Activates AMP-activated Protein Kinase Activation Research Articles

Uncovering crosstalk between AMPK and the ATF4-Integrated Stress Response

The reprograming of cellular metabolism in cancer cells is directly inter-related to their capacity for uncontrolled growth. As such, targeting of mitochondrial function has been explored as a therapeutic strategy in cancer. However, inhibition of mitochondria inevitably leads to activation of cellular stress pathways including the integrated stress response (ISR). The ISR includes as a key feature, increased activity of activating transcription factor 4 (ATF4), which goes on to up-regulate a range of gene targets to restore cellular homeostasis. Inhibition of mitochondria also can impair bioenergetic and biosynthetic capacity, which might activate AMP-activated protein kinase (AMPK), a cellular energy sensor that also orchestrates metabolic homeostasis. While the ISR and AMPK both critically regulate metabolism in cancer cells, the link between these mechanisms remains unclear. To address this gap, we tested effects of various energetic stress conditions in MEF cell systems deficient in AMPK activity. To comprehensively impair metabolic networks, we used nutrient starvation treatments in combination with silencing of the protein OPA1 which maintains organisational integrity of the mitochondrial cristae. Our results suggest that AMPK may be involved in suppressing the ISR upon mitochondrial disruption. To further explore, we took the alternative approach to test a range of mitochondrial targeting drugs in wildtype or AMPK-deficient MEF. To gain insight into roles of AMPK in the ISR and related adaptive gene responses, we analysed resulting transcriptomes. These results further revealed a regulatory link between AMPK and the ATF4-ISR, showing that AMPK loss hypersensitizes the cell to energetic stress. Our work uncovering the crosstalk between AMPK and the ATF4-ISR has potential to reveal novel interactions that could suggest metabolic liabilities that may be relevant for targeting in cancer cells.

Radix Rehmanniae Praeparata extracts ameliorate hepatic ischemia-reperfusion injury by restoring lipid metabolism in hepatocytes

Ethnopharmacological relevanceHepatic ischemia/reperfusion injury (HIRI) is a common occurrence during or after liver surgery, representing a major cause for postoperative complications or increased morbidity and mortality in liver diseases. Rehmanniae Radix Praeparata (RRP) is a traditional Chinese medicine frequently used and has garnered extensive attention for its therapeutic potential treating cardiovascular and hepatic ailments. Recent studies have indicated the possibility of RRP in regulating lipid accumulation and apoptosis in hepatocytes. Aim of the studyThis study aimed to investigate the specific mechanisms by which RRP may impede the progression of HIRI through the regulation of lipid metabolism. Materials and methodsHigh-performance liquid chromatography (HPLC) was used to identify the major components of RRP water extract. C57BL/6J mice were orally given RRP at doses of 2.5 g/kg, 5 g/kg, and 10 g/kg for a duration of 7 days before undergoing HIRI surgery. Furthermore, we established a lipid-loaded in vitro model by exposing hepatocytes to oleic acid and palmitic acid (OAPA). The anti-HIRI effect of RRP was determined through transcriptomics and various molecular biology experiments. ResultsAfter identifying active ingredients in RRP, we observed that RRP exerted lipid-lowering and hepatoprotective effects on HIRI mice and OAPA-treated hepatocytes. RRP activated AMP-activated protein kinase (AMPK) and inhibited mammalian target of rapamycin (mTOR), which further on the one hand, inhibited the cleavage and activation of sterol regulatory element binding protein 2 (SREBP2) by limiting the movement of SREBPs cleavage-activating protein (SCAP)-SREBP2 complex with the help of endoplasmic reticulum lipid raft-associated protein 1 (ERLIN1) and insulin-induced gene 1 (INSIG1), and on the other hand, promoted liver X receptor α (LXRα) nuclear transportation and subsequent cholesterol efflux. Meanwhile, the anti-lipotoxic effect of RRP can be partly reversed by an LXRα inhibitor but largely blocked by the application of compound C, an AMPK inhibitor. ConclusionOur study elucidated that RRP served as a potential AMPK activator to alleviate HIRI by blocking SREBP2 activation and cholesterol synthesis, while also activating LXRα to facilitate cholesterol efflux. These findings shed new light on the potential therapeutic use of RRP for improving HIRI.

