Autophagic Activity Research Articles

GLP‑1 receptor agonist protects palmitate-induced insulin resistance in skeletal muscle cells by up-regulating sestrin2 to promote autophagy

In this study, we aimed to determine whether liraglutide could effectively reduce insulin resistance (IR) by regulating Sestrin2 (SESN2) expression in L6 rat skeletal muscle cells by examining its interactions with SESN2, autophagy, and IR. L6 cells were incubated with liraglutide (10–1000 nM) in the presence of palmitate (PA; 0.6 mM), and cell viability was detected using the cell counting kit-8 (CCK-8) assay. IR-related and autophagy-related proteins were detected using western blotting, and IR and autophagy-related genes were analyzed using quantitative real-time polymerase chain reaction. Silencing SESN2 was used to inhibit the activities of SESN2. A reduction in insulin-stimulated glucose uptake was observed in PA-treated L6 cells, confirming IR. Meanwhile, PA decreased the levels of GLUT4 and phosphorylation of Akt and affected SESN2 expression. Further investigation revealed that autophagic activity decreased following PA treatment, but that liraglutide reversed this PA-induced reduction in autophagic activity. Additionally, silencing SESN2 inhibited the ability of liraglutide to up-regulate the expression of IR-related proteins and activate autophagy signals. In summary, the data showed that liraglutide improved PA-induced IR in L6 myotubes by increasing autophagy mediated by SESN2.

Zwitterionic betaines over HEPES as the new generation biocompatible pH buffers for cell culture.

Good's buffers have been widely applied in cell/organ culture over the past half a century as biocompatible pH stabilizers. However, the emergence of severe adverse effects, such as cellular uptake, lysosomal autophagic activation, and visible light-induced cytotoxicity, raises serious questions over its biocompatibility while underlying mechanism was unclear. Here we report that riboflavin (RF, component of cell culture medium) generates 1O2, ·OH, and O2 •- under visible light exposure during regular cell manipulation. These short half-life reactive oxygen species (ROS) react with tertiary amine groups of HEPES, producing 106.6μM of H2O2. Orders of magnitude elevated half-life of ROS in the medium caused severe cytotoxicity and systematic disorder of normal cell functions. We have further designed and validated zwitterionic betaines as the new generation biocompatible organic pH buffers, which is able to completely avoid the adverse effects that found on HEPES and derivate Good's buffers. These findings may also open a new avenue for zwitterionic betaine based materials for biomedical applications.

AdipoRon alleviates fatty acid–induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes by promoting autophagy

During the transition period in dairy cows, high circulating concentrations of nonesterified fatty acids (NEFA) increase hepatic lipid deposits and are considered a major pathological factor for liver damage. We investigated whether AdipoRon, a synthetic small-molecule agonist of adiponectin receptors 1 and 2 shown to prevent liver lipid accumulation in nonruminants, could alleviate NEFA-induced lipid accumulation and mitochondrial dysfunction. Bovine hepatocytes were isolated from 5 healthy Holstein female newborn calves (1 d of age, 30-40 kg, fasting), and independently isolated hepatocytes from at least 3 different calves were used for each subsequent experiment. The composition and concentration of NEFA used in this study were selected according to hematological criteria of dairy cows with fatty liver or ketosis. First, hepatocytes were cultured with various concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 12 h. In a second experiment, hepatocytes were treated with AdipoRon at different concentrations (0, 5, 25, or 50 μM for 12 h) and times (25 μM for 0, 6, 12, or 24 h) with or without NEFA (1.2 mM) treatment. In the last experiment, hepatocytes were treated with AdipoRon (25 μM), NEFA (1.2 mM), or both for 12 h after treatment with or without the autophagy inhibitor chloroquine. Hepatocytes treated with NEFA had increased protein abundance of sterol regulatory element-binding protein 1c (SREBP-1c) and mRNA abundance of acetyl-CoA carboxylase 1 (ACACA), and decreased protein abundance of peroxisome proliferator-activated receptor α (PPARA), proliferator-activated receptor gamma coactivator-1 α (PGC-1α), mitofusin 2 (MFN2), cytochrome c oxidase subunit IV (COX IV), and mRNA abundance of carnitine palmitoyltransferase 1A (CPT1A), along with lower ATP concentrations. AdipoRon treatment reversed these effects, suggesting this compound had a positive effect on lipid metabolism and mitochondrial dysfunction during the NEFA challenge. In addition, upregulated expression of microtubule-associated protein 1 light chain 3-II (LC3-II, encoded by MAP1LC3) and downregulated expression of sequestosome-1 (SQSTM1, also called p62) indicated that AdipoRon enhanced autophagic activity in hepatocytes. The fact that chloroquine impeded the beneficial effects of AdipoRon on lipid accumulation and mitochondrial dysfunction suggested a direct role for autophagy during NEFA challenge. Our results suggest that autophagy is an important cellular mechanism to prevent NEFA-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes, which is consistent with other studies. Overall, AdipoRon may represent a promising therapeutic agent to maintain hepatic lipid homeostasis and mitochondrial function in dairy cows during the transition period.

