatment options. Moreover, its prevalence is doubled in type 2 diabetes mellitus (T2DM). Kaempferol (KAP) is a flavonoid compound that has been suggested to have beneficial effects on NAFLD, but studies on the mechanism are lacking, especially in the diabetic state. Herein, we investigated the effect of KAP on NAFLD associated with T2DM and its underlying mechanism in vitro and in vivo. The results of in vitro studies indicated that KAP treatment (10−8–10−6 M) significantly reduced lipid accumulation in oleic acid-induced HepG2 cells. Moreover, in the T2DM animal model of db/db mice, we confirmed that KAP (50 mg/kg) significantly reduced lipid accumulation and improved liver injury. Mechanistic studies in vitro and in vivo showed that Sirtuin 1 (Sirt1)/AMP-activated protein kinase (AMPK) signal was involved in KAP regulation of hepatic lipid accumulation. KAP treatment activated Sirt1 and AMPK, upregulated the levels of fatty acid oxidation-related protein proliferator activated receptor gamma coactivator 1α (PGC1α); and downregulated lipid synthesis-related proteins, including acetyl-coA carboxylase (ACC), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1 (SREBP1). Furthermore, the curative effect of KAP on lipid accumulation was abolished by siRNA-mediated knockdown of either Sirt1 or AMPK. Collectively, these findings suggest that KAP may be a potential therapeutic agent for NAFLD associated with T2DM by regulating hepatic lipid accumulation through activation of Sirt1/AMPK signaling.

AMPK negatively regulates RANKL-induced osteoclast differentiation by controlling oxidative stress

AMP-activated protein kinase (AMPK) is a crucial energy sensor of cellular metabolism under various metabolic stresses, such as oxidative stress and inflammation. AMPK deficiency increases osteoclast numbers and reduces bone mass; however, the precise mechanisms remain unclear. This study aimed to clarify the mechanistic connection between AMPK and osteoclast differentiation, and the potential role of AMPK in the anti-resorptive effects of several phytochemicals. We found that receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation, osteoclastic gene expression, and activation of mitogen-activated protein kinase (MAPK) and NF-κB were promoted in cells transfected with AMPK siRNA. AMPK knockdown led to defective synthesis of heme oxygenase-1, an antioxidant enzyme, and the upstream mediator, nuclear factor erythroid-2-related factor 2. Furthermore, treatment with N-acetyl-l-cysteine, an antioxidant, abolished osteoclast differentiation and MAPK/NF-κB activation induced by AMPK knockdown. AMPK activators, hesperetin, gallic acid, resveratrol, and curcumin, suppressed osteoclast differentiation via the activation of AMPK. These results suggest that AMPK inhibits RANKL-induced osteoclast differentiation by enhancing antioxidant defense system and regulating oxidative stress. AMPK activation by dietary-derived phytochemicals may be effective for the treatment of bone diseases.

Lactobacillus plantarum postbiotics trigger AMPK-dependent autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome activation.

We previously found that Lactobacillus plantarum (LP)-derived postbiotics protected animals against Salmonella infection, but the molecular mechanism remains obscure. This study clarified the mechanisms from the perspective of autophagy. Intestinal porcine epithelial cells (IPEC-J2) were pretreated with LP-derived postbiotics (the culture supernatant, LPC; or heat-killed bacteria, LPB), and then challenged with Salmonella enterica Typhimurium (ST). Results showed that LP postbiotics markedly triggered autophagy under ST infection, as indicated by the increased LC3 and Beclin1 and the decreased p62 levels. Meanwhile, LP postbiotics (particularly LPC) exhibited a strong capacity of inhibiting ST adhesion, invasion and replication. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) led to a significant decrease of autophagy and the aggravated infection, indicating the importance of autophagy in LP postbiotics-mediated Salmonella elimination. LP postbiotics (especially LPB) significantly suppressed ST-induced inflammation by modulating inflammatory cytokines (the increased interleukin (IL)-4 and IL-10, and decreased tumor necrosis factor-α (TNF), IL-1β, IL-6 and IL-18). Furthermore, LP postbiotics inhibited NOD-like receptor protein 3 (NLRP3)inflammasome activation, as evidenced by the decreased levels of NLRP3, Caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Deficits in autophagy resulted in an increase of inflammatory response and inflammasome activation. Finally, we found that both LPC and LPB triggered AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy, and this was further confirmed by AMPK RNA interference. The intracellular infection and NLRP3 inflammasome were aggravated after AMPK knockdown. In summary, LP postbiotics trigger AMPK-mediated autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome in IPEC-J2 cells. Our findings highlight the effectiveness of postbiotics, and provide a new strategy for preventing Salmonella infection.

