腺苷-磷酸激活蛋白激酶激活剂研究进展

腺苷—磷酸激活蛋白激酶（AMPK）是调控能量代谢的重要激酶,在代谢障碍、心血管疾病及肿瘤等疾病的病理进程中都有重要的调节作用。对AMPK的结构及其生理调节作用进行介绍,并重点综述AMPK间接激活剂和直接激活剂的研究进展,旨在为AMPK激活剂的深入开发提供参考。     

蛋白激酶AMPK高效制备及活性检测方法的建立

目的 建立高效的腺苷酸依赖的蛋白激酶（AMPK）的制备方法,并建立1种基于酶联免疫吸附（ELISA）法的AMPK的激酶活性检测方法以用于药物筛选。方法 利用大肠杆菌表达系统,将AMPK的α、β和γ 3个亚基与钙调素依赖性蛋白激酶β(CAMKK β)共表达,重组AMPK被CAMKK β磷酸化修饰并激活,进而纯化得到具有激酶活性的重组AMPK蛋白复合体。根据AMPK底物乙酰辅酶A羧化酶（ACC）的磷酸化修饰位点（Ser79）设计合成生物素标记的多肽（SAMS）作为AMPK激酶的底物。在ATP存在的情况下AMPK对生物素标记的SAMS多肽进行磷酸化修饰,反应产物转移到链霉亲和素包被的酶标板中,SAMS多肽通过链霉亲和素结合到酶标板。以能够识别SAMS磷酸化修饰的抗体作为一抗进行ELISA检测,SAMS的磷酸化修饰水平反应AMPK的激酶活性。结果 利用大肠杆菌表达系统成功得到有激酶活性的AMPK蛋白复合体,建立了ELISA检测AMPK的激酶活性方法。通过对AMPK激活剂A-769662检测,证明该方法可以用于AMPK激活剂/抑制剂的筛选。结论 通过构建大肠杆菌共表达系统,成功获得了AMPK激酶...     

瑞舒伐他汀对内皮细胞中组织因子途径抑制物表达的影响

目的探讨瑞舒伐他汀对组织因子途径抑制物(TF-PI)表达的调节作用及机制。方法培养人脐静脉内皮细胞(HUVECs),给予多种剂量的瑞舒伐他汀和(或)肿瘤坏死因子-α(TNF-α)、磷酸腺苷激活的蛋白激酶(AMPK)抑制剂与激活剂处理细胞,利用实时定量PCR、Western blot技术检测内皮细胞中TFPI表达的变化。结果 TNF-α下调内皮细胞中TFPI的的表达水平,而这一作用被瑞舒伐他汀所阻断。而且,瑞舒伐他汀呈时间及剂量依赖性上调TFPI的mRNA与蛋白水平。AMPK抑制剂———Compoud C则可逆转瑞舒伐他汀对TFPI表达的上调作用,而AMPK激动剂———Aicar则具有与瑞舒伐他汀相似的上调TFPI的作用。结论瑞舒伐他汀通过激活AMPK途径上调血管内皮细胞中TFPI的表达。     

AMPK信号通路在糖尿病心肌病中的研究进展

AMP依赖的蛋白激酶(AMPK)是调节生物能量恒定性的重要传感分子,能激活上游激酶LKB1、Ca MKKβ和Tak1等。AMP/ATP比值可调节AMPK的活性,从而控制能量代谢的分解与合成通路,骨骼肌收缩与心肌缺血时能够激活AMPK,调节脂肪细胞的代谢和血管功能,促进葡萄糖转运和脂肪酸氧化。排除动脉粥样硬化、高血压及其他潜在病因,由糖尿病引发的心肌结构与功能异常的一种独立的原发病,称之为糖尿病心肌病,可引起患者死亡。由于血糖水平、脂肪酸氧化和糖原代谢受AMPK的调节,因此,在DCM形成过程中AMPK起着至关重要的作用。该文综述了AMPK信号通路在糖尿病心肌病发生、发展过程中的作用。     