Effects of oral β-caryophyllene (BCP) treatment on perioperative neurocognitive disorders: Attenuation of neuroinflammation associated with microglial activation and reinforcement of autophagy activity in aged mice

Perioperative neurocognitive disorders (PND) are a constellation of cognitive impairments that arise following surgical procedures and anesthesia, with a higher incidence in elderly patients. PND is deeply entwined with microglia-mediated neuroinflammation and disrupted autophagy. β-caryophyllene (BCP) is a natural terpene that occurs widely in dietary plants, and possesses robust anti-inflammatory properties by selectively activating CB2 receptors (CB2R). Accordingly, the present study endeavors to investigate the potential of BCP in ameliorating PND in aged mice, by mitigating hippocampal neuroinflammation and improving autophagy. In this study, an abdominal surgery was utilized to induce perioperative neurocognitive disorders (PND) in aged mice. BCP was administered orally at a dosage of 200 mg/kg for seven consecutive days prior to the scheduled surgery. In order to explore the relationship between BCP and CB2 receptors (CB2R), a co-administration of intraperitoneal injections of the CB2R antagonist AM630 was implemented, 30 min preceding the oral gavage of BCP. Postoperative cognitive functions were assessed using Morris water maze (MWM) tests. The extent of hippocampal inflammation was examined by measuring the microglial marker Iba-1 protein levels, Iba-1 and GFAP immunoactivity, as well as IL-1β and IL-6 concentrations. Evaluation of autophagy activity was conducted based on the ratio of LC3B2/LC3B1 and protein levels of Beclin-1, p62, and phospho-mTOR (p-mTOR). After being orally administered BCP, the compromised behavioral performance of abdominal surgical interventions on aged mice was alleviated. This was evident by the extended escape latency, reduced time spent in the target quadrant, and fewer platform crossings observed through MWM testing. While hippocampal CB2R mRNA or protein expression remained unaffected by the abdominal surgical procedure, their levels were significantly upregulated in mice that were administered BCP. Moreover, the oral administration of BCP was able to reduce neuroinflammation in response to microglia activation, as evidenced by the decreased levels of Iba-1 protein and immunoactivity, as well as the reduction of IL-1β and IL-6 concentrations. Additionally, BCP intensified autophagic activity, as detected by increased LC3B2/LC3B1 ratio and Beclin-1 protein levels, coupled with decreased levels of p62 and p-mTOR in the hippocampus of aged mice. Conversely, the treatment of AM630 ameliorated the suppressive effect of BCP triggered by the neuroinflammation caused by microglial activation post-surgery in aged mice (increased Iba-1 protein levels and immunoactivity, accompanied by higher IL-1β and IL-6 concentrations). Furthermore, the pro-autophagy effect of BCP on aged mice following surgery was partially blocked by AM630, culminating in decreased LC3B2/LC3B1 ratio and Beclin-1 protein levels. However, the levels of p62 and p-mTOR remained unchanged by AM630. Our investigation unveils the remarkable therapeutic benefits of oral BCP administration for managing PND in aged mice through the attenuation of neuroinflammation associated with microglial activation and the fortification of autophagy activity. Hence, BCP holds great promise as a formidable candidate englobing various potential physiological mechanisms that would mitigate cognitive decline associated with aging.