Mammalian target of differentiation-inducing factor-1 is mitochondrial malate dehydrogenase for activation of AMP-activated protein kinase and induction of mitochondrial fission

AimsDifferentiation-inducing factor-1 (DIF-1) is a polyketide produced by Dictyostelium discoideum that inhibits growth and migration, while promoting the differentiation of Dictyostelium stalk cells through unknown mechanisms. DIF-1 localizes in stalk mitochondria. In addition to its effect on Dictyostelium, DIF-1 also inhibits growth and migration, and induces mitochondrial fission followed by mitophagy in mammalian cells, at least in part by activating AMP-activated protein kinase (AMPK). In a previous study, we found that DIF-1 binds to mitochondrial malate dehydrogenase (MDH2) and inhibits its activity in HeLa cells. In the present study, we investigated whether MDH2 serves as a pharmacological target of DIF-1 in mammalian cells. Main methodsTo examine the enzymatic activity of MDH, mitochondrial morphology, and molecular mechanisms of DIF-1 action, we conducted an MDH reverse reaction assay, immunofluorescence staining, western blotting, and RNA interference using mammalian cells such as human umbilical vein endothelial cells, human cervical cancer cells, mouse endothelial cells, and mouse breast cancer cells. Key findingsDIF-1 inhibited mitochondrial but not cytoplasmic MDH activity. Similar to DIF-1, LW6, an authentic MDH2 inhibitor, induced phosphorylation of AMPK, resulting in the phosphorylation of acetyl-CoA carboxylase (ACC) and the dephosphorylation of p70 S6 kinase with approximately the same potency. DIF-1 and LW6 induced mitochondrial fission. Furthermore, MDH2 knockdown using siRNA reproduced the DIF-1 action on the AMPK signaling and mitochondrial morphology. Conversely, an AMPK inhibitor prevented DIF-1-induced mitochondrial fission. SignificanceWe propose that MDH2 is a mammalian target of DIF-1 for the activation of AMPK and induction of mitochondrial fission.

O-GlcNAc Modification Is a Promising Therapeutic Target for Diabetic Retinopathy.

Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.

Carnosic Acid against Lung Cancer: Induction of Autophagy and Activation of Sestrin-2/LKB1/AMPK Signalling.

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and 3H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo.

The opposite role of lactate dehydrogenase a (LDHA) in cervical cancer under energy stress conditions

Due to insufficient and defective vascularization, the tumor microenvironment is often nutrient-depleted. LDHA has been demonstrated to play a tumor-promoting role by facilitating the glycolytic process. However, whether and how LDHA regulates cell survival in the nutrient-deficient tumor microenvironment is still unclear. Here, we sought to investigate the role and mechanism of LDHA in regulating cell survival and proliferation under energy stress conditions. Our results showed that the aerobic glycolysis levels, cell survival and proliferation of cervical cancer cells decreased significantly after inhibition of LDHA under normal culture condition while LDHA deficiency greatly inhibited glucose starvation-induced ferroptosis and promoted cell proliferation and tumor formation under energy stress conditions. Mechanistic studies suggested that glucose metabolism shifted from aerobic glycolysis to mitochondrial OXPHOS under energy stress conditions and LDHA knockdown increased accumulation of pyruvate in the cytosol, which entered the mitochondria and upregulated the level of oxaloacetate by phosphoenolpyruvate carboxylase (PC). Importantly, the increase in oxaloacetate production after absence of LDHA remarkably activated AMP-activated protein kinase (AMPK), which increased mitochondrial biogenesis and mitophagy, promoted mitochondrial homeostasis, thereby decreasing ROS level. Moreover, repression of lipogenesis by activation of AMPK led to elevated levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which effectively resisted ROS-induced cell ferroptosis and enhanced cell survival under energy stress conditions. These results suggested that LDHA played an opposing role in survival and proliferation of cervical cancer cells under energy stress conditions, and inhibition of LDHA may not be a suitable treatment strategy for cervical cancer.

