Daphnetin triggers ROS-induced cell death and induces cytoprotective autophagy by modulating the AMPK/Akt/mTOR pathway in ovarian cancer

BackgroundOvarian cancer is one of the most common gynecological malignancies in the world. Daphnetin (Daph) was previously reported to possess antitumor potential, but its potential and molecular mechanisms in ovarian cancer remain poorly understood.PurposeIn the current study, we aimed to explore the antitumor effect and detailed mechanisms of Daph in ovarian cancer cells.MethodsThe cytotoxic effect of Daph on ovarian cells was determined in vitro and in vivo. Cell growth, proliferation, apoptosis and ROS generation were measured by CCK8 assays, colony formation assays and flow cytometry. Western blotting was used to evaluate the related signal proteins. Immunofluorescence and transmission electron microscopy were used to evaluate markers of autophagy and autophagic flux. The antitumor effects were observed in the A2780 xenograft model. Moreover, Daph-induced autophagy was observed by enhanced LC3-II accumulation and endogenous LC3 puncta, and an autophagy inhibitor further enhanced the antitumor efficacy of Daph, which indicated that the cytoprotective role of autophagy in ovarian cancer.ResultsWe found that Daph exhibited antitumor effects by inducing ROS-dependent apoptosis in ovarian cancer, which could be reversed by N-acetyl cysteine (NAC). The AMPK/Akt/mTOR pathway was involved in Daph-mediated cytoprotective autophagy, and when Daph-mediated the expression level of AMPK and autophagy were blocked, there was robust inhibition of cell proliferation and induction of apoptosis. In addition, in the A2780 xenograft model, combined treatment with Daph and an autophagy inhibitor showed obvious synergetic effects on the inhibition of cell viability and promotion of apoptosis, without any side effects.ConclusionOur results suggest that Daph triggers ROS-induced cell apoptosis and induces cytoprotective autophagy by modulating the AMPK/Akt/mTOR pathway. Moreover, the combination of Daph and autophagy inhibitor may be a potential therapeutic strategy for ovarian cancer.     

The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy

AMP-activated protein kinase (AMPK) is a crucial energy sensor and plays a key role in integration of cellular functions to maintain homeostasis. Despite this, it is largely unknown whether targeting the AMPK pathway can be used as a therapeutic strategy for infectious diseases. Herein, we show that AMPK activation robustly induces antibacterial autophagy, which contributes to antimicrobial defense against Mycobacterium tuberculosis (Mtb). AMPK activation led to inhibition of Mtb-induced phosphorylation of the mechanistic target of rapamycin (MTOR) in macrophages. In addition, AMPK activation increased the genes involved in oxidative phosphorylation, mitochondrial ATP production, and biogenesis in Mtb-infected macrophages. Notably, peroxisome proliferator-activated receptor-gamma, coactivator 1α (PPARGC1A) was required for AMPK-mediated antimicrobial activity, as well as enhancement of mitochondrial function and biogenesis, in macrophages. Further, the AMPK-PPARGC1A pathway was involved in the upregulation of multiple autophagy-related genes via CCAAT/enhancer binding protein (C/EBP), β (CEBPB). PPARGC1A knockdown inhibited the AMPK-mediated induction of autophagy and impaired the fusion of phagosomes with MAP1LC3B (LC3B) autophagosomes in Mtb-infected macrophages. The link between autophagy, mitochondrial function, and antimicrobial activity was further demonstrated by studying LysMCre-mediated knockout of atg7, demonstrating mitochondrial ultrastructural defects and dysfunction, as well as blockade of antimicrobial activity against mycobacteria. Collectively, our results identify the AMPK-PPARGC1A axis as contributing to autophagy activation leading to an antimicrobial response, as a novel host defense mechanism.     

Nano-TiO<Subscript>2</Subscript> induces autophagy to protect against cell death through antioxidative mechanism in podocytes

Autophagy is a cellular pathway involved in degradation of damaged organelles and proteins in order to keep cellular homeostasis. It plays vital role in podocytes. Titanium dioxide nanoparticles (nano-TiO₂) are known to induce autophagy in cells, but little has been reported about the mechanism of this process in podocytes and the role of autophagy in podocyte death. In the present study, we examined how nano-TiO₂ induced authophagy. Besides that, whether autophagy could protect podocytes from the damage induced by nano-TiO₂ and its mechanism was also investigated. Western blot assay and acridine orange staining presented that nano-TiO₂ significantly enhanced autophagy flux in podocytes. In addition, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were involved in such process. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that upregulated level of autophagy induced by rapamycin in high concentration nano-TiO₂-treated podocytes could significantly reduce the level of oxidative stress and alleviate podocyte death. Downregulating the level of autophagy with 3-methyladenine had the opposite effects. These findings indicate that nano-TiO₂ induces autophagy through activating AMPK to inhibit mTOR in podocytes, and such autophagy plays a protecting role against oxidative stress on the cell proliferation. Changing autophagy level may become a new treatment strategy to relieve the damage induced by nano-TiO₂ in podocytes.     