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy.

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 µM) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

Autophagic activation in porcine oocytes is independent of meiotic progression.

In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.

Recent insights into autophagy and metals/nanoparticles exposure.

Some anthropogenic pollutants, such as heavy metals and nanoparticles (NPs), are widely distributed and a major threat to environmental safety and public health. In particular, lead (Pb), cadmium (Cd), chromium (Cr), arsenic (As), and mercury (Hg) have systemic toxicity even at extremely low concentrations, so they are listed as priority metals in relation to their significant public health burden. Aluminum (Al) is also toxic to multiple organs and is linked to Alzheimer's disease. As the utilization of many metal nanoparticles (MNPs) gradually gain traction in industrial and medical applications, they are increasingly being investigated to address potential toxicity by impairing certain biological barriers. The dominant toxic mechanism of these metals and MNPs is the induction of oxidative stress, which subsequently triggers lipid peroxidation, protein modification, and DNA damage. Notably, a growing body of research has revealed the linkage between dysregulated autophagy and some diseases, including neurodegenerative diseases and cancers. Among them, some metals or metal mixtures can act as environmental stimuli and disturb basal autophagic activity, which has an underlying adverse health effect. Some studies also revealed that specific autophagy inhibitors or activators could modify the abnormal autophagic flux attributed to continuous exposure to metals. In this review, we have gathered recent data about the contribution of the autophagy/mitophagy mediated toxic effects and focused on the involvement of some key regulatory factors of autophagic signaling during exposure to selected metals, metal mixtures, as well as MNPs in the real world. Besides this, we summarized the potential significance of interactions between autophagy and excessive reactive oxygen species (ROS)-mediated oxidative damage in the regulation of cell survival response to metals/NPs. A critical view is given on the application of autophagy activators/inhibitors to modulate the systematic toxicity of various metals/MNPs.

Perillaldehyde Functions as a Potential Antifungal Agent by Triggering Metacaspase-Independent Apoptosis in Botrytis cinerea.

Botrytis cinerea, the causal agent of gray mold, is an important plant pathogen causing preharvest and postharvest diseases. Due to the extensive use of commercial fungicides, fungicide-resistant strains have emerged. Natural compounds with antifungal properties are widely present in various kinds of organisms. Perillaldehyde (PA), derived from the plant species Perilla frutescens, is generally recognized as a potent antimicrobial substance and to be safe to humans and the environment. In this study, we demonstrated that PA could significantly inhibit the mycelial growth of B. cinerea and reduced its pathogenicity on tomato leaves. We also found that PA had a significant protective effect on tomato, grape, and strawberry. The antifungal mechanism of PA was investigated by measuring the reactive oxygen species (ROS) accumulation, the intracellular Ca2+ level, the mitochondrial membrane potential, DNA fragmentation, and phosphatidylserine exposure. Further analyses revealed that PA promoted protein ubiquitination and induced autophagic activities and then triggered protein degradation. When the two metacaspase genes, BcMca1 and BcMca2, were knocked out from B. cinerea, all mutants did not exhibit reduced sensitivity to PA. These findings demonstrated that PA could induce metacaspase-independent apoptosis in B. cinerea. Based on our results, we proposed that PA could be used as an effective control agent for gray mold management. IMPORTANCE Botrytis cinerea causes gray mold disease, is considered one of the most important dangerous pathogens worldwide, and leads to severe economic losses worldwide. Due to the lack of resistant varieties of B. cinerea, gray mold control has mainly relied on application of synthetic fungicides. However, long-term and extensive use of synthetic fungicides has increased fungicide resistance in B. cinerea and is harmful to humans and the environment. In this study, we found that perillaldehyde has a significant protective effect on tomato, grape, and strawberry. We further characterized the antifungal mechanism of PA on B. cinerea. Our results indicated that PA induced apoptosis that was independent of metacaspase function.