Regulation of the intestinal Na+/H+ exchanger NHE3 by AMP-activated kinase is dependent on phosphorylation of NHE3 at S555 and S563.

Electroneutral NaCl transport by Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and decreased NHE3 activity contributes to diarrhea. Patients with diabetes often experience gastrointestinal adverse effects and medications are often a culprit for chronic diarrhea in type 2 diabetes (T2D). We have shown previously that metformin, the most widely prescribed drug for the treatment of T2D, induces diarrhea by inhibition of Na+/H+ exchanger 3 (NHE3) in rodent models of T2D. Metformin was shown to activate AMP-activated protein kinase (AMPK), but AMPK-independent glycemic effects of metformin are also known. The current study is undertaken to determine whether metformin inhibits NHE3 by activation of AMPK and the mechanism by which NHE3 is inhibited by AMPK. Inhibition of NHE3 by metformin was abolished by knockdown of AMPK-α1 or AMPK-α2. AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) phosphorylated NHE3 at S555. S555 is the primary site of phosphorylation by protein kinase A (PKA), but AMPK phosphorylated S555 independently of PKA. Using Mass spectrometry, we found S563 as a newly recognized phosphorylation site in NHE3. Altering either S555 or S563 to Ala was sufficient to block the inhibition of NHE3 activity by AMPK. NHE3 inhibition is dependent on ubiquitination by the E3 ubiquitin ligase Nedd4-2 and metformin was shown to induce NHE3 internalization via Nedd4-2-mediated ubiquitination. AICAR did not increase NHE3 ubiquitination when S555 or S563 was mutated. We conclude that AMPK activation inhibits NHE3 activity and NHE3 inhibition is associated with phosphorylation of NHE3 at S555 and S563.NEW & NOTEWORTHY We show that AMP-activated protein kinase (AMPK) phosphorylates NHE3 at S555 and S563 to inhibit NHE3 activity in intestinal epithelial cells. Phosphorylation of NHE3 by AMPK is necessary for ubiquitination of NHE3.

Veronica persica ameliorates acetaminophen-induced murine hepatotoxicity via attenuating oxidative stress and inflammation

Excess acetaminophen (APAP) commonly causes severe acute liver injury (ALI), characterized by oxidative stress, pro-inflammatory responses, and hepatocyte damage. Veronica persica (VP) is a traditional medicine with antioxidant and anti-inflammatory properties. There is a paucity of information on its medicinal value, especially its potential mechanisms for alleviating ALI. This study aimed to clarify the ameliorative effects and intracellular mechanisms of VP on APAP-induced ALI via attenuating oxidative stress and inflammation. Mice were given VP for 7 days before exposure to APAP (300 mg/kg). The HPLC and radical scavenging assay found that VP contains 12 phenolic acids and 6 flavonoids, as well as show robust antioxidant capacity. In the APAP-induced ALI model, pre-treatment with VP significantly reduces APAP-induced hepatotoxicity by observing improved hepatocyte pathological injury and further confirmed by serum biochemical indicator. Also, the reduction of TUNEL-positive regions and the regulation of Bcl-2-associated X protein indicated that VP attenuates hepatocytotoxicity. Moreover, VP pre-intervention inhibits the formation of liver pro-inflammatory cytokines, the expression of inflammatory response genes, and increases in myeloperoxidase (MPO) in APAP-exposed mice. The elevated reduced glutathione (GSH) levels and decreased oxidative stress markers indicate that VP reduces APAP-promoted oxidative stress. Further study revealed that VP inhibited the phosphorylation of NF-κB/STAT3 cascade, blocked ERK and JNK phosphorylation, and activated AMP-activated protein kinase (AMPK). To sum up, this study demonstrated that VP exists hepatoprotective abilities on APAP-induced ALI, primarily by suppressing the phosphorylation of NF-κB/STAT3 cascade and ERK-JNK and inducing AMPK activation to alleviate oxidative stress and inflammation.

Gossypetin prevents the progression of nonalcoholic steatohepatitis by regulating oxidative stress and AMP-activated protein kinase.

