scholarly journals Apigenin and Luteolin Regulate Autophagy by Targeting NRH-Quinone Oxidoreductase 2 in Liver Cells

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 776
Author(s):  
Elzbieta Janda ◽  
Concetta Martino ◽  
Concetta Riillo ◽  
Maddalena Parafati ◽  
Antonella Lascala ◽  
...  
Keyword(s):  
Potential Role ◽  
Autophagic Flux ◽  
Ampk Activation ◽  
Amp Kinase ◽  
Quinone Oxidoreductase ◽  
Ampk Phosphorylation ◽  
Autophagy Induction ◽  
Ampk Activity ◽  
Dietary Flavonoids

Dietary flavonoids stimulate autophagy and prevent liver dysfunction, but the upstream signaling pathways triggered by these compounds are not well understood. Certain polyphenols bind directly to NRH-quinone oxidoreductase 2 (NQO2) and inhibit its activity. NQO2 is highly expressed in the liver, where it participates in quinone metabolism, but recent evidence indicates that it may also play a role in the regulation of oxidative stress and autophagy. Here, we addressed a potential role of NQO2 in autophagy induction by flavonoids. The pro-autophagic activity of seven flavonoid aglycons correlated perfectly with their ability to inhibit NQO2 activity, and flavones such as apigenin and luteolin showed the strongest activity in all assays. The silencing of NQO2 strongly reduced flavone-induced autophagic flux, although it increased basal LC3-II levels in HepG2 cells. Both flavones induced AMP kinase (AMPK) activation, while its reduction by AMPK beta (PRKAB1) silencing inhibited flavone-induced autophagy. Interestingly, the depletion of NQO2 levels by siRNA increased the basal AMPK phosphorylation but abrogated its further increase by apigenin. Thus, NQO2 contributes to the negative regulation of AMPK activity and autophagy, while its targeting by flavones releases pro-autophagic signals. These findings imply that NQO2 works as a flavone receptor mediating autophagy and may contribute to other hepatic effects of flavonoids.

AMPK activation with AICAR provokes an acute fall in plasma [K+]

2008 ◽  
Vol 294 (1) ◽  
pp. C126-C135 ◽  
Author(s):  
Dan Zheng ◽  
Anjana Perianayagam ◽  
Donna H. Lee ◽  
M. Douglas Brannan ◽  
Li E. Yang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Infusion Rate ◽  
Extracellular Fluid ◽  
Ampk Activation ◽  
Conscious Rats ◽  
Significant Fall ◽  
Ampk Phosphorylation ◽  
Atp Levels ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK), activated by an increase in intracellular AMP-to-ATP ratio, stimulates pathways that can restore ATP levels. We tested the hypothesis that AMPK activation influences extracellular fluid (ECF) K+ homeostasis. In conscious rats, AMPK was activated with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) infusion: 38.4 mg/kg bolus then 4 mg·kg−1·min−1 infusion. Plasma [K+] and [glucose] both dropped at 1 h of AICAR infusion and [K+] dropped to 3.3 ± 0.04 mM by 3 h, linearly related to the increase in muscle AMPK phosphorylation. AICAR treatment did not increase urinary K+ excretion. AICAR lowered [K+] whether plasma [K+] was chronically elevated or lowered. The K+ infusion rate needed to maintain baseline plasma [K+] reached 15.7 ± 1.3 μmol K+·kg−1·min−1 between 120 and 180 min AICAR infusion. In mice expressing a dominant inhibitory form of AMPK in the muscle (Tg-KD1), baseline [K+] was not different from controls (4.2 ± 0.1 mM), but the fall in plasma [K+] in response to AICAR (0.25 g/kg) was blunted: [K+] fell to 3.6 ± 0.1 in controls and to 3.9 ± 0.1 mM in Tg-KD1, suggesting that ECF K+ redistributes, at least in part, to muscle ICF. In summary, these findings illustrate that activation of AMPK activity with AICAR provokes a significant fall in plasma [K+] and suggest a novel mechanism for redistributing K+ from ECF to ICF.