High glucose inhibits neural differentiation by excessive autophagy <em>via</em> peroxisome proliferator-activated receptor gamma

The high prevalence of prediabetes and diabetes globally has led to the widespread occurrence of severe complications, such as diabetic neuropathy, which is a result of chronic hyperglycemia. Studies have demonstrated that maternal diabetes can lead to neural tube defects by suppressing neurogenesis during neuroepithelium development. While aberrant autophagy has been associated with abnormal neuronal differentiation, the mechanism by which high glucose suppresses neural differentiation in stem cells remains unclear. Therefore, we developed a neuronal cell differentiation model of retinoic acid induced P19 cells to investigate the impact of high glucose on neuronal differentiation in vitro. Our findings indicate that high glucose (HG) hinders neuronal differentiation and triggers excessive. Furthermore, HG treatment significantly reduces the expression of markers for neurons (Tuj1) and glia (GFAP), while enhancing autophagic activity mediated by peroxisome proliferator-activated receptor gamma (PPARγ). By manipulating PPARγ activity through pharmacological approaches and genetically knocking it down using shRNA, we discovered that altering PPARγ activity affects the differentiation of neural stem cells exposed to HG. Our study reveals that PPARγ acts as a downstream mediator in high glucose-suppressed neural stem cell differentiation and that refining autophagic activity via PPARγ at an appropriate level could improve neuronal differentiation efficiency. Our data provide novel insights and potential therapeutic targets for the clinical management of gestational diabetes mellitus.

α-Mangostin Exhibits a Therapeutic Effect on Spinal Cystic Echinococcosis by Affecting Glutamine Metabolism.

Spinal cystic echinococcosis, a severely neglected, rare disease, is characterized by high morbidity, disability, and mortality in prevalent regions. Due to the high-risk nature of surgical treatment and the ineffectiveness of conventional drugs, there is an unmet need for novel safe and effective drugs for the treatment of this disease. In this study, we examined the therapeutic effects of α-mangostin for spinal cystic echinococcosis, and explored its potential pharmacological mechanism. The repurposed drug exhibited a potent in vitro protoscolicidal effect and significantly inhibited the evolution of larval encystation. Moreover, it demonstrated a remarkable anti-spinal cystic echinococcosis effect in gerbil models. Mechanistically, we found that α-mangostin intervention led to intracellular depolarization of mitochondrial membrane potential and reactive oxygen species generation. In addition, we observed elevated expression of autophagic proteins, aggregation of autophagic lysosomes, activated autophagic flux, and disrupted larval microstructure in protoscoleces. Further metabolite profiling showed that glutamine was imperative for autophagic activation and anti-echinococcal effects mediated by α-mangostin. These results suggest that α-mangostin is a potentially valuable therapeutic option against spinal cystic echinococcosis through its effect on glutamine metabolism.

Plasma-activated medium triggers immunomodulation and autophagic activity for periodontal regeneration.

Periodontitis is an infection-induced inflammation, evidenced by an increase in inflammatory macrophage infiltration. Recent research has highlighted the role of plasma-activated medium (PAM) as a regulator of the innate immune system, where macrophages are the main effector cells. This study therefore aims to investigate the immunomodulatory effects of PAM on macrophages and its potential applications for periodontitis management. PAM was generated using an argon jet and applied to culture macrophages. Proinflammatory macrophage markers were significantly reduced after PAM stimulation, and this was correlated with the activation of autophagy via the Akt signaling pathway. Further investigations on the proregenerative effects of PAM-treated macrophages on periodontal ligament cells (PDLCs) revealed a significant increase in the expression of osteogeneis/cementogenesis-associated markers as well as mineralization nodule formation. Our findings suggest that PAM is an excellent candidate for periodontal therapeutic applications.