腺苷酸活化蛋白激酶在肺部疾病中的作用

腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)是一种广泛参与人体内能量代谢活动的蛋白激酶。新近的研究发现,AMPK可调控多种疾病发生、发展过程中的病理环节。近年来,AMPK与肺部疾病的关系愈发受到人们的重视,目前研究认为AMPK对肺癌、支气管哮喘及肺动脉高压等肺部疾病的治疗具有潜在的作用。该文就AMPK的生物学特性及其对多种肺部疾病的潜在治疗作用进展做一综述。     

腺苷酸活化蛋白激酶在卵巢肿瘤中的表达及临床意义

目的探讨腺苷酸活化蛋白激酶(AMPK)在卵巢肿瘤组织中的表达及临床意义。方法收集手术标本存档蜡块56份。其中,正常卵巢组织6份,卵巢良性上皮性肿瘤组织20份,卵巢癌组织30份。用免疫组化比较各组蜡块标本AMPKα1、AMPKα2及p-AMPK蛋白表达。结果AMPKα1蛋白表达于细胞的胞浆;卵巢癌组织中AMPKα1蛋白的阳性表达率为30%,与卵巢良性肿瘤组织的35%和正常卵巢组织的33%相仿(P>0.05)。AMPKα2蛋白也表达于胞浆,少量表达于胞核;其在卵巢癌中的阳性表达率为80%,明显高于卵巢良性肿瘤组织的45%和正常卵巢组织的33%(P<0.05)。卵巢癌中p-AMPK蛋白表达明显低于卵巢良性肿瘤组织和正常卵巢组织(P<0.05)。AMPKα1和AMPKα2蛋白的表达与不同临床病理指标之间无明显相关性。结论卵巢癌组织中AMPKα2蛋白高表达,表明AMPK信号传导途径在卵巢癌发病机制中有重要作用。     

FUNDC1通过调控线粒体分裂影响高糖损伤H9c2心肌细胞凋亡的机制研究

目的 探讨含FUN14域蛋白1（FUNDC1）在高糖损伤的H9c2心肌细胞中通过调控线粒体分裂影响心肌细胞凋亡的作用机制。方法 体外培养H9c2心肌细胞并建立高糖损伤模型,分别进行细胞转染,使用腺苷酸活化蛋白激酶（AMPK）激活剂5-氨基-4-咪唑羧基酰胺核苷（AICAR）后,分为正常对照组（CTRL组）、高糖组（HG...     

EMAP-Ⅱ通过LKB1/AMPK/mTOR信号通路增加BTB的通透性

目的研究内皮-单核细胞激活多肽(EMAP-Ⅱ)对体外血肿瘤屏障(BTB)通透性的影响及可能机制。方法建立体外BTB模型,EMAP-Ⅱ处理后,millipore电阻测量系统检测跨内皮细胞阻抗(TEER)值,采用Western blot方法检测p LKB1/LKB1、p AMPK/MAPK和p-m TORC1/m TORC1的蛋白表达水平;分别应用LKB1抑制剂radicicol、AMPK抑制剂compound C和m TORC1激活剂IGF1进行预处理后再给予EMAPⅡ,检测体外BTB模型的TEER值,Western blot方法检测紧密连接相关蛋白ZO-1和occludin的蛋白表达水平。结果与EMAP-Ⅱ0 h组相比,EMAP-Ⅱ可明显降低体外BTB模型的TEER值,增加p-LKB1/LKB1和p-AMPK/AMPK的表达水平,降低p-m TORC1/m TORC1的表达水平,以上指标在EMAP-Ⅱ作用1 h时效果最明显;radicicol、compound C和IGF1预处理能够部分阻断EMAP-Ⅱ的作用,使体外BTB模型的TEER值及紧密连接相关蛋白ZO-1和occludin的表达水平明显增加。结论 EMAP-Ⅱ能明显增加体外BTB模型通透性,其机制可能与激活LKB1/AMPK/m TOR信号通路相关。     

