腺苷酸活化蛋白激酶:肿瘤治疗新靶点

腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)是真核细胞内发现的一类与细胞能量代谢有关激酶家族中的一员,被称之为"能量感应器".当细胞内AMP/ATP值升高时,AMPK被激活.研究发现,在肿瘤细胞中,活化的AMPK可协同相关抑癌因子调节细胞周期、细胞凋亡以及蛋白质合成,最终影响细胞的增殖.因而,AMPK可以通过感应细胞能量水平的变化来调节细胞增殖.这给肿瘤治疗提供了一定的启示,即以肿瘤细胞能量代谢特点而探寻抑制肿瘤细胞增殖的途径.     

AMPK激活剂抑制PDGF诱导的大鼠血管平滑肌细胞增殖的机制研究

目的: 观察血小板源性生长因子(PDGF)及一磷酸腺苷激活的蛋白激酶(AMPK)激活剂5-氨基咪唑-4-甲酰胺核糖核苷(AICAR)干预后血管平滑肌细胞(VSMCs)的增殖变化,探讨PDGF对平滑肌细胞的促增殖效应及激活AMPK抑制增殖机制。 方法: SD大鼠主动脉血管平滑肌细胞经PDGF及AICAR干预24 h、48 h、72 h后,分为A组(AICAR)、P组(PDGF)、A+P组(AICAR+PDGF)和对照组,4个组用MTT法测量细胞的增殖情况;并检测不同AICAR作用时间下(30 min、1 h、3 h、6 h、12 h)AMPK的活化情况和mTOR 的蛋白活性,以及上述各组中AMPK活化和mTOR 蛋白活性。结果: (1)与对照组相比,PDGF干预组的MTT值显著增加(P<0.05),AMPK激活组能显著抑制PDGF诱导的MTT值的增加效应(P<0.05);(2) AICAR可诱导细胞AMPK的磷酸化水平增加(P<0.05),AICAR的诱导效应有随药物干预时间增加而逐渐增强的趋势;(3)与对照组相比, AICAR干预后p-mTOR表达活性显著减弱(P<0.05),随着药物干预时间延长,p-mTOR表达也呈逐渐减弱的趋势;(4)各组干预12 h后分别检测p-AMPK表达强度,与对照组比较,P组显著减弱(P<0.01),A+P组显著增强(P<0.01),而A+P组与P组比较,A+P组强于P组(P<0.01),A组较对照组显著增强(P<0.01);检测p-mTOR表达强度,与对照组比较,P组显著增强(P<0.05),A+P组较低(P<0.05),A+P组低于P组(P<0.05),A组低于对照组(P<0.05)。结论: PDGF刺激能促进VSMCs增殖,该促增殖效应可被AMPK激活剂AICAR所抑制;细胞mTOR活性下调可能参与AMPK活化诱导的抑制VSMCs增殖的作用。     

AMPK，SIRT1与能量代谢

在调节细胞能量状态的蛋白激酶级联反应中,AMP 激活的蛋白激酶(AMPK) 是其中枢组成部分,AMPK的活性受AMP/ATP比值的调节。沉默信息调节因子2相关酶1（SIRT1）作为一种NAD+依赖的组蛋白去乙酰化酶,同样在调节细胞能量代谢中起着重要作用。AMPK与SIRT1相互起调控作用,阐明AMPK与SIRT1作用的上下游信号通路有可能成为新的治疗代谢疾病作用靶点。     

哇巴因对乳腺癌MCF7细胞能量代谢的影响研究

目的检测哇巴因对人乳腺癌MCF7细胞能量代谢的影响,初步探索相关作用机制。方法联合使用ATP检测系统及海马生化分析仪检测哇巴因作用后细胞内ATP水平及线粒体呼吸作用的变化;使用葡萄糖氧化酶法检测哇巴因对MCF7细胞葡萄糖吸收水平的影响;使用AMPK活性检测探针检测AMPK活性的变化;通过Western blot实验初步检测哇巴因对MCF7细胞内相关激酶蛋白水平的影响。结果哇巴因能够时间依赖性地降低MCF7细胞内ATP水平,剂量依赖性地降低线粒体呼吸作用,同时25~50 nmol/L的哇巴因作用24 h后能够促进葡萄糖吸收。该化合物可激活AMPK活性,在12 h表现出最佳活性。Western blot实验结果证实哇巴因能够升高AMPK和底物蛋白乙酰辅酶A羧化酶(ACC)的磷酸化水平,抑制Na+/K+-ATP酶α1亚型(NKAα1)的表达。结论哇巴因可抑制人乳腺癌MCF7细胞的线粒体氧化呼吸作用,促进糖酵解。哇巴因对肿瘤代谢的影响可能与其调控AMPK活性有关。     