scholarly journals CMTM7 as a novel molecule of ATG14L-Beclin1-VPS34 complex enhances autophagy by Rab5 to regulate tumorigenicity

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Baocai Liu ◽  
Yinliang Lu ◽  
Tingting Zhang ◽  
Xinyue Yu ◽  
Qian Wang ◽  
...  
Keyword(s):  
Early Stage ◽  
Xenograft Model ◽  
Vital Role ◽  
Autophagic Flux ◽  
In Vivo Experiments ◽  
Transmission Electron ◽  
Autophagy Induction

Abstract Background CMTM7 is a tumor suppressor that positively regulates EGFR degradation by promoting Rab5 activation, and plays a vital role in tumor progression. Rab5 forms complexes with Beclin1 and VPS34, and acts in the early stage of autophagy. However, the affects of CMTM7 on autophagy and its mechanism are still unclear. Methods The effect of CMTM7 on autophagy induction was confirmed by western blotting, confocal microscopy and transmission electron microscopy. Co-immunoprecipitation was used to analyse the interaction of CMTM7 with autophagy initiation complex and Rab5. The xenograft model in nude mice was used to elucidate the function of CMTM7 in tumorigenicity and autophagy in vivo. Results In this study, we first demonstrated that CMTM7 facilitated the initiation of autophagosome formation, which consequently promoted the subsequent multistage process of autophagic flux, i.e. from autophagosome assembly till autolysosome formation and degradation. Confocal and co-immunoprecipitation showed that CMTM7 interacted with Rab5, VPS34, Beclin1, and ATG14L, but not with ULK1, UVRAG and LC3B. CMTM7 also increased the activity of ATG14L-linked VPS34 complex and its association with Rab5. Both in vitro and in vivo experiments demonstrated that knockdown of CMTM7 enhanced tumor growth by impairing autophagy. Conclusion These findings highlighted the role of CMTM7 in the regulation of autophagy and tumorigenicity, revealing it as a novel molecule that is associated with the interaction of Rab5 and ATG14L-Beclin1-VPS34 complex.

scholarly journals Prefused lysosomes cluster on autophagosomes regulated by VAMP8

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Qixin Chen ◽  
Mingang Hao ◽  
Lei Wang ◽  
Linsen Li ◽  
Yang Chen ◽  
...  
Keyword(s):  
Potential Role ◽  
Autophagic Flux ◽  
Snare Protein ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Fusion Probability ◽  
Autophagosome Maturation ◽  
Factor Attachment Protein Receptor

AbstractLysosome–autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes—vesicle-associated membrane protein 8 (VAMP8)—plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.

scholarly journals ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 475 ◽  
Author(s):  
Tahira Anwar ◽  
Xiaonan Liu ◽  
Taina Suntio ◽  
Annika Marjamäki ◽  
Joanna Biazik ◽  
...  
Keyword(s):  
Beclin 1 ◽  
Autophagic Flux ◽  
Wild Type ◽  
Autophagosome Formation ◽  
Double Knockout ◽  
Exact Role ◽  
Initiation Phase ◽  
Cytoplasmic Material ◽  
Autophagy Induction

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.

scholarly journals Regulation of glucose transport by the AMP-activated protein kinase

2004 ◽  
Vol 63 (2) ◽  
pp. 205-210 ◽  
Author(s):  
Nobuharu Fujii ◽  
William G. Aschenbach ◽  
Nicolas Musi ◽  
Michael F. Hirshman ◽  
Laurie J. Goodyear
Keyword(s):  
Protein Kinase ◽  
Glucose Transport ◽  
Glycogen Content ◽  
Acid Oxidation ◽  
Ampk Activation ◽  
Muscle Biology ◽  
Ampk Activity ◽  
Energy Sensing ◽  
Amp Activated Protein Kinase

The AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated during exercise and muscle contraction as a result of acute decreases in ATP:AMP and phosphocreatine:creatine. Physical exercise increases muscle glucose uptake, enhances insulin sensitivity and leads to fatty acid oxidation in muscle. An important issue in muscle biology is to understand whether AMPK plays a role in mediating these metabolic processes. AMPK has also been implicated in regulating gene transcription and, therefore, may function in some of the cellular adaptations to training exercise. Recent studies have shown that the magnitude of AMPK activation and associated metabolic responses are affected by factors such as glycogen content, exercise training and fibre type. There have also been conflicting reports as to whether AMPK activity is necessary for contraction-stimulated glucose transport. Thus, during the next several years considerably more research will be necessary in order to fully understand the role of AMPK in regulating glucose transport in skeletal muscle.

scholarly journals Activated CAMKKβ-AMPK Signaling Promotes Autophagy in a Spheroid Model of Ovarian Tumour Metastasis

2020 ◽  
Author(s):  
Jeremi Laski ◽  
Bipradeb Singha ◽  
Xu Wang ◽  
Yudith Ramos Valdés ◽  
Olga Collins ◽  
...  
Keyword(s):  
Cell Survival ◽  
Autophagic Flux ◽  
Quiescent State ◽  
Spheroid Formation ◽  
Ampk Signaling ◽  
Tumour Metastasis ◽  
Compound C ◽  
Autophagy Induction ◽  
Ampk Activity

Abstract Background A hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids. Results We observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence microscopy using the mCherry-eGFP-LC3B reporter. A complementary approach using pharmacologic agents Compound C and CAMKKβ inhibitor STO-609 to inhibit AMPK activity both yielded a potent blockade of autophagic flux as well. However, direct activation of AMPK in EOC cells using oligomycin and metformin was insufficient to induce autophagy. STO-609 treatment of EOC spheroids resulted in reduced viability in 7 out of 9 cell lines, but with no observed effect in non-malignant FT190 cell spheroids. Conclusions Our results support the premise that CAMKKβ-mediated AMPK activity is required, at least in part, to regulate autophagy induction in EOC spheroids and support cell viability in this in vitro model of EOC metastasis.

AMPK activation is not critical in the regulation of muscle FA uptake and oxidation during low-intensity muscle contraction

2005 ◽  
Vol 288 (3) ◽  
pp. E592-E598 ◽  
Author(s):  
Marcella A. Raney ◽  
Alice J. Yee ◽  
Mark K. Todd ◽  
Lorraine P. Turcotte
Keyword(s):  
Electrical Stimulation ◽  
Fatty Acid ◽  
Muscle Contraction ◽  
Ampk Activation ◽  
Acetyl Coa Carboxylase ◽  
Low Intensity ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Synergistic Increase

To determine the role of AMP-activated protein kinase (AMPK) activation on the regulation of fatty acid (FA) uptake and oxidation, we perfused rat hindquarters with 6 mM glucose, 10 μU/ml insulin, 550 μM palmitate, and [14C]palmitate during rest (R) or electrical stimulation (ES), inducing low-intensity (0.1 Hz) muscle contraction either with or without 2 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). AICAR treatment significantly increased glucose and FA uptake during R ( P < 0.05) but had no effect on either variable during ES ( P > 0.05). AICAR treatment significantly increased total FA oxidation ( P < 0.05) during both R (0.38 ± 0.11 vs. 0.89 ± 0.1 nmol·min−1·g−1) and ES (0.73 ± 0.11 vs. 2.01 ± 0.1 nmol·min−1·g−1), which was paralleled in both conditions by a significant increase and significant decrease in AMPK and acetyl-CoA carboxylase (ACC) activity, respectively ( P < 0.05). Low-intensity muscle contraction increased glucose uptake, FA uptake, and total FA oxidation ( P < 0.05) despite no change in AMPK (950.5 ± 35.9 vs. 1,067.7 ± 58.8 nmol·min−1·g−1) or ACC (51.2 ± 6.7 vs. 55.7 ± 2.0 nmol·min−1·g−1) activity from R to ES ( P > 0.05). When contraction and AICAR treatment were combined, the AICAR-induced increase in AMPK activity (34%) did not account for the synergistic increase in FA oxidation (175%) observed under similar conditions. These results suggest that while AMPK-dependent mechanisms may regulate FA uptake and FA oxidation at rest, AMPK-independent mechanisms predominate during low-intensity muscle contraction.