AMPK与肝脏糖异生研究进展

腺苷酸活化蛋白激酶(AMPK)是一种广泛参与多种代谢调节的激酶,是主要的细胞“能量感受器”。AMPK通过α亚基的Thr172发生磷酸化而激活,通过抑制脂质合成相关转录因子以及肝脏糖异生来控制脂质合成以及血糖稳定。肝脏糖异生的调控过程中,通过激素及转录因子如 、 、 、 、 等的参与,最后将信号传达至糖异生的二个关键性限速酶基因 与 ,起到调控作用。激活的AMPK能抑制糖异生转录因子及相关关键酶的有效表达,阻止肝脏发生糖异生,进而下降血糖浓度。本次综述旨在讨论与总结AMPK在肝脏糖脂代谢中的作用,重点分析AMPK与肝脏糖异生信号通路转录因子之间的联系。     

Central AMPK contributes to sleep homeostasis in mice

AMP-activated protein kinase (AMPK) is an energy-sensing molecular signal involved in glucose and lipid metabolism. The known interaction of sleep with energy metabolism led us to investigate the role of central AMPK in sleep homeostasis. Sleep deprivation (SD) for 6 h increased p-AMPK protein in the hypothalamus and also increased the mRNA level of Ca²⁺/calmodulin (CaM)-dependent protein kinase kinase β (CaMKK2), an activator of AMPK, and carnitine palmitoyltransferase 1 (CPT1), a downstream signaling factor of AMPK. Central injection of compound C (CC), an inhibitor of AMPK, suppressed EEG delta power during NREM sleep, while 5-aminoimidazole-4-carboxamide riboside (AICAR), an activator of AMPK, enhanced EEG delta power. The treatment of both CC and AICAR attenuated rebound responses of delta power in NREM sleep after SD. These results indicate that central AMPK is involved in the regulation of sleep depth and sleep homeostasis.     

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside alleviated carbon tetrachloride-induced acute hepatitis in mice

AMP-activated protein kinase (AMPK) is one of the principal cellular energy sensors participating in maintenance of energy balance but recent evidences also suggested that AMPK might be involved in the regulation of inflammation. In the present study, the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) was used to investigate the potential roles of AMPK in carbon tetrachloride (CCl₄)-induced acute hepatitis. The experimental data indicated that treatment with AICAR significantly decreased the elevation of plasma aminotransferases and alleviated hepatic histological abnormalities in CCl₄-exposed mice. Treatment with AICAR also inhibited the increase of myeloperoxidase (MPO), the induction of TNF-α, IL-6, inducible nitric oxide synthase (iNOS), nitric oxide and the upregulation of matrix metalloproteinase 2 (MMP-2), MMP-3 and MMP-9 in mice exposed to CCl₄. These effects were associated with suppressed nuclear accumulation of NF-κB p65. These results indicated that the AMPK activator AICAR effectively suppressed the inflammatory responses and alleviated liver damage induced by CCl₄, implying that AMPK activation might be beneficial for ameliorating inflammation-based liver damage.     

AMPK-independent down-regulation of cFLIP and sensitization to TRAIL-induced apoptosis by AMPK activators

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a TNF superfamily member that is being considered as a new strategy in anticancer therapy because of its ability to induce apoptosis, alone or in combination with other stimuli, in many cancer cells. AMP-activated protein kinase (AMPK) is an evolutionarily conserved key regulator of cellular energy homeostasis that protects the cell from energy depletion and stress by activating several biochemical pathways that lead to the conservation, as well as generation, of ATP. Here we report that a number of AMPK activators, including the small molecule activator A-769662, markedly sensitize TRAIL-resistant breast cancer cells to TRAIL-induced apoptosis. However, silencing AMPKα1 expression with siRNA or over-expression of DN-AMPKα1 does not inhibit AICAR, glucose deprivation, phenformin or A-769662-induced sensitization to TRAIL. Furthermore, the expression of constitutively active AMPK subunits does not sensitize resistant breast cancer cells to TRAIL-induced apoptosis. The cellular FLICE-inhibitory proteins (cFLIP_L and cFLIP_S) were significantly down-regulated following exposure to AMPK activators through an AMPK-independent mechanism. Furthermore, in cells over-expressing cFLIP_L, sensitization to TRAIL by AMPK activators was markedly reduced. In summary, our results indicate that AMPK activators facilitate the activation by TRAIL of an apoptotic cell death program through a mechanism independent of AMPK and dependent on the down-regulation of cFLIP levels.     