低氧诱导因子1对心脏保护作用的研究进展

低氧诱导因子1可调控其下游相关因子表达,从而促进心肌梗死区局部血管形成、促进细胞增殖、改善细胞能量代谢并能抑制细胞凋亡,进而增加有效心肌细胞数量,改善心肌梗死后心室重构。为缺血性心脏病的治疗提供了新的思路。     

AMPK与代谢综合征

能量代谢失衡是肥胖和代谢综合征的主要原因。在调节细胞能量状态的蛋白激酶级联反应中,AMP激活的蛋白激酶(AMPK)是其中枢组成部分。AMPK的活性受AMP/ATP比值的调节。应激反应可通过ATP的产生减少或利用增加,使细胞内AMP/ATP的比值增加,从而激活AMPK。激活的AMPK可激发一系列的反应来恢复细胞内的能量平衡。AMPK可启动分解代谢途径,如脂肪酸氧化和糖酵解,从而增加ATP的产生,同时关闭合成代谢途径,如脂肪酸合成和蛋白合成,减少ATP的消耗。AMPK不仅可以在细胞水平作为能量的感受器,还可以通过激素和细胞因子,如瘦素、脂联素和ghrelin来参与调节机体的能量消耗和能量摄入。新近的研究发现二甲双胍类抗糖尿病药物可以激活AMPK,调节糖代谢,提示AMPK有可能在预防机体发生代谢性疾病,如代谢综合征、肥胖和2型糖尿病中起作用。因而,阐明AMPK在不同组织及整体水平上对能量贮存与消耗的调节是今后该领域的研究热点,并且AMPK的级联反应有可能成为治疗肥胖和代谢综合征的有效靶标。     

亚硒酸钠通过AMPK/mTOR通路调控白血病NB4细胞凋亡

 目的 研究亚硒酸钠对白血病NB4细胞中AMPK及其下游靶蛋白对细胞凋亡的调控作用。方法 分别用亚硒酸钠、AICAR和AMPK干扰序列处理NB4细胞。用Western blot检测细胞AMPK、mTOR及其下游蛋白P70S6K的磷酸化水平及激活和干扰AMPK后AMPK、mTOR及P70S6K的磷酸化水平、流式细胞术检测NB4细胞凋亡率;免疫共沉淀法检测AMPK和mTOR的相互作用。结果 亚硒酸钠可以上调NB4细胞中AMPK的磷酸化水平,下调mTOR及P70S6K的磷酸化水平;AICAR和亚硒酸钠单独处理具有类似的促进NB4细胞凋亡的效果;干扰AMPK后,mTOR及P70S6K的磷酸化水平上升,亚硒酸钠对NB4细胞的促凋亡作用被拮抗;AMPK和mTOR有直接相互作用。结论 亚硒酸钠可通过激活AMPK表达,抑制mTOR及P70S6K,促进NB4细胞凋亡。     

重编程为心肌细胞过程中的能量代谢的研究进展

急性心肌梗死的发病率逐年上升,通过重编程细胞移植,可以促进梗死区心肌细胞再生、防治心室重构和改善心功能.但重编程效率低下,如何提高重编程的效率是目前研究的难点.代谢与细胞生命过程密切相关,心肌利用能量代谢的形式维持心脏内环境稳定和组织结构更新的物质基础,线粒体动力学参与调节能量代谢.但心肌细胞直接重编程过程中代谢机制尚未清楚.     