Acrylamide induces human chondrocyte cell death by initiating autophagy‑dependent ferroptosis.

Acrylamide (ACR) is formed during heat treatment of foodstuffs and ACR may serve as a probable malignant neoplastic disease agent in all organs and tissues of the human body. However, it is unknown if ACR is associated with ankylosing spondylitis (AS) pathogenesis. Cell viability and proliferation were determined using CCK-8 assay and EdU staining. Flow cytometry was used to determine cell death and cell cycle arrest. Intracellular lipid reactive oxygen species, Fe2+ and mitochondrial membrane potential (MMP) were analyzed using a C11-BODIPY581/591 fluorescent probe, FerroOrange staining and a JC-1 MMP Assay kit, respectively. The present study showed that ACR decreased chondrocyte cell viability in a dose-dependent manner and that ACR significantly promoted chondrocyte senescence. ACR also elevated the expression of cell cycle arrest-associated proteins, including p53, cyclin-dependent kinase inhibitor 1 and cyclin-dependent kinase inhibitor protein, in human chondrocytes. Similarly, DNA damage was also enhanced following ACR treatment in chondrocytes. In addition, the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) and the autophagy inhibitor 3-methyladenine abolished ACR-induced cell death in chondrocytes. ACR was shown to activate autophagic flux and induce mitochondrial dysfunction by increasing the MMP. Western blot analysis of ferroptosis-related proteins demonstrated that ACR decreased the expression of glutathione peroxidase 4, solute carrier family 7 member 11, transferrin receptor protein 1 and ferritin heavy chain 1 in chondrocytes whereas Fer-1 abolished these effects. ACR treatment significantly elevated the phosphorylation levels of AMP-activated protein kinase (AMPK) and serine/threonine-protein kinase ULK1 in human chondrocytes. Notably, the effect of ACR was diminished by knockdown of AMPK, as evidenced by reduced lipid reactive oxygen species accumulation and Fe2+ levels. Hence, ACR inhibited cell proliferation and contributed to cell death by inducing autophagy-dependent ferroptosis while promoting autophagy by activating AMPK-ULK1-mTOR signaling in human chondrocytes. It was hypothesized that the presence of ACR in foodstuffs may increase the risk of AS and that decreasing ACR in food products is of importance.

Thalidomide Attenuates Mast Cell Activation by Upregulating SHP-1 Signaling and Interfering with the Action of CRBN.

Allergy is a chronic inflammatory disease, and its incidence has increased worldwide in recent years. Thalidomide, which was initially used as an anti-emetic drug but was withdrawn due to its teratogenic effects, is now used to treat blood cancers. Although the anti-inflammatory and immunomodulatory properties of thalidomide have been reported, little is known about its influence on the mast cell-mediated allergic reaction. In the present study, we aimed to evaluate the anti-allergic activity of thalidomide and the underlying mechanism using mouse bone marrow-derived mast cells (BMMCs) and passive cutaneous anaphylaxis (PCA) mouse models. Thalidomide markedly decreased the degranulation and release of lipid mediators and cytokines in IgE/Ag-stimulated BMMCs, with concurrent inhibition of FcεRI-mediated positive signaling pathways including Syk and activation of negative signaling pathways including AMP-activated protein kinase (AMPK) and SH2 tyrosine phosphatase-1 (SHP-1). The knockdown of AMPK or SHP-1 with specific siRNA diminished the inhibitory effects of thalidomide on BMMC activation. By contrast, the knockdown of cereblon (CRBN), which is the primary target protein of thalidomide, augmented the effects of thalidomide. Thalidomide reduced the interactions of CRBN with Syk and AMPK promoted by FcεRI crosslinking, thereby relieving the suppression of AMPK signaling and suppressing Syk signaling. Furthermore, oral thalidomide treatment suppressed the PCA reaction in mice. In conclusion, thalidomide suppresses FcεRI-mediated mast cell activation by activating the AMPK and SHP-1 pathways and antagonizing the action of CRBN, indicating that it is a potential anti-allergic agent.