Ginkgo biloba extract (EGb-761) confers neuroprotection against ischemic stroke by augmenting autophagic/lysosomal signaling pathway

Ginkgo biloba extract (EGb-761) is well-recognized to have neuroprotective properties. Meanwhile, autophagy machinery is extensively involved in the pathophysiological processes of ischemic stroke. The EGb-761 is widely used in the clinical treatment of stroke patients. However, its neuroprotective mechanisms against ischemic stroke are still not fully understood. The present study was conducted to uncover whether the pharmacological effects of EGb-761 can be executed by modulation of the autophagic/lysosomal signaling axis. A Sprague-Dawley rat model of ischemic stroke was established by middle cerebral artery occlusion (MCAO) for 90 min, followed by reperfusion. The EGb-761 was then administered to the MCAO rats once daily for a total of 7 days. Thereafter, the penumbral tissues were acquired to detect proteins involved in the autophagic/lysosomal pathway including Beclin1, LC-3, SQSTM1/p62, ubiquitin, cathepsin B, and cathepsin D by western blot and immunofluorescence, respectively. Subsequently, the therapeutic outcomes were evaluated by measuring the infarct volume, neurological deficits, and neuron survival. The results showed that the autophagic activities of Beclin1 and LC3-II in neurons were markedly promoted by 7 days of EGb-761 therapy. Meanwhile, the autophagic cargoes of insoluble p62 and ubiquitinated proteins were effectively degraded by EGb-761-augmented lysosomal activity of cathepsin B and cathepsin D. Moreover, the infarction size, neurological deficiencies, and neuron death were also substantially attenuated by EGb-761 therapy. Taken together, our study suggests that EGb-761 exerts a neuroprotective effect against ischemic stroke by promoting autophagic/lysosomal signaling in neurons at the penumbra. Thus, it might be a new therapeutic target for treating ischemic stroke.

The comparative effectiveness of metformin and risperidone in a rat model of valproic acid-induced autism, Potential role for enhanced autophagy

RationaleRisperidone is the first antipsychotic to be approved by Food and Drug Administration (FDA) for treating autism spectrum disorder (ASD). The potential efficacy of metformin in preventing and/or controlling ASD behavioral deficits was also recently reported. Suppression of hippocampus autophagy was suggested as a potential pathologic mechanism in ASD.ObjectivesIs metformin’s ability to improve ASD clinical phenotype driven by its autophagy-enhancing properties? And does hippocampus autophagy enhancement underlie risperidone’s efficacy as well? Both questions are yet to be answered.MethodsThe effectiveness of metformin on alleviation of ASD-like behavioral deficits in adolescent rats exposed prenatally to valproic acid (VPA) was compared to that of risperidone. The potential modulatory effects of risperidone on hippocampal autophagic activity were also assessed and compared to those of metformin.ResultsMale offspring exposed to VPA during gestation exhibited marked anxiety, social impairment and aggravation of stereotyped grooming; such deficits were efficiently rescued by postnatal risperidone or metformin therapy. This autistic phenotype was associated with suppressed hippocampal autophagy; as evidenced by reduced gene/dendritic protein expression of LC3B (microtubule-associated proteins 1 light chain 3B) and increased somatic P62 (Sequestosome 1) protein aggregates. Interestingly, compared to risperidone, the effectiveness of metformin in controlling ASD symptoms and improving hippocampal neuronal survival was well correlated to its ability to markedly induce pyramidal neuronal LC3B expression while lowering P62 accumulation.ConclusionsOur work highlights, for the first time, positive modulation of hippocampus autophagy as potential mechanism underlying improvements in autistic behaviors, observed with metformin, as well as risperidone, therapy.

