The role of autophagy in liver cancer: Molecular mechanisms and potential therapeutic targets

Autophagy is an evolutionarily conserved pathway for degradation of cytoplasmic proteins and organelles via lysosome. Proteins coded by the autophagy-related genes (Atgs) are the core molecular machinery in control of autophagy. Among the various biological functions of autophagy identified so far, the link between autophagy and cancer is probably among the most extensively studied and is often viewed as controversial. Autophagy might exert a dual role in cancer development: autophagy can serve as an anti-tumor mechanism, as defective autophagy (e.g., heterozygous knockdown Beclin 1 and Atg7 in mice) promotes the malignant transformation and spontaneous tumors. On the other hand, autophagy functions as a protective or survival mechanism in cancer cells against cellular stress (e.g., nutrient deprivation, hypoxia and DNA damage) and hence promotes tumorigenesis and causes resistance to therapeutic agents. Liver cancer is one of the common cancers with well-established etiological factors including hepatitis virus infection and environmental carcinogens such as aflatoxin and alcohol exposure. In recent years, the involvement of autophagy in liver cancer has been increasingly studied. Here, we aim to provide a systematic review on the close cross-talks between autophagy and liver cancer, and summarize the current status in development of novel liver cancer therapeutic approaches by targeting autophagy. It is believed that understanding the molecular mechanisms underlying the autophagy modulation and liver cancer development may provoke the translational studies that ultimately lead to new therapeutic strategies for liver cancer.     

Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1^−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.     

Time-Point Dependent Activation of Autophagy and the UPS in SOD1G93A Mice Skeletal Muscle

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by a selective loss of motor neurons together with a progressive muscle weakness. Albeit the pathophysiological mechanisms of the disease remain unknown, growing evidence suggests that skeletal muscle can be a target of ALS toxicity. In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue. In this study we investigated the activation of autophagy and the UPS as well as apoptosis in the skeletal muscle from SOD1G93A mice along disease progression. Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages. The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy. Furthermore, no apoptosis activation was observed along disease progression. The combined data provided clear evidence for the first time that there is a time-point dependent activation of autophagy and UPS in the skeletal muscle from SOD1G93A mice.     

The Zinc Transporter,Slc39a7 (Zip7) Is Implicated in Glycaemic Control in Skeletal Muscle Cells

Dysfunctional zinc signaling is implicated in disease processes including cardiovascular disease, Alzheimer''s disease and diabetes. Of the twenty-four mammalian zinc transporters, ZIP7 has been identified as an important mediator of the ‘zinc wave’ and in cellular signaling. Utilizing siRNA targeting Zip7 mRNA we have identified that Zip7 regulates glucose metabolism in skeletal muscle cells. An siRNA targeting Zip7 mRNA down regulated Zip7 mRNA 4.6-fold (p = 0.0006) when compared to a scramble control. This was concomitant with a reduction in the expression of genes involved in glucose metabolism including Agl, Dlst, Galm, Gbe1, Idh3g, Pck2, Pgam2, Pgm2, Phkb, Pygm, Tpi1, Gusb and Glut4. Glut4 protein expression was also reduced and insulin-stimulated glycogen synthesis was decreased. This was associated with a reduction in the mRNA expression of Insr, Irs1 and Irs2, and the phosphorylation of Akt. These studies provide a novel role for Zip7 in glucose metabolism in skeletal muscle and highlight the importance of this transporter in contributing to glycaemic control in this tissue.     

电针对快速衰老小鼠肝脏自噬及脂质代谢的影响

目的:研究电针对快衰老小鼠(SAMP8)肝脏中自噬相关因子LC3-Ⅱ、Beclin1、Atg7、P62表达的影响,探讨电针改善衰老小鼠肝脏脂质代谢的机制.方法:30周龄雄性SAMP8小鼠随机分为模型组、药物组、电针组,每组7只,以同周龄抗快速老化SAMR1小鼠7只作为对照组.对照组和模型组动物常规饲养4周,不进行任何干...     