Acute spinal cord injury in rats should target activated autophagy

2014 ◽  
Vol 20 (5) ◽  
pp. 568-577 ◽  
Author(s):  
Hongping Hou ◽  
Lihai Zhang ◽  
Licheng Zhang ◽  
Peifu Tang
Keyword(s):  
Electron Microscopy ◽  
Spinal Cord ◽  
Caspase 3 ◽  
Potential Role ◽  
Autophagic Flux ◽  
Autophagosome Formation ◽  
Expression Levels ◽  
Cord Injury ◽  
Microscopy Images

Object Autophagy is a cellular mechanism of maintaining balance between protein synthesis and degradation; the latter can be induced by starvation and neurodegenerative disease. Spinal cord injury (SCI) induces necrosis and apoptosis. Autophagic flux has not yet been defined, especially the potential role of autophagy in relation to apoptosis in different tissue cells. The object of this study was to investigate the occurrence of autophagic flux and the potential role of autophagy and apoptosis post-SCI in rats. Methods Following creation of SCI in rats, activation of autophagic flux was detected at the protein (LC3, beclin1, and p62) and mRNA (beclin1) levels and on electron microscopy images. Distribution of LC3, colocalization of activated caspase-3, and changes in expression levels of bcl-2 and Bax were assessed to investigate the potential role of autophagy and apoptosis. Sprague-Dawley rats were used, and T9–10 hemitransection was performed. Expression levels of LC3, beclin1, p62, bcl-2, and Bax were assessed by Western blot analysis, and beclin1 mRNA levels were assessed by reverse transcription–polymerase chain reaction. Distribution of LC3 and colocalization of activated caspase-3 were analyzed by immunohistochemistry. Autophagosome formation was investigated by electron microscopy. Results The authors found a dramatic elevation in LC3 and beclin1 levels near the scar region. Using double staining, they observed that upregulation of LC3 started at 4 hours in neurons and at 3 days in astrocytes after SCI. Confocal images indicated that the percentage of neurons with apoptosis was reduced, while the percentage of astrocytes with apoptosis was high at 4 hours, 8 hours, and 1 day post-SCI but decreased sharply at 3 days. Electron microscopy images provided evidence of autophagosome formation. Elimination of p62 indicated occurrence of autophagic flux. Expression levels of bcl-2 and Bax were increased and decreased, respectively, near the injury site. Conclusions The results of this research demonstrated that autophagic flux is activated after SCI. Potentially, inhibition of apoptosis could be a target to promote neural recovery.

scholarly journals Activated CAMKKb-AMPK Signaling Promotes Autophagy in a Spheroid Model of Ovarian Tumour Metastasis

2020 ◽  
Author(s):  
Jeremi Laski ◽  
Bipradeb Singha ◽  
Xu Wang ◽  
Yudith Ramos Valdés ◽  
Olga Collins ◽  
...  
Keyword(s):  
Cell Survival ◽  
Autophagic Flux ◽  
Quiescent State ◽  
Spheroid Formation ◽  
Ampk Signaling ◽  
Tumour Metastasis ◽  
Compound C ◽  
Autophagy Induction ◽  
Ampk Activity