Effects of Cilostazol on Angiogenesis in Diabetes through Adiponectin/Adiponectin Receptors/Sirtuin1 Signaling Pathway.

Cilostazol is an antiplatelet agent with vasodilating effects that functions by increasing the intracellular concentration of cyclic adenosine monophosphate. We have previously shown that cilostazol has favorable effects on angiogenesis. However, there is no study to evaluate the effects of cilostazol on adiponectin. We investigated the effects of cilostazol on angiogenesis in diabetes in vitro and in vivo through adiponectin/adiponectin receptors (adipoRs) and the sirtuin 1 (SIRT1)/AMP-activated protein kinase (AMPK) signaling pathway. Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were cocultured under high glucose (HG) conditions. Adiponectin concentrations in the supernatants were significantly increased when HASMCs were treated with cilostazol but not significantly changed when only HUVECs were treated with cilostazol. Cilostazol treatment enhanced the expression of SIRT1 and upregulated the phosphorylation of AMPK in HG-treated HUVECs. By sequential knockdown of adipoRs, SIRT1, and AMPK, our data demonstrated that cilostazol prevented apoptosis and stimulated proliferation, chemotactic motility, and capillary-like tube formation in HG-treated HUVECs through the adipoRs/SIRT1/AMPK signaling pathway. The phosphorylation of downstream signaling molecules, including acetyl-CoA carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), was downregulated when HUVECs were treated with a SIRT1 inhibitor. In streptozotocin-induced diabetic mice, cilostazol treatment could improve blood flow recovery 21-28 days after inducing hindlimb ischemia as well as increase the circulating of CD34+CD45dim cells 14-21 days after operation; moreover, these effects were significantly attenuated by the knockdown of adipoR1 but not adipoR2. The expression of SIRT1 and phosphorylation of AMPK/ACC and Akt/eNOS in ischemic muscles were significantly attenuated by the gene knockdown of adipoRs. Cilostazol improves HG-induced endothelial dysfunction in vascular endothelial cells and enhances angiogenesis in diabetic mice by upregulating the expression of adiponectin/adipoRs and its SIRT1/AMPK downstream signaling pathway.

AMPK/MFF Activation: Role in Mitochondrial Fission and Mitophagy in Dry Eye.

To assess the role of mitochondrial morphology and adenosine monophosphate-activated protein kinase (AMPK)/mitochondrial fission factor (MFF) in dry eye and the underlying mechanisms. Immortalized human corneal epithelial cells (HCECs) and primary HCECs were cultured under high osmotic pressure (HOP). C57BL/6 female mice were injected subcutaneously with scopolamine. Quantitative real-time PCR was used to measure mRNA expression. Protein expression was assessed by western blot and immunofluorescence staining. Mitochondrial morphology was observed by confocal microscopy and transmission electron microscopy. First, HOP induced mitochondrial oxidative damage to HCECs, accompanied by mitochondrial fission and increased mitophagy. Then, AMPK/MFF pathway proteins were increased consequent to HOP-induced energy metabolism dysfunction. Interestingly, the AMPK pathway promoted mitochondrial fission and mitophagy by increasing the recruitment of dynamin-related protein 1 (DRP1) to the mitochondrial outer membrane in the HOP group. Moreover, AMPK knockdown attenuated mitochondrial fission and mitophagy due to HOP in HCECs. AMPK activation triggered mitochondrial fission and mitophagy. Mitochondrial fission of HCECs stressed by HOP was mediated via MFF phosphorylation. MFF knockdown reversed mitochondrial fragmentation and mitophagy in HCECs treated with HOP. Inhibition of MFF protected HCECs against oxidative damage, cell death, and inflammation in the presence of HOP. Finally, we detected mitochondrial fission and AMPK pathway activation in vivo. The AMPK/MFF pathway mediates the development of dry eye by positively regulating mitochondrial fission and mitophagy. Inhibition of mitochondrial fission can alleviate oxidative damage and inflammation in dry eye and may provide experimental evidence for treating dry eye.