压力超载状态血管重构调控的研究进展

压力超载状态下的血管重构是一个非常复杂的病理过程。血管重构的中心环节是血管平滑肌细胞的增殖、凋亡和重塑。本文综述了血管壁张力，血管壁剪切应力及血管收缩肽在这一过程中的作用及其机制     

Ad-apoptin通过AMPK/ACC信号通路抑制肺癌细胞脂代谢北大核心CSCD

目的:探讨溶瘤腺病毒Ad-apoptin对非小细胞肺癌细胞脂代谢的影响及分子机制。方法:CCK-8法检测细胞活性;JC-1染色和Annexin V-FITC/PI检测细胞凋亡;Western blot检测脂代谢和凋亡相关蛋白;siRNA干扰肺癌细胞AMPK的表达;Co-IP检测Ad-apoptin与AMPK蛋白相互间作用;建立移植瘤小鼠模型,检测肿瘤的生长,并通过免疫组化检测TUNEL、Ki-67、p-AMPK和FASN蛋白表达量。结果:Ad-apoptin对肺癌细胞抑制率具有浓度依赖性(P<0.05),显著诱导细胞凋亡(P<0.05),降低细胞脂质累积,下调三酰甘油(TG)和总胆固醇(TC)含量,上调p-AMPK和p-ACC;敲低AMPK可显著减弱Ad-apoptin诱导的凋亡,而抑制ACC可显著增强Ad-apoptin诱导的凋亡。在体内试验中,Ad-apoptin能抑制体内肿瘤生长,同时也能抑制细胞增殖,并促进细胞凋亡。结论:Ad-apoptin以AMPK为靶点,通过AMPK/ACC途径,抑制肿瘤增殖和脂代谢并促进细胞凋亡。     

LKB1 and AMPK: central regulators of lymphocyte metabolism and function

When T cells encounter foreign antigen and appropriate costimulatory signals from professional antigen-presenting cells (APCs), they initiate a coordinated program of rapid proliferation and differentiation, leading to the development of activated T cells with specific effector functions tailored toward pathogen clearance or control. One of the fundamental programs that underpin T-cell proliferation and function is the regulation of cellular metabolism. Recent efforts to identify the signal transduction pathways that regulate T-cell metabolism have led to the identification of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK) as key regulators of T-cell metabolism. LKB1 and AMPK are part of an evolutionarily conserved signal transduction pathway that monitors cellular energy status. AMPK senses bioenergetic fluctuations in cells and works in concert with LKB1 to maintain cellular energy homeostasis by promoting catabolic pathways of ATP production and limiting processes that consume ATP. Recent data indicate that LKB1 and AMPK can influence diverse aspects of T-cell biology beyond metabolism, including T-cell development, peripheral T-cell homeostasis, and T-cell effector function. In this review, we focus on the regulation of lymphocyte metabolism by this energy-sensing pathway and discuss its influence on T-cell function.     

Inhibition of the LKB1–AMPK pathway by the Epstein–Barr virus‐encoded LMP1 promotes proliferation and transformation of human nasopharyngeal epithelial cells

The association of Epstein–Barr virus (EBV) infection with the development of nasopharyngeal carcinoma (NPC) is well established. Latent membrane protein 1 (LMP1), the major oncogene encoded by EBV, is believed to play a crucial role in NPC pathogenesis by virtue of its ability to constitutively activate multiple cell signalling pathways. The LKB1–AMPK pathway is a master regulator of cellular metabolism that, via modulation of energy metabolism, has tumour suppressor activity. In this study we identify a novel ability of LMP1 to inhibit the LKB1–AMPK pathway through phosphorylation of LKB1 at serine 428 with subsequent suppression of the phosphorylation of AMPK and its substrates, ACC and Raptor. We show that MEK/ERK–MAPK signalling, activated by the CTAR1 domain of LMP1, is responsible for LKB1–AMPK inactivation. In addition, reactivation of AMPK signalling by AMPK activator, AICAR, abolished LMP1‐induced cellular transformation (proliferation and anchorage‐independent growth) in nasopharyngeal epithelial cells. Immunohistochemical staining revealed that a low level of phosphorylated AMPK is common in primary NPC specimens, and that this correlated significantly with the expression of LMP1. AICAR treatment inhibited the proliferation and anchorage‐independent growth of NPC cells as well as potentiating the cytotoxic effect of the chemotherapeutic drug 5‐fluorouracil. The current findings demonstrate that LMP1‐mediated AMPK inactivation contributes to the proliferation and transformation of epithelial cells, thereby implicating the LKB1–AMPK pathway in the EBV‐driven pathogenesis of NPC. Our findings also suggest that AMPK activators could be used to enhance the efficacy of conventional chemotherapeutic agents in the treatment of local and metastatic NPC. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