Nonalcoholic steatohepatitis (NASH) is severe liver metabolic disorder, however, there are still no effective and safe drugs for its treatment. Previous clinical trials used various therapeutic approaches to target individual pathological mechanisms, but these approaches were unsuccessful because of the complex pathological causes of NASH. Combinatory therapy in which two or more drugs are administered simultaneously to patients with NASH, however, it carries the risk of side effects associated with each individual drug. To solve this problem, we identified gossypetin as an effective dual-targeting agent that activates AMP-activated protein kinase (AMPK) and decreases oxidative stress. Administration of gossypetin decreased hepatic steatosis, lobular inflammation and liver fibrosis in the liver tissue of mice with CDHFD and MCD diet-induced NASH. Gossypetin functioned directly as anti-oxidant agents, decreasing hydrogen peroxide and palmitate-induced oxidative stress in the AML12 cells and liver tissue of MCD diet fed mice without regulating the antioxidant response factors. In addition, gossypetin acted as a novel AMPK activator by binding to the allosteric drug and metabolite (ADaM) site, which stabilizes the activated structure of AMPK. Our findings demonstrate that gossypetin has the potential to serve as a novel therapeutic agent for NAFLD/NASH. Significance Statement This study demonstrates that gossypetin has preventive effect to progression of NASH as a novel AMPK activator and antioxidants. Our findings implicate that simultaneous activation of AMPK and oxidative stress using gossypetin has the potential to serve as a novel therapeutic approach for NAFLD/NASH patients.

Mannose antagonizes GSDME-mediated pyroptosis through AMPK activated by metabolite GlcNAc-6P

Pyroptosis is a type of regulated cell death executed by gasdermin family members. However, how gasdermin-mediated pyroptosis is negatively regulated remains unclear. Here, we demonstrate that mannose, a hexose, inhibits GSDME-mediated pyroptosis by activating AMP-activated protein kinase (AMPK). Mechanistically, mannose metabolism in the hexosamine biosynthetic pathway increases levels of the metabolite N-acetylglucosamine-6-phosphate (GlcNAc-6P), which binds AMPK to facilitate AMPK phosphorylation by LKB1. Activated AMPK then phosphorylates GSDME at Thr6, which leads to blockade of caspase-3-induced GSDME cleavage, thereby repressing pyroptosis. The regulatory role of AMPK-mediated GSDME phosphorylation was further confirmed in AMPK knockout and GSDMET6E or GSDMET6A knock-in mice. In mouse primary cancer models, mannose administration suppressed pyroptosis in small intestine and kidney to alleviate cisplatin- or oxaliplatin-induced tissue toxicity without impairing antitumor effects. The protective effect of mannose was also verified in a small group of patients with gastrointestinal cancer who received normal chemotherapy. Our study reveals a novel mechanism whereby mannose antagonizes GSDME-mediated pyroptosis through GlcNAc-6P-mediated activation of AMPK, and suggests the utility of mannose supplementation in alleviating chemotherapy-induced side effects in clinic applications.

Inhibition of the LRRC8A channel promotes microglia/macrophage phagocytosis and improves outcomes after intracerebral hemorrhagic stroke.

Promoting microglial/macrophage (M/Mφ) phagocytosis accelerates hematoma clearance and improves the prognosis of intracerebral hemorrhagic stroke (ICH). Cation channels such as Piezo1 modulate bacterial clearance by regulating M/Mφ. Whether LRRC8A, an anion channel, affects M/Mφ erythrophagocytosis and functional recovery after ICH was investigated here. We found that LRRC8A is highly expressed on M/Mφ in the perihematomal region of ICH mice. Conditional knockout of Lrrc8a in M/Mφ or treatment with an LRRC8A channel blocker accelerated hematoma clearance, reduced neuronal death, and improved functional recovery after ICH. Mechanistically, the LRRC8A channel inhibition promoted M/Mφ phagocytosis by activating AMP-activated protein kinase (AMPK), thereby inducing nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and increasing Cd36 transcription. Our findings illuminate the regulation of M/Mφ phagocytosis by the LRRC8A channel via the AMPK-Nrf2-CD36 pathway after ICH, suggesting that LRRC8A is a potential target for hematoma clearance in ICH treatment.