Elevated p62/SQSTM1 determines the fate of autophagy-deficient neural stem cells by increasing superoxide

Autophagy plays important roles in many biological processes, but our understanding of the mechanisms regulating stem cells by autophagy is limited. Interpretations of earlier studies of autophagy using knockouts of single genes are confounded by accumulating evidence for other functions of many autophagy genes. Here, we show that, in contrast to Fip200 deletion, inhibition of autophagy by deletion of Atg5, Atg16L1, or Atg7 does not impair the maintenance and differentiation of postnatal neural stem cells (NSCs). Only Fip200 deletion, but not Atg5, Atg16L1, or Atg7 deletion, caused p62/sequestome1 aggregates to accumulate in NSCs. Fip200 and p62 double conditional knockout mice demonstrated that p62 aggregate formation triggers aberrant superoxide increases by impairing superoxide dismutase functions. By comparing the inhibition of autophagy by deletion of Atg5, Atg16L1, or Atg7 with Fip200 deletion, we revealed a critical role of increased p62 in determining the fate of autophagy-deficient NSCs through intracellular superoxide control.     

Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization

Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

Autophagy inhibits oxidative stress and tumor suppressors to exert its dual effect on hepatocarcinogenesis

The role of autophagy in carcinogenesis is controversial and apparently complex. By using mice with hepatocyte-specific knockout of Atg5, a gene essential for autophagy, we longitudinally studied the role of autophagy in hepatocarcinogenesis. We found that impairing autophagy in hepatocytes would induce oxidative stress and DNA damage, followed by the initiation of hepatocarcinogenesis, which could be suppressed by the antioxidant N-acetylcysteine. Interestingly, these mice developed only benign tumors with no hepatocellular carcinoma (HCC), even after the treatment with diethylnitrosamine, which induced HCC in wild-type mice. The inability of mice to develop HCC when autophagy was impaired was associated with the induction of multiple tumor suppressors including p53. Further analysis indicated that the induction of p53 was associated with the DNA-damage response. Tumorigenesis studies using an established liver tumor cell line confirmed a positive role of autophagy in tumorigenesis and a negative role of p53 in this process when autophagy was impaired. Our studies thus demonstrate that autophagy is required to maintain healthy mitochondria and to reduce oxidative stress and DNA damage to prevent the initiation of hepatocarcinogenesis. However, once hepatocarcinogenesis has been initiated, its presence is also required to suppress the expression of tumor suppressors to promote the development of HCC.Autophagy (i.e., macroautophagy) is important for cells to remove protein aggregates and damaged organelles. Its dysfunction can cause a variety of diseases including cancers.^{1, 2} However, its role in carcinogenesis is apparently complex, as it has been shown in different reports to positively or negatively regulate carcinogenesis.^{3, 4} Autophagy apparently can function as a tumor suppressor, as the gene encoding Beclin-1, a component of the phosphatidylinositol-3-kinase class III (PI3KC3) complex that is essential for the initiation of autophagy, is often monoallelically deleted or mutated in breast, ovarian and prostate cancers.⁵ Frameshift mutations in Atg2B, Atg5, Atg9B and Atg12 autophagy genes are also often found in gastric and colorectal cancers with microsatellite instability.⁶ The tumor suppressor role of autophagy is further supported by the studies using mouse models. It has been shown that the monoallelic deletion of the Beclin-1 gene in mice induced tumor lesions in various tissues,⁷ Atg4C-knockout (KO) mice had increased susceptibility to carcinogens for the development of fibrosarcomas⁸ and the systemic mosaic KO of Atg5 and the liver-specific KO of Atg7 in mice led to the development of benign liver adenomas.^{9, 10}Autophagy has also been shown to promote tumor growth. It has been shown that autophagy can enhance the survival of tumor cells in the hypoxic regions of solid tumors.¹¹ It has also been shown that in cells expressing oncogenic Ras, autophagy is required to promote tumorigenesis by maintaining oxidative metabolism or facilitating glycolysis.^{12, 13} Moreover, it has also been demonstrated that the suppression of autophagy by the expression of FIP200, a component of the ULK1-Atg13-FIP200-Atg101 complex that is essential for the induction of autophagy, could suppress mammary tumorigenesis induced by the polyomavirus middle T antigen in mice.¹⁴ These observations indicated a protumorigenic role of autophagy.In this report, we used mice with liver-specific KO of Atg5 (L-Atg5-KO) to study the role of autophagy in carcinogenesis. We found that abolishing the expression of Atg5 impaired autophagy in the liver and led to oxidative DNA damage and the development of benign hepatic tumors with no visible carcinoma. This inability to develop hepatocellular carcinoma (HCC) was correlated with the induction of tumor suppressors, which negatively regulate the progression of tumorigenesis when autophagy was impaired.     