Potent carotenoid astaxanthin expands the anti-cancer activity of cisplatin in human prostate cancer cells

Prostate cancer (PCa) is the second most common type of cancer and the sixth cause of death in men worldwide. Radiotherapy and immunotherapy are commonly used in treating PCa, but understanding the crosstalk mechanisms of carcinogenesis and new therapeutic approaches is essential for supporting poor diagnosis and existing therapies. Astaxanthin (ASX) is a member of the xanthophyll family that is an oxygenated derivative of carotenoids whose synthesis is in plant extracts from lycopene. ASX has protective effects on various diseases, such as Parkinson's disease and cancer by showing potent antioxidant and anti-inflammatory properties. However, there is an ongoing need for a detailed investigation of the molecular mechanism of action to expand its therapeutic use. In the present study, we showed the new regulatory role of ASX in PCa cells by affecting the unfolded protein response (UPR) signaling, autophagic activity, epithelial-mesenchymal transition (EMT) and regulating the expression level ofangiogenesis-related protein vascular endothelial growth factor A (VEGF-A), proto-oncogene c-Myc and prostate-specific antigen (PSA). Additionally, we determined that it exhibited synergistic action with cisplatin and significantly enhanced apoptotic cell death in PCa cells. Present findings suggest that ASX may be a potent adjuvant therapeutic option in PCa treatment when used alone or combined with chemotherapeutics. Schematic illustration of the biochemical activity of astaxanthin and its combination with cisplatin.

Kinetics of macroautophagy in lung adenocarcinoma (A549) cells exposed to nanoparticles

Macroautophagy is an intracellular process helping to maintain normal cellular function. Dysregulation of autophagy is recognized in certain diseases, but it is unknown how maintenance of cellular homeostasis might be affected by the kinetics of autophagy in response to various stimuli. In this study, we measured autophagy kinetics in A549 cells exposed to nanoparticles (NP) (including ambient ultrafine particles (UFP)) and/or Rapamycin. A549 cells were apically exposed (t=0-24hrs) in four experimental settings: (1) control exposure only to culture fluid; (2) exposure to polystyrene NP (PNP), UFP or Rapamycin; (3) concurrent exposure to PNP and Rapamycin or UFP and Rapamycin; and, (4) sequential (in both orders) exposure to a first agent (PNP, UFP or Rapamycin) at t=0-5hrs, followed by exposure to an additional agent (Rapamycin after PNP (or UFP) and PNP (or UFP) after Rapamycin) at t=5-24hrs. Autophagic activity was assessed using fluorescence intensity of DAPRed (a marker for autophagosomes and autolysosomes; 0.1 μM; Dojindo; Rockville, MD) or BacMam RFP-GFP-LC3B (Thermo Fisher; Waltham, MA) in the presence or absence of 40 μM chloroquine measured over the entire cell volume of live single cells using confocal laser scanning microscopy. Autophagic flux was calculated as the difference of observed fluorescence intensity of DAPRed or RFP-GFP-LC3B in the presence and absence of chloroquine. Autophagic flux increased in all experimental modalities, including both single agent and double agent exposures, and reached steady state in all cases ~2 times control from ~8 to 24 hrs, suggesting the presence of an upper limit to autophagic activity. Rapamycin exposure alone induced a rapid rise in autophagic flux, peaking at ~3hrs post exposure, followed by a lowered steady state autophagic flux for up to 24hrs. Exposure to PNP or UFP alone also increased autophagic flux, gradually reaching steady state at ~10-24hrs. During concurrent double exposure to Rapamycin and PNP or UFP at t=0, the steady state level of autophagic flux remained comparable to that observed with single exposures to Rapamycin, PNP or UFP for up to 24 hrs. Under sequential exposure conditions, the steady state level of autophagic flux did not exceed the steady state level observed in single or concurrent exposures. In summary, exposure to nanoparticles activates macroautophagy in a time-dependent manner in A549 cells. Autophagic activity demonstrated comparable steady state levels after exposure to single or dual stimuli, indicating an effective upper limit of autophagic flux in A549 cells under our experimental conditions. These findings suggest that environmental stressors might exert their harmful effects, at least in part, by limiting available autophagic capacity, thereby making NP-exposed cells more susceptible to secondary injury due to autophagic overload. Funding: NIH; WRMPPF This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HIF-1α/Malat1/miR-141 Axis Activates Autophagy to Increase Proliferation, Migration, and Invasion in Triple-negative Breast Cancer.