Endothelial SIRT-1 has a critical role in the maintenance of capillarization in brown adipose tissue

Brown adipose tissue (BAT) has critical roles in thermogenesis and systemic metabolism. Capillary rarefaction was reported to develop in BAT with dietary obesity, and previous studies showed that suppression of vascular endothelial growth factor A (VEGF-A) reduced capillary density in BAT, promoting the functional decline of this organ. Capillarization is regulated through the balance between angiogenesis and vasculogenesis on the one hand and apoptosis of endothelial cells (ECs) on the other; however, the role of EC apoptosis in BAT remained to be explored. In studies testing the role of boysenberry polyphenols (BoyP) in BAT, we found that BoyP decreased EC apoptosis, enhanced capillarization in BAT, and ameliorated dietary BAT dysfunction, which was associated with the upregulation of nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin 1 (SIRT-1) in ECs. Our studies suggest that EC SIRT-1 would be one of the potential targets of BoyP that contributes to BAT capillarization and function.

Effect of caffeine on mitochondrial biogenesis in the skeletal muscle - A narrative review.

Caffeine is one of the most widely used substances as recreational drug for performance-enhancement in sport, underpinned by a strong evidence base. Although the effects of caffeine are widely investigated within the scope of performance physiology, the molecular effects of caffeine within skeletal muscle remain unclear. Evidence from invitro and invivo models suggest that caffeine regulates the glucose metabolism in the skeletal muscle. Moreover, caffeine seems to stimulate CaMKII, PPARδ/β, AMPK and PGC1α, classical markers of exercise-adaptations, including mitochondrial biogenesis and mitochondrial content. This review summarizes evidence to suggest caffeine-effects within skeletal muscle fibers, focusing on the putative role of caffeine on mitochondrial biogenesis to explore whether caffeine supplementation might be a strategy to enhance mitochondrial biogenesis.

Porcine epidemic diarrhoea virus (PEDV) infection activates AMPK and JNK through TAK1 to induce autophagy and enhance virus replication

ABSTRACT Autophagy plays an important role in defending against invading microbes. However, numerous viruses can subvert autophagy to benefit their replication. Porcine epidemic diarrhoea virus (PEDV) is an aetiological agent that causes severe porcine epidemic diarrhoea. How PEDV infection regulates autophagy and its role in PEDV replication are inadequately understood. Herein, we report that PEDV induced complete autophagy in Vero and IPEC-DQ cells, as evidenced by increased LC3 lipidation, p62 degradation, and the formation of autolysosomes. The lysosomal protease inhibitors chloroquine (CQ) or bafilomycin A and Beclin-1 or ATG5 knockdown blocked autophagic flux and inhibited PEDV replication. PEDV infection activated AMP-activated protein kinase (AMPK) and c-Jun terminal kinase (JNK) by activating TGF-beta-activated kinase 1 (TAK1). Compound C (CC), an AMPK inhibitor, and SP600125, a JNK inhibitor, inhibited PEDV-induced autophagy and virus replication. AMPK activation led to increased ULK1S777 phosphorylation and activation. Inhibition of ULK1 activity by SBI-0206965 (SBI) and TAK1 activity by 5Z-7-Oxozeaenol (5Z) or by TAK1 siRNA led to the suppression of autophagy and virus replication. Our study provides mechanistic insights into PEDV-induced autophagy and how PEDV infection leads to JNK and AMPK activation.

HMGB1 is a mediator of cuproptosis-related sterile inflammation.

Cuproptosis is a recently recognized modality of cell death driven by intracellular copper-dependent mitochondrial stress. However, the mediators of the sterile inflammatory response to cuproptotic death are undetermined. Here, we report that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern, is released by cuproptotic cells to initiate inflammation. Mechanically, copper accumulation-induced adenosine triphosphate (ATP) depletion activates AMP-activated protein kinase (AMPK) to promote HMGB1 phosphorylation, resulting in increased extracellular release. In contrast, genetic (using RNAi) or pharmacologic (using dorsomorphin) inhibition of AMPK activation limits cuproptosis and HMGB1 release. Functionally, the ability of HMGB1-deficient cuproptotic cells to promote advanced glycosylation end product-specific receptor (AGER, also known as RAGE)-dependent inflammatory cytokine production is greatly reduced. Thus, HMGB1 is a key immune mediator of cuproptosis-initiated sterile inflammation.