AMP-activated protein kinase regulates lymphocyte responses to metabolic stress but is largely dispensable for immune cell development and function

AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, represents an energy sensor able to adapt cellular metabolism in response to nutritional environmental variations. TCR stimulation activates AMPK, a regulatory event that is known to stimulate ATP-producing processes, possibly in anticipation of the increased energetic needs associated with cell division and expression of effector function. Taking advantage of the selective expression of the AMPKalpha1 catalytic subunit in lymphoid cells, we have analyzed the in vitro and in vivo capacity of lymphocytes lacking AMPK activity (AMPKalpha1-KO cells) to respond to metabolic stress and to initiate and sustain an immune response. AMPKalpha1-KO cells displayed increasing sensitivity to energetic stress in vitro, and were found unable to maintain adequate ATP levels in response to ATP synthase inhibition. These cells were, however, able to respond to antigen stimulation in vitro, as shown by optimal proliferation and cytokine production. Similarly, AMPKalpha1-KO mice were fully immunocompetent in vivo and displayed normal cell proliferation, humoral, cytotoxic and delayed-type hypersensitivity (DTH) responses following antigen injection. In conclusion, AMPK represents an important enzyme allowing lymphocytes to resist a mild energy crisis in vitro, but is largely dispensable for activation and expression of effector function in response to antigen stimulation.     

AMPA type glutamate receptor mediates neurotransmission at turtle vestibular calyx synapse

AMPK is a serine/threonine protein kinase, which serves as an energy sensor in all eukaryotic cell types. Published studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumour cells. These actions of AMPK appear to be mediated through multiple mechanisms including regulation of the cell cycle and inhibition of protein synthesis, de novo fatty acid synthesis, specifically the generation of mevalonate as well as other products downstream of mevalonate in the cholesterol synthesis pathway. Cell cycle regulation by AMPK is mediated by up-regulation of the p53–p21 axis as well as regulation of TSC2–mTOR (mammalian target of rapamycin) pathway. The AMPK signalling network contains a number of tumour suppressor genes including LKB1, p53, TSC1 and TSC2, and overcomes growth factor signalling from a variety of stimuli (via growth factors and by abnormal regulation of cellular proto-oncogenes including PI3K, Akt and ERK). These observations suggest that AMPK activation is a logical therapeutic target for diseases rooted in cellular proliferation, including atherosclerosis and cancer. In this review, we discuss about exciting recent advances indicating that AMPK functions as a suppressor of cell proliferation by controlling a variety of cellular events in normal cells as well as in tumour cells.     

Metabolomics of the effect of AMPK activation by AICAR on human umbilical vein endothelial cells

AMP-activated protein kinase (AMPK) is a metabolic master switch expressed in a great number of cells and tissues. AMPK is thought to modulate the cellular response to different stresses that increase cellular AMP concentration. The adenosine analog, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is an AMPK activator used in many studies to assess the effects of AMPK activation on cellular metabolism and function. However, the effect of AICAR on cell metabolism reaches many different pathways and metabolites, some of which do not seem to be fully related to AMPK activation. We have now for the first time used NMR metabolomics on human umbilical vein endothelial cells (HUVEC) for the study of the global metabolic impact of AMPK activation by AICAR. In our study, incubation with AICAR activates AMPK and is associated with, among others, broad metabolic alterations in energy metabolism and phospholipid biosynthesis. Using NMR spectroscopy and metabolic network tools, we analyzed the connections between the different metabolic switches activated by AICAR. Our approach reveals a strong interconnection between different phospholipid precursors and oxidation by-products. Metabolomics profiling is a useful tool for detecting major metabolic alterations, generating new hypotheses and provides some insight about the different molecular correlations in a complex system. The present study shows that AICAR induces metabolic effects in cell metabolism well beyond energy production pathways.     