AMPK mediates a pro-survival autophagy downstream of PARP-1 activation in response to DNA alkylating agents

In this study we aim to elucidate the signaling pathway and biological function of autophagy induced by MNNG, a commonly used DNA alkylating agent. We first observed that MNNG is able to induce necrotic cell death and autophagy in Bax?/? Bak?/? double knockout MEFs. We analyzed the critical role of PARP-1 activation and ATP depletion in MNNG-mediated cell death and autophagy via AMPK activation and mTOR suppression. We provide evidence that suppression of AMPK blocks MNNG-induced autophagy and enhances cell death, suggesting the pro-survival function of autophagy in MNNG-treated cells. Taken together, data from this study reveal a novel mechanism in controlling MNNG-mediated autophagy via AMPK activation downstream of PARP-1 activation and ATP depletion.     

Calcineurin suppresses AMPK-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress

Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H₂O₂) treatment. However, prolonged (12 h) H₂O₂ treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H₂O₂ treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.     

Humanin-induced autophagy plays important roles in skeletal muscle function and lifespan extension

BackgroundAutophagy, a highly conserved homeostatic mechanism, is essential for cell survival. The decline of autophagy function has been implicated in various diseases as well as aging. Although mitochondria play a key role in the autophagy process, whether mitochondrial-derived peptides are involved in this process has not been explored.MethodsWe developed a high through put screening method to identify potential autophagy inducers among mitochondrial-derived peptides. We used three different cell lines, mice, c.elegans, and a human cohort to validate the observation.ResultsHumanin, a mitochondrial-derived peptide, increases autophagy and maintains autophagy flux in several cell types. Humanin administration increases the expression of autophagy-related genes and lowers accumulation of harmful misfolded proteins in mice skeletal muscle, suggesting that humanin-induced autophagy potentially contributes to the improved skeletal function. Moreover, autophagy is a critical role in humanin-induced lifespan extension in C. elegans.ConclusionsHumanin is an autophagy inducer.General significanceThis paper presents a significant, novel discovery regarding the role of the mitochondrial derived peptide humanin in autophagy regulation and as a possible therapeutic target for autophagy in various age-related diseases.     

Akt/AMPK/mTOR pathway was involved in the autophagy induced by vitamin E succinate in human gastric cancer SGC-7901 cells

Vitamin E succinate (VES), a derivative of vitamin E, is a promising cancer chemopreventive agent that inhibits tumor promotion by inducing apoptotic cell death. The effects of VES on autophagy, an intricate programmed process which helps cells survive in some stressed situations by degrading some cytoplasmic material, are unclear. When human gastric cancer cells SCG-7901 were exposed to VES, both the level of microtubule-associated protein 1 light chain 3 and the yeast ATG6 homolog Beclin-1 increased, and related autophagy genes were activated, thereby suggesting that autophagy was induced by VES. We also observed that VES-induced autophagy was accompanied by the activation of AMP-activated protein kinases (AMPK). VES-induced autophagy decreased when AMPK was inhibited by using small interfering RNA (siRNA), thereby suggesting that VES-induced autophagy is mediated by AMPK. Moreover, further studies revealed that the decreased activity of mammalian target of rapamycin (mTOR) and its downstream targets P70S6K and 4EBP-1 were involved in VES-activated autophagy associated with AMPK activation. The experiments also showed that the activity of protein kinases B (Akt)-mTOR axis was inhibited by VES. VES-induced AMPK activation could be attenuated by Akt activation. Overall, our studies demonstrated that AMPK was involved in the VES-induced autophagy. Crosstalk exists between AMPK and the Akt/mTOR axis. The results elucidated the mechanism of VES-induced autophagy in human gastric cancer cells.     

Smooth muscle-specific HuR knockout induces defective autophagy and atherosclerosis

Human antigen R (HuR) is a widespread RNA-binding protein involved in homeostatic regulation and pathological processes in many diseases. Atherosclerosis is the leading cause of cardiovascular disease and acute cardiovascular events. However, the role of HuR in atherosclerosis remains unknown. In this study, mice with smooth muscle-specific HuR knockout (HuR^SMKO) were generated to investigate the role of HuR in atherosclerosis. HuR expression was reduced in atherosclerotic plaques. As compared with controls, HuR^SMKO mice showed increased plaque burden in the atherosclerotic model. Mechanically, HuR could bind to the mRNAs of adenosine 5′-monophosphate-activated protein kinase (AMPK) α1 and AMPKα2, thus increasing their stability and translation. HuR deficiency reduced p-AMPK and LC3II levels and increased p62 level, thereby resulting in defective autophagy. Finally, pharmacological AMPK activation induced autophagy and suppressed atherosclerosis in HuR^SMKO mice. Our findings suggest that smooth muscle HuR has a protective effect against atherosclerosis by increasing AMPK-mediated autophagy.Subject terms: Cardiovascular diseases, Pathogenesis     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

AMP-activated protein kinase and autophagy

Autophagy is inhibited by TOR-dependent signaling. Interruption of signalling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. However, inactivation of TOR by AMPK has yielded controversial results in the literature with regard to its effect on autophagy: activation of autophagy in yeast but inhibition in hepatocytes. In a recent study, carried out with hepatocytes, HT-29 cells, and HeLa cells, the possible role of AMPK in the control of mammalian autophagy was reexamined. The data suggest that in mammalian cells, as in yeast, AMPK is required for autophagy.     

Therapeutic strategies targeting AMPK-dependent autophagy in cancer cells

Macroautophagy is a health-modifying process of engulfing misfolded or aggregated proteins or damaged organelles, coating these proteins or organelles into vesicles, fusion of vesicles with lysosomes to form autophagic lysosomes, and degradation of the encapsulated contents. It is also a self-rescue strategy in response to harsh environments and plays an essential role in cancer cells. AMP-activated protein kinase (AMPK) is the central pathway that regulates autophagy initiation and autophagosome formation by phosphorylating targets such as mTORC1 and unc-51 like activating kinase 1 (ULK1). AMPK is an evolutionarily conserved serine/threonine protein kinase that acts as an energy sensor in cells and regulates various metabolic processes, including those involved in cancer. The regulatory network of AMPK is complicated and can be regulated by multiple upstream factors, such as LKB1, AKT, PPAR, SIRT1, or noncoding RNAs. Currently, AMPK is being investigated as a novel target for anticancer therapies based on its role in macroautophagy regulation. Herein, we review the effects of AMPK-dependent autophagy on tumor cell survival and treatment strategies targeting AMPK.     

AMPK-independent induction of autophagy by cytosolic Ca2+ increase

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca²⁺ can induce autophagy by inhibition of mTOR via the CaMKK-α/β-mediated activation of AMPK. Here we demonstrate that Ca²⁺ signaling can additionally induce autophagy independently of the Ca²⁺-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca²⁺ induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca²⁺-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca²⁺ by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca²⁺-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca²⁺ flux modifications. Importantly, using AMPK-α1^?/?α2^?/? MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-α/β inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca²⁺-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.     

Role of AMPK in atherosclerosis via autophagy regulation

Atherosclerosis is characterized by the accumulation of lipids and deposition of fibrous elements in the vascular wall, which is the primary cause of cardiovascular diseases. Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor of energy metabolism that regulates multiple physiological processes, including lipid and glucose metabolism and the normalization of energy imbalances. Overwhelming evidence indicates that AMPK activation markedly attenuates atherosclerosis development. Autophagy inhibits cell apoptosis and inflammation and promotes cholesterol efflux and efferocytosis. Physiological autophagy is essential for maintaining normal cardiovascular function. Increasing evidence demonstrates that autophagy occurs in developing atherosclerotic plaques. Emerging evidence indicates that AMPK regulates autophagy via a downstream signaling pathway. The complex relationship between AMPK and autophagy has attracted the attention of many researchers because of this close relationship to atherosclerosis development. This review demonstrates the role of AMPK and autophagy in atherosclerosis. An improved understanding of this interrelationship will create novel preventive and therapeutic strategies for atherosclerosis.     

High glucose suppresses autophagy through the AMPK pathway while it induces autophagy via oxidative stress in chondrocytes

Diabetes (DB) is a risk factor for osteoarthritis progression. High glucose (HG) is one of the key pathological features of DB and has been demonstrated to induce apoptosis and senescence in chondrocytes. Autophagy is an endogenous mechanism that can protect cells against apoptosis and senescence. The effects of HG on autophagy in cells including chondrocytes have been studied; however, the results have been inconsistent. The current study aimed to elucidate the underlying mechanisms, which could be associated with the contrasting outcomes. The present study revealed that HG can induce apoptosis and senescence in chondrocytes, in addition to regulating autophagy dynamically. The present study demonstrated that HG can cause oxidative stress in chondrocytes and suppress the AMPK pathway in a dose-dependent manner. Elimination of oxidative stress by Acetylcysteine, also called N-acetyl cysteine (NAC), downregulated autophagy and alleviated HG-stimulated apoptosis and senescence, while activation of the AMPK signaling pathway by AICAR not only upregulated autophagy but also alleviated HG-stimulated apoptosis and senescence. A combined treatment of NAC and AICAR was superior to treatment with either NAC or AICAR. The study has demonstrated that HG can suppress autophagy through the AMPK pathway and induce autophagy via oxidative stress in chondrocytes.Subject terms: Autophagy, Bone, Endocrine system and metabolic diseases     

Quercetin attenuates renal ischemia/reperfusion injury via an activation of AMP-activated protein kinase-regulated autophagy pathway

Renal ischemia/reperfusion (I/R) is a major cause of acute renal failure. Quercetin, a flavonoid antioxidant, presents in many kinds of food. The molecular mechanism of quercetin on renal protection during I/R is still unclear. Here, we investigated the role of AMP-activated protein kinase (AMPK)-regulated autophagy in renal protection by quercetin. To investigate whether quercetin protects renal cells from I/R-induced cell injury, an in vitro model of I/R and an in vivo I/R model were used. Cell apoptosis was determined by propidium iodide/annexin V staining. Western blotting and immunofluorescence were used to determine the autophagy. AMPK expression was inhibited with appropriate short hairpin RNA (shRNA). In cultured renal tubular cell I/R model, quercetin decreased the cell injury, up-regulated the AMPK phosphorylation, down-regulated the mammalian target of rapamycin (mTOR) phosphorylation and activated autophagy during I/R. Knockdown of AMPK by shRNA transfection decreased the quercetin-induced autophagy but did not affect the mTOR phosphorylation. In I/R mouse model, quercetin decreased the increased serum creatinine level and altered renal histological score. Quercetin also increased AMPK phosphorylation, inhibited the mTOR phosphorylation and activated autophagy in the kidneys of I/R mice. These results suggest that quercetin activates an AMPK-regulated autophagy signaling pathway, which offers a protective effect in renal I/R injury.     

Dexamethasone-induced autophagy mediates muscle atrophy through mitochondrial clearance

Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin–proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent atrophy program remains unknown. Here, we show that dexamethasone stimulates an early activation of autophagy in L6 myotubes depending on protein kinase, AMPK, and glucocorticoid receptor activity. Dexamethasone increases expression of several autophagy genes, including ATG5, LC3, BECN1, and SQSTM1 and triggers AMPK-dependent mitochondrial fragmentation associated with increased DNM1L protein levels. This process is required for mitophagy induced by dexamethasone. Inhibition of mitochondrial fragmentation by Mdivi-1 results in disrupted dexamethasone-induced autophagy/mitophagy. Furthermore, Mdivi-1 increases the expression of genes associated with the atrophy program, suggesting that mitophagy may serve as part of the quality control process in dexamethasone-treated L6 myotubes. Collectively, these data suggest a novel role for dexamethasone-induced autophagy/mitophagy in the regulation of the muscle atrophy program.     

Morus alba leaves ethanol extract protects pancreatic islet cells against dysfunction and death by inducing autophagy in type 2 diabetes

BackgroundProtection of pancreatic islet cells against dysfunction or death by regulating autophagy is considered to be an effective method for treatment of type 2 diabetes mellitus (T2DM). Morus alba leaves (mulberry leaves), a popular herbal medicine, have been used for prevention of T2DM since ancient times.PurposeThis study aimed to clarify whether Morus alba leaves ethanol extract (MLE) could protect islet cells in vivo and in vitro by regulating autophagy in T2DM, and explore the possible mechanism of action.MethodsThe main chemical constituents in MLE were analyzed by HPLC. The T2DM rat model was induced via high-fat diet combined with peritoneal injection of low-dose streptozotocin, and MLE was administered by oral gavage. Fasting blood glucose (FBG) and plasma insulin were measured, and homeostatic model assessment of β cell function (HOMA-β) and insulin resistance (HOMA-IR) were determined. The histomorphology of pancreas islets was evaluated by haematoxylin and eosin staining. In palmitic acid (PA)-stressed INS-1 rat insulinoma cells, cell viability was assayed by an MTT method. Expression of the autophagy-related proteins LC3 I/II, p62, p-AMPK and p-mTOR in islet tissues and INS-1 cells was evaluated by western blotting or immunohistochemistry analysis.ResultsThe four main chemical constituents in MLE were identified as chlorogenic acid, rutin, isoquercitrin and quercitrin. MLE ameliorated hyperglycemia, insulin resistance and dyslipidemia of T2DM rats with prominent therapeutic effect. Further study indicated that MLE observably improved islet function, alleviated islet injury of T2DM rats, and inhibited PA-induced INS-1 cell death. On the other hand, MLE significantly induced autophagy in islet cells both in vivo and in vitro, and autophagy inhibitors abolished its therapeutic effect on T2DM rats and protective effect on islet cells. Apart from this, MLE markedly activated the AMPK/mTOR pathway in INS-1 cells, and the AMPK inhibitor prevented the autophagy induction ability of MLE.ConclusionTogether, MLE could protect islet cells against dysfunction and death by inducing AMPK/mTOR-mediated autophagy in T2DM, and these findings provide a new perspective for understanding the treatment mechanism of Morus alba leaves against T2DM.     

Hypothalamic AMPK-induced autophagy increases food intake by regulating NPY and POMC expression

Hypothalamic AMP-activated protein kinase (AMPK) plays important roles in the regulation of food intake by altering the expression of orexigenic or anorexigenic neuropeptides. However, little is known about the mechanisms of this regulation. Here, we report that hypothalamic AMPK modulates the expression of NPY (neuropeptide Y), an orexigenic neuropeptide, and POMC (pro-opiomelanocortin-α), an anorexigenic neuropeptide, by regulating autophagic activity in vitro and in vivo. In hypothalamic cell lines subjected to low glucose availability such as 2-deoxy-d-glucose (2DG)-induced glucoprivation or glucose deprivation, autophagy was induced via the activation of AMPK, which regulates ULK1 and MTOR complex 1 followed by increased Npy and decreased Pomc expression. Pharmacological or genetic inhibition of autophagy diminished the effect of AMPK on neuropeptide expression in hypothalamic cell lines. Moreover, AMPK knockdown in the arcuate nucleus of the hypothalamus decreased autophagic activity and changed Npy and Pomc expression, leading to a reduction in food intake and body weight. AMPK knockdown abolished the orexigenic effects of intraperitoneal 2DG injection by decreasing autophagy and changing Npy and Pomc expression in mice fed a high-fat diet. We suggest that the induction of autophagy is a possible mechanism of AMPK-mediated regulation of neuropeptide expression and control of feeding in response to low glucose availability.     

Mitochondrial ALDH2 protects against lipopolysaccharide-induced myocardial contractile dysfunction by suppression of ER stress and autophagy

Lipopolysaccharide (LPS), an essential component of outer membrane of the Gram-negative bacteria, plays a pivotal role in myocardial anomalies in sepsis. Recent evidence depicted an essential role for mitochondrial aldehyde dehydrogenase (ALDH2) in cardiac homeostasis. This study examined the effect of ALDH2 on endotoxemia-induced cardiac anomalies. Echocardiographic, cardiac contractile and intracellular Ca²⁺ properties were examined. Our results indicated that LPS impaired cardiac contractile function (reduced fractional shortening, LV end systolic diameter, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration, oxidation of SERCA, and intracellular Ca²⁺ mishandling), associated with ER stress, inflammation, O₂⁻ production, increased autophagy, CAMKKβ, phosphorylated AMPK and suppressed phosphorylation of mTOR, the effects of which were significantly attenuated or negated by ALDH2. LPS promoted early endosomal formation (as evidenced by RAB4 and RAB5a), apoptosis and necrosis (MTT and LDH) while decreasing late endosomal formation (RAB7 and RAB 9), the effects were reversed by ALDH2. In vitro study revealed that LPS-induced SERCA oxidation, autophagy and cardiac dysfunction were abrogated by ALDH2 activator Alda-1, the ER chaperone TUDCA, the autophagy inhibitor 3-MA, or the AMPK inhibitor Compound C. The beneficial effect of Alda-1 against LPS was nullified by AMPK activator AICAR or rapamycin. CAMKKβ inhibition failed to rescue LPS-induced ER stress. Tunicamycin–induced cardiomyocyte dysfunction was ameliorated by Alda-1 and autophagy inhibition, the effect of which was abolished by rapamycin. These data suggested that ALDH2 protected against LPS-induced cardiac anomalies via suppression of ER stress, autophagy in a CAMKKβ/AMPK/mTOR-dependent manner.