Critical Reanalysis of the Mechanisms Underlying the Cardiorenal Benefits of SGLT2 Inhibitors and Reaffirmation of the Nutrient Deprivation Signaling/Autophagy Hypothesis.

SGLT2 (sodium-glucose cotransporter 2) inhibitors produce a distinctive pattern of benefits on the evolution and progression of cardiomyopathy and nephropathy, which is characterized by a reduction in oxidative and endoplasmic reticulum stress, restoration of mitochondrial health and enhanced mitochondrial biogenesis, a decrease in proinflammatory and profibrotic pathways, and preservation of cellular and organ integrity and viability. A substantial body of evidence indicates that this characteristic pattern of responses can be explained by the action of SGLT2 inhibitors to promote cellular housekeeping by enhancing autophagic flux, an effect that may be related to the action of these drugs to produce simultaneous upregulation of nutrient deprivation signaling and downregulation of nutrient surplus signaling, as manifested by an increase in the expression and activity of AMPK (adenosine monophosphate-activated protein kinase), SIRT1 (sirtuin 1), SIRT3 (sirtuin 3), SIRT6 (sirtuin 6), and PGC1-α (peroxisome proliferator-activated receptor γ coactivator 1-α) and decreased activation of mTOR (mammalian target of rapamycin). The distinctive pattern of cardioprotective and renoprotective effects of SGLT2 inhibitors is abolished by specific inhibition or knockdown of autophagy, AMPK, and sirtuins. In the clinical setting, the pattern of differentially increased proteins identified in proteomics analyses of blood collected in randomized trials is consistent with these findings. Clinical studies have also shown that SGLT2 inhibitors promote gluconeogenesis, ketogenesis, and erythrocytosis and reduce uricemia, the hallmarks of nutrient deprivation signaling and the principal statistical mediators of the ability of SGLT2 inhibitors to reduce the risk of heart failure and serious renal events. The action of SGLT2 inhibitors to augment autophagic flux is seen in isolated cells and tissues that do not express SGLT2 and are not exposed to changes in environmental glucose or ketones and may be related to an ability of these drugs to bind directly to sirtuins or mTOR. Changes in renal or cardiovascular physiology or metabolism cannot explain the benefits of SGLT2 inhibitors either experimentally or clinically. The direct molecular effects of SGLT2 inhibitors in isolated cells are consistent with the concept that SGLT2 acts as a nutrient surplus sensor, and thus, its inhibition causes enhanced nutrient deprivation signaling and its attendant cytoprotective effects, which can be abolished by specific inhibition or knockdown of AMPK, sirtuins, and autophagic flux.

Abstract 6160: Tie2-mediated AMPK activation by ferritin-based protein C nanoparticles inhibits advanced prostate cancer development through induction of vasodilation