Role of the AMPK pathway in promoting autophagic flux via modulating mitochondrial dynamics in neurodegenerative diseases: Insight into prion diseases

Neurons are highly energy demanding cells dependent on the mitochondrial oxidative phosphorylation system. Mitochondria generate energy via respiratory complexes that constitute the electron transport chain. Adenosine triphosphate depletion or glucose starvation act as a trigger for the activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK is an evolutionarily conserved protein that plays an important role in cell survival and organismal longevity through modulation of energy homeostasis and autophagy. Several studies suggest that AMPK activation may improve energy metabolism and protein clearance in the brains of patients with vascular injury or neurodegenerative disease. Mild mitochondrial dysfunction leads to activated AMPK signaling, but severe endoplasmic reticulum stress and mitochondrial dysfunction may lead to a shift from autophagy towards apoptosis and perturbed AMPK signaling. Hence, controlling mitochondrial dynamics and autophagic flux via AMPK activation might be a useful therapeutic strategy in neurodegenerative diseases to reinstate energy homeostasis and degrade misfolded proteins. In this review article, we discuss briefly the role of AMPK signaling in energy homeostasis, the structure of AMPK, activation mechanisms of AMPK, regulation of AMPK, the role of AMPK in autophagy, the role of AMPK in neurodegenerative diseases, and finally the role of autophagic flux in prion diseases.     

A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway

BACKGROUND: The mammalian target of rapamycin (mTOR) regulates multiple cellular functions including translation in response to nutrients, especially amino acids. AMP-activated protein kinase (AMPK) modulates metabolism in response to energy demand by responding to changes in AMP. RESULTS: The treatment of SV40-immortalized human corneal epithelial cells (HCE-T cells) with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), widely used as an AMPK activator, inhibits p70 S6k activities. Altered glucose availability, which regulates AMPK activity, also modulates the activity of p70 S6k. AICAR treatment also inhibits phosphorylation of Thr-412 in the p70 S6 kinase (p70 S6k), which is indispensable for the activity. Furthermore, over-expression of mutant AMPK subunits by stable expression in rabbit pulmonary fibroblast cell lines (PS120 cells) also modulates p70 S6k activity. The insensitivity of the rapamycin-resistant p70 S6k variant to AICAR treatment suggests that the inhibition of p70 S6k is mediated through a common effector, supporting a model whereby mTOR and its downstream effector are controlled by AMPK. CONCLUSION: These results indicate that the AMPK and mTOR signalling pathways are possibly linked. In addition to the mTOR signal acting as a priming switch that modulates p70 S6k activation, AMPK appears to provide an overriding switch linking p70 S6k regulation to cellular energy metabolism.     

AMPK通过抑制STING调节干扰素免疫应答通路的研究

目的探索腺苷酸活化蛋白激酶(AMPK)与cGAS-STING通路之间的联系及其在先天免疫中扮演的角色。方法利用CRISPR/Cas9技术、蛋白质印迹、RT-qPCR等方法,探究AMPK对DNA相关免疫通路的调控机制。结果在HT-DNA和cGAMP刺激下,AMPK-/-细胞株的IFN-β的表达量明显高于野生型细胞株,但这种变化在RNA信号通路中并不明显;激活AMPK可以抑制细胞内的DNA信号通路;在DNA信号通路中,AMPK-/-细胞株相较于野生型细胞株,STING在RNA和蛋白水平上都明显升高,即AMPK对cGAS-STING通路的抑制很可能是通过抑制STING起作用。结论AMPK在调节cGAS-STING介导的干扰素免疫应答中起重要作用。     

AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function

AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains cellular energy homeostasis. It arose very early during eukaryotic evolution, and its ancestral role may have been in the response to starvation. Recent work shows that the kinase is activated by increases not only in AMP, but also in ADP. Although best known for its effects on metabolism, AMPK has many other functions, including regulation of mitochondrial biogenesis and disposal, autophagy, cell polarity, and cell growth and proliferation. Both tumor cells and viruses establish mechanisms to down-regulate AMPK, allowing them to escape its restraining influences on growth.