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.     

The CD40-autophagy pathway is needed for host protection despite IFN-Γ-dependent immunity and CD40 induces autophagy via control of P21 levels

Autophagy degrades pathogens in vitro. The autophagy gene Atg5 has been reported to be required for IFN-γ-dependent host protection in vivo. However, these protective effects occur independently of autophagosome formation. Thus, the in vivo role of classic autophagy in protection conferred by adaptive immunity and how adaptive immunity triggers autophagy are incompletely understood. Employing biochemical, genetic and morphological studies, we found that CD40 upregulates the autophagy molecule Beclin 1 in microglia and triggers killing of Toxoplasma gondii dependent on the autophagy machinery. Infected CD40(-/-) mice failed to upregulate Beclin 1 in microglia/macrophages in vivo. Autophagy-deficient Beclin 1(+/-) mice, mice with deficiency of the autophagy protein Atg7 targeted to microglia/macrophages as well as CD40(-/-) mice exhibited impaired killing of T. gondii and were susceptible to cerebral and ocular toxoplasmosis. Susceptibility to toxoplasmosis occurred despite upregulation of IFN-γ, TNF-α and NOS2, preservation of IFN-γ-induced microglia/macrophage anti-T. gondii activity and the generation of anti-T. gondii T cell immunity. CD40 upregulated Beclin 1 and triggered killing of T. gondii by decreasing protein levels of p21, a molecule that degrades Beclin 1. These studies identified CD40-p21-Beclin 1 as a pathway by which adaptive immunity stimulates autophagy. In addition, they support that autophagy is a mechanism through which CD40-dependent immunity mediates in vivo protection and that the CD40-autophagic machinery is needed for host resistance despite IFN-γ.     

Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation

Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.     

SIRT1 attenuates sepsis-induced acute kidney injury via Beclin1 deacetylation-mediated autophagy activation

Our previous studies showed that silent mating-type information regulation 2 homologue-1 (SIRT1, a deacetylase) upregulation could attenuate sepsis-induced acute kidney injury (SAKI). Upregulated SIRT1 can deacetylate certain autophagy-related proteins (Beclin1, Atg5, Atg7 and LC3) in vitro. However, it remains unclear whether the beneficial effect of SIRT1 is related to autophagy induction and the underlying mechanism of this effect is also unknown. In the present study, caecal ligation and puncture (CLP)-induced mice, and an LPS-challenged HK-2 cell line were established to mimic a SAKI animal model and a SAKI cell model, respectively. Our results demonstrated that SIRT1 activation promoted autophagy and attenuated SAKI. SIRT1 deacetylated only Beclin1 but not the other autophagy-related proteins in SAKI. SIRT1-induced autophagy and its protective effect against SAKI were mediated by the deacetylation of Beclin1 at K430 and K437. Moreover, two SIRT1 activators, resveratrol and polydatin, attenuated SAKI in CLP-induced septic mice. Our study was the first to demonstrate the important role of SIRT1-induced Beclin1 deacetylation in autophagy and its protective effect against SAKI. These findings suggest that pharmacologic induction of autophagy via SIRT1-mediated Beclin1 deacetylation may be a promising therapeutic approach for future SAKI treatment.Subject terms: Macroautophagy, Acetylation     