Abstract Background: A hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids.Results: We observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence microscopy using the mCherry-eGFP-LC3B reporter. A complementary approach using pharmacologic agents Compound C and CAMKKb inhibitor STO-609 to inhibit AMPK activity both yielded a potent blockade of autophagic flux as well. However, direct activation of AMPK in EOC cells using oligomycin and metformin was insufficient to induce autophagy. STO-609 treatment of EOC spheroids resulted in reduced viability in 7 out of 9 cell lines, but with no observed effect in non-malignant FT190 cell spheroids.Conclusions: Our results support the premise that CAMKKb-mediated AMPK activity is required, at least in part, to regulate autophagy induction in EOC spheroids and support cell viability in this in vitro model of EOC metastasis.

scholarly journals Hyperosmotic Stress Initiates AMPK-Independent Autophagy and AMPK- and Autophagy-Independent Depletion of Thioredoxin 1 and Glyoxalase 2 in HT22 Nerve Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-10
Author(s):  
Alcir Luiz Dafre ◽  
Ariana Ern Schmitz ◽  
Pamela Maher
Keyword(s):  
Ribosomal Protein ◽  
Phosphorylation Site ◽  
Nerve Cells ◽  
Hyperosmotic Stress ◽  
Ampk Activation ◽  
Mtor Inhibition ◽  
Ampk Phosphorylation ◽  
Autophagy Induction ◽  
Thioredoxin 1 ◽  
Glyoxalase 2

Background. Hyperosmotic stress is an important pathophysiologic condition in diabetes, severe trauma, dehydration, infection, and ischemia. Furthermore, brain neuronal cells face hyperosmotic stress in ageing and Alzheimer’s disease. Despite the enormous importance of knowing the homeostatic mechanisms underlying the responses of nerve cells to hyperosmotic stress, this topic has been underrepresented in the literature. Recent evidence points to autophagy induction as a hallmark of hyperosmotic stress, which has been proposed to be controlled by mTOR inhibition as a consequence of AMPK activation. We previously showed that methylglyoxal induced a decrease in the antioxidant proteins thioredoxin 1 (Trx1) and glyoxalase 2 (Glo2), which was mediated by AMPK-dependent autophagy. Thus, we hypothesized that hyperosmotic stress would have the same effect. Methods. HT22 hippocampal nerve cells were treated with NaCl (37, 75, or 150 mM), and the activation of the AMPK/mTOR pathway was investigated, as well as the levels of Trx1 and Glo2. To determine if autophagy was involved, the inhibitors bafilomycin (Baf) and chloroquine (CQ), as well as ATG5 siRNA, were used. To test for AMPK involvement, AMPK-deficient mouse embryonic fibroblasts (MEFs) were used. Results. Hyperosmotic stress induced a clear increase in autophagy, which was demonstrated by a decrease in p62 and an increase in LC3 lipidation. AMPK phosphorylation, linked to a decrease in mTOR and S6 ribosomal protein phosphorylation, was also observed. Deletion of AMPK in MEFs did not prevent autophagy induction by hyperosmotic stress, as detected by decreased p62 and increased LC3 II, or mTOR inhibition, inferred by decreased phosphorylation of P70 S6 kinase and S6 ribosomal protein. These data indicating that AMPK was not involved in autophagy activation by hyperosmotic stress were supported by a decrease in pS555-ULK1, an AMPK phosphorylation site. Trx1 and Glo2 levels were decreased at 6 and 18 h after treatment with 150 mM NaCl. However, this decrease in Trx1 and Glo2 in HT22 cells was not prevented by autophagy inhibition by Baf, CQ, or ATG5 siRNA. AMPK-deficient MEFs under hyperosmotic stress presented the same Trx1 and Glo2 decrease as wild-type cells. Conclusion. Hyperosmotic stress induced AMPK activation, but this was not responsible for its effects on mTOR activity or autophagy induction. Moreover, the decrease in Trx1 and Glo2 induced by hyperosmotic stress was independent of both autophagy and AMPK activation.

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