The mechanism of metastasis-associated lung adenocarcinoma transcript 1 (Malat1) in triple-negative breast cancer (TNBC) is still unclear. This study aimed to investigate the role of miR-141-3p and Malat1 in autophagy in TNBC under hypoxia. The expression levels of Malat1 and miR-141-3p were detected via quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of hypoxia-inducible factor 1α (HIF-1α), HIF-2α, MMP9, p62 and LC3 were determined via western blotting. A Cell Counting Kit-8 assay was used to detect cell viability, while a Transwell assay to detect cell proliferation and invasion. A luciferase assay was used to confirm the relationship between Malat1 and miR-141-3p. A significant increase was observed in the expression level of Malat1 and the autophagic activity in TNBC tissues and cells. The expression level of Malat1 was higher in a hypoxic environment, which can significantly promote the proliferation, migration, and invasion of TNBC cells by activating autophagy. HIF-1α, but not HIF-2α, was identified to induce the upregulation of Malat1 in TNBC cells. The dual-luciferase assay results identified a miR-141-binding site in Malat1. Malat1 knockdown and miR-141-3p overexpression were demonstrated to significantly inhibit autophagy, thereby inhibiting cell proliferation, invasion, and migration. Moreover, hypoxia can inhibit the effect of miR-141-3p on TNBC cells. miR-141-3p could suppress autophagy and inhibit proliferation, migration, and invasion by targeting Malat1 in TNBC cells under hypoxia. The existence of the HIF-1α/Malat1/miR-141 axis plays a vital role in the development of TNBC and may be a target for the diagnosis and treatment of TNBC.

The hippo kinases MST1/2 in cardiovascular and metabolic diseases: A promising therapeutic target option for pharmacotherapy.

Cardiovascular diseases (CVDs) and metabolic disorders are major components of noncommunicable diseases, causing an enormous health and economic burden worldwide. There are common risk factors and developmental mechanisms among them, indicating the far-reaching significance in exploring the corresponding therapeutic targets. MST1/2 kinases are well-established proapoptotic effectors that also bidirectionally regulate autophagic activity. Recent studies have demonstrated that MST1/2 influence the outcome of cardiovascular and metabolic diseases by regulating immune inflammation. In addition, drug development against them is in full swing. In this review, we mainly describe the roles and mechanisms of MST1/2 in apoptosis and autophagy in cardiovascular and metabolic events as well as emphasis on the existing evidence for their involvement in immune inflammation. Moreover, we summarize the latest progress of pharmacotherapy targeting MST1/2 and propose a new mode of drug combination therapy, which may be beneficial to seek more effective strategies to prevent and treat CVDs and metabolic disorders.

A comprehensive review of the relationship between autophagy and sorafenib-resistance in hepatocellular carcinoma: ferroptosis is noteworthy.