AMPK activator C24 inhibits hepatic lipogenesis and ameliorates dyslipidemia in HFHC diet-induced animal models

Dyslipidemia is a chronic metabolic disease characterized by elevated levels of lipids in plasma.Recently,various studies demonstrate that the increased activity of adenosine 5′-monophosphate-activated protein kinase(AMPK)causes health benefits in energy regulation.Thus,great efforts have been made to develop AMPK activators as a metabolic syndrome treatment.In the present study,we investigated the effects of the AMPK activator C24 on dyslipidemia and the potential mechanisms.We showed that C24(5-40μM)dose-dependently increased the phosphorylation of AMPKαand acetyl-CoA carboxylase(ACC),and inhibited lipogenesis in HepG2 cells.Using compound C,an AMPK inhibitor,or hepatocytes isolated from liver tissue-specific AMPK knockout AMPKα1α2fl/fl;Alb-cre mice(AMPK LKO),we demonstrated that the lipogenesis inhibition of C24 was dependent on hepatic AMPK activation.In rabbits with high-fat and high-cholesterol diet-induced dyslipidemia,administration of C24(20,40,and 60 mg·kg^?1·d^?1,ig,for 4 weeks)dose-dependently decreased the content of TG,total cholesterol(TC),and low-density lipoprotein cholesterol(LDL-C)in plasma and played a role in protecting against hepatic dysfunction by decreasing lipid accumulation.A lipid-lowering effect was also observed in high-fat and high-cholesterol diet-fed hamsters.In conclusion,our results demonstrate that the small molecular AMPK activator C24 alleviates hyperlipidemia and represents a promising compound for the development of a lipid-lowering drug.     

AMPK/mTOR信号通路在肿瘤中的研究现状

肿瘤是信号传递过程中发生紊乱,导致细胞无限增生而形成的恶性结果.针对异常传递通路的深入研究,以发现可用的高效作用靶点是目前关于肿瘤治疗的研究热点.单磷酸腺苷活化蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTOR)信号通路在细胞和个体水平维持能量及氧化还原平衡方面起着举足轻重的作用,信号传递异常可能打破这种平衡而引发肿瘤.该文将对AMPK/mTOR信号通路及其激动剂在肿瘤中的研究现状进行综述.     

δ-Opioid receptors stimulate the metabolic sensor AMP-activated protein kinase through coincident signaling with G(q/11)-coupled receptors

AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-D-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G(q/11)-coupled receptors, potentiating Ca(2+) release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake.     

Vascular AMPK as an attractive target in the treatment of vascular complications of obesity

The key for the survival of all organisms is the regulation and control of energy metabolism. Thus, several strategies have evolved in each tissue in order to balance nutrient supply with energy demand. Adenosine monophosphate-activated protein kinase (AMPK) is now recognized as a key participant in energy metabolism. It ensures an appropriate energetic supply by promoting energy conserving pathways in detriment of anabolic processes not essential for cell survival. Vascular AMPK plays a critical role in the regulation of blood flow and vascular tone through several mechanisms, including vasodilation by stimulating nitric oxide release in endothelial cells. Since obesity leads to endothelial damage and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. In the present review we focus on the role of vascular AMPK in both endothelial and smooth muscle cells, paying special attention to its dysregulation in obesity- and high-fat diet-related complications, as well as to the mechanisms and benefits of vascular AMPK activation.