Abstract Prostate cancer (PCa) is one of the most common malignancies and the second-leading cause of cancer-related mortality in men in Western countries. Androgen deprivation therapy (ADT) is an initial systemic therapy for advanced PCa, but almost all cancer eventually becomes castration resistant. Recently, we introduced ferritin-based protein C nanoparticles (PCNs), known as TFG and TFMG, which enhance normalization of the tumor vasculature. However, the exact underlying mechanism how PCNs induces AMP-activated protein kinase (AMPK) activation and anti-tumor activity in castration-resistant prostate cancer (CRPC) remains unknown. Here, we investigated the role of PCNs for CRPC development in endothelial cells. We found that TFMG inhibits prostate cancer castration resistance by activating AMPK through the Tie2-mediated signaling pathway. TFMG treatment increased AMPK phosphorylation at Thr172 and acetyl-CoA carboxylase (ACC) at Ser79 in EA.hy926 cells. TFMG treatment also significantly increased the phosphorylation of endothelial nitric oxide synthase (eNOS) with inducing nitric oxide (NO) production in EA.hy926 cells. In addition, TFMG treatment induced the vasodilatory effect in isolated rat mesenteric resistance arteries (MRAs), which was blocked by NO synthase inhibitor L-NAME. Interestingly, these effects were all abolished by pretreatment with Compound C (an AMPK inhibitor), rebastinib (selective Tie2 Inhibitor), AMPK knockdown using siRNA, or dominant negative AMPKα recombinant adenovirus. Finally, we found that TFMG functionally abrogates CRPC development in mouse model. In summary, TFMG exerted vasodilatation through activating Tie2/AMPK/eNOS signaling in endothelial cells, which leads to the overcoming vasoconstriction induced ADT. This study provides the potential value of TFMG in vasodilation of blood vessels leading suppression of CRPC development. Citation Format: You Mie Lee, Ji-Hak Jeong, Hyunha Jang, Jihye You. Tie2-mediated AMPK activation by ferritin-based protein C nanoparticles inhibits advanced prostate cancer development through induction of vasodilation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6160.

AMP-activated kinase regulates porcine reproductive and respiratory syndrome virus infection in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen in the pig industry worldwide. Many viruses manipulate their cellular metabolism to replicate themselves and cause infection. A conserved cellular energy sensor, 5'-AMP-activated protein kinase (AMPK), maintains cellular energy homeostasis. We found that PRRSV infection caused significant AMPK activation in a time-dependent manner via the ROS-calcium/calmodulin-dependent protein kinase-2 pathway. RNA interference-mediated AMPK knockdown could increase PRRSV replication in MARC-145 cells, suggesting that AMPK contributed to PRRSV infection regulation. Moreover, investigation of the effect of AMPK activity on PRRSV replication showed that PRRSV replication could be suppressed by the pharmacological agonists 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside and A769662. Conversely, an AMPK inhibitor, compound C, markedly enhanced PRRSV infection. Furthermore, the AMPK agonist A769662 was found to exert no effect on PRRSV entry, assembly, and release, suggesting that A769662 may hinder the PRRSV genome replication in MARC-145 cells. In conclusion, AMPK may be a promising antiviral drug target against PRRSV infection.

AMP-activated protein kinase re-sensitizes A549 to paclitaxel via up-regulating solute carrier organic anion transporter family member 1B3 expression

Paclitaxel (PTX) is a common antineoplastic drug whose functionality is often restricted by drug resistance. Solute carrier organic anion transporter family member 1B3 (SLCO1B3) is a PTX influx transporter and its low expression has been proved to be relevant with PTX resistance. It has been widely reported that AMP-activated protein kinase (AMPK) could re-sensitize tumor cells to PTX. Our gene array result demonstrates AMPK up-regulated SLCO1B3. In this paper, we have tried to explain the relationships between PTX, SLCO1B3 and AMPK. First, we have verified the proliferative inhibition of PTX on A549 and found that PTX could inhibit A549 cells proliferation. Then, we have explored the relationship between SLCO1B3 and PTX: SLCO1B3 expression significantly decreased when A549 cells were treated with PTX or in A549 PTX resistant cells (A549-PTX) and the intracellular PTX concentration in A549-PTX was also lower. When treated with metformin/LKB1, both SLCO1B3 expression and intracellular PTX concentration have increased. Knockdown of AMPK has induced decreased SLCO1B3 expression. Moreover, in vitro and in vivo experiments have showed that metformin not only obviously inhibited A549-PTX tumor xenograft and A549-PTX proliferation alone, but also enhanced PTX efficacy to A549-PTX and this may be relevant to SLCO1B3. To verify it, we have treated A549 cells with AMPK both activators and an inhibitor, and then found that AMPK activators could weaken the PTX effect in inhibiting SLCO1B3 while its inhibitor has opposite effect. With knockdown of SLCO1B3, the effect of AMPK in re-sensitizing A549 to paclitaxel has decreased. To sum up, activation of AMPK can up-regulate SLCO1B3 expression, enhance the sensitivity of A549 cells to PTX, providing a new way to re-sensitize PTX resistance.