Aspiletrein A Induces Apoptosis Cell Death via Increasing Reactive Oxygen Species Generation and AMPK Activation in Non-Small-Cell Lung Cancer Cells.

Lung cancer remains a leading cause of death in cancer patients, and deregulation of apoptosis is a serious concern in clinical practice, even though therapeutic intervention has been greatly improved. Plants are a versatile source of biologically active compounds for anticancer drug discovery, and aspiletrein A (AA) is a steroidal saponin isolated from Aspidistra letreae that has a potent cytotoxic effect on various cancer cell lines. In this study, we investigated and determined the underlying molecular mechanism by which AA induces apoptosis. AA strongly induced apoptosis in NSCLC cells by mediating ROS generation and thereby activating AMP-activated protein kinase (AMPK) signaling. Consequently, downstream signaling and levels of phosphorylated mTOR and Bcl-2 were significantly decreased. Pretreatment with either an antioxidant, N-acetylcysteine, or an AMPK inhibitor, compound C, could reverse the apoptosis-inducing effect and counteract the effect of AA on the AMPK signaling pathway. Decreased levels of Bcl-2 were due to AA-mediating Bcl-2 degradation via a ROS/AMPK/mTOR axis-dependent proteasomal mechanism. Consistently, the apoptotic-inducing effect of AA was also observed in patient-derived malignant lung cancer cells, and it suppressed an in vitro 3D-tumorigenesis. This study identified the underlying mechanism of AA on lung cancer apoptosis, thereby facilitating potential research and development of this compound for further clinical implications.

LACC1 contributes to inflammation and cognitive disorder after stroke via the AMPK/NLRP3 pathway.

The current study aimed to investigate the effects of LACC1 on cognitive disorder due to stroke, as well as its underlying mechanism. LACC1 promoted inflammation and aggravated cognitive impairment in a mouse model of stroke. In an in vitro model of stroke, inhibition of LACC1 reduced inflammation and ROS‑induced oxidative stress by activating AMP‑activated protein kinase (AMPK) expression and suppressing NLPR3 expression. Furthermore, our studies revealed that inhibition of AMPK activity reduced the effects of si‑LACC1 on cognitive disorder in mice after stroke via the AMPK/NLPR3 pathway. AMPK activation also reduced the effects of LACC1 on inflammation and ROS‑induced oxidative stress via the NLPR3 pathway in the in vitro model that we evaluated. Our study suggests that LACC1‑aggravated inflammation causes cognitive impairment after stroke via the AMPK/NLRP3 pathway, which may provide a new therapeutic target for stroke and other neurological diseases and their associated complications. In sum, we identified an important role and regulatory mechanism for LACC1 in maintaining stroke‑induced cognitive disorder via the AMPK/NLRP3 pathway.

In Vivo Effects of AICAR and Prodrug 39 (P39) on Anabolic Pathways Following Refeeding in Skeletal Muscle