Patients with hepatocellular carcinoma (HCC) bear a heavy burden of disease and economic burden but have fewer treatment options. Sorafenib, a multi-kinase inhibitor, is the only approved drug that can be used to limit the progression of inoperable or distant metastatic HCC. However, enhanced autophagy and other molecular mechanisms after sorafenib exposure further induce drug resistance in HCC patients. Sorafenib-associated autophagy also generates a series of biomarkers, which may represent that autophagy is a critical section of sorafenib-resistance in HCC. Furthermore, many classic signaling pathways have been found to be involved in sorafenib-associated autophagy, including the HIF/mTOR signaling pathway, endoplasmic reticulum stress, and sphingolipid signaling, among others. In turn, autophagy also provokes autophagic activity in components of the tumor microenvironment, including tumor cells and stem cells, further impacting sorafenib-resistance in HCC through a special autophagic cell death process called ferroptosis. In this review, we summarized the latest research progress and molecular mechanisms of sorafenib-resistance-associated autophagy in detail, providing new insights and ideas for unraveling the dilemma of sorafenib-resistance in HCC.

Actin cytoskeletal reorganization is involved in hyperosmotic stress-induced autophagy in tubular epithelial cells

Tubular epithelial cells are routinely exposed to severe changes in osmolarity. Although the autophagic activity of cells is an indispensable process to maintain cellular homeostasis and respond to stressors, the effect of hyperosmotic stress on autophagic activity in tubular epithelial cells remains unknown. The aim of this study was to determine the effect of hyperosmotic stress on autophagy in rat kidney tubular epithelial cells focusing on the role of actin and microtubule cytoskeletons. Normal rat kidney (NRK)-52E cells exposed to mannitol-induced hyperosmotic stress. As a result, NRK-52E cells showed elevated protein levels of the autophagosome marker LC3-II, indicating enhancement of the autophagic flux. Hyperosmotic stress also transiently decreased cell volume and caused the reorganization of actin and microtubule cytoskeletal structures in NRK-52E cells. The inhibition of the actin cytoskeleton reorganization by cytochalasin D impaired the increase in the levels of LC3-II; however, disassembly of the microtubules following treatment with nocodazole did not affect the increase. These results indicate that hyperosmotic stress can induce autophagy mediated by the reorganization of the actin cytoskeleton in tubular epithelial cells.

Contribution of HIF-1α/BNIP3-mediated autophagy to lipid accumulation during irinotecan-induced liver injury

Irinotecan is a topoisomerase I inhibitor which has been widely used to combat several solid tumors, whereas irinotecan therapy can induce liver injury. Liver injury generally leads to tissue hypoxia, and hypoxia-inducible factor-1α (HIF-1α), a pivotal transcription factor, mediates adaptive pathophysiological responses to lower oxygen condition. Previous studies have reported a relationship between HIF-1α and autophagy, and autophagy impairment is a common characteristic in a variety of diseases. Here, irinotecan (50 mg/kg) was employed on mice, and HepG2 and L-02 cells were cultured with irinotecan (10, 20 and 40 μM). In vivo study, we found that irinotecan treatment increased final liver index, serum aminotransferase level and hepatic lipid accumulation. Impaired autophagic flux and activation of HIF-1α/BNIP3 pathway were also demonstrated in the liver of irinotecan-treated mice. Moreover, irinotecan treatment significantly deteriorated hepatic oxidative stress, evidenced by increased MDA and ROS contents, as well as decreased GSH-Px, SOD and CAT contents. Interestingly, protein levels of NLRP3, cleaved-caspase 1 and IL-1β were enhanced in the liver of mice injected with irinotecan. In vitro study, irinotecan-treated HepG2 and L-02 cells also showed impaired autophagic flux, while HIF-1α inhibition efficaciously removed the accumulated autophagosomes induced by irinotecan. Additionally, irinotecan treatment aggravated lipid accumulation in HepG2 and L-02 cells, and HIF-1α inhibition reversed the effect of irinotecan. Furthermore, HIF-1α inhibition weakened irinotecan-induced NLRP3 inflammasome activation in HepG2 cells. Taken together, our results suggest that irinotecan induces liver injury by orchestrating autophagy via HIF-1α/BNIP3 pathway, and HIF-1α inhibition could alleviate irinotecan-induced lipid accumulation in HepG2 and L-02 cells, which will provide a new clue and direction for the prevention of side effects of clinical chemotherapy drugs.