Knockdown of Atg7 suppresses Tumorigenesis in a murine model of liver cancer

Hepatocellular Carcinoma (HCC) is the most common type of primary liver cancer in adults and a leading cause of cancer-related deaths worldwide. Studies have shown that autophagy is significantly involved in carcinogenesis, in particular, driven by activated RAS signaling. Autophagy related 7 (Atg7) is a critical component for the formation of autophagosome and required for autophagy processes. We investigated the role of autophagy in RAS-driven tumorigenesis in the liver, via the knockdown of Atg7 in the model. Transposon vectors encoding short hairpin RNAs targeting Atg7 (Atg7 shRNA) were constructed. Inhibition of autophagy via Atg7 knockdown was tested in Hep3B cells cultured in nutrient-starved medium. Formation of autophagosome was suppressed in nutrient-starved Hep3B cells expressing Atg7 shRNA, demonstrating that it efficiently inhibited autophagy in HCC cells. Transposons encoding Atg7 shRNA were mixed with those expressing HRAS^G12V and p53 shRNA, and subsequently used for hydrodynamic injection to 5-week-old C57BL/6 mice. Tumorigenesis in livers induced by HRAS^G12V and p53 shRNA was significantly suppressed by Atg7 knockdown. The inhibition of autophagy led to a decreased proliferation of cancer cells, as determined by Ki-67 staining. Our data indicate that knockdown of Atg7 led to a significant decrease in tumorigenesis in a murine HCC model induced by activated RAS. Inhibition of autophagosome formation is expected to be a therapeutic option for liver cancer.     

Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis

Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 β release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD⁺:NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis.     

A Non-canonical MEK/ERK Signaling Pathway Regulates Autophagy via Regulating Beclin 1