Exercise training is known to elicit many beneficial adaptations. However, the mechanisms by which this happens are not entirely understood. During muscle contraction, increased intracellular AMP levels mediate many exercise‐induced adaptations by activating AMP‐activated protein kinase (AMPK), among other targets. AMP mimicry may, therefore, be an attractive pharmacological approach to stimulate some of exercise’s benefits. The prodrug AICAR is a well‐established AMPK activator when phosphorylated in the cell to form ZMP, an AMP mimetic. However, it has limited pharmacological potential due to poor pharmacokinetics. Prodrug 39 (P39) uses an alternative approach to deliver ZMP to the cell. The purpose of this study was to compare AMPK and P39 effects on blood glucose levels, AMPK activation, and anabolic signaling in the fasted and refed states. C57BL/6 mice were subjected to a 12 hour fast, followed by 1 hour of refeeding. Following the 12‐hour fast, blood glucose was measured, and mice were injected with saline, AICAR or P39 (400 mg/kg BW). Mice were immediately transferred to cages with food, except for a fasted saline group. After 1 hour of refeeding, food was removed from cages. Blood glucose was measured 25 minutes later, and mice were sacrificed. Gastrocnemius‐plantaris‐soleus complex (GPS) muscles were quick‐frozen for protein analysis. Fasting blood glucose levels were 80 ± 4 mg/dL prior to refeeding and were elevated to 144 ± 8 mg/dL (p<0.0001) after food reintroduction. Both AICAR and P39 caused a significant reduction in blood glucose compared to refed controls (p<0.0001, 54 ± 9, and 56 ± 10 mg/dL, respectively). In GPS muscle, AMPK phosphorylation status was unaffected by any treatment. However, ACC phosphorylation, a downstream target of AMPK, was ~3‐fold higher in AICAR‐treated animals compared to refed controls (p<0.01), with no significant effect of P39. Refeeding induced an increase in anabolic signaling, as indicated by an increase in S6 phosphorylation (~4‐fold, p<0.01) and a trend for increased eIF4E‐binding protein (4E‐BP1) phosphorylation (p = 0.1). This increase was entirely prevented in AICAR and P39‐treated animals. Overall, these results suggest that AICAR and P39 are similarly effective in lowering blood glucose and dampening anabolic signaling following refeeding. However, AICAR was more effective in activating AMPK signaling in skeletal muscle at this dose.

Effect of Long-Term Supplementation with Acetic Acid on the Skeletal Muscle of Aging Sprague Dawley Rats

Mitochondrial function in skeletal muscle, which plays an essential role in oxidative capacity and physical activity, declines with aging. Acetic acid activates AMP-activated protein kinase (AMPK), which plays a key role in the regulation of whole-body energy by phosphorylating key metabolic enzymes in both biosynthetic and oxidative pathways and stimulates gene expression associated with slow-twitch fibers and mitochondria in skeletal muscle cells. In this study, we investigate whether long-term supplementation with acetic acid improves age-related changes in the skeletal muscle of aging rats in association with the activation of AMPK. Male Sprague Dawley (SD) rats were administered acetic acid orally from 37 to 56 weeks of age. Long-term supplementation with acetic acid decreased the expression of atrophy-related genes, such as atrogin-1, muscle RING-finger protein-1 (MuRF1), and transforming growth factor beta (TGF-β), activated AMPK, and affected the proliferation of mitochondria and type I fiber-related molecules in muscles. The findings suggest that acetic acid exhibits an anti-aging function in the skeletal muscles of aging rats.

Chronic Treatment with Metformin Has No Disrupting Effect on the Hepatic Circadian Clock in Mice.

Background and Objectives: The antidiabetic agent metformin is known to activate AMP-activated protein kinase (AMPK) in various tissues. Because AMPK can modulate intracellular circadian clocks through regulating the stability of clock components, a single dose of metformin has been reported to affect circadian clocks in the peripheral tissues. In this study, therefore, we investigated whether chronic treatment with metformin causes the impairment of circadian clocks, especially if given at an inappropriate time. Materials and Methods: Non-diabetic C57BL/6J mice were allowed access to food only during 4 h at the beginning of the dark period, and repeatedly i.p. injected with a nearly maximum non-toxic dose of metformin, once daily either at 4 h after the beginning of the dark period or at the beginning of the light period. Diabetic ob/ob mice were given free access to food and treated with metformin in drinking water. Results: Under the controlled feeding regimen, 8-day treatment with metformin did not alter the mRNA expression rhythms of clock genes in both liver and adipose tissue of C57BL/6J mice, regardless of dosing time. In addition, chronic treatment with metformin for 2 weeks affected hepatic AMPK activation rhythm but did not disrupt the circadian clocks in the liver and adipose tissues of the ob/ob mice. Conclusions: These results mitigate concerns that treatment with metformin impairs peripheral circadian clocks, although confirmation is needed in humans.