Unc-51-like Kinase 1 Research Articles

Dual targeting of NUAK1 and ULK1 using the multitargeted inhibitor MRT68921 exerts potent antitumor activities

Utilizing oxidative stress has recently been regarded as a potential strategy for tumor therapy. The NUAK family SNF1-like kinase 1 (NUAK1) is a critical component of the antioxidant defense system and is necessary for the survival of tumors. Therefore, NUAK1 is considered an attractive therapeutic target in cancer. However, antioxidant therapy induced elevated ROS levels to activate the Unc-51-like kinase 1 (ULK1) pathway to promote protective autophagy and ULK1-dependent mitophagy. Thus, the combined inhibition of NUAK1 and ULK1 showed a strong synergistic effect in different tumor types. Herein, the potential antitumor activities of a dual NUAK1/ULK1 inhibitor MRT68921 were evaluated in both tumor cell lines and animal models. MRT68921 significantly kills tumor cells by breaking the balance of oxidative stress signals. These results highlight the potential of MRT68921 as an effective agent for tumor therapy.

Diurnal Physical Activity Regulates Skeletal Muscle Ulk-1 Phosphorylation In Tumor Bearing Male Mice

Introduction: Cancer cachexia is characterized by severe muscle mass loss associated with increased autophagy. Unc-51 like kinase 1 (ULK1) plays a primary role in initiating autophagy. Under conditions of energetic stress, AMPK activates ULK1 at serine 555 to initiate autophagosome formation for removal of damaged proteins. Daily physical activity and feeding behaviors in mice exhibit diurnal fluctuations that can impact skeletal muscle ULK1 activation. PURPOSE: We investigated the effect of diurnal regulation of skeletal muscle ULK1 phosphorylation in tumor bearing mice. We also examined the impact of increased physical activity on skeletal muscle ULK1 phosphorylation in the cancer environment. METHODS: Free living male C57BL/6 (B6; N=24) and ApcMin/+ (MIN; N=22) mice were single housed without wheel access. An additional cohort of B6 (B6+W;N=16) and MIN (MIN+W;N=19) mice were given wheel access for 2-weeks. To examine diurnal fluctuation, all mice were sacrificed at 7:00AM or 7:00PM under ad libitum conditions. ULK1 was measured in the gastrocnemius muscle as phosphorylation to total ratio at serine 555 by western blot. RESULTS: Free living MIN mice exhibited body weight loss (p<0.001) and reduced gastrocnemius mass (p<0.001) when compared to B6 mice. In the MIN free living mouse, ULK1 phosphorylation was associated with greater body weight loss (R= -0.505; p=0.023) and reduced cage activity (R= -0.548; p=0.012). ULK1 phosphorylation in MIN and MIN+W mice exhibited diurnal fluctuation, being increased at 7AM when compared to 7PM. This diurnal variation was not present in B6 or B6+W mice. Interestingly, regardless of sacrifice time ULK1 phosphorylation was inversely related to total wheel distance (R=-0.498; p=0.049). CONCLUSION: ULK1 demonstrated diurnal fluctuation in MIN mice but not in B6 mice. This change in ULK1 phosphorylation was associated to body weight loss and reduced activity. These data suggest that ULK1 phosphorylation is dependent on the time of day in the tumor environment, further research is warranted to examine if this induction of ULK1 is sufficient to induce autophagy.

Comparison of intermittent fasting and exercise on skeletal muscle autophagy activation and fat reduction

To observe the effect of weight control of intermittent fasting in different time course (14 d, 28 d)and explore its effect on skeletal muscle mass and autophagy. Sixty SD rats (male) were randomly divided into sedentary group (Sed), intermittent fasting group (InF) and exercise group (Exe), 20 rats of each group, intervention duration is 14 d and 28 d. Animals of InF group were fasted every other day, animals of Exe group underwent aerobic exercise on treadmill, the weight was recorded every week. The body fat mass was recorded by DEXA and then the fat mass index was calculated. The wet weight of bilateral soleus muscle was weighed and wet weight index was calculated. Immunofluorescence was used to detect laminin and light chain 3 (LC3), which reflect muscle fiber cross-sectional area and autophagosome respectively. Transmission electron microscopy was used to observe the number and morphology of autophagosomes. The expression levels of autophagy related proteins Unc-51 like kinase 1 (ULK1), LC3, sequestosome1 protein (p62), AMP activated protein kinase (AMPKα) and p-AMPKα (Thr172) were detected by Western blot. ① From the 7th day of intervention, the body weight of rats in InF and Exe groups was significantly lower than that in Sed group, and the body weight of InF group was significantly lower than that in Exe group (P＜0.01). After 28 days of intervention, the fat mass index in InF and Exe groups was significantly lower than that in Sed group, the fat mass index of InF group was significantly lower than that in Exe group (P＜0.05).② After 28 days of intervention, the cross-sectional of muscle fibers in Exe group was significantly larger than that in Sed and InF groups (P＜0.01). ③The expressions of AMPKα, p-AMPKα (Thr172) and ULK1 in InF and Exe groups were significantly higher than those in Sed group (P＜0.05). However, at 14 days, only InF group showed the increase of LC3-II/LC3-I ratio and the decrease of p62 level (P＜0.05). The same indicators of Exe group were only significant changed at 28 d. ① Intermittent fasting is superior to exercise in controlling the growth of body weight and body fat in rats. ② In terms of skeletal muscle autophagy activation, the length of intervention required for intermittent fasting (14 days) is shorter than aerobic exercise (28 days).

BET Protein Inhibition Prolongs Cardiac Transplant Survival via Enhanced Myocardial Autophagy.

Graft rejection continues to be a major barrier to long-term engraftment after transplantation. Autophagy plays an important role in cardiac injury pathogenesis. The bromodomain and extraterminal protein inhibitor (S)-tert-butyl2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (JQ1) inhibits inflammatory responses. However, the beneficial effect of JQ1 on transplant and the potential role of autophagy in the protective effect of graft survival are yet to be investigated. Syngeneic or allogeneic heterotopic heart transplantation was performed using C57BL/6 or BALB/c donors for C57BL/6 recipients through different treatments. Some mice were used to observe the survival of the grafts. The other mice were euthanized on the third, fifth, and seventh days after surgery. The graft samples were taken for cytokines and autophagy pathway analyses. Our study revealed that JQ1 treatment prolonged cardiac allograft survival. JQ1 increased the expression levels of liver kinase beta 1, autophagy-specific gene 5, and microtubule-associated protein light chain3-II (LC3-II) and potentiated the phosphorylation of AMP-activated protein kinase, unc-51-like kinase 1 (ULK1), and autophagy-specific gene 14 in allografts. A conditional autophagy-specific gene 5 deletion donor was utilized to abrogate the effect induced by JQ1. The combined use of JQ1 with bafilomycin A1 partially reversed the effect of JQ1, suggesting that autophagy is involved in the signaling pathway in graft survival. JQ1 downregulated the expression of inflammatory cytokines, such as interleukin-6, interleukin-1β, tumor necrosis factor-α, and interferon-γ, which was abrogated when autophagy was inhibited. JQ1 prolonged cardiac allograft survival by potentiating myocardial autophagy through the liver kinase beta 1-AMP-activated protein kinase-ULK1 signaling pathway and inhibiting the subsequent release of inflammatory cytokines. This result might provide novel insights for extending transplant survival.

ULK1 inhibition as a targeted therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia

BackgroundIn acute myeloid leukemia (AML), internal tandem duplication mutations in the FLT3 tyrosine kinase receptor (FLT3-ITD) are associated with a dismal outcome. Although uncoordinated 51-like kinase 1 (ULK1), which plays a central role in the autophagy pathway, has emerged as a novel therapeutic target for various cancers, its role in FLT3-ITD AML remains elusive. In this study, we evaluated the effects of ULK1 inhibition on leukemia cell death in FLT3-ITD AML.MethodWe evaluated ULK1 expression and the levels of apoptosis and autophagy following ULK1 inhibition in FLT3-ITD AML cell lines and investigated the mechanism underlying apoptosis induced by ULK1 inhibition. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc).ResultsFLT3-ITD AML cells showed significantly higher ULK1 expression than FLT3-wild-type (WT) AML cells. Two ULK1 inhibitors, MRT 68921 and SBI-0206965, induced apoptosis in FLT3-ITD AML cells, with relatively minimal effects on FLT3-WT AML cells and normal CD34-positive cells. Apoptosis induction by ULK1 inhibition was associated with caspase pathway activation. Interestingly, ULK1 inhibition paradoxically also induced autophagy, showing synergistic interaction with autophagy inhibitors. Hence, autophagy may act as a prosurvival mechanism in FLT3-ITD AML cells. FLT3-ITD protein degradation and inhibition of the ERK, AKT, and STAT5 pathways were also observed in FLT3-ITD AML cells following treatment with ULK1 inhibitors.ConclusionULK1 is a viable drug target and ULK1 inhibition may represent a promising therapeutic strategy against FLT3-ITD AML.

Sestrin 2 attenuates sepsis-associated encephalopathy through the promotion of autophagy in hippocampal neurons.

Sepsis‐associated encephalopathy (SAE) has typically been associated with a poor prognosis. Although sestrin 2 (SESN2) plays a crucial role in metabolic regulation and the stress response, its expression and functional roles in SAE are still unclear. In the present study, SAE was established in mice through caecal ligation and puncture (CLP). The adeno‐associated virus 2 (AAV2)‐mediated SESN2 expression (ie overexpression and knockdown) system was injected into the hippocampi of mice with SAE, and subsequently followed by electron microscopic analysis, the Morris water maze task and pathological examination. Our results demonstrated an increase of SESN2 in the hippocampal neurons of mice with SAE, 2‐16 hours following CLP. AAV2‐mediated ectopic expression of SESN2 attenuated brain damage and loss of learning and memory functions in mice with SAE, and these effects were associated with lower pro‐inflammatory cytokines in the hippocampus. Mechanistically, SESN2 promoted unc‐51‐like kinase 1 (ULK1)‐dependent autophagy in hippocampal neurons through the activation of the AMPK/mTOR signalling pathway. Finally, AMPK inhibition by SBI‐0206965 blocked SESN2‐mediated attenuation of SAE in mice. In conclusion, our findings demonstrated that SESN2 might be a novel pharmacological intervention strategy for SAE treatment through promotion of ULK1‐dependent autophagy in hippocampal neurons.

Ethanol Extract of Brucea javanica Seed Inhibit Triple-Negative Breast Cancer by Restraining Autophagy via PI3K/Akt/mTOR Pathway.

Triple-negative breast cancer (TNBC) is an aggressive disease with worst prognosis than other subtypes of breast cancer. Owing to the lack of hormone receptors and HER2 expression on TNBC cells, patients do not have targeted therapy options available with other breast cancer subtypes. Extensive efforts have been made to identify novel therapeutics against TNBC. Interestingly, recent studies had shown that plant-derived natural products could modulate the autophagy and induce the breast cancer cells death. Seed of Brucea javanica has been used as an important traditional Chinese medicine against cancers. In the present study, the anti-breast cancer potential of ethanol crude extracts from B. javanica seed (BJE) was explored. Data demonstrated that BJE could inhibit the TNBC cell line MDA-MB-231 proliferation and induced apoptosis. In the cells exposed to BJE, protein expressions of UNC-51-like kinase-1 (ULK1) and Beclin-1 and the ratio of light chain 3 II/I (LC3 II/I) were reduced, while the expression of p62 was increased, indicating an inhibition on autophagy. Moreover, BJE promoted the phosphorylation of mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase (PI3K), and Akt in MDA-MB-231. BJE also suppressed the MDA-MB-231 tumor growth in vivo. Coincide with the results in vitro, autophagy in the tumor tissue was weakened as indicated by decreased ratio of LC 3 II/I and Beclin-1 accompanied by enhanced phosphorylation of mTOR, which confirmed that autophagy restraint via the PI3K/Akt/mTOR signaling pathway contributes to the suppression by BJE. Notably, no noticeable toxicity in non-targeted organs was found, including small intestine, liver, and kidney. Taken together, this study revealed anti-breast cancer activity of BJE based on autophagy restraint, highlighting its clinical importance as a novel natural agent against TNBC.

Identification of a phosphorylation site on Ulk1 required for genotoxic stress-induced alternative autophagy

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy.

Artemisinin Attenuated Atherosclerosis in High-Fat Diet–Fed ApoE−/− Mice by Promoting Macrophage Autophagy Through the AMPK/mTOR/ULK1 Pathway

Artemisinin is an endoperoxide sesquiterpene lactone from Artemisia annua L with multiple beneficial effects, including anti-inflammation, antioxidant, and vascular protection. Recent studies have found that inflammation along with autophagy deficiency in macrophages is the possible reason for foam cell accumulation in the intima, which leads to atherosclerotic plaque formation. The primary aims of this study were to explore the inhibiting effect of artemisinin on atherosclerosis in high-fat diet-fed ApoE mice and investigate the probable mechanism. Artemisinin (50 and 100 mg/kg, intragastric administration) treatment effectively inhibited foamy macrophage transformation and decreased atherosclerotic plaque formation in atherosclerotic mice. Moreover, artemisinin promoted AMP-activated protein kinase (AMPK) activation, inhibited mammalian target of rapamycin (mTOR) and uncoordinated-51-like kinase 1 (ULK1) phosphorylation, and increased LC-3II accumulation and P62 degradation, thereby enhancing macrophage autophagy. Besides, the inhibiting effect of artemisinin on mTOR and ULK1 phosphorylation could be abrogated by AMPK knockdown, suggesting AMPK was the essential target of artemisinin on promoting macrophage autophagy. Our study indicated that artemisinin alleviated atherosclerotic lesions by accelerating macrophage autophagy through the AMPK/mTOR/ULK1 pathway.

VEGF Triggers Transient Induction of Autophagy in Endothelial Cells via AMPKα1.

AMP-activated protein kinase (AMPK) is activated by vascular endothelial growth factor (VEGF) in endothelial cells and it is significantly involved in VEGF-induced angiogenesis. This study investigates whether the VEGF/AMPK pathway regulates autophagy in endothelial cells and whether this is linked to its pro-angiogenic role. We show that VEGF leads to AMPKα1-dependent phosphorylation of Unc-51-like kinase 1 (ULK1) at its serine residue 556 and to the subsequent phosphorylation of the ULK1 substrate ATG14. This triggers initiation of autophagy as shown by phosphorylation of ATG16L1 and conjugation of the microtubule-associated protein light chain 3B, which indicates autophagosome formation; this is followed by increased autophagic flux measured in the presence of bafilomycin A1 and by reduced expression of the autophagy substrate p62. VEGF-induced autophagy is transient and probably terminated by mechanistic target of rapamycin (mTOR), which is activated by VEGF in a delayed manner. We show that functional autophagy is required for VEGF-induced angiogenesis and may have specific functions in addition to maintaining homeostasis. In line with this, inhibition of autophagy impaired VEGF-mediated formation of the Notch intracellular domain, a critical regulator of angiogenesis. Our study characterizes autophagy induction as a pro-angiogenic function of the VEGF/AMPK pathway and suggests that timely activation of autophagy-initiating pathways may help to initiate angiogenesis.

Abstract P5-05-12: Small molecule targeting regulated cell death pathways in treating triple negative breast cancer

Abstract The absence of hormone and Her2 receptor expression in triple negative breast cancer (TNBC) precludes the use of targeted therapy available for other breast cancer subtypes, and cytotoxic chemotherapy such as paclitaxel and doxorubicin remains the mainstay of treatment for TNBC. However, patients with TNBC have higher relapse rates, with residual disease after chemotherapy and shorter overall survival, leaving space for further improvement beyond these cytotoxic chemotherapies. We have identified a new chemical entity, VMY- BC-1, that potently blocks growth in TNBC cell lines compared to other breast cancer subgroups or normal-like breast epithelial cells. Our molecule reduces invasion and induces apoptosis in TNBC cell lines. VMY-BC-1 is orally bioavailable and has promising pharmacological properties, and has demonstrated TNBC growth inhibition in vivo. Quantitative proteomics with tandem mass tag (TMT) and Ingenuity Pathway Analysis (IPA) identified significantly altered pathways upon treatment with VMY-BC-1 in the human TNBC cell line, MDA-MB-231. These include activation of the apoptosis, autophagy, and DNA damage pathways, and inhibition of the mTOR1, PI3K, and AKT pathways (Z score = 0.156, p value =1.97E-10). Thermal proteome profiling (TPP) analysis identified proteins interacting with VMY-BC-1 that relate to activated autophagy and DNA damage pathways, such as UNC-51 like kinase 1 (ULK1) and ataxia-telangiectasia mutated (ATM). Importantly, in line with our in vitro observations, in addition to a significantly lower expression of ULK1 on TNBC (T-test; p value = 2.127E-10), we observed that patients with tumors with high ULK1 expression have a larger relapse-free survival (n=3,951; HR=0.77 [0.69-0.86]; log rank; p=5.1E-06). Moreover, our small molecules activated autophagy mediated signaling pathways through ULK1 phosphorylation. These data suggest that autophagy and DNA damage pathways could be therapeutically targeted in TNBC. Citation Format: Venkata Mahidhar Yenugonda, Ariana Waters, Sivaramakrishna Yadavalli, Diego Marzese, Surojeet Senugupta, Elmar Nurmammadov, Yueqin Quan, Natsuko Nomura, Annamarie Allnutt, Santosh Kesari. Small molecule targeting regulated cell death pathways in treating triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-12.

Sympathetic innervation suppresses the autophagic-lysosomal system in brown adipose tissue under basal and cold-stimulated conditions.

The sympathetic nervous system (SNS) activates cAMP signaling and promotes trophic effects on brown adipose tissue (BAT) through poorly understood mechanisms. Because norepinephrine has been found to induce antiproteolytic effects on muscle and heart, we hypothesized that the SNS could inhibit autophagy in interscapular BAT (IBAT). Here, we describe that selective sympathetic denervation of rat IBAT kept at 25°C induced atrophy, and in parallel dephosphorylated forkhead box class O (FoxO), and increased cathepsin activity, autophagic flux, autophagosome formation, and expression of autophagy-related genes. Conversely, cold stimulus (4°C) for up to 72 h induced thermogenesis and IBAT hypertrophy, an anabolic effect that was associated with inhibition of cathepsin activity, autophagic flux, and autophagosome formation. These effects were abrogated by sympathetic denervation, which also upregulated Gabarapl1 mRNA. In addition, the cold-driven sympathetic activation stimulated the mechanistic target of rapamycin (mTOR) pathway, leading to the enhancement of protein synthesis, evaluated in vivo by puromycin incorporation, and to the inhibitory phosphorylation of Unc51-like kinase-1, a key protein in the initiation of autophagy. This coincided with a higher content of exchange protein-1 directly activated by cAMP (Epac1), a cAMP effector, and phosphorylation of Akt at Thr308, all these effects being abolished by denervation. Systemic treatment with norepinephrine for 72 h mimicked most of the cold effects on IBAT. These data suggest that the noradrenergic sympathetic inputs to IBAT restrain basal autophagy via suppression of FoxO and, in the setting of cold, stimulate protein synthesis via the Epac/Akt/mTOR-dependent pathway and suppress the autophagosome formation, probably through posttranscriptional mechanisms.NEW & NOTEWORTHY The underlying mechanisms related to the anabolic role of sympathetic innervation on brown adipose tissue (BAT) are unclear. We show that sympathetic denervation activates autophagic-lysosomal degradation, leading to a loss of mitochondrial proteins and BAT atrophy. Conversely, cold-driven sympathetic activation suppresses autophagy and stimulates protein synthesis, leading to BAT hypertrophy. Given its high-potential capacity for heat production, understanding the mechanisms that contribute to BAT mass is important to optimize chances of survival for endotherms in cold ambients.

A Review of ULK1-Mediated Autophagy in Drug Resistance of Cancer.

The difficulty of early diagnosis and the development of drug resistance are two major barriers to the successful treatment of cancer. Autophagy plays a crucial role in several cellular functions, and its dysregulation is associated with both tumorigenesis and drug resistance. Unc-51-like kinase 1 (ULK1) is a serine/threonine kinase that participates in the initiation of autophagy. Many studies have indicated that compounds that directly or indirectly target ULK1 could be used for tumor therapy. However, reports of the therapeutic effects of these compounds have come to conflicting conclusions. In this work, we reviewed recent studies related to the effects of ULK1 on the regulation of autophagy and the development of drug resistance in cancers, with the aim of clarifying the mechanistic underpinnings of this therapeutic target.

Increased autophagy mediates the adaptive mechanism of the mammary gland in dairy cows with hyperketonemia

Hyperketonemia is a metabolic disease in dairy cows, associated with negative nutrition balance (NNB) induced by low dry matter intake (DMI) and increased nutrient requirements. Hyperketonemia could induce metabolic stress, which might indirectly affect mammary tissue. Autophagy is a highly conserved physiological process that results in the turnover of intracellular material, and is involved in maintaining cellular homeostasis under the challenge of metabolic stress induced by NNB. The aim of this study was to investigate the autophagy status and autophagy-related pathways AMP-activated kinase α (AMPKα) and mechanistic target of rapamycin (mTOR) in the mammary glands of dairy cows with hyperketonemia. Cows with hyperketonemia [CWH, n = 10, blood β-hydroxybutyrate (BHB) concentration 1.2 to 3.0 mmol/L] and cows without hyperketonemia (CWOH, n = 10, BHB < 1.2 mmol/L) from 3 to 12 DIM were randomly selected from the herd. The mammary tissue and blood samples were collected from these cows between 0630 and 0800 h, before feeding, at 3 to 12 d in milk. Serum concentrations of glucose, BHB, and fatty acids were determined using an autoanalyzer with commercial kits between 0630 and 0800 h, before feeding. Concentrations of fatty acids, BHB (median and interquartile range: CWH, 2.44 and 1.3, 2.82 mM; CWOH, 0.49 and 0.41, 0.57 mM), and milk fat were greater in CWH. The DMI, glucose concentration, milk production, and milk protein levels were lower in CWH. The mRNA abundance of autophagosome formation-related gene, beclin 1 (BECN1), phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), autophagy-related gene (ATG) 5, ATG7, ATG12, microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3) and sequestosome-1 (SQSTM1, also called p62) were greater in the mammary glands of CWH. The protein abundance of LC3-II and phosphorylation level of Unc-51-like kinase 1 (ULK1) were greater in CWH, but the total ubiquitinated proteins and protein abundance of p62 were lower. Transmission electron microscopy showed an increased number of autophagosomes in the mammary glands of CWH. Furthermore, the phosphorylation of AMPKα was greater, but the phosphorylation of mTOR was lower in the mammary glands of CWH. These results indicate that activity of mTOR pathways and autophagy activity, and upregulation of AMPKα, may be response mechanisms to mitigate metabolic stress induced by hyperketonemia in the mammary glands of dairy cows.

SIRT3 Acts as a Positive Autophagy Regulator to Promote Lipid Mobilization in Adipocytes via Activating AMPK.

Obesity is increasing at an alarming rate worldwide, which is characterized by the excessive accumulation of triglycerides in adipocytes. Emerging evidence has demonstrated that macroautophagy and chaperone-mediated autophagy (CMA) regulate lipid mobilization and play a key role in energy balance. Sirtuin 3 (SIRT3) is an NAD+-dependent deacetylase, which is important in regulating macroautophagy and lipid metabolism. It is still unknown whether SIRT3 modulates macroautophagy and CMA in adipocytes. The current study found that macroautophagy was dynamically regulated during 3T3-L1 adipocyte differentiation, which coincided with SIRT3 expression. In mature adipocytes, overexpression of SIRT3 activated macroautophagy, mainly on lipid droplets (LDs), through activating the AMP-activated protein kinase (AMPK)-unc-51-like kinase 1 (ULK1) pathway, which in turn resulting in smaller LD size and reduced lipid accumulation. Moreover, SIRT3 overexpression induced the formation of perilipin-1 (PLN1)-heat shock cognate 71 kDa protein (HSC70)-lysosome-associated membrane protein 2 (LAMP2) complex, to activate CMA and cause the instability of LDs in adipocytes. In summary, we found SIRT3 is a positive regulator of macroautophagy and CMA in adipocytes, which might be a promising therapeutic target for treatment of obesity and its related metabolic dysfunction.

Podocytes maintain high basal levels of autophagy independent of mtor signaling

ABSTRACT While constant basal levels of macroautophagy/autophagy are a prerequisite to preserve long-lived podocytes at the filtration barrier, MTOR regulates at the same time podocyte size and compensatory hypertrophy. Since MTOR is known to generally suppress autophagy, the apparently independent regulation of these two key pathways of glomerular maintenance remained puzzling. We now report that long-term genetic manipulation of MTOR activity does in fact not influence high basal levels of autophagy in podocytes either in vitro or in vivo. Instead we present data showing that autophagy in podocytes is mainly controlled by AMP-activated protein kinase (AMPK) and ULK1 (unc-51 like kinase 1). Pharmacological inhibition of MTOR further shows that the uncoupling of MTOR activity and autophagy is time dependent. Together, our data reveal a novel and unexpected cell-specific mechanism, which permits concurrent MTOR activity as well as high basal autophagy rates in podocytes. Thus, these data indicate manipulation of the AMPK-ULK1 axis rather than inhibition of MTOR as a promising therapeutic intervention to enhance autophagy and preserve podocyte homeostasis in glomerular diseases. Abbreviations: AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy related; BW: body weight; Cq: chloroquine; ER: endoplasmic reticulum; ESRD: end stage renal disease; FACS: fluorescence activated cell sorting; GFP: green fluorescent protein; i.p.: intra peritoneal; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PLA: proximity-ligation assay; PRKAA: 5ʹ-AMP-activated protein kinase catalytic subunit alpha; RPTOR/RAPTOR: regulatory associated protein of MTOR, complex 1; RFP: red fluorescent protein; TSC1: tuberous sclerosis 1; ULK1: unc-51 like kinase 1

Mechanistic target of rapamycin-mediated autophagy is involved in the alleviation of lipopolysaccharide-induced acute lung injury in rats

Acute lung injury (ALI) is a complex clinical syndrome with high morbidity and mortality rates. Autophagy is an adaptive process that plays a complex role in ALI. The aim of this study was to investigate the effects of autophagy on lipopolysaccharide (LPS)-induced lung injury by establishing a rat ALI model and to further explore the possible mechanisms involved. Rats were pretreated with the autophagy inhibitor 3-methyladenine (3-MA) or the autophagy activator rapamycin before they were challenged with the intratracheal instillation of LPS (5 mg/kg). The level of autophagy in the lung tissue was detected. Lung injury and vascular permeability were assessed. The role of the mechanistic target of rapamycin (mTOR)-mediated Unc-51-like kinase 1 (ULK1) and the class III PI3 kinase VPS34 in autophagy regulation was examined. LPS challenge induced autophagy and rapamycin pretreatment enhanced autophagy activity in LPS-induced ALI rats. LPS caused severe lung injury and high pulmonary vascular permeability, which could be alleviated by enhancing autophagy. In addition, the inhibition of mTOR upregulated the expression of ULK1 and VPS34 and thus increased LPS-induced autophagy. Autophagy plays a protective role in LPS-induced ALI, and enhancing autophagy via the inhibition of mTOR alleviates lung injury and pulmonary barrier function. Moreover, mTOR negatively mediates ULK1 and VPS34 to regulate LPS-induced autophagy in rats.

Schisandrin A protects against lipopolysaccharide-induced mastitis through activating Nrf2 signaling pathway and inducing autophagy

Schisandrin A (Sch A), a dibenzocyclooctadiene lignan extracted from Schisandra chinensis (Turcz.) Baill., has anti-oxidant and anti-inflammatory effects, but the effect on masitits has not been studied. Therefore, we investigated the effect of Sch A in cell and mouse models of lipopolysaccharide (LPS)-induced mastitis. Studies in vivo showed that Sch A reduced LPS-induced mammary injury and the production of pro-inflammatory mediators. Sch A also decreased the levels of pro-inflammatory mediators and activated nuclear factor-E2 associated factor 2 (Nrf2) signaling pathway in mouse mammary epithelial cells (mMECs). The Nrf2 inhibitor partially abrogated the downregulation of Sch A on LPS-induced inflammatory response. In addition, LPS stimulation suppressed autophagy, while both Sch A and the autophagy inducer rapamycin activated autophagy in mMECs, which down-regulated inflammatory response. Sch A also restrained LPS-induced phosphorylation of mammalian target of rapamycin (mTOR) and activated AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1). In summary, these results suggest that Sch A exerts protective effects in LPS-induced mastitis models by activating Nrf2 signaling pathway and inducing autophagy and the autophagy is initiated by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway.

DDIS-37. SMALL MOLECULE AUTOPHAGY ACTIVATORS IN GROWTH INHIBITION OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is one of the most lethal human tumors, and despite considerable advances in multimodality treatments consisting of surgical removal, radiation and chemotherapy, overall survival is 14 to 16 months after diagnosis. Therefore, there is a need to target non-apoptotic death pathways and inhibit pro-survival signaling mechanisms that contribute to both tumorigenesis and treatment resistance. Depending on tumor type and environment, autophagy modulation plays an important role in tumor cell survival and inhibition pathways. Increasing evidence supports that chemical modulation of autophagy inhibition and activation holds a therapeutic potential and these studies have led to the initiation of multiple clinical trials combining chemotherapeutic agents and autophagy inhibitors and activators for various cancer types. However, recent studies have also demonstrated a therapeutic potential for enhancers of autophagy in GBM. This clearly demonstrates the need for further investigation into the autophagy induced mechanism of GBM, and the development of better treatments. We have identified a new chemical entity VMY-BC-1 that showed promising effects in both serum and patient-derived glioma cell lines. Drug target engagement studies identified an autophagy induction protein, UNC-51 like kinase 1 (ULK1), as the main target of VMY-BC-1, in which VMY-BC-1 covalently bound to the protein’s active pocket and increased ULK1 phosphorylation.ULK1 is a component of the ULK1 complex (ULK1-ATG13-FIP200) that is essential for inducing autophagy, a lysosome-dependent mechanism of intracellular degradation for maintaining cellular homeostasis. Additionally, our molecule cross talks to the apoptotic and DNA damage pathways, which could potentially overcome treatment resistance.VMY-BC-1 is orally bioavailable, brain penetrant, and inhibits cell invasion and tumor growth in GBM flank models, making this small molecule an ideal candidate for further development. We hypothesize that activating ULK1-modulated autophagy will be a therapeutic strategy for GBM growth inhibition

Tomorou attenuates progression of rheumatoid arthritis through alteration in ULK-1 independent autophagy pathway in collagen induced arthritis mice model

Rheumatoid arthritis (RA) is a multifactorial disease which is complicated by apoptosis resistance. Autophagy is one of the key mechanisms which are involved in the development of resistance to apoptosis as well as to the standard therapies against RA. Aberration in autophagy and apoptosis homeostasis results in the development of oxidative stress thus complicates the pathogenesis of RA. In the given study, tomorou, an indigenous herb of Hunza-Nagar Valley, has been evaluated for its pro-apoptotic, anti-inflammatory, and anti-rheumatic activity. Several major classes of bioactive phytochemicals including steroids, terpenoids, phenols, flavonoids, and essential oils have been detected in the aqueous and ethyl acetate extracts of tomorou through phytochemical analysis. Plant extracts depicted enhanced free radical scavenging activity through di-phenyl-2-picryl hydrazyl hydrate (DPPH) assay and ameliorated the symptoms of arthritis in collagen induced arthritic (CIA) mice model. Moreover, the 6 week extract treatment resulted in the reduction of IL-6 serum levels thus making it an effective anti-inflammatory agent. Upregulation of microtubule-associated proteins light chain 3b (LC3b) and downregulation of UNC51-like kinase 1 (ULK-1) in arthritic mice proposed a ULK-1 independent non-canonical autophagy pathway. Treatment with extracts upregulated the expression of caspase 3 which in turn inhibited the activity of LC3b thus altering the autophagy pathway. However, ULK-1 expression was restored to normal in aqueous extract treated group whereas it was upregulated in ethyl acetate extract treated group. On the other hand, a novel LC3b-independent autophagy pathway was observed in mice treated with ethyl acetate extract due to ULK-1 upregulation. Despite of significantly high IL-6 levels, the arthritic symptoms waned off which suggested the participation of IL-6 in LC3b-independent autophagy pathway in the extract prepared in ethyl acetate. Conclusively, the study established pro-apoptotic, antioxidant, anti-inflammatory and anti-rheumatic activity of tomorou and suggested an intricate autophagy pathway shift.