Autophagy-essential proteins are the molecular basis of protective or destructive autophagy machinery. However, little is known about the signaling mechanisms governing these proteins and the opposing consequences of autophagy in mammals. Here we report that a non-canonical MEK/ERK module, which is positioned downstream of AMP-activated protein kinase (AMPK) and upstream of tuberous sclerosis complex (TSC), regulates autophagy by regulating Beclin 1. Depletion of ERK partially inhibited autophagy, whereas specific inhibition on MEK completely inhibited autophagy. MEK could bypass ERK to promote autophagy. Basal MEK/ERK activity conferred basal Beclin 1 by preventing disassembly of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. Activation of MEK/ERK by AMPK upon autophagy stimuli disassembled mTORC1 via binding to and activating TSC but disassembled mTORC2 independently of TSC. Inhibition of mTORC1 or mTORC2 by transiently or moderately activated MEK/ERK caused moderately enhanced Beclin 1 resulting in cytoprotective autophagy, whereas inhibition of both mTORC1 and mTORC2 by sustained MEK/ERK activation caused strongly pronounced Beclin 1 leading to cytodestructive autophagy. Our findings thus propose that the AMPK-MEK/ERK-TSC-mTOR pathway regulation of Beclin 1 represents different thresholds responsible for a protective or destructive autophagy.Autophagy is an evolutionally conserved machinery involving the degradation and turnover of cytoplasmic material in lysosomes. Autophagy plays a role in cellular homeostasis (1), antiaging (2–4), development (1, 5), protection of the genome (6), and regulation of cell size (7). Autophagy may act as a means of defense against bacterium and virus invasion and be linked to various diseases including cancer (8–10), cardiomyopathy (11), and neurodegenerative disorders (12).Autophagy starts with the formation of an autophagosome, enclosed within a double membrane that engulfs part of the cytoplasm. During periods of autophagy stimuli, cells respond to either maintain the metabolism essential for survival or execute cell death. Autophagy-essential proteins (Atg)² are the molecular basis of autophagy machinery. About 30 Atg proteins in yeast and 10 in mammals have been identified. In yeast, the protein kinase target of rapamycin (TOR) mediates autophagy via Atg1-Atg13 kinase complex. Atg1 interacts with multiple components of the autophagic machinery through direct association, phosphorylation, and/or intracellular localization (13, 14).In mammalian systems, autophagosomes fuse with lysosomes to generate autophagolysosomes, which undergo a maturation process by fusing with endocytic compartments and lysosomes (15). Because it is not known how the Atg1 homolog acts in mammals, a different mechanism may be involved in regulating autophagy. Beclin 1/Atg6, microtubule-associated protein 1 light chain 3 (LC3)/Atg8, Atg5, Atg12, and Atg13 are essential for autophagosome formation in mammalian species (5, 16–20). Atg7 and Atg3 are required in the conjugation reaction between Atg12 and Atg5 and in the lipidation of LC3. During the formation of autophagosomes in mammalian cells, LC3 is lipidated via a ubiquitylation-like system (17, 21), generating a soluble form, LC3-I. LC3-I is further modified to a membrane-bound form, LC3-II, which is subsequently localized to autophagosomes and autolysosomes until being degraded by the lysosome.Beclin 1 was initially isolated as a B-cell lymphoma-2 (Bcl2)-interacting tumor suppressor in mammalian cells (22). Overexpression of Bcl2 attenuates the formation of the kinase complex Beclin 1-class III phosphatidylinositol 3-kinase (PI3KC3) essential for the formation of autophagosomes (23). The UV radiation resistance-associated gene tumor suppressor and the activating molecule in Beclin 1-regulated autophagy protein 1 (Ambra 1) were identified as new Beclin 1-binding partners that also regulate autophagy by regulating the Beclin 1-PI3KC3 kinase complex. Association of Beclin 1 with PI3KC3 is negatively regulated by Bcl2 (22) and positively regulated by UV radiation resistance-associated gene tumor suppressor and Ambra 1 (24, 25). Beclin 1 is homoallelically deleted in many human tumors. A decreased Beclin 1 level causes defective autophagy and breast cancer, but restoration of Beclin 1 induces autophagy and inhibits tumorigenicity of human breast cancer cells (18). These reports evidence the dependence on Beclin 1 for a functional autophagy mechanism.Diverse signaling pathways have been reported in the regulation of autophagy in mammalian cells (26, 27). In contrast to yeast, mammalian cells regulate autophagy via both class I and class III PI3K. Class I PI3K plays an inhibitory role, whereas class III PI3K kinase complex, which includes Beclin 1, plays a stimulatory role in autophagy by promoting the nucleation of autophagic vesicles (28, 29). A recent study also indicates that hVps15 is required in regulation of class III PI3K in mammalian cells (30). However, the signaling mechanisms controlling autophagy-essential proteins, in particular Beclin 1, and the opposing consequences of autophagy remain to be resolved.Our present studies identified and positioned a non-canonical MEK/ERK pathway downstream of AMPK and upstream of TSC and mTOR. This MEK/ERK module regulated autophagy via regulating the Beclin 1 level through the AMPK-MEK/ERK-TSC-mTOR pathway. Moderately enhanced Beclin 1 by transient or moderate activation of MEK/ERK and subsequent inhibition on mTORC1 and mTORC2 individually caused protective autophagy. Strongly pronounced Beclin 1 by sustained or strong activation of MEK/ERK followed by dual inhibition on mTORC1 and mTORC2 caused destructive autophagy. Our results thus reveal interesting Beclin 1 thresholds in regulating autophagy.     

Enhanced Gastrointestinal Expression of Cytosolic Malic Enzyme (ME1) Induces Intestinal and Liver Lipogenic Gene Expression and Intestinal Cell Proliferation in Mice

The